Title of Invention: BASE-PROTECTED OLIGONUCLEOTIDE
Technical Field
5 [OOOl]
The present invention relates to a particular
oligonucleotide comprising a protected base and a production
method of an oligonucleotide using the same. In addition, the
present invention relates to a particular branched chain-
10 containing aromatic protecting group, an oligonucleotide having
a 3'-hydroxyl group protected by said protecting group, and a
production method of an oligonucleotide using the
oligonucleotide having a 3'-hydroxyl group protected by said
protecting group.
15 Background Art
[0002]
The synthesis method of oligonucleotide includes a
phosphate triester method, an H-phosphonate method, a
phosphoramidite method and the like, and solid phase synthesis
20 (solid phase method) using a phosphoramidite method is most
widely used at present (non-patent document 1). The solid
phase method is advantageous from the aspect of speed, since
process has been optimized and automation has progressed.
However, it is associated with defects in that scaling-up is
25 limited due to facility restriction, reagents and starting
materials are used in excess, and confirmation of the progress
status of the reaction in an intermediate step, analysis of
intermediate structure and the like are difficult.
[0003]
The synthesis methods of oligonucleotide by a liquid
phase method have also been studied. Generally, however,
treatments after each reaction are performed by a method
including (1) directly concentrating the reaction mixture,
followed by isolation and purification by silica gel column
35 chromatography, (2) extracting with a solvent such as methylene
chloride, chloroform and the like, washing with an aqueous
solution, concentrating, purifying by silica gel column
chromatography, and the like, and the operation is complicated
and the yield is low. In particular, a large-scale, rapid
5 synthesis of a long oligonucleotide is difficult, and the
methods are impractical as industrial production processes.
[0004]
In recent years, a pseudo-solid phase method-like
approach has been reported as an attempt to solve the
l o respective defects of the liquid phase method and the solid
phase method, and an oligonucleotide production method using a
soluble polymer such as monomethoxypolyethylene glycol (MPEG)
and the like as a protecting group is disclosed as one example
thereof (non-patent document 2 ) . However, while synthetic
15 examples of up to 20mer DNA are disclosed, a crystallization
isolation operation is essential for each reaction, and the
progress status of the reaction and the like are difficult to
confirm, since MPEG molecule itself is not a unimolecule.
[0005]
20 In addition, as a pseudo-solid phase method-like method,
a production method of oligonucleotide including use of an
ionic liquid as a protecting group has been reported, and
Synthetic Examples of DNA up to pentamers are shown (non-patent
document 3). However, the method is inferior to a solid phase
25 method in the speed and efficiency, since a crystallization
isolation operation is essential for each reaction and an
operation time is necessary therefor.
[0006]
Furthermore, a synthesis method of oligonucleotide
30 comprising use of a hydrophobic group-linked nucleoside is
disclosed (patent document 1). While it has been reported that
the method affords synthesis of 2lmer oligonucleotide, it is
markedly complicated, since the method requires solidification
isolation in every step of deprotection of 5'-protecting group,
35 coupling and oxidation.
Document List
Patent Document
[0007]
Patent Document 1: JP-A-2010-275254
5 Non-Patent Documents
[0008]
Non-Patent Document 1: S. L. Beaucage, D. E. Bergstorm, G. D.
Glick, R. A. Jones, Current Protocols in Nucleic Acid
Chemistry; John Wiley & Sons (2000)
10 Non-Patent Document 2: Nucleic Acid Res., 1990, Vol. 18, No. 11,
3155-3159
Non-Patent Document 3: J. Org. Chem., 2006, Vol. 71, No.20,
7907-7910
Summary of the Invention
15 Problems to be Solved by the Invention
[0009]
The problem of the present invention is provision of a
protected nucleotide for elongation, which can afford a
production method of oligonucleotide by a phosphoramidite
20 method, which enables efficient purification in a high yield by
a liquid-liquid extraction operation.
Means of Solving the Problems
[OOlO]
As a result of the intensive studies, the present
25 inventors have found that the above-mentioned problem can be
achieved by a particular oligonucleotide comprising a protected
base, which resulted in the completion of the present invention.
The present invention includes the following.
[OOll]
30 [I] An oligonucleotide comprising a protected base, which is
represented by the formula (I) :
[OOlZ]
[0013]
wherein q is any i n t e g e r of not l e s s than 0;
~ a s e *i n the number of q+l are each independently a nucleic
5 acid base protected by a group having a C5-30 s t r a i g h t chain or
branched chain alkyl group and/or a C5-30 s t r a i g h t chain or
branched chain alkenyl group;
P' is a hydrogen atom, or a temporary protecting group
removable under acidic conditions;
10 X is a hydrogen atom, an optionally protected hydroxyl group, a
halogen atom or an organic group crosslinked with the 4-
p o s i t i o n carbon atom;
X' i n the number of q are each independently a hydrogen atom,
an optionally protected hydroxyl group, a halogen atom or an
15 organic group crosslinked with the 4-position carbon atom;
p2 i n the number of q+l are each independently a protecting
group removable under basic conditions;
R~~ i n the number of q are each independently an oxygen atom or
a s u l f u r atom; and
20 Re and Rf are each independently a C1-6 alkyl group, or a 5- or
6-membered saturated cyclic amino group formed together with
the adjacent nitrogen atom.
[0014]
[ Z ] The oligonucleotide comprising a protected base o f [ I ] ,
wherein q is 0.
[3] The oligonucleotide comprising a protected base of [ I ] or
5 [ Z ] , wherein the group having a C5-30 s t r a i g h t chain or branched
chain a l k y l group and/or a C5-30 s t r a i g h t chain or branched
c h a i n a l k e n y l group is
a group represented by the formula ( k ) :
[0015]
0
[0016]
wherein * i n d i c a t e s t h e bonding p o s i t i o n t o a n u c l e i c a c i d
base;
RZ7 is a C5-30 s t r a i g h t chain or branched chain a l k y l group or a
15 C5-30 s t r a i g h t chain or branched c h a i n a l k e n y l group,
a group represented by the formula (1) :
[0017]
[0018]
20 wherein * i n d i c a t e s the bonding p o s i t i o n t o a n u c l e i c acid
base;
Q1 is -0-, -S- or - N R ~ O - wherein R30 is a hydrogen atom or a C1-22
a l k y l group;
Rc and Rd are each independently a hydrogen atom or a C1-22 a l k y l
25 group; and
R~~ is a C5-30 s t r a i g h t chain or branched chain a l k y l group or a
C5-30 s t r a i g h t chain or branched c h a i n a l k e n y l group,
a group represented by the formula (m) :
[0019]
[0020]
wherein * indicates the bonding p o s i t i o n t o a nucleic acid
base;
5 1 is an integer of 1 t o 5;
Q2 i n the number of 1 are each independently a s i n g l e bond, or
-0-, -S-, -0C (=O) -, -C (=O) 0-, -0-CH2-, -NH-, -NHC (=O) -, -
C (=O) NH-, -NH-CH2- or -CH2-;
R2' i n the number of 1 are each independently a C5-30 s t r a i g h t
lo chain or branched chain alkyl group or a C5-30 s t r a i g h t chain or
branched chain alkenyl group;
r i n g C is a benzene ring or a cyclohexane ring, each optionally
having, i n addition t o Q~R" i n the number of 1 and *c=o, a
s u b s t i t u e n t selected from the group consisting of a halogen
15 atom, a C1-6 alkyl group optionally s u b s t i t u t e d by one or more
halogen atoms, and a C1-6 alkoxy group optionally s u b s t i t u t e d by
one or more halogen atoms, or
a group represented by the formula (s) :
[0021]
[0022]
wherein * indicates the position a t which an imino bond is
formed with an amino group of a nucleic acid base; and
R~~ and R~~ are each independently a C5-30 s t r a i g h t chain or
25 branched chain alkyl group or a C5-30 s t r a i g h t chain or branched
chain a1 kenyl group.
[0023]
[ 4 ] The oligonucleotide comprising a protected base of [3],
wherein R ~ R~2*,, R2' i n the number of 1, R~~ and R~~ are each
30 independently a branched chain alkyl group or branched chain
alkenyl group selected from the group consisting of a
2,6,10,14-tetramethylpentadecyl group, a 2,6,10-
trimethylundecyl group, a 2,2,4,8,10,10-hexamethyl-5-undecyl
group, a 2,6,10-trimethylundeca-1,5,9-trienyl group, a 2,6-
5 dimethylheptyl group, a 2,6-dimethylhept-5-enyl group, a 2,6-
dimethylhepta-1,5-dienyl group, a 9-nonadecyl group, a 12-
methyltridecyl group, an 11-methyltridecyl group, an 11-
methyldodecyl group, a 10-methylundecyl group, an 8-heptadecyl
group, a 7-pentadecyl group, a 7-methyloctyl group, a 3-
lo methyloctyl group, a 3,7-dimethyloctyl group, a 3-methylheptyl
group, a 3-ethylheptyl group, a 5-undecyl group, a 2-heptyl
group, a 2-methyl-2-hexylbgroup, a 2-hexyl group, a 3-heptyl
group, a 4-heptyl group, a 4-methyl-pentyl group, a 3-methylpentyl
group, and a 2,4,4-trimethylpentyl group; or a straight
15 chain alkyl group selected from the group consisting of a
tetradecyl group, a tridecyl group, a dodecyl group, an undecyl
group, a decyl group, a nonyl group, an octyl group, a heptyl
group, a hexyl group, and a pentyl group.
[0024]
20 [5] The oligonucleotide comprising a protected base of any one
of [I] to [4], wherein the C5-30 straight chain or branched
chain alkyl group and/or C5-30 straight chain or branched chain
alkenyl group is a C5-30 branched chain alkyl group and/or a C5-
30 branched chain alkenyl group.
25 [0025]
[6] The oligonucleotide comprising a protected base of any one
of [I] to [5], wherein P' is a monomethoxytrityl group or a
dimethoxytrityl group.
[0026]
30 [7] A method of producing an oligonucleotide, comprising using
the oligonucleotide comprising a protected base of any one of
[I1 to [GI
[8] A method of producing an n+p-mer oligonucleotide,
comprising
35 (2) a step of condensing a p-mer oligonucleotide comprising a
protected base (p is any integer of one or more) wherein the
3'-hydroxyl group is phosphoramidited, the 5'-hydroxyl group is
protected by a temporary protecting group removable under
acidic conditions, and the nucleic acid base is protected by a
5 group having a C5-30 straight chain or branched chain alkyl
group and/or a C5-30 straight chain or branched chain alkenyl
group, with an n-mer oligonucleotide (n is an integer of one or
more) wherein the 5'-hydroxyl group is not protected and the
3'-hydroxyl group is protected, by forming a phosphite triester
10 bond via the 5'-hydroxyl group thereof.
[0027]
[9] The production method of [8], wherein p is 1.
[lo] The production method of [8] or [9], further comprising
the following step (3):
15 (3) a step of converting the phosphite triester bond of the
n+p-mer oligonucleotide obtained in the condensation step to a
phosphate triester bond or a thiophosphate triester bond by
adding an oxidizing agent or a sulfurizing agent to the
reaction mixture obtained in the condensation step (2).
20 [0028]
[ll] The production method of any one of [8] to [lo], further
comprising the following step (1) :
(1) a step of removing the temporary protecting group removable
under acidic conditions of the 5'-hydroxyl group by reacting,
25 in a non-polar solvent prior to the cbndensation step (2)' an
n-mer oligonucleotide wherein the 3'-hydroxyl group is
protected, and the 5'-hydroxyl group is protected by a
temporary protecting group, with an acid.
[0029]
30 [I21 The production method of [Ill, wherein the step (1) is
performed in the presence of at least one kind of cation
scavenger selected from a pyrrole derivative and an indole
derivative, and further comprises a step of neutralization with
an organic base after removal of the temporary protecting group
35 of the 5'-hydroxyl group.
[0030]
[13] The production method of any one of [lo] to [12], further
comprising the following step (4):
(4) a step of isolating the n+p-mer oligonucleotide from the
5 reaction mixture obtained in step (3) by an extraction
operation alone.
[14] The method of [13], further comprising the following step
10 (5) a step of removing all the protecting groups of the n+p-mer
oligonucleotide obtained in step (4).
[0032]
[15] The production method of any one of [8] to [14] , wherein
the p-mer oligonucleotide comprising a protected base, wherein
15 the 3'-hydroxyl group is phosphoramidited, the 5'-hydroxyl
group is protected by a temporary protecting group removable
under acidic conditions, and the nucleic acid base is protected
by a group having a C5-30 straight chain or branched chain alkyl
group and/or a C5-30 straight chain or branched chain alkenyl
20 group, is the oligonucleotide comprising a protected base of
any one of [I] to [6] .
[0033]
[16] The production method of any one of [8] to [15], wherein
the 3'-hydroxyl group of the n-mer oligonucleotide is protected
25 by a group represented by the formula (111):
[0034]
-L-Y-z
[0035]
wherein
30 L is a group represented by the formula (al) :
[0036]
0
[0037]
* *
wherein * shows the bonding position to Y; indicates the
bonding position to a 3'-hydroxy group of the nucleotide;
L1 is an optionally substituted divalent C1-22 hydrocarbon
5 group; and
* *
L2 is a single bond, or a group represented by C (=O)N( R2)- R1-
* * ***
N(R3)*** wherein shows the bondingposition to L1, shows
the bonding position to C=O, R1 is an optionally substituted C1-
22 alkylene group, and R2 and R3 are each independently a
10 hydrogen atom or an optionally substituted C1-22 alkyl group, or
R2 and R3 are optionally joined to form an optionally
substituted C1-22 alkylene bond,
Y is an oxygen atom or NR wherein R is a hydrogen atom, an
alkyl group or an aralkyl group, and
15 Z is a group represented by the formula (a2):
[0038]
[0039]
wherein * shows the bonding position to Y;
20 R4 is a hydrogen atom, or when Rb is a group represented by the
following formula (a3), R4 is optionally a single bond or -0-
in combination with R6 to form a fluorenyl group or a xanthenyl
group together with ring B;
Q in the number of k are each independently a single bond, or -
25 0-, -S-, -0C (=O) -, -NHC (=O) - or -NH-;
R5 in the number of k are each independently an organic group
having at least one aliphatic hydrocarbon group having one or
more branched chains and the total carbon number of not less
than 14 and not more than 300;
30 k is an integer of 1 to 4;
ring A optionally further has, in addition to R4, Q R ~ in the
number of k and *C (R,) (Rb), a substituent selected from the
group consisting of a halogen atom, a C1-6 alkyl group
optionally substituted by one or more halogen atoms, and a C1-6
alkoxy group optionally substituted by one or more halogen
5 atoms;
R, is a hydrogen atom; and
Rb is a hydrogen atom, or a group represented by the formula
(a3) :
wherein * indicates a bonding position;
j is an integer of 0 to 4;
Q in the number of j are each independently as defined above;
15 R7 in the number of j are each independently an organic group
having at least one aliphatic hydrocarbon group having one or
more branched chains and the total carbon number of not less
than 14 and not more than 300;
R6 is a hydrogen atom, or optionally a single bond or -0- in
20 combination with R4 to form a fluorenyl group or a xanthenyl
group together with ring A; and
ring B optionally further has, in addition to QR~ in the number
of j and R6, a substituent selected from the group consisting
of a halogen atom, a C1-6 alkyl group optionally substituted by
25 one or more halogen atoms, and a C1-6 alkoxy group optionally
substituted by one or more halogen atoms.
[17] The production method of any one of [8] to [16], wherein
at least one nucleic acid base of the n-mer oligonucleotide is
30 protected by a group having a C5-30 straight chain or branched
chain alkyl group and/or a C5-30 straight chain or branched
chain alkenyl group.
[18] The production method of any one of [8] to [17], wherein
the group having a C5-30 straight chain or branched chain alkyl
group and/or a C5-30 straight chain or branched chain alkenyl
group is a group represented by the formula (k):
5 [0044]
[0045]
wherein * indicates the bonding position to a nucleic acid
base; and
lo R~~ is a C5-30 straight chain or branched chain alkyl group or a
C5-30 straight chain or branched chain alkenyl group,
a group represented by the formula (1) :
[004 61
15 [0047]
wherein * indicates the bonding position to a nucleic acid
base;
Q1 is -0-, -S- or -NR"- wherein R" is a hydrogen atom or a C1-22
alkyl group;
20 Rc and Rd are each independently a hydrogen atom or a C1-22 alkyl
group; and
RZ8 is a C5-30 straight chain or branched chain alkyl group or a
C5-30 straight chain or branched chain alkenyl group,
a group represented by the formula (m) :
25 [0048]
0
[0049]
wherein * indicates the bonding position to a nucleic acid
base;
1 is an integer of 1 to 5;
Q2 in the number of 1 are each independently a single bond, or
-0-, -S-, -0C (=O) -, -C (=O) 0-, -0-CH2-, -NH-, -NHC (=O) -, -
5 C (=O)NH-, -NH-CH2- or -CH2-;
R" in the number of 1 are each independently a C5-30 straight
chain or branched chain alkyl group or a C5-30 straight chain or
branched chain alkenyl group; and
ring C is a benzene ring or a cyclohexane ring each optionally
10 having, in addition to (12R2' in the number of 1 and *c=o, a
' substituent selected from the group consisting of a halogen
atom, a C1-6 alkyl group optionally substituted by one or more
halogen atoms, and a C1-6 alkoxy group optionally substituted by
one or' more halogen atoms) , or
15 a group represented by the formula (s) :
[0050]
[0051]
wherein * indicates the position at which an imino bond is
20 formed with an amino group of a nucleic acid base; and
R~~ and R~~ are each independently a C5-30 straight chain or
branched chain alkyl group or a C5-30 straight chain or branched
chain a1 kenyl group.
[0052]
25 [I91 The production method of [18], wherein R ~ R~ ~,R~ 2' ,in the
number of 1, R~~ and R~~ are each independently a branched chain
alkyl group or branched chain alkenyl group selected from the
group consisting of a 2,6,10,14-tetramethylpentadecyl group, a
2,6,10-trimethylundecyl group, a 2,2,4,8,10,10-hexamethyl-5-
30 undecyl group, a 2,6,10-trimethylundeca-l,5,9-trienyl group, a
2,6-dimethylheptyl group, a 2,6-dimethylhept-5-enyl group, a
2,6-dimethylhepta-l,5-dienyl group, a 9-nonadecyl group, a 12-
methyltridecyl group, an 11-methyltridecyl group, an 11-
methyldodecyl group, a 10-methylundecyl group, an 8-heptadecyl
group, a 7-pentadecyl group, a 7-methyloctyl group, a 3-
methyloctyl group, a 3,7-dimethyloctyl group, a 3-methylheptyl
group, a 3-ethylheptyl group, a 5-undecyl group, a 2-heptyl
5 group, a 2-methyl-2-hexyl group, a 2-hexyl group, a 3-heptyl
group, a 4-heptyl group, a 4-methyl-pentyl group, a 3-methylpentyl
group, and a 2,4,4-trimethylpentyl group; or a straight
chain alkyl group selected from the group consisting of a
tetradecyl group, a tridecyl group, a dodecyl group, an undecyl
10 group, a decyl group, a nonyl group, an octyl group, a heptyl
group, a hexyl group, and a pentyl group.
[0053]
[20] The production method of any one of [8] to [19], wherein
the C5-30 straight chain or branched chain alkyl group and/or C5-
15 30 straight chain or branched chain alkenyl group are/is a C5-30
branched chain alkyl group and/or a C5-30 branched chain alkenyl
group.
[0054]
[21] A pharmaceutical product comprising the oligonucleotide
20 produced by the production method of any one of [7] to [20].
[0055]
[22] An oligonucleotide protected by a branched chaincontaining
aromatic group, which is represented by the formula
wherein
m is any integer of 0 or more;
ase el in the number of m+l are each independently an optionally
5 protected nucleic acid base;
P' is a hydrogen atom, or a temporary protecting group
removable under acidic conditions;
X is a hydrogen atom, an optionally protected hydroxyl group, a
halogen atom or an organic group crosslinked with the 4-
10 position carbon atom;
X' in the number of m are each independently a hydrogen atom,
an optionally protected hydroxyl group, a halogen atom or an
organic group crosslinked with the 4-position carbon atom;
p2 in the number of m are each independently a protecting group
15 removable under basic conditions;
R~~ in the number of m are each independently an oxygen atom or
a sulfur atom;
L is a group represented by the formula (al) :
[0058]
0
* *
wherein * shows the bonding position to Y; indicates the
bonding position to a 3'-hydroxy group of the nucleotide;
L1 is an optionally substituted divalent C1-22 hydrocarbon
25 group; and
* *
L2 is a single bond, or a group represented by C(=O)N(R2) -R1-
* * ***
N(R3)***w herein shows the bonding position to L1, shows
the bonding position to C=O, R1 is an optionally substituted C1-
22 alkylene group, and R2 and R3 are each independently a
30 hydrogen atom or an optionally substituted C1-22 alkyl group, or
R2 and R3 are optionally joined to form an optionally
substituted C1-22 alkylene bond,
Y is an oxygen atom or NR wherein R is a hydrogen atom, an
alkyl group or an aralkyl group,. and
Z is a group represented by the formula (a2):
[0060]
[0061]
wherein * shows the bonding position to Y;
R4 is a hydrogen atom, or when Rb is a group represented by the
following formula (a3) , R4 is optionally a single bond or -0-
10 in combination with R6 to form a fluorenyl group or a xanthenyl
group together with ring B;
Q in the number of k are each independently a single bond, or -
0-, -S-, -0C (=O) -, -NHC (=O) - or -NH-;
R5 in the number of k are each independently an organic group
15 having at least one aliphatic hydrocarbon group having one or
more branched chains, and the total carbon number of not less
than 14 and not more than 300;
k is an integer of 1 to 4;
ring A optionally further has, in addition to R4, Q R ~ in the
20 number of k and *c(Ra) (Rb), a substituent selected from the
group consisting of a halogen atom, a C1-6 alkyl group
optionally substituted by one or more halogen atoms, and a C1-6
alkoxy group optionally substituted by one or more halogen
atoms;
25 Ra is a hydrogen atom; and
Rb is a hydrogen atom, or a group represented by the formula
(a3) :
[0062]
[0063]
wherein * indicates the bonding position;
j is an integer of 0 to 4;
5 Q in the number of j are each independently as defined above;
R7 in the number of j are each independently an organic group
having at least one aliphatic hydrocarbon group having one or
more branched chains and the total carbon number of not less
than 14 and not more than 300;
10 R6 is a hydrogen atom, or optionally a single bond or -0- in
combination with R4 to form a fluorenyl group or a xanthenyl
group together with ring A; and
ring B optionally further has, in addition to Q R ~ in the number
of j and R6, substituent (s) selected from the group consisting
15 of a halogen atom, a C1-6 alkyl group optionally substituted by
one or more halogen atoms, and a C1-6 alkoxy group optionally
substituted by one or more halogen atoms.
[0064]
[23] The oligonucleotide of [22], wherein m is 0.
20 [24] The oligonucleotide of [22] or [23], wherein R5 and R7 are
each independently a 3,7,11,15-tetramethylhexadecyl group, a
3,7,ll-trimethyldodecyl group, a 2,2,4,8,10,10-hexamethyl-5-
dodecanoyl group, a 3,4,5-tri (3', 7', 11' ,15'-
tetramethylhexadecyloxy)benzyl group, or a 3,5-
25 di(3',7',11',15'-tetramethylhexadecyloxy)benzyl group.
[0065]
[25] The oligonucleotide of [22] or [23], wherein -L-Y-Z is
selected from the group consisting of a 2-{2,4-di(Z1,3'-
dihydrophytyloxy)benzylaminocarbonyl)ethylcarbonyl group; a
30 3,5-di (2' ,3' -dihydrophytyloxy) benzylsuccinyl group; a 4- (2' ,3' -
dihydrophytyloxy)benzylsuccinyl group; a 2-{I-[(2-chloro-5-
(2' ,3'-
dihydrophytyloxy)phenyl)]benzylaminocarbonyl}ethylcarbonyl
group; a 3,4,5-tri(2',3'-dihydrophytyloxy)benzylsuccinyl group;
a 2-{3,4,5-tri (2',3'-
dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group; a 2-
5 {4-(2',3'-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl
group; a 2 - ( 2 - [ 3 ' , 4 ' , 5 ' - t r i ( 2 " , 3 " -
dihydrophytyloxy)benzyloxy]-4-
methoxybenzylaminocarbonyl}ethylcarbonyl group; a 2-{4-(2',3'-
dihydrophytyloxy)-2-methoxybenzylaminocarbonyl}ethylcarbonyl
10 group; a 4-(2',3'-dihydrophytyloxy)-2-methylbenzylsuccinyl
group; a 2- (4- (2', 3' -dihydrophytyloxy) -2-
methylbenzylaminocarbonyl}ethylcarbonyl group; a 4-
[2,2,4,8,lO,lO-hexamethyl-5-dodecanoylamino]benzylsuccinyl
group; a 2-(4- [2,2,4,8,10,10-hexamethyl-5-
15 dodecanoylamino]benzylaminocarbonyl}ethylcarbonyl group; a 4-
(3,7,11-trimethyldodecyloxy)benzylsuccinyl group; a 2-{4-
(3,7,11-trimethyldodecyloxy)benzylaminocarbonyl}ethylcarbonyl
group; a 2-{3,5-di(2',3'-
dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group; a 2-
20 (1- [ Z , 3,4-tri (2', 3'-
dihydrophytyloxy)phenyl]benzylaminocarbonyl}ethylcarbonyl
group; a 2-{1-[4-(2',3'-dihydrophytyloxy)phenyl]-4'-(2',3'-
dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group; a
3,4,5-tris [3,4,5-tri(Zf,3'-
25 dihydrophytyloxy)benzyl]benzylsuccinyl group; and a 2-{3,4,5-
tris [3,4,5-tri (2', 3'-
dihydrophytyloxy)benzyl]benzylaminocarbonyl}ethylcarbonyl group.
[0066]
[26] The oligonucleotide of any one of [22] to [25], wherein at
30 least one of the nucleic acid bases is protected by a group
having a C5-30 straight chain or branched chain alkyl group
and/or a C5-30 straight chain or branched chain alkenyl group.
[27] A protecting group of nucleotide 3'-hydroxyl group, which
is represented by the formula (111) :
35 [0067]
-L-Y-z (m)
[0068]
wherein
L is a group represented by the formula (al) :
5 [0069]
0
[0070]
* *
wherein * shows the bonding position to Y; indicates the
bonding position to a 3'-hydroxy group of the nucleotide;
l o L1 is an optionally substituted divalent C1-22 hydrocarbon
group; and
* *
L2 is a single bond, or a group represented by c(=o)N(R~)-R1-
* * ***
N(R3)*** wherein shows the bonding position to L1, shows
the bonding position to C=O, R1 is an optionally substituted C1-
15 22 alkylene group, and R2 and R3 are each independently a
hydrogen atom or an optionally substituted C1-22 alkyl group, or
R2 and R3 are optionally joined to form an optionally
substituted C1-22 alkylene bond,
Y is an oxygen atom or NR wherein R is a hydrogen atom, an
20 alkyl group or an aralkyl group, and
Z is a group represented by the formula (a2):
[0071]
[0072]
25 wherein * shows the bonding position to Y;
R4 is a hydrogen atom, or when Rb is a group represented by the
following formula (a3), R4 is optionally a single bond or -0-
in combination with R6 to form a fluorenyl group or a xanthenyl
group together with ring B;
Q in the number of k are each independently a single bond, or -
0-, -S-, -OC(=O)-, -NHC(=O)- or -NH-;
R5 in the number of k are each independently an organic group
5 having at least one aliphatic hydrocarbon group having one or
more branched chains and the total carbon number of not less
than 14 and not more than 300;
k is an integer of 1to 4;
ring A optionally further has, in addition to R4, Q R ~ in the
10 number of k and *c(R,) (Rb), a substituent selected from the
group consisting of a halogen atom, a C1-6 alkyl group
optionally substituted by one or more halogen atoms, and a C1-6
alkoxy group optionally substituted by one or more halogen
atoms;
15 R, is a hydrogen atom; and
Rb is a hydrogen atom, or a group represented by the formula
20 [0074]
wherein * indicates the bonding position;
j is an integer of 0 to 4;
Q in the number of j are each independently as defined above;
R7 in the number of j are each independently an organic group
25 having at least one aliphatic hydrocarbon group having one or
more branched chains and the total carbon number of not less
than 14 and not more than 300;
R6 is a hydrogen atom, or optionally a single bond or -0- in
combination with R4 to form a fluorenyl group or a xanthenyl
30 group together with ring A; and
ring B optionally further has, in addition to Q R ~ in the number
of j and R6, a substituent selected from the group consisting
of a halogen atom, a C1-6 alkyl group optionally substituted by
one or more halogen atoms, and a CIw6 alkoxy group optionally
substituted by one or more halogen atoms.
[0075]
[28] The protecting group of [27], wherein R5 and R7 are each
5 independently a 3,7,11,15-tetramethylhexadecyl group, a 3,7,11-
trimethyldodecyl group, a 2,2,4,8,10,10-hexamethyl-5-dodecanoyl
group, a 3,4,5-tri (3', 7', 11' ,15'-tetramethylhexadecyloxy) benzyl
group, or a 3,5-di (3', 7', 11' ,15' -tetramethylhexadecyloxy) benzyl
group.
lo [0076]
[29] The protecting group of [27], which is selected from the
group consisting of a 2-{2,4-di (2', 3' -
dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group; a
3,5-di (2' ,3' -dihydrophytyloxy) benzylsuccinyl group; a 4- (2' ,3' -
15 dihydrophytyloxy)benzylsuccinyl group; a 2-{I-[(2-chloro-5-
(2',3'-
dihydrophytyloxy)phenyl)]benzylaminocarbonyl}ethylcarbonyl
group; a 3,4,5-tri (2' ,3' -dihydrophytyloxy) benzylsuccinyl group;
a 2-{3,4,5-tri(2',3'-
20 dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group; a 2-
{4-(2',3'-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl
group; a 2 - ( 2 - [ 3 ' , 4 ' , 5 ' - t r i ( 2 " , 3 " -
dihydrophytyloxy) benzyloxy] -4-
methoxybenzylaminocarbonyl}ethylcarbonyl group; a 2-{4-(2',3'-
25 dihydr0phytyloxy)-2-methoxybenzylaminocarbonyl}ethylcarbonyl
group; a 4-(2',3'-dihydrophytyloxy)-2-methylbenzylsuccinyl
group; a 2- { 4- (2' ,3' -dihydrophytyloxy) -2-
methylbenzylaminocarbonyl}ethylcarbonyl group; a 4-
[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]benzy1succiny1
30 group; a 2-(4- [2,2,4,8,10,10-hexamethyl-5-
dodecanoylamino]benzylaminocarbonyl}ethylcarbonyl group; a 4-
(3,7,11-trimethyldodecyloxy)benzylsuccinyl group; a 2-14-
(3,7,11-trimethyldodecyloxy)benzylaminocarbonyl}ethylcarbonyl
group; a 2-{3,5-di (2', 3'-
35 dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group; a 2-
(1-[2,3,4-tri (2',3'-
dihydrophytyloxy)phenyl]benzylaminocarbonyl}ethylcarbonyl
group; a 2-{1-[4-(2',3'-dihydrophytyloxy)phenyl]-4'-(2',3'-
dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group; a
5 3'4'5-tris[3,4,5-tri(2',3'-
dihydrophytyloxy)benzyl]benzylsuccinyl group; and a 2-(3'4'5-
tris [3,4,5-tri (2'' 3'-
dihydrophytyloxy)benzyl]benzylaminocarbonyl}ethylcarbonyl group.
[0077]
lo [30] A method of producing an n'+p'-mer oligonucleotide
comprising :
(2') a step of condensing a p'-mer oligonucleotide (p' is any
integer of one or more) wherein the 3'-hydroxyl group is
phosphoramidited, the 5'-hydroxyl group is protected by a
15 temporary protecting group removable under acidic conditions,
and the nucleic acid base is optionally protected, with an n'-
mer oligonucleotide (n' is any integer of one or more) wherein
the 5'-hydroxyl group is not protected, and the 3'-hydroxyl
group is protected by the protecting group according to any one
20 of [27] to [29], by forming a phosphite triester bond via the
5' -hydroxyl group thereof.
[0078]
[31] The production method of [30], wherein p' is 1.
[32] The production method of [30] or [31], further comprising
25 the following step (3' ) :
(3') a step of converting the phosphite triester bond of the
nr+p'-mer oligonucleotide obtained by the condensation step to
a phosphate triester bond or a thiophosphate triester bond by
adding an oxidizing agent or a sulfurizing agent to the
30 reaction mixture obtained in the condensation step (2').
[0079]
[33] The production method of any one of [30] to [32], further
comprising the following step (1' ) :
(1') a step of removing a temporary protecting group removable
35 under acidic conditions of the 5'-hydroxyl group by reacting,
in a non-polar solvent prior to the condensation step (2')' the
n'-mer oligonucleotide wherein the 3'-hydroxyl group is
protected by the protecting group according to any one of [27]
to [29], and the 5'-hydroxyl group is protected by the
5 temporary protecting group, with an acid.
[0080]
[34] The production method of [33], wherein step (1') is
performed in the presence of at least one kind of cation
scavenger selected from a pyrrole derivative and an indole
lo derivative, and further comprises a step of neutralization with
an organic base after removal of the temporary protecting group
of the 5 ' -hydroxyl group.
[0081]
[35] The production method of any one of [32] to [34], further
15 comprising the following step (4') :
(4') a step of isolating the n'+p'-mer oligonucleotide from the
reaction mixture obtained in step (3') by an extraction
operation alone.
[0082]
20 [36] The production method of [35], further comprising the
following step (5' ) :
(5') a step of removing all the protecting groups of the n'+p'-
mer oligonucleotide obtained in step (4').
Effect of the Invention
25 [0083]
Using the particular oligonucleotide comprising a
protected base of the present invention, a production method of
an oligonucleotide by a phosphoramidite method, which enables
efficient purification in a high yield by a liquid-liquid
30 extraction operation, can be provided.
Using the oligonucleotide comprising a protected base,
particularly an oligonucleotide comprising a branched chainprotected
base, of the present invention, liposolubility and
solubility in an organic solvent (particularly, non-polar
35 solvent) of an intermediate oligonucleotide obtained in each
step of the nucleotide elongation reaction are strikingly
improved to enable isolation and purification by an extraction
operation alone, and therefore, a complicated, time-consuming
operation such as solidification isolation and the like is not
5 necessary, the speed increases, and the efficiency and
producibility are strikingly improved.
Furthermore, since an elongated oligonucleotide can be
isolated and purified by an extraction operation alone, an
elongation reaction in the next cycle can be sequentially
lo performed without taking out the resultant product from the
reaction apparatus, whereby an oligonucleotide can be produced
continuously in one pot.
It has been clarified that, as another embodiment of the
present invention, the same kind of solubility improving effect
15 can also be obtained by protecting a nucleotide 3'-hydroxyl
group with a protecting group having a particular structure of
a branched chain-containing aromatic group, and further, a
synergistic effect can be obtained by a combined use with an
oligonucleotide comprising a protected base.
20 Description of Embodiments
[0084]
The present invention relates to a novel oligonucleotide
comprising a protected base, wherein the 3'-hydroxyl group is
phosphoramidited, the 5'-hydroxyl group is protected by a
25 temporary protecting group removable under acidic conditions,
and the nucleic acid base is protected by a protecting group
having a C5-30 straight chain or branched chain alkyl group
and/or a C5-30 straight chain or branched chain alkenyl group.
In another embodiment, the present invention relates to a
30 production method of an oligonucleotide comprising using an
oligonucleotide comprising a protected base wherein the nucleic
acid base is protected by a group having a C5-30 straight chain
or branched chain alkyl group and/or a C5-30 straight chain or
branched chain alkenyl group, which preferably includes the
35 following step (2) :
(2) a step of condensing a p-mer oligonucleotide comprising a
protected base (p is any integer of one or more) wherein the
3'-hydroxyl group is phosphoramidited, the 5'-hydroxyl group is
protected by a temporary protecting group removable under
5 acidic conditions, and the nucleic acid base is protected by a
group having a C5-30 straight chain or branched chain alkyl
group and/or a C5-30 straight chain or branched chain alkenyl
group, with an n-mer oligonucleotide (n is an integer of one or
more) wherein the 5'-hydroxyl group is not protected and the
10 3'-hydroxyl group is protected, by forming a phosphite triester
bond via the 5'-hydroxyl group thereof to give an n+p-mer
oligonucleotide.
[0085]
In a still another embodiment, the present invention
15 relates to a novel oligonucleotide protected by a branched
chain-containing aromatic group, wherein the 3'-hydroxyl group
is protected by a particular branched chain-containing aromatic
protecting group, and the 5'-hydroxyl group is protected by a
temporary protecting group removable under acidic conditions.
20 In a yet another embodiment, the present invention.
relates to a particular branched chain-containing aromatic
protecting group used for protecting the nucleotide 3'-hydroxyl
group.
[0086]
25 In another embodiment, furthermore, the present invention
relates to a production method of an oligonucleotide,
comprising using an oligonucleotide wherein the 3'-hydroxyl
group is protected by the aforementioned branched chaincontaining
aromatic protecting group, and preferably includes
30 the following step (2' ) :
(2') a step of condensing a p'-mer oligonucleotide (p' is any
integer of one or more) wherein the 3'-hydroxyl group is
phosphoramidited, the 5'-hydroxyl group is protected by a
temporary protecting group removable under acidic conditions,
35 and the nucleic acid base is optionally protected, with an n'-
mer oligonucleotide (n' is any integer of one or more) wherein
the 5'-hydroxyl group is not protected, and the 3'-hydroxyl
group is protected by the aforementioned branched chaincontaining
aromatic protecting group, by forming a phosphite
5 triester bond via the 5'-hydroxyl group thereof to give an
n'+p'-mer oligonucleotide.
Explanations are given below.
100871
1. Explanation of terms
10 Unless otherwise specified in the sentences, any
technical terms and scientific terms used in the present
specification, have the same meaning as those generally
understood by those of ordinary skill in the art the present
invention belongs to. Any methods and materials similar or
15 equivalent to those described in the present specification can
be used for practicing or testing the present invention, and
preferable methods and materials are described in the following.
All publications and patents referred to in the specification
are hereby incorporated by reference so as to describe and
20 disclose constructed products and methodology described in, for
example, publications usable in relation to the described
invention.
[0088]
In the present specification, the "nucleoside" means a
25 compound wherein a nucleic acid base is bonded to the 1'-
position of a sugar (e.g., ribose, 2-deoxyribose, ribose
crosslinked between the 2-position and the 4-position and the
like) by N-glycosidation.
Examples of the ribose wherein the 2-position and the 4-
30 position are crosslinked include compounds represented by the
following formulas.
[0089]
In the present specification, the "nucleic acid base" is
not particularly limited as long as it can be used for the
5 synthesis of nucleic acid and includes, for example, a
pyrimidine base such as cytosyl group, uracil group, thyminyl
group and the like, and a purine base such as adenyl group,
guanyl group and the like.
Moreover, in addition to the above-mentioned groups, a
10 modified nucleic acid base (e.g., a 8-bromoadenyl group, a 8-
bromoguanyl group, a 5-bromocytosyl group, a 5-iodocytosyl
group, a 5-bromouracil group, a 5-iodouracil group, a 5-
fluorouracil group, a 5-methylcytosyl group, a 8-oxoguanyl
group, a hypoxanthinyl group etc.), which is a nucleic acid
1s base substituted by any 1 to 3 substituents (e.g., a halogen
atom, an alkyl group, an aralkyl group, an alkoxy group, an
acyl group, an alkoxyalkyl group, a hydroxy group, an amino
group, a monoalkylamino group, a dialkylamino group, a carboxy
group, a cyano group, a nitro group etc. ) at any position (s) ,
20 are also encompassed in the "nucleic acid base".
[0091]
In the present specification, the "halogen atom" means a
fluorine atom, a chlorine atom, a bromine atom or iodine atom.
[0092]
In the present specification, examples of the
"hydrocarbon group" include an aliphatic hydrocarbon group, an
aromatic-aliphatic hydrocarbon group, a monocyclic saturated
hydrocarbon group, an aromatic hydrocarbon group and the l i k e .
S p e c i f i c examples t h e r e o f i n c l u d e a monovalent group such as an
a l k y l group, a n a l k e n y l group, an alkynyl group, a cycloalkyl
group, an a r y l group, an a r a l k y l group, an acyl group and the
5 l i k e , and a d i v a l e n t group derived therefrom.
[0093]
In the present s p e c i f i c a t i o n , examples of the "alkyl
(group)" include a l i n e a r or branched chain a l k y l group having
one or more carbon atoms. When the carbon number is not
lo p a r t i c u l a r l y l i m i t e d , it is preferably a C1-lo a l k y l group, more
preferably a C1-6 a l k y l group. When t h e carbon number is not
p a r t i c u l a r l y l i m i t e d , f o r example, methyl, e t h y l , propyl,
isopropyl, b u t y l , i s o b u t y l , sec-butyl, t e r t - b u t y l , pentyl,
hexyl and the l i k e are p r e f e r a b l e , and methyl and e t h y l a r e
15 p a r t i c u l a r l y p r e f e r a b l e .
[0094]
In t h e present s p e c i f i c a t i o n , t h e "aralkyl (group)" means
a C7-20 a r a l k y l group, preferably a C7-16 a r a l k y l group (a C6-10
a r ~ l - C ~a l-k~y l group) .
20 Preferable s p e c i f i c examples include benzyl, 1-
phenylethyl, 2-phenylethyl, 1-phenylpropyl, naphthylmethyl, 1-
naphthylethyl, 1-naphthylpropyl and t h e l i k e , and benzyl is
p a r t i c u l a r l y p r e f e r a b l e .
[0095]
25 In the present s p e c i f i c a t i o n , examples of the "alkoxy
(group)" include an alkoxy group having one or more carbon
atoms. When the carbon number is not p a r t i c u l a r l y l i m i t e d , it
is preferably a C1-lo alkoxy group, more preferably a C1-6 alkoxy
group. When t h e carbon number is not p a r t i c u l a r l y l i m i t e d ,
30 methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, secbutoxy,
tert-butoxy, pentyloxy, hexyloxy and t h e l i k e a r e
p r e f e r a b l e , and methoxy and ethoxy a r e p a r t i c u l a r l y p r e f e r a b l e .
[0096]
In the present s p e c i f i c a t i o n , examples of the "acyl
35 (group)" include a l i n e a r or branched chain C1-6 alkanoyl group,
a C7-13 aroyl group and the l i k e . Specific examples thereof
include f ormyl, acetyl, n-propionyl, isopropionyl, n-butyryl,
i s o b u t y r y l , pivaloyl, v a l e r y l , hexanoyl, benzoyl, naphthoyl,
l e v u l i n y l and the l i k e , each of which is optionally s u b s t i t u t e d .
5 [0097]
I n t h e present s p e c i f i c a t i o n , examples of the "alkenyl
(group)" include a l i n e a r or branched chain C2-6 alkenyl group
and the l i k e . Preferable examples thereof include vinyl, 1-
propenyl, a l l y l , isopropenyl, butenyl, isobutenyl and the l i k e .
10 Among them, a C2-C4 alkenyl group is p r e f e r a b l e .
[0098]
In the present s p e c i f i c a t i o n , examples of the "alkynyl
(group)" include a C2-6 alkynyl group and the l i k e . Preferable
examples thereof include ethynyl, 1-propynyl, 2-propynyl, 1-
15 butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-
pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-
hexynyl, 5-hexynyl and t h e l i k e . Among them, a C2-C4 alkynyl
group is p r e f e r a b l e .
[0099]
I n t h e present s p e c i f i c a t i o n , the 'cycloalkyl (group)"
means a cyclic alkyl group, and examples thereof include
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
cyclooctyl and the l i k e . Among them, a C3-C6 cycloalkyl group
such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and
25 the l i k e is preferable, and cyclohexyl is p a r t i c u l a r l y
p r e f e r a b l e .
[ O l O O ]
In the present s p e c i f i c a t i o n , the "aryl (group)" means a
monocyclic aromatic or polycyclic (fused) hydrocarbon group.
30 Specific examples thereof include a C6-14 a r y l group such as
phenyl, 1-naphthyl, 2-naphthyl, biphenylyl, 2-anthryl and the
l i k e , and the l i k e . Among them, a C6-lo a r y l group is more
preferably and phenyl is p a r t i c u l a r l y p r e f e r a b l e .
[ O l O l ]
In the present s p e c i f i c a t i o n , the "organic group having a
hydrocarbon group" means a group having the aforementioned
"hydrocarbon group", and the moiety other than the "hydrocarbon
group" of t h e "organic group having a hydrocarbon group" can be
determined f r e e l y . For example, t h e organic group o p t i o n a l l y
5 has, as a l i n k e r , a moiety such as -0-, -S-, -COO-, -0CONH-, -
CONH- and t h e l i k e .
[0102]
In the present s p e c i f i c a t i o n , examples of t h e "alkylene
(group)" include a l i n e a r or branched chain alkylene group
10 having one or more carbon atoms. When t h e carbon number is not
p a r t i c u l a r l y l i m i t e d , it is p r e f e r a b l y a Cl-lo alkylene group,
more preferably a C1-6 alkylene group. When t h e carbon number
is not p a r t i c u l a r l y l i m i t e d , f o r example, methylene, ethylene,
propylene, butylene, pentylene, hexylene and t h e l i k e a r e
15 p r e f e r a b l e , and methylene and ethylene a r e p a r t i c u l a r l y
p r e f e r a b l e .
[0103]
2. Oligonucleotide comprising a p r o t e c t e d base
The oligonucleotide comprising a protected base t o be
20 used f o r oligonucleotide synthesis i n t h e present invention can
be obtained by p r o t e c t i n g a n u c l e i c acid base of a nucleotide
wherein t h e 3'-hydroxyl group is phosphoramidited and the 5'-
hydroxyl group is protected by a temporary p r o t e c t i n g group
removable under a c i d i c conditions by a group having a C5-30
25 s t r a i g h t chain or branched chain a l k y l group and/or a C5-30
s t r a i g h t chain or branched chain alkenyl group, and can provide
a production method of an oligonucleotide s u i t a b l e f o r l i q u i d
phase s y n t h e s i s , since l i p o s o l u b i l i t y and s o l u b i l i t y i n an
organic solvent ( p a r t i c u l a r l y , non-polar solvent) of an
30 intermediate oligonucleotide is remarkably improved.
From t h e aspect of s o l u b i l i t y i n an organic solvent, t h e
group having a C5-30 s t r a i g h t chain or branched chain a l k y l
group and/or a C5-30 s t r a i g h t chain or branched chain alkenyl
group a r e / i s p r e f e r a b l y a C5-30 branched chain a l k y l group
35 and/or a C5-30 branched chain alkenyl group.
[0104]
As a solvent that shows improved solubility of an
oligonucleotide by the oligonucleotide comprising a protected
base of the present specification, a non-polar solvent is
5 preferable.
In the present specification, examples of the Yon-polar
solvent" include halogenated solvents such as chloroform,
dichloromethane, 1,2-dichloroethane and the like; aromatic
solvents such as benzene, toluene, xylene, mesitylene and the
1 0 like; ester solvents such as ethyl acetate, isopropyl acetate
and the like ; aliphatic solvents such as hexane, pentane,
heptane, octane, nonane, cyclohexane and the like; non-polar
ether solvents such as diethyl ether, cyclopentyl methyl ether,
tert-butyl methyl ether and the like can be mentioned. Two or
15 more kinds of these solvents may be used in a mixture at an
appropriate ratio. Among them, aromatic solvents, aliphatic
solvents and a combination thereof are preferable, benzene,
toluene, hexane, pentane, heptane, nonane, cyclohexane, and a
combination thereof and the like are preferable, toluene,
20 heptane, nonane and a combination thereof are more preferable,
and toluene, heptane and a combination thereof are particularly
preferable.
[0105]
Particularly, in the object method for production of an
25 oligonucleotide, an oligonucleotide comprising a protected base,
which is preferable for achieving speeding up, high yield and
high efficiency, is, for example, a novel compound represented
by the following formula (I) (hereinafter sometimes to be
referred to as the compound (I) of the present invention).
30 When q is 0, the oligonucleotide comprising a protected
base, which is represented by the formula (I), is understood as
"nucleoside comprising a protected base".
[0106]
Formula (I) :
35 [0107]
[0108]
wherein q is any i n t e g e r of not l e s s than 0;
~ a s ien~ th e number of q+l are each independently a nucleic
s acid base protected by a group having a C5-30 s t r a i g h t chain or
branched chain alkyl group and/or a C5-30 s t r a i g h t chain or
branched chain alkenyl group;
P' is a hydrogen atom, or a temporary protecting group
removable under a c i d i c conditions;
lo X is a hydrogen atom, an optionally protected hydroxyl group, a
halogen atom or an organic group crosslinked with the 4-
p o s i t i o n carbon atom;
X' i n the number of q are each independently a hydrogen atom,
an optionally protected hydroxyl group, a halogen atom or an
1s organic group crosslinked with the 4-position carbon atom;
p2 i n the number of q+l are each independently a protecting
group removable under basic conditions;
R~~ i n the number of q are each independently an oxygen atom or
a s u l f u r atom; and
20 Re and Rf are each independently a C1-6 alkyl group, or a 5- or
6-membered saturated cyclic amino group formed together with
the adjacent nitrogen atom.
[0109]
q is any integer of 0 or more, preferably 0. While the
upper limit of q is not particularly set, it is preferably 49
or less, more preferably 29 or less, further preferably 19 or
less, still more preferably 4 or less, still further preferably
5 2 or less and particularly preferably 1.
The groups X and X' in the number of q at the 2-position
of ribose residues constituting the oligonucleotide comprising
a protected base, which is the compound (I) of the present
invention, are each independently a hydrogen atom, an
lo optionally protected hydroxyl group, a halogen atom or an
organic group crosslinked with the 4-position carbon atom.
The protecting group of the "optionally protected
hydroxyl group" is not particularly limited and, for example,
any protecting groups described in Greene's PROTECTIVE GROUPS
15 IN ORGANIC SYNTHESIS, 4th ed., JOHN WILLY&SONS (2006) and the
like can be mentioned. Specific examples thereof include a
methyl group, a benzyl group, a p-methoxybenzyl group, a tertbutyl
group, a methoxymethyl group, a methoxyethyl group, a 2-
tetrahydropyranyl group, an ethoxyethyl group, a cyanoethyl
20 group, a cyanoethoxymethyl group, a phenylcarbamoyl group, a
1,l-dioxothiomorpholine-4-thiocarbamoyl group, an acetyl group,
a pivaloyl group, a benzoyl group, a trimethylsilyl group, a
triethylsilyl group, a triisopropylsilyl group, a tertbutyldimethylsilyl
group, a [(triisopropylsilyl)oxy]methyl
25 (Tom) group, an 1-(4-chloropheny1)-4-ethoxypiperidin-4-yl
(Cpep) group and the like. Among these, a triethylsilyl group,
a triisopropylsilyl group and a tert-butyldimethylsilyl group
are preferable. From the aspects of economic efficiency and
easy availability, a tert-butyldimethylsilyl group is
30 particularly preferable.
[OllO]
As the halogen atom for X or X', a fluorine atom, a
chlorine atom and the like are preferable, and a fluorine atom
is more preferable.
While the "organic group crosslinked with the 4-position
carbon atom" for X or X' is not particularly limited as long as
the 2-position and the 4-position of the nucleoside is
crosslinked, for example, a C2-7 alkylene group can be mentioned.
The alkylene group may be interrupted at one or more moieties
5 (preferably 1 or 2 moieties) by a linker selected' from, for
example, -0-, - N R ~ ~(- Ri~s ~a h ydrogen atom or a C1-6 alkyl
group) , -S-, -CO-, -COO-, -OCONR~~-( Ri~s ~a h ydrogen atom or a
C1-6 alkyl group), -c0NR3'- (R3' is a hydrogen atom or a C1-6
alkyl group) and the like.
10 Preferable examples of the "organic group crosslinked
with the 4-position carbon atom" include -0Ri (Ri is a C1-6
alkylene group crosslinked with the 4-position), -o-NR~~-R(~Rj
is a C1-6 alkylene group crosslinked with the 4-position, and
R~~ is as defined above), -0-Rk-0-R1 (Rk is a C1-6 alkylene
15 group, and R1 is a C1-6 alkylene group crosslinked with the 4-
position) and the like. The C1-6 alkylene groups for Ri, Rj, Rk
and R1 are preferably each independently a methylene group or
an ethylene group.
As the "organic group crosslinked with the 4-position
20 carbon atom", -0-CH2-, -0-CH2-CH2-, -o-NR~C~H-2 - ( Ri~s a~s
defined above), -0-CH2-0-CH2- and the like are preferable, and
-0-CH2-, -0-CH2-CH2-, -0-NH-CH2-, -0-NMe-CH2-, -0-CH2-0-CH2- (in
all of which the left side binds to the 2-position and the
right side binds to the 4-position) and the like are more
25 preferable .
[Olll]
The temporary protecting group P' that can be used as the
5'-hydroxyl-protecting group of the compound (I) of the present
invention is not particularly limited as long as it can be
30 deprotected under acidic conditions and can be used as a
hydroxyl-protecting group. Examples thereof include a trityl
group, 9-(9-pheny1)xanthenyl group, a 9-phenylthioxanthenyl
group, di (C1-6 alkoxy) trityl groups such as a l,l-bis (4-
methoxypheny1)-1-phenylmethyl group and the like,
35 monomethoxytrityl group such as mono (C1-18 alkoxy) trityl groups
such as a 1-(4-methoxypheny1)-1,l-diphenylmethyl group and the
like. Among these, a monomethoxytrityl group and a
dimethoxytrityl group are preferable, and a dimethoxytrityl
group is more preferable, in view of easiness of deprotection
5 and easy availability.
[0112]
The 5- or 6-membered saturated cyclic amino group formed
by Re and Rf together with the adjacent nitrogen atom may have,
as a ring-constituting atom besides nitrogen atom, one of
lo oxygen atom or sulfur atom. For example, a pyrrolidinyl group,
a piperidinyl group, a morpholinyl group, a thiomorpholinyl
group, an N-methylpyrazinyl group and the like can be mentioned.
As Re and Rf, an isopropyl group is preferable.
[0113]
The protecting group p2 in the number of q+l that can be
used as a protecting group of phosphoramidite or a protecting
group of a nucleotide phosphate group of the compound (I) of
the present invention is not particularly limited as long as
it can be deprotected under base conditions and used as a
20 hydroxyl-protecting group. A group represented by -CH2CH2WG
(WG is an electron-withdrawing group) is preferable, and a
cyano group is preferable as WG. R~~ in the number of q is
each independently an oxygen atom or a sulfur atom, preferably
an oxygen atom.
25 [0114]
While the "group having a C5-30 straight chain or branched
chain alkyl group and/or a C5-30 straight chain or branched
chain alkenyl group" of the "nucleic acid base protected by a
group having a C5-30 straight chain or branched chain alkyl
30 group and/or a C5-30 straight chain or branched chain alkenyl
group" for ~ a s ein~ t he number of q+l is not particularly
limited as long as it is a protecting group having one or more
"C5-30 straight chain or branched chain alkyl group and/or C5-30
straight chain or branched chain alkenyl groupN in the
35 molecular structure thereof, a group represented by the
following formula (k), (l), (m) or (s) is preferable.
[0115]
A group represented by the formula (k):
wherein * indicates the bonding position to a nucleic acid
base;
RZ7 is a C5-30 straight chain or branched chain alkyl group or a
10 C5-30 straight chain or branched chain alkenyl group,
a group represented by the formula (1) :
wherein * indicates the bonding position to a nucleic acid
base ;
Q1 is -0-, -S- or - N R ~ O - wherein R30 is a hydrogen atom or a C 1 - ~ ~
alkyl group;
20 Rc and Rd are each independently a hydrogen atom or a C 1 - ~ a~l kyl
group;
R~~ is a C5-30 straight chain or branched chain alkyl group or a
C5-30 straight chain or branched chain alkenyl group,
a group represented by the formula (m):
wherein * indicates the bonding position to a nucleic acid
base ;
1 is an i n t e g e r of 1 - 5;
Q2 i n t h e number of 1 a r e each independently a s i n g l e bond, or
-0-, -S-, -0C (=O) -, -C (=O) 0-, -0-CH2-, -NH-, -NHC (=O) -, -
5 (=O) NHC-, -NH-CH2- or -CH2-;
R" i n the number of 1 a r e each independently a C5-30 s t r a i g h t
chain or branched chain a l k y l group or a C5-30 s t r a i g h t chain or
branched chain alkenyl group;
r i n g C is a benzene r i n g or a cyclohexane r i n g each o p t i o n a l l y
10 having, i n a d d i t i o n t o Q ~ iRn t~he ~num ber of 1 and *c=o, a
s u b s t i t u e n t s e l e c t e d from the group c o n s i s t i n g of a halogen
atom, a C1-6 a l k y l group o p t i o n a l l y s u b s t i t u t e d by one o r more
halogen atoms, and a C1-6 alkoxy group o p t i o n a l l y s u b s t i t u t e d by
one or more halogen atoms, or
15 a group represented by t h e formula ( s ) :
[0122]
wherein * i n d i c a t e s t h e p o s i t i o n a t which an imino bond is
20 formed with an amino group of a n u c l e i c acid base; and
R~~ and R~~ a r e each independently a C5-30 s t r a i g h t chain or
branched chain a l k y l group or a C5-30 s t r a i g h t chain or branched
chain alkenyl group.
[0124]
25 In t h e formula (1) , Q1 is p r e f e r a b l y -0-, and Rc and Rd
a r e each p r e f e r a b l y a hydrogen atom.
In t h e formula ( m ) , 1 is p r e f e r a b l y 1, Q2 is p r e f e r a b l y -
0-, and r i n g C is p r e f e r a b l y a benzene r i n g .
[0125]
30 The lower l i m i t of t h e carbon atoms of t h e "C5-30 s t r a i g h t
chain or branched chain a l k y l group" and nC5-30 s t r a i g h t chain
o r branched chain alkenyl group" of the nC5-30 s t r a i g h t chain or
branched chain a l k y l group or a C5-30 s t r a i g h t chain or branched
chain alkenyl groupff f o r R ~ R~ ~, R~2' ,i n the number of 1, R~~
and R~~ i n the formulas ( k ) , (l), (m) and ( s ) , r e s p e c t i v e l y , is
5 or more, preferably 16 or more, more preferably 18 or more.
The upper l i m i t of the carbon number is 30 or l e s s , preferably
5 25 o r l e s s , more preferably 20 o r l e s s .
[0126]
While the "C5-30 s t r a i g h t chain or branched chain a l k y l
group or C5-30 s t r a i g h t chain or branched chain alkenyl groupff
f o r R ~ R~2', , R2' i n the number of 1, R~~ or R~~ is not
10 p a r t i c u l a r l y limited, a branched chain a l k y l group represented
by the following formula (n) or (e' ) is p r e f e r a b l e .
[0127]
The carbon numbers, the number of repeat u n i t s (n19, n2o
or n21) and the l i k e i n the d e f i n i t i o n of each symbol i n the
15 formulas (n) and (e' ) are shown f o r convenience, and can be
appropriately changed within the above-mentioned d e f i n i t i o n
range t o achieve the t o t a l carbon number of 5 or more
(preferably 16 or more, more preferably 18 or more) and 30 or
l e s s (preferably 25 o r l e s s , more preferably 20 or l e s s ) . In
20 the following, the formulas (n) and ( e ' ) are successively
explained.
[0128]
The formula (n) is as follows.
A branched chain a l k y l group represented by
25 [0129]
[0130]
wherein * shows a bonding position;
n19 i s an i n t e g e r of 2 t o 6;
30 R30 and R31 i n the number of n19 are each independently a
hydrogen atom or a C1-4 a l k y l group;
X6 i n the number of n19 are each independently a s i n g l e bond or
a C1-4 alkylene group; and
R~~ is a hydrogen atom or a C1-4 alkyl group; and
R~~ is a C1-4 alkyl group,
provided that R30 and R31 are not hydrogen atoms at the same
5 time, and when n19 is 2, R32 is a C1-4 alkyl group.
[0131]
In the group of the formula (n), a group wherein
R30 and R31 in the number of n19 are each independently a
hydrogen atom, a methyl group or an ethyl group;
10 X6 in the number of n19 are each independently a single bond, a
methylene group or an ethylene group;
R~~ is a hydrogen atom, a methyl group or an ethyl group; and
R~~ is a methyl group or an ethyl group
is preferable, provided that R30 and R31 are not hydrogen atoms
15 at the same time, and when n19 is 2, R32 is a methyl group or an
ethyl group.
[0132]
Examples of more preferable group of the formula (n)
include a 2,6,10,14-tetramethylpentadecyl group, a 2,6,10-
20 trimethylundecyl group, a 2,6-dimethylheptyl group and the like.
[0133]
A branched chain alkyl group represented by the formula
(el ) :
[0134]
[0135]
wherein * shows a bonding position;
1220 is an integer of 1 to 5;
1121 is an integer of 1 to 5;
30 RZ0 and R21 in the number of n20 are each independently a
hydrogen atom or a C1-4 alkyl group;
X3 in the number of n2o are each independently a single bond or
a C1-4 alkylene group;
R2' and R~~ in the number of n21 are each independently a
hydrogen atom or a C1-4 alkyl group;
X5 in the number of n2l are each independently a single bond or
5 a C1-4 alkylene group;
X4 is a single bond or a C1-4 alkylene group; and
R17, R18, R19, R'~, RZ5 and R'~ are each independently a hydrogen
atom or a C1-4 alkyl group,
provided that R20 and R21 and/or R~~ and R~~ are not hydrogen
10 atoms at the same time, and when nzo+n21 is 2, two or more of R17,
R18 and R19 are each independently a C1-4 alkyl group, or two or
more of R ~ R~~~, an d RZ6 are each independently a C1-4 alkyl group.
[0136]
A group of the formula (e'), wherein
15 n20 is an integer of 1 to 5;
n2l is an integer of 1 to 5;
R20 and R" in the number of n20 are each independently a
hydrogen atom, a methyl group or an ethyl group;
X3 in the number of n20 are each independently a single bond, a
20 methylene group or an ethylene group;
R2' and R~~ in the number of n21 are each independently a
hydrogen atom, a methyl group or an ethyl group;
X5 in the number of n2l are each independently a single bond, a
methylene group or an ethylene group;
25 X4 is a single bond, a methylene group or an ethylene group;
R17, R18, R19, R ~ R~~~, an d R~~ are each independently a hydrogen
atom or a C1-4 alkyl group is preferable, provided that R20 and
R21 and/or R~~ and R~~ are not hydrogen atoms at the same time,
and when nzo+nzl is 2, two or more of R17, R18 and R19 are each
30 independently a C1-4 alkyl group, or two or more of R", R~~ and
R" are each independently a C1-4 alkyl group.
[0137]
A group of the formula (ef ) , wherein
n20 is an integer of 1 to 5;
35 n21 is an integer of 1 to 5;
RZ0 and R21 in the number of n20 are each independently a
hydrogen atom or a methyl group;
X3 in the number of n2o are each independently a single bond or
a methylene group;
5 R~~ and R~~ in the number of 1121 are each independently a
hydrogen atom or a methyl group;
X5 in the number of n21 are each independently a single bond or
a methylene group;
X4 is a single bond or a methylene group; and
lo R17, R18, R19, R ~ R~~~, an d R~~ are methyl groups, provided that
R20 and R21 and/or R~~ and R~~ are not hydrogen atoms at the same
time, is particularly preferable.
[0138]
More preferable group of the formula (e') includes a
15 2,2,4,8,lO, 10-hexamethyl-5-undecyl group and the like.
[0139]
Other preferable examples of the nC5-30 straight chain or
branched chain alkyl group or C5-30 straight chain or branched
chain alkenyl group" for R ~ R~Z8,, R 2' in the number of 1 , R~~
20 or R~~ include a branched chain alkyl group and a branched
chain alkenyl group selected from the group consisting of a
2,6,10,14-tetramethylpentadecyl group, a 2,6,10-
trimethylundecyl group, a 2,2,4,8,10,10-hexamethyl-5-undecyl
group, a 2,6,10-trimethylundeca-1,5,9-trienyl group, a 2,6-
25 dimethylheptyl group, a 2,6-dimethylhept-5-enyl group, a 2,6-
dimethylhepta-1,5-dienyl group, a 9-nonadecyl group, a 12-
methyltridecyl group, an 11-methyltridecyl group, an 11-
methyldodecyl group, a 10-methylundecyl group, an 8-heptadecyl
group, a 7-pentadecyl group, a 7-methyloctyl group, a 3-
30 methyloctyl group, a 3,7-dimethyloctyl group, a 3-methylheptyl
group, a 3-ethylheptyl group, a 5-undecyl group, a 2-heptyl
group, a 2-methyl-2-hexyl group, a 2-hexyl group, a 3-heptyl
group, a 4-heptyl group, a 4-methyl-pentyl group, a 3-methylpentyl
group, and a 2,4,4-trimethylpentyl group; and a straight
35 chain alkyl group selected from the group consisting of a
t e t r a d e c y l group, a t r i d e c y l group, a dodecyl group, an undecyl
group, a decyl group, a nonyl group, an octyl group, a heptyl
group, a hexyl group, and a pentyl group.
[0140]
Examples of the nucleic acid base protected by a group
having a C5-30 s t r a i g h t chain or branched chain alkyl group
and/or a C5-30 s t r a i g h t chain or branched chain alkenyl group
include the following formulas (Al) - (Al2)
[014 11
wherein each symbol i s as defined above.
A method of introducing a group having a C5-30 straight
chain or branched chain alkyl group and/or a C5-30 straight
chain or branched chain alkenyl group into a nucleic acid base
can be performed according to a known method described in
5 Greene's PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 4th Edition,
published by JOHN WILLY & SONS (2006), ORGANIC LETTERS, 2005,
Vol. 7, NO. 24, 5389-5392, JOURNAL OF AMERICAN CHEMICAL SOCIETY,
1982, Vol. 104, 1316-1319 and the like and using, as a
protecting reagent, an activated derivative of the protecting
10 group.
Examples of the activated derivative of the group having
a C5-30 straight chain or branched chain alkyl group and/or a C5-
30 straight chain or branched chain alkenyl group include a
compound wherein a halogen atom (e.g., chlorine atom, bromine
15 atom etc.) is bonded to * in a group represented by the abovementioned
formula (k) , (1) or (m) ; a symmetric acid anhydride
wherein two groups represented by the above-mentioned formula
(k) or (m) are bonded to an oxygen atom at *; a mixed acid
anhydride wherein a group represented by the above-mentioned
20 formula (k) or (m) is bonded to other acyl group (e.g.,
isobutyryl group) at *, a compound represented by (Me0)2CHN
R ~ a~ndR t~he ~li ke.
The activated derivative of the protecting group is
available as a commercially available product, or can be
25 produced by a method known per se or a method analogous thereto
[0143]
A preferable embodiment of the compound represented by
the formula (I) of the present invention is a compound of the
formula (I) , wherein
30 q is 0;
as el is a cytosyl group, a uracil group, a thyminyl group, an
adenyl group, or a guanyl group, each of which is optionally
protected by a group having a C5-30 straight chain or branched
chain alkyl group and/or a C5-30 straight chain or branched
35 chain alkenyl group;
P' is a di (C1-6 alkoxy) trityl group, or a mono (C1-6 alkoxy) trityl
group;
X is a hydrogen atom, an optionally protected hydroxyl group, a
fluorine atom, -0Ri (Ri is as defined above), -o-NR'~-R~ (Rj
5 and R'~ are as defined above, or -0-Rk-0-R1 (Rk and R1 is as
defined above) ;
Re and Rf are each independently a C1-6 alkyl group; and
P' is a group represented by -CH2CH2WG (WG is an electronwithdrawing
group) .
10 [0144]
Another preferable embodiment of the compound represented
by the formula (I) of the present invention is a compound of
the formula (I), wherein
q is 0;
15 Base' is a cytosyl group, a uracil group, a thyminyl group, an
adenyl group, or a guanyl group, each of which is optionally
protected by a group having a C5-'0 straight chain or branched
chain alkyl group and/or a C5-'0 straight chain or branched
chain a1 kenyl group;
20 P' is a dimethoxytrityl group or a monomethoxytrityl group;
X is a hydrogen atom, an optionally protected hydroxyl group, a
fluorine atom, -0-CH2-, -0-CH2-CH2-, or -O-NR'~-CH~- (R'~i s as
defined above) or -0-CH2-0-CH2- (in all of which the left side
binds to the 2-position and the right side binds to the 4-
25 position) ;
Re and Rf are each an isopropyl group; and
P2 is a group represented by -CH2CH2WG (WG is an electronwithdrawing
group) .
[0145]
30 A still another preferable embodiment of the compound
represented by the formula (I) of the present invention is a
compound of the formula (I), wherein
q is 0;
Base2 is a cytosyl group, a uracil group, a thyminyl group, an
35 adenyl group, or a guanyl group, each of which is optionally
protected by a group having a C5-30 straight chain or branched
chain alkyl group and/or a C5-30 straight chain or branched
chain a1 kenyl group;
P' is a dimethoxytrityl group;
5 X is a hydrogen atom, a methoxy group, a tertbutyldimethylsilyloxy
group, a fluorine atom, -0-CH2-, -0-CH2-
CH2-, -0-NH-CH2-, -0-NMe-CH2- or -0-CH2-0-CH2- (in all of which
the left side binds to the 2-position and the right side binds
to the 4-position);
10 Re and Rf are each an isopropyl group; and
p2 is a group represented by -CH2CH2CN.
3. Production method of oligonucleotide comprising protected
base
15 Of the oligonucleotides comprising a protected base
represented by the following formula (I) of the present
invention, an oligonucleoside comprising a protected base
represented by the formula (I,), wherein q is 0, can be
produced according to a known method (M.H. Caruthers et al.,
20 Method in Enzymology 1987, 154, 287-313; S. L. Beaucage and M.
H. Caruthers, Tetrahedron Letters 1981, 22, 1859-1862)
comprising reacting a nucleoside represented by the formula
(Ia), wherein the 5'-hydroxyl group is protected by a temporary
protecting group P' and the nucleic acid base is protected by a
25 group having a C5-30 straight chain or branched chain alkyl
group and/or a C5-30 straight chain or branched chain alkenyl
group, with a phosphoramiditing reagent represented by the
following formula (0) or (p)
[0147]
30
Hal % l N J f m-p: m-P{
N
%' -f &,N, R f
(o) @I
[0148]
wherein Hal is a halogen atom, and other symbols are as defined
above.
5 [0149]
A compound represented by the formula (Ia) to be used as
a starting material can be produced from a corresponding
nucleoside wherein the nucleic acid base is not protected, by
protecting the nucleic acid base with a "group having a C5-30
lo straight chain or branched chain alkyl group and/or a C5-30
straight chain or branched chain alkenyl group" according to
the above-mentioned "method for introducing a group having a
C5-30 straight chain or branched chain alkyl group and/or a C5-30
straight chain or branched chain alkenyl group into a nucleic
15 acid base". The corresponding nucleoside wherein the nucleic
acid base is not protected is available as a commercially
available product, or can be produced according to a method
known per se or a method analogous thereto.
[0150]
The nucleoside comprising a protected base represented by
the formula (1') of the present invention can also be produced
by a known method (ORGANIC LETTERS, 2005, Vol. 7, No. 24, 5389-
5392) comprising reacting a nucleoside represented by the
formula (Ib), wherein the 3'-hydroxyl group is phosphoramidited,
25 the 5'-hydroxyl group is protected by a temporary protecting
group PI, and the nucleic acid base is not protected, with a
protecting reagent having a C5-30 straight chain or branched
chain alkyl group and/or a C5-30 straight chain or branched
chain a1 kenyl group.
[0151]
5 [0152]
wherein Base' is an unprotected nucleic acid base, and other
symbols are as defined above.
[0153]
A compound represented by the formula (Ib) to be used as
10 a starting material can be produced by deprotecting a
corresponding nucleoside wherein the nucleic acid base is
protected by a protecting group conventionally used for nucleic
acid synthesis (e.g., acetyl group, phenoxyacetyl group, pisopropylphenoxyacetyl
group, benzoyl group, isobutyryl group
15 etc.) according to a known method described in ORGANIC LETTERS,
2005, Vol. 7, No. 24, 5389-5392 and the like. The
corresponding nucleoside wherein the nucleic acid base is
protected by a protecting group conventionally used for nucleic
acid synthesis is available as a commercially available product,
20 or can be produced according to a method known per se or a
method analogous thereto.
[0154]
An oligonucleotide comprising a protected base
represented by of the formula (I) wherein q is 1 or more can be
25 produced by appropriately applying, for example, the method
described in Aust. J. Chem. 2010, 63, 227-235 and the
production method of oligonucleoside described in the present
specification to produce a corresponding oligonucleotide
comprising a protected base wherein the 3'-hydroxyl group is
protected, removing the 3'-hydroxyl-protecting group, and
reacting the resulting compound with a phosphoramiditing
reagent represented by the formula (0) or (p) according to a
known method (M.H. Caruthers et al., Method in Enzymology 1987,
5 154, 287-313; S.L. Beaucage and M.H. Caruthers, Tetrahedron
Letters 1981, 22, 1859-1862.) .
[0155]
Hal
&-P<
wherein p4 is a nucleotide 3' -hydroxyl-protecting group, and
other symbols are as defined above.
As the protecting group of nucleotide 3'-hydroxyl group
for p4, a protecting group capable of deprotection under the
15 conditions under which the 5'-hydroxyl-protecting group P', the
protecting group when the nucleic acid base has a protecting
group and a phosphate-protecting group p2 are not deprotected
is used. For example, a protecting group capable of
deprotection with hydrazine can be mentioned. Preferable
examples of the protecting group capable of deprotection with
hydrazine include a levulyl group and the like (see Aust. J.
Chem. 2010, 63, 227-235).
4. Production method of oligonucleotide
Next, the production method of oligonucleotide relating
to the present invention (hereinafter to be also referred to as
"the production method of the present inventionff) is explained.
The production method of the present invention
characteristically comprises using the aforementioned
oligonucleotide comprising a protected base. To be specific, a
production method of an n+p-mer oligonucleotide from an n-mer
oligonucleotide is explained. For example, when n=l, an n-mer
oligonucleotide is to be understood as "nucleoside", when p=l,
a p-mer oligonucleotide comprising a protected base is to be
understood as "nucleoside comprising a protected base", and an
n+p-mer oligonucleotide is to be understood as "dinucleoside".
The production method of the present invention preferably
includes the following step (2):
[0158]
(2) a step of condensing a p-mer oligonucleotide comprising a
protected base (p is any integer of one or more) wherein the
3'-hydroxyl group is phosphoramidited, the 5'-hydroxyl group is
protected by a temporary protecting group removable under
acidic conditions, and the nucleic acid base is protected by a
group having a C5-30 straight chain or branched chain alkyl
group and/or a C5-30 straight chain or branched chain alkenyl
group, with an n-mer oligonucleotide (n is an integer of one or
more) wherein the 5'-hydroxyl group is not protected and the
3'-hydroxyl group is protected, by forming a phosphite triester
bond via the 5'-hydroxyl group thereof to give an n+p-mer
oligonucleotide.
[0159]
The production method of the present invention preferably
further includes the following step (3), by which the phosphite
5 triester bond of the n+p-mer oligonucleotide obtained in step
(2) is converted to a phosphate triester bond or thiophosphate
triester bond.
(3) a step of converting the phosphite triester bond of the
n+p-mer oligonucleotide obtained in the condensation step to a
lo phosphate triester bond or a thiophosphate triester bond by
adding an oxidizing agent or a sulfurizing agent to the
reaction mixture obtained in the condensation step (2).
[0160]
!
The production method of the present invention preferably
15 further includes the following step (I), whereby an n-mer
oligonucleotide wherein the 5'-hydroxyl group is not protected
and the 3'-hydroxyl group is protected, which is used in step
(2), is prepared:
(1) a step of removing the temporary protecting group removable
20 under acidic conditions of the 5'-hydroxyl group by reacting,
in a non-polar solvent prior to the condensation step (2), an
n-mer oligonucleotide wherein the 3'-hydroxyl group is
protected, and the 5'-hydroxyl group is protected by a
temporary protecting group, with an acid.
Step (1) is preferably performed in the presence of at
least one kind of cation scavenger selected from a pyrrole
derivative and an indole derivative, and further includes a
step of removing the temporary protecting group of the 5'-
hydroxyl group and neutralizing the compound with an organic
30 base. This enables continuous performance of steps (1) , (2)
and (3) in a solution, and an oligonucleotide wherein
nucleoside in the number of p has elongated can be isolated and
purified by an extraction operation alone.
[0161]
Furthermore, by including the following step (4), an n+pmer
oligonucleotide is purified by removing an excess starting
material and by-product conveniently and effectively, without
the need for complicated solidification-isolation, and can be
led to the next step without taking out the resultant product
from the reaction vessel:
(4) a step of isolating the ntp-mer oligonucleotide from the
reaction mixture obtained in step (3) by an extraction
operation alone.
[0162]
When the amount of the by-product can be controlled by
the management of equivalent amounts of the starting materials
and the control of the reaction, it is preferable to repeat
step (1) to step (3) as a basic unit, and include step (4).
Moreover, since occurrence of by-product can be strictly
managed and controlled and highly pure oligonucleotide can be
obtained, it is preferable to repeat step (1) to step (4) as a
basic unit.
By repeating such cycle in the liquid phase method, the
final oligonucleotide can be produced in one-pot, without
changing the reaction vessel.
[0163]
In the production method of the present invention, an
oligonucleotide can be isolated and produced by further
including step (5) :
(5) a step of removing all the protecting groups of the ntp-mer
oligonucleotide obtained in step (4).
n is an integer of one or more. While the upper limit
thereof is not particularly limited, it is generally 100 or
less, preferably 75 or less, more preferably 50 or less, and
still more preferably 30 or less
p is an integer of one or more, preferably 1. While the
upper limit thereof is not particularly limited, it is
preferably 50 or less, more preferably 30 or less, more
preferably 20 or less, still more preferably 5 or less, and
particularly preferably 3 or less.
[0164]
4-1. Explanation of "n-mer oligonucleotide"
First of all, the n-mer oligonucleotide to be used as a
starting material of steps (1) and (2) is explained.
The n-mer oligonucleotide to be used in step (1) is, for
example, an n-mer oligonucleotide represented the following
formula (i) wherein P' is a temporary protecting group
removable under acidic conditions, the 3'-position hydroxyl
group is protected and the 5'-position hydroxyl group is
lo protected by a temporary protecting group removable under
acidic conditions. The n-mer oligonucleotide to be used in
step (2) shows, for example, an n-mer oligonucleotide
represented by the following formula (ii) wherein the 5'-
position hydroxyl group is not protected, and the 3'-position
15 hydroxyl group is protected.
[0166]
wherein m is any integer of not less than 0, Base in the number
20 of m+l are each independently an optionally protected nucleic
acid base, R~~ in the number of m are each independently an
oxygen atom or a sulfur atom, p2 in the number of m are each
independently a protecting group removable under basic
conditions, p3 is a nucleotide 3' -hydroxyl-protecting group, Xf
25 in the number of mtl are each independently as defined for X,
and other symbols are as defined above.
While the upper limit of m is not particularly limited,
it is generally 99 or less, preferably 74 or less, more
preferably 49 or less, still more preferably 29 or less.
The "protecting group removable under basic conditions"
5 for p2 is as defined for p2 in the formula (I) .
Each symbol in the formulas (i) and (ii) is explained
below.
[0167]
4-2. Explanation of 'nucleotide 3'-hydroxyl-protecting group"
The "nucleotide 3'-hydroxyl-protecting group" for p3 in
the formulas (i) and (ii) is not particularly limited as long
as it is a group stable under acidic conditions capable of
removing the 5'-hydroxyl-protecting group, and can dissolve an
n-mer oligonucleotide in a non-polar reaction solvent so that
15 the reaction will proceed in steps (1) and (2). It is
preferably a group represented by the following formula (111')
[0168]
-L-Y-2' (m)
[0169]
20 wherein
L is a group represented by the formula (al):
[0170]
25 [0171]
* *
wherein * shows the bonding position to Y; indicates the
bonding position to a 3'-hydroxy group of the nucleotide;
L1 is an optionally substituted divalent C1-22 hydrocarbon
group; and
30 L2 is a single bond, or a group represented by **c(=O)N(R2-)R 1-
* * ***
N(R3) *** wherein shows the bonding position to L1, shows
the bonding position to C=O, R1 is an optionally substituted C1-
22 alkylene group, and R2 and R3 are each independently a
hydrogen atom or an optionally substituted C1-22 alkyl group, or
R2 and R3 are optionally joined to form an optionally
substituted C1-22 alkylene bond,
5 Y is an oxygen atom or NR wherein R is a hydrogen atom, an
alkyl group or an aralkyl group, and
Z' is an organic group having a hydrocarbon group.
[0172]
A preferable embodiment of the linker L represented by
10 the above-mentioned formula (al) is a group wherein, in the
formula (al) ,
L1 is an ethylene group or CH2-0-1, 4-phenylene-0-CH2; and
* *
L2 is a single bond, or a group represented by c (=o)N( R2)- R1-
* * ***
N(R3)*** wherein shows the bondingposition to L1, shows
15 the bonding position to C=O, R1 is a C1-6 alkylene group, and R2
and R' are each independently a hydrogen atom or an optionally
substituted C1-6 alkyl group, or R2 and R3 are optionally joined
to form an optionally substituted C1-6 alkylene bond.
[0173]
20 Another preferable embodiment of the linker L represented
by the above-mentioned formula (al) is a group wherein, in the
formula (al) ,
Ll is an ethylene group; and
L2 is a single bond.
25 [0174]
Another preferable embodiment of the linker L represented
by the above-mentioned formula (al) is a group wherein, in the
formula (al) ,
Ll is an ethylene group; and
30 the moiety N (R2)- R1-N( R3) for L2 is a piperazinylene group.
[0175]
Another preferable embodiment of the linker L represented
by the above-mentioned formula (al) is a group wherein, in the
formula (al),
35 L1 is an ethylene group; and
* * * *
L2 is a group represented by C (=O) N (R2) -R'-N (R3) *** wherein
***
shows the bonding p o s i t i o n t o L1, shows t h e bonding p o s i t i o n
t o C=O, R' is a pentylene group or a hexylene group, and R2 and
R3 a r e each independently a hydrogen atom or a methyl group.
5 [0176]
A p a r t i c u l a r l y preferable example of t h e above-mentioned
l i n k e r L is a succinyl group since it is economical and e a s i l y
a v a i l a b l e .
[0177]
Y i n the above-mentioned formula (111') is an oxygen atom,
or NR wherein R is a hydrogen atom, an a l k y l group or an
a r a l kyl group.
[0178]
In t h e present s p e c i f i c a t i o n , the "alkyl group" f o r R is
15 a C1-30 a l k y l group, preferably a C1-10 a l k y l group, more
preferably a C1-6 a l k y l group. S p e c i f i c p r e f e r a b l e examples
thereof include methyl, e t h y l , propyl, isopropyl, butyl,
i s o b u t y l , sec-butyl, t e r t - b u t y l and the l i k e , and methyl and
e t h y l are p a r t i c u l a r l y p r e f e r a b l e .
20 [0179]
In t h e present s p e c i f i c a t i o n , the "aralkyl group" f o r R
is a C7-30 a r a l k y l group, preferably a C7-20 a r a l k y l group, more
preferably a C7-16 a r a l k y l group (a C6-lo a r ~ l - C ~a l-k~y l group).
S p e c i f i c p r e f e r a b l e examples thereon include benzyl, 1-
25 phenylethyl, 2-phenylethyl, 1-phenylpropyl, a-naphthylmethyl,
1- (a-naphthyl) e t h y l , 2- (a-naphthyl) e t h y l , 1- (a-naphthyl) propyl,
P-naphthylmethyl, 1- (P-naphthyl) e t h y l , 2- (P-naphthyl) e t h y l , 1-
(P-naphthy1)propyl and the l i k e , and benzyl is p a r t i c u l a r l y
preferable .
30 [0180]
R is preferably a hydrogen atom, a C1-6 a l k y l group or a
C7-16 a r a l k y l group, more preferably a hydrogen atom, methyl,
e t h y l or benzyl, p a r t i c u l a r l y preferably a hydrogen atom.
[0181]
Y is preferably an oxygen atom or NH.
[0182]
Examples of the "organic group having a hydrocarbon
groupff for Z' include a C1-6 alkyl group such as methyl, ethyl,
tert-butyl and the like, benzyl, p-nitrobenzyl, p-methoxybenzyl,
5 diphenylmethyl, allyl, 1,l-dimethyl-2-phenyl-ethyl, 2,4-
dimethoxybenzyl, bis(4-methoxypheny1)methyl and the like. In
addition, as the organic group having a hydrocarbon group, a
group having a branched chain is preferable. When a group
having a branched chain is used, liposolubility and solubility
10 in a solvent (particularly, non-polar solvent) of an n-mer
oligonucleotide and an n+p-mer oligonucleotide can be improved,
step (1) can be performed smoothly, and an n+p-mer
oligonucleotide can be easily transferred into a non-polar
solvent in the extraction-isolation step of the below-mentioned
15 step (4).
Preferable examples of the "group having a branched
chain" for Z' include a group represented by the formula (a2):
[0183]
[0184]
wherein * shows the bonding position to Y;
R4 is a hydrogen atom, or when Rb is a group represented by the
following formula (a3), then R4 shows, in combination with R6,
25 a single bond or -0- to optionally form a fluorenyl group or a
xanthenyl group together with ring B;
Q in the number of k are each independently a single bond, -0-,
-S-, -0C (=O) -, -NHC (=O) - or -NH-;
R5 in the number of k are each independently an organic group
30 having at least one aliphatic hydrocarbon group having one or
more branched chains and the total carbon number of not less
than 14 and not more than 300;
k is an integer of 1 to 4;
ring A optionally further has, in addition to R4, QR5 in the
number of k and *C (R,) (Rb), a substituent selected from the
5 group consisting of a halogen atom, a C1-6 alkyl group
optionally substituted by one or more halogen atoms, and a C1-6
alkoxy group optionally substituted by one or more halogen
atoms;
R, is a hydrogen atom; and
10 Rb is a hydrogen atom, or a group represented by the formula
(a3) :
[0185]
15 [0186]
wherein * indicates the bonding position;
j is an integer of 0 to 4;
Q in the number of j are each independently as defined above;
R7 in the number of j are each independently an organic group
20 having at least one aliphatic hydrocarbon group having one or
more branched chains and the total carbon number of not less
than 14 and not more than 300;
R6 is a hydrogen atom, or shows, in combination with R4, a
single bond or -0- to form a fluorenyl group or a xanthenyl
25 group together with ring A; and
ring B optionally further has, in addition to QR7 in the number
of j and R6, a substituent selected from the group consisting
of a halogen atom, a C1-6 alkyl group optionally substituted by
one or more halogen atoms, and a C1-6 alkoxy group optionally
30 substituted by one or more halogen atoms.
[0187]
That is, a preferable one embodiment of the 3'-hydroxylprotecting
group in the n-mer oligonucleotide in the present
invention is represented by the following formula (111):
[0188]
5 -L-Y-z
[0189]
wherein
L and Y are as defined above, and
Z is the formula (a2):
10 [0190]
wherein each symbol is as defined above.
15 [0192]
When the nucleotide 3'-hydroxyl-protecting group is -L-YZ'
and Z' is a 'group having branched chain", preferably a
group represented by the formula (a2), then a solvent, wherein
solubility of the nucleoside or oligonucleotide is improved, is
20 preferably a non-polar solvent.
[0193]
Preferable embodiments of L and Y in the above-mentioned
formula (111) are similar to those of the above-mentioned
formula (111' ) .
25 [0194]
The preferable embodiment for Z' in the above-mentioned
formula (111' ) , that is, a group represented by the formula
(a2) for Z in the above-mentioned formula (111) is a particular
benzyl group (in the formula (a2), both R, and Rb are hydrogen
30 atoms, and R~ is a hydrogen atom); a particular diphenylmethyl
group (in the formula (a2), R, is a hydrogen atom, R~ is a
hydrogen atom, k is 1 t o 3, and Rb is a group represented by
t h e formula (a3) wherein R6 is a hydrogen atom, and j is 0 or
1) ; a p a r t i c u l a r fluorenyl group ( i n t h e formula (a2) , R, is a
hydrogen atom, k i s 1, Rb is a group represented by t h e formula
5 (a3) wherein j is 0, and R6 shows, t o g e t h e r with R4, a s i n g l e
bond t o form a f l u o r i n e r i n g t o g e t h e r with r i n g A ) ; a
p a r t i c u l a r xanthenyl group ( i n t h e formula (a2) , R, is a
hydrogen atom, k is 1, Rb is a group represented by t h e formula
(a3) wherein j is 0, and R6 shows -0- t o g e t h e r with R4 t o form
l o a xanthine r i n g together with r i n g A ) .
[0195]
In t h e formula (111), t h e "organic group having a t l e a s t
one a l i p h a t i c hydrocarbon group having one or more branched
chains and a t o t a l carbon number of not less than 1 4 and not
15 more than 300" f o r R5 and R7 is an organic group having a t
l e a s t one a l i p h a t i c hydrocarbon group having one o r more
branched chains i n a molecular s t r u c t u r e t h e r e o f , and a t o t a l
carbon number of not less than 1 4 and not more than 300.
[0196]
20 The "branched chain" of t h e " a l i p h a t i c hydrocarbon group
having one or more branched chains" is a s t r a i g h t o r branched
s a t u r a t e d a l i p h a t i c hydrocarbon group. Preferred is a C1-6
a l k y l group, more p r e f e r r e d is a C1-4 a l k y l group, and s t i l l
more p r e f e r r e d is a methyl group or an e t h y l group. In
25 a d d i t i o n , t h e 'branched chain" is o p t i o n a l l y s u b s t i t u t e d by one
or more halogen atoms.
[0197]
The " a l i p h a t i c hydrocarbon group" of t h e " a l i p h a t i c
hydrocarbon group having one or more branched chains" is a
30 s t r a i g h t s a t u r a t e d or unsaturated a l i p h a t i c hydrocarbon group,
a C2-C300 a l k y l group (preferably, a C3-Cloo a l k y l group, more
p r e f e r a b l y , a C3-Cso a l k y l group) , a C2-C300 alkenyl group
( p r e f e r a b l y , a C3-Cleo alkenyl group, more p r e f e r a b l y , a C3-Cso
alkenyl group) o r a C2-C300 alkynyl group ( p r e f e r a b l y , a C3-Cloo
35 alkynyl group, more p r e f e r a b l y , a C3-Cso alkynyl group).
[0198]
The moiety of the "aliphatic hydrocarbon group having one
or more branched chains" of the "organic group having at least
one aliphatic hydrocarbon group having one or more branched
5 chains, and a total carbon number of not less than 14 and not
more than 300" is not particularly limited, and it may be
present at the terminal (monovalent group), or other site (e.g.,
divalent group) .
[0199]
Specific examples of the "aliphatic hydrocarbon group
having one or more branched chains" include a monovalent group
having one or more branched chain(s) of a branched isomer of a
propyl group, a butyl group, a pentyl group, a hexyl group, a
heptyl group, an octyl group, a nonyl group, a decyl group, an
15 undecyl group, a dodecyl group (a lauryl group), a tridecyl
group, a myristyl group, a cetyl group, a stearyl group, an
arachyl group, a behenyl group, an oleyl group, a linolyl group,
a lignoceryl group and the like, and a divalent group derived
therefrom, preferably, a 3,7,11-trimethyldodecyl group, a
20 3,7,11,15-tetramethylhexadecyl group (hereinafter sometimes to
be referred to as a 2,3-dihydrophytyl group), a 2,2,4,8,10,10-
hexamethylundecan-5-yl group, and the like.
[0200]
When plural "aliphatic hydrocarbon groups having one or
25 more branched chains" are in the "organic group having at least
one aliphatic hydrocarbon group having one or more branched
chains, and a total carbon number of not less than 14 and not
more than 300", each may be same or different.
[ 02011
The moiety other than the "aliphatic hydrocarbon group
having one or more branched chains" of the "organic group
having at least one aliphatic hydrocarbon group having one or
more branched chains, and a total carbon number of not less
than 14 and not more than 300" can be set freely. For example,
35 the group optionally has moieties such as -0-, -S-, -CO-, -NH-,
-COO-, -0CONH-, -CONH-, -NHCO-, hydrocarbon group (monovalent
group or divalent group) and the like. Examples of the
"hydrocarbon group" include an aliphatic hydrocarbon group, an
aromatic aliphatic hydrocarbon group, a monocyclic saturated
5 hydrocarbon group, an aromatic hydrocarbon group and the like.
Specifically, for example, monovalent groups such as an alkyl
group, an alkenyl group, an alkynyl group, a cycloalkyl group,
an aryl group, an aralkyl group and the like, and divalent
groups derived therefrom are used. As the "alkyl group", a C1-6
lo alkyl group and the like are preferable and, for example,
methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,
tert-butyl, pentyl, hexyl and the like can be mentioned. As
the "alkenyl group", a C2-6 alkenyl group and the like are
preferable and, for example, vinyl, 1-propenyl, allyl,
15 isopropenyl, butenyl, isobutenyl and the like can be mentioned.
As the "alkynyl group", a C2-6 alkynyl group and the like are
preferable and, for example, ethynyl, propargyl, 1-propynyl and
the like can be mentioned. As the 'cycloalkyl group", a C3-6
cycloalkyl group and the like are preferable and, for example,
20 cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl can be
mentioned. As the "aryl group", for example, a C6-14 aryl group
and the like are preferable and, for example, phenyl, 1-
naphthyl, 2-naphthyl, biphenylyl, 2-anthryl and the like can be
mentioned. Of these, a C6-lo aryl group is more preferable, and
25 phenyl is particularly preferable. As the "aralkyl group", a
C7-20 aralkyl group is preferable and, for example, benzyl, 1-
phenylethyl, 2-phenylethyl, 1-phenylpropyl, naphthylmethyl, 1-
naphthylethyl, 1-naphthylpropyl and the like can be mentioned.
Of these, a C7-16 aralkyl group (C6-lo aryl-C1-6 alkyl group) is
30 more preferable, and benzyl is particularly preferable. The
"hydrocarbon group" is optionally substituted by a substituent
selected from a halogen atom (a chlorine atom, a bromine atom,
a fluorine atom, an iodine atom), an 0x0 group and the like.
[0202]
Z has a Q R ~ group in the number of k. Here, Q is a
single bond, or -0-, -S-, -C (=O) 0-, -C (=O) NH- or -NH-,
preferably 0. The Q R ~ group i n the number of k may be the same
or d i f f e r e n t .
[0203]
In Z, the "organic group having a t l e a s t one a l i p h a t i c
hydrocarbon group having one or more branched chains, and a
t o t a l carbon number of not l e s s than 1 4 and not more than 300"
f o r R5 and R7 preferably has a t o t a l carbon number of not l e s s
than 1 4 , preferably not l e s s than 16, more preferably not l e s s
10 than 18. The "organic group having a t l e a s t one a l i p h a t i c
hydrocarbon group having one or more branched chains, and a
t o t a l carbon number of not l e s s than 1 4 and not more than 300"
f o r R5 and R7 preferably has a t o t a l carbon number of not more
than 300, preferably not more than 200, more preferably not
15 more than 160. In addition, i n the compound of i n the present
invention, while a t o t a l number of the branched chain of the
"organic group having a t l e a s t one a l i p h a t i c hydrocarbon group
having one or more branched chains, and a t o t a l carbon number
of not l e s s than 1 4 and not more than 300" f o r R5 and R7 is not
20 p a r t i c u l a r l y limited, it preferably has a t o t a l number of the
branched chain of preferably two or more, more preferably not
l e s s than 3, more preferably not l e s s than 4, more preferably
not l e s s than 8, more preferably not l e s s than 10. When the
t o t a l number of the branched chain is higher, an
25 oligonucleotide wherein the 3'-hydroxyl group is protected by
s a i d protecting group becomes an o i l showing good s o l u b i l i t y i n
various organic solvents ( p a r t i c u l a r l y , non-polar solvents)
even when the oligonucleotide chain becomes long.
[0204]
As the "organic group having a t l e a s t one a l i p h a t i c
hydrocarbon group having one or more branched chains, and a
t o t a l carbon number of not l e s s than 1 4 and not more than 300"
f o r R~ and R7, a group having the same or d i f f e r e n t divalent
groups represented by the formula ( b ) :
35 [0205]
wherein * shows a bonding p o s i t i o n with the adjacent atom;
5 R8 and R9 are each independently a hydrogen atom or a C1-4 alkyl
group;
X1 is a single bond, or a C1-4 alkylene group,
provided t h a t R8 and R9 are not hydrogen atoms a t the same time,
i s preferable and, for example, a group represented by any of
lo the following formulas (c) t o ( e ) can be mentioned.
[0207]
The carbon number, repeat unit number (ml, no t o n2) and
the l i k e i n t h e d e f i n i t i o n of each symbol i n the formulas (c)
t o (e) are shown f o r convenience, and can be changed as
15 appropriate within the range defined above, so t h a t the t o t a l
number of the carbon w i l l be not l e s s than 1 4 (preferably not
l e s s than 16, more preferably not l e s s than 18) and not more
than 300 (preferably not more than 200, more preferably not
more than 160) . I n t h e following, the formulas ( c ) t o ( e ) a r e
20 successively explained.
[0208]
The formula ( c ) is as described below.
[0209]
[ 02101
wherein* shows a bonding position t o Q;
R1° and R" are both hydrogen atoms, or show =O i n combination;
no is an integer of 2 t o 40;
30 R12 and R13 i n the number of no are each independently a
hydrogen atom or a C1-4 alkyl group;
X2 i n the number of no are each independently a s i n g l e bond or
a C1-4 alkylene group;
R14 i s a hydrogen atom or a C1-4 alkyl group; and
5 R15 i s a C1-4 alkyl group;
provided t h a t R12 and R13 are not hydrogen atoms a t the same
time, and when no is 2, R14 is a Cl-4 alkyl group.
[0211]
In the group of the formula ( c ) , a group wherein
l o R1° and R1l are both hydrogen atoms;
no is an i n t e g e r of 2 t o 40;
R12 and R13 i n the number of no are each independently a
hydrogen atom, a methyl group or an ethyl group;
X2 i n the number of no are each independently a s i n g l e bond, a
15 methylene group or an ethylene group; and
R14 is a hydrogen atom, a methyl group or an ethyl group
i s preferable, provided t h a t R" and R13 are not hydrogen atoms
a t the same time, and when no is 2, R14 is methyl or an ethyl
group.
20 [0212]
More preferable group of the formula ( c ) is a group of a
branched isomer having a carbon number of 1 4 t o 160, of a
myristyl group, a c e t y l group, a s t e a r y l group, an arachyl
group, a behenyl group and the l i k e . Of these, a 2,3-
25 dihydrophytyl group, a 3,7,11-trimethyldodecyl group and a
2,2,4,8,10,10-hexamethyl-5-dodecanoyl group a r e p a r t i c u l a r l y
p r e f e r a b l e .
[0213]
The formula (d) is as described below.
30 [0214]
wherein * shows a bonding position to Q;
0R16 in the number of ml are each independently hydroxyl group
substituted by a group represented by the formula (c); and
ml is an integer of 1 to 3.
5 The group represented by the formula (c) is the same as
the group represented by the above-mentioned formula (c) except
that * does not show a bonding position to Q but shows a
bonding position to 0.
[0216]
10 In the group of the formula (d), R16 is more preferably a
group of a branched isomer having a carbon number of 14 to 30
of a myristyl group, a cetyl group, a stearyl group, an arachyl
group, a behenyl group and the like. Of these, a 2,3-
dihydrophytyl group and a 3,7,11-trimethyldodecyl group are
15 particularly preferable.
[0217]
The formula (e) is as described below.
[0218]
wherein * shows a bonding position to Q;
nl is an integer of 1 to 10;
n2 is an integer of 1 to 10;
25 R20 and R21 in the number of nl are each independently a
hydrogen atom or a C1-4 alkyl group;
X3 in the number of nl are each independently a single bond or
a C1-4 alkylene group;
R~~ and R~~ in the number of 1-22 are each independently a
30 hydrogen atom or a C1-4 alkyl group;
X5 in the number of n2 are each independently a single bond or
a C1-4 alkylene group;
X4 is a single bond or a C1-4 alkylene group; and
R17, R", R19, R ~ R~~~, an d R~~ are each independently a hydrogen
atom or a C1-4 alkyl group,
provided that R20 and R21 and/or R~~ and R~~ are not hydrogen
5 atoms at the same time, and when nl+n2 is 2, two or more of R17,
R1' and R19 are each independently a C1-4 alkyl group, or two or
more of R ~ R~~~, an d R~~ are each independently a C1-4 alkyl group.
[0220]
A group of the formula (e), wherein
lo nl is an integer of 1 to 5;
n2 is an integer of 1 to 5;
R20 and R21 in the number of nl are each independently a
hydrogen atom, a methyl group or an ethyl group;
X3 in the number of nl are each independently a single bond, a
15 methylene group or an ethylene group;
R~~ and R~~ in the number of n2 are each independently a
hydrogen atom, a methyl group or an ethyl group;
X5 in the number of n2 are each independently a single bond, a
methylene group or an ethylene group;
20 X4 is a single bond, a methylene group or an ethylene group;
and
R17, R", R19, R ~ R~~~, an d R~~ are each independently a hydrogen
atom or a C1-4 alkyl group is more preferable,
provided that R20 and R21 and/or R~~ and R~~ are not hydrogen
25 atoms at the same time, and when nl+n2 is 2, two or more of R17,
R18 and R19 are each independently a C1-4 alkyl group, or two or
more of R ~ R~~~, an d R~~ are each independently a C1-4 alkyl group.
[0221]
A group of the formula (e), wherein
30 nl is an integer of 1 to 5;
n2 is an integer of 1 to 5;
R20 and R21 in the number of nl are each independently a
hydrogen atom or a methyl group;
X3 in the number of nl are each independently a single bond or
35 a methylene group;
R~~ and R~~ i n the number of n2 are each independently a
hydrogen atom or a methyl group;
X5 i n the number of n2 are each independently a s i n g l e bond or
a methylene group;
5 X4 is a single bond or a methylene group; and
R17, R19, R ~ R~Z5, a nd RZ6 are each a methyl group, provided
t h a t RZ0 and R21 and/or R~~ and RZ3 are not hydrogen atoms a t the
same time is p a r t i c u l a r l y p r e f e r a b l e .
Specific examples of the "organic group having a t l e a s t
one a l i p h a t i c hydrocarbon group having one or more branched
chains, and a t o t a l carbon number of not l e s s than 1 4 and not
more than 300" f o r R5 and R7 include the following groups,
wherein * i n each group shows a bonding p o s i t i o n , n3 i n the
15 formula is an i n t e g e r of not l e s s than 3, and n4 can be
a p p r o p r i a t e l y a d j u s t e d so t h a t the t o t a l carbon number of the
group w i l l be not l e s s than 1 4 and not more than 300.
[0223]
[0224]
S p e c i f i c p r e f e r a b l e examples of the "organic group having
at least one aliphatic hydrocarbon group having one or more
branched chains, and a total carbon number of not less than 14
and not more than 300" for R5 and R7 include the following
groups :
5 3,7,11,15-tetramethylhexadecyl group;
3,7,11-trimethyldodecyl group;
2,2,4,8,10,10-hexamethyl-5-dodecanoyl group;
3,4,5-tri (3', 7', llr,1 5' -tetramethylhexadecyloxy)b enzyl group;
and
lo 3,5-di (3', 7' ,11' ,15' -tetramethylhexadecyloxy)b enzyl group.
[0225]
Preferable examples of protecting group represented by
the formula (111' ) or the formula (111) of the present
invention include the following benzylsuccinyl group, or
15 diphenylmethylsuccinyl group, which are not to be construed as
limiting the present invention:
2-{2,4-di(2',3'-
dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group; 3,5-
di (2 ,3' -dihydrophytyloxy) benzylsuccinyl group; 4- (2', 3' -
20 dihydrophytyloxy)benzylsuccinyl group; 2-{I-[(2-chloro-5-
group; 3,4,5-tri(2',3'-dihydrophytyloxy)benzylsuccinyl group;
2-{3,4,5-tri (2',3'-
25 dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group; 2-(4-
(2',3'-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl
group; 2-(2- [3', 4', 5'-tri (2' ' ,3' ' -dihydrophytyloxy) benzyloxy] -
4-methoxybenzylaminocarbonyl}ethylcarbonyl group; 2-{4-(2',3'-
dihydrophytyloxy)-2-methoxybenzylaminocarbonyl}ethylcarbonyl
30 group; 4-(2',3'-dihydrophytyloxy)-2-methylbenzylsucciny1 group;
2-(4- (2', 3'-dihydrophytyloxy) -2-
methylbenzylaminocarbonyl}ethylcarbonyl group; 4-
[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]benzylsuccinyl
group; 2-{4- [2,2,4,8,10,10-hexamethyl-5-
35 dodecanoylamino]benzylaminocarbonyl}ethylcarbonyl group; 4-
(3,7,11-trimethyldodecyloxy)benzylsuccinyl group; 2-{4-(3,7,11-
trimethyldodecyloxy)benzylaminocarbonyl}ethylcarbonyl group; 2-
{3,5-di (2',3'-
dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group; 2-{I-
5 [2,3,4-tri(2',3'-
dihydrophytyloxy)phenyl]benzylaminocarbonyl}ethylcarbonyl
group; 2-{1-[4-(2',3'-dihydrophytylo~y)phenyl]-4~-(2~,3~-
dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group;
3,4,5-tris [3,4,5-tri(2',3'-
10 dihydrophytyloxy) benzyl] benzylsuccinyl group; and 2- { 3,4,5-
tris [3,4,5-tri (2', 3'-
dihydrophytyloxy)benzyl]benzylaminocarbonyl}ethylcarbonyl group.
[0226]
4-3. Explanation of "optionally protected nucleic acid base"
The "optionally protected nucleic acid base" represented
by Base in the formulas (i) and (ii) means, for example, that
an amino group may be protected in an adenyl group, a guanyl
group or a cytosyl group, which is a nucleic acid base having
an amino group, or an imido group may be protected in a thymyl
20 group, or an uracil group, which is a nucleic acid base having
a cyclic imido group, a nucleic acid base wherein the amino
group therein is protected by a protecting group sustainable
under the deprotection conditions of the 5'-position is
preferable. The "amino-protecting group" and "imide-protecting
25 group" are not particularly limited, and examples thereof
include the protecting groups described in Greene's PROTECTIVE
GROUPS IN ORGANIC SYNTHESIS, 4th edition, JOHN WILLY&SONS, 2006
and the like. Specific examples of the "amino-protecting
group" include a pivaloyl group, a pivaloyloxymethyl group, a
30 trifluoroacetyl group, a phenoxyacetyl group, a 4-
isopropylphenoxyacetyl group, a 4-tert-butylphenoxyacetyl group,
an acetyl group, a benzoyl group, an isobutyryl group, a
dimethylformamidinyl group, a 9-fluorenylmethyloxycarbonyl
group and the like. Among them, a phenoxyacetyl group, a 4-
35 isopropylphenoxyacetyl group, an acetyl group, a benzoyl group,
an isobutyryl group and a dimethylformamidinyl group are
preferable. In addition, the carbonyl group of the nucleic
acid base is optionally protected, and can be protected, for
example, by reacting phenol, 2,5-dichlorophenol, 3-chlorophenol,
5 3,5-dichlorophenol, 2-formylphenol, 2-naphthol, 4-methoxyphenol,
4-chlorophenol, 2-nitrophenol, 4-nitrophenol, 4-
acetylaminophenol, pentafluorophenol, 4-pivaloyloxybenzyl
alcohol, 4-nitrophenethyl alcohol, 2-(methylsulfonyl)ethanol,
2- (phenylsulfo nyl)e thanol, 2-cyanoethanol, 2-
10 (trimethylsilyl)ethanol, dimethylcarbamoyl chloride,
diethylcarbamoyl chloride, ethylphenylcarbamoyl chloride, 1-
pyrrolidinecarbonyl chloride, 4-morpholinecarbonyl chloride,
diphenylcarbamoyl chloride and the like. In some cases, the
carbonyl-protecting group does not need to be particularly
15 introduced.
[0227]
Another preferable embodiment of the protecting group of
the nucleic acid base includes a "group having a C5-30 straight
chain or branched chain alkyl group and/or a C5-30 straight
20 chain or branched chain alkenyl group". The "group having a
C5-30 straight chain or branched chain alkyl group and/or a C5-30
straight chain or branched chain alkenyl group" is as defined
for the "group having a C5-30 straight chain or branched chain
alkyl group and/or a C5-30 straight chain or branched chain
25 alkenyl group" of the 'nucleic acid base protected by a group
having a C5-30 straight chain or branched chain alkyl group
and/or a C5-30 straight chain or branched chain alkenyl group"
for ~ a s ein~ t he above-mentioned formula (I).
Of these, a "group having a C5-30 branched chain alkyl
30 group and/or a C5-30 branched chain alkenyl group" is preferable,
and a "group having a C5-30 branched chain alkyl group" is more
preferable.
Protection of a nucleic acid base by a "group having a
C5-30 straight chain or branched chain alkyl group and/or a C5-30
35 straight chain or branched chain alkenyl group" further confers
l i p o s o l u b i l i t y and s o l u b i l i t y i n an organic solvent
( p a r t i c u l a r l y , non-polar solvent) t o an n-mer oligonucleotide,
which is advantageous f o r the synthesis of a long chain
oligonucleotide.
5 [0228]
A t l e a s t one n u c l e i c a c i d base of an n-mer
oligonucleotide is preferably protected by a group having a C5-
30 s t r a i g h t chain or branched chain alkyl group and/or a C5-30
s t r a i g h t chain or branched chain alkenyl group, and more
10 preferably protected by a group having a C5-30 branched chain
a l k y l group and/or a C5-30 branched chain alkenyl group.
In t h i s case, a t l e a s t one nucleic acid base of the n-mer
oligonucleotide only needs t o be protected by the protecting
group, or a l l nucleic acid bases i n the number of n may be
15 protected by the protecting group, or a p a r t thereof may be
protected by the protecting group and o t h e r n u c l e i c acid bases
may be protected by a protecting group conventionally used i n
the f i e l d of nucleic acid synthesis ( e . g . , pivaloyl group,
pivaloyloxymethyl group, t r i f l u o r o a c e t y l group, phenoxyacetyl
20 group, 4-isopropylphenoxyacetyl group, 4-tertbutylphenoxyacetyl
group, acetyl group, benzoyl group,
isobutyryl group, dimethylformamidinyl group, 9-
fluorenylmethyloxycarbonyl group e t c . ) .
[0229]
25 Thus, when a l l or a p a r t of the nucleic acid bases
represented by Base a r e / i s protected by a group having a C5-30
s t r a i g h t chain or branched chain alkyl group and/or a C5-30
s t r a i g h t chain or branched chain alkenyl group, the obtained
n+p-mer oligonucleotide shows f u r t h e r improved l i p o s o l u b i l i t y
30 and s o l u b i l i t y i n an organic solvent ( p a r t i c u l a r l y , non-polar
s o l v e n t ) , which f a c i l i t a t e s the extraction operation i n the
next step ( 4 ) and enables synthesis of an oligonucleotide
having a higher degree of polymerization.
In the nucleic acid base i n the number of n, the r a t i o of
35 protection by a group having a C5-30 s t r a i g h t chain or branched
chain alkyl group and/or a C5-30 straight chain or branched
chain alkenyl group can be appropriately set so that the n-mer
oligonucleotide shows sufficient solubility in an organic
solvent (particularly, non-polar solvent).
5 [0230]
Preferable examples of the "group having a C5-30 straight
chain or branched chain alkyl group and/or a C5-30 straight
chain or branched chain alkenyl group" and "group having a C5-30
branched chain alkyl group and/or a C5-30 branched chain alkenyl
10 group" are as explained for the aforementioned formula (I).
[0231]
4-4. Oligonucleotide protected by a branched chain-containing
aromatic group
A more preferable embodiment of the n-mer oligonucleotide
15.in the present invention is a novel oligonucleotide represented
by the following formula (11), wherein, in the aforementioned
formulas (i) and (ii) , a 3' -hydroxyl-protecting group
represented by p3 is a group represented by the aforementioned
formula (111): -L-Y-Z (sometimes to be referred to as "branched
20 chain-containing aromatic protecting group" in the present
specification) (sometimes to be referred to as an
"oligonucleotide protected by a branched chain-containing
aromatic group" in the present specification).
When m is 0, the oligonucleotide protected by a branched
25 chain-containing aromatic group, which is represented by the
formula (11), is understood to mean a "nucleoside protected by
a branched chain-containing aromatic group".
[0232]
Since the oligonucleotide protected by a branched chain-
30 containing aromatic group of the present invention is easily
soluble in a non-polar solvent superior in the partitioning
operability, which is a reaction solvent in the production
method of the present invention, it is extremely useful as a
novel compound usable for a production method of
35 oligonucleotide wherein the reaction in each step can be
performed smoothly, and the final resultant product can be
obtained without crystallization and isolation of each
intermediate but via an extraction separation alone (also
referred to as one-pot synthesis method).
5 [0233]
The compound should be clearly differentiated from the
protecting group having a straight chain structure described in
JP-A-2010-275254 since it shows good solubility particularly in
heptane as a representative solvent of non-polar solvents.
In addition, as compared to conventional liquid phase
methods, since the compound permits stable dissolution and
transfer into a nonpolar solvent irrespective of the degree of
polymerization (sequence and chain length) of oligonucleotide,
it is advantageous in that the isolation and purification step
15 can be simplified as for the steps, and high purity and high
yield can be ensured as a total view.
[0234]
While the lower limit of the solubility
(=solute/ (solventfsolute) ~100) (mass %) of a nucleoside
20 protected by a branched chain-containing aromatic group wherein
m is 0 in heptane at 20°C is not particularly limited as long
as the binding to a reaction substrate and the reaction
thereafter proceed, it is preferably 1 mass %, more preferably
2 mass %, further preferably 5 mass %, still more preferably 10
25 mass %, especially preferably 25 mass %, particularly
preferably 50 mass %.
[0235]
The upper limit of the solubility
(=solute/( solvent+solute)~ 100)( mass %) of a nucleoside
30 protected by a branched chain-containing aromatic group wherein
m is 0 in .heptane at 20°C is preferably 80 mass %, more
preferably 85 mass %, further preferably 90 mass %, still more
preferably 95 mass %, particularly preferably 98 mass %, since
the reaction can proceed stably irrespective of the industrial
35 progress degree of the reaction.
[0236]
In the present specification, the "solubility" means the
percentage (mass % ) of the mass of solute relative to the total
mass of the solvent and the solute when the solute is saturated
5 in the solvent.
[0237]
The formula (11) :
[0238]
r
',
L-Y-Z
10 [0239]
wherein
m is an integer of 0 or more;
ase el in the number of m+l are each independently an optionally
protected nucleic acid base;
15 P' is a hydrogen atom, or a temporary protecting group
removable under acidic conditions;
X is a hydrogen atom, an optionally protected hydroxyl group, a
halogen atom or an organic group crosslinked with the 4-
position carbon atom;
20 X' in the number of m are each independently a hydrogen atom,
an optionally protected hydroxyl group, a halogen atom or an
organic group crosslinked with the $-position carbon atom;
p2 in the number of m are each independently a protecting group
removable under basic conditions;
25 R~~ in the number of m are each independently an oxygen atom or
a sulfur atom; and
L, Y and Z are as defined above.
[0240]
In compound (11) of the present invention, p-mer
5 oligonucleotide wherein the 3'-hydroxyl group is
phosphoramidited and the 5'-hydroxyl group is protected by a
temporary protecting group is bonded via oxygen atom of 5'-
hydroxyl group to form m+l+p-mer oligonucleotide.
The compound (11) wherein m is 0 of the present invention
10 is a starting compound of 3'-terminal in the oligonucleotide
synthesis. In addition, the compound of the present invention
also encompasses a compound wherein 5'-hydroxyl group is not
protected (P' is a hydrogen atom) in a wide sense.
[0241]
The definition, preferable embodiments and the like of P',
P2, X, X' and R~~ are similar to those of the above-mentioned
compound (I).
The definition, preferable embodiments and the like of m
are similar to those of the above-mentioned compound (i) and
20 (ii) .
[0242]
The preferable embodiments of L, Y and Z in the formula
(11) are similar to those of L, Y and Z in the aforementioned
formula (111' ) or (111) .
25 [0243]
The "optionally protected nucleic acid base" represented
by Base' is similar to that in the aforementioned formulas (i)
and (ii) .
[0244]
Since the oligonucleotide protected by a branched chaincontaining
aromatic group, which is represented by the formula
(11), is conferred with sufficient liposolubility by a branched
chain-containing aromatic protecting group, it is not
necessary; however, Base' is optionally protected by a group
35 having a C5-30 straight chain or branched chain alkyl group
and/or a C5-30 s t r a i g h t chain or branched chain alkenyl group,
preferably a group having a C5-30 branched chain alkyl group
and/or a C5-30 branched chain alkenyl group t o f u r t h e r improve
the l i p o s o l u b i l i t y .
Preferable examples of the "group having a C5-30 s t r a i g h t
chain or branched chain alkyl group and/or a C5-30 s t r a i g h t
chain or branched chain alkenyl group" and the "group having a
C5-30 branched chain alkyl group and/or a C5-30 branched chain
alkenyl group" are as mentioned above.
10 [0245]
A preferable embodiment of the compound represented by
the formula (11) of the present invention is a compound of the
formula ( I I a ) , wherein
m is 0;
15 ase el is a cytosyl group, a u r a c i l group, a thyminyl group, an
adenyl group, or a guanyl group, each of which is optionally
protected;
P1 is a d i (C1-6 alkoxy) t r i t y l group, or a mono (C1-6 alkoxy) t r i t y l
group;
20 X is a hydrogen atom, an optionally protected hydroxyl group,
f l u o r i n e atom, -0Ri ( R i is as defined above), - o - N R ~ ~ - R ~ ( R j
and R~~ are as defined above), or -0-Rk-O-R1 (Rk and R1 are as
defined above); and
L-Y-Z is the combination of each group shown as a preferable
25 embodiment i n the aforementioned formula (111') o r t h e formula
(111).
[0246]
Another preferable embodiment of the compound represented
by the formula (11) of the present invention is a compound of
30 the formula ( I I b ) , wherein
~ a s e ' i s a cytosyl group, a u r a c i l group, a thyminyl group, an
adenyl group, or a guanyl group, each of which is optionally
protected;
35 P1 is a dimethoxytrityl group or a monomethoxytrityl group;
X is a hydrogen atom, an optionally protected hydroxyl group,
fluorine atom, -0-CH2-, -0-CH2-CH2-, or -O-NR~~-CH(~-R i~s ~as
defined above), -0-CH2-O-CH2- (in all of which the left side
binds to the 2-position and the right side binds to the 4-
5 position); and
L-Y-Z is the combination of each group shown as a preferable
embodiment in the aforementioned formula (111' ) or the formula
(111).
102471
10 A still another preferable embodiment of the compound
represented by the formula (11) of the present invention is a
compound of the formula (IIc), wherein
m is 0;
ase el is a cytosyl group, a uracil group, a thyminyl group, an
15 adenyl group, or a guanyl group, each of which is optionally
protected;
P' is a dimethoxytrityl group;
X is a hydrogen atom, methoxy group, tert-butyldimethylsilyloxy
group, fluorine atom, -0-CH2-, -0-CH2-CH2-, -0-NH-CH2-, -0-NMe-
20 CH2-, -0-CH2-O-CH2- (in all of which the left side binds to the
2-position and the right side binds to the 4-position); and
L-Y-Z is the combination of each group shown as a preferable
embodiment in the aforementioned formula (111') or the formula
4-5. Production method of oligonucleotide protected by branched
chain-containins aromatic sroup
While the production method of a nucleoside protected by
a branched chain-containing aromatic group represented by the
30 formula (II'), which is an oligonucleotide protected by a
branched chain-containing aromatic group represented by the
formula (11) wherein m is 0, is not particularly limited, it
can be produced by a method known per se (Richard T. Pon et al.,
Nucleic Acids Research 2004, 32, 623-631) or a method analogous
35 thereto.
When a starting compound has a substituent (e.g.,
hydroxyl group, amino group, carboxy group) that influences the
reaction, the starting compound is generally protected in
advance by a suitable protecting group according to a known
5 method and then subjected to the reaction. Such protecting
group can be removed after the reaction by a known method such
as an acid treatment, an alkali treatment, a catalytic
reduction and the like.
A general production method of a nucleoside protected by
10 a branched chain-containing aromatic group of the abovementioned
formula (11') wherein L is a succinyl group is shown
below.
[0249]
15 [0250]
wherein each symbol is as defined above.
Nucleoside (q) wherein 5'-hydroxyl group is protected by
protecting group P' is reacted with succinic anhydride in the
presence of a base to give compound (r) wherein succinic acid
20 is introduced into 3'-hydroxyl group. A nucleoside protected
by a branched chain-containing aromatic group represented by
the formula (11') can be obtained by dehydration condensation
of compound (r) with Z-Y-H in the presence of a condensing
agent.
25 [0251]
The conversion step of the above-mentioned nucleoside (q)
to compound (r) is advantageously performed in a solvent inert
to the reaction. While such solvent is not particularly
limited as long as the reaction proceeds, halogenated
30 hydrocarbon solvents such as dichloromethane, 1,2-
dichloroethane, chloroform, carbon tetrachloride and the like,
aromatic hydrocarbon solvents such as benzene, toluene, xylene
and the like, aliphatic hydrocarbon solvents such as pentane,
hexane, heptane, octane and the like, ether solvents such as
5 diethyl ether, tetrahydrofuran, cyclopentyl methyl ether and
the like, and mixed solvents thereof are preferable. Of these,
dichloromethane and chloroform are particularly preferable.
[0252]
While the base is not particularly limited, for example,
lo an organic base mentioned below can be used, with preference
given to triethylamine.
[0253]
The above-mentioned dehydrating condensation step is
advantageously performed in a solvent inert to the reaction.
15 While such solvent is not particularly limited as long as the
reaction proceeds, halogenated hydrocarbon solvents such as
dichloromethane, 1,2-dichloroethane, chloroform, carbon
tetrachloride and the like, aromatic hydrocarbon solvents such
as benzene, toluene, xylene and the like, or aliphatic
20 hydrocarbon solvents such as pentane, hexane, heptane, octane
and the like, and mixed solvents thereof are preferable. Of
these, dichloromethane and chloroform are particularly
preferable.
[0254]
25 Examples of the condensing agent used for the
condensation reaction of compound (r) with Z-Y-H include
dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC),
N-ethyl-N'-3-dimethylaminopropylcarbodiimide and hydrochloride
thereof (EDC HCl), (benzotriazol-l-
30 yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBop), O-
(benzotriazol-l-y1)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TBTU) , l- [bis (dimethylamino)m ethylene] -5-
chloro-1H-benzotriazolium-3-oxide hexafluorophosphate (HCTU),
O-benzotriazole-N,N,Nf,N'-tetramethyluronium
35 hexafluorophosphate (HBTU) and the like. Of these, HBTU, HCTU,
N-ethyl-N'-3-dimethylaminopropylcarbodiimide and hydrochloride
thereof (EDC HC1) are preferable.
LO2551
The amount of the condensing agent to be used is 1 to 10
5 mol, preferably 1 to 5 mol, per 1 mol of compound (r) . The
amount of Z-Y-H to be used is 1 to 10 mol, preferably 1 to 5
mol, per 1 mol of compound (r). While the reaction temperature
is not particularly limited as long as the reaction proceeds,
it is preferably -lO°C to 50°C, more preferably O°C to 30°C.
lo The reaction time is 30 min to 70 hr.
LO2561
A compound of the above-mentioned formula (11') wherein L
is other than a succinyl group can also be produced by
performing a reaction similar to the above-mentioned production
1s method except that a corresponding acid anhydride, a
corresponding dicarboxylic halide, an activated ester of
corresponding dicarboxylic acid and the like is used instead of
succinic anhydride.
A compound of the above-mentioned formula (11) wherein m
20 is one or more can be produced by repeating the 5'-terminal
elongation process according to the following production method
of the present invention and using a compound represented by
the formula (11' ) as starting material.
[0257]
25 4-6. Production method of Z-Y-H (alcohol or amine)
While the production method of an alcohol compound or an
amine compound represented by the formula: Z-Y-H, which is a
starting compound used for the production of an oligonucleotide
protected by a branched chain-containing aromatic group, is not
30 particularly limited, for example, it can be produced by the
following steps.
[0258]
Step
(Q"-.or s, C02, NH)
Step
-
/ Step - _____)
1)Step (d-3)
Step
wherein Q' is -0-, -S-, -C (=O) 0- or -NH-, R, is a hydrogen atom,
an ORh group (wherein Rh is an alkyl group such as a ClU6 alkyl
5 group and the like, an aralkyl group such as benzyl group and
the like, and the like) or a group represented by the formula
(a3) :
102601
wherein each symbol is as defined above, Y1 is a leaving group
such as a halogen atom and the like, and other symbol is as
defined above.
[0262]
step (a)
5 In this step, an R5 group is introduced into a QfH group
wherein Qf is -0-, -S-, -C(=O)O- or -NH- of a compound
represented by the formula (IV) (hereinafter to be abbreviated
as compound (IV)) to give a compound represented by the formula
(IVa) (hereinafter to be abbreviated as compound (IVa)).
10 When Q' is -0-, -S- or -NH-, the reaction is carried out
in a solvent that does not influence the reaction, in the
presence or absence of a base and using a halide corresponding
to an R5 group (chloride, bromide or iodide), a carboxylic acid
or an acid halide corresponding to an R5 group or
15 alkylsulfonyloxylation product (e.g., methanesulfonyloxylation
product etc.) or an arylsulfonyloxylation product (e.g., ptoluenesulfonyloxylation
product etc.) corresponding to an R5
group. In addition, when Q' is -0-, the reaction can be
carried out under the conditions of Mitsunobu reaction
20 including reacting compound (IV) with hydroxide corresponding
to an R5 group in the presence of triphenylphosphine and
diisopropyl azodicarboxylate,. Furthermore, when Q' is -
C (=O) 0-, for example, compound (IVa) can be synthesized by
reacting compound (IV) with amine or hydroxide corresponding to
25 an R5 group in the presence of the below-mentioned condensing
agent.
102631
Examples of the base include alkali metal salt such as
sodium carbonate, sodium hydrogen carbonate, potassium
30 carbonate, sodium hydride, potassium hydride, potassium tertbutoxide
and the like; amines such as pyridine, triethylamine,
N,N-dimethylaniline, 1,8-diazabicyclo[5.4.0]undec-7-ene etc.,
and the like. Of these, sodium carbonate, potassium carbonate,
sodium hydride and the like are preferable.
35 [0264]
Examples of the solvent include aromatic hydrocarbons
such as toluene, xylene and the like; ethers such as
tetrahydrofuran, dioxane and the like; amides such as
dimethylformamide, dimethylacetamide and the like; halogenated
5 hydrocarbons such as chloroform, dichloromethane and the like;
nitriles such as acetonitrile and the like, N-methylpyrrolidone,
and a mixture thereof. Of these, dimethylformamide,
tetrahydrofuran, toluene, N-methylpyrrolidone and the like are
preferable.
lo lo2651
The reaction temperature is preferably 50°C to 150°C,
more preferably 60°C to 130°C. The reaction time is preferably
2 to 30 hr, more preferably 3 to 10 hr.
[0266]
1s step (b)
In this step, compound (IVa) is reduced to give a
compound represented by the formula (I-a) (hereinafter to be
abbreviated as compound (I-a)). The reduction reaction can be
performed by a method using a reducing agent.
20 [0267]
Examples of the reducing agent to be used for the
reduction reaction include metal hydride (sodium borohydride,
lithium borohydride, sodium cyanoborohydride, sodium
triacetoxyborohydride, dibutylaluminum hydride, aluminum
25 hydride, lithium aluminum hydride, etc. ) and the like. Of
these, sodium borohydride, dibutylaluminum hydride and the like
are preferable.
lo2681
The reaction is performed in a solvent that does not
30 influence the reaction. Examples of the solvent include
alcohols such as methanol, ethanol and the like; ethers such as
diethyl ether, tetrahydrofuran, dioxane and the like; aromatic
hydrocarbons such as toluene, xylene and the like; and a
mixture thereof. Of these, tetrahydrofuran, toluene and the
35 like are preferable.
The reaction temperature is preferably O°C to 100°C, more
preferably 30°C to 70°C, and the reaction time is preferably 1
to 24 hr, more preferably 2 to 5 hr.
5 step (c)
In this step, compound (IVa) (in the formula (IVa), R, is
not a hydrogen atom or an ORh group) is reduced in the same
manner as in the above-mentioned step (b).
[0270]
10 step (d-1)
In this step, compound (IVa) (in the formula (IVa), Rg is
a hydrogen atom) is oximated to give a compound represented by
the formula (If-a) (hereinafter to be abbreviated as compound
(1'-a) ) .
15 [0271]
The oximation reaction includes reacting compound (IVa)
with hydroxylamine acid addition salt in a solvent that does
not influence the reaction in the presence of a base.
[0272]
20 Examples of the hydroxylamine acid addition salt include
mineral acid salts such as hydrochloride, sulfate, nitrate and
the like, organic acid salts such as acetate, trifluoroacetate,
methanesulfonate, trifluoromethanesulfonate, p-toluenesulfonate
etc., and the like, and hydrochloride is particularly
25 preferable .
[0273]
Examples of such base include alkali metal salts such as
potassium hydroxide, sodium hydroxide, sodium hydrogen
carbonate, potassium carbonate and the like; organic amines
30 such as pyridine, triethylamine, diisopropylethylamine, N,Ndimethylaniline,
1,8-diazabicyclo [5.4.0] undec-7-ene etc . , and
the like. Of these, triethylamine, diisopropylethylamine and
the like are preferable.
[0274]
Examples of the solvent include halogen solvents such as
chloroform, dichloromethane and the like; aromatic hydrocarbons
such as toluene, xylene and the like; ethers such as
tetrahydrofuran, dioxane and the like; and/or a mixture thereof.
Of these, dichloromethane, chloroform, toluene and the like are
5 preferable.
The reaction temperature is preferably 10°C to 100°C,
more preferably 20°C to 60°C, and the reaction time is
preferably 0.5 to 30 hr, more preferably 2 to 20 hr.
[0275]
lo step (d-2)
In this step, compound (1'-a) is reduced by a catalytic
hydrogenation reaction in the presence of a metal catalyst such
as palladium-carbon, Raney-nickel and the like, or by a
reducing agent such as metal hydride and the like, which is
1s similar to those in the aforementioned step (b), to give a
compound represented by the formula (I-b) (hereinafter to be
abbreviated as compound (I-b)), which is the compound of the
present invention.
[0276]
20 Compound (I-b) can also be produced from step (d-3) via
step (d-4) and step (d-5).
[0277]
step (d-3)
In this step, compound (I-a) is halogenated with, for
25 example, a chlorinating agent such as acetyl chloride, thionyl
chloride and the like or, for example, a brominating agent such
as acetyl bromide, phosphorus tribromide,
diphenylphosphine/bromine and the like to give a compound
represented by the formula (1'-b) (hereinafter to be
30 abbreviated as compound (I' -b) ) .
Examples of the solvent include halogenated hydrocarbons
such as chloroform, dichloromethane, and the like; aromatic
hydrocarbons such as toluene, xylene, and the like; ethers such
as tetrahydrofuran, dioxane, and the like; and a mixture
35 thereof. Of these, chloroform, tetrahydrof uran, toluene, and
the like are preferable.
The reaction temperature is preferably 10°C to 150°C,
more preferably 30°C to 80°C, and the reaction time is
preferably 0.5 to 30 hr, more preferably 2 to 20 hr.
5 [0278]
step (d-4)
In this step, compound (1'-b) is azidated with an
azidating agent such as sodium azide and the like to give a
compound represented by the formula (1'-c) (hereinafter to be
10 abbreviated as compound (I' -c) ) .
The reaction includes reacting compound (1'-b) with an
azidating agent in a solvent that does not influence the
reaction.
Examples of the solvent include halogenated hydrocarbons
15 such as chloroform, dichloromethane, and the like; aromatic
hydrocarbons such as toluene, xylene, and the like; ethers such
as tetrahydrofuran, dioxane, and the like; amides such as N,Ndimethylformamide
and the like; and a mixture thereof. Of
these, chloroform, N,N-dimethylformamide, and the like are
20 preferable.
The reaction temperature is preferably 10°C to 150°C,
more preferably 20°C to 100°C, and the reaction time is
preferably 0.5 to 30 hr, more preferably 2 to 20 hr.
25 step (d-5)
In this step, compound (1'-c) is aminated to give
compound (I-b) .
The reaction is carried out by reacting compound (Ir-c)
with triphenylphosphine in a solvent that does not influence
30 the reaction in the presence of water or catalytic
hydrogenation.
The amount of triphenylphosphine to be used is preferably
1 to 10 mol, particularly preferably 1 to 5 mol, per 1 mol of
compound (1'-c) .
The amount of water to be used is preferably 1 to 10 mol,
particularly preferably 1 to 5 mol, per 1 mol of compound (Ifc)
Examples of the solvent include aromatic hydrocarbons
such as toluene, xylene, and the like; ethers such as
5 tetrahydrofuran, dioxane, and the like; and a mixture thereof.
Of these, toluene, tetrahydrofuran, and the like are preferable.
The reaction temperature is preferably 10°C to 150°C,
more preferably 20°C to 100°C, and the reaction time is
preferably 0.5 to 30 hr, more preferably 2 to 20 hr.
10 [0280]
step (d-6)
In this step, compound (1'-b) is reacted with RNH2
(wherein R is as defined above) to give a compound represented
by the formula (I-c) (hereinafter to be abbreviated as compound
15 (I-c)), which is the compound of the present invention wherein
Y is an -NHR group.
The reaction includes reacting compound (If-b) with amine
represented by R-NH2 in a solvent that does not influence the
reaction in the presence of, where necessary, for example, a
20 base such as tertiary amine (triethylamine,
diisopropylethylamine etc.) and the like.
Examples of the solvent include aromatic hydrocarbons
such as toluene, xylene, and the like; ethers such as
tetrahydrofuran, dioxane, and the like; and, halogen solvents
25 such as chloroform, dichloromethane, and the like and a mixture
thereof. Of these, toluene, tetrahydrofuran, chloroform, and
the like are preferable.
The reaction temperature is generally 10°C to 100°C,
preferably 20°C to 60°C, and the reaction time is generally 0.5
30 to 30 hr, preferably 2 to 20 hr.
[0281]
step (d-7)
In this step, compound (I-d) is reacted with a compound
having a -CONH2 group or a -OCONH2 group, and treated with a
35 base to give compound (I-e).
The reaction of compound (I-d) with a compound having a -
CONH2 group or a -OCONH2 group is carried out in a solvent that
does not influence the reaction and under an acid catalyst.
Examples of the acid catalyst include methanesulfonic
5 acid, trifluoromethanesulfonic acid, toluenesulfonic acid and
the like. Of these, methanesulfonic acid and toluenesulfonic
acid are preferable.
The amount of the acid catalyst to be used is preferably
0.05 to 0.5 mol, particularly preferably 0.1 to 0.3 mol, per 1
10 mol of compound (I-d) .
Examples of the compound having a -CONH2 group or a -
OCONH2 group include Fmoc-NH2, HCONH2, CF3CONH2, AcNH2, EtOCONH2,
Cbz-NH2 and the like. Of these, Fmoc-NH2, EtOCONH2 and the like
are preferable.
Here, the "Fmoc-I' means a 9-f luorenylmethoxycarbonyl
group (hereinafter to be also referred to as a Fmoc group), and
"Cbz-" means a benzyloxycarbonyl group (hereinafter to be also
referred to as a Cbz group) .
[0282]
20 The R5 forming-reagent to be used as a starting compound
of step (a) [i.e., hydroxide, halide, an alkylsulfonyloxylation
product (e.g., methanesulfonyloxylation product etc.) or an
arylsulfonyloxylation product (e.g., p-toluenesulfonyloxylation
product etc.) corresponding to R5 group] may be a commercially
25 available product. In addition, the R5 forming-reagent can be
produced by, for example,
(1) halogenation, alkylsulfonyloxylation or
arylsulfonyloxylation of hydroxide corresponding to an R5 group,
30 (2) reduction reaction of unsaturated hydroxide corresponding
to an R5 group (e.g., catalytic hydrogenation reaction in the
presence of a metal catalyst such as platinum-carbon (Pt/C),
palladium-carbon (pd/C) , rhodium-carbon (Rh/C) , Raney-nickel
etc. and the like), and subsequently halogenation,
35 alkylsulfonyloxylation or arylsulfonyloxylation.
[0283]
In the production of the R5 forming-reagent, examples of
the reagent to be used for conversion to a leaving group from a
hydroxyl group include, in addition to halogenating agent such
5 as chlorinating agent (thionyl chloride, N-chlorosuccinimide
(NCS) and the like) , brominating agent (hydrobromic acid,
acetyl bromide, N-bromosuccinimide (NBS), phosphorus tribromide,
diphenylphosphine/bromine and the like) and the like,
alkylsulfonylating agent such as methanesulfonyl chloride,
lo trifluoromethanesulfonyl chloride and the like,
arylsulfonylating agent such as benzenesulfonyl chloride, ptoluenesulfonyl
toluenesulfonyl chloride et~.a nd the like. Of these, thionyl
chloride, hydrobromic acid and the like are preferable, which
are the halogenating agents.
15 [0284]
The reaction is performed in a solvent that does not
influence the reaction. Examples of the solvent include water,
halogenated hydrocarbons such as chloroform, dichloromethane
and the like; aromatic hydrocarbons such as benzene, toluene,
20 xylene and the like; nitriles such as acetonitrile,
propionitrile and the like; ethers such as tetrahydrofuran,
1,4-dioxane, diethyl ether and the like. Of these, water,
halogenated hydrocarbons such as chloroform and the like are
preferable.
25 The reaction temperature is preferably 10 to 120°C, more
preferably 50 to 100°C, and the reaction time is preferably 1
to 72 hr, more preferably 3 to 24 hr.
[0285]
The compound represented by Z-Y-H wherein the
30 aforementioned Q is a single bond can be also produced by, for
example, the following method. That is, introduction of an R5
group onto a benzene ring can be carried out by
(1) Friedel-Crafts reaction using halide corresponding to an R5
group (chloride, bromide, or iodide), carboxylic acid or acid
35 halide corresponding to an R5 group,
(2) a method comprising subjecting a compound corresponding to
the above-mentioned compound (11) (a compound wherein a Q'H
group is substituted by a -CHO group) to carbon homologation by
a Wittig reaction and, followed by catalytic hydrogenation and
5 the like, or
(3) conventional organic synthesis reaction such as cross
coupling using a metal catalyst and the like.
[028 61
In each scheme above, the carbon number of an organic
lo group for R ~ ,th e kind of halogen atom, reaction reagents and
the like are shown for the sake of convenience, and can be
appropriately changed within the scope of the above-mentioned
definitions.
15 4-7. Explanation of "oligonucleotide comprising a protected
base wherein the 3'-hydroxyl group is phosphoramidited, the 5'-
hydroxyl group is protected by a temporary protecting group
removable under acidic conditions, and the nucleic acid base is
protected by a group having a C5-30 straight chain or branched
20 chain alkyl group and/or a C5-30 straight chain or branched
chain alkenyl group"
The "p-mer oligonucleotide comprising a protected base (p
is an integer of one or more) wherein the 3'-hydroxyl group is
phosphoramidited, the 5'-hydroxyl group is protected by a
25 temporary protecting group removable under acidic conditions,
and the nucleic acid base is protected by a group having a C5-30
straight chain or branched chain alkyl group and/or a C5-30
straight chain or branched chain alkenyl group" used in step
(2) is not particularly limited as long as it satisfies the
30 structure requirements.
"3'-hydroxyl group is phosphoramidited" means that the
oligonucleotide 3'-hydroxyl group is modified by, for example,
a phosphoramiditing group represented by -P ( 0 ~(N~R,)Rf ) wherein
35 each symbol is as defined above.
The definitions, examples and preferable embodiments of
P', Re and Rf are as explained for the above-mentioned formula
The definitions, examples and preferable embodiments of
5 the "temporary protecting group removable under acidic
conditions" are as explained for the above-mentioned formula
(1)
The definitions, examples and preferable embodiments of
the "group having a C5-30 straight chain or branched chain alkyl
10 group and/or a C5-30 straight chain or branched chain alkenyl
group" are as explained for the above-mentioned formula (I).
As the p-mer oligonucleotide comprising a protected base
used in step (Z), an oligonucleotide comprising a protected
base represented by the above-mentioned formula (I) is
15 preferable.
[028 91
4-8. Explanation of steps (1) - (5)
- -
While steps (1) - (5) are explained below by reference to
the formulas (i) , (ii) , (iii) and the like for convenience,
20 they are not limited thereby.
[0290]
step (1) (deprotection step)
In this step, before condensation step (Z), in a nonpolar
solvent, temporary protecting group P1 (P1 is a temporary
25 protecting group removable under acidic conditions) of the 5'-
terminal hydroxyl group of an n-mer oligonucleotide (i) wherein
the 3'-hydroxyl group is protected, and the 5'-hydroxyl group
is protected by a temporary protecting group removable under
acidic conditions is removed by reaction with an acid
30 (deprotection step) .
[0291]
cation
scavenger
2 ) organic
base
[0292]
wherein each symbol is as defined above.
[0293]
5 This step is performed in a solvent that does not
influence the reaction. Since a higher solubility of the
solvent is expected to afford superior reactivity, a non-polar
solvent showing high solubility of the n-mer oligonucleotide of
the present invention is preferably selected. Specifically,
10 examples thereof include halogenated solvents such as
chloroform, dichloromethane, 1,2-dichloroethane and the like;
aromatic solvents such as benzene, toluene, xylene, mesitylene
and the like; ester solvents such as ethyl acetate, isopropyl
acetate and the like; aliphatic solvents such as hexane,
15 pentane, heptane, octane, nonane, cyclohexane and the like;
non-polar ether solvents such as diethyl ether, cyclopentyl
methyl ether, tert-butyl methyl ether and the like. Two or
more kinds of these solvents may be used in a mixture in an
appropriate ratio. In addition, the above-mentioned non-polar
20 solvent may be mixed with a polar solvent at an appropriate
ratio, such as nitrile solvents such as acetonitrile,
propionitrile and the like, amide solvents such as N,Ndimethylformamide,
N,N-dimethylacetamide, N-methylpiperidone
and the like, as long as n-mer oligonucleotide is dissolved.
25 Of these, aromatic solvents, aliphatic solvents, or a
combination of these is preferable, benzene, toluene, hexane,
pentane, heptane, nonane, cyclohexane or a combination of these
is preferable, toluene, heptane, nonane or a combination of
these is more preferable, and toluene and heptane or a
combination of these is particularly preferable.
[0294]
In this step, the concentration of n-mer oligonucleotide
(i) in a solvent is not particularly limited as long as the
oligonucleotide is dissolved, it is preferably 1 to 30 mass%.
[0295]
To continuously perform the deprotection step, subsequent
10 condensation step, and oxidation step in a solution, it is
preferable to use a cation scavenger in this step during or
after the removal reaction of a temporary protecting group P'
of 5'-hydroxyl group in n-mer oligonucleotide (i).
[0296]
15 While the cation scavenger is not particularly limited as
long as re-protection (returning to starting material) with the
removed protecting group P' and side reaction with the
deprotected functional group do not proceed, pyrrole
derivatives such as pyrrole, 2-methylpyrrole, 3-methylpyrrole,
20 2,3-dimethylpyrrole, 2,4-dimethylpyrrole and the like; and
indole derivatives such as indole, 4-methylindole, 5-
methylindole, 6-methylindole, 7-methylindole, 5,6-
dimethylindole, 6,7-dimethylindole and the like can be used.
Since a good cation trap effect can be obtained, pyrrole, 3-
25 methylpyrrole, 2,4-dimethylpyrrole, indole, 4-methylindole, 5-
methylindole, 6-methylindole, 7-methylindole, 5,6-
dimethylindole and 6,7-dimethylindole are preferable, pyrrole,
3-methylpyrrole and indole are more preferable, pyrrole and
indole are more preferable, and pyrrole is particularly
30 preferable.
[0297]
The amount of cation scavenger to be used in this step is
1 to 50 mol, preferably 5 to 20 mol, per 1 mol of n-mer
oligonucleotide (i) .
35 [0298]
While the acid t o be used i n t h i s step is not
p a r t i c u l a r l y limited as long as good deprotection can be
achieved, t r i f l u o r o a c e t i c acid, dichloroacetic acid,
trifluoromethanesulfonic acid, t r i c h l o r o a c e t i c acid,
5 methanesulfonic acid, hydrochloric acid, a c e t i c acid, ptoluenesulfonic
acid and the l i k e are preferably used.
Since good reaction can be achieved, t r i f l u o r o a c e t i c acid,
dichloroacetic acid, trifluoromethanesulfonic acid and
t r i c h l o r o a c e t i c acid are more preferable, t r i f l u o r o a c e t i c acid,
10 dichloroacetic acid and trifluoromethanesulfonic acid are more
preferable, t r i f l u o r o a c e t i c acid and dichloroacetic acid are
s t i l l more preferable, and t r i f l u o r o a c e t i c acid is p a r t i c u l a r l y
preferable. These acids may be diluted with the abovementioned
non-polar solvent. When the aforementioned acid is
15 used, it may be combined with a p a r t i c u l a r base t o
appropriately adjust the a c i d i t y before use.
[0299]
The amount of the acid t o be used i n t h i s step is 1 t o
100 mol, preferably 1 t o 40 mol, per 1 mol of n-mer
20 oligonucleotide (i).
[0300]
While the reaction temperature i n t h i s step is not
p a r t i c u l a r l y limited as long as the reaction proceeds, it is
preferably -lO°C t o 50°C, more preferably O°C t o 40°C. While
2s the reaction time varies depending on the kind of n-mer
oligonucleotide t o be used, the kind of acid, the kind of
solvent, the reaction temperature and the l i k e , it is 5 min t o
5 hr.
[0301]
30 To continuously perform the deprotection step, subsequent
condensation step, and oxidation step i n a solution, it is
preferable t o remove the temporary protecting group of the 5'-
hydroxy group i n t h i s step and neutralize the compound with an
organic base.
The organic base t o be used for n e u t r a l i z a t i o n is not
particularly limited as long as it can neutralize the abovementioned
acids, and the obtained salt can function as a
condensing agent. Since the reaction proceeds smoothly,
pyridine, benzimidazole, 1,2,4-triazole, N-phenylimidazole, 2-
5 amino-4,6-dimethylpyrimidine, 1,lO-phenanthroline, imidazole,
N-methylimidazole, 2-chlorobenzimidazole, 2-bromobenzimidazole,
2-methylimidazole, 2-phenylbenzimidazole, N-phenylbenzimidazole
and 5-nitrobenzimidazole are preferable, pyridine,
benzimidazole, 1,2,4-triazole, N-phenylimidazole, Nlo
methylimidazole, 2-amino-4,6-dimethylpyrimidine and 1,lOphenanthroline
are more preferable, pyridine, benzimidazole,
1,2,4-triazole and N-phenylimidazole are further preferable,
pyridine, benzimidazole and 1,2,4-triazole are still more
preferable, and pyridine is particularly preferable.
15 [0302]
The amount of the organic base to be used in this step is
1 to 10 mol, preferably 1 to 3 mol, per 1 mol of acid.
[0303]
A particularly preferable combination of an acid and an
20 organic base in this step is that of trifluoroacetic acid and
pyridine and/or N-methylimidazole.
[0304]
step (2) (condensation step)
In this step, an n-mer oligonucleotide (ii) wherein the
2s 5'-hydroxyl group is not protected, and the 3'-hydroxyl group
is protected is condensed with a p-mer oligonucleotide
comprising a protected base (iii) wherein the 3'-hydroxyl group
is phosphoramidited, the 5'-hydroxyl group is protected by a
temporary protecting group removable under acidic conditions,
30 and the nucleic acid base is protected by a group having a C5-30
straight chain or branched chain alkyl group and/or a C5-30
straight chain or branched chain alkenyl group.
[0305]
[0306]
wherein X' means the same as X, and other symbols are as
defined above.
5 [0307]
In this step, the n-mer oligonucleotide (ii) is not
particularly limited, and one obtained in the aforementioned
step (1) can be preferably used. In this case, a p-mer
oligonucleotide comprising a protected base (iii) only needs to
l o be added directly to the reaction mixture after step (I),
without isolating the n-mer oligonucleotide (ii) . In this
condensation step, the salt (e.g., pyridine trifluoroacetate),
which is formed by the acid added during deprotection step (1)
and the organic base added during neutralization reaction, acts
as a condensing agent. Therefore, steps (1) and (2)
5 continuously performed in a solution provide advantages of not
only omission of an isolation operation but also improved
reaction efficiency.
[0308]
The reaction efficiency can also be improved by adding a
10 condensing agent (e.g., pyridine trifluoroacetate, tetrazole,
5-benzylthio-1H-tetrazole, 4,5-dicyanoimidazole etc.) in this
condensation reaction.
[0309]
In this step, moreover, when the acidity of the reaction
15 mixture becomes high, a side reaction removing temporary
protecting group P' may occur. Therefore, N-methylimidazole is
preferably added to suppress acidification of the reaction
mixture.
[0310]
20 The amount of N-methylimidazole to be added to adjust the
acidity is 0.1 to 1 mol, preferably 0.5 mol, per 1 mol of
organic base used for neutralization.
[0311]
As the p-mer oligonucleotide comprising a protected base
25 (iii) used in this step, an oligonucleotide comprising a
protected base represented by the above-mentioned formula (I)
can be preferably used.
[0312]
This step is performed in a solvent that does not
30 influence the reaction. Specifically, a non-polar solvent
similar to the one used in the aforementioned step (1) can be
mentioned. For efficient activation of a phosphoramidite group
of the p-mer oligonucleotide comprising a protected base (iii),
a mixture of the above-mentioned non-polar solvent and a polar
35 solvent, for example, nitrile solvents such as acetonitrile,
propionitrile and the like; ketone solvents such as acetone, 2-
butanone and the like; N,N-dimethylformamide, N,Ndimethylacetamide,
polar ether solvents such as l,4-dioxane,
tetrahydrofuran and the like, amide solvents such as N-
5 methylpyrrolidone and the like; sulfoxide solvents such as
dimethyl sulfoxide and the like, and the like at an appropriate
ratio is preferably used as long as the n-mer oligonucleotide
(ii) after removal of the temporary protecting group of the 5'-
hydroxyl group can be dissolved.
In this case, as the polar solvent, amide solvent,
nitrile solvent, and a combination thereof are preferable,
acetonitrile, N,N-dimethylformamide, N-methylpiperidone, and a
combination thereof are more preferable, and acetonitrile is
particularly preferable.
The polar solvent may be added as a solution of a p-mer
oligonucleotide comprising a protected base (iii), a condensing
agent and the like.
[0313]
The amount of a p-mer oligonucleotide comprising a
20 protected base (iii) to be used is 1 to 10 mol, preferably 1 to
5 mol, per 1 mol of an n-mer oligonucleotide (ii).
[0314]
While the reaction temperature is not particularly
limited as long as the reaction proceeds, 0°C to 100°C is
25 preferable, and 20°C to 50°C is more preferable. While the
reaction time varies depending on the kind of n-mer
oligonucleotide to be condensed, the reaction temperature and
the like, it is 30 min to 24 hr.
30 step ( 3 ) (oxidation step or sulfurization step)
The n+p-mer oligonucleotide (iv) obtained in step (2) is
reacted with an oxidizing agent or sulfurizing agent to convert
the phosphite triester bond in the n+p-mer oligonucleotide (iv)
to a phosphate triester bond or a thiophosphate triester bond.
35 [0316]
oxidizing
agent or
sulfurizing
agent
[0317]
wherein the symbols are as defined above.
[0318]
This step can be simply performed by directly adding an
oxidizing agent or sulfurizing agent to the reaction mixture
after step (2), without isolating the n+p-mer oligonucleotide
(iv) obtained in step (2).
[0319]
While the "oxidizing agent" to be used in this step is
5 not particularly limited as long as it can oxidize a phosphite
triester bond into a phosphate triester bond without oxidizing
other moieties, iodine, (IS) - (+) - (10-
camphorsulfonyl)oxaziridine, tert-butyl hydroperoxide (TBHP),
2-butanone peroxide, 1,l-dihydroperoxycyclododecane,
10 bis(trimethylsilyl)peroxide, m-chloroperbenzoic acid or
hydrogen peroxide is preferably used. Since good oxidation
reaction can be achieved, iodine, (IS) - (+) - (10-
camphorsulfonyl)oxaziridine, tert-butyl hydroperoxide, 2-
butanone peroxide and 1,l-dihydroperoxycyclododecane are more
15 preferable, iodine, (IS) - (+) - (10-camphorsulfonyl)o xaziridine,
tert-butyl hydroperoxide and 2-butanone peroxide are more
preferable, iodine and tert-butyl hydroperoxide are still more
preferable, and iodine is particularly preferable. The
oxidizing agent can be used after diluting with a suitable
20 solvent to achieve a concentration of 0.05 to 2M. While the
dilution solvent is not particularly limited as long as it is
inert to the reaction, for example, pyridine, THF,
dichloromethane, water, nonane and a mixed solvent of any of
them can be mentioned. Of these, for example,
25 iodine/water/pyridine - THF, iodine/pyridine - acetic acid,
peroxide (TBHP)/dichloromethane, tert-butyl
hydroperoxide/nonane or hydrogen peroxide/potassium
iodide/phosphoric acid buffer are preferably used.
[0320]
The "sulfurizing agent" to be used in this step is not
particularly limited as long as it can convert a phosphite
triester bond to a thiophosphate triester bond, 3-((N,Ndimethylaminomethylidene)
amino)-3H-1,2,4-dithiazole-5-thione
(DDTT), 3H-1,2-benzodithiol-3-one-1,l-dioxide (Beaucage
35 reagent), 3H-1,2-benzodithiol-3-one, phenylacetyl disulfide
(PADS), tetraethylthiuram disulfide (TETD), 3-amino-1,2,4-
dithiazole-5-thione (ADTT) or sulfur is preferably used. Since
a good reaction proceeds, 3- ( (N, Ndimethylaminomethylidene)
amino)-3H-1,2,4-dithiazole-5-thione
5 (DDTT), 3H-1,2-benzodithiol-3-one-1,l-dioxide (Beaucage
reagent), 3H-1,2-benzodithiol-3-one and phenylacetyl disulfide
(PADS) are more preferable, 3- ( (N,Ndimethylaminomethylidene)
amino)-3H-1,2,4-dithiazole-5-thione
and 3H-1,2-benzodithiol-3-one-1,l-dioxide are further
10 preferable, and 3- ( (N, N-dimethylaminomethylidene) amino) -3H-
1,2,4-dithiazole-5-thione is particularly preferable. The
sulfurizing agent can be used after diluting with a suitable
solvent to achieve a concentration of 0.05 to 2M. While the
dilution solvent is not particularly limited as long as it is
15 inert to the reaction, for example, dichloromethane,
acetonitrile, pyridine and a mixed solvent of any of them can
be mentioned.
[0321]
The amount of the oxidizing agent or sulfurizing agent to
20 be used is 1 to 50 mol, preferably 1 to 5 mol, per 1 mol of the
n+p-mer oligonucleotide (iv) obtained in step (2) .
[0322]
While the reaction temperature is not particularly
limited as long as the reaction proceeds, 0°C to 100°C is
25 preferable, and 20°C to 50°C is more preferable. While the
reaction time varies depending on the kind of n+p-mer
oligonucleotide (iv), the kind of oxidizing agent or
sulfurizing agent to be used, the reaction temperature and the
like, it is 1 min to 3 hr.
30 [0323]
step (4) (extraction isolation step)
This step is a method of isolating and purifying an n+pmer
oligonucleotide (v) from a reaction mixture containing n+pmer
oligonucleotide (v) having a phosphate triester bond or a
35 thiophosphate triester bond, which is obtained from step (3),
by an extraction operation alone.
[0324]
While the extraction operation is not particularly
limited, it is preferably performed by adding a polar solvent
5 and/or a non-polar solvent as necessary to the reaction mixture
obtained in step (3), partitioning the mixture between polar
solvent - non-polar solvent, and transferring the n+p-mer
oligonucleotide to the non-polar solvent. When the reaction is
performed by mixing a non-polar solvent with a polar solvent in
10 step (21, the reaction mixture is preferably partitioned by
adding a non-polar solvent.
The extraction operation can remove impurities such as
the remaining starting materials, reagents (e.g., acid, cation
scavenger, organic base, p-mer oligonucleotide wherein 3,-
15 hydroxyl group is phosphoramidited and the 5'-hydroxyl group is
protected by a temporary protecting group removable under
acidic conditions, oxidant and sulfurizing agent) and the like
into a polar solvent.
[0325]
20 Examples of the non-polar solvent to be added as
necessary to transfer an n+p-mer oligonucleotide into a nonpolar
solvent in this step include halogenated solvents such as
chloroform, dichloromethane, 1,2-dichloroethane and the like;
aromatic solvents such as benzene, toluene, xylene, mesitylene
25 and the like; ester solvents such as ethyl acetate, isopropyl
acetate and the like; aliphatic solvents such as hexane,
pentane, heptane, octane, nonane, cyclohexane and the like;
non-polar ether solvents such as diethyl ether, cyclopentyl
methyl ether, tert-butyl methyl ether and the like. Two or
30 more kinds of these solvents may be used in a mixture in an
appropriate ratio. Of these, aromatic solvents, aliphatic
solvents, or a combination of these is preferable, benzene,
toluene, hexane, pentane, heptane, nonane, cyclohexane or a
combination of these is preferable, toluene, heptane, nonane or
35 a combination of these is more preferable, toluene, heptane or
a combination of these is further preferable, and heptane is
particularly preferable.
Examples of the polar solvent to be added as necessary to
transfer impurities in this step into a polar solvent include
5 alcohol solvent methanol, ethanol, isopropanol and the like,
nitrile solvents such as acetonitrile, propionitrile and the
like, ketone solvents such as acetone, 2-butanone and the like,
polar ether solvents such as 1,4-dioxane, tetrahydrofuran and
the like, amide solvents such as N,N-dimethylformamide, N,Nlo
dimethylacetamide, N-methylpiperidone and the like, sulfoxide
solvents such as dimethyl sulfoxide and the like, water and the
like, and a mixed solvent of two or more kinds of these. Of
these, amide solvents, nitrile solvents, and a combination of
these are preferable, acetonitrile, N,N-dimethylformamide, N-
15 methylpiperidone, and a combination of these are more
preferably used. The polar solvent in the present invention is
particularly preferably acetonitrile from the practical aspects.
[0326]
The impurity can be removed by removing polar solvents
20 after partitioning between polar solvent - non-polar solvent.
Moreover, the impurity remaining in a small amount can be
further removed by adding a polar solvent to a non-polar
solvent after removal of the polar solvent, stirring the
mixture and removing the polar solvent by partitioning (washing
25 in the present invention).
While the number of washing with a polar solvent is not
particularly limited, it may be repeated until the impurity in
the non-polar solvent layer decreases to the extent the
nucleotide elongation reaction in the next cycle is not
30 inhibited by the analysis of the non-polar solvent by thin
layer silica gel chromatography, high performance liquid
chromatography and the like.
[0327]
The polar solvent to be used for partitioning and washing
35 may contain water to improve partitioning performance from the
non-polar solvent.
In this case, the water content of the polar solvent is
preferably 1 - 10%(v/v), more preferably 3 - 8%(v/v). When the
water content is too low, the partitioning performance may be
5 insufficient, and when the water content is too high, the
solubility of the byproduct, the remaining starting material,
reagent and the like to be removed in a polar solvent trends to
decrease to degrade the removal efficiency.
[0328]
The n+p-mer oligonucleotide (v) can be isolated by
concentrating the non-polar solvent layer after washing with a
polar solvent. In this case, nucleotide elongation can be
repeated in one-pot by adding the solvent and reagent for the
next cycle to a reaction vessel containing the concentrate.
Alternatively, it is also possible to apply, without
concentration, the non-polar solvent layer after washing to the
nucleotide elongation in the next cycle.
[0329]
The production method of oligonucleotide of the present
20 invention can afford the object long oligonucleotide with high
purity and in a high yield by repeating the above-mentioned
steps (1) to (4) a desired number of times.
[0330]
step (5) (deprotection, oligonucleotide isolation step)
25 In the production method of oligonucleotide of the
present invention, deprotection is performed after step (4)
according to the kind and properties of the protecting group,
whereby oligonucleotide is isolated. All protecting groups can
be removed from oligonucleotide according to the deprotection
30 method described in Greene's PROTECTIVE GROUPS IN ORGANIC
SYNTHESIS, 4th ed., JOHN WILLY&SONS (2006) and the like. To be
specific, nucleotide 3'-hydroxyl-protecting group, as well as
phenoxyacetyl group, acetyl group, a group having a C5-30
straight chain or branched chain alkyl group and/or a C5-30
35 straight chain or branched chain alkenyl group and the like,
which are nucleic acid base-protecting groups, cyanoethyl group
bonded to phosphate group and the like in the present invention
can all be removed by treating with aqueous ammonia/ethanol
solution, aqueous ammonia/aqueous methylamine solution,
5 ethylenediamine and the like. In addition, nucleotide 5'
hydroxyl-protecting group can be removed by a treatment with
the acid used in step (1) or an appropriately diluted solution
of such acid.
Since oligonucleotide without a protecting group is
10 easily degraded by an enzyme, oligonucleotide is preferably
isolated under appropriate air contamination control.
[0331]
The progress of the reaction in each of the abovementioned
steps can be confirmed by a method similar to
15 conventional liquid phase organic synthesis reaction. That is,
the reaction can be traced by thin layer silica gel
chromatography, high performance liquid chromatography and the
like.
[ 0 3 3 2 ]
The oligonucleotide obtained by step (4) or step (5) can
also be led to a desired oligonucleotide derivative by further
applying an organic synthesis reaction.
5. Explanation of steps (I' ) - (5')
25 Another embodiment of the production method of
oligonucleotide of the present invention is a method including
the following step (2' ) .
(2' ) A step of producing an n'+pf -mer oligonucleotide by
condensing a pf-mer oligonucleotide (p' is an integer of one or
30 more) wherein the 3'-hydroxyl group is phosphoramidited, the
5'-hydroxyl group is protected by a temporary protecting group
removable under acidic conditions, and the nucleic acid base is
optionally protected, with an n'-mer oligonucleotide (n' is an
integer of one or more) wherein the 5'-hydroxyl group is not
35 protected, and the 3'-hydroxyl group is protected by a
protecting group represented by the above-mentioned formula
(111) to form a phosphite triester bond via the 5'-hydroxyl
group thereof.
[0334]
This method preferably further includes the following
step ( 3 ' ) ' wherein the phosphite triester bond of the n'+p'-mer
oligonucleotide obtained in step (2') is converted to a
phosphate triester bond or a thiophosphate triester bond:
(3') a step of adding an oxidizing agent or a sulfurizing agent
10 to the reaction mixture obtained in the condensation step (2')
to convert the phosphite triester bond of the n'+pf-mer
oligonucleotide obtained in the condensation step to a
phosphate triester bond or a thiophosphate triester bond.
[0335]
This method preferably further includes the following
step (1' ) by which an n'-mer oligonucleotide used in step (2' )
wherein the 5'-hydroxyl group is not protected and the 3'-
hydroxyl group is protected by a protecting group represented
by the above-mentioned formula (111) is prepared.
20 (1') a step of removing a temporary protecting group removable
under acidic conditions of the 5'-hydroxyl group by reacting,
in a non-polar solvent prior to the condensation step (2')' the
n'-mer oligonucleotide wherein the 3'-hydroxyl group is
protected by the protecting group represented by the above-
25 mentioned formula (111) , and the 5' -hydroxyl group is protected
by the temporary protecting group, with an acid.
Step (1') is preferably performed in the presence of at
least one kind of cation scavenger selected from a pyrrole
derivative and an indole derivative, and further includes a
30 step of neutralization with an organic base after removal of
the temporary protecting group of the 5'-hydroxyl group. As a
result, steps (1' ) (2' ) and (3' ) can be continuously performed
in a liquid, and an oligonucleotide wherein nucleoside in the
number of p' is elongated can be isolated and purified by an
35 extraction operation alone.
[0336]
Furthermore, by including the following s t e p ( 4 ' ) , an
n'+p'-mer oligonucleotide is p u r i f i e d by removing an excess
s t a r t i n g material and by-product conveniently and e f f e c t i v e l y ,
5 without the need f o r complicated s o l i d i f i c a t i o n - i s o l a t i o n , and
can be led t o the next step without taking out the r e s u l t a n t
product from the r e a c t i o n v e s s e l :
( 4 ' ) a step of i s o l a t i n g t h e n'+pr-mer oligonucleotide from the
reaction mixture obtained i n step (3') by an extraction
10 operation alone.
[0337]
When the amount of the by-product generated can be
controlled by the management of equivalent of the s t a r t i n g
materials and controlling the reaction, it is preferable t o
15 repeat step (1') t o step ( 3 ' ) as a basic u n i t , which includes
s t e p ( 4 ' ) .
Since the generation of by-product can be s t r i c t l y
managed and controlled and highly pure oligonucleotide can be
obtained, it is preferable t o repeat step (1') t o step ( 4 ' ) as
20 a basic u n i t .
By repeating such cycle i n t h e l i q u i d phase method, the
f i n a l oligonucleotide can be produced i n one-pot, without
changing the r e a c t i o n v e s s e l .
[0338]
2s In the production method of the present invention,
oligonucleotide can be i s o l a t e d and produced by f u r t h e r
including step (5' ) :
( 5 ' ) a s t e p of removing a l l the protecting groups of the n'+prmer
oligonucleotide obtained i n step ( 4 ) .
30 n' is an integer of one or more. While the upper l i m i t
of n is not p a r t i c u l a r l y limited, it is generally 100 o r l e s s ,
preferably 75 or l e s s , more preferably 50 o r l e s s , and more
preferably 30 or l e s s
p' is an integer of one or more, preferably 1. While the
35 upper l i m i t of p is not p a r t i c u l a r l y limited, it is preferably
50 or less, more preferably 30 or less, more preferably 20 or
less, still more preferably 5 or less, and particularly
preferably 3 or less.
[0339]
The n' -mer oligonucleotide used in step (I' ) is, for
example, an nf-mer oligonucleotide represented the following
formula (if) wherein P' is a temporary protecting group
removable under acidic conditions, the 3'-hydroxyl group is
protected by a protecting group represented by the above-
10 mentioned formula (111) and the 5'-hydroxyl group is protected
by a temporary protecting group removable under acidic
conditions, and the n'-mer oligonucleotide used in step (2') is,
for example, an n'-mer oligonucleotide represented by the
following formula (ii') wherein the 5'-hydroxyl group is not
15 protected, and the 3'-hydroxyl group is protected by a
protecting group represented by the above-mentioned formula
\ L-Y-Z I L-Y-Z
20 [0341]
wherein m' is any integer of not less than 0, and each of other
symbols is as defined above.
While the upper limit of m' is not particularly limited,
it is generally 99 or less, preferably 74 or less, more
25 preferably 49 or less, more preferably 29 or less.
The definition, example and preferable embodiment of each
of other symbols are the same as those in the explanation on
the formulas (i) and (ii) .
[0342]
The 'a p'-mer oligonucleotide wherein the 3'-hydroxyl
5 group is phosphoramidited, the 5'-hydroxyl group is protected
by a temporary protecting group removable under acidic
conditions, and the nucleic acid base is optionally protected"
used in step (2') is not particularly limited as long as the
structural requirements are satisfied.
10 The "3'-hydroxyl group is phosphoramidited" means that
oligonucleotide 3'-hydroxyl group is modified by, for example,
a phosphoramidite group represented by -P ( 0 ~(~NR,)Rf ) wherein
each syrnbol is as defined above.
The definitions,. examples and preferable embodiments of
15 p2, Re and Rf are as explained for the above-mentioned formula
The definitions, examples and preferable embodiments of
"temporary protecting group removable under acidic conditions"
are as explained for ,the above-mentioned formula (I) .
20 The protecting group of the "nucleic acid base is
optionally protected" are the same as the protecting groups
exemplified for the "optionally protected nucleic acid base"
for Base in the above-mentioned formulas (i) and (ii).
The protecting group is preferably a group free of a C5-30
25 straight chain or branched chain alkyl group and/or a C5-30
straight chain or branched chain alkenyl group and, for example,
pivaloyl group, pivaloyloxymethyl group, trifluoroacetyl group,
phenoxyacetyl group, 4-isopropylphenoxyacetyl group, 4-tertbutylphenoxyacetyl
group, acetyl group, benzoyl group,
30 isobutyryl group, dimethylformamidinyl group, 9-
fluorenylmethyloxycarbonyl group and the like are preferable.
Of these, phenoxyacetyl group, 4-isopropylphenoxyacetyl group,
acetyl group, benzoyl group, isobutyryl group, and
dimethylformamidinyl group are more preferable.
35 [OM31
Examples of the pr-mer oligonucleotide having optionally
protected nucleic acid base wherein the 3'-hydroxyl group is
phosphoramidited, and the 5'-hydroxyl group is protected by a
temporary protecting group removable under acidic conditions
5 include the following formula (iii') .
[0345]
10 wherein
Base is optionally substituted nucleic acid base, q' is any
integer of not less than 0, and other symbol is as defined
above.
"optionally substituted nucleic acid base" for Base is as
15 defined for the "optionally protected nucleic acid base" for
Base in the formulas (i) and (ii) .
q' is any integer of 0 or more, preferably 0. While the
upper limit of q' is not particularly limited, it is preferably
49 or less, more preferably 29 or less, more preferably 19 or
20 less, still more preferably 4 or less, still more preferably 2
or less, and particularly preferably 1.
[0346]
Steps (I' ) - (5' ) can be performed under similar
conditions as in the above-mentioned steps (1) - (5) by reading
the formulas (i) , (ii) and (iii) as the formulas (i' ) , (ii' )
and (iii' ) , respectively.
6. Use of olisonucleotide
The oligonucleotide produced by the present invention can
5 be used for various applications such as various veterinary
pharmaceutical products (RNA, DNA, oligonucleic acid medicine,
etc.) for human or animal, functional food, food for specified
health uses, food, chemical product, polymer material for human
or industrial use, and the like.
10 Examples
[0347]
The present invention is explained in more detail in
the following by referring to Preparation Examples and
Examples, which are not to be construed as limiting the
15 scope of the present invention. The reagents, apparatuses
and materials used in the present invention are commercially
available unless otherwise specified. In the present
specification, when indicated by abbreviation, each indication
is based on the abbreviation of the IUPAC-IUB Commission on
20 Biochemical Nomenclature or conventional abbreviations in the
art.
[0348]
The yield in the following Preparation Examples and
Examples shows mol/mol%. Unless particularly specified, '%"
2s means "mass %" in the present specification. In addition, the
ratio of the solvent in the following Preparation Examples and
Examples is volume ratio. For 'H-NMR spectrum,
tetramethylsilane was used as the internal standard, and CDC13
was used as a measurement solvent. NMR spectrum was measured
30 using Bruker AVANCE AV300 (300 MHz) nuclear magnetic resonance
apparatus or Bruker AVANCE 400 (400 MHz) nuclear magnetic
resonance apparatus.
For electrospray ionization liquid chromatography/mass
spectrometry (hereinafter to be abbreviated as LC/MS), flow
35 injection analysis (FIA) (solvent: 0.1 mol/l TEAA buffer pH 7.0,
acetonitrile, ionization mode: ESI, ion node: negative, mass
analyzer: quadrupole, fragmentor voltage: 200V) was performed
using 6130 Quadrupole LC/MS (Agilent Technologies).
For quadrupole mass spectrometry, flow injection analysis
5 (FIA) (solvent: acetonitrile, ionization mode: ESI, ion mode:
positive-negative, mass analyzer: quadrupole, fragmentor
voltage: 71V) was performed using ZQ2000 (manufactured by Nihon
Waters K.K.).
[034 91
10 The abbreviations used in the following Preparation
Examples and Examples are as described below. When nucleic
acid base of nucleoside is protected, the protecting group is
shown in superscript after each nucleoside.
dT : 2' -deoxythymidine
15 dC : 2' -deoxycytidine
dG: 2' -deoxyguanosine
dA: 2' -deoxyadenosine
U(M) : 2'-methoxyuridine
U (F) : 2' -f luorouridine
20 (LNA) T : 2' -0,4' -C-methylenethyrnidine
DMTr: 4,4'-dimethoxytrityl
PA: (2-cyanoethy1)-N,N-diisopropylphosphoramidite
suc : succinyl
TPB: 3,4,5-tris (3,7,11,15-tetramethyl-1-hexadecanyloxy)b enzyl
25 (same as 3,4,5-tris(2,3-phyty1oxy)benzyl)
Phy: 2,3-dihydrophytyl (wherein "2,3-dihydrophytyl" means
"3,7,11,15-tetramethyl-1-hexadecanyl")
PhyOM: (3,7,11,15-tetramethyl-1-hexadecanoyloxym)e thyl
(same as 2,3-phytyloxyrnethyl)
30 4-Cit-Bz: 4-(3,7-dimethyl-1-octyloxy)benzoyl
(same as 4-(dihydrocitronellyloxy)-benzyl)
2Et-Hex: 2-ethyl-1-hexanoyl
3,5,5-Me3Hex: 3,5,5-trimethyl-1-hexanoyl
Me6Dodecanoyl: 2,2,4,8,10,10-hexamethyl-5-dodecanoyl
35 2-HepUndecanoyl: 2-heptyl-1-undecanoyl
Myr: tetradecanoyl
(same as myristoyl)
(same as N.N-bis-dihydrocitronellyl-methylene)
Bz: benzoyl
ibu: isobutyryl
[0350]
10 Preparation Example 1: Synthesis of 2,3-dihydrophytol
Phytol (10.00 g, 33.7 mmol) was dissolved in methanol,
P~/C (2%, 1.00 g) was suspended therein and the suspension was
stirred overnight under a hydrogen atmosphere. After
completion of the reaction, the suspension was filtered to
15 remove pt/C, and the filtrate was concentrated to give 2,3-
dihydrophytol. This was used for the next reaction without
purification.
Preparation Example 2: Synthesis of 2,3-dihydrophytyl bromide
2,3-Dihydrophytol (33.7 mmol) was suspended in 48%
hydrobromic acid (100 ml), concentrated sulfuric acid (0.17 ml)
was added dropwise and the mixture was stirred at 100°C
overnight. The reaction mixture was cooled to room temperature,
extracted with hexane (200 ml), and washed twice with 5%
25 aqueous sodium hydrogen carbonate solution (70 ml) and once
with 20% brine (70 ml). The organic layer was dried over
sodium sulfate, and the solvent in the filtrate was evaporated.
The obtained residue was purified by silica gel column
chromatography (short column, hexane alone) to give 2,3-
30 dihydrophytyl bromide ("2,3-dihydrophytyl group" is sometimes
to be referred to as "Phy" hereunder) (10.41 g, 28.8 mmol, 85%
vs. phytol).
[0352]
Preparation Example 3: Synthesis of 3,7,11-trimethyldodecan-1-
Using Farnesol (3.00 g, 13.5 mmol) and in the same manner
as in Preparation Example 1, 3,7,11-trimethyldodecan-1-01 was
obtained. This was used for the next reaction without
purification.
5 LO3531
Preparation Example 4: Synthesis of 1-bromo-3,7,11-
trimethyldodecane
Using 3,7,11-trimethyldodecan-1-01 obtained in
Preparation Example 3 and in the same manner as in Preparation
10 Example 2, 1-bromo-3,7,11-trimethyldodecane (2.98 g, 10.2 mmol,
76% vs. Farnesol) was obtained.
[0354]
Preparation Example 5: Synthesis of 1-[(2-chloro-5-(2,3-
dihydrophyty1oxy)phenyl)l-1-phenylmethanamine
15 To 2,3-dihydrophytyl bromide (1.02 g, 2.82 mmol) were
added DMF (15 ml), 2-chloro-5-hydroxybenzophenone (0.99 g, 4.23
mmol) and K2CO3 (0.78 g, 5.64 mmol), and the mixture was
stirred at 90°C for 3 hr. The reaction mixture was cooled to
room temperature, ethyl acetate (25 ml) and 1 mol/l
20 hydrochloric acid (25 ml) were added and the mixture was
stirred to allow layer separation. The aqueous layer was
separated and discarded. The organic layer was washed twice
with purified water (25 ml), and the organic layer was
evaporated under reduced pressure to give 2-chloro-5-(2,3-
25 dihydrophyty1oxy)benzophenone.
To the aforementioned 2-chloro-5-(2,3-
dihydrophyty1oxy)benzophenone were added chloroform (20 ml),
methanol (2 ml) and sodium borohydride (440 mg, 11.6 mmol), and
the mixture was stirred at 50°C overnight. The reaction
30 mixture was cooled to room temperature, 1 mol/l hydrochloric
acid (15 ml) was added dropwise in an ice bath to decompose
unreacted sodium borohydride. The aqueous layer was discarded,
and the organic layer was washed twice with purified water (10
ml). The organic layer was evaporated under reduced pressure,
35 and moisture was azeotropically distilled with acetonitrile to
SPM anpTsa1 ay& =axnssaxd pampa2 xapun paqexodena SPM 1aAe1
aueqday ayq pue ( ~5u7:) uoTqnTos aITxqTuoqa3e snoanbe %OS-(IUI sz
OE) aueqday y q s~auTq~ aa xyq paqexedas-p~nbp~u~e 'paqexode~a
SPM JHL 'axnqexaduaq uoox oq pa1003 seM axnqxy uoTq3eax
ayA =xy z xoj 3,05 qe paxxTqs seM axnqxTu ayq pue '(TOW
07:'~' 5u €18) au?ydsoyd~Auayd-pq pue ( ~2u) xaqeM pa-g-pnd
'(~u0 2) JH& pappe axaM aueyqau~Auayd(~Auayd(Axo~AqAydoxpAyoz~ p
-E'z)-~-o~oI~~-z)]-~-poa~uToTZq~u-au~a xoje ayq o&
= a u e y q a u ~ ~ u a y d ( ~ ~ u a y d ( ~ ~ ~ ~ ~ q A y d o ~ p ~ y ~ p - ~ ~ ~ -z)]-1-op~ze-1 an-$ oq axnssaxd pampa2 xapun paqexode~a SPM
xaAe7: 3-pebxo ay& = ( ~5u1) xaqeM pa-g~xnd y q a~3TMq~ pu e '(~u
OE) xaqeM pa-g-pnd y qa3~uo ~pay seM seM axnqxTu ayq pue 'pappe Gr
axaM ( ~0u2) auexay pue ( ~0u2) aqeqax ~Ayqa' axnqexaduaq uoox
seM axnqx-p ayq pue '(TOW 1.~1'b ur 9 8 ~ )ap Tze unTpos pue
( p 51) JMa pappe axaM aueyqau~Auayd(~Auayd(Axo~AqAydoxpAy~p
-E'z)-~-o~oT~=>-z)]-~-o~poauTo~~=q>u-au~a xojea yq. o& or
-aueyqau~~uayd(~Auayd(Axo~AqAydoxpAy~p
-~'~)-5-oxo~y3-~)]-1-oxo~ya3n-~1 5oq auanToq
y qpa~TTT~qsT p A~p3~doxqoazsee M apTxoTy3 ~Auo~ybqu? u~euax '
ayq pue 'axnssaxd pampa1 xapun paqexodena seM quaqos
ay& =axnqexaduaq uoox oq paToo3 seM axnqxru uoTq3eax ay& =xy s
P xoj 3,05 qe paxxTqs seM axnqxTu ayq pue (TOW 1 '~u€0 - 1)
~ P T ~ O T W~A uorrlqP UP (TO& 655 '7:d EP) m a '(TU OZ) u ~ o ~ o ~ o ~ r l 3
mmol) and potassium carbonate (7.82 g, 56.6 mmol) , and the
mixture was stirred at 80°C for 5 hr. The reaction mixture was
cooled to room temperature, ethyl acetate (300 ml) and 1 mol/l
hydrochloric acid (100 ml) were added and the mixture was
5 stirred to allow layer separation. The aqueous layer was
separated and discarded. The organic layer was washed twice
with purified water (100 ml), and the organic layer was
evaporated under reduced pressure to give 4,4'-bis(2,3-
dihydrophyty1oxy)benzophenone oil. This was dissolved in
10 chloroform (60 ml) and methanol (10 ml), sodium borohydride
(4.49 g, 119 mmol) was added, and the mixture was stirred at
60°C for 3 hr. To the reaction mixture was added 1 mol/l
hydrochloric acid (80 ml), and the mixture was concentrated.
Ethyl acetate (100 ml) was added, and the mixture was washed
15 successively with 1 mol/l hydrochloric acid and water. The
organic layer was concentrated to give 4,4'-bis(2,3-
dihydrophyty1oxy)benzhydryl alcohol oil.
Preparation Example 7: Synthesis of 3,4,5-tri(2,3-
20 dihydrophyty1oxy)benzyl alcohol
2,3-Dihydrophytyl bromide (40.6 g, 112 mmol), methyl
gallate (5.90 g, 32.0 mmol) and potassium carbonate (22.14 g,
160 mmol) were suspended in DMF (400 ml), and the mixture was
stirred at llO°C overnight. The reaction mixture was extracted
25 with hexane (800 ml), washed with 1 mol/l hydrochloric acid
(400 ml), 5% aqueous sodium hydrogen carbonate solution (400
ml) and 20% brine (400 ml), dried over sodium sulfate and the
solvent in the filtrate was evaporated to give methyl 3,4,5-
tri (2,3-dihydrophytyloxy)b enzoate (29.3 g, yield 93%) .
The aforementioned methyl 3,4,5-tri(2,3-
dihydrophyty1oxy)benzoate (29.3 g; 30.0 mmol) was dissolved in
THF (400 ml), and diisobutylaluminum hydride (DIBAL) (1.0 mol/l
toluene solution, 96 ml, 96 mmol) was added dropwise over 30
min under a nitrogen atmosphere at O°C. After stirring at room
35 temperature overnight, 0.2 mol/l hydrochloric acid (50 ml) was
added dropwise at O°C to quench the reaction. The solvent was
evaporated to about half, and the residue was dissolved in
ethyl acetate (600 ml). The mixture was washed three times
with 1 mol/l hydrochloric acid (300 ml), once with 5% aqueous
5 sodium hydrogen carbonate solution (300 ml), and once with 20%
brine (300 ml), and dried over sodium sulfate. The solvent in
the filtrate was evaporated to give 3,4,5-tri (2,3-
dihydrophyty1oxy)benzyl alcohol (26.8 g, yield 94%).
[0357]
lo Preparation Example 8 : Synthesis of 3,4,5-tri (2,3-
dihydrophyty1oxy)benzyl amine
3,4,5-Tri (2,3-dihydrophytyloxy)b enzyl chloride ( 6.46 g,
6.63 mmol) was dissolved in DMF-chloroform (60+20 ml), sodium
azide (861 mg, 13.2 mmol) was added and the mixture was stirred
15 at 70°C for 2 hr. The reaction mixture was cooled to room
temperature, ethyl acetate (160 ml) was added, and the mixture
was washed twice with water (80 ml) and three times with 20%
brine (50 ml), and dried over sodium sulfate. The solvent in
the filtrate was evaporated to give 3,4,5-tri (2,3-
20 dihydrophyty1oxy)benzyl azide oil, which was directly used for
the next step.
The aforementioned 3,4,5-tri (2,3-dihydrophytyloxy)b enzyl
azide oil was dissolved in THF (80 ml), water (1.19 ml, 66.1
mmol) and triphenylphosphine (1.91 g, 7.28 mmol) were added and
25 the mixture was stirred at 70°C for 1 hr. After cooling to
room temperature, the solvent was evaporated, and the residue
was dissolved in heptane (160 ml). The mixture was washed
three times with 50% aqueous acetonitrile solution (50 ml) and
twice with 20% brine (50 ml), and dried over sodium sulfate.
30 The solvent in the filtrate was evaporated. The obtained
residue was purified by silica gel column chromatography
(hexane:ethyl acetate=5:l-+chloroform:methanol:aqueous
ammonia=100 : 10 : 1) to give 3,4,5-tri (2,3-dihydrophytyloxy)b enzyl
amine (5.31 g, 5.32 mmol, yield 80% vs. chloride product) .
35 [0358]
Preparation Example 9 : Synthesis of 3,5-bis (2,3-
dihydrophyty1oxy)benzyl alcohol
2,3-Dihydrophytyl bromide (895 mg, 2.48 mmol) , methyl
3,5-dihydroxybenzoate (204 mg, 1.21 mmol), and potassium
5 carbonate(513 mg, 3.71 mmol) were suspended in DMF (10 ml), and
the suspension was stirred at 100°C for 7 hr. The reaction
mixture was extracted with ethyl acetate (30 ml), and the
extract was washed three times with 1 mol/l hydrochloric acid
(10 ml) and 20% brine (10 ml), and dried over sodium sulfate.
l o The solvent in the filtrate was evaporated to give methyl 3,5-
bis (2,3-dihydrophytyloxy)b enzoate (0.78 g, yield 92%) .
The aforementioned methyl 3,5-bis(2,3-
dihydrophytyloxy) benzoate (0.70 g, 1.00 mmol) was dissolved in
THF (10 ml), and lithium aluminum hydride (2.0 mol/l THF
15 solution, 1.2 ml, 2.4 mmol) was added dropwise under a nitrogen
atmosphere at O°C. After stirring at room temperature for 5 hr,
water was added dropwise at O°C to quench the reaction. The
solution was dissolved in ethyl acetate (30 ml), and the
mixture was washed three times with 1 mol/l hydrochloric acid
20 (10 ml), and once with 20% brine (20 ml), and dried over sodium
sulfate. The solvent in the filtrate was evaporated. The
obtained residue was purified by silica gel column
chromatography (hexane alone+hexane:ethyl acetate=5:l) to give
3,5-bis (2,3-dihydrophytyloxy)b enzyl alcohol (0.61 g, yield 90%) .
25 [0359]
Preparation Example 10: Synthesis of 4-(2,3-
dihydrophyty1oxy)benzyl alcohol
2,3-Dihydrophytyl bromide (600 mg, 1.66 mmol), 4-
hydroxybenzaldehyde (223 mg, 1.83 mmol) and potassium carbonate
30 (344 mg, 2.49 mmol) were suspended in DMF (6 ml) , and the
suspension was stirred at 60°C for 3 days. The reaction
mixture was cooled to room temperature, and extracted with
ethyl acetate (30 ml). The extract was washed three times with
1 mol/l hydrochloric acid (6 ml), three times with 5% aqueous
35 sodium hydrogen carbonate solution (6 ml), and once with 20%
brine (6 ml), and dried over sodium sulfate. The solvent in
the filtrate was evaporated. The obtained residue was purified
by silica gel column chromatography (hexane:ethyl
acetate=15 : 1+5: 1) to give 4- (2,3-dihydrophytyloxy)b enzaldehyde
5 (640 mg, yield 100% vs. 2,3-dihydrophytyl bromide).
The aforementioned 4-(2,3-dihydrophyty1oxy)benzaldehyde
(640 mg, 1.66 mmol) was dissolved in THF-methanol mixed
solution (7t0.3 ml), sodium borohydride (110 mg, 90%, 2.62
mmol) was added at O°C, and the mixture was stirred at room
10 temperature for 30 min. The reaction mixture was cooled to O°C,
and the reaction was quenched with 1 mol/l hydrochloric acid.
Ethyl acetate (30 ml) was added and the mixture was washed
three times with 1 mol/l hydrochloric acid (5 ml), three times
with 5% aqueous sodium hydrogen carbonate solution (5 ml), and
15 once with 20% brine (5 ml), and dried over sodium sulfate. The
solvent in the filtrate was evaporated to give 4-(2,3-
dihydrophyty1oxy)benzyl alcohol (619 mg, 1.53 mmol, yield 92%
vs. 2,3-dihydrophytyl bromide).
[0360]
20 Preparation Example 11: Synthesis of 4-(2,3-
dihydrophyty1oxy)benzyl amine
4- (2,3-Dihydrophytyloxy)b enzyl alcohol ( 619 mg, 1.53
mmol) was dissolved in chloroform (6 ml), thionyl chloride (167
p1, 2.29 mmol) was added and the mixture was stirred for 5 hr.
25 After completion of the reaction, the solvent was evaporated to
give 4- (2,3-dihydrophytyloxy)- benzyl chloride oil, which was
directly used for the next step.
The aforementioned 4- (2,3 -dihydro~hytyloxyb)e nzyl
chloride (1.53 mmol) was dissolved in DMF-CHC13 mixed solvent
30 (6+3 ml), sodium azide (298 mg, 4.58 mmol) was added and the
mixture was stirred at 70°C overnight. The reaction mixture
was cooled to room temperature, ethyl acetate (20 ml) was added,
and the mixture was washed 5 times with water (10 ml) and dried
over sodium sulfate. The solvent in the filtrate was
35 evaporated to give 4-(2,3-dihydrophyty1oxy)benzyl azide (632 mg,
yield 96% vs. 4-(2,3-dihydrophyty1oxy)benzyl alcohol).
The aforementioned 4-(2,3-dihydrophyty1oxy)benzyl azide
(632 mg, 1.47 mmol) was dissolved in THF (6 ml) , water (265 p1,
14.7 mmol) and triphenylphosphine (424 mg, 1.62 mmol) were
5 added and the mixture was stirred at 70°C overnight. After
cooling to room temperature, the solvent was evaporated, and
the residue was dissolved in hexane (10 ml), and the mixture
was washed 3 times with 50% aqueous acetonitrile solution (5
ml). The solvent was evaporated. The obtained residue was
lo purified by silica gel column chromatography (hexane:ethyl
give 4- (2,3-dihydrophytyloxy)be nzyl amine (555 mg, 1.37 mmol,
yield 94%).
15 Preparation Example 12: Synthesis of 2-methoxy-4-(2,3-
dihydrophyty1oxy)benzyl amine
2,3-Dihydrophytyl bromide (2.00 g, 5.53 mmol), 2-methoxy-
4-hydroxybenzaldehyde (884 mg, 5.81 mmol) and potassium
carbonate (1.15 g, 8.32 mmol) were suspended in DMF (20 ml),
20 and the suspension was stirred at 80°C overnight. The reaction
mixture was cooled to room temperature, and extracted with
ethyl acetate (50 ml). The extract was washed three times with
1 mol/l hydrochloric acid (20 ml), three times with 5% aqueous
sodium hydrogen carbonate solution (20 ml), and once with 20%
25 brine (20 ml), and dried over sodium sulfate. The solvent in
the filtrate was evaporated. The obtained residue was purified
by silica gel column chromatography (hexane:ethyl acetate=20:l)
to give 2-methoxy-4-(2,3-dihydrophyty1oxy)benzaldehyde oil,
which was used for the next step.
30 The aforementioned 2-methoxy-4-(2,3-
dihydrophytyloxy)benzaldehyde, and hydroxylamine hydrochloride
(1.15 g, 16.5 mmol) were suspended in dichloromethane (25 ml),
triethylamine (3.84 ml, 27.7 mmol) was added at O°C and the
suspension was stirred at room temperature for 3 hr. To the
35 reaction mixture was added chloroform (30 ml) and the mixture
was washed three times with 1 mol/l hydrochloric acid (15 ml),
three times with 5% aqueous sodium hydrogen carbonate solution
(15 ml), and once with 20% brine (15 ml), and the solvent was
evaporated to give 2-methoxy-4-(2,3-
5 dihydrophytyloxy)benzaldoxime. After confirmation of the
structure by NMR, it was used for the next step.
The aforementioned 2-methoxy-4-(2,3-
dihydrophytyloxy)benzaldoxime was dissolved in methanol-THE'
mixed solvent (20+10 ml) , 10% palladium-carbon (K) (200 mg) was
10 added and the mixture was stirred under a hydrogen atmosphere
at room temperature overnight. The solvent in the filtrate was
evaporated. The obtained residue was purified by silica gel
column chromatography (chloroform:methanol:aqueous
ammonia=100:10:1) to give 2-methoxy-4-(2,3-
15 dihydrophyty1oxy)benzyl amine (1.87 g, 4.31 mmol, yield 78% vs.
2,3-dihydrophytyl bromide) .
[0362]
Preparation Example 13: Synthesis of 4-(2,3-dihydrophyty1oxy)-
2-methylbenzyl alcohol
20 To methanol (10 ml) was added dropwise thionyl chloride
(1.92 ml, 26.3 mmol) at O°C, 4-hydroxy-2-methylbenzoic acid
(2.00 g, 13.1 mmol) was added, and the mixture was stirred at
60°C overnight. After completion of the reaction, the solvent
was evaporated, and the residue was dissolved in ethyl acetate
25 (20 ml). The mixture was washed twice with 5% aqueous sodium
hydrogen carbonate solution (10 ml), once with 1 mol/l
hydrochloric acid (10 ml), and once with water (10 ml), and the
solvent was evaporated to give methyl 4-hydroxy-2-
methylbenzoate (2.24 g, yield 100%) .
30 The aforementioned methyl 4-hydroxy-2-methylbenzoate (269
mg, 1.62 mmol) , 2,3-dihydrophytyl bromide (389 mg, 1.08 mmol)
and potassium carbonate(297 mg, 2.15 mmol) were suspended in
DMF (5 ml), and the suspension was stirred at 90°C for 5 hr.
The reaction mixture was cooled to room temperature, extracted
35 with hexane-ethyl acetate (10+10 ml), and washed once with 1
mol/l hydrochloric acid (15 ml) and twice with water (10 ml).
The solvent was evaporated to give methyl 4-(2,3-
dihydrophyty1oxy)-2-methylbenzoate. After confirmation of the
structure by NMR, it was used for the next step.
The aforementioned methyl 4-(2,3-dihydrophyty1oxy)-2-
methylbenzoate (1.08 mmol) was dissolved in THF (6 ml), DIBAL
(LON, 4.9 ml, 4.9 mmol) was added, and the mixture was stirred
at room temperature for 100 min. The reaction mixture was
cooled to O°C, and the reaction was quenched with 1 mol/l
10 hydrochloric acid (15 ml) . Hexane (10 ml) and ethyl acetate
(10 ml) were added to allow liquid-separation, and the mixture
was washed once with 0.5 mol/l hydrochloric acid (10 ml) and
once with water (10 ml), and the solvent was evaporated to give
4-(2,3-dihydrophyty1oxy)-2-methylbenzyl alcohol.
Preparation Example 14 : synthesis of 4- (2,3-dihydrophytyloxy)-
2-methylbenzyl amine
4-(2,3-Dihydrophyty1oxy)-2-methyl-benzyl alcohol (1.08
mmol) was dissolved in chloroform (8 ml), thionyl chloride (393
20 p1, 5.38 mmol) was added and the mixture was stirred at 50°C
for 4.5 hr. After completion of the reaction, the solvent was
evaporated to give 4-(2,3-dihydrophyty1oxy)-2-methylbenzyl
chloride. After confirmation of the structure by NMR, it was
used for the next step.
25 The aforementioned 4-(2,3-dihydrophyty1oxy)-2-
methylbenzyl chloride (1.08 mmol) was dissolved in DMF (6 ml),
sodium azide (350 mg, 5.38 mmol) was added and the mixture was
stirred at 70°C overnight. The reaction mixture was cooled to
room temperature, hexane (10 ml) and ethyl acetate (5 ml) were
30 added, and the mixture was washed 3 times with water (10 ml).
The solvent in the filtrate was evaporated to give 4-(2,3-
dihydrophyty1oxy)-2-methylbenzyl azide. After confirmation of
the structure by NMR, it was used for the next step.
The aforementioned 4- (2,3-dihydrophytyloxy)- 2-
35 methylbenzyl azide (1.08 mmol) was dissolved in THF (10 ml) ,
water (2 ml) and triphenylphosphine (565 mg, 2.15 mol) were
added and the mixture was stirred at 60°C for 3 hr. After
cooling to room temperature, the solvent was evaporated, and
the residue was dissolved in heptane (10 ml), and washed 3
times with 50% aqueous acetonit.rile solution (10 ml). The
solvent was evaporated and the obtained residue was purified by
silica gel column chromatography (hexane:ethyl
acetate=5:l+chloroform:methanol:aqueous amonia=50:5:1) to
give 4- (2,3-dihydrophytyloxy)- 2-methylbenzyl amine (281 mg,
0.67 mol, yield 62% vs . 2,3-dihydrophytyl bromide) .
[0364]
Preparation Example 15: Synthesis of 2,2,4,8,10,10-hexamethyl-
5-dodecanoic acid (4-hydroxymethy1)phenylamide
2,2,4,8,10,10-Hexamethyl-5-dodecanoic acid (2.81 g, 9.88
mol), 4-aminobenzyl alcohol (1.00 g, 8.12 mol) and 3,4-
dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt) (133 mg,
0.812 mol) were suspended in chloroform (10 ml), EDC HC1 (2.05
g, 10.7 mol) was added at O°C, and the suspension was stirred
at room temperature overnight. The solvent was removed, and
the residue was purified by silica gel column chromatography
(hexane:ethyl acetate=Z:l) to give 2,2,4,8,10,10-hexamethyl-5-
dodecanoic acid . (4-hydroxymethyl)p henylamide (2.69 g, 6.67 mol,
yield 82%) .
Preparation Example 16: Synthesis of 4-(3,7,11-
trimethyldodecyloxy)benzyl alcohol
obtained in Reference Example 4 was dissolved in DMF (5 ml), 4-
hydroxybenzyl alcohol (0.85 g, 6.85 mol) and potassium
carbonate (1.42 g, 10.3 mol) were added and the mixture was
stirred at 120°C overnight. The reaction mixture was cooled to
room temperature, extracted with chloroform (50 ml), and the
extract was washed three times with 1 mol/l hydrochloric acid
(30 ml), once with 5% aqueous sodium hydrogen carbonate
solution (30 ml), and once with purified water (30 ml). The
solvent of the organic layer was evaporated to give 4-(3,7,11-
trimethyldodecyloxy) benzyl alcohol (1.09 g, 3.2 6 mmol, yield
95% vs. 1-bromo-3,7,ll-trimethyldodecane).
[0366]
5 Preparation Example 17: Synthesis of 3,7,11,15-tetramethyl-1-
hexadecanoic acid
3,7,11,15-Tetramethyl-hexadecan-1-01 (8.96 g, 30.0 mmol)
was dissolved in a mixe.d solvent of acetone (360 ml) and acetic
acid (180 ml), a solution of anhydrous chromic acid (7.27 g,
10 72.7 mmol) in water (9.0 ml) was added dropwise, and the
mixture was stirred at room temperature for 1 hr. A solution
of sodium disulfite (100 g, 526 mmol) in water (450 ml) was
added to the reaction mixture after completion of the reaction,
the mixture was stirred at room temperature overnight and
15 extracted 6 times with diethyl ether (135 ml). The solvent of
the obtained organic layer was evaporated under reduced
pressure. Diethylether (450 ml) and water (150 ml) were added
to the oil after concentration to allow partitioning, and the
aqueous layer was extracted 3 times with ethyl acetate (150 ml).
20 The combined organic layer was dried over anhydrous sodium
sulfate, and the solvent was evaporated under reduced pressure.
The oil (10.1 g) after concentration was purified by
chromatography (silica gel; 250 g, eluate; 50:1-+3:1 hexaneethyl
acetate) to give the title compound (6.78 g, 72.3%) as a
25 pale-blue oil.
Preparation Example 18: Synthesis of 3,7,11,15-tetramethyl-1-
hexadecanovl chloride
The compound (2.81 g, 9.0 mmol) synthesized in
30 Preparation Example 17 was dissolved in anhydrous chloroform
(4.5 ml), thionyl chloride (1.31 ml, 18.0 mmol) was added and
the mixture was stirred at room temperature for 1 hr. After
completion of the reaction, the solvent was evaporated under
reduced pressure and the obtained oil (3.07 g) was directly
3 used for the next reaction as the title compound.
Preparation Example 19: Synthesis of chloromethyl 3,7,11,15-
The compound (6.56 g, 21.0 mol) synthesized in
Preparation Example 17 was dissolved in anhydrous chloroform
(10 ml), thionyl chloride (3.06 ml, 42.0 mol) was added and
the mixture was stirred at room temperature for 1 hr. After
completion of the reaction, the solvent was evaporated under
reduced pressure, and the obtained oil was added dropwise to a
mixed solid of 90% para-formaldehyde (840 mg, 25.2 mol) and
zinc chloride (42.9 mg, 0.32 mol) over 30 min under cooling in
an ice bath. The obtained mixed solution was stirred with
heating at 60°C for 5 hr and allowed to cool to room
temperature. 10% Aqueous sodium hydrogen carbonate solution
(30 ml) and dichloromethane (15 ml) were added to allow layer
separation, and the mixture was further extracted twice with an
equal amount of dichloromethane. The combined organic layer
was washed with saturated brine (30 ml), dried over anhydrous
sodium sulfate and the solvent was evaporated under reduced
pressure. The oil (7.40 g) after concentration was purified by
chromatography (silica gel; 120 g, eluate; 10:lhexanedichloromethane)
to give the title compound (5.01 g, 66.1%) as
a colorless oil.
Preparation Example 20: Synthesis of 4-(3,7-dimethyl-1-
octyloxy)benzoic acid
(1) Synthesis of 1-bromo-3,7-dimethyloctane
3,7-Dimethyloctan-1-01 (10.0 g, 63.2 mol) was suspended
in 48% aqueous hydrobromic acid solution, concentrated sulfuric
acid (0.17 ml) was added dropwise and the mixture was stirred
at 100°C for 16 hr. The reaction mixture was allowed to cool
to room temperature, extracted with hexane (200 ml), and washed.
twice with 5% aqueous sodium hydrogen carbonate solution (100
ml) and once with 20% brine (100 ml) . The organic layer was
dried over sodium sulfate, and the solvent in the filtrate was
evaporated to give the title compound (13.3 g, 95.1%) as a
colorless oil.
(2) Synthesis of methyl [4-(3,7-dimethyl-l-octyloxy)]benzoate
Under an argon atmosphere, potassium carbonate (12.5 g,
5 90.3 mol) was suspended in anhydrous N,N-dimethylformamide
(100 ml) , l-bromo-3,7-dimethyloctane (13.3 g, 60.1 mrnol) and
methyl (4-hydroxy)b enzoate (8.74 g, 57.4 mol) were added, and
the mixture was stirred at 70°C for 16 hr. The reaction
mixture was filtered to remove potassium carbonate. Water (50
10 ml) was added and the mixture was extracted with hexane (250
ml). The extract was washed successively with 1.0 mol/l
aqueous hydrochloric acid solution (100 ml), aqueous sodium
hydrogen carbonate solution (100 ml) and saturated brine (100
ml). The obtained organic layer was dried over sodium sulfate,
15 filtered and concentrated under reduced pressure to give the
title compound (16.1 g, 95.7%) as a colorless oil.
(3) Synthesis of 4-(3,7-dimethyl-l-octyloxy)benzoic acid
Methyl [4- (3,7-dimethyl-1-octyloxy) ] benzoate (16.1 g,
54.9 mmol) was dissolved in 1,4-dioxane (300 ml), 50% aqueous
20 potassium hydroxide solution (25 ml) was added and the mixture
was stirred at 100°C for 6 hr. To acidify the reaction mixture,
concentrated hydrochloric acid was added dropwise, and the
mixture was extracted with ethyl acetate (200 ml), and washed
with 10% aqueous sodium hydroxide solution (100 ml) and
25 saturated brine (100 ml). The organic layer was dried over
sodium sulfate, filtered and concentrated under reduced
pressure to give the title compound (14.2 g, 92.8%) as a white
solid.
[0370]
30 Preparation Example 21 : Synthesis of 5' -0- (4,4' -
- -
dimethoxytrityl)- 2' -deoxycytidine-3' -0-[ (2-cyanoethyl)- N,N -
diisopropylphosphoramidite]
Under an argon atmosphere, 5' -0- (4,4'-dimethoxytrityl) -
~~-acet~l--2d'eo xycytidine-3~-[ (2-cyanoethyl)- N,N -
35 diisopropylphosphoramidite] (3.5 g, 4.5 mrnol) was dissolved in
2.0 mol/l ammonia/methanol solution (40 ml) and the mixture was
stirred at room temperature for 2 hr. The reaction mixture was
concentrated under reduced pressure to give the title compound
(3.4 g) quantitatively.
Preparation Example 22: Synthesis of 5'-0-(4,4'-
diisopropylphosphoramidite]
Under an argon atmosphere, 5' -0- (4,4' -dimethoxytrityl) -
10 N ~(-2 -methyl-1-oxopropyl)- 2' -deoxyguanosine-3' -0-[ (2-
cyanoethyl) -N, N-diisopropylphpsphoramidite] (5.0 g, 5.9 mmol)
was dissolved in 2.0 mol/l ammonia/methanol solution (60 ml)
and the mixture was stirred at room temperature overnight. The
reaction mixture was concentrated under reduced pressure, and
15 the obtained oil was purified by silica gel column
chromatography to give the title compound (4.6 g)
quantitatively.
(Preparation of amidine-type protecting group reagent)
20 Preparation Example 23: Synthesis of N,N-di(3,7-dimethylocty1)
formamide dimethylacetal
(1) Synthesis of 3,7-dimethyl-1-octyl bromide
48% HBr (200 ml) and concentrated sulfuric acid (0.46 ml)
were added to 3,7-dimethyl-1-octanol (21.0 g, 157.9 mmol) , and
25 the mixture was heated overnight. After allowing to cool to
room temperature, and the mixture was extracted with hexane.
The organic layer was washed with 5% aqueous sodium hydrogen
carbonate solution and saturated brine, dried and concentrated
to give the title compound (28.7 g, 82.1%) .
30 (2) Synthesis of di(3,7-dimethyl-octy1)benzyl amine
Benzyl amine (7.1 ml, 64.8 mmol), potassium carbonate
(17.9 g, 129.6 mmol) and the compound (28.7 g, 129.6 mmol)
obtained in Preparation Example 23-(1) were dissolved in dry
acetonitrile (80 ml) and the mixture was heated overnight. The
35 reaction mixture was concentrated, dichloromethane (200 ml) was
added and the mixture was washed with water. The organic layer
was concentrated and the obtained oil was purified by silica
gel column chromatography to give the title compound (13.2 g,
52.2%) .
5 (3) Synthesis of N, N -di (3,7-dimethyl-octyl)a mine
The compound (13.2 g, 33.8 mmol) obtained in Preparation
Example 23-(2) was dissolved in ethanol (150 ml), 5% palladium
carbon (53% wetted, 2.86 g) was added and the mixture was
stirred overnight under a hydrogen atmosphere. The reaction
10 mixture was filtered through celite to remove the palladium
catalyst, and the filtrate was concentrated to give the title
compound (10.0 g, 99.3%) quantitatively.
(4) Synthesis of N,N -di (3,7-dimethyl-octyl)f ormamide
dimethvlacetal
To the compound (7.0 g, 23.4 mmol) obtained in
Preparation Example 23-(3) were added N,N-dimethylformamide
dimethyl acetal(2.8 g, 23.4 mmol) and a catalytic amount of
pyridinium p-toluenesulfonate, and the mixture was heated to
160°C and stirred overnight. The reaction mixture was
20 evaporated under reduced pressure to give the title compound
(1.4 g, 16%) .
[0373]
Preparation Example 24: Synthesis of 2,3,4-tris(2,3-
dihydrophyty1oxy)benzhydryl amine
25 (1) Synthesis of 2,3,4-tris (2,3-dihydrophytyloxy)b enzophenone
Under an argon atmosphere, 2,3,4-trihydroxybenzophenone
(0.94 g, 4.07 mmol) , 2,3-dihydrophytyl bromide (6.01 g, 16.6
mmol) and potassium carbonate (2.57 g, 138.2 mmol) were added
to anhydrous N,N-dimethylformamide (25 ml), and the mixture was
30 stirred at 80°C overnight. The reaction mixture was allowed to
cool to room temperature, extracted with hexane (50 ml), washed
successively with 1 mol/l aqueous hydrochloric acid (20 ml), 5%
aqueous sodium hydrogen carbonate solution (20 ml) and
saturated brine (20 ml), and dried over sodium sulfate. The
35 solvent in the filtrate was evaporated and the obtained residue
was purified by silica gel column chromatography (hexane:ethyl
acetate=100:0 - 95/5(v/v)). The object fractions were
collected and concentrated to give the title compound (3.90 g,
88.9%) as an oil.
5 (2) Synthesis of 2,3,4-tris (2,3-dihydrophytyloxy)b enzhydryl
alcohol
3.64 mmol) synthesized in Preparation Example 24- (1) was
dissolved in a mixed solvent of chloroform (35 ml) and methanol
10 (3.5 ml) , sodium borohydride (0.41 g, 10.9 mmol) was added, and
the mixture was stirred at 45OC for 2 hr. After completion of
the reaction, 0.1 mol/l aqueous hydrochloric acid was added
dropwise to decompose unreacted sodium borohydride, and the
mixture was washed with 1.0 mol/l aqueous hydrochloric acid.
15 The organic layer was dried over sodium sulfate and filtered,
and the filtrate was concentrated under reduced pressure. The
solvent in the filtrate was evaporated and the obtained residue
was purified by silica gel column chromatography (hexane:ethyl
acetate=98:2 - 90/10(v/v)). The object fractions were
20 collected and concentrated to give the title compound (3.56 g,
91.0%) as an oil.
(3) Synthesis of N- (9-f luorenylmethoxycarbonyl) -2,3 , 4-tris (2 , 3-
dihydrophyty1oxy)benzhydryl amine - -
Under an argon atmosphere, 2,3,4-tris(2,3-
25 dihydrophyty1oxy)benzhydryl alcohol (3.56 g, 3.31 mmol)
synthesized in Preparation Example 24-(2) and 9-fluorenylmethyl
carbarnate (1.42 g, 5.96 mmol) were dissolved in anhydrous
toluene (40 ml) at 50°C, methanesulfonic acid (64 p1, 993 pol)
was added and the mixture was stirred at 100°C for 2 hr. The
30 reaction mixture was allowed to cool to room temperature,
washed with 5% aqueous sodium hydrogen carbonate solution (20
ml) and saturated brine (20 ml), and dried over sodium sulfate.
The solvent in the filtrate was evaporated and the obtained
residue was purified by silica gel column chromatography
35 (hexane: ethyl acetate=98 : 2 - 90/10 (v/v) ) . The object fractions
were collected and concentrated to give the title compound
(4.00 g, 93.3%) as an oil.
(4) Synthesis of 2,3,4-tris(2,3-dihydrophyty1oxy)benzhydryl
amine
N- (9-Fluorenylmethoxycarbonyl)- 2,3,4-tris (2,3-
dihydrophyty1oxy)benzhydryl amine (4.00 g, 3.09 mmol)
synthesized in Preparation Example 24-(3) was dissolved in a
mixed solvent of chloroform (30 ml) and acetonitrile (15 ml),
20% piperidine [1-methyl-2-pyrrolidone solution] (30.5 ml, 61.8
10 mmol) was added, and the mixture was stirred at room
temperature for 30 min. After completion of the reaction,
ethyl acetate (60 ml), hexane (30 ml) and water (10 ml) were
added to allow layer separation. The organic layer was washed
with saturated brine and dried over sodium sulfate. The
15 solvent in the filtrate was evaporated and the obtained residue
was purified by silica gel column chromatography (hexane:ethyl
acetate=100:0 - 80/20(v/v)). The object fractions were
collected and concentrated to give the title compound (2.92 g,
88.2%) as an oil.
20 [0374]
Preparation Example 25: Synthesis of 4,4'-bis(2,3-
dihydrophyty1oxy)benzhydryl amine
(1) Synthesis of N- (9-f luorenylmethoxycarbonyl) -bis-4- (2,3-
dihydrophyty1oxy)benzhydryl amine
25 To 4,4' -bis (2,3-dihydrophytyloxy)b enzhydryl alcohol (3.80
g, 4.89 mmol) described in Preparation Example 6 were added
toluene (50 ml) and 9-fluorenylmethyl carbamate (2.11 g, 8.81
mmol), and the mixture was dissolved by heating to 50°C.
Methanesulfonic acid (95.3 p1, 1.47 mrnol) was added and the
30 mixture was stirred at 100°C for 2 hr. The completion of the
reaction was confirmed and the reaction mixture was allowed to
cool to room temperature. 5% Aqueous sodium hydrogen carbonate
solution (20 ml) was added and the mixture was stirred. After
partitioning, the organic layer was further washed with water
35 (20 ml) and saturated brine (20 ml). The organic layer was
evaporated under reduced pressure to give the title compound
(5.10 g, quant).
(2) Synthesis of 4,4'-bis(2,3-dihydrophyty1oxy)benzhydryl amine
N- (9-Fluorenylmethoxycarbonyl)- 4-bis (2,3-
5 dihydrophyty1oxy)benzhydryl amine (5.10 g, 5.27 mmol) obtained
in Preparation Example 25-(1) was dissolved in a mixed solvent
of chloroform (50 ml) and acetonitrile (25 ml), 20% piperidine
[1-methyl-2-pyrrolidone solution] (52.1 ml, 105.4 mol) was
added, and the mixture was stirred at room temperature for 30
l o min. After completion of the reaction, ethyl acetate (100 ml),
hexane (60 ml) and water (15 ml) were added to allow layer
separation. The organic layer was washed with saturated brine
and dried over sodium sulfate. The solvent in the filtrate was
evaporated and the obtained residue was purified by silica gel
15 column chromatography (hexane:ethyl acetate=100:0 - 30/70v/v)).
The object fractions were collected and concentrated to give
the title compound (3.56 g, 86.8%) as an oil.
[0375]
Preparation Example 26: Synthesis of 3,5-bis (2,3-
20 dihydrophytyloxy) benzyl amine
(1) Synthesis of 3,5-bis (2,3-dihydrophytyloxy)b enzyl chloride
Under an argon atmosphere, 3,5-bis (2,3-
dihydrophyty1oxy)benzyl alcohol (4.70 g, 6.70 mmol) described
in Preparation Example 9 was dissolved in chloroform (34 ml),
25 pyridine (a few drops) and thionyl chloride (0.97 ml, 13.4
mmol) were added, and the mixture was stirred at room
temperature for 90 min. The reaction mixture was concentrated
under reduced pressure to give the title compound (4.93 g,
quant) as an oil.
30 (2) Synthesis of 3,5- bis (2,3-dihydrophytyloxy)b enzyl azide
3,5-Bis (2,3-dihydrophytyloxy)b enzyl chloride (4.93 g,
6.85 rnmol) obtained in Preparation Example 26-(1) was dissolved
in a mixed solvent of chloroform (27 ml) and N,Ndimethylformamide
(81 ml), sodium azide (0.90 g, 13.7 mmol) was
35 added and the mixture was stirred at 80°C for 2.5 hr. After
131
completion of the reaction, the reaction mixture was allowed to
cool to room temperature, ethyl acetate (250 ml) and purified
water (180 ml) were added to allow layer separation, and the
organic layer was washed with purified water (180 ml) and
5 saturated brine (130 ml). The organic layer was dried over
sodium sulfate and filtered to give the title compound (4.79 g,
96.4%).
(3) Synthesis of 3,5-bis (2,3-dihydrophytyloxy)b enzyl amine
Under an argon atmosphere, 3,5-bis (2,3-
10 dihydrophytyloxy) benzyl azide (4.7 9 g, 6.60 mmol) obtained in
Preparation Example 26-(2) was dissolved in anhydrous
tetrahydrofuran (33 ml), lithium aluminum hydride (0.50 g, 13.2
mmol) was added under ice-cooling, and the reaction mixture was
stirred at room temperature for 2 hr. To the reaction mixture
15 after completion of the reaction were added dropwise 1 mol/l
aqueous hydrochloric acid (25 ml) and ethyl acetate (50 ml) to
allow layer separation, and the aqueous layer was extracted
with ethyl acetate (50 ml). The organic layers were combined,
washed twice with water (90 ml), and further washed with 5%
20 aqueous sodium hydrogen carbonate solution (90 ml) and
saturated brine (90 ml). The organic layer was dried over
magnesium sulfate, and the filtrate was concentrated under
reduced pressure and purified by silica gel column
chromatography to give the title compound (3.29 g, 71.1%) as an
25 oil.
[037 61
Preparation Example 27 : Synthesis of 5' -0- (4,4' -
cyanoethy1)-N,N-diisopropylphosphoramidite]
30 (1) Synthesis of 5' -0- (4,4' -dimethoxytrityl) -2' -deoxy-2' -
fluorouridine
An operation of dissolving 2'-Deoxy-2'-fluorouridine
(3.00 g, 12.2 mmol) in dry pyridine, followed by concentration
under reduced pressure was repeated 3 times to perform
35 dehydrative azeotropic distillation. Thereafter, under an
argon atmosphere, the reaction mixture was dissolved in dry
pyridine (120 ml) , 4,4'-dimethoxytrityl chloride (4.55 g, 13.4
mmol) was added, and the mixture was stirred at room
temperature for 3 hr. The completion of the reaction was
5 confirmed, and ethyl acetate (150 ml) and water (60 ml) were
added to the reaction mixture to allow layer separation. The
organic layer was washed 3 times with 5% aqueous sodium
hydrogen carbonate solution (20 ml), washed with water (20 ml)
and saturated brine (20 ml), and the obtained organic layer was
10 dried over sodium sulfate. The solvent in the filtrate was
evaporated and the obtained residue was purified by silica gel
column chromatography (hexane:ethyl acetate = 50:50 -
0/100(v/v), containing 1% triethylamine). The object fractions
were collected and concentrated to give the title compound
15 (8.48 g, quant).
[0377]
(2) Synthesis of 5' -0- (4,4' -dimethoxytrityl) -2' -deoxy-2'-
diisopropylphosphoramidite]
(3.00 g, 5.47 mmol) obtained in Preparation Example 27- (1) was
dissolved in anhydrous dichloromethane (50 ml) under an argon
atmosphere, N,N-diisopropylethylamine (0.55 ml, 3.18 mmol), 1Htetrazole
(0.45 g, 6.45 mmol) and 2-cyanoethyl-N,N,N1 ,N' -
25 tetraisopropylphosphordiamidite (1.92 g, 6.3 6 mmol) were added,
and the mixture was stirred at room temperature overnight. The
completion of the reaction was confirmed, 5% aqueous sodium
hydrogen carbonate solution (20 ml) was added to the reaction
mixture to allow layer separation, and the organic layer was
30 washed with saturated brine (20 ml). The organic layer was
dried over sodium sulfate, the filtrate was concentrated and
the obtained crude product was purified by silica gel
chromatography (hexane:ethyl acetate=75:25 - 30/70(v/v),
containing 3% triethylamine). The object fractions were
35 collected and concentrated to give the title compound (2.76 g,
Example 1: Synthesis of 5'-0- (4,4' -dimethoxytrityl) -2' -
deoxythymidin-3'-yl-0- [3,4,5-tris (3,7,11,15-tetramethyl-l-
' hexadecanyloxy) benzyl] succinate [5' -0-DMTr-dT-suc-TPB]
(1) Synthesis of 5'-0-(4,4'-dimethoxytrity1)-2'-deoxythymidine-
3' -0-succinate
Under an argon atmosphere, 5' -0- (4,4' -dimethoxytrityl) -
2'-deoxythymidine (4.98 g, 9.14 mmol) and tetrahydrofuran-2,5-
dione (1.39 g, 13.9 mmol) were dissolved in anhydrous
dichloromethane (100 ml), triethylamine (3.80 ml, 27.3 mmol)
was added and the mixture was stirred at room temperature for
16 hr. The reaction mixture after completion of the reaction
was washed 3 times with 2.0 mol/l aqueous triethylammonium
phosphate solution (70 ml), and the organic layer was dried
over sodium sulfate, filtered and concentrated under reduced
pressure. The obtained residue was azeotropically distilled 3
times with toluene (10 ml) to quantitatively give a
triethylamine salt of the title compound (7.15 g) as a white
solid.
(2) Synthesis of 5' -0- (4,4' -dimethoxytrityl) -2'-deoxythymidin-
3'-yl-0- [3,4,5-tris (3,7,11,15-tetramethyl-lhexadecanyloxy)
benzyl] succinate [5' -0-DMTr-dT-suc-TPB]
The compound synthesized in Example 1 -(I) (6.55 g, 8.78
mmol) and 3,4,5-tris (3,7,11,15-tetramethyl-lhexadecany1oxy)
benzyl alcohol (5.19 g, 5.16 mmol) synthesized
in Preparation Example 7 were dissolved in anhydrous
dichloromethane (15 ml) , 2- (1H-benzotriazol-l-yl) 1,1,3,3-
tetramethyluronium hexafluorophosphate [HBTU] (11.8 g, 30.8
mmol) and N,N-diisopropylethylamine (5.53 ml, 31.2 mmol) were
added, and the mixture was stirred at room temperature for 1 hr.
The mixture was washed with saturated aqueous sodium hydrogen
carbonate solution and saturated brine, and the obtained
organic layer was dried over sodium sulfate and filtered. The
filtrate was concentrated under reduced pressure and the
obtained residue was purified by silica gel column
chromatography (hexane/ethyl acetate, l%v/v triethylamine) to
give the title compound (3.83 g, 45.3%) as a viscous solid.
5 Example 2: Synthesis of 5'-0- (4,4'-dimethoxytrityl) -2'-
Using a triethylamine salt (1.45 g, 1.94 mmol) of 5'-0-
(4f4f-dimethoxytrityl)-2f-deoxythpidine-3f-O-succinateafn d
lo 3,4,5-tris (3,7,11,15-tetramethyl-l-hexadecanyloxy)b enzyl amine
(1.02 g, 1.10 mmol) synthesized in Preparation Example 8, and
in the same manner as in Example 1 -(2), the title compound
(1.22 g, 72.4%) was obtained as a viscous solid.
15 Example 3: Synthesis of 5' -0- (4, 4'-dimethoxytrityl) -N~-
[0382]
Under an argon atmosphere, 5' -0- (4,4' -dimethoxytrityl) -
2' -deoxycytidine-3' -0-[ (2-cyanoethyl)- N,N -
diisopropylphosphoramidite] (1.46 g, 2.00 mmol) was dissolved
25 in anhydrous tetrahydrofuran (10 ml), N,N-diisopropylethylamine
(720 p1, 4.00 mrnol) and the compound synthesized in Preparation
Example 18 (990 mg, 3.00 mrnol) were added, and the mixture was
stirred at room temperature for 3 hr. After completion of the
reaction, ethyl acetate (60 ml) and 5% aqueous sodium hydrogen
5 carbonate solution (15 ml) were added to the reaction mixture
to allow phase separation, and the aqueous phase was extracted
with ethyl acetate (30 ml). The combined organic phase was
dried over anhydrous sodium sulfate, and the solvent was
evaporated under reduced pressure. The concentrated oil (2.70
10 g) was purified by chromatography (silica gel; 50 g, eluate; 1%
triethylamine-containing 10:1+1:1 hexane-ethyl acetate) to
give the title compound (1.08 g, 52.6%) as a colorless oil.
[0383]
'H-NMR(~OOMHZC, D C ~:~ 6)0 .84 (d, 6H, J=6.6Hz), 0.86 (d, 6H,
15 J=6.6Hz), 0.97 (d, 3H, J=6.6Hz), 1.00-1.41 (m, 32H), 1.46-1.56
(m, lH), 1.94-2.18 (m, 2H), 2.21-2.33 (m, lH), 2.34-2.46 (m,
2H), 2.62 (t, lH, J=6.3Hz), 2.67-2.83 (m, lH), 3.34-3.67 (m,
5H), 3.69-3.86 (m, lH), 3.80 (s, 3H), 3.81 (s, 3H), 4.20-4.24
(m, lH), 4.55-4.66 (m, lH), 6.22-6.29 (m, lH), 6.82-6.87 (m,
20 4H), 7.10-7.14 (m, lH), 7.21-7.33 (m, 7H), 7.36-7.42 (m, 2H),
7.94 (brs, lH), 8.17-8.29 (m, 1H)
3 1 ~ - ~ ~ ~ ( 1 6: 6~ 1~50~.0~, ,150~.6~ ~ 1 3 )
m/z (ESI-MS): Anal. Calc. for C59HB6N50BP1:0 23.6. Found 1022.3
(M-H) -
Example 4: Synthesis of N3- [4- (3,7-dimethyl-1-
octyloxy ) benzoyl] -2 -deoxythymidine
(1) Synthesis of 4-(3,7-dimethyl-1-octyloxy)benzoyl chloride
Under an argon atmosphere, 4-(3,7-dimethyl-1-
30 octyloxy)benzoic acid was dissolved in anhydrous chloroform (25
ml) , and after ice-cooling, thionyl chloride (6.72 ml, 92.7
mmol) was added dropwise. The reaction mixture was stirred at
room temperature overnight and concentrated under reduced
pressure to give the title compound as an oil. The present
35 compound was directly used for the next step.
(2) Synthesis of 3',5'-0-bis (trimethylsilyl) -N3- [4- (3,7-
dimethyl-1-octyloxy)benzoyl]2'-deoxythymidine
2' -Deoxythymidine (5.00 g, 20.6 mol) was azeotropically
distilled 3 times with anhydrous pyridine (10 ml), and under an
5 argon atmosphere, the mixture was dissolved in anhydrous
pyridine (60 ml) , N,N-diisopropylethylamine (17.9 ml, 103 mol)
and trimethylsilyl chloride (6.50 ml, 51.5 rnmol) were added,
and the mixture was stirred at room temperature for 30 min.
After stirring, an oil of the aforementioned 4-(3,7-dimethyl-1-
10 octyloxy)benzoyl chloride was added dropwise over 25 min, and
thereafter the mixture was stirred at room temperature for 4 hr.
The reaction mixture after completion of the reaction was icecooled,
potassium dihydrogen phosphate (17 g) and water (80 ml)
were added, and the mixture was stirred for 5 min. The mixture
15 was extracted with diethyl ether (100 ml), washed with
saturated aqueous potassium dihydrogen phosphate solution (50
ml) and saturated brine (50 ml), the obtained organic layer was
dried over sodium sulfate and filtered, and the filtrate was
concentrated under reduced pressure to give the title compound.
20 The present compound was directly used for the next step.
(3) Synthesis of N3- [4- (3, 7-dimethyl-1-octyloxY) benzoyl] -2' -
deoxythymidine
3',5' -0-bis- (trimethylsilyl) -N3- [4- (3, 7-dimethyl-1-
octyloxy)benzoyl]-2'-deoxythymidine was dissolved in a mixed
25 solvent of chloroform (70 ml) and methanol (70 ml) ,
trifluoroacetic acid (350 p1) was added, and the mixture was
stirred at room temperature for 30 min. After completion of
the reaction, the solvent was evaporated and dissolved again in
ethyl acetate (150 ml). The mixture was washed with 5% aqueous
30 sodium hydrogen carbonate solution (75 ml) and saturated brine
(75 ml), and the organic layer was dried over sodium sulfate
and filtered. The solvent in the filtrate was evaporated and
the obtained residue was purified by silica gel column
chromatography (dichloromethane/methanol) to give the title
35 compound (7.10 g, 68.5%) as a white solid.
[0385]
Example 5 : Synthesis of 5' -0- (4,4' -dimethoxytrityl) -N3- [4- (3,7-
Under an argon atmosphere, N3- [4- (3,7-dimethyl-1-
5 octyloxy) benzoyl] -2' -deoxythymidine (7.10 g, 14.1 mmol) was
azeotropically distilled 3 times with anhydrous pyridine (10
ml) and dissolved in anhydrous pyridine (130 ml). 4,4'-
Dimethoxytrityl chloride (4.83 g, 14.3 mmol) was added, and the
mixture was stirred overnight. Water (130 ml) was added to the
lo reaction mixture after completion of the reaction, and the
mixture was extracted with diethyl ether (260 ml) and washed
with water (130 ml). The organic layer was dried over sodium
sulfate and filtered, the filtrate was concentrated, and the
obtained residue was purified by silica gel column
15 chromatography (dichloromethane/methanol, l%v/v triethylamine)
to give the title compound (10.2 g, 90.1%) as a white solid.
[038 61
Example 6: Synthesis of 5'-0-(4,4'-dimethoxytrityl)-N3-[4-(3,7-
dimethyl-l-octyloxy)benzoyl]-2'-deoxythyidine-3~-0-[(2-
20 cyanoethyl) -N, N-diisopropylphosphoramidite] [5' -0-DMTr-dT '4-Cit-
BZ) -PA]
Under an argon atmosphere, 5' -0- (4,4' -dimethoxytrityl) -
N3- [4-( 3,7-dimethyl-1-octyloxy)b enzoyl]- 2' -deoxythymidine ( 6.47
g, 8.03 mmol) was azeotropically distilled 3 times with
2s anhydrous acetonitrile (10 ml) and dissolved in anhydrous
dichloromethane (40 ml) . Under ice-cooling, N, Ndiisopropylethylamine
(5.60 ml, 32. O mmol) was added, and a
solution of chloro-2-cyanoethyl-N,N-diisopropylphosphoramidite
(2.36 ml, 10.0 mmol) in .dichloromethane (40 ml) was added
30 dropwise over 20 min. The reaction mixture was stirred at room
temperature for 30 min and concentrated under reduced pressure,
and the obtained residue was purified by silica gel column
chromatography (hexane/ethyl acetate, 3%v/v triethylamine) to
give the title compound (7.32 g, 91.0%) as a white solid.
35 [0387]
Example 7: Synthesis of 5'-0-(4,4'-dimethoxytrity1)-3'-0-
Under an argon atmosphere, potassium carbonate (2.10 g,
5 15.2 mmol) was suspended in anhydrous N,N-dimethylformamide (50
ml), the compound synthesized in Preparation Example 19 (3.65 g,
10.1 mmol) and 5' -0- (4,4' -dimethoxytrityl) -3' -0-levulinoyl-2' -
deoxythymidine (3.25 g, 5.06 mmol) were dissolved therein, and
the mixture was stirred at 40°C for 24 hr. The reaction
10 mixture was filtered, water (50 ml) was added, and the mixture
was extracted twice with diethyl ether (100 ml). The organic
layer was washed with water (100 ml), dried over sodium sulfate,
filtered and concentrated under reduced pressure. The obtained
residue was purified by silica gel column chromatography
15 (dichloromethane, l%v/v triethylamine) to give the title
compound (4.80 g, 98%) as a pale-yellow viscous solid.
Example 8 : Synthesis of 5' -0- (4,4' -dimethoxytrityl) -N3-
20 deoxythymidine
The compound synthesized in Example 7 (4.80 g, 4.96 mmol)
was dissolved in a mixed solvent of pyridine (40 ml) and acetic
acid (10 ml) , anhydrous hydrazine (244 p1, 7.71 mmol) was added,
and the mixture was stirred at room temperature for 30 min.
25 Acetylacetone (1.07 ml, 10.3 mmol) was added and the mixture
was stirred at room temperature for 5 min. Diethyl ether (100
ml) was added, and the mixture was washed successively with 10%
aqueous hydrogen sulfate potassium solution (50 ml), 10%
aqueous sodium hydrogen carbonate solution (50 ml) and
30 saturated brine (50 ml). The organic layer was concentrated to
give the title compound (3.58 g, 83.0%) as a pale-yellow
viscous solid.
Example 9: Synthesis of 5' -0- (4,4' -dimethoxytrityl) -N3-
diisopropylphosphoramidite] [5' - o - D M T ~ - ~PTA 1~ ~ ~ ~ ~ -
Under an argon atmosphere, the compound synthesized in
Example 8 (3.58 g, 4.12 mmol) was azeotropically distilled 3
5 times with anhydrous acetonitrile (10 ml) and dissolved in
anhydrous dichloromethane (22 ml) . N, N-diisopropylethylamine
(2.49 ml, 16.5 mmol) was added dropwise, and chloro-2-
cyanoethyl-N,N-diisopropylphosphoramidite (1.15 ml, 5.15 mmol)
was dissolved in anhydrous dichloromethane (22 ml) was added
10 dropwise over20 min. After stirring at room temperature for
30 min, the mixture was concentrated under reduced pressure and
the obtained residue was purified by silica gel column
chromatography (hexane/ethyl acetate=8 /2,3%v/v triethylamine)
to give the title compound (3.90 g, 88.6%) as a pale-yellow
1s viscous solid.
Example 10 : Synthesis of 5' -0- (4,4' -dimethoxytrityl) -N~-
diisopropylphosphoramidite] [5' - o - D M T ~ - ~PTA ~1 ~ ~ ~ ~ -
Under an argon atmosphere, 5'-0-(4,4'-dimethoxytrity1)-
diisopropylphosphoramidite] (1.00 g, 1.34 mmol) was dissolved
in anhydrous N,N-dimethylformamide (15 ml), potassium carbonate
2s (279 mg, 2.02 mmol) and (3,7,11,15-tetramethyl-1-
hexadecanoyloxy)methyl chloride (970 mg, 2.69 mmol) were added,
and the mixture was stirred at room temperature for 16 hr. The
reaction mixture was filtered, water (20 ml) was added and the
mixture was extracted with diethyl ether (50 ml). The organic
30 layer was dried over sodium sulfate and filtered, the filtrate
was concentrated under reduced pressure, and the obtained
residue was purified by silica gel column chromatography (ethyl
acetate/hexane, 3%v/v triethylamine) to give the title compound
(929 mg, 65.0%) as a pale-yellow viscous solid.
35 [0391]
Example 11: Synthesis of 5' -0- (4,4' -dimethoxytrityl) -N~-
Under an argon atmosphere, 5' -0- (4,4' -dimethoxytrityl) -
2'-deoxyguanosine-3'-0-[(2-cyanoethy1)-N,Ndiisopropylphosphoramidite]
(1.52 g, 1.98 mmol) was dissolved
in anhydrous tetrahydrofuran (20.ml), N,N-diisopropylethylamine
(696 p1, 4-00 mmol) was added, and 3,7,11,15-tetramethyl-1-
lo hexadecanoyl chloride (993 mg, 3.00 mmol) synthesized in
Preparation Example 18 was added dropwise. After stirring at
room temperature for 1.5 hr, the reaction mixture was
concentrated and the obtained residue was purified by silica
gel column chromatography (dichloromethane/methanol, l%v/v
15 triethylamine) to give the title compound (1.73 g, 82.0%) as a
pale-yellow viscous solid.
[0392]
Example 12 : Synthesis of 5' -0- (4,4' -dimethoxytrityl) -N~-
-
20 [(2-cyanoethy1)-N,N-diisopropylphosphoramidite] [5'-0-DMTrdlPhy-
PA]
Using 5' -0- (4,4' -dimethoxytrityl) -2' -deoxyadenosine-3' -0-
mmol) and in the same manner as in Example 11, the title
25 compound (1.12 g, 45.8%) was obtained as a pale-yellow viscous
solid.
Example 13: Synthesis of deoxythymidinyl-[3'-+5']-
deoxythymidinyl- [3'+5' ] -deoxythymidine (5' -d [TTT] -3' )
30 (1) Synthesis of 5' - O - D M T ~ - ~ T ~ ~ ~ ~ ~ - ~ T - S U C - N H - T P B
Under an argon atmosphere, 5'-0-DMTr-dT-suc-NH-TPB (206 '
mg, 127 pol) synthesized in Example 2 was dissolved in a mixed
solvent of anhydrous heptane (650 p1) and anhydrous toluene
(650 p1) , trifluoroacetic acid (26.0 pl, 350 pol) and 1H-
35 pyrrole (17.5 p1, 254 pol) were added, and the mixture was
stirred for 5 min. The completion of the deprotection was
confirmed by thin layer chromatography, pyridine (28.3 p1, 350
pol) and N-methylimidazole (13.9 p1, 175 pol) were added, and
the mixture was stirred for 5 min. To the reaction mixture
5 after neutralization was added the compound synthesized in
Example 10 (271 mg, 254 pol) dissolved in 0.25 mol/l 5-
(benzylthio)- lH-tetrazole/acetonitrile solution (1.0 ml) , and
the mixture was stirred for 10 min. 0.2 mol/l Iodine
pyridine/tetrahydrofuran/water=49/49/2 solution (1.27 ml) was
10 added and the mixture was stirred for 5 min. To the reaction
mixture after completion of the reaction was added heptane (5.0
ml) to allow phase separation. The lower layer was extracted,
washed with a mixed solution of acetonitrile (1.0 ml) and water
(80 pl), and the obtained organic layer was concentrated under
15 reduced pressure to give the title compound (291 mg, 99.5%) as
a viscous solid.
(2 ) Svnthesis of 5' - o - D M T ~ - ~ T ~ ~ ~ ~ ~ - ~ T ~ ~ ~ ~ ~ - ~ T - s u c - N H - T P B
- -- -- --
Using the compound synthesized in Example 13-(1) (291 mg,
126 pol) and the compound synthesized in Example 10 (271 mg,
20 254 pol), and in the same manner as in Example 13- (I), the
title compound (369 mg, 98.0%) was obtained as a viscous solid.
(3 ) Synthesis of deoxythymidinyl- [3' +5' ] -deoxythymidinyl-
[3'-+5']-deoxythymidine (5'-d[TTT]-3')
The compound synthesized in Example 13-(2) and a solution
25 (4.0 ml) of 28% aqueous ammonia solution:40% aqueous
methylamine-solution=l:l were placed in an autoclave, the
mixture was heated at 65OC for 16 hr, and concentrated by a
rotary evaporator under reduced pressure. The mixture was
adsorbed to C-18 reversed-phase cartridge column and washed
30 with 0.1 mol/L aqueous ammonium acetate solution. A
dimethoxytrityl group bonded to the hydroxyl group at the 5'-
terminal was deprotected with 2% aqueous trifluoroacetic acid
solution and eluted with 20% aqueous acetonitrile solution to
give the title compound.
35 m/z (ESI-MS): Anal. Calc. for C30H40N6019P28:5 0.2. Found 849.1 (MExample
14: Synthesis of deoxythymidinyl- [3'+5' ] -
deoxythymidinyl- [3' +5' ] -deoxythymidine (5' -d [TTT] -3' )
5 (1) Synthesis of 5' -0-DMT~-(d4-~Ci t-BZ' -dT-suc-NH-TPB
Using 5'-0-DMTr-dT-suc-NH-TPB synthesized in Example 2
(206 mg, 127 pol) and the compound synthesized in Example 6
(256 mg, 254 pol), and in the same manner as in Example 13-(I),
the title compound (273 mg, 96.0%) was obtained as a viscous
lo solid.
(2) Synthesis of 5' -0-DMT~-(d4-~Cit 'BZ' -dT (4-Cit-Bz) -dT-SUC-NH-TPB
Using the compound synthesized in Example 14-(1) (273 mg,
122 pol) and the compound synthesized in Example 6 (256 mg,
254 pol), and in the same manner as in Example 13-(I), the
15 title compound (339 mg, 97.5%) was obtained as a viscous solid.
(3) Synthesis of deoxythymidinyl- [3' +5' ] -deoxythymidinyl-
[3'+5' ] -deoxythymidine (5' -d [TTT] -3' )
Using the compound synthesized in Example 14-(2) and in
the same manner as in Example 13-(3), the title compound was
20 obtained.
m/z(ESI-MS) : Anal-Calc. for C30H40N6019P28:5 0.18. Found 849.1
(M-H) -
Example 15: Synthesis of deoxyadenylyl-[3'+5']-deoxyadenylyl-
25 [3'+5']-deoxythymidine (5'-d[AAT]-3')
(1) Synthesis of 5' - O - D M T ~ - ~ A ~ ~ ~ - ~ T - S U C - N H - T P B
Under an argon atmosphere, 5'-0-DMTr-dT-suc-NH-TPB (213
mg, 131 pol) synthesized in Example 2 was dissolved in a mixed
solvent of anhydrous heptane (700 p1) and anhydrous toluene
30 (700 p1) , trifluoroacetic acid (28.0 p1, 377 pol) and 1Hpyrrole
(18.1 p1, 262 pol) were added, and the mixture was
stirred for 5 min. The completion of the deprotection was
confirmed by thin layer chromatography, pyridine (30.5 p1, 377
pol) and N-methylimidazole (15.0 p1, 189 pol) were added, and
35 the mixture was stirred for 5 min. To the reaction mixture
after neutralization was added the compound synthesized in
Example 12 (275 mg, 262 pol) dissolved in a mixed solvent of
0.25 mol/l 5-(benzy1thio)-1H-tetrazole/acetonitrile solution
(1.0 ml) and anhydrous toluene (300 pl), and the mixture was
5 stirred for 10 min. 5.78 mol/l tert-Butyl hydroperoxide/nonane
solution (45.3 p1) was added and the mixture was stirred for 5
min. To the reaction mixture after completion of the reaction
was added heptane (4.0 ml) to allow layer separation. The
lower layer was extracted and washed with a mixed solution of
10 acetonitrile (1.0 ml) and water (80 p1) , and the obtained
organic layer was concentrated under reduced pressure to give
the title compound (301 mg) as a viscous solid quantitatively.
(2 ) Synthesis of 5' - O - D M T ~ - ~ A ~ ~ ~ - ~ A ~ ~ ~ - ~ T - S U C - N H - T P B
Using the compound synthesized in Example 15-(1) (301 mg,
15 131 pol) and the compound synthesized in Example 12 (275 mg,
262 pol) , and in the same manner as in Example 15- (1) , the
title compound (387 mg) was obtained as a viscous solid
quantitatively.
(3) Synthesis of deoxyadenylyl- [3'-+5' ] -deoxyadenylyl- [3'-+5' ] -
20 deoxythymidine (5' -d [AAT] -3' )
The compound synthesized in Example 15-(2) and 28%
aqueous ammonia solution (4.0 ml) were placed in an autoclave,
the mixture was heated at 65OC for 16 hr, and concentrated by a
rotary evaporator under reduced pressure. The concentrated
25 solution was adsorbed to C-18 reversed-phase cartridge column
and washed with 0.1 mol/L aqueous ammonium acetate solution. A
dimethoxytrityl group bonded to the hydroxyl group at the 5'-
terminal was deprotected with 2% aqueous trifluoroacetic acid
solution and eluted with 20% aqueous acetonitrile solution to
30 give the title compound.
m/z (ESI-MS) :Anal. Calc. for C30H38N12015:P 826 8.21.F ound 8 67.1 (M-H)-
[ 0 3 9 61
Example 16: Synthesis of deoxyguanosinyl- [3'-+5' ] -
deoxyguanosinyl- [3' -5' ] -deoxythymidine (5' -d [GGT] -3' )
35 (1) Synthesis of ~ ~ - o - D M T ~ - ~ G ~ ~ ~ - ~ T - ~ ~ ~ - N H - T P B
Using 5' -0-DMTr-dT-suc-NH-TPB (201 mg, 124 pol)
synthesized in Example 2 and the compound (266 mg, 250 pol)
synthesized in Example 11, and in the same manner as in Example
15-(I), the title compound (303 mg) was obtained as a viscous
5 solid quantitatively.
(2 ) Synthesis of 5, - O - D M T ~ - ~ G ~ ~ ~ - ~ G ~ ~ ~ - ~ T - S U C - N H - T P B
Under an argon atmosphere, using the compound synthesized
in Example 16-(1) (303 mg, 124 pol) and the compound
synthesized in Example 11 (265 mg, 249 pol), and in the same
lo manner as in Example 15- (1) , the title compound (400.0 mg) was
obtained as a viscous solid quantitatively.
(3) Synthesis of deoxyguanosinyl-[3f+5f]-de~~yguanosinyl-
[3' +5' ] -deoxythymidine (5' -d [GGT] -3' )
Using the compound synthesized in Example 16-(2) (23.8 mg,
15 7.37 mmol) and in the same manner as in Example 15-(3), the
title compound was obtained.
m/z(ESI-MS) : Anal.Calc. for C30H38N12017P29:0 0.20. Found
20 Example 17 : Synthesis of deoxycytidinyl- [3' +5' ] -
deoxycytidinyl- [3 +5 'l-deoxythymidine (5' -d [CCT] -3' )
(1) Synthesis of 5' - O - D M T ~ - ~ C ~ ~ ~ - ~ T - S U C - N H - T P B
Under an argon atmosphere, using 5'-0-DMTr-dT-suc-NH-TPB
(203 mg, 125 pol) synthesized in Example 2 and the compound
25 synthesized in Example 3 (260 mg, 254 pol), and in the same
manner as in Example 15-(I), the title compound (366.9 mg) was
obtained as a viscous solid quantitatively.
(2 ) Synthesis of 5 ' - O - D M T ~ - ~ C ~ ~ ~ - ~ C ~ ~ ~ - ~ T - S U C - N H - T P B
Under an argon atmosphere, using the compound synthesized
30 in Example 17-(1) (367 mg, 125 pol) and the compound
synthesized in Example 3 (262 mg, 256 pol), and in the same
manner as in Example 15- (I), the title compound (394 mg) was
obtained as a viscous solid quantitatively.
(3) Synthesis of deoxycytidinyl- [3' +5 'l-deoxycytidinylUsing
the compound synthesized in Example 17-(2) (20.8 mg,
6.59 mrnol) and in the same manner as in Example 13-(3), the
title compound was obtained.
m/z(ESI-MS): Anal.Calc. for C28H38N8017P28:2 0.2. Found 819.1(M-
5 H)-
[0398]
Example 18: Synthesis of ~ ~ - o - D M T ~ - ~ T " ~ ~ ~ - ~ T ~ ~ ~ lo (1) Under an argon atmosphere, 5'-O-DMTr-dT-suc-NH-TPB (208 mg,
128 pol) synthesized in Example 2 was dissolved in a mixed
solvent of anhydrous heptane (650 p1) and anhydrous toluene
(650 pl), trifluoroacetic acid (26.0 p1, 350 pol) and 1Hpyrrole
(17.7 p1, 256 pol) were added, and the mixture was
15 stirred for 5 min. The completion of the deprotection was
confirmed by thin layer chromatography, pyridine (28.3 p1, 350
pol) and N-methylimidazole (13.9 p1, 175 pol) were added, and
the mixture was stirred for 5 min. To the reaction mixture
after neutralization was added the compound synthesized in
20 Example 9 (273 mg, 256 pol) dissolved in 0.25 mol/l 5-
(benzy1thio)-1H-tetrazole/acetonitrile solution (1.0 ml), and
the mixture was stirred for 10 min. 5.78 mol/l tert-Butyl
hydroperoxide/nonane solution (383 pol, 66.3 p1) was added and
the mixture was stirred for 5 min. The reaction mixture after
25 completion of the reaction was partitioned by adding heptane
(3.0 ml), the lower layer was extracted, washed with a mixed
solution of acetonitrile (1.0 ml) and water (80 p1) , and the
obtained organic layer was concentrated under reduced pressure
to give a compound same as that synthesized in Example 14-(1)
30 (291 mg, 98.8%) as a viscous solid.
(2) By repeating a similar operation 18 times, the title
compound (1.67 g, 89.6%) was obtained as an orange solid.
[0399]
Example 19: Synthesis of 5' -0-( 4,4' -dimethoxytrityl)- N~-(2 -
35 ethyl-l-hexanoyl)- 2 ' -deoxycytidine-3' -0-[ (2-cyanoethyl)- N,N -
diis opropylphosphoramidit el [5 ' -0-DMTr-dC '2Et-Hex' - PA]
Under an argon atmosphere, 5' -0- (4,4' -dimethoxytrityl) -
2' -deoxycytidine-3' -0-[ (2-cyanoethyl)- N,N -
diisopropylphosphoramidite] (20.4 g, 28.0 mmol) was dissolved
5 in tetrahydrofuran (200 ml) , N, N-diisopropylethylamine (7.4 g,
57.0 mmol) and 2-ethyl-l-hexanoic anhydride (11.4 g, 42.0 mmol)
were added, and the mixture was stirred at room temperature for
3 days. To the reaction mixture after completion of the
reaction were added ethyl acetate and water, and the mixture
10 was extracted. The ethyl acetate layer was washed with 5%
aqueous sodium hydrogen carbonate solution and saturated brine.
The organic layer was dried over anhydrous sodium sulfate, the
solvent was evaporated under reduced pressure, and the obtained
crude product was purified by silica gel column chromatography
15 (eluate; 1% triethylamine-containing 10:1+1:1 heptane-ethyl
acetate) to give the title compound (15.9 g, 66.5%).
[0400]
Example 20: Synthesis of 5' -0-( 4,4' -dimethoxytrityl)- N~-(3 ,5,5-
trimethyl-l-hexanoyl)-2'-deoxycytidine-3~-0-[(2-cyanoethyl)-
20 N,N -diisopropylphosphoramidite] [5' - ~ - D M T ~ - ~ c ' ~ 'P~A]' ~ - ~ ~ ~ ~ ~ ~ (1) Synthesis of N ~(-3 ,5,5-trimethyl-l-hexanoyl)- 2'-
deoxycyt idine
Suspending 2'-Deoxycytidine (2.3 g, 10.0 mmol) in dry
pyridine, followed by concentration under reduced pressure was
25 repeated 3 times to perform dehydrative azeotropic distillation,
the concentrate was suspended again in dry pyridine (60 ml),
and trimethylsilyl chloride (6.4 ml, 50 mmol) was added
dropwise over 5 min. To the reaction mixture was added 3,5,5-
trimethyl-l-hexanoyl chloride (9.5 ml, 50.0 mmol) over 5 min.
30 After completion of the reaction, under ice-cooling, aqueous
ammonia (25 ml) was added and the mixture was reacted for 20
min. The reaction mixture was concentrated under reduced
pressure. Water (150 ml) was added to the concentrate, and the
mixture was extracted 3 times with ethyl acetate (100 ml). The
35 organic layer was concentrated and the obtained oil was
purified by silica gel column chromatography to give the title
compound (4.7 g).
(2) Synthesis of 5' -0- (4,4' -dimethoxytrityl)- N~-(3 ,5,5-
trimethyl-l-hexanoy1)-2'-deoxycytidine
Dissolving the compound obtained in Example 20-(1) (4.7
g) in dry pyridine, followed by concentration under reduced
pressure was repeated 3 times to perform dehydrative azeotropic
distillation, and the concentrate was dissolved in dry pyridine
(40 ml) . 4,4'-Dimethoxytrityl chloride (3.8 g, 11.0 mmol) was
10 added and the mixture was stirred for 30 min. Water (100 ml)
was added to the reaction mixture and the mixture was extracted
3 times with ethyl acetate (100 ml). The organic layer was
further washed with water and concentrated under reduced
pressure, and the obtained oil was purified by silica gel
15 column chromatography to give the title compound (5.2 g, 77.3%).
(3) Synthesis of 5'-0- (4,4'-dimethoxytrityl)- N~-(3 ,5,5-
trimethyl-l-hexanoyl)-2'-deoxycytidine-3~-0-[(2-cyanoethyl)-
N, N-diisopropylphosphoramidite] [5' - o - D M T ~ - ~ c ' ~ ' ~P'A]~ - ~ ~ ~ ~ ~ ~ ' -
Dissolving the compound obtained in Example 20-(2) in dry
20 acetonitrile (5 ml), followed by concentration under reduced
pressure was repeated 3 times to perform dehydrative azeotropic
distillation and the concentrate was dissolved in dry
dichloromethane (30 ml) . N,N-diisopropylethylamine (5.4 ml,
30.9 mmol) was added dropwise over 5 min, and a solution of
25 chlor0-2-cyan0ethyl-N,N-diisopropylphosphoramidite (2.2 ml, 9.7
mmol) in dichloromethane (30 ml) was added dropwise over 15 min.
After reaction at room temperature for 30 min, the reaction
mixture was concentrated under reduced pressure, and the
obtained oil was purified by silica gel column chromatography
30 (dichloromethane/methanol/triethylamine = 94 /3/3) . The
fractions containing the object product were concentrated to
dryness to give the title compound (3.7 g, 55.3%) as a white
solid.
35 Example 21: Synthesis of 5' -0- (4,4' -dimethoxytrityl) -N'-
(2,2,4,8,10,10-hexamethy1-5-dodecanoy1)-2'-deoxycytidine-3'-0-
[ (2-cyanoethyl)- N,N -diisopropylphosphoramidite] [5' -0-DMTrdC
(Me 6Dodecanoyl) -PA1
According to the method described in Example 19, the
5 title compound (16.5 g, 47.3%) was prepared from 5'-0- (4,4'-
dimethoxytrityl) -2 ' -deoxycytidine-3' -0- [ (2-cyanoethyl) -N, Ndiisopropylphosphoramidite]
(25.6 g, 35.1 mmol) using
2,2,4,8,10,10-hexamethyl-5-dodecanoyl chloride (16.5 g, 54.5
mmol) as an acylating agent.
Example 22: Synthesis of 5'-0- (4,4' -dimethoxytrityl)- N~-(2 -
diisopropylphosphoramidite] [5' - o - D M T ~ - ~ c '- P~A]- ~ ~ ~ ~ ~ ~ ~ According to the method described in Example 19, 5'-0-
N, N-diisopropylphosphoramidite] (1.8 g, 2.5 mmol) was reacted
with 2-heptyl-1-undecanoic anhydride (2.1 g, 3.8 mmol) as an
acylating agent. Since the reaction proceeds slowly, 2-heptyl-
1-undecanoyl chloride (1.2 g, 3.8 mmol) was added again as an
20 acylating agent, and the mixture was reacted for 30 min. After
completion of the reaction, the mixture was extracted with
chloroform, and the organic layer was washed with 5% aqueous
sodium hydrogen carbonate solution, concentrated and purified
by silica gel column chromatography to prepare the title
25 compound (1.7 g, 67.5%).
Example 23 : Synthesis of 5' -0-( 4,4' -dimethoxytrityl)- N~-(2 -
diisopropylphosphoramidite] [ 5' -0-DMTr-dc '2-HexDecan0y1) - PA]
According to the method described in Example 19, the
title compound (12.6 g, 47.0%) was prepared from 5'-0-(4,4'-
dimethoxytrityl) -2' -deoxycytidine-3' -0- [ (2-cyanoethyl) -N, Ndiisopropylphosphoramidite]
(20.2 g, 28.0 mmol), using 2-hexyl-
1-decanoylchloride (11.4 g, 41.0 mmol) as an acylating agent, .
Example 24 : Synthesis of 5' -0- (4,4' -dimethoxytrityl) - ~ ~ - n -
diisopropylphosphoramidite] [5' -O-DMT~-~C~~'-PA]
According to the method described in Example 19, the
5 title compound (1.3 g, 49.2%) was prepared from 5'-0-(4,4'-
dimethoxytrityl) -2' -deoxycytidine-3' -0-[ (2-cyanoethyl)- N,N -
diisopropylphosphoramidite] (2.0 g, 2.7 mrnol), using the
corresponding tetradecanoyl chloride (751 mg, 3.0 mmol) as an
acylating agent.
Example 25 : Synthesis of 5' -0- (4,4' -dimethoxytrityl) -N~-
[bis(3,7-dimethyl-octyl)amino-methylene]-2'-deoxyguanosine-3'-
0- [ (2-cyanoethyl)- N,N-diisopropylphosphoramidite] [5' -0-DMTrdG
(N, N-Cit2-methylene) -PA1
Suspending 5' -0- (4,4' -dimethoxytrityl) -2' -deoxyguanosine-
3' -0-[ (2-cyanoethyl)- N,N -diisopropylphosphoramidite] (1.0 g,
1.3 mmol) obtained in Preparation Example 22 in dry pyridine,
followed by concentration under reduced pressure was repeated
twice, the concentrate was further dissolved in toluene,
20 concentrated under reduced pressure, and subjected to
dehydrative azeotropic distillation. Thereafter, in dry
methanol (2.6 ml), the concentrate was reacted with the
compound synthesized in Preparation Example 23 overnight and
concentrated under reduced pressure, and the obtained oil was
25 purified by silica gel column chromatography to give the title
compound (184 mg, 13.1%) .
Example 2 6 : Synthesis of dABZ-d~BZ-dABZ-d~-d~ibU-d~BZ-dABZ-d~-d~-
30 (1) Synthesis of dT-dT-suc-NH-TPB
Under an argon atmosphere, 5'-0-DMTr-dT-suc-NH-TPB (225
mg, 139 pol) was dissolved in anhydrous cyclopentyl methyl
ether (1.0 ml) , and 1H-pyrrole (9.6 p1, 139 pol) was added.
Furthermore, 20 p1 of a solution (2.0 ml) of
35 trif luoromethanesulfonic acid (24.4 p1, 2.78 pol) in
cyclopentyl methyl ether, prepared separately, was added, and
the mixture was stirred at room temperature for 10 min. The
completion of the deprotection was confirmed by thin layer
chromatography, and the mixture was neutralized with a solution
5 (14.0 p1, 176 pol) of 0.2 mol/l N-methylimidazole in
cyclopentyl methyl ether. dT-CE Phosphoramidite (5'-0-(4,4'-
dimethoxytrityl) deoxythymidine-3' - [O- (2-cyanoethyl)- (N,Ndiisopropyl)]-
phosphoramidite) (207 mg, 278 pol) dissolved in
0.3 mol/l 4,5-dicyanoimidazole/acetonitrile solution was added
10 and the mixture was stirred for 10 min. Thereafter, pH 6.8
phosphoric acid buffer (1.0 ml), aqueous hydrogen peroxide (47
1 , 417 pol) and potassium iodide (23.1 mg, 139 pol) were
added to the reaction mixture, and the mixture was stirred for
15 min. The reaction mixture was washed with 10% aqueous
15 sodium thiosulfate solution. To the obtained organic layer
were added 1H-pyrrole (9.6 p1, 139 pol) and trifluoroacetic
acid (206 p1, 2.8 rnmol) and the mixture was stirred for 10 min.
The organic layer was washed with 10% aqueous potassium
hydrogen sulfate solution, 10% aqueous sodium hydrogen
20 carbonate solution and brine. The obtained organic layer was
concentrated under reduced pressure to give the title compound
(316 mg) as a viscous solid quantitatively.
(2) Svnthesis of ~A~'-~T-~T-SUC-NH-TPB
The compound obtained in Example 26- (1) (316 mg) was
25 dissolved in dichloromethane (2.0 ml), dA-CE phosphoramidite
(5' -0-( 4,4' -dimethoxytrityl)- ~~-benzo~l--2de'0 x~adenosine-3-~
[O- (2-cyanoethyl)- (N,N -diisopropyl) ] -phosphoramidite) (239 mg,
278 pol) dissolve in 0.3 mol/l 4,5-
dicyanoimidazole/acetonitrile solution was added, and the
30 mixture was stirred for 10 min. Thereafter, pH 6.8 phosphoric
acid buffer (1.0 ml), potassium iodide (20.0 mg, 97.4 pol) and
aqueous hydrogen peroxide (16.0 p1, 139 pol) were added to the
reaction mixture, and the mixture was stirred for 15 min. The
reaction mixture was washed with 10% aqueous sodium thiosulfate
35 solution. To the obtained organic layer were added 1H-pyrrole
(9.6 p1, 139 pol) and trifluoroacetic acid (206 p1, 2.8 mmol)
and the mixture was stirred for 10 min. The organic layer was
washed with 10% aqueous potassium hydrogen sulfate solution,
10% aqueous sodium hydrogen carbonate solution and brine. The
5 obtained organic layer was concentrated under reduced pressure
to give the title compound (442 mg) as a viscous solid
quantitatively.
( 3 ) Synthesis of dABZ-d~BZ-dABZ-d~-d~ibU-d~BZ-dABZ-d~-d~-suc-~~-~~~
The title compound was synthesized by repeating the
10 method described in Example 26-(2).
Example 27: Synthesis of ~ ' - O - D M T ~ - ~ C-'dC~ (2~~~t--H~~-Xd)~T -~s)uc -NHTPB
(1) Svnthesis of 5' - o - D M T ~ - ~ c '-d~T~-s~u-c-~N~H-~T)PB
Using 5' -0-DMTr-dT-suc-NH-TPB (207 mg, 127 pol) and the
phosphoramidite monomer synthesized in Example 19 (336 mg, 393
pol), and in the same manner as in Example 15-(I), the title
compound (226 mg, 84.9%) was obtained as a viscous solid.
(2) Svnthesis of 5 ' -0-DMT~-dc'2 Et-Hex)- d ~ ( ~-dT~-s~uc--N~H-T~PB~ ) --
Under conditions similar to those of Example 27-(I),
cytidine derivative was elongated by further using the
phosphoramidite monomer synthesized in Example 19 to give the
title compound (249 mg, 90.0%) .
thin layer chromatography: Rf 0.18 (eluent = ethyl acetate)
25 [0408]
Example 28 : Synthesis of 5' - ~ - D M T ~ - ~ c ' ~-d'C~ (3'' 5' 5~--~e~3~~e-x~ ) ~ ~ ~ )
dC (3' 5' 5-Me3Hex) -dC (3 5' 5-Me3Hex) -dT-SUC-NH-TPB
Based on the method described in Example 27, the title
compound (314 mg, 72.2%) was obtained from 5' -0-DMTr-dT-suc-NH-
30 TPB (198.6 mg, 122 pol) by repeating elongation of cytidine
derivative 4 times using the phosphoramidite monomer described
in Example 20.
thin layer chromatography: Rf0.21 (eluent =
toluene/acetonitrile = 9/l(v/v))
35 [0409]
Example 2 9 : Synthesis of 5' - o - D M T ~ - ~ c (- d~~-(~ -~ ~ ~ ~ ~ ~ ~ ~ TPB
Based on the method described in Example 27, the title
compound (210 mg, 49.1%) was obtained from 5'-0-DMTr-dT-suc-NHTPB
(203 mg, 125 pol) by repeating elongation of cytidine
derivative 9 times using the phosphoramidite monomer described
in Example 22.
10 thin layer chromatography: Rf 0.13 (eluent = ethyl acetate)
Example 30 : Synthesis of 5' - o - D M T ~ - ~ c ~ ~ ' - ~ c ~ ~ ~ - ~ c ~ ~ ' - ~ c ~ ~ ~ NH-TPB
Based on the method described in Example 27, the title
15 compound (199 mg, 82.2%) was obtained from 5'-0-DMTr-dT-suc-NHTPB
(102 mg, 63.1 pol) by repeating elongation of cytidine
derivative 4 times using the-phosphoramidite monomer described
in Example 24.
thin layer chromatography: Rf 0.14 (eluent = ethyl acetate)
Example 31 : Synthesis of 5' - o - D M T ~ - ~ c- d(C (M~e6 D~ode~cano~yl)~- ~ ~ ~ ~ ~ Based on the method described in Example 27, 5'-0-DMTr-
25 ~ c ~ ~ ~ - ~ c ~ ~ ~ - ~ c ~ ~ ~ - ~ c ~( 5~3~ 4 -mg~,T 9-7s .9u%) c w-aNs Hob-tTaiPneBd
from 5' -0-DMTr-dT-suc-NH-TPB (210 mg, 129 pol) by repeating
elongation of cytidine derivative 4 times using ~ ' - o - D M T ~ - ~ c ~ ~ ~ -
PA. Furthermore, based on the method described in Example 27,
the title compound (598 mg, 64.6%) was obtained by repeating
30 elongation of cytidine derivative 5 times using 5'-0-DMTrdC(
Me6Dodecanoyl) -PA described in Example 21.
[0412]
Example 32: Synthesis of 2'-deoxycytidine-[3'+5']-2'-
deoxycytidine- [3'+5' ] -2' -deoxycytidine- [3'+5' ] -2' -
35 deoxycytidine- [3' +5' ] -2' -deoxycytidine- [3' +5' ] -deoxycytidine[
3'+5' ] -2'-deoxycytidine- [3'+5' ] -2'-deoxycytidine- [3'+5' ] -2'-
deoxycytidine- [3' +5' ] -2' -deoxythymidine [5' -d (CCCCCCCCCT)- 3' ]
To the compound synthesized in Example 31 (20.0 mg, 2.8
pol) were added 40% aqueous methylamine solution (2.0 ml) and
5 28% aqueous ammonia (2.0 ml), and the mixture was reacted in an
autoclave at 65OC for 1 hr. The reaction mixture was
concentrated with a rotary evaporator under reduced pressure,
adsorbed on C-18 reversed-phase cartridge column, and washed
with 0.1 mol/l aqueous ammonium acetate solution. The
10 dimethoxytrityl group bonded to the 5'-terminal hydroxyl group
was removed with aqueous 2% trifluoroacetic acid solution, and
the mixture was eluted with 20% aqueous acetonitrile solution
to give the title compound.
IEX-HPLC (DNA Pac PA200(4~250 mm)), flow rate 1 ml/min, eluent
15 A 20 mM Tris-HC1 (pH 7.5)' eluent B 400 mM NaC104/20 mM Tris-
HC1 (pH 7.5)' gradient 20% to 70% for 30 min, h=260 nm: RT =
6.68 min (94.0 area%)
MALDI-TOF/MS : 28 43.87 [M-HI -
[0413]
20 Example 33: Synthesis of 5'-0- (4'4'-dimethoxytrityl) -2'-
deoxythymidin-3' -yl-N- [2 3 4-tris (2Jdihydrophytyloxy)
benzhydryl]succinamate
Using a triethylamine salt (3.52 g, 4.63 rnmol) of 5'-0-
(4,4'-dimethoxytrityl)-2'-deoxythymidine-3'-O-succinate and
25 2'3'4-tris (2'3-dihydrophytyloxy) benzhydryl amine (2.92 g, 2.73
mmol) obtained in Preparation Example 24, and in the same
manner as in Example 1 -(2), a filtrate of the reaction
solution was obtained. Said filtrate was concentrated under
reduced pressure and the obtained oil was purified by silica
30 gel column chromatography (hexane:ethyl acetate = 75:25 -
50/50(v/v), containing 3% triethylamine) to give the title
compound (3.60 g, 77.6%) as an oil.
[0414]
Example 34: Synthesis of 5'-0- (4'4'-dimethoxytrityl) -2'-
35 deoxythymidin-3' -yl-N- [4,4' -bis (2'3-
dihydrophytyloxy)benzhydryl]succinamate
Using a triethylamine salt (2.88 g, 3.79 mmol) of 5'-0-
(4'4' -dimethoxytrityl) -2' -deoxythymidine- 0-succinate and
4'4' -bis (2'3-dihydrophytyloxy) benzhydryl amine (1.73 g, 2.23
5 mmol) obtained in Preparation Example 25, and in the same
manner as in Example 1 -(2), a filtrate of the reaction
solution was obtained. Said filtrate was concentrated under
reduced pressure and the obtained oil was purified by silica
gel column chromatography (hexane:ethyl acetate = 100:O -
lo 30/70 (v/v) , containing 3% triethylamine) to give the title
compound (1.87 g, 59.8%) as an oil.
[0415]
Example 35: Synthesis of 5' -0- (4'4' -dimethoxytrityl) -2' -
deoxythymidin-3' -yl-N- [3,5-bis (2'3-
15 dihydrophytyloxy) benzyl] succinamate
Using a triethylamine salt (2.84 g, 3.74 mmol) of 5'-0-
(4'4 ' -dimethoxytrityl) -2' -deoxythymidine- 0-succinate and
3'5-bis (2'3-dihydrophytyloxy) benzyl amine (1.54 g, 2.20 mol)
obtained in Preparation Example 26, and in the same manner as
20 in Example 1 -(2), a filtrate of the reaction solution was
obtained. Said filtrate was concentrated under reduced
pressure and the obtained oil was purified by silica gel column
chromatography (hexane:ethyl acetate = 90:10 - 20/80(v/v),
containing 1% triethylamine) to give the title compound (0.97 g,
25 33%) as an oil.
Example 36: Synthesis of 2' -deoxythymidinyl- [3'+5' ] -2' -
deoxythymidine (5'-d[TT] -3' ) using 5' -0- (4'4'-dimethoxytrityl) -
30 dihydrophytyloxy)benzhydryl]succinamate
(1) Synthesis of 5' -0- (4'4' -dimethoxytrityl) -2' -deoxythymidine-
3' - [0- (2-cyanoethyl) ] phosphoryl-2' -deoxythymidin-3' -yl-N-
[2,3,4-tris (2'3-dihydrophytyloxy) benzhydryl] succinamate
Using the compound synthesized in Example 33 (199.0 mg,
35 117.1 pol) and a dT-CE phosphoramidite reagent (5'-0-(4'4'-
dimethoxytrityl) deoxythymidine-3' - [O- (2-cyanoethyl)- (N,N -
diisopropyl) ] -phosphoramidite) (223.8 mg, O. 30 mmol) , and in
the same manner as in Example 18-(I), the title compound (213.8
mg, 88.1%) was obtained as a viscous oil.
5 (2 ) Synthesis of 2' -deoxythymidinyl- [3' +5' ] -2' -deoxythymidine
(5' -d [TT] -3' )
[0417]
[0418]
10 To the compound synthesized in Example 36-(1) (about 50
mg) was added a mixture (5.0 ml) of 28% aqueous ammonia
solution:40% aqueous methylamine solution = l:l(v/v), and the
mixture was incubated at room temperature for 1 hr. The
reaction mixture was concentrated in a rotary evaporator, the
15 obtained crude product was adsorbed to C-18 reversed-phase
cartridge column and washed with 0.1 mol/l aqueous ammonium
acetate solution. The dimethoxytrityl group bonded to the 5'-
terminal hydroxyl group was removed with aqueous 2%
trifluoroacetic acid solution, and the mixture was eluted with
20 20% aqueous acetonitrile solution to give the title compound.
m/z (ESI-MS): Anal. Calc. for C20H27N4012P5:4 6.12 Found 545.1 (MH)
-
Example 37 : Synthesis of 2' -methoxyuridinyl- [3' +5' ] -2' -
25 deoxythymidine (5'-U(M)dT-3') using 5'-0-(4'4'-
dimethoxytrityl) -2' -deoxythymidin-3' -yl-N- [2,3,4-tris (2'3-
dihydrophytyloxy) benzhydryl ] succinamate
[0420]
(1) Synthesis of 5' -0- (4'4' -dimethoxytrityl) -2' -methoxyuridine-
5 3' - [O- (2-cyanoethyl) ] phosphoryl-2 ' -deoxythymidin-3' -yl-N-
[2,3,4-tris(2,3-dihydrophytyloxy)benzhydryl]succinamate
Using the compound synthesized in Example 33 (200.4 mg,
117.9 pol) and a 2'-OMe-U-CE phosphoramidite reagent (5'-0-
lo (N,N-diisopropyl)] - phosphoramidite) (222.3 mg, 0.30 mmol) , and
in the same manner as in Example 18-(I), the title compound
(219.1 mg, 89.2%) was obtained as a viscous oil.
[0421]
(2) Synthesis of 2'-methoxyuridinyl-[3'+5']-2'-deoxythymidine
15 (5' -U (MI dT-3' I
[0423]
Using the compound synthesized in Example 37-(1) (about
20 50 mg) and in the same manner as in Example 36-(2)' the title
compound was obtained.
m/z (ESI-MS): Anal. Calc. for C20H27N4013P5:6 2.13 Found 561.1 (MH)
-
[0424]
25 Example 38 : Synthesis of 2' -deoxythymidinyl- [3'+5' ] -2' -
deoxythymidine (5' -d [TT] -3' ) using 5' -0- (4'4' -dimethoxytrityl) -
(1) Synthesis of 5' -0- (4'4' -dimethoxytrityl) -2' -deoxythymidine-
Using the compound synthesized in Example 34 (200.1 mg,
0.14 mmol) and a 2' -dT-CE phosphoramidite reagent (5' -0- (4'4' -
dimethoxytrityl) -2' -deoxythymidine-3' - [o- (2-cyanoethyl)- (N,N -
10 diisopropyl)]-phosphoramidite) (267.3 mg, 0.36 mmol), and in
the same manner as in Example 18-(I), the title compound (154.3
mg, 61.0%) was obtained as a viscous oil.
(2 ) Synthesis of 2' -deoxythymidinyl- [3' +5' ] -2' mdeoxythymidine
15 (5' -d [TT] -3' )
[0428]
Using the compound synthesized in Example 38-(1) (about
20 50 mg) and in the same manner as in Example 36-(2)' the title
compound was obtained.
m/z (ESI-MS): Anal. Calc. for C20H27N4012P5:4 6.12 Found 545.1 (M-
2s Example 39: Synthesis of 2' -methoxyuridinyl- [3'+5' ] -2' -
deoxythymidine (5'-U(M)dT-3') using 5'-0-(4'4'-
dimethoxytrityl) -2' -deoxythymidin-3'-yl-N- [4,4'-bis (2'3-
dihydrophytyloxy) benzhydryl ] succinamat e
[0430]
(1) Synthesis of 5' -0- (4'4' -dimethoxytrityl) -2 ' -methoxyuridinebis
(2'3-dihydrophytyloxy) benzhydryl] succinamate
Using the compound synthesized in Example 34 (201.7 mg,
0.14 mmol) and a 2'-OMe-U-CE phosphoramidite reagent (5'-0-
(4'4 ' -dimethoxytrityl) -2' -methoxyuridine-3' - [O-( 2-cyanoethyl)-
10 (N,N-diisopropyl)] - phosphoramidite) (278.9 mg, 0.37 mmol) , and
in the same manner as in Example 18-(I), the title compound
(137.3 mg, 53.3%) was obtained as a viscous oil.
(2) Synthesis of 2' -methoxyuridinyl- [3'+5' ] -2' ~deoxythymidine
15 (5' -U (M) dT-3' )
[0433]
20 Using the compound synthesized in Example 39-(1) (about
50 mg) and in the same manner as in Example 36-(2)' the title
compound was obtained.
m/z (ESI-MS): Anal.C alc. for C20H27N4013P5:6 2.13 Found 561.1 (MH)
-
25 [0434]
Example 40 : Synthesis of 2' -deoxythymidinyl- [3'+5' ] -2' -
159
deoxythymidine (5'-d[TT] -3' ) using 5'-0- (4'4' -dimethoxytrityl) -
5 (1) Synthesis of 5' -0- (4'4' -dimethoxytrityl) -2' -deoxythymidinebis
(2'3-dihydrophytyloxy) benzyl] succinamate
Using the compound synthesized in Example 35 (196.7 mg,
0.15 mmol) and a 2' -dT-CE phosphoramidite reagent (5 ' -0- (4'4' -
10 dimethoxytrityl) -2' -deoxythymidine-3' - [o- (2-cyanoethyl)- (N,N -
diisopropyl) 1,-phosphoramidite) (288.2 mg, O. 39 mmol) , and in
the same manner as in Example 18- (I), the title compound (76.7
mg, 30.5%) was obtained as a viscous oil.
15 (2) Synthesis of 2' -deoxythymidinyl- [3'+5' ] -2' -deoxythymidine
[0438]
20 Using the compound synthesized in Example 40-(1) (about
50 mg) and in the same manner as in Example 36-(2)' the title
compound was obtained.
m/z (ESI-MS): Anal. Calc. for C20H27N4012P5:4 6.12 Found 545.1 (MH)
-
[0439]
Example 41: Synthesis of 2'-deoxy-2'-fluorouridiny1[3'-+5']-2'-
deoxythymidine (5'-U(F)dT-3') using 5'-0-(4'4'-
dimethoxytrityl) -2' -deoxythymidin-3' -yl-N- [3,4,5-tris (2'3-
[0440]
(1) Synthesis of 5'-0- (4'4'-dimethoxytrityl) -2'-deoxy-2'-
fluorouridine-3'-[0-(2-cyanoethyl)]phosphoryl-2'-deoxythpidin-
3' -yl-N- [3,4,5-tris (2,3-dihydrophytyloxy)b enzyl] succinamate
10 Using the compound synthesized in Example 2 (202.4 mg,
0.13 mmol) and the 2' -F-U-CE phosphoramidite reagent (5' -0-
(4,4 ' -dimethoxytrityl) -2' -deoxy-2 ' -fluorouridine-3' - [O- (2-
cyanoethyl) - (N, N-diisopropyl) ] -phosphoramidte) (291.5 mg, 0.39
mmol) obtained in Preparation Example 27, and in the same
15 manner as in Example 18-(I), the title compound (198.9 mg,
78.8%) was obtained as a viscous oil.
[0441]
(2) Synthesis of 2' -deoxy-2 ' -fluorouridiny1[3' -+5' ] -2' -
deoxvthvmidine (5' -U (F) dT-3' )
[0443]
Using the compound synthesized in Example 41-(1) (about
50 mg) and in the same manner as in Example 36-(2), the title
25 compound was obtained.
m/z (ESI-MS) :Anal. Calc. for C19H24FN40125P5:0 .11 Found 549.1 (M-H)-
Example 42 : Synthesis of 2' -0'4' -C-methylenethymidinyl[3'+5' ] -
2'-deoxythymidine (5'-(LNA)TdT-3') using 5'-0-(4'4'-
5 dimethoxytrityl) -2' -deoxythymidin-3'-yl-N- [3,4,5-tris (2'3-
dihydrophytyloxy)benzyl]succinamate
[0445]
(1) Synthesis of 5'-0- (4'4'-dimethoxytrityl) -2'-0'4'-Cmethylenethymidine-
3' - [O-( 2-cyanoethyl)] p hosphoryl-2' -
10 deoxythymidin-3' -yl-N- [3,4,5-tris (2'3-
dihydrophytyloxy)benzyl]succinamate
Using the compound synthesized in Example 2 (197.8 mg,
0.13 rnrnol) and an LNA-T-CE phosphoramidite reagent (5' -0- (4'4' -
dimethoxytrityl) -2' -0'4' -C-methylenethymidine-3' - [0- (2-
1s cyanoethyl) - (N, N-diisopropyl) ] -phosphoramidite) (250.7 mg, 0.32
mmol), and in the same manner as in Example 18-(I), the title
compound (220.3 mg, 88.3%) was obtained as a viscous oil.
LO4461
(2) Synthesis of 2'-0'4'-C-methylenethymidinyl[3'+5' ] -2'-
20 deoxythymidine (5' - (LNA)T dT-3 ' )
To the compound synthesized in Example 42-(1) (about 50
mg) was added a solution (5.0 ml) of 10% (v/v) tert-butylamine
dissolved in 28% aqueous ammonia solution, and the mixture was
heated to 65OC in an autoclave and reacted overnight. The
5 reaction mixture was concentrated in a rotary evaporator, the
obtained crude product was adsorbed to C-18 reversed-phase
cartridge column, and washed with 0.1 mol/l aqueous ammonium
acetate solution. The dimethoxytrityl group bonded to the 5'-
terminal hydroxyl group was removed with aqueous 2%
10 trifluoroacetic acid solution, and the mixture was eluted with
20% aqueous acetonitrile solution to give the title compound.
m/z(~S1-MS): Anal.Calc. for C21H27N4013P5:7 4.13 Found 573.1(MH)
-
15 Example 43: Synthesis of 5'-0- (4, 4'-dimethoxytrityl) -N'- [4-
(2,3-dihydrophytyloxy)b enzoyl] -2' -0,4' -C-methylenethymidine-3' -
(1) Synthesis of 4- (2,3-dihydrophytyloxy)b enzoic acid
In the same manner as in Preparation Example 20, the
20 title compound was prepared using 2,3-dihydrophytyl bromide and
methyl (4-hydroxy)benzoate described in Preparation Example 2.
'H-NMR(~OOMHZC,D C~') : 6 0.83-0.98 (m, 15H), 1.01-1.92 (m, 24H),
4.01-4.12 (m, 2H), 6.91-6.96 (m, 2H), 8.03-8.08 (m, 2H)
(2) Synthesis of 4-(2,3-dihydrophyty1oxy)benzoyl chloride
25 The benzoic acid compound prepared in the above-mentioned
Example 43-(1) was converted to the corresponding acid chloride
in the same manner as in Example 4-(1) to give an oily compound.
The present compound was directly used for the next step.
(3) Synthesis of 5'-0- (4,4'-dimethoxytrityl) -N'- [4- (2,3-
30 dihydrophytyloxy) benzoyl] -2' -0,4' -C-methylenethymidine-3' -0-
[(2-cyanoethy1)-N,N-diisopropylphosphoramidite]
An operation of dissolving commercially available LNA-TCE
phosphoramidite reagent (5' -0- (4,4' -dimethoxytrityl) -2 ' -
0,4' -C-methylenethymidine-3' - [O-( 2-cyanoethyl)- (N,N -
35 diisopropyl) ] -phosphoramidite) (701.7 mg, O. 91 mmol) in
anhydrous pyridine (5.0 ml), followed by concentration under
reduced pressure was repeated 3 times to perform dehydrative
azeotropic distillation of adhesive water. Thereafter, under
an argon atmosphere, the reaction mixture was dissolved in dry
5 pyridine (8.0 ml) , N, N-diisopropylethylamine (0.17 ml, 1.12
mmol) and acid chloride (0.6 g, 1.36 mmol) prepared in the
above-mentioned Example 42-(2) were added, and the mixture was
stirred at room temperature for 9 hr. Ethyl acetate (50 ml)
and water (10 ml) were added to the reaction mixture to allow
10 layer separation. The organic layer was washed 3 times with
10% aqueous sodium hydrogen carbonate solution and once with
brine (10 ml), and the obtained organic layer was dried over
sodium sulfate. The filtered filtrate was concentrated and the
obtained crude product was purified by silica gel column
15 chromatography (hexane:ethyl acetate = 100:O - 30/70(v/v),
containing 1% triethylamine) . The object fractions were
collected and concentrated, and the concentrated residue was
azeotropically distilled with toluene and dried to solidness to
give the title compound (0.77 g, 65.5%) .
Example 44 : Synthesis of 2' -0'4' -C-methylenethymidinyl[3' -35, ] -
2' -deoxythymidine (5' - (LNA) TdT-3' )
(1) Synthesis of 5' -0- (4'4' -dimethoxytrityl) -2' -0,4' -CUsing
the compound synthesized in Example 2 (200 mg, 0.12
mmol) and the phosphoramidite monomer synthesized in Example 43
30 (362 mg, 0.31 mmol), and in the same manner as in Example 18-
(I), the title compound (295 mg, 99.4%) was synthesized.
(2) Synthesis of 2'-0'4'-C-methylenethymidinyl[3'+5' ] -2' -
deoxythymidine (5' - (LNA) TdT-3' )
The compound (about 20 mg) synthesized in Example 44-(1)
was treated according to a method similar to that in Example 32,
5 the title compound was obtained by deprotection and cutout from
the anchor.
m/z(ESI-MS) : Anal.Calc. for C21H27N4013P5:7 4.13 Found 573.1(M-
10 Comparative Example 1 : Synthesis of 5' -0- (4,4' -
dimethoxytrityl) -2' -deoxythymidine-3' - [0- (2-
cyanoethyl)] phosphoryl-~2-isobutyryl-2-'d eoxyguanosine-3' - [O-
15 cyanoethyl) ] phosphoryl-2' -deoxythymidine-3' - [O- (2-
cyanoethyl) ] phosphoryl-2' -deoxythymidin-3' -yl- [3,4,5-
Under an argon atmosphere, 5' -0- (4,4' -dimethoxytrityl) -
20 hexadecanyloxy) benzyl] succinate (509 mg, 313 pol) was
dissolved in a mixed solvent of anhydrous heptane (3.6 ml) and
anhydrous toluene (3.6 ml), trifluoroacetic acid (279 p1, 3.76
mmol) and 1H-pyrrole (217 p1, 3.13 mmol) were added, and the
mixture was stirred for 15 min. The completion of the
deprotection was confirmed by thin layer chromatography,
acetonitrile (1.2 ml) , pyridine (304 p1, 3.76 mmol) and Nmethylimidazole
(149 p1, 1.88 mmol) were added, and the mixture
5 was stirred for 5 min. To the reaction mixture after
neutralization was added a solution (1.2 ml) of 5'-0- (4,4'-
dimethoxytrityl)-2'-deoxythymidine-3'-0-(2-cyanoethyl-N,Ndiisopropylphosphoramidite)
(466 mg, 626 pol) in acetonitrile,
and the mixture was stirred for 1 hr. 2,6-Lutidine (350 pl),
10 N-methylimidazole (350 p1) and acetic anhydride (350 p1) were
added and the mixture was stirred for 5 min. 1.OM iodine
pyridine/THF/H20 solution (1.25 ml) was added and the mixture
was stirred at room temperature for 10 min. The reaction
mixture after completion of the reaction was partitioned by
15 adding heptane (7.2 ml) and water (480 p1) , the lower layer was
extracted, and the heptane layer was washed with acetonitrile
containing water, and the obtained organic layer was
concentrated under reduced pressure to give 5'-0-(4,4'-
dimethoxytrityl) -2' -deoxythymidine-3' - [O- (2-
20 cyanoethyl) ] phosphoryl-deoxythymidin-3' -yl- [3,4,5-
tris(3,7,11,15-tetramethyl-l-hexadecanyloxy)benzyl]succinate
(620 mg, 99.8%) .
According to the nucleic acid base sequence of the object
product, a similar operation was repeated using the
2s corresponding commercially available phosphoramidite monomer to
synthesize the title compound. When deoxyguanosine was
elongated, the resultant product showed insufficient
liposolubility, and the yield after extraction operation
decreased to 90%. When deoxythymidine was further elongated,
30 it could not be dissolved in a heptane/toluene mixed solvent,
and the yield after reaction and extraction decreased to 85%.
The following Tables show phosphoramidite monomers used in each
stage and the yield thereof.
[0456]
Table 1
Comparative Example 2 : Synthesis of 5' -0- (4'4 ' -
Monomer
5' -0- (4'4' -dimethoxytrityl) -
deoxythymidine-3' -0- (2-
cyanoethyl-N,Ndiisopropylphosphoramidite)
~~-benzo~l--50'-( 4'4' -
dimethoxytrity1)-deoxyadenosine-
3' -0-( 2-cyanoethyl-N,N -
diisopropylphosphoramidite)
~~-benzo~l--50'-( 4'4' -
dimethoxytrity1)-deoxycytidine-
3' -0- ( 2-cyanoethyl-N, Ndiisopropylphosphoramidite)
~~-isobut~r~l-0--5( '4' 4' -
dimethoxytrityl) -deoxyguanosine-
3' -0- ( 2-cyanoethyl-N, Ndiisopropylphosphoramidite)
5' -0- (4'4' -dimethoxytrityl) -
deoxythymidine-3' -0- (2-
cyanoethyl-N,Ndiisopropylphosphoramidite)
dimethoxytrityl) -2' -deoxythymidin-3' -yl-N- (3'4'5-
tris (octadecyloxy)b enzyl) succinamate
product
DMTr-dTT-suc-TPB
DMTr-d (ABZTT-)s uc-
TPB
DMTr-d (c~'A~'TT) -
suc-TPB
DMTr-d (G~~~C~'A~'-T T)
suc-TPB
DMTrd
( T G ~ ~ ~ c ~-~SUAC-~ ~ T T )
TPB
(1) Synthesis of 5' -0- (4'4' -dimethoxytrityl) -2' -deoxythymidine-
Yield
99.8%
99.2%
98.3%
90.3%
85.2%
Under an argon atmosphere, 5' -0- (4'4' -dimethoxytrityl) -
2'-deoxythymidine (5.00 g, 9.18 mmol), succinic anhydride (1.38
g, 13.8 rnmol) and triethylamine (3.85 mL, 27.5 mmol) were
dissolved in dichloromethane (95 mL), and the mixture was
stirred at room temperature for 8 hr. The completion of the
reaction was confirmed by thin layer chromatography, and the
reaction mixture was partitioned and washed three times with
2.OM phosphoric acid-triethylamine buffer (pH 7.50). The
organic layer was evaporated under reduced pressure to give a
5 triethylamine salt (7.02 g, 98%) of 5'-0- (4,4' -
dimethoxytrity1)deoxythymidine-3'-0-succnate as a colorless
frothy solid.
(2) Synthesis of 5' -0- (4,4' -dimethoxytrityl) -2' -deoxythymidin-
3' -yl-N- (3,4,5-tris( octadecyloxy)b enzyl) succinamate
A triethylamine salt (1.45 g, 1.94 mmol) of 5'-0- (4,4'-
dimethoxytrityl)-2'-deoxythymidine-3'-O-succinate and 3,4,5-
tris (octadecyloxy)b enzyl amine (1.02 g, 1.10 mmol) were
dissolved in anhydrous dichloromethane (15 mL) , 2- (1Hbenzotriazol-
1-y1)-1,1,3,3-tetramethyluronium
15 hexafluorophosphate [HBTU] (2.53 g, 6.60 mmol) and N, Ndiisopropylethylamine
(1.17 mL, 6.60 mmol) were added, and the
mixture was stirred at room temperature for 1 hr. The
disappearance of the starting material was confirmed by thin
layer chromatography, and the mixture was washed with saturated
20 aqueous sodium hydrogen carbonate solution and saturated brine.
The organic layer was dried over sodium sulfate and filtered,
and the filtrate was concentrated under reduced pressure.
Methanol was added to the concentrated solution, the mixture
was filtered, and the obtained solid was purified by silica gel
25 column chromatography (dichloromethane/methanol, 1% v/v
triethylamine) to give the title compound (1.22 g, 72.4%) as a
white solid.
[0458]
In the following, Table 2 to Table 13 show the structural
30 formulas and compound data of the compounds produced in
Preparation Examples 1 to 27, Examples 1, 2, 4 to 12, 19 to 25,
33 to 35, 43, and Comparative Example 2.
[0459]
Table 2
Prep.
Ex- 1
Prep.
EXPrep.
Ex. 3
Prep.
Ex. 4
Prep.
EX. 5
>$e*Tdx:d5-3T/-%&>-emyi. -: . ... . . : \ & : ? < r F ~ s $ J H
.I I i
1 ~ - ~ ~ ~ ( 3 0 0 ~ ~ ~ ) : 6 0 . 8 50~- 0, m. 9, ~3 e( 1) , 0 . 9 8 - 1 . 7 0 ( 2 4 ~ , b r , m ,-~- e ~ ~ t - J - [ ~ ~ ~ -HM e13-Cl&CH2-OH) ,3.62-3.75(2H1-Ct&-OH) -y xA>-:.Y -+c-eI"+OH: , ,: - - f p 7 \ ~ + ; r Y p < x B ~
I.
1 ~ - ~ ~ ~ ( 3 0 0 ~ ~ z ) : 6 0 . 7 95- 0~.,9m2,(~1e ) , 0 . 9 5 - 1 . 9 5 ( 2 4 ~ , b r , m-,- ~ e ~ ~ ~ - [ ~ ~ ~ &
-HM e13-Cl&CH2-Br),3.35-3.52(2H,-Ct-i2-Br) ~ ~ " r " " " . .
.
H-NMR(~OOMHZ):~.I0 9-1.43(m,24H), 1,48-1. 66(m15H),3.63-3.70(m,2H)
r‘u‘rmTj8!+ 4?%,,Hl$ .. L F % 4 3 k.. ~ ... B. r
..g,.$f.(=aF:T t. ." : 1
1H -NMR(300MHz):61 .I2 -1.43(m,24H), 1.48-1 .70(m14H),1 .84-1 .90(m,l H),3.36-
49(m ,2H)
A , . 1WHk $a
J'.; ..:
. 0 %7.,$. :
. I:,
'Oqb. pb, * . %J*&+&/<.,J.&J,
2-chloro-5-(2,3-dihydrophytyloxy)benzophenone
H-NMR(30OMHz):60.75-0.90(15 H,m1Me),0.95-1.7 0(24H,b r,Me2CH-[C3tis-CH
e]3-C~CH2-0-Ar),3.82-3.92(2H,brl-O-C~-C1,H3g)168H9,(dl, J=8.3Hz,C3-HJ,7
5-7.80(7H1m,C4,6-H,Ph-H)
2-chloro-5-(2,3-dihydrophytyloxy) benzhydrol
H - ~ ~ ~ ( 3 0 0 ~ ~ z ) : 6 0 . 8 2 - 05~. 9,m0,(M1e )1, .00-1 .90(24H,br,Me2CH-[C3H6-CB
e]~-C~2CH~-O-Ar),3.88-4.00(2H,brI-O-C&-ClHH3g, )sl, 5A.~9-8c(~l o H - ~ ~ ) , ~
5-6.90(1 H,m,C3-H),7.10-7.45(7H1m,C4,6-ti,Ph-H)
I -chloro-I -[(2-chloro-5-(2,3-dihydrophytyloxy)phenyl)phenylmethane
1 H-NMR(300MHz):60.80-0.90(15H,m,Me), 1.00-1 .90(24H,br,Me2Ct-J-[C3H6-CH
e]3-C~2CH~-0-Ar),3.88-4.05(2H,m,-0-C&-C19H3g),648H(,ld ,J=I .~HZ,A~CH
Ph),6.77(1 H1d,J=8.7Hz,C3-H),7.1 0-7.55(7H,m,C4,6-ti,Ph-HJ
I -azido-l-[(2-chloro-5-(2,3'-dihydrophyt)phenyl)phenylmethane
'H-NMR(~OOMHZ):~O.~~-05.~9,m5,M(1e J,0.95-1 .85(24H,br,Me2CH-[C3H6-CH
e]3-CbCH2-O-Ar),3.75-4. 02(2H, m,-0-Ch-Cl 9H39)r5.90-61.0 (1 H, m , A ~ - c ~ ~ N ~ - P
),6.79(~~,d,~=9.0~z,~3-~},7.10-7.50(7~,m,~4,6-l-l,~h-~)
1 -[(2-chloro-5-(2',3'-dihydrophytyloxy)phenyl)]-I -phenylmethanamine ' H-NM ~ ( 3 0~0z):~60 .85-0.95(15~,m,Me),0.95-1.85(24Hb,r ,Me2CH-[C3Hs-CH
e]3-C~CH~-0-Ar),3.85-4.00(2H,brl-O-C~-CHg,Hs),A,r5-C.H4N3H(l2- Ph),6
5(1 H,~,J=~.~Hz,c~-H),0~-7.I. 50(7~,rn,~&6-~,~h-I-J
[0460]
Table 3
Prep.
Ex. 6
Prep.
Ex. 7
Prep.
Ex. 8
Prep.
Ex. 9
Prep.
EX. 10
:,a< q!?:
. .
!?&a? ....
. - ., .
ppw*?:s;. r.;. .u. .LJ..
'~ - ~ ~ ~ ( 3 0 0 ~ ~ ~ ) : 6 0 . 8 6 - 0 . 9 0 ( 2 4I.~ 1,0m-1,. 4M0(e4)8,H ,br,Me2CH-[C3Hl-CUM
e]3-Ct-l&H2-O-Ar),2.03(l H,s,0H),3.90-3.94(4H,m,-O-C&-ClgH39),5.76(1 H,s,Ar-
CHN3-Ph),6.85(4H,m,C3-H),7.20-7.26(4H,m,C4,6-H,Ph-H)
0.
. . . . . . . . . . . . .
.-.
' ~ - ~ ~ ~ ( 3 0 0 ~ ~ z ) : 6 0 . 8~5=(63.63~~z,,t~,e ),0.94(9~,t,~=6.3~z,~e),1.00-2.00(7
2H ,br,m,MeiCH-[C3BE-CHMe]3-C&CH2-0-Ar),3.93-4.07(6H ,m,-Ch-0-Ar),4.60(
2H,s,-O-C&-OH),6.57(2H,s,C2,6-H)
..........
' ~ - ~ ~ ~ ( 3 0 0 ~ ~ z ) : 6 0 . 8 5 ( 3 6 ~ , t , ~ = 6 . 3 ~ z , ~ e ) , 0 . 9 3 ( 9 ~ , t , ~ = 6 . 3 ~ z , ~ e ) , l . 0 0 - 2 . 0 0 ( 7
2H,br,m,Me2C~-[C~~-C~e]~-C~H~-O-Ar),3.79(2Hls,benzyl-)O,3(68 5-4l
~,m,-~l&-0-~r),6.52(2~,s,C2,6-H)-
.r .. ...
'~-~~~(3.00~~z):60.80-0.90(24~,m,~e),0..93(6~,,~=63z,~e),1.00-1.90(48
H, br,m, Me2CH-[C&-CHMe13-Ct&CH2-0-Ar), 3.92-4.02(4H ,m,C19H39-Cl&-0-Ar)
,4.62(2H,slAr-C~-OH),6.38(1H,t,J=2.1Hz,C4-H),6.50(2H,d,J=2.OHz,C2,6-~)
4-(2,3-dihydrophytyloxy) benzaldehyde
' ~ - ~ ~ ~ ( 3 0 0 ~ H ~ ) : 6 0 . 8 2 -20H.,8m9,~(e1) ,0.95(3H,d,~=6.4Hz,~e),1.00-1.95(24
H, rn,MezCH-[C&-CHMe]3-Cl-&CH2-O-Ar),4.03-4. I 3(2H,m,-0-C&-C19H39),4.62
(2~,s,~r-~~-0~),6.99(2~,m,~3,5-~),7.83(2~,mH,,S~,2C,H6O-) H)., 9.88(1
4-(2,3-dihydrophytyloxy)benzyl alcohol
'H-~~~(300~Hz):60.81-0.90(12H,m,Me),0.94(3H,d,~=6.4Hz,~e),1.00-1.90(24
HI m, Me 2Ct-J-[C3t&-CHMeI3-Ct&C H2-0-Ar),3 .94-4. 0 5 ) , 6 . 8 9
(2H,mIC3,5-H),7.28(2H,m,C2,6-ti)
[0461]
[Table 41
Prep.
Ex. 11
Prep.
EX. 12
prep.
EX. 13
H">-T'' oP+~h-F)=-=~-J~-y %y -+ *3'Ak[tap-&- H ~ N ~ ~ - 11 -,l OPhy
4-(2,3-di hydrophyty1oxy)benzyl azide
1~-~~~(300~~z):60.81-0.90(12~,m,~e),0.94(3~l.0d0l-1~.=906(.244 ~zl~e)ll
HlmlMe~C~-[C~~E-C~Me]3-C&CH2-O-Ar),3.94-4.04(2H,m,-0-C~-ClsH3s)l4.26
(2H,s,Ar-C~-N3),6.90(2H,d,J=8.6Hz,C3,5-~),7.23(2H1d1J=8.6Hz,C2,6-~
4-(2,3-dihydrophytyloxy)benzylamine
1 ~ - ~ ~ ~ ( 3 0 0 ~ ~ z ) :26~0,.~t8,= 66(.10 ~ z , ~ e ) 0 . 9 4 (J3=~~.,~dH z,M~).,0I0 -1.90(
24H,m,Me2C~-[C3~E-C~Me]3-C~CH2-O-Ar)1338O(2H,s,Ar-C~-NH2),3.92-4.04(
2H,rn,-O-C~-ClsH3s),6.87(2H,d,J=8.6Hz,C (2H,d,J=8.6Hz1C2,6-HJ
* QMe 0 OMe QMe OMe
Hf% -k+\b-* H\q\- H2?7k Gphy
%&OH OP~Y- -4 ~ h y ,++.
2-methoxy-4-(2,3-di hydrophytyloxy)benzaldoxime
'H-NMR(300MHz):60.82-0.92(12H,m,Me),0.95(3H,dlJ=6.4Hz,.0M0e-)1,.19 5(24
Hlm,Me2C~-[C3~E-C~Me]3-C&CH2-O-Ar)I3.83(3H~l397-4.10(2H,m,-OC~-
ClsH39),6.44(1H ,d1J=2.2Hz,C3-HJ,6.49(1H ,dd,J=2.2,8.6H~,C5-HJ~175, (1H ,s
,-CHN0HJ17.62(1H ,d1J=8.6Hz,C6-HJ,8,41( IH ,s,-CHNOH)
2-methoxy-4-(2,3-dihydrophytyloxy)benzylamine
'~-~~~(300~~z):60.80-0.90(12~,m,~e),0.94(3~,d,~=6.4~z,~e),1.00-1.90(24
H,m,Me2C~-[C3~~-C~Me]3-C~CH2-0-Ar),3.74(2Hls1Ar-C~-NH2)l3.82(3HlslO
Me),3.90-4.05(2H,m,-0-Cl&-C1sH39)H1ld6d6,4J2=(2l.3 ,8.I Hz,C5-HJ,6.46(1 H,d,J
~.2 .H1z 1*C3-HJ,7.09(1 H,d, Jz8.1 Hz,C6-HJ - ..3& __IC Me~s _e_)_
/ QH -1 cri3 4* QPhy UPhy
methyl 4-h ydroxy-2-methyl benzoate
H-NMR(300MHz):62.57(3H,s,C2-Me)l3386(3H,sl-COOl5.68(l H ,s, br,-OHJ16
.66-6.72(2H,m1C3,5-~,7.89(H1, ddIJ=2.4,6.9Hz,C6-HJ
methyl 4-(2,3-di hydrophyty1oxy)-2-methyl benzoate
H-NMR(300MHz):60.85(12H,t,J=6.6Hz,Me),0.94(3H,d,J=6.6Hzl~e), I .00-1.90(
24H,m,Me2C~-[C~~-C~Me]~-C~CH2-O-Ar),2.59(3H,s,C2-~,3.85(3HIsI-CO
O~),4.02(2H,dt,J=3.0,6.7HzIO-C~-C19H39)l6.65-6.76(2HlmlC3l5-~)l7.85-7.96
(1 H,mlC6-I-l)
4-(2,3-dihydrophyty1oxy)-2-methylbenzyl alcohol
'H-NMR(300MHz):60.86(12H,t1J=6.3Hz,~e),0.94(3H,d,J=6.4Hz,~e)I, .0 0-1.90(
24H,m,Me2C~-[C3~-C~Me]3-C~CH2-O-Ar),2.36(3H,s,C2-Me),3.98(2H,dt,J=3.
0,6.7Hz,0-C~-C1sH3s),4.63(2H,Jd=, 4.7Hz1Ar-Cl&-OH),6.60-6.77(2H1m,C3,5-l
),7.15-7.25(1 H,rn,C6-H)
[0462]
[Table 51
Prep.
EX. 14
Prep.
Ex. 15
Prep.
Ex. 16
Prep.
EX. l7
H.+h &Lo;;- cr* - : .;.-=$ I " OPhy OPhy ~ ~ 0 ~ h y
4-(2,3-dihydrophyty1oxy)-2-methybl enzyl chloride
'H-NMR(300~Hz):60.86(1 2Hlt,J=6.3Hz,Me),0.93(3H,d,J=6.3Hz,Me),l .00-1.90(
24Hlm,Me2C~-[C3~s-C~Me]3-C~2CH2-0-Ar)l2.40(3Hls,C2-l3.97(2H,dt,J=2.
7,6.7H~,0-Cl&-C~~H~~),4.59(2H,s,Ar-C~-CI),6H.,6d9d,(J =2.4,8.3H~,C5-HJ~6~7
4(1 H,dlJ=2.3HzlC3-HJ,7.21 (1 Hld,J=8.3Hz,C6-HJ
4-(2,3-di hydr0phytyloxy)-2-methyl benzyl azide
1~-~~~(300~~z):60.86(12~,tl~=6.3~z,~e),0.94(.030~-1l.9d0,( ~=6.3~z,~e),l
24H,m,Me~C-[C3~-C~Me]3-Cl&CH2-O-Ar),2.3,3.98(2Hldt,J=3.
0,6.6Hz,0-Cl&-C19H39)J4.28(2HlslAr-C~(1- HN ,3d)dJ16J.=721. 6,8.2Hz,C5-HJ16,
77(1 Hld,J=2.3Hz,C3-H),7. 15(1 H,dlJ=8.3Hz,C6-HJ
4-(2,3-dihydrophyty1oxy)-2-methylbenzylamine
H-NMR(300MHz):60.86(1 2H1t,J=6.3Hz,Me),0.93(3H,d,J=6.6Hz1Me), 1.00-1.90(
24H1m ,Me2C~-[C3~-C~Me]rC~CH2-O-Ar)l2.32(3H,s,C2-Me),3.79(2,sH,A r-C
~-NH~),3.97(2H,dt,J=3.0,6.7H~,0-C~-C~~(IH H~,d~d),J~=62..67,8 .2Hz,C5-HJl
668-6.75(2~,br,~3,5-H),7H.1,d7lJ(=18 .7Hz,C6-l4)
cpf-f ~--+jN H2fl,
'~-~~~(300~~z):60.92-0.99(m,24~.)0,91(.m0J16-H1) ,1. I9 -1 .23(m,4H),4.58(s1
2H),7.21 (d,2HJJ=6Hz),7.43(d,2HJ, =gHz),7.53-7.66( 1H )
H0
fivBr
I
Q--'i"~7N
1~-~~~(300~~z):60.81-02~.)8,19.(0m81-1.13 7 (m11 2H), 1.48-1. 83(ml5H),3.96-
4.02(m,2H),4.62(s,2H)l6689(d,2H,J=9Hz),7.29(d,2H,J=9Hz)
OH
'~-~~~(400~~z,~~~1~):~0.84(d,6~,~=6.6~z)~0.87(d,6~~~=6.6~z)~=6.6Hz),1 .00-1 .44(m121H ),1.47-1.58(mIl H),1.91-1 .99(m11~ ),2.15(ddd,Hl ,J=l5.
0,8.2,2.OHz),2.36(ddd,I HlJ=15.0,5,9,1 .9Hz)
LO4631
[Table 61
Prep.
Ex- 18
Prep.
Ex l9
prep-
EX. 20
Prep.
EX. 21
Prep.
Ex. 22,
Prep.
Ex. 23
CI
1~-~~~(400~~z,~~~l~):60.84(d,6~,~=6.6~z)l~.87(dl6~l~=6.6~=6.6Hz),1.00-1.44(m121H),1.47-1.58(m.1911H-1).,919 (m,lH),2.15(ddd,l H,J=15.
0,8.2,2.0Hz),2.36(ddd11 HIJ=l 5.0,5.9,1 .9Hz)
0-CI
1~-~~~(400~~~,~~~~~):60.84(d,6~~= 6,.~6=~6~ ).,60 .~9z6)( dl,~3.~8, ~7 (dl6~1
=6.6Hz),1.01-1.42(m,20H),1.45-1.57(m,1H),1.93-2.02(m11H),2.18(ddd,1H,J=14.
9,8.1 , I .8Hz),2.37(ddd,1H,J=14.9,5.9,1.8Hz),5.70(d,lH,J=6.OHz),5.71(d,l H,J=6.
OHz)
Hob 0
A 4 v ~2 M a ! of N 1 NH2
n~+oF.N ?k
A
' 1 ~ - ~ ~ ~ ( 4 0 0 ~ ~ z , ~ ~ ~ 1 3 ) : 8 0 . 8 7 ( d , 6 ~ ,
J=6.6Hz),0.95(dl3H,J=6.5Hz),1 .12-I .38
(m,6H), 1 50-1.63(m,3H), 1.82-1 .90(m,
1 H),404-4.1 I (m.1 H),6.93(dI2Hl
J=8.8Hz),8.06(d,2Hl J=8.8Hz)
'H -NMR(~OOMHZ,CDC~~).:~I6 (Om~, ~~-I
12H),2.14-2.35(m,1H),2.42(t,lH,
J=6.5Hz),2.55-2.74(m,2H),3.30-3.40
(m,l H),3.45-3.66(mI4H),3.70-3.87(mI7H),
4. I 0-4.1 4(m, I H),4.52-4.69(mI I H),5.35
(dl I H, J=7.2Hi'),6.27-6.38(mll H),6.79
-6.88(m,4H),7.20-7.35(ml7H),7.35-7.44
(m12H),7.94,8.03(2d,1 HIJ=7.4Hz)
3 1 ~ - ~ ~ ~ ( 1 2 0 ~ ~ ~ , ~ ~1~491.13 ) : 6 1 4 9 . 7 ,
H-NMR(~OOMHZ,CDCI~):.0&4~- 1.19 (m1
12H),2.44-2.61 (mI3H),2.72-2.85(mI1 H),
3.29-3.40(mI2H),3.53-3.90(m11 0H),4.24
(s,l H),4.70-4.73(m11H ),6.02(brsI2H),
6.21 (tll H, J=7.02Hz),6.78(ddl4H,J=2.70,
8.64Hz),7.1 4-7.30(mI7H),7.38-7.41
(m,2H),7.65(dJl H,J=4.05Hz)
31~-~~~(160~~~,~~~13):6149.5,149.3
[0464]
[Table 71
t
Prep.
Ex. 24
Prep.
EX. 25
Prep-
Ex. 26
@ ' HO OH HN,Fm~~ '*m, 'hy0 > @ 6 h Y 0 OPhy
O H
O p h ~ O p h y ~ OPhy
YO OPhy 'hy0 ' Phy
P ~ Y P ~ Y
2,3,4-tris(2,3-dihydrophytyloxy)benzophenone
"H-NMR(~OOMHZ,CDC~~):~.9O5.(~m~,I- II7 H),3.88-3.95(m12H),3.97-4.1I ( mI4H
),6.71 (d,l HIJ=8.7Hz),7. 14(d11H ,J=8.6Hz),7.38-7.44(m,2H),7.50-7.55(m,1 H),7.7
6-7.79(m,2H)
21314-tri~(2,3-dihydrophytyloxy)benzhydrylalcohol
'H-NM~R(400MHz,CDCI3):60.78-1.9 5(m,117H),3.05-3.1 O(m,l H),3.68-3.78(m,l H.
),3.93-4.05(m15H),5.89-5.94(mll H),6.61 (d,l HI J=8.6Hz),6.82-6.86(mll H),7.20-7.
28(m,l H),7.29-7.34(mI2H),7.35-7.39(m,2H)
N-(9-fl~orenylmethoxycarbonyl)-2,3,4-tris(2~3-
dihydrophytyloxy)benzhydrylamine
'H-NMR(400MHzICDC13):60.83-1 .90(m,117H),3.37-3.45(m,1 H),3.92-4.06(m,5H
),4.22-4.28(m1 1 H),4.35-4.42(m, 1 H),4.42-4.50(m, 1 H),5.90-5.98(m, I H),6.0 1 -6.08(
m, I H),6.63(dI I H, J=8.5Hz),6.89-6.94(mI 1 H),7. I 4-7.43(m,9H),7.58-7.65(mI2H),7
.73-7.78(mI2H)
2,3,4-tris(2,3-dihydrophytyloxy)benzhydrylamine
'H-NMR(400MHz,CDC13):60.83-1 .90(m,1 17H),3.77-3.91 (mI2H),3.91 -4,02(m,4H
),5.40(s,1H),6.60(d,1H1J=8.6Hz),6.90(d,1H,J=8.4Hz),7.20-7.37(m,5H)
JJ%J, ,&& PhyO OPhy PhyO OPhy PhyO *A .OPhy
N-(9-fluorenylmethoxycarbonyl)-bis-4-(2,3-
dihydrophytyloxy)benzhydrylamine
'H-NMR(400MHz,CDCI3):60.83-1. 85(m,78~),3.90-4.02(m,4~),4.18-4.25(mH,l ),
4.35-4.44(m12H),5.20-5.30(m,l H),5.83-5.91 (m,l H),6.84(d,4HIJ=8.9Hz),7.1 I (d,4
H, J=8.6Hz),7.24-7.43(m,4H),7.53-7.62(m,2H),7.7O-7.78(ml2H)
4,4'-bis(2,3-dihydrophytyloxy)benzhydrylamine
'H-NMR(400MHz,CDC1~)60.80-0.93(m,30H),1.01-1.8 5(m,52H),3.93-3.98(mI4H)
,5.1 2(s,I H),6.83(d,4H,J=8.7Hz),7.24(dl4H,J=8.9Hz)
c l ~ O p h Y N 3 ~ 0 P h Y H d " P " h ~ '
\ C \ * \ * OPhy OPhy OPhy OPhy
3,5-bis(2,3-dihydrophytyloxy)benzyl chloride
'H-NMR(400MHzICDC13):b0.83-1 .85(m,78H),3.90-4.02(m,4H)14450(s,2H),6.40(d
, I HIJ=2.2Hz),6.51 (d,2H, J=2.2Hz)
3,5- bis(2,3-di hydrophytyloxy) benzyl azide
'H-NMR(400MHz,CDCI3):60.83-1 .85(m,78H),3.90-4.02(m14H)14.50(s12H)16.40-6
.45(m,3H)
3,5-bis(2,3-dihydrophytyloxy)benzyl amine
'H-NMR(400MHz,CDC13):60.82-1. 88(m,78H),3.79(~,2H),3~92-4,0(1m l4H),6.34(t,
I H, J=2.2Hz),6.45(dl2H, J=2.2Hz)
[0465]
[Table 81
Prep.
EX. 27
Ex. 1
OH F OH F NC,,Ao'P\N
A
: 5'-0-(4,41-dimethoxyf rityl)-2'-deoxy-2'-fluorouridine
'~-~~~(400~~z,~~~l~):63.50-3.64(m,2~),3.IH8 )0,4(.4s9,-46.6~0)(m~4, .08(m
1 H),5.03(dd,l H,J=4.2,51 .2Hz);5.33(dl1 H,J=8.2Hz),6.07(d,l H,
J=l4.9Hz),6.83-7.40(m, 13H),7.90(d11H , J=8.2Hz)
5'-0-(4,4'-dimetho~rityl)-2'-deoxy-2'-fluorouridine-31-O-[(2-cyanoethyl)-NN-l
diisopropylphosphoramidite]
1H -NMR(400MHz,CDC13):bO099-1.3 2(m, 12H),2.40-2.65(m12H),3.40-3.93(m,6H),
3.80(m16H),4.20-4.30(Hm)l,4l. 55-4.80(m, 1H ),5.00-5.23(m, IH ),5.24-5.29(m, IH )
,6.07(d,2H1J=16 .1 Hz),6.82-7.44(m113H),7.92-8.0I4H ()m l
3 1 ~ - ~60~~~~z,(~1~~ 1~):6149.7,150.3
'~-~~~(400~~z,~~~l~):60.83-0.97(m,45~)~0.98-(m,75~),2.38-2.48(m~2~),0-2.71 (m14H),3.39-3.51( m,2H),3.79(s16H),3.85-4.20(m16H),4.13-4.15(m,H'l ),5.4
0-5.50(m, 1H ),6.38-6.45(m11H ),6.54(~,2H),6.84(d,4H,J=8.9Hz),7.20-7.39(m,9H)~
7.60(s,l H)
Ex. 2
H - N M R ( ~ O O M H ~ , C D C ~ ) : ~ ~ . ~1~. 0-0O-2..0~0~(m(,~75, H~)~,2H.4)2-,2 .48(m12H
),2.52(t,2H,J=6.6Hz),2.71(t,2H,J=6.6Hz),3.79(s,6H),3.47(ddt,2H,J=24156ll6.2
Hz),3.91-4.00(m,6H),4.13-4.17(m,1H),5.46-5.50(m11 H),6.40-6.45(ml1 H),6.47(s,
2H),6.82-6.85(m14H),7.22-7.39(m19H),7.61( d,l H,J=1.2Hz)
[0466]
[Table 91
Ex. 4
Ex. 6
Ex. 5
Ex. 7
' H-NMR(~ooMHz,cDcI~):~o.~1~2-~o).,~0~.9(4~(,d ,3~,~=6.6~z),1..04-1.58(m,2
1H),1.89-2.01(m,IH),2.1 1(ddd,1H,J=2.6,8.5,14.9Hz),2.19(s,3H),2.31-2.37(m11H
),2.42-2.51 (m,2H),2.57(t,2H,J=6.2Hz),2.75(dt,2HlJ=2.4,6.4M),3.45(dd,l H,J=2.
4,10.5Hz),3.48(dd,l H1J=2.4,10.5Hz),3.79(s,6H),4.12-4.15(m1Hl ),5.46-5.50(m1l
H),5.98(t,2H,J=7.3Hz),6.46(dd1l H1J=5.7,8.8H~),6.81-6.85(rn,4H),7.14-7.39(m,9
H),7.64(d1I H,J=1 .I Hz)
[0467]
[Table 101
Ex. 8
Ex. 9
Ex. 10
Ex. 11
OCf
H - N M R ( ~ o o M H z , c D c ~ ~ ) : ~ ~I.2 ~~~),-0o..9~3~((d~~,3 ~~~=6I.. 062f-l1z.5)~6( m,2 '
4H),1.88-1 .99(m,1 H),2.1 O(ddd,l H,J=3.0,6.9,1 5.9Hz),2.31-2.36(m12H),2.42(ddd1
1 H,J=3.0,6.9,15.9Hz),3.38(dd,1H1J=3.0,10.5Hz),3.49(dd,1 H,J=3.OI10.5Hz),3.75
-3.81 (m,2H),3.79(sI6H),4.04(dt,1 HIJ=3.1,6.2Hz),4.56-4.60(m,I H),5.98(dtl2H,J=
1.9,l I.2 Hz),6.42(dt11H ,J=I .2,7.3Hz),6.72-6.78(m11H),6.82-6.86(m,4H),7.10-7.4
I (m,9H),7.60(d,l H,J=I .2Hz)
0
N C * ~ , ~ * ~A
h
m/z(ESI-MS):Anal.
Calc.for HB9N4OI0P:
1068.6.Found 1 069.4(M-I-H)+
h"-*:-
* '"
MceO.~.N A
A
' H - N M R ( ~ O ~ M H Z , C D C ~ ~ ) :I~2 ~~.)~~0 9- O4 (.d~J,~3=(~~~,.~ ~ HIZ.0 )3 -1 .58(m13
7H),1.94-1.97(m1lH ),2.07-2.14(m11fl),2.29-2.37(m1H1 ),2.41 (t,1 HIJ=6.3Hz),2.46
-2.59(m,1H),2.61(t,1H,J=6.3Hz),3.31-3.35(m11H),3.47-3.64(m,4H),3.74-3.85(m,
I H),3.78,3.79(2~,6H),4.14,4.18(2d,I HIJ=2.2Hz),4.63-4.69(m1l H),5.98(s12H),6.4
0-6.46(m11H ),6.82-6.86(mI4H),7.1 5-7.18 (mI2H),7.23-7.32(m,5H),7.39-(m7.,24 1
H),7.63,7.68(2dl1 H,J=I .I Hz)
3 1 ~ - ~ ~ ~ ( 4 0 0 ~ ~ ~ , ~ ~5~0.3~ 3 ) : 6 1 4 9 . 7 , 1
Dms~ wF2 4 z ~ 4 L u
N C e O + P - NL
A
I H-NMR(400MHz,CDC13):60.83-0.87(m,27H)ll .09-I .52(mI22H),2.08-2.1 9(m,1 H
),2.43-2.49(m,2H),2.62(tl1 H1J=6,3Hz),2.70-2,76(m,I H),2.86-2.95(ml1 H),3.29-3.
88(ml6H),3.77(s,6H),4.26-4.30(m11 H),4.73-4.76(m11H ),6.27-6.30(m,l H),6.75-7.
42(m,l 3H),7.85,7,86(2S11 H)
3 1 ~ - ~ ~ ~ ( 4 0 0 ~ ~ ~ , ~15~0.~0 ~ ~ ) : 6 1 4 9 . 9 ,
m/z(ESI-MS):Anal.Calc.for C60H86N708P:10 63.6.Found 1062.4(M-H)-
' [0468]
[Table 111
Ex. 12
0 ??U ""wN ? .
NC,,., J,
1
1 ~ - ~ ~ ~ ( 4 0 0 ~ ~ z , ~ ~ ~ 1 3 ) : 6 0 . 8 2 - 0 . 8 7 ( m , 2 4J~=)~, 0..~9H31z(.0d)2 ,-31.~55,( m,2
2H),1.87-1.93(m1l H),1.99-2.02(m,I H),2.22-2.26(m,l H),2.38-2.49(ml1 H),2.45(t,
1 H1J=6.5Hz),2.60(t,l H,J=6.5Hz),3.31-3:42(m,2H),3.57-3;63(m,2H),3.66-3.86(ml
2H),3.76,3.77(2~,6H),4.26-4.33(m,IH ),4.70-4.78(m, IH ),6.41 -6.49(m1I H ),6.76-6.
81 (mI4H),7. 15-7.48(m;9H),7.97,7.99(2s,I H),8.61,8.85(2s1I H )
31P -NMR(~OOMHZ,CDCI~):~4I9 .9,150.0
m/z(ESI-MS):Anal.Calc.for CsoHesN707P:10 47.6.Found 10 46.3(~-H)-
' 1 ~ - ~ ~ ~ ( 3 0 0 ~ ~ z , ~ ~ ~ ~ 3 ) : 6 ~ . 8 3 - ~ (m,6H), 1.03-1.35(m11 6H),1.42-1.73
(mI4H),2.10 -2.33(m12H),2.44(t,1H ,
J=6.5Hz),2.62(t, 1 H1J=6.3Hz),2.68-2.85
(m,l H),3.30-3.90(m16H),3.81(s,6H),
4.20-4.24(m11H ),4.53-4.65(m11H ),
6.1 8-6.28(m,1 H),6.82-6.89(m14H),
7.1 3-7.44(m,9H),7.79(brs11 H),
8.1 9,8.29(2d,I H1J=7.4Hz)
[0469]
[Table 121
Ex. 22
Ex. 23
H-NMR(~OOMHZ,CDCI~):~~.~~-O.~~
(m,6H),I .05-1.20(m,12H),I .21-1.75
(m,28H),2.07-2.85(m,5H),3.34-3.88
(m,6H),3.80,3.81(2~,6H),4.20-4.24
(m,1 H),4.53-4.66(m,IH),6.23-6.30
(m, I H),6.82-6.88(m,4H),7.12-7.42
(m,IOH),7.89-7.99(m,IH),8.18,8.27
(2d, 1 H,J=7.4Hz)
A
QhIe
3 1 ~ - ~O~MH~Z,(CD1C ~~)5:~0I.0 ~150.6
H-NMR(~OOMHZ,CDC~~-0).:9~3 ~.~I
(m,6H),1.05-1.20(m,12H),1.20-1.80
(m,24H),2.18-2.85(m,5H),3.32-3.88
(m,6H),3.80,3.81(2~,6H),4..19-4.24
(m,l H),4.53-4.68(m,1 H),6.22-6.31
(m,1 H),6.81-6.88(m,4H),7.13-7.45
(m,IOH),8.02-8.12(m,lH),8.19,8.29
(2d,l H,J=7.5Hz)
3 1 ~ - ~2 0~~ ~~~(, ~1~ ~ 1 ~ ) : 6 1 4 9 . 2 , 1 4 9 . 8
-Oe92(m,3H),1.0 3-1.20(m, 12H),
I .20-1.78(m,22H),2.23-2.85
(m,6H),3.34-3.87(m,6H),3.80,
3.81 (2~,6H),4.20-4.24(m1, H ),
4.55-4.66(m, 1 H),6.22-6.30
(m, I H),6.80-6.87(m,4H),7.08
-7.42(m,gH),7.83(brs,I H),
8.1 9,8.29(2d11H ,J=7.5Hz)
P-NMR(1 60MHz,CDC13):
61 50.0,150.5
H-NMR(~OOMHZ,CDC~~):~~.~~
0
EX. 25
EX. 33
H-NMR(~OOMHZ,CDCI~):~O.~~
-0.91 (m,12H),0.92-0.98(m,6H),
1 -07-1 .72(m128H),2.45-2.65(m,
4H),3.27-3.95(ml 12H),4.22-4.28
(m, I H),4.63-4.72(m, 1 H),6.36
-6.45(m,I H),6.77-6.85(m,4H),7.15
-7.34(m,l3H),7.71-7.74(m,l H),
A 8.41 (brs, I H),8.58-8.64(m, 1 H)
P-NMR(I 6OMHz,CDCI3):
6149.9,150.1
DMTrO
H-NMR(~OOMHZ,CDCI~):.~93~ .~~-I
(m, 120H),2.40-2.83(m,6H),3.20-3.33
(m,l H),3.46(rn,2H),3.78(~,6H),3.98
(m,5H),4.13(brs,1H),5.50(m,lH),
6.23-6.28(m, I H),6.40-6.46(m,l H),
6.59-6.63(mJ1 H),6.74-7.43(m,20H),
7.58-7.63(m,I H),8.28(brs, 1 H)
[0470]
[Table 131
5 [0471]
Experimental Example 1: Solubility test of nucleoside with
Ex 34
.
EX. 35
EX. 43
Camp-
EX.
protected 3'-hydroxyl group
The solubility (=solute / (solvent +solute ) ~100) (mass %)
at 20°C, shown in the following Table 2, of the compound
l o representing the present invention, thymidine wherein the 5,-
DM+xT0 ro"P
._. 04 -- o
/ OPhy
DMT,.~$o .
o,+Tophy
/
OPhy
'H-NMR(~ooMHZ,CDC~~):~O.~~-O.~~
(m,30H), I -00-I .85(m,51 H),2.40-2.45
(mI2H),2.52-2.58(m,2H),2.67-2.74
(ml2H),3.45(d,2H,J=2.3Hz),3.78(s,6H),
3.85-3.97(m,4H),4.lO(brs,lH),5.48
(m,l H),6.09(dd,2H,J=7.9,13.6Hz),
6.40(m11 H),6.78-7.41 (m,21 H),7.60
(m,l H),8.l4(brsl1 H)
'H-NMR(~ooMHZ,CDCI~):~~.~~-O.~~
(mI30H),1 .00-I .85(m,51 H),2.40-2.55
(m,4H),2.67-2.74(m,2H),3.43-3.50
(m,l H),3.78(~,6H),3.88-3.97(m14H),
4.1 4(m,l H),4.35(d,I HIJ=5.5Hz),
56.435-56..5405((mml,14 H),56.7820-57.7470(m,l 3HH),) ,
7.60(m, I H),7.94(brsll H) '
f l b o 0
DMTrOQ
Nc-oJ\N~
A
IH -NMR(400MHz1CDC13):60.83-0.95(m1, 5H),0.97-I .93(m,39H),2.35-2.40(ml IH
),2.52-2.63(m, I H),3.37-3.6O(m,6H),3.78-3.90(m,8H),4.03-4.1 3(mI2H),4.33-4.42(
m,1 H),4.59(dll H,J=26,5Hz),5.67(dl1 HIJ=2.2Hz),6.81-6.88(m14H),6.90-6.95(m,2
H),4.01-4.1.2(m,2H),6.91-6.96(m,2H),7.23-7.36(m17H),7,42-7.49(m,2H),7.68-7.7
4(m, I H),8.03-8.08(mI2H)
TLC: Rf=O.5O(dichloromethane : methanol = 4: I
' ~ - ~ ~ ~ ( 4 0 0 ~ ~ z ) : ~ 0 . 8 9 ( t , 9 ~ , ~ = 7 . 0 ~ z ~ ( o c t d e c l 0 x y ) ) , 1 . 2 5 - 1 . 7 9 ( m , 1 0 2 H , -
CH2-(octadecyloxy)), I .35(s,3H,N5-CH3-thymidine)l2,45(m,2H,2'-thymidine)l2.5
I (m,2H,succinyl),2.70(m,2H,succinyl),3.46(m12H,5~-thymidine),3.79(s,6H,~3~0
-DMTr),3.79-3.95(m,6HlBn-0-CH2-),4.1 5(m11H ,4'-thyrnidine),4.32(d12H~, = 5 . 5 ~
2,-NH-CH2-benzyl),5.47(ml I H,3'-thyrnidine),5.72(d,2Hj J=5.5Hzf-NH-CH2~benzy
1),6.41 (m,l~,1'-~hymidine),6.45(s,2~,-benzy1),6.83(d,4~,~=9.0~z,~~~r),7.24-7.
38(m79H,DMTr),7.61(s , IH ,N6-thymidine),7.95(br.s1N3-NH-thymidine)
hydroxyl group is protected by dimethoxytrityl group, and the
3'-hydroxyl group is protected by the branched chain-containing
aromatic protecting group (Example 2), and thymidine wherein
the 5'-hydroxyl group is protected by dimethoxytrityl group,
5 and the 3'-hydroxyl group is protected by the corresponding
group containing the straight chain structure (Comparative
Example 2) as a comparison target thereof was measured.
[Solubility measurement method]
1) A solute (100 parts by mass) was added to a solvent (100
10 parts by mass) to saturation at 20°C.
2) When the solute remained by visual observation, the
supernatant was quantitatively analyzed under the following
HPLC conditions, and the concentration was determined and taken
as the solubility (=solute/ (solvent +solute) ~100()m ass %) .
15 3) When the solute was absent by visual observation, the
solubility was >50 mass %.
[HPLC analysis conditions]
use instrument: Hitachi high performance liquid chromatography
LaChrom Elite L-2000 series
20 column: YMC-PACK 5 p 150 x 4.6 rnrn
column temperature: 40°C
eluent : THF/CH3CN/H20
flow rate: 1.0 ml/min
[0472]
[Table 141
[0473]
5 The nucleoside protected by a branched chain-containing
aromatic group wherein the nucleoside 3'-hydroxyl group is
protected by a branched chain-containing aromatic protecting
group of the present invention was found to show a remarkable
solubility in heptane (the most representative non-polar
10 solvent) which is preferably used for reaction solvents and
extraction solvents in the present invention, and a solvent of
an appropriate mixture of heptane with toluene (non-polar
solvent with different polarity from heptane) or acetonitrile
(polar solvent), as compared to a nucleoside protected by a
15 group having a straight chain structure similar to that of a
known nucleotide 3'-hydroxyl-protecting group (described in JPA-
2010-275254) .
Industrial Applicability
[0474]
20 Using the particular oligonucleotide comprising a
protected base of the present invention, a production method of
oligonucleotide by a phosphoramidite method wherein the
oligonucleotide can be purified by a liquid-liquid extraction
Solvent
hept ane
hept ane/
toluene = 1/1
hept ane/
toluene/
acetonitrile
= l/l/l
Example 2
OPhy
> 50% by mass
> 50% by mass
> 50%by mass
Comparative Example 2
OC18H37
0% by mass
22.3% by mass
13.7% by mass
operation efficiently and in a high yield can be provided.
Using the particular oligonucleotide comprising a
protected base of the present invention, liposolubility and
solubility in an organic solvent (particularly, non-polar
5 solvent) of an intermediate oligonucleotide obtained in each
step of a nucleotide elongation reaction are strikingly
improved to enable isolation and purification by an extraction
operation alone, and therefore, a complicated, time-consuming
operation such as solidification isolation and the like is not
10 required, the speed increases, and the efficiency and
producibility of synthesis of oligonucleotide with high
polymerization degree is strikingly improved.
Furthermore, by using the (01igo)nucleotide comprising a
protected base of the present invention, which is imparted with
15 liposoluble and solubility in organic solvents (particularly,
non-polar solvents), adding a particular cation scavenger
during or after deprotection of the 5'-terminal hydroxyl group
protected by a temporary protecting group, applying a
neutralization treatment after completion of the deprotection
20 reaction, and using a particular oxidizing agent or sulfurizing
agent in an oxidation step or a sulfurization step, for a
nucleotide elongation reaction including (1) a deprotection
step of 5'-terminal hydroxyl group protected by a temporary
protecting group, (2) an elongation step of 5'-terminal by the
25 addition of an oligonucleotide comprising a protected base, and
(3) a phosphite triester moiety oxidizing step or sulfurizing
step, steps (I), (2) and (3) can be performed in a liquid and
oligonucleotide, which is an elongated nucleotide, can be
isolated and purified by an extraction operation alone, and
30 therefore, the elongation reaction in the next cycle can be
sequentially performed without taking out the resultant product
from the reaction apparatus, whereby oligonucleotide can be
produced continuously in one pot.
[0475]
35 In addition, since in the production method of
oligonucleotide of the present invention, the oligonucleotide
can be stably dissolved in or transferred to a non-polar
solvent irrespective of the sequence and chain length of
oligonucleotide even as compared to conventional liquid phase
5 methods, it is advantageous in that the isolation and
purification step can be simplified as for the steps, and high
purity and high yield can be ensured as a total view.
This application is based on a patent application Nos.
2012-033429 and 2012-254718 filed in Japan, the contents of
l o which are incorporated in full herein.

CLAIMS
1. An oligonucleotide comprising a protected base, which is
represented by the formula (I) :
wherein q is any integer of not l e s s than 0;
~ a s e "i n the number of q+l are each independently a nucleic
acid base protected by a group having a C5-30 s t r a i g h t chain or
branched chain alkyl group and/or a C5-30 s t r a i g h t chain or
10 branched chain alkenyl group;
P' is a hydrogen atom, or a temporary protecting group
removable under acidic conditions;
X is a hydrogen atom, an optionally protected hydroxyl group, a
halogen atom or an organic group crosslinked with the 4-
15 position carbon atom;
X' i n the number of q are each independently a hydrogen atom,
an optionally protected hydroxyl group, a halogen atom or an
organic group crosslinked with the $-position carbon atom;
p2 i n the number of q+l are each independently a protecting
20 group removable under basic conditions;
R~~ i n the number of q are each independently an oxygen atom or
a sulfur atom; and
Re and Rf are each independently a alkyl group, or a 5- or
6-membered saturated cyclic amino group formed together with
the adjacent nitrogen atom.
5 2. The oligonucleotide comprising a protected base of according
to claim 1, wherein q is 0.
3. The oligonucleotide comprising a protected base according to
claim 1 or 2, wherein the group having a C5-30 straight chain or
lo branched chain alkyl group and/or a C5-30 straight chain or
branched chain alkenyl group is
a group represented by the formula (k) :
wherein * indicates the bonding position to a nucleic acid
15 base;
R~~ is a C5-30 straight chain or branched chain alkyl group or a
C5-30 straight chain or branched chain alkenyl group,
a group represented by the formula (1) :
20 wherein * indicates the bonding position to a nucleic acid
base;
Q1 is -0-, -S- or - N R ~ O - wherein R30 is a hydrogen atom or a C1-22
alkyl group;
Rc and Rd are each independently a hydrogen atom or a C1-22 alkyl
25 group; and
R2* is a C5-30 straight chain or branched chain alkyl group or a
C5-30 straight chain or branched chain alkenyl group,
a group represented by the formula (m):
wherein * indicates the bonding position to a nucleic acid
base;
1 is an integer of 1 to 5;
5 Q2 in the number of 1 are each independently a single bond, or
-0-, -S-, -0C (=O) -, -C (=O) 0-, -0-CH2-, -NH-, .-NHC (=O) -, -
C (=O) NH-, -NH-CH2- or -CH2-;
RZ9 in the number of 1 are each independently a C5-30 straight
chain or branched chain alkyl group or a C5-30 straight chain or
10 branched chain alkenyl group;
ring C is a benzene ring or a cyclohexane ring, each optionally
having, in addition to Q ~ iRn t~he ~num ber of 1 and *c=o, a
substituent selected from the group consisting of a halogen
atom, a C1-6 alkyl group optionally substituted by one or more
15 halogen atoms, and a C1-6 alkoxy group optionally substituted by
one or more halogen atoms, or
a group represented by the formula (s) :
wherein * indicates the position at which an imino bond is
20 formed with an amino group of a nucleic acid base; and
R~~ and R~~ are each independently a C5-30 straight chain or
branched chain alkyl group or a C5-30 straight chain or branched
chain a1 kenyl group.
25 4. The oligonucleotide comprising a protected base according to
claim 3, wherein R ~ R~2*,, R2 ' in the number of 1, R~~ and R~~ are
each independently a branched chain alkyl group or branched
chain alkenyl group selected from the group consisting of a
2,6,10,14-tetramethylpentadecyl group, a 2,6,10-
30 trimethylundecyl group, a 2,2,4,8,10,10-hexamethyl-5-undecyl
group, a 2,6,10-trimethylundeca-1,5,9-trienyl group, a 2,6-
dimethylheptyl group, a 2,6-dimethylhept-5-enyl group, a 2,6-
dimethylhepta-1,5-dienyl group, a 9-nonadecyl group, a 12-
methyltridecyl group, an 11-methyltridecyl group, an 11-
5 methyldodecyl group, a 10-methylundecyl group, an 8-heptadecyl
group, a 7-pentadecyl group, a 7-methyloctyl group, a 3-
methyloctyl group, a 3,7-dimethyloctyl group, a 3-methylheptyl
group, a 3-ethylheptyl group, a 5-undecyl group, a 2-heptyl
group, a 2-methyl-2-hexyl group, a 2-hexyl group, a 3-heptyl
10 group, a 4-heptyl group, a 4-methyl-pentyl group, a 3-methylpentyl
group, and a 2,4,4-trimethylpentyl group; or a straight
chain alkyl group selected from the group consisting of a
tetradecyl group, a tridecyl group, a dodecyl group, an undecyl
group, a decyl group, a nonyl group, an octyl group, a heptyl
15 group, a hexyl group, and a pentyl group.
5. The oligonucleotide comprising a protected base according to
any one of claims 1 to 4, wherein the C5-30 straight chain or
branched chain alkyl group and/or C5-30 straight chain or
20 branched chain alkenyl group is a C5-30 branched chain alkyl
group and/or a C5-30 branched chain alkenyl group.
6. The oligonucleotide comprising a protected base according to
any one of claims 1 to 5, wherein P' is a monomethoxytrityl
25 group or a dimethoxytrityl group..
7. A method of producing an ntp-mer oligonucleotide, comprising
(2) a step of condensing a p-mer oligonucleotide comprising a
protected base (p is any integer of one or more) wherein the
30 3'-hydroxyl group is phosphoramidited, the 5'-hydroxyl group is
protected by a temporary protecting group removable under
acidic conditions, and the nucleic acid base is protected by a
group having a C5-30 straight chain or branched chain alkyl
group and/or a C5-30 straight chain or branched chain alkenyl
35 group, with an n-mer oligonucleotide (n is an integer of one or
more) wherein the 5'-hydroxyl group is not protected and the
3'-hydroxyl group is protected, by forming a phosphite triester
bond via the 5'-hydroxyl group thereof.
5 8. The production method according to claim 7, wherein p is 1.
9. The production method according to claim 7 or 8, further
comprising the following step (3) :
(3) a step of converting the phosphite triester bond of the
lo n+p-mer oligonucleotide obtained in the condensation step to a
phosphate triester bond or a thiophosphate triester bond by
adding an oxidizing agent or a sulfurizing agent to the
reaction mixture obtained in the condensation step (2).
15 10. The production method according to any one of claims 7 to 9,
further comprising the following step (1) :
(1) a step of removing the temporary protecting group removable
under acidic conditions of the 5'-hydroxyl group by reacting,
in a non-polar solvent prior to the condensation step (2)' an
20 n-mer oligonucleotide wherein the 3'-hydroxyl group is
protected, and the 5'-hydroxyl group is protected by a
temporary protecting group, with an acid.
11. The production method according to claim 10, wherein the
25 step (1) is performed in the presence of at least one kind of
cation scavenger selected from a pyrrole derivative and an
indole derivative, and further comprises a step of
neutralization with an organic base after removal of the
temporary protecting group of the 5'-hydroxyl group.
30
12. The production method according to any one of claims 9 to
11, further comprising the following step (4) :
(4) a step of isolating the n+p-mer oligonucleotide from the
reaction mixture obtained in step (3) by an extraction
35 operation alone.
13. The method according to claim 12, further comprising the
following step (5) :
(5) a step of removing all the protecting groups of the n+p-mer
5 oligonucleotide obtained in step (4).
14. The production method according to any one of claims 7 to
13, wherein the p-mer oligonucleotide comprising a protected
base, wherein the 3'-hydroxyl group is phosphoramidited, the
10 5'-hydroxyl group is protected by a temporary protecting group
removable under acidic conditions, and the nucleic acid base is
protected by a group having a C5-30 straight chain or branched
chain alkyl group and/or a C5-30 straight chain or branched
chain alkenyl group, is the oligonucleotide comprising a
15 protected base according to any one of claims 1 to 6.
15. The production method according to any one of claims 7 to
14, wherein the 3'-hydroxyl group of the n-mer oligonucleotide
is protected by a group represented by the formula (111) :
20 - L - Y - z (111)
wherein
L is a group represented by the formula (al):
* *
wherein * shows the bonding position to Y; indicates the
25 bonding position to a 3'-hydroxy group of the nucleotide;
L1 is an optionally substituted divalent C1-22 hydrocarbon
group; and
L2 is a single bond, or a group represented by **c(=0)N(R2)-R1-
* * ***
N (R3)** * wherein shows the bonding position to L1, shows
30 the bonding position to C=O, R1 is an optionally substituted C1-
22 alkylene group, and R2 and R3 are each independently a
hydrogen atom or an optionally substituted C1-22 alkyl group, or
R2 and R3 are optionally joined to form an optionally
substituted C1-22 alkylene bond,
Y is an oxygen atom or NR wherein R is a hydrogen atom, an
alkyl group or an aralkyl group, and
5 Z is a group represented by the formula (a2) :
%\*/Rb
wherein * shows the bonding position to Y;
R4 is a hydrogen atom, or when Rb is a group represented by the
following formula (a3), R4 is optionally a single bond or -0-
10 in combination with R6 to form a fluorenyl group or a xanthenyl
-group together with ring B;
Q in the number of k are each independently a single bond, or -
0-, -S-, -0C (=O) -, -NHC (=O) - or -NH-;
R5 in the number of k are each independently an organic group
15 having- at least one aliphatic hydrocarbon group having one or
more branched chains and the total carbon number of not less
than 14 and not more than 300;
k is an integer of 1 to 4;
ring A optionally further has, in addition to R4, Q R ~ in the
20 number of k and *C (R,) (Rb), a substituent selected from the
group consisting of a halogen atom, a C1-6 alkyl group
optionally substituted by one or more halogen atoms, and a C1-6
alkoxy group optionally substituted by one or more halogen
atoms;
25 Ra is a hydrogen atom; and
Rb is a hydrogen atom, or a group represented by the formula
(a3) :
wherein * indicates a bonding position;
j is an integer of 0 to 4;
Q in the number of j are each independently as defined above;
R7 in the number of j are each independently an organic group
having at least one aliphatic hydrocarbon group having one or
5 more branched chains and the total carbon number of not less
than 14 and not more than 300;
R6 is a hydrogen atom, or optionally a single bond or -0- in
combination with R4 to form a fluorenyl group or a xanthenyl
group together with ring A; and
10 ring B optionally further has, in addition to QR" in the number
of j and Rb, a substituent selected from the group consisting
of a halogen atom, a C1-6 alkyl group optionally substituted by
one or more halogen atoms, and a C1-6 alkoxy group optionally
substituted by one or more halogen atoms.
16. The production method according to any one of claims 7 to
15, wherein at least one nucleic acid base of the n-mer
oligonucleotide is protected by a group having a C5-30 straight
chain or branched chain alkyl group and/or a C5-30 straight
20 chain or branched chain alkenyl group.
17. The production method according to any one of claims 7 to
16, wherein the C5-30 straight chain or branched chain alkyl
group and/or C5-30 straight chain or branched chain alkenyl
25 group are/is a C5-30 branched chain alkyl group and/or a C5-30
branched chain alkenyl group.
18. An oligonucleotide protected by a branched chain-containing
aromatic group, which is represented by the formula (11):
L-Y-z
wherein
m is any integer of 0 or more;
~ase' in the number of m+l are each independently an optionally
5 protected nucleic acid base;
P' is a hydrogen atom, or a temporary protecting group
removable under acidic conditions;
X is a hydrogen atom, an optionally protected hydroxyl group, a
halogen atom or an organic group crosslinked with the 4-
lo position carbon atom;
X' in the number of m are each independently a hydrogen atom,
an optionally protected hydroxyl group, a halogen atom or an
organic group crosslinked with the 4-position carbon atom;
p2 in the number of m are each independently a protecting group
15 removable under basic conditions;
R~~ in the number of m are each independently an oxygen atom or
a sulfur atom;
L is a group represented by the formula (al):
* *
20 wherein * shows the bonding position to Y; indicates the
bonding position to a-3'-hydroxy group of the nucleotide;
L1 is an optionally substituted divalent C1-22 hydrocarbon
group; and
L2 is a single bond, or a group represented by **c( =O)N(R2)- R1-
* * * * *
N (R3)** * wherein shows the bonding position to L1, shows
the bonding position to C=O, R1 is an optionally substituted C1-
22 alkylene group, and R2 and R3 are each independently a
5 hydrogen atom or an optionally substituted C1-22 alkyl group, or
R2 and R3 are optionally joined to form an optionally
substituted C1-22 alkylene bond,
Y is an oxygen atom or NR wherein R is a hydrogen atom, an
alkyl group or an aralkyl group, and
10 Z is a group represented by the formula (a2) :
wherein * shows the bonding position to Y;
R4 is a hydrogen atom, or when Rb is a group represented by the
following formula (a3), R4 is optionally a single bond or -0-
15 in combination with R6 to form a fluorenyl group or a xanthenyl
group together with ring B;
Q in the number of k are each independently a single bond, or -
0-, -S-, -0C (=O) -, -NHC (=O) - or -NH- ;
R5 in the number of k are each independently an organic group
20 having at least one aliphatic hydrocarbon group having one or
more branched chains, and the total carbon number of not less
than 14 and not more than 300;
k is an integer of 1to 4;
ring A optionally further has, in addition to R4, Q R ~ in the
25 number of k and *c(R,) (Rb), a substituent selected from the
group consisting of a halogen atom, a C1-6 alkyl group
optionally substituted by one or more halogen atoms, and a C1-6
alkoxy group optionally substituted by one or more halogen
atoms;
30 R, is a hydrogen atom; and
Rb is a hydrogen atom, or a group represented by the formula
wherein * indicates the bonding position;
j is an integer of 0 to 4;
5 Q in the number of j are each independently as defined above;
R7 in the number of j are each independently an organic group
having at least one aliphatic hydrocarbon group having one or
more branched chains and the total carbon number of not less
than 14 and not more than 300;
10 R6 is a hydrogen atom, or optionally a single bond or -0- in
combination with R4 to form a fluorenyl group or a xanthenyl
group together with ring A; and
ring B optionally further has, in addition to QR~ in the number
of j and R6, substituent (s) selected from the group consisting
15 of a halogen atom, a C1-6 alkyl group optionally substituted by
one or more halogen atoms, and a C1-6 alkoxy group optionally
substituted by one or more halogen atoms.
19. Theoligonucleotide according to claim 18, wherein m is 0.
20. The oligonucleotide according to claim 18 or 19, wherein at
least one of the nucleic acid bases is protected by a group
having a C5-30 straight chain or branched chain alkyl group
and/or a C5-30 straight chain or branched chain alkenyl group.
21. A method of producing an nf+p'-mer oligonucleotide
comprising:
(2') a step of condensing a p'-mer oligonucleotide (p' is any
integer of one or more) wherein the 3'-hydroxyl group is
30 phosphoramidited, the 5'-hydroxyl group is protected by a
temporary protecting group removable under acidic conditions,
and the nucleic acid base is optionally protected, with an nfmer
oligonucleotide (n' is any integer of one or more) wherein
the 5'-hydroxyl group is not protected, and the 3'-hydroxyl
group is protected by the protecting group represented by the
formula (111) :
5 -L-Y-z (III)
wherein
L is a group represented by the formula (al):
* *
wherein * shows the bonding position to Y; indicates the
10 bonding position to a 3'-hydroxy group of the nucleotide;
L1 is an optionally substituted divalent C1-22 hydrocarbon
group; and
* *
L2 is a single bond, or a group represented by C (=O)N( R2)- R1-
* * ***
N(R~)**w*h erein shows the bonding position to L1, shows
15 the bonding position to C=O, R1 is an optionally substituted C1-
22 alkylene group, and R2 and R3 are each independently a
hydrogen atom or an optionally substituted C1-22 alkyl group, or
R2 and R3 are optionally joined to form an optionally
substituted C1-22 alkylene bond,
20 Y is an oxygen atom or NR wherein R is a hydrogen atom, an
alkyl group or an aralkyl group, and
Z is a group represented by the formula (a2) :
wherein * shows the bonding position to Y;
25 R4 is a hydrogen atom, or when Rb is a group represented by the
following formula (a3), R4 is optionally a single bond or -0-
in combination with R6 to form a fluorenyl group or a xanthenyl
group together with ring B;
Q in the number of k are each independently a single bond, or -
0-, -S-, -0C (=O) -, -NHC (=O) - or -NH-;
R5 in the number of k are each independently an organic group
having at least one aliphatic hydrocarbon group having one or
5 more branched chains and the total carbon number of not less
than 14 and not more than 300;
k is an integer of 1 to 4;
ring A optionally further has, in addition to R4, Q R ~ in the
number of k and *c(R,) (Rb), a substituent selected from the
10 group consisting of a halogen atom, a C1-6 alkyl group
optionally substituted by one or more halogen atoms, and a C1-6
alkoxy group optionally substituted by one or more halogen
atoms;
R, is a hydrogen atom; and
15 Rb is a hydrogen atom, or a group represented by the formula
(a3) :
wherein * indicates the bonding position;
j is an integer of 0 to 4;
20 Q in the number of j are each independently as defined above;
R' in the number of j are each independently an organic group
having at least one aliphatic hydrocarbon group having one or
more branched chains and the total carbon number of not less
than 14 and not more than 300;
25 R6 is a hydrogen atom, or optionally a single bond or -0- in
combination with R4 to form a fluorenyl group or a xanthenyl
group together with ring A; and
ring B optionally further has, in addition to QR~ in the number
of j and R6, a substituent selected from the group consisting
30 of a halogen atom, a C1-6 alkyl group optionally substituted by
one or more halogen atoms, and a C1-6 alkoxy group optionally
substituted by one or more halogen atoms, by forming a
phosphite triester bond via the 5'-hydroxyl group thereof.