SPECIFICATION
METHOD FOR PRODUCING OLIGONUCLEOTIDE
TECHNICAL FIELD OF THE INVENTION
[0001]
5 The present invention relates to a production method of
particular oligonucleotide. Moreover, the present invention
relates to a particular pseudo solid phase protecting group,
and a particular nucleoside.
BACKGROUND OF THE INVENTION
10 [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
(solid phase method) using a phosphoramidite method is most
15 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
limited due to facility restriction, reagents and starting
20 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.
On the other hand, synthesis methods of oligonucleotide
by a liquid phase method have also been studied. However,
25 since the operation is complicated and the yield is low, a
large-scale, rapid synthesis of long oligonucleotide is
difficult.
[0003]
In recent years, in an attempt to solve the respective
30 defects of the liquid phase method and the solid phase method,
an oligonucleotide production method using
monomethoxypolyethylene glycol (MPEG) as a protecting group is
disclosed (non-patent documents 2 to 4). However, while
synthetic examples of up to 20mer DNA are disclosed, a
35 crystallization isolation operation is essential for each
1
reaction, and the progress status of the reaction and the like
are difficult to confirm, since MPEG molecule itself is not a
unimolecule.
[0004]
5 In the meantime, a synthesis method of oligonucleotide
comprising use of a hydrophobic group-linked nucleoside is
disclosed (patent document 1). While it has been reported that
the method affords synthesis of 21mer oligonucleotide, the
number of steps is markedly high and they are complicated,
10 since a crystallization isolation operation is repeated in
every step of deprotection of 5'-protecting group, coupling
and oxidation.
[Document List]
[patent document]
15 [0005]
patent document 1: JP-A-2010-275254
[non-patent documents]
[0006]
non-patent document 1: S. L. Beaucage, D. E. Bergstorm, G. D.
20 Glick, R. A. Jones, Current Protocols in Nucleic Acid
Chemistry; John Wiley & Sons (2000)
non-patent document 2: Nucleic Acid Res., 1990, Vol.18, No.11,
3155-3159
non-patent document 3: Nucleic Acid Res., 1993, Vol.21, No.5,
25 1213-1217
non-patent document 4: Bioconjugate Chem., 1997, Vol.8, No.6,
793-797
[SUMMARY OF THE INVENTION]
Problems to be Solved by the Invention
30 [0007]
The present inventors have conducted intensive studies in
view of the aforementioned problems and found that,
particularly, in the synthesis method of oligonucleotide as
described in patent document 1, which uses a hydrophobic
35 group-linked nucleoside, methanol used as a scavenger of a
2
free protecting group (i.e., cation) in the deprotection step
becomes an inhibitory substance for the coupling reaction in
the next step, and therefore, needs to be completely removed
and, for this object, a complicated operation such as a
5 crystallization isolation step, an azeotropic distillation
removal step and the like is essential.
In addition, it was clarified that when a cation
scavenger other than methanol exemplified in patent document 1
is used, a side reaction such as re-protection of 5'-hydroxyl
10 group and the like occurs in the neutralization step of an
acid used as a deprotecting agent, and that the acid needs to
be removed by crystallization isolation rather than
neutralization.
Accordingly, the problem of the present invention is to
15 provide an industrially advantageous production method without
multistep procedures including a crystallization isolation
step, which is an essential step in a synthesis method of
oligonucleotide using a hydrophobic group-linked nucleoside.
More specifically, it is provision of a method of
20 producing an n+p-mer oligonucleotide (n and p are each
independently an integer of 1 or more) efficiently in a high
yield which comprises use of, as a starting material, an n-mer
oligonucleotide wherein the 3'-terminal hydroxyl group is
protected by a pseudo solid phase protecting group, and the
25 5'-terminal hydroxyl group is protected by a temporary
protecting group, and performing (1) a deprotection step of
the 5'-terminal hydroxyl group protected by a temporary
. protecting group, (2) a 5'-terminal elongation step by the
addition of a p-mer oligonucleotide wherein the 3'-position is
30 phosphoramidited, and (3) an oxidation step or a sulfurization
step of a phosphite triester moiety, wherein each step does
not include crystallization and isolation, and is continuously
performed in a solution.
Means of Solving the Problems
35 [0008]
3
As a result of the intensive studies, the present
inventors have found that the above-mentioned problems can be
solved by adding a particular cation scavenger during or after
deprotection of a 5'-terminal hydroxyl group protected by a
5 temporary protecting group, applying a neutralization
treatment with an organic base after completion of the
deprotection reaction, and applying an oxidation treatment or
sulfurization treatment using an oxidizing agent or
sulfurizing agent.
10 The present invention includes the following.
[1] A method of producing an oligonucleotide comprising the
following steps (1) to (3):
(1) a step of reacting, in a non-polar solvent, an n-mer
oligonucleotide (n is an integer of one or more) wherein the
15 3'-hydroxyl group is protected by a pseudo solid phase
protecting group, and the 5'-hydroxyl group is protected by a
temporary protecting group removable under acidic conditions,
an acid, and at least one kind of cation scavenger selected
from a pyrrole derivative and an indole derivative to remove
20 the temporary protecting group of the 5'-hydroxyl group, and
neutralizing the reaction mixture with an organic base,
(2) a step of adding, to the reaction mixture after the
neutralization in step (1), a p-mer oligonucleotide (p is an
integer of one or more) wherein the 3'-hydroxyl group is
25 phosphoramidited, and the 5'-hydroxyl group is protected by a
temporary protecting group removable under acidic conditions
to allow condensation with the n-mer oligonucleotide obtained
in step (1), wherein the temporary protecting group of the 5'-
hydroxyl group is removed, by forming a phosphite triester
30 bond via the 5'-hydroxyl group thereof, and
(3) a step of adding an oxidizing agent or a sulfurizing agent
to the reaction mixture obtained in step (2) to convert the
phosphite triester bond of the n+p-mer oligonucleotide
obtained in step (2) to a phosphate triester bond or a
35 thiophosphate triester bond.
4
[2] The method of the above-mentioned [1], wherein p is 1.
[3] The method of the above-mentioned [1] or [2], further
comprising the following step (4) :
(4) a step of adding a polar solvent to the reaction mixture
5 obtained in step (3) to precipitate the n+p-mer
oligonucleotide and obtaining same by solid-liquid separation.
[4] The method of the above-mentioned [3], further comprising
the following step (5):
(5) a step of removing all the protecting groups of the n+pio
mer oligonucleotide obtained in step (4).
[5] The method of any one of the above-mentioned [1] to [4],
wherein the temporary protecting group removable under acidic
conditions is a dimethoxytrityl group or a monomethoxytrityl
group.
is [6] The method of any one of the above-mentioned [1] to [5],
wherein the non-polar solvent is a solvent selected from the
group consisting of a halogenated solvent, an aromatic solvent,
an ester solvent, an aliphatic solvent, a non-polar ether
solvent, and a combination thereof.
20 [7] The method of any one of the above-mentioned [1] to [5],
wherein the non-polar solvent is a solvent selected from the
group consisting of dichloromethane, chloroform, 1,2-
dichloroethane, benzene, toluene, xylene, mesitylene, hexane,
pentane, heptane, nonane, cyclohexane, ethyl acetate,
25 isopropyl acetate, tert-butyl methyl ether, cyclopentyl methyl
ether, and a combination thereof.
[8] The method of any one of the above-mentioned [3] to [7],
wherein the polar solvent is an alcohol solvent or a nitrile
solvent.
30 [9] The method of any one of the above-mentioned [3] to [7],
wherein the polar solvent is methanol or acetonitrile.
[10] The method of any one of the above-mentioned [1] to [9],
wherein the pyrrole derivative or the indole derivative is at
least one kind selected from the group consisting of pyrrole,
35 3-methylpyrrole, 2,4-dimethylpyrrole, indole, 4-methylindole,
5
5-methylindole, 6-methylindole, 7-methylindole, 5,6-
dimethylindole and 6,7-dimethylindole.
[11] The method of any one of the above-mentioned [1] to [10],
wherein the oxidizing agent is iodine, (IS)-(+)-(10-
5 camphorsulfonyl)oxaziridine, tert-butyl hydroperoxide, 2-
butanone peroxide, 1,1-dihydroperoxycyclododecane,
bis(trimethylsilyl)peroxide or m-chloroperbenzoic acid.
[12] The method of any one of the above-mentioned [1] to [10],
wherein the sulfurizing agent is 3-((N,N-
10 dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione,
3H-1,2-benzodithiol-3-one-l,1-dioxide, 3H-1,2-benzodithiol-3-
one, phenylacetyl disulfide, tetraethylthiuram disulfide, 3-
amino-1,2,4-dithiazole-5-thione or sulfur.
[13] The method of any one of the above-mentioned [1] to [12],
15 wherein the acid is trifluoroacetic acid, dichloroacetic acid,
trifluoromethanesulfonic acid, trichloroacetic acid,
methanesulfonic acid, hydrochloric acid, acetic acid or ptoluenesulfonic
acid.
[14] The method of any one of the above-mentioned [1] to [13],
20 wherein the organic base is at least one kind selected from
the group consisting of pyridine, 2,4,6-trimethylpyridine,
benzimidazole, 1,2,4-triazole, N-phenylimidazole, 2-amino-4,6-
dimethylpyrimidine, 1,10-phenanthroline, imidazole, Nmethylimidazole,
2-chlorobenzimidazole, 2-bromobenzimidazole,
25 2-methylimidazole, 2-phenylbenzimidazole, Nphenylbenzimidazole
and 5-nitrobenzimidazole.
[15] A method of producing an oligonucleotide by an improved
continuous phosphoramidite method, comprising using at least
one kind of cation scavenger selected from a pyrrole
30 derivative and an indole derivative in a deprotection step.
[16] A pseudo solid phase protecting group represented by the
formula (I):
[0009]
-L-Y-Z (I)
35 [0010]
6
wherein
L is a group represented by the formula (al)
[0011]
o
**
L1
*
(a1)
O
5 [0012]
wherein * shows the bonding position to Y;
** indicates the bonding position to a group to be
protected;
Li is an optionally substituted divalent C1-22 hydrocarbon
10 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 Li,
shows the bonding position to C=0, R1 is an optionally
substituted C1-22 alkylene group, and R2 and R3 are each
15 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
20 Z is a group represented by the formula (a2):
[0013]
(a2)
[0014]
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;
R5 in the number of k are each independently an organic
group having an aliphatic hydrocarbon group having 10 or
more carbon atoms;
5 k is an integer of 1 to 4;
ring A optionally further has, in addition to OR5 in the
number of k, substituent(s) selected from the group
consisting of a halogen atom, a Ci_6 alkyl group optionally
substituted by a halogen atom, and a Ci_6 alkoxy group
io optionally substituted by a halogen atom;
Ra is a hydrogen atom; and
Rb is a hydrogen atom, or a group represented by the formula
(a3):
[0015]
15 (a3)
[0016]
wherein * shows a bonding position;
j is an integer of 0 to 4;
R7 in the number of j are each independently an organic
20 group having an aliphatic hydrocarbon group having 10 or
more carbon atoms;
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
25 ring B optionally further has, in addition to OR7 in the
number of j, substituent (s) selected from the group
consisting of a halogen atom, a Ci_6 alkyl group
optionally substituted by a halogen atom, and a Ci_6
alkoxy group optionally substituted by a halogen atom.
30 [17] A nucleotide represented by the formula (II):
[0017]
8
£
Base
L—Y—Z
[0018]
wherein
m is an integer of 0 or more,
5 Base in the number of m+1 are each independently an optionally
protected nucleic acid base,
Q is a hydrogen atom, or a temporary protecting group
removable under acidic conditions,
X is a hydrogen atom, a halogen atom, or an optionally
io protected hydroxyl group,
X' in the number of m are each independently a hydrogen atom,
a halogen atom, or an optionally protected hydroxyl group,
R8 in the number of m are each independently an oxygen atom or
a sulfur atom,
15 WG in the number of m are each independently an electronwithdrawing
group,
L is a group represented by the formula (al):
[0019]
o
* * "\ ^ |_9
^ • s^ —. *
•1
o
(a1)
20 [0020]
wherein * shows the bonding position to Y;
indicates the bonding position to a 3'-hydroxy group of
the nucleotide;
Li is an optionally substituted divalent Ci_22 hydrocarbon
group; and
5 L2 is a single bond, or a group represented by **C (=0)N (R2) -
R1-N(R3)*** wherein ** shows the bonding position to Li,
shows the bonding position to C=0, R1 is an optionally
substituted C1-22 alkylene group, and R2 and R3 are each
independently a hydrogen atom or an optionally substituted
10 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):
15 [0021]
va
.R4
5v OR*) (a2)
[0022]
wherein* shows the bonding position to Y;
R4 is a hydrogen atom, or when Rb is a group represented by
20 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;
R5 in the number of k are each independently is an organic
group having an aliphatic hydrocarbon group having 10 or
25 more carbon atoms;
k is an integer of 1 to 4;
ring A optionally further has, in addition to OR5 in the
number of k, substituent(s) selected from the group
consisting of a halogen atom, a C1-6 alkyl group optionally
30 substituted by a halogen atom, and a Ci_6 alkoxy group
optionally substituted by halogen atom;
10
•3
Ra is a hydrogen atom; and
Rb is a hydrogen atom, or a group represented by the formula
(a3) :
[0023]
wherein shows a bonding position;
j is an integer of 0 to 4;
R7 in the number of j are each independently is an
io organic group having an aliphatic hydrocarbon group
having 10 or more carbon atoms;
R6 is a hydrogen atom, or optionally a single bond or -Clin
combination with R4 to form a fluorenyl group or a
xanthenyl group together with ring A; and
15 ring B optionally further has, in addition to OR7 in the
number of j, substituent(s) selected from the group
consisting of a halogen atom, a Ci_6 alkyl group
optionally substituted by a halogen atom, and a Ci_6
alkoxy group optionally substituted by halogen atom.
20 [18] The nucleotide of the above-mentioned [17], wherein m is
0.
[19] The nucleotide of the above-mentioned [17] or [18],
wherein
L in the formula (II) is a succinyl group, and
25 R5 and/or R7 are/is an alkyl group having 10 to 40 carbon atoms.
[20] The nucleotide of the above-mentioned [17] or [18],
wherein
L in the formula (II) is a succinyl group, and
Ra and Rb are both hydrogen atoms, and
30 R5 is an alkyl group having 10 to 40 carbon atoms.
[21] The nucleotide of the above-mentioned [17] or [18],
wherein
L in the formula (II) is a succinyl group, and
11
R5 and/or R7 are/is an alkyl group having 12 to 30 carbon atoms.
[22] The nucleotide of the above-mentioned [17] or [18],
wherein
L in the formula (II) is a succinyl group, and
5 Y-Z is a group selected from the group consisting of
a 3,4,5-tri(octadecyloxy)benzyloxy group,
a 3,5-di(docosyloxy)benzyloxy group,
a 3,5-di[3',4',5'-tri(octadecyloxy)benzyloxy]benzyloxy group,
a 3,4,5-tri[3',4',5'-tri(octadecyloxy)benzyloxy]benzyloxy
io group,
a 3,4,5-tri(octadecyloxy)benzylamino group,
a 2,4-di(docosyloxy)benzylamino group,
a 3,5-di(docosyloxy)benzylamino group,
a di(4-docosyloxyphenyl)methylamino group,
15 a 4-methoxy-2-[3',4',5'-tri(octadecyloxy)benzyloxy]benzylamino
group,
a 4-methoxy-2-[3' ,4',5'-
tri(octadecyloxy)cyclohexylmethyloxy]benzylamino group,
a 2,4-di(dodecyloxy)benzylamino group,
20 a phenyl(2,3,4-tri(octadecyloxy)phenyl)methylamino group,
a di[4-(12-docosyloxydodecyloxy)phenyl]methylamino group,
a 3,5-di[3',4',5'-tri(octadecyloxy)benzyloxy]benzylamino group,
and
a 3,4,5-tri[3',4',5'-tri(octadecyloxy)benzyloxy]benzylamino
25 group.
[23] The nucleotide of any one of the above-mentioned [17] to
[22], wherein Q is a monomethoxytrityl group or a
dimethoxytrityl group.
Effect of the Invention
30 [0025]
A method of producing an n+p-mer oligonucleotide
efficiently in a high yield can be provided, which includes
use of an n-mer oligonucleotide, wherein the 3'-terminal
hydroxyl group is protected by a pseudo solid phase protecting
35 group, and the 5'-terminal hydroxyl group is protected by a
12
temporary protecting group, as a starting material, and (1) a
deprotection step of the 5'-terminal hydroxyl group protected
by a temporary protecting group, (2) a 5'-terminal elongation
step by the addition of a p-mer oligonucleotide wherein the
5 3'-position is phosphoramidited, and (3) an oxidation step or
a sulfurization step of a phosphite triester moiety, by
continuously performing the steps in this order in a solution,
adding a particular cation scavenger during or after the
deprotection of a 5'-terminal hydroxyl group protected by a
io temporary protecting group, applying a neutralization
treatment after completion of the deprotection reaction, and
using a particular oxidizing agent or sulfurizing agent in the
oxidation step or sulfurization step.
Description of Embodiments
15 [0026]
The present invention provides a method of producing an
n+p-mer oligonucleotide efficiently in a high yield, which
includes use of, as a starting material, an n-mer
oligonucleotide wherein the 3'-terminal hydroxyl group is
20 protected by a pseudo solid phase protecting group, and the
5'-terminal hydroxyl group is protected by a temporary
protecting group, and (1) a deprotection step of the 5'-
terminal hydroxyl group protected by a temporary protecting
group, (2) a 5'-terminal elongation step by the addition of a
25 p-mer oligonucleotide wherein the 3'-position is
phosphoramidited, and (3) an oxidation step or a sulfurization
step of a phosphite triester moiety, by continuously
performing the steps in this order in a solution, adding a
particular cation scavenger during or after the deprotection
30 of a 5'-terminal hydroxyl group protected by a temporary
protecting group, applying a neutralization treatment after
completion of the deprotection reaction, and using a
particular oxidizing agent or sulfurizing agent in the
oxidation step or sulfurization step.
35 [0027]
13
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
5 invention belongs to. Any methods and materials similar or
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
10 specification are hereby incorporated by reference so as to
describe and disclose constructed products and methodology
described in, for example, publications usable in relation to
the described invention.
[0028]
15 In the present specification, the "nucleoside" to be the
constituent unit of oligonucleotide means a compound wherein a
nucleic acid base is bonded to the 1'-position of a sugar
(e.g., ribose) by N-glycosidation.
[0029]
20 In the present specification, the "nucleic acid base" is
not particularly limited as long as it can be used for the
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,
25 guanyl group and the like. The "optionally protected nucleic
acid base" 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, and
a nucleic acid base wherein the amino group therein is
30 protected by a protecting group sustainable under the
deprotection conditions of the 5'-position is preferable. The
"amino-protecting group" is not particularly limited, and
examples thereof include the protecting groups described in
PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 3rd edition, JOHN
35 WILLY&SONS, 1999 and the like. Specific examples of the
14
"amino-protecting group" include a pivaloyl group, a
pivaloyloxymethyl group, a trifluoroacetyl group, a
phenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a 4-
tert-butylphenoxyacetyl group, an acetyl group, a benzoyl
5 group, an isobutyryl group, a dimethylformamidinyl group, a 9-
fluorenylmethyloxycarbonyl group and the like. Among them, a
phenoxyacetyl group, a 4-isopropylphenoxyacetyl group, an
acetyl group, a benzoyl group, an isobutyryl group and a
dimethylformamidinyl group are preferable. In addition, the
io carbonyl group of the nucleic acid base is optionally
protected, and can be protected, for example, by reacting
phenol, 2,5-dichlorophenol, 3-chlorophenol, 3,5-dichlorophenol,
2-formylphenol, 2-naphthol, 4-methoxyphenol, 4-chlorophenol,
2-nitrophenol, 4-nitrophenol, 4-acetylaminophenol,
15 pentafluorophenol, 4-pivaloyloxybenzyl alcohol, 4-
nitrophenethyl alcohol, 2-(methylsulfonyl)ethanol, 2-
(phenylsulfonyl)ethanol, 2-cyanoethanol, 2-
(trimethylsilyl)ethanol, dimethylcarbamoyl chloride,
diethylcarbamoyl chloride, ethylphenylcarbamoyl chloride, 1-
20 pyrrolidinecarbonyl chloride, 4-morpholinecarbonyl chloride,
diphenylcarbamoyl chloride and the like. In some cases, the
carbonyl-protecting group does not need to be particularly
introduced. Moreover, in addition to the above-mentioned
groups, a modified nucleic acid base (e.g., a 8-bromoadenyl
25 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
base substituted by any 1 to 3 substituents (e.g., a halogen
30 atom, an alkyl group, an aralkyl group, an alkoxy group, an
acyl group, an alkoxyalkyl group, a hydroxy group, an amino
group, monoalkylamino, dialkylamino, carboxy, cyano, nitro
etc.) at any position(s), are also encompassed in the "nucleic
acid base".
35 [0030]
15
In the present specification, the "halogen atom" means a
fluorine atom, a chlorine atom, a bromine atom or iodine atom.
[0031]
In the present specification, examples of the "alkyl
5 (group)" include a linear or branched chain alkyl group having
one or more carbon atoms. When the carbon number is not
particularly limited, it is preferably a Ci_i0 alkyl group, more
preferably a Ci-6 alkyl group. When the carbon number is not
particularly limited, for example, methyl, ethyl, propyl,
10 isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl,
hexyl and the like are preferable, and methyl and ethyl are
particularly preferable.
[0032]
In the present specification, the "aralkyl (group)" means
15 a C7_2o aralkyl group, preferably a C7-i6 aralkyl group (a C6-10
aryl-Ci_6 alkyl group). Preferable specific examples include
benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl,
naphthylmethyl, 1-naphthylethyl, 1-naphthylpropyl and the like,
and benzyl is particularly preferable.
20 [0033]
In the present specification, examples of the "alkoxy
(group)" include an alkoxy group having one or more carbon
atoms. When the carbon number is not particularly limited, it
is preferably a Ci_i0 alkoxy group, more preferably a Ci_6 alkoxy
25 group. When the carbon number is not particularly limited,
methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, secbutoxy,
tert-butoxy, pentyloxy, hexyloxy and the like are
preferable, and methoxy and ethoxy are particularly preferable.
[0034]
30 In the present specification, examples of the "acyl
(group)" include a linear or branched chain Ci_6 alkanoyl group,
a C7-13 aroyl group and the like. Specific examples thereof
include formyl, acetyl, n-propionyl, isopropionyl, n-butyryl,
isobutyryl, pivaloyl, valeryl, hexanoyl, benzoyl, naphthoyl,
35 levulinyl and the like, each of which is optionally
16
substituted.
[0035]
In the present specification, examples of the "alkenyl
(group)" include a linear or branched chain C2-6 alkenyl group
5 and the like. Preferable examples thereof include vinyl, 1-
propenyl, allyl, isopropenyl, butenyl, isobutenyl and the like
Among them, a C2-C4 alkenyl group is preferable.
[0036]
In the present specification, examples of the "alkynyl
10 (group)" include a C2-6 alkynyl group and the like. Preferable
examples thereof include ethynyl, 1-propynyl, 2-propynyl, 1-
butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-
pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-
hexynyl, 5-hexynyl and the like. Among them, a C2-C4 alkynyl
15 group is preferable.
[0037]
In the present specification, the "cycloalkyl (group)"
means a cyclic alkyl group, and examples thereof include
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
20 cyclooctyl and the like. Among them, a C3-C6 cycloalkyl group
such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and
the like is preferable, and cyclohexyl is particularly
preferable.
[0038]
25 In the present specification, the "aryl (group)" means an
aromatic monocyclic or polycyclic (fused) hydrocarbon group.
Specific examples thereof include a C6_i4 aryl group such as
phenyl, 1-naphthyl, 2-naphthyl, biphenylyl, 2-anthryl and the
like, and the like. Among them, a C6-10 aryl group is more
30 preferably and phenyl is particularly preferable.
[0039]
In the present specification, examples of the
"hydrocarbon group" include an aliphatic hydrocarbon group, an
aromatic-aliphatic hydrocarbon group, a monocyclic saturated
35 hydrocarbon group, an aromatic hydrocarbon group and the like.
17
£ Specific examples thereof include a monovalent group such as
an alkyl group, an alkenyl group, an alkynyl group, a
cycloalkyl group, an aryl group, an aralkyl group and the like,
and a divalent group derived therefrom.
5 [0040]
In the present specification, the "organic group having a
hydrocarbon group" means a group having the aforementioned
"hydrocarbon group", and the moiety other than the
"hydrocarbon group" of the "organic group having a hydrocarbon
10 group" can be determined freely. For example, the organic
group optionally has, as a linker, a moiety such as -0-, -S-,
-COO-, -OCONH-, -CONH- and the like.
[0041]
[Pseudo solid phase protecting group]
is The pseudo solid phase protecting group used in the
present invention is not particularly limited as long as it is
a protecting group simultaneously satisfying the reactivity
and easiness of work-up, by binding to a reactive substrate to
solubilize same in a non-polar solvent, thus enabling reaction
20 in the liquid phase, and forming precipitation upon addition
of a polar solvent to enable solid-liquid separation, and
stable under acidic conditions capable of removing the 5'-
terminal hydroxyl-protecting group. While examples of the
pseudo solid phase protecting group include a group disclosed
25 in non-patent documents 2 to 4, patent document 1 and the like,
particularly, a pseudo solid phase protecting group
represented by the following formula (I) is preferable, since
a high yield can be achieved in the objective production
method of oligonucleotide.
30 [0042]
A group represented by the formula (I):
[0043]
-L-Y-Z (I)
[0044]
35 wherein
L is a group (linker) represented by the formula (al]
[0045]
0
**
L< ^*
O
(a1)
[0046]
5 wherein shows the bonding position to Y;
** indicates the bonding position to a group to be
protected;
Li is an optionally substituted divalent C1-22 hydrocarbon
group; and
10 L2 is a single bond, or a group (linker) represented by
**C (=0)N (R2) -R1-N (R3) *** wherein ** shows the bonding position
to Li, *** shows the bonding position to C=0, R1 is an
optionally substituted Ci_22 alkylene group, and R2 and R3 are
each independently a hydrogen atom or an optionally
15 substituted C1-22 alkyl group, or R2 and R3 are optionally
joined to form an optionally substituted C1-22 alkylene bond,
Y is a single bond, 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):
20 [0047]
kt^OR5>* ^^
25
[0048]
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;
R5 in the number of k are each independently an organic
19
group having an aliphatic hydrocarbon group having 10 or
more carbon atoms;
k is an integer of 1 to 4;
ring A optionally further has, in addition to OR5 in the
5 number of k, substituent(s) selected from the group
consisting of a halogen atom, a Ci_6 alkyl group optionally
substituted by a halogen atom, and a Ci_6 alkoxy group
optionally substituted by a halogen atom;
Ra is a hydrogen atom; and
io Rb is a hydrogen atom, or a group represented by the formula
(a3) :
[0049]
15 wherein * shows a bonding position;
j is an integer of 0 to 4;
R7 in the number of j are each independently an organic
group having an aliphatic hydrocarbon group having 10 or
more carbon atoms;
20 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 OR7 in the
number of j, substituent(s) selected from the group
25 consisting of a halogen atom, a Ci_6 alkyl group
optionally substituted by a halogen atom, and a Ci_6
alkoxy group optionally substituted by a halogen atom,
or
a group represented by the formula (a2'):
30 [0051]
20
^(0R5')k.
(a2')
[0052]
wherein shows a bonding position;
R5' in the number of k' are each independently is an organic
5 group having an aliphatic hydrocarbon group having 10 or
more carbon atoms;
k' is an integer of 1 to 4; and
ring C optionally further has, in addition to OR5' in the
number of k', substituent(s) selected from the group
10 consisting of a halogen atom, a Cis alkyl group optionally
substituted by a halogen atom, and a Ci-6 alkoxy group
optionally substituted by a halogen atom.
[0053]
Examples of a group to be protected by the pseudo solid
15 phase protecting group represented by the formula (I) include
a hydroxy group, an amino group and the like, particularly a
3'-hydroxy group of a nucleoside or a nucleotide is preferable.
Preferable embodiment of the linker L represented by the
above-mentioned formula (al) is a group wherein, in the
20 formula (al) ,
Li is an ethylene group or CH2-0-l,4-phenylene-0-CH2; and
L2 is a single bond, or a group represented by **C (=0) N (R2) -R1-
N(R3)*** wherein ** shows the bonding position to Li, *** shows
the bonding position to C=0, R1 is an optionally substituted Ci_
25 6 alkylene group, and R2 and R3 are each independently a
hydrogen atom or an optionally substituted Ci_6 alkyl group, or
R2 and R3 are optionally joined to form an optionally
substituted Ci_6 alkylene bond.
[0054]
30 Another preferable embodiment of the linker L represented
by the above-mentioned formula (al) is a group wherein, in the
formula (al) ,
21
Li i s an ethylene group; and
L2 i s a single bond.
[0055]
Another preferable embodiment of the linker L represented
5 by the above-mentioned formula (al) is a group wherein, in the
formula (al),
Li is an ethylene group; and
the moiety N (R2) -R^N (R3) of L2 is a piperazinylene group.
[0056]
io Another preferable embodiment of the linker L represented
by the above-mentioned formula (al) is a group wherein, in the
formula (al),
Li is an ethylene group; and
L2 is a group represented by **C (=0) N (R2) -Rx-N (R3) *** wherein
15 shows the bonding position to Li, shows the bonding position
to C=0, R1 is a pentylene group or a hexylene group, and R2 and
R3 are each independently hydrogen atom or a methyl group.
[0057]
A particularly preferable example of the above-mentioned
20 linker L is a succinyl group since it is economical and easily
available.
[0058]
Preferable embodiment of Y is an oxygen atom, or NR
wherein R is a hydrogen atom, an alkyl group or an aralkyl
25 group.
R is preferably a hydrogen atom, a Ci_6 alkyl group or a
C7-i6 aralkyl group, more preferably a hydrogen atom, methyl,
ethyl or benzyl, particularly preferably a hydrogen atom.
[0059]
30 Preferable embodiment of Z is a group represented by the
formula (a2).
Preferable embodiment of Z represented by the abovementioned
formula (a2) is a group wherein, in the formula (a2),
Ra and Rb are both hydrogen atoms;
35 R4 is a hydrogen atom,
22
R5 in the number of k are each independently is an organic
group having an aliphatic hydrocarbon group having 10 or more
carbon atoms (e.g., C10-40 alkyl group); and
k is an integer of 1 to 3.
5 [0060]
Another preferable embodiment of Z represented by the
above-mentioned formula (a2) is a group wherein, in the
formula (a2),
k is an integer of 1 to 3;
10 Ra and Rb are both hydrogen atoms;
R4 is a hydrogen atom;
R5 in the number of k are each independently benzyl group
having 1 to 3 aliphatic hydrocarbon groups having 10 or more
carbon atoms, or a cyclohexyl group having 1 to 3 aliphatic
15 hydrocarbon groups having 10 or more carbon atoms; and
ring A optionally further has, in addition to OR5 in the number
of k, substituent(s) selected from the group consisting of a
halogen atom, a Ci-6 alkyl group optionally substituted by a
halogen atom, and a C1-6 alkoxy group optionally substituted by
20 a halogen atom.
[0061]
Another preferable embodiment of Z represented by the
above-mentioned formula (a2) is a group wherein, in the
formula (a2),
25 Ra is a hydrogen atom; and
Rb is a group represented by the above-mentioned formula (a3)
wherein * shows a bonding position; j is an integer of 0 to 3;
R7 in the number of j are each independently a C10-40 alkyl
group; and R4 and R6 are both hydrogen atoms.
30 [0062]
Another preferable embodiment of Z represented by the
above-mentioned formula (a2) is a group wherein, in the
formula (a2),
Ra is a hydrogen atom;
35 Rb is a group represented by the above-mentioned formula (a3)
23
wherein * shows a bonding position; j is an integer of 0 to 3;
R7 in the number of j are each independently a C10-40 alkyl
group;
R6 is joined with R4 of ring A to form a single bond or -0-,
5 and therefore, ring A and ring B form a fluorenyl group or a
xanthenyl group in combination.
[0063]
The pseudo solid phase protecting group represented by
the above-mentioned formula (I) is preferably a group
10 difficult to cleave under acidic conditions that permit
removal of the protecting group of 5'-terminal hydroxyl and
easy to cleave under basic conditions.
[0064]
Representative examples of the pseudo solid phase
15 protecting group include a group wherein, in the abovementioned
of the formula (I),
L is a group represented by the above-mentioned formula (al)
(preferably a succinyl group etc.), and
Y-Z is the following group:
20 a 3,4,5-tri(octadecyloxy)benzyloxy group,
a 3,5-di(docosyloxy)benzyloxy group,
a 3,5-di[3',4',5'-tri(octadecyloxy)benzyloxy]benzyloxy group,
a 3,4,5-tri[3',4',5'-tri(octadecyloxy)benzyloxy]benzyloxy
group,
25 a 3,4,5-tri(octadecyloxy)benzylamino group,
a 2,4-di(docosyloxy)benzylamino group,
a 3,5-di(docosyloxy)benzylamino group,
a di(4-docosyloxyphenyl)methylamino group,
a 4-methoxy-2-[3',4',5'-tri(octadecyloxy)benzyloxy]benzylamino
30 group,
a 4-methoxy-2-[3',4',5'-
tri(octadecyloxy)cyclohexylmethyloxy]benzylamino group,
a 2,4-di(dodecyloxy)benzylamino group,
a phenyl(2,3,4-tri(octadecyloxy)phenyl)methylamino group,
35 a di[4-(12-docosyloxydodecyloxy)phenyl]methylamino group,
24
^p%
^ ^ ^ j
a 3,5-di[3',4',5'-tri(octadecyloxy)benzyloxyJbenzylanri.no group,
or
a 3,4,5-tri[3f,4',5'-tri(octadecyloxy)benzyloxylbenzylamino
group.
5 [0065]
[Nucleotide wherein the 3'-hydroxyl group is protected by a
pseudo solid phase protecting group and the 5'-hydroxyl group
is protected by a temporary protecting group]
The nucleotide suitable for pseudo solid phase synthesis
10 can be produced by bonding a pseudo solid phase protecting
group used in the present invention to a 3'-hydroxyl group of
nucleotide.
A particularly preferable nucleotide to achieve a high
yield in the objective production method of oligonucleotide is,
15 for example, a compound represented by the following formula
(II) (hereinafter sometimes to be referred to as the compound
of the present invention).
[0066]
The formula (II):
20 [0067]
Q—O
WG.
Base
o //
R8
^CX
m
(M)
Base
^
0
\ L—Y—Z
[0068]
wherein
m is an integer of 0 or more,
25 Base in the number of m+1 are each independently an optionally
25
protected nucleic acid base,
Q is a hydrogen atom or a temporary protecting group removable
under acidic conditions,
X is a hydrogen atom, a halogen atom or an optionally
5 protected hydroxyl group,
X' in the number of m are each independently a hydrogen atom,
a halogen atom, or an optionally protected hydroxyl group,
R8 in the number of m are each independently an oxygen atom or
a sulfur atom,
10 WG in the number of m are each independently an electronwithdrawing
group,
Y is a single bond, an oxygen atom or NR wherein R is a
hydrogen atom, an alkyl group or an aralkyl group, and
L and Z are as defined above, except that for the formula
15 (la) indicates the bonding position to a 3'-hydroxy group of
the nucleoside.
[0069]
Of the compounds of the present invention, a compound of
the formula (II) wherein Y is an oxygen atom or NR wherein R
20 is a hydrogen atom, an alkyl group or an aralkyl group is a
novel compound.
[0070]
In the compound of the present invention, p-mer
oligonucleotide (p is an integer of one or more) wherein the
25 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+1+p-mer
oligonucleotide (p is an integer of one or more).
When m is 0, the compound of the present invention is to
30 be understood as "nucleoside", which 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 (Q is a hydrogen
atom) in a wide sense.
35 [0071]
26
m is an integer of 0 or more, preferably 0. While the
upper limit of m is not particularly limited, it is preferably
4 9 or less, more preferably 2 9 or less, and further preferably
19 or less.
5 Group X and X' in the number of m at the 2-position of
ribose residue constituting nucleotide, which is the compound
of the present invention, are each independently a hydrogen
atom, a halogen atom or an optionally protected hydroxyl group.
As the halogen atom, a fluorine atom or a chlorine atom
io is preferable, and a fluorine atom is more preferable.
While the protecting group of the "optionally protected
hydroxyl group" is not particularly limited, for example, any
protecting group described in PROTECTIVE GROUPS IN ORGANIC
SYNTHESIS, 3rd ed., JOHN WILLY&SONS (1999) and the like can be
15 mentioned. Specifically, methyl group, benzyl group, pmethoxybenzyl
group, tert-butyl group, methoxymethyl group,
methoxyethyl group, 2-tetrahydropyranyl group, ethoxyethyl
group, cyanoethyl group, cyanoethoxymethyl group,
phenylcarbamoyl group, 1,l-dioxothiomorpholine-4-thiocarbamoyl
20 group, acetyl group, pivaloyl group, benzoyl group,
trimethylsilyl group, triethylsilyl group, triisopropylsilyl
group, tert-butyldimethylsilyl group,
[(triisopropylsilyl)oxy]methyl(Tom) group, 1-(4-chlorophenyl)-
4-ethoxypiperidin-4-yl(Cpep) group and the like can be
25 mentioned. Among these, triethylsilyl group, triisopropylsilyl
group and tert-butyldimethylsilyl group are preferable. From
the aspects of economic efficiency and easy availability,
tert-butyldimethylsilyl group is particularly preferable.
[0072]
30 Temporary protecting group Q that can be used as 5'-
hydroxyl-protecting group of the compound of the present
invention is not particularly limited as long as it can be
deprotected under acidic conditions and can be used as a
hydroxyl-protecting group and, for example, trityl group, 9-
35 (9-phenyl)xanthenyl group, 9-phenylthioxanthenyl group, di(Ci_6
27
alkoxy)trityl groups such as 1,1-bis(4-methoxyphenyl)-1-
phenylmethyl group (dimethoxytrityl group) and the like,
mono(Ci-i8 alkoxy) trityl groups such as 1-(4-methoxyphenyl)-1,1-
diphenylmethyl group (monomethoxytrityl group) and the like
5 can be mentioned. Among these, monomethoxytrityl group and
dimethoxytrityl group are preferable, and dimethoxytrityl
group is more preferable, from the aspects of easy
deprotection and easy availability.
R8 in the number of m are each independently an oxygen
10 atom or a sulfur atom, preferably an oxygen atom.
WG in the number of m are each independently an electronwithdrawing
group. Examples of the electron-withdrawing group
include a cyano group, a nitro group and the like, preferably
a cyano group.
is [0073]
The "organic group having an aliphatic hydrocarbon group
having 10 or more carbon atoms" for R5 or R7 in the present
specification is a monovalent organic group having an
aliphatic hydrocarbon group having a carbon number of 10 or
20 more in the molecule structure thereof.
[0074]
The "aliphatic hydrocarbon group" of the "aliphatic
organic group having a hydrocarbon group" is a straight chain
or branched saturated or unsaturated aliphatic hydrocarbon
25 group, and an aliphatic hydrocarbon group having a carbon
number of 10 or more is preferable, an aliphatic hydrocarbon
group having a carbon number of 10 to 40 is more preferable,
and an aliphatic hydrocarbon group having a carbon number of
12 to 30 is particularly preferable.
30 The position of the "aliphatic hydrocarbon group" of the
"aliphatic organic group having a hydrocarbon group" is not
particularly limited, and may be present on the terminal
(monovalent group) or a position other than the terminal (for
example, divalent group).
35 [0075]
28
As the "aliphatic hydrocarbon group", a monovalent group
such as a linear or branched chain alkyl group having a carbon
number of not less than 10, a linear or branched chain alkenyl
group and the like, and a divalent group induced therefrom can
5 be mentioned. Of these, an alkyl group having a carbon number
of 10 to 40 is preferable, and an alkyl group having a carbon
number of 12 to 30 is particularly preferable. Specific
examples of the "aliphatic hydrocarbon group" include
monovalent groups such as decyl group, dodecyl group, tridecyl
io group, myristyl group, cetyl group, stearyl group, oleyl group,
linoleyl group, arachyl group, behenyl group, isostearyl group
and the like and divalent groups induced therefrom.
[0076]
The moiety other than the "aliphatic hydrocarbon group"
is of the "aliphatic organic group having a hydrocarbon group"
can be optionally determined. For example, as a linker, a
moiety such as -0-, -S-, -COO-, -OCONH- and -CONH-, as well as
a hydrocarbon group (monovalent group or divalent group) and
the like may be present. Examples of the "hydrocarbon group"
20 include an aliphatic hydrocarbon group, an aromatic-aliphatic
hydrocarbon group, a monocyclic saturated hydrocarbon group,
an aromatic hydrocarbon group and the like, and specific
examples thereof include monovalent groups such as an alkyl
group, an alkenyl group, an alkynyl group, a cycloalkyl group,
25 an aryl group, an aralkyl group and the like and a divalent
group induced therefrom. As "alkyl group", "alkenyl group",
"alkynyl group", "cycloalkyl group", "aryl group", or "aralkyl
group" as the moiety other than "aliphatic hydrocarbon group",
those similar to the aforementioned groups can be mentioned.
30 The "hydrocarbon group" is optionally substituted by a
substituent selected from a halogen atom (chlorine atom,
bromine atom, fluorine atom, iodine atom), a Ci_6 alkyl group
optionally substituted by one or more halogen atoms, an oxo
group and the like.
35 [0077]
29
In the present specification, R constituting Y in the
formula (II) is a hydrogen atom, an alkyl group or an aralkyl
group, preferably a hydrogen atom, a Ci-6 alkyl group or a C7-16
aralkyl group, more preferably a hydrogen atom, methyl, ethyl
5 or benzyl, and particularly preferably a hydrogen atom.
[0078]
The "aliphatic organic group having a hydrocarbon group"
for "R5 (group)" and/or "R7 (group)" constituting Z in the
above-mentioned formula (II) may contain plural "aliphatic
10 hydrocarbon groups" due to a branch and the like. When plural
"aliphatic hydrocarbon groups" are present in the "aliphatic
organic group having a hydrocarbon group", they may be the
same or different.
[0079]
15 The lower limit of the total carbon number of the
"aliphatic organic group having a hydrocarbon group" for
"R5 (group)" and/or "R7 (group)" constituting Z in the abovementioned
formula (II) is preferably 10 or more, more
preferably 12 or more, further preferably 14 or more, still
20 more preferably 18 or more, and particularly preferably 30 or
more. On the other hand, the upper limit of the total carbon
number of the "aliphatic organic group having a hydrocarbon
group" for "R5 (group)" and/or "R7 (group)" is preferably 200 or
less, more preferably 150 or less, further preferably 120 or
25 less, still more preferably 100 or less, especially preferably
80 or less, and particularly preferably 60 or less. When the
carbon number is higher, the crystallinity of the compound of
the present invention in a polar solvent is fine even when
oligonucleotide has a long chain.
30 [0080]
A preferable embodiment of Y-Z in the formula (II) is the
same as the preferable embodiment of Y-Z in the aforementioned
formula (I).
[0081]
35 A preferable embodiment of the compound represented by
30
the formula (II) of the present invention is a compound of the
formula (II), wherein m is 0,
Base is a cytosyl group, a uracil group, a thyminyl group, an
adenyl group, or a guanyl group, each of which is optionally
5 protected;
Q is a di(Ci_6 alkoxy) trityl group, or a mono (Ci_6 alkoxy) trityl
group;
X is a hydrogen atom, a halogen atom, or an optionally
protected hydroxyl group; and
10 L-Y-Z is the combination of each group shown as a preferable
embodiment in the aforementioned formula (I).
[0082]
Another preferable embodiment of the compound represented
by the formula (II) of the present invention is a' compound of
15 the formula (II), wherein m is 0,
Base is a cytosyl group, a uracil group, a thyminyl group, an
adenyl group, or a guanyl group, each of which is optionally
protected;
Q is a dimethoxytrityl group or a monomethoxytrityl group;
20 X is a hydrogen atom, a halogen atom, or an optionally
protected hydroxyl group; and
L-Y-Z is the combination of each group shown as a preferable
embodiment in the aforementioned formula (I).
[0083]
25 A still another preferable embodiment of the compound
represented by the formula (II) of the present invention is a
compound of the formula (II), wherein m is 0,
Base is a cytosyl group, a uracil group, a thyminyl group, an
adenyl group, or a guanyl group, each of which is optionally
30 protected;
Q is a dimethoxytrityl group;
X is a hydrogen atom, a fluorine atom, a methoxy group, an
acetoxy group, or a tert-butyldimethylsilyloxy group; and
L-Y-Z is the combination of each group shown as a preferable
35 embodiment in the aforementioned formula (I).
31
compound.
[0084]
[Production method of precursor (Z-Y-H) (alcohol or amine) of
pseudo solid phase protecting group]
5 While the production method of a precursor of the
aforementioned pseudo solid phase protecting group is not
particularly limited, it can be produced from a starting
material compound according to a method known per se (e.g.,
Bull. Chem. Soc. Jpn. 2001, 74, 733-738, JP-A-2000-44493,
io WO2006/104166, WO2007/034812, WO2007/122847, WO2010/113939
etc.) or a method analogous thereto.
A compound to be used as a starting material compound,
for example, a halide corresponding to R5 and R7 constituting Z
in the formula (II) and the like is a commercially available
15 product, or can be produced according to a method known per se
or a method analogous thereto.
[0085]
While the production method of the compound represented
by the formula (II) of the present invention wherein m is 0,
20 hereinafter referred to as "the formula (Ila)", is not
particularly limited, it can be produced from the abovementioned
precursor of a pseudo solid phase protecting group
by a method known per se (Richard T. Pon et al., Nucleic Acids
Research 2004, 32, 623-631) or a method analogous thereto.
25 The precursor (Z-Y-H) of the pseudo solid phase
protecting group can be produced by a method known per se or a
method analogous thereto, as mentioned above. When a starting
material compound has a substituent (e.g., hydroxyl group,
amino group, carboxy group) that influences the reaction, the
30 starting material compound is generally protected in advance
by a suitable protecting group according to a known 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
35 like.
32
A general production method of a compound of the abovementioned
formula (Ila) wherein L is a succinyl group is shown
below.
[0086]
0
Y-0 QO^ Base Q0 ^ Base
QCX . . o 1 ^ 1
y—< ~ °v
x
o-
Z—Y—H
Q X
OH X >—, O condensing h—v 0
(a) f 1) acid,
fa L cation scavenger
W G A \ i " WG
• / T ] ^o Base ^o rga n | Cbase i A. \ i WG /v > 1
V V n > n Base 2) organic base V V / / r o Base
'J i n i l o I '/Jml A I
0)
[0101]
37
wherein R in the number of m is an oxygen atom or a sulfur
atom, WG in the number of m is an electron-withdrawing group
(e.g., cyano group), X' in the number of m is each
independently as defined for X, and other symbols are as
5 defined above.
[0102]
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
10 solvent showing high solubility of the compound of the present
invention is preferably selected. Specifically, 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
15 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 more kinds of
20 these solvents may be used in a mixture in an appropriate
ratio. In addition, the above-mentioned non-polar 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,N-dimethylformamide, N,N-
25 dimethylacetamide, N-methylpiperidone and the like, as long as
n-mer oligonucleotide is dissolved. Among them,
dichloromethane, chloroform, 1,2-dichloroethane, benzene,
toluene, xylene, mesitylene, hexane, pentane, heptane, nonane,
cyclohexane, ethyl acetate, isopropyl acetate, tert-butyl
30 methyl ether, cyclopentyl methyl ether, combinations thereof
and the like are preferable, and chloroform, dichloromethane
and toluene are particularly preferable.
[0103]
In this step, the concentration of n-mer oligonucleotide
35 (i) in a solvent is not particularly limited as long as the
38
oligonucleotide is dissolved, it is preferably 1 to 30 wt%.
[0104]
To continuously perform the deprotection step, subsequent
condensation step, and oxidation step in a solution, it is
5 essential to use a cation scavenger in this step during or
after the removal reaction of a temporary protecting group Q
of 5'-hydroxyl group in n-mer oligonucleotide (i).
[0105]
While the cation scavenger is not particularly limited as
10 long as re-protection (returning to starting material) with
the removed protecting group Q and side reaction with the
deprotected functional group do not proceed, pyrrole
derivatives such as pyrrole, 2-methylpyrrole, 3-methylpyrrole,
2,3-dimethylpyrrole, 2,4-dimethylpyrrole and the like; and
15 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-
methylpyrrole, 2,4-dimethylpyrrole, indole, 4-methylindole, 5-
20 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
preferable.
25 [0106]
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).
[0107]
30 While the acid to be used in this step is not
particularly limited as long as good deprotection can be
achieved, trifluoroacetic acid, dichloroacetic acid,
trifluoromethanesulfonic acid, trichloroacetic acid,
methanesulfonic acid, hydrochloric acid, acetic acid, p-
35 toluenesulfonic acid and the like are preferably used. Since
39
good reaction can be achieved, trifluoroacetic acid,
dichloroacetic acid, trifluoromethanesulfonic acid and
trichloroacetic acid are more preferable, trifluoroacetic acid,
dichloroacetic acid and trifluoromethanesulfonic acid are more
5 preferable, trifluoroacetic acid and trifluoromethanesulfonic
acid are still more preferable, and trifluoroacetic acid is
particularly preferable. These acids may be diluted with the
above-mentioned non-polar solvent. When the aforementioned
acid is used, it may be combined with a particular base to
10 appropriately adjust the acidity before use.
[0108]
The amount of the acid to be used in this step is 1 to
100 mol, preferably 1 to 40 mol, per 1 mol of n-mer
oligonucleotide (i).
is [0109]
While the reaction temperature in this step is not
particularly limited as long as the reaction proceeds, it is
preferably -10°C to 50°C, more preferably 0°C to 40°C. While
the reaction time varies depending on the kind of n-mer
20 oligonucleotide to be used, the kind of acid, the kind of
solvent, the reaction temperature and the like, it is 5 min to
5 hr.
[0110]
When an acid used as a deprotecting agent is present in
25 the condensation step of the next step, deprotection of
protecting group Q of p-mer oligonucleotide (iii) wherein the
5'-hydroxyl group is protected by temporary protecting group Q,
and the 3'-hydroxy1 group is phosphoramidited is induced.
Therefore, the acid needs to be removed or neutralized. Since
30 condensation step is continuously performed in the reaction
mixture in the present invention, neutralization by an organic
base is performed.
The organic base to be used for neutralization is not
particularly limited as long as it can neutralize the above-
35 mentioned acids, and the obtained salt can function as a
40
condensing agent. Since the reaction proceeds smoothly,
pyridine, 2,4,6-trimethylpyridine, benzimidazole, 1,2,4-
triazole, N-phenylimidazole, 2-amino-4,6-dimethylpyrimidine,
1,10-phenanthroline, imidazole, N-methylimidazole, 2-
5 chlorobenzimidazole, 2-bromobenzimidazole, 2-methylimidazole,
2-phenylbenzimidazole, N-phenylbenzimidazole and 5-
nitrobenzimidazole are preferable, pyridine, 2,4,6-
trimethylpyridine, benzimidazole, 1,2,4-triazole, Nphenylimidazole,
N-methylimidazole, 2-amino-4,6-
io dimethylpyrimidine and 1,10-phenanthroline are more preferable,
pyridine, 2,4,6-trimethylpyridine, benzimidazole, 1,2,4-
triazole and N-phenylimidazole are further preferable,
pyridine, 2,4,6-trimethylpyridine, benzimidazole and 1,2,4-
triazole are still more preferable, and pyridine, 2,4,6-
15 trimethylpyridine and benzimidazole are particularly
preferable.
[0111]
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.
20 [0112]
A particularly preferable combination of acid and organic
base in this step is trifluoroacetic acid and pyridine,
trifluoroacetic acid and 2,4,6-trimethylpyridine or
trifluoromethanesulfonic acid and benzimidazole.
25 [0113]
step (2) (condensation step)
In this step, n-mer oligonucleotide (ii) wherein the 5'-
hydroxyl group is deprotected, which is obtained in the
aforementioned step (1), and p-mer oligonucleotide (iii) (q is
30 any integer of 0 or more, when q=0, it is nucleoside) wherein
the 5'-hydroxyl group is protected by temporary protecting
group Q, and the 3'-hydroxy1 group is phosphoramidited are
condensed.
As the p-mer oligonucleotide (iii) wherein the 5'-
35 hydroxyl group is protected by temporary protecting group Q,
41
the 3'-hydroxyl group is phosphoramidited, the compound
wherein p is 1 (i.e., nucleoside wherein the 5'-hydroxyl group
is protected by temporary protecting group Q, and the 3'-
hydroxyl group is phosphoramidited) is preferable.
5 While the upper limit of q is not particularly limited,
it is preferably 49 or less, more preferably 29 or less,
further preferably 19 or less, still more preferably 4 or less,
and particularly preferably 2 or less.
[0114]
42
Base
Base
R9 ^R1
Base
L—Y—Z
[0115]
wherein X' is as defined for X, R9 and R10 are each
independently an alkyl group, or a 5- or 6-membered saturated
5 cyclic amino group formed together with the adjacent nitrogen
atom. The saturated cyclic amino group optionally has one
oxygen atom or sulfur atom as a ring-constituting atom besides
nitrogen atom. Other symbols are as defined above.
[0116]
43
This step can be simply performed by directly adding pmer
oligonucleotide (iii) wherein the 5'-hydroxy1 group is
protected by temporary protecting group Q, and the 3'-hydroxyl
group is phosphoramidited to the reaction mixture after step
5 (1) without isolation of n-mer oligonucleotide (ii) wherein
5'-hydroxyl group is deprotected, which is obtained in the
aforementioned step (1). 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
io during neutralization reaction, acts as a condensing agent.
Therefore, steps (1) and (2) continuously performed in a
solution provide advantages of not only omission of an
isolation operation but also improved reaction efficiency.
When the condensation reaction proceeds slowly, the reaction
15 efficiency can also be further improved by additionally adding
a separate condensing agent (e.g., pyridine trifluoroacetate,
tetrazole, 5-benzylthio-lH-tetrazole, 4,5-dicyanoimidazole
etc.).
[0117]
20 In this step, moreover, when the acidity of the reaction
mixture becomes high, a side reaction removing temporary
protecting group Q may occur. Therefore, N-methylimidazole is
preferably added to suppress acidification of the reaction
mixture.
25 [0118]
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.
[0119]
30 The p-mer oligonucleotide (iii) wherein the 5'-hydroxyl
group is protected by temporary protecting group Q, and the
3'-hydroxyl group is phosphoramidited to be used in this step
is, for example, p-mer oligonucleotide wherein a group
represented by the following formula:
35 [0120]
44
WG
R9'%10
[0121]
wherein each symbol is as defined above, is bonded. As the
nucleic acid base of the p-mer oligonucleotide, the groups as
5 defined above can be mentioned.
[0122]
The p-mer oligonucleotide (iii) wherein the 5'-hydroxyl
group is protected by temporary protecting group Q, and the
3'-hydroxyl group is phosphoramidited to be used in this step
io can be produced by a known method (M.H. Caruthers et al.,
Method in Enzymology 1987, 154, 287-313; S.L. Beaucage and M.H.
Caruthers, Tetrahedron Letters 1981, 22, 1859-1862), including
reacting p-mer oligonucleotide wherein the 5'-hydroxyl group
is protected by temporary protecting group Q, and the 3'-
15 hydroxyl group is not protected with a phosphoramiditing
reagent represented by the following formulas (c) or (d);
[0123]
WG
(C)
[0124]
• N , >10
20 In the formula (c) , (d) , R11 is a halogen atom, and other
symbols are as defined above.
[0125]
This step is performed in a solvent that does not
influence the reaction. Specifically, a non-polar solvent
25 similar to the one used in the aforementioned step (1) can be
mentioned. The above-mentioned non-polar solvent may be mixed
with nitrile solvents such as acetonitrile, propionitrile and
the like; ketone solvents such as acetone, 2-butanone and the
45
like; amide solvents such as N,N-dimethylformamide, N,Ndimethylacetamide,
N-methylpyrrolidone and the like; polar
ether solvents such as 1,4-dioxane, tetrahydrofuran and the
like; sulfoxide solvents such as dimethylsulfoxide and the
5 like at an appropriate ratio, as long as n-mer oligonucleotide
(ii) wherein the temporary protecting group of 5'-hydroxyl
group is removed can be dissolved.
In this case, as the polar solvent, amide solvent,
nitrile solvent, and a combination thereof are preferable,
10 acetonitrile, N,N-dimethylformamide, N-methylpiperidone, and a
combination thereof are more preferable, and acetonitrile is
particularly preferable.
A polar solvent may be added as a solution of p-mer
oligonucleotide (iii) wherein the 5'-hydroxyl group is
15 protected by temporary protecting group Q, and the 3'-hydroxyl
group is phosphoramidited, a condensing agent and the like.
[0126]
The amount of p-mer oligonucleotide (iii) wherein the 5'
hydroxyl group is protected by temporary protecting group Q,
20 and the 3'-hydroxyl group is phosphoramidited to be used is 1
to 10 mol, preferably 1 to 5 mol, per 1 mol of the n-mer
oligonucleotide (ii) obtained in step (1), wherein the
temporary protecting group of 5'-hydroxyl group is removed.
[0127]
25 While the reaction temperature is not particularly
limited as long as the reaction proceeds, 0°C to 100°C is
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
30 the like, it is 5 min to 24 hr.
[0128]
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
35 convert the phosphite triester bond in the n+p-mer
46
oligonucleotide (iv) to a phosphate triester bond or a
111 thiophosphate triester bond.
[0129]
47
'^p Q- "°\ Base
-0~
d x-
WG.
O
//
R8
WG.
tX Base
^
oxidizing agent or
sulfurizing agent
O X'
O—P- 0 Base
-O-
(iv) >
& x'
WG.
Q- ~ 0 \ Base
R8 XX
m
Base
CX
d x1
O X
L—Y—Z
WG.
Base
[0130]
wherein each symbol is as defined above,
48
[0131]
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
5 (iv) obtained in step (2).
[0132]
While the "oxidizing agent" to be used in this step is
not particularly limited as long as it can oxidize a phosphite
triester bond into a phosphate triester bond without oxidizing
10 other moieties, iodine, (IS)-(+)-(10-
camphorsulfonyl)oxaziridine, tert-butyl hydroperoxide (TBHP),
2-butanone peroxide, 1,1-dihydroperoxycyclododecane,
bis(trimethylsilyl)peroxide or m-chloroperbenzoic acid is
preferably used. Since good oxidation reaction can be achieved,
15 iodine, (IS)-(+)-(10-camphorsulfonyl)oxaziridine, tert-butyl
hydroperoxide, 2-butanone peroxide and 1,1-
dihydroperoxycyclododecane are more preferable, iodine, (1S)-
(+)-(10- camphorsulfonyl)oxaziridine, tert-butyl hydroperoxide
and 2-butanone peroxide are more preferable, iodine and tert-
20 butyl hydroperoxide are still more preferable, and iodine is
particularly preferable. The oxidizing 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 inert to the reaction, pyridine, THF,
25 dichloromethane, water and a mixed solvent of any of them can
be mentioned. Among them, for example,
iodine/water/pyridine—THF or iodine/pyridine—acetic acid,
peroxide (TBHP)/dichloromethane or tert-butyl
hydroperoxide/nonane is preferably used.
30 [0133]
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
35 (DDTT), 3H-1,2-benzodithiol-3-one-l,1-dioxide (Beaucage
49
reagent), 3H-1,2-benzodithiol-3-one, phenylacetyl disulfide
(PADS), tetraethylthiuram disulfide (TETD), 3-amino-l,2,4-
dithiazole-5-thione (ADTT) or sulfur is preferably used. Since
a good reaction proceeds, 3-((N,N-
5 dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione
(DDTT), 3H-l,2-benzodithiol-3-one-l,1-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
io and 3H-1,2-benzodithiol-3-one-l,1-dioxide are further
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
15 dilution solvent is not particularly limited as long as it is
inert to the reaction, for example, dichloromethane,
acetonitrile, pyridine and a mixed solvent of any of them can
be mentioned.
[0134]
20 The amount of the oxidizing agent or sulfurizing agent to
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).
[0135]
While the reaction temperature is not particularly
25 limited as long as the reaction proceeds, 0°C to 100°C is
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
30 like, it is 1 min to 3 hr.
[0136]
step (4) (precipitation and solid-liquid separation step)
In this step, a polar solvent is added to a reaction
mixture containing n+p-mer oligonucleotide (v) having a
35 phosphate triester bond or a thiophosphate triester bond,
50
which is obtained in step (3), to allow precipitation of the
n+p-mer oligonucleotide (v), and the precipitate is obtained
by solid-liquid separation.
[0137]
5 Examples of the polar solvent used to precipitate the
object product n+p-mer oligonucleotide (v) in this step
include alcohol solvents such as methanol, ethanol,
isopropanol and the like; nitrile solvents such as
acetonitrile, propionitrile and the like; ketone solvents such
10 as acetone, 2-butanone and the like; polar ether solvents such
as 1,4-dioxane, tetrahydrofuran and the like; amide solvents
such as dimethylformamide, dimethylacetamide, Nmethylpiperidone
and the like, sulfoxide solvents such as
dimethylsulfoxide and the like; water etc., and mixed solvent
is of two or more kinds thereof. Among them, alcohol solvents and
nitrile solvents are preferably used, and methanol and
acetonitrile are more preferably used. The polar solvent in
the present invention is preferably methanol, particularly
from the practical aspects.
20 The polar solvent may contain water to minimize the loss
of the object product in a polar solvent. Particularly, when
acetonitrile is used as a polar solvent, the object product
tends to dissolve in the polar solvent to increase the loss.
Using acetonitrile containing a small amount of water, the
25 loss can be minimized.
In this case, the content of water in the polar solvent
is preferably 1 to 10% (v/v), more preferably 3 to 8% (v/v).
When the water content is too low, the loss of the object
product in a polar solvent tends to increase, and when the
30 water content is too high, removal of unnecessary substances
such as excess monomer etc. into a polar solvent tends to be
insufficient.
[0138]
In precipitation where iodine is used as an oxidizing
35 agent, the color development due to iodine can be removed by
51
using a solution of methanol, which is a precipitation solvent,
saturated with sodium thiosulfate (hypo), and therefore, n+pmer
oligonucleotide (v) wherein the 5'-hydroxyl group is
protected can be isolated with high purity.
5 [0139]
In precipitation where a sulfurizing agent is used, n+pmer
oligonucleotide (v) wherein the 5'-hydroxyl group is
protected can be isolated with high purity by using a solution
of methanol, which is a precipitation solvent, saturated with
10 a reducing agent such as a trivalent phosphite reagent (e.g.,
trimethylphosphite, triethylphosphite, tris(2-
carboxyethyl)phosphine etc.), hypo and the like.
[0140]
The production method of oligonucleotide of the present
is invention can afford the object oligonucleotide with high
purity and in a high yield by repeating the above-mentioned
steps (1) to (4) a desired number of times.
[0141]
step (5) (deprotection, oligonucleotide isolation step)
20 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
25 method described in PROTECTIVE GROUPS IN ORGANIC SYNTHESIS,
3rd ed., JOHN WILLY&SONS (1999) and the like. To be specific,
the pseudo solid phase protecting group in the present
invention, as well as the protecting group of nucleic acid
base such as phenoxyacetyl group, acetyl group and the like,
30 and cyanoethyl group and the like for protection of phosphate
backbone can all be removed by a treatment with aqueous
ammonia, aqueous ammonia/ethanol solution, or a mixture of
aqueous ammonia and aqueous methylamine solution. In addition,
nucleotide 5' hydroxyl-protecting group can be removed by a
35 treatment with the acid used in step (1) or an appropriately
52
diluted solution of such acid.
Since oligonucleotide without a protecting group is
easily degraded by an enzyme, oligonucleotide is preferably
isolated under appropriate air contamination control.
5 [0142]
The progress of the reaction in each of the abovementioned
steps can be confirmed by a method similar to
conventional liquid phase organic synthesis reaction. That is,
the reaction can be traced by thin layer silica gel
10 chromatography, high performance liquid chromatography and the
like.
[0143]
The oligonucleotide obtained by step (4) or step (5) can
also be led to a desired oligonucleotide derivative by further
15 applying an organic synthesis reaction.
The oligonucleotide produced by the present invention can
be used for various applications such as various human or
veterinary pharmaceutical products (RNA, DNA, oligonucleic
acid medicine, etc.), functional food, specified health food,
20 food, chemical product, polymer material for human or
industrial use, and the like.
Examples
[0144]
The present invention is explained in more detail in
25 the following by referring to Examples, which are not to be
construed as limiting the 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
30 abbreviation, each indication is based on the abbreviation of
the IUPAC-IUB Commission on Biochemical Nomenclature or
conventional abbreviations in the art.
[0145]
Reference Example 1: Synthesis of 3,4,5-
35 tris (octadecyloxy)benzyl amine
53
[0146]
[0147]
(1) Synthesis of 3,4,5-tris(octadecyloxy)benzyl chloride
5 Under an argon atmosphere, 3,4,5-tris(octadecyloxy)benzyl
alcohol (10.0 g, 11.0 mmol) was dissolved in dichloromethane
(100 niL) , N,N-dimethylformamide (84.0 (J.L, 1.10 mmol) and
thionyl chloride (1.20 mL, 16.4 mmol) were added, and the
mixture was stirred at 30°C for 90 min. After completion of
10 the reaction, the reaction mixture was concentrated under
reduced pressure, acetonitrile was added, and the precipitated
solid was filtered. The obtained solid was dried under reduced
pressure to give the title compound (10.0 g, 98.2%) as a white
solid.
15 (2) Synthesis of 3,4,5-tris(octadecyloxy)benzyl azide
3,4,5-Tris(octadecyloxy)benzyl chloride (4.02 g, 4.31
mmol) was dissolved in a mixed solvent of chloroform (40 mL)
and N,N-dimethylformamide (120 mL), sodium azide (418 mg, 6.42
mmol) was added, and the mixture was stirred at 80°C for 3 hr.
20 After completion of the reaction, the reaction mixture was
cooled to room temperature and washed with purified water (150
mL) . The organic layer was dried over sodium sulfate and
filtered. The filtrate was concentrated under reduced pressure,
methanol was added thereto, and the precipitated solid was
25 collected by filtration. The obtained solid was dried under
reduced pressure to give the title compound (3.92 g, 97.4%) as
a white solid.
(3) Synthesis of 3,4,5-tris(octadecyloxy)benzyl amine
Under an argon atmosphere, 3,4,5-tris(octadecyloxy)benzyl
30 azide (3.00 g, 3.21 mmol) was dissolved in dehydrating
tetrahydrofuran (35 mL) and, under ice-cooling, lithium
aluminum hydride (162 mg, 4.30 mmol) was added. The reaction
54
mixture was stirred at room temperature for 2 hr. After
completion of the reaction, purified water (2 mL) and 4.0
mol/L aqueous sodium hydroxide solution (2 mL) were added
dropwise to the reaction mixture to decompose unreacted
5 lithium aluminum hydride, and the reaction mixture was
filtered through celite. The filtrate was concentrated under
reduced pressure, acetonitrile was added, and the precipitated
solid was collected by filtration and dried under reduced
pressure to quantitatively give the title compound (3.09 g) as
io a white solid.
TLC:Rf=0.80 (dichloromethane:hexane=l:1)
1H-NMR(400MHz): 6 0.88 (t, 9H, J=l .0Hz, H3C,-octadecyloxy),
1.27-1.46 (m, 96H, -CH2-octadecyloxy), 1.70-1.82 (m, 6H, -CH*-
octadecyloxy), 3.76 (s, 2H, HsN-CH^-benzyl) , 3.92 (t, 2H,
15 J=6.6Hz, -0-CH2-octadecyloxy) , 3.97 (t, 4H, J-=6.6Hz, -O-CHgoctadecyloxy),
6.50 (s, 2H, -benzyl)
[0148]
Reference Example 2: Synthesis of 3,5-bis(docosyloxy)benzyl
alcohol
20 [0149]
OC22H45
[0150]
(1) Synthesis of methyl [3,5-bis(docosyloxy)]benzoate
Potassium carbonate (19.9 g, 144 mmol) was suspended in
25 dehydrating N,N-dimethylformamide (200 mL), 1-bromodocosane
(15.6 g, 4 6.7 mmol) and methyl (3,5-dihydroxy)benzoate (3.60 g
21.4 mmol) were added, and the mixture was stirred at 90°C
overnight. After completion of the reaction, the reaction
mixture was poured into purified water (800 mL), the mixture
30 was stirred for 1 hr, and the precipitated solid was collected
by filtration. The obtained solid was dissolved again in
55
dichloromethane (800 mL), and the insoluble material was
filtered off. The filtrate was concentrated under reduced
pressure, and acetone was added again to precipitate a solid.
The solid was collected by filtration and slurry-washed in
5 methanol. After filtration, the obtained solid was dried under
reduced pressure to give the title compound (13.9 g, 96.5%) as
a white solid.
(2) Synthesis of 3,5-bis(docosyloxy)benzyl alcohol
Under an argon atmosphere, methyl [3,5-
10 bis(docosyloxy)Jbenzoate (2.03 g, 2.59 mmol) was dissolved in
dehydrating tetrahydrofuran (50 mL) and, under ice-cooling,
lithium aluminum hydride (148 mg, 3.93 mmol) was added, and
the mixture was stirred at 40°C for 1 hr. Ethyl acetate was
added dropwise to decompose unreacted lithium aluminum hydride.
is Diethyl ether was added, and the mixture was washed with
aqueous hydrochloric acid. The organic layer was concentrated
under reduced pressure, methanol was added to the concentrated
solution, and the precipitated solid was collected by
filtration. The obtained solid was dried under reduced
20 pressure to give the title compound (1.73 g, 88.0%) as a white
solid.
TLC: Rf=0.80 (ethyl acetate:hexane=l:4)
1H-NMR(400MHz): 5 0.88 (t, 6H, J=7.0Hz, H^C-docosyloxy), 1.25-
1.80 (m, 84H, -CH^-docosyloxy), 3.93 (t, 4H, J=6.6Hz, -O-CHg-
25 docosyloxy), 4.62 (d, 2H, J=6.1Hz, H0-CH2-benzyl) , 6.38 (s, IH,
-benzyl), 6.50 (s, 2H, -benzyl)
[0151]
Reference Example 3: Synthesis of 3,5-bis(docosyloxy)benzyl
amine
30 [0152]
OC22H45
56
[0153]
The title compound was synthesized from 3,5-
bis(docosyloxy)benzyl alcohol according to the method of
Reference Example 1.
5 TLC: Rf=0.50 (dichloromethane:hexane=l:4)
^-NMR(400MHz): 5 0.88 (t, 6H, J=7.0Hz, HaC-docosyloxy), 1.25-
1.79 (m, 84H, -CH2-docosyloxy) , 3.79 (s, 2H, H2N-CH2-benzyl) ,
3.93 (t, 4H, J=6.6Hz, -0-CH2-docosyloxy) , 6.34 (s, 1H, -benzyl),
6.44 (s, 2H, -benzyl)
10 [0154]
Reference Example 4: Synthesis of 2,4-bis(docosyloxy)benzyl
alcohol
[0155]
OC22H45
OC22H45
15 [0156]
(1) Synthesis of 2,4-bis(docosyloxy)benzaldehyde
Under an argon atmosphere, 2,4-dihydroxybenzaldehyde
(3.00 g, 21.7 mmol), potassium carbonate (30.0 g, 217 mmol)
and 1-bromodocosane (17.3 g, 44.5 mmol) were added to
20 dehydrating N,N-dimethylformamide (150 mL), and the mixture
was stirred at 70°C overnight. After completion of the
reaction, the reaction mixture was poured into purified water
(1 L ) , the mixture was stirred for 1 hr, and the precipitated
solid was collected by filtration. The obtained solid was
25 slurry-washed in methanol and filtered, and the obtained solid
was dried under reduced pressure to give the title compound
(16.3 g, 99.3%) as a white solid.
(2) Synthesis of 2,4-bis(docosyloxy)benzyl alcohol
Under an argon atmosphere, 2,4-
30 bis(docosyloxy)benzaldehyde (2.99 g, 3.96 mmol) was dissolved
in a mixed solvent of dehydrating tetrahydrofuran (60 mL) and
57
methanol (6.0 mL), sodium borohydride (375 mg, 9.91 mmol) was
added, and the mixture was stirred at 40°C for 2 hr. After
completion of the reaction, purified water (2.0 mL) was added
dropwise to decompose unreacted sodium borohydride and the
5 mixture was filtered through celite. The filtrate was
concentrated under reduced pressure, methanol was added, and
the precipitated solid was collected by filtration. The
obtained solid was dried under reduced pressure to give the
title compound (2.77 g, 92.9%) as a white solid.
20 TLC: Rf=0.80 (ethyl acetate:hexane=l:4)
XH-NMR(400MHz): 5 0.88 (t, 6H, J=7.0Hz, HsC-docosyloxy), 1.25-
1.80 (m, 84H, -CH2-docosyloxy) , 2.23 (t, 1H, J=6.5Hz, HObenzyl),
3.94 (t, 2H, J=6.6Hz, -O-CH^-docosyloxy), 3.98 (t, 2H,
J=6.6Hz, -0-CH2-docosyloxy) , 4.61 (d, 2H, J=6.5Hz, HO-CHg-
15 benzyl), 6.42 (d, 1H, J=8.2Hz, -benzyl), 6.45 (s, 1H, -benzyl),
7.12 (d, 1H, J=8.2Hz, -benzyl)
[0157]
Reference Example 5: Synthesis of 2 , 4 - b i s ( d o c o s y l o x y ) b e n z yl
amine
20 [0158]
OC22H45
H 2 N ^ X V l
OC22H45
[0159]
The t i t l e compound was s y n t h e s i z e d from 2 , 4 -
b i s ( d o c o s y l o x y ) b e n z y l alcohol according t o the method of
25 Reference Example 1.
TLC: Rf=0.40 (dichloromethane:methanol=9:1)
XH-NMR(4 00MHz): 5 0.88 (t, 6H, J=7.0Hz, HsC-docosyloxy), 1.25-
1.81 (m, 84H, -CH2-docosyloxy) , 3.75 (s, 2H, H2N-CH2-benzyl) ,
3.91-3.97 (m, 4H, -O-CHg-docosyloxy), 6.39 (d, 1H, J=8.2Hz, -
30 b e n z y l ) , 6.44 (s, 1H, - b e n z y l ) , 7.07 (d, 1H, J=8.2Hz, -benzyl)
[0160]
58
Reference Example 6: Synthesis of 2,3,4-
tris(octadecyloxy)benzhydryl amine
[0161]
\ ^
HoN
OC18H37
OC18H37
OC18H37
5 [0162]
(1) Synthesis of 2, 3, 4-tris(octadecyloxy)benzophenone
Under an argon atmosphere, 2,3,4-trihydroxybenzophenone
(1.61 g, 7.00 mmol), 1-bromooctadecane (7.32 g, 22.0 mmol) and
potassium carbonate (4.35 g, 31.5 mmol) were added to
10 dehydrating N,N-dimethylformamide (30 mL), and the mixture was
stirred at 80°C overnight. After completion of the reaction,
purified water (200 mL) was added to the reaction mixture, the
mixture was stirred for 1 hr, and the precipitated solid was
collected by filtration. The obtained solid was slurry-washed
15 in methanol and, after filtration, dried under reduced
pressure to give the title compound (6.31 g, 91.1 %) as a
white solid.
(2) Synthesis of 2,3,4-tris(octadecyloxy)benzhydryl alcohol
2,3,4-Tris(octadecyloxy)benzophenone (3.00 g, 3.04 mmol)
20 was dissolved in a mixed solvent of chloroform (30 mL) and
methanol (10 mL), sodium borohydride (360 mg, 9.51 mmol) was
added, and the mixture was stirred at 45°C for 2 hr. After
completion of the reaction, 0.1 mol/L aqueous hydrochloric
acid was added dropwise to decompose unreacted sodium
25 borohydride and the mixture was washed with 1.0 mol/L aqueous
hydrochloric acid. 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, and the precipitated solid was collected by
59
filtration and dried under reduced pressure to give the title
compound (2.98 g, 99.0%) as a white solid.
(3) Synthesis of N-(9-fluorenylmethoxycarbonyl)-2,3,4-
tris(octadecyloxy)benzhydryl amine
5 Under an argon atmosphere, 2,3,4-
tris(octadecyloxy)benzhydryl alcohol (2.44 g, 2.46 mmol) and
9-fluorenylmethylcarbamate (1.06 g, 4.41 mmol) were dissolved
in dehydrating toluene (40 mL) at 50°C, methanesulfonic acid
(51.4 \xL, 740 |jmol) was added, and the mixture was stirred at
io 100°C overnight. After completion of the reaction, the
reaction mixture was concentrated under reduced pressure,
methanol was added, and the precipitated solid was collected
by filtration. The obtained solid was dried under reduced
pressure to give the title compound (3.37 g, 98.1%) as a white
is solid.
(4) Synthesis of 2,3,4-tris(octadecyloxy)benzhydryl amine
N-(9-Fluorenylmethoxycarbonyl)-2,3,4-
tris(octadecyloxy)benzhydryl amine (3.37 g, 2.89 mmol) was
dissolved in a mixed solvent of chloroform (30 mL) and
20 acetonitrile (15 mL), 20% piperidine [l-methyl-2-pyrrolidone
solution] (28.6 mL, 57.9 mmol) was added, and the mixture was
stirred at room temperature for 30 min. After completion of
the reaction, the reaction mixture was concentrated under
reduced pressure, methanol was added, and the precipitated
25 solid was collected by filtration. The obtained solid was
dried under reduced pressure to give the title compound (2.80
g, 97.9%) as a white solid.
TLC: Rf=0.60 (dichloromethane:methanol=9:1)
1H-NMR(4 00MHz): 5 0.88 (t, 9H, J=7.0Hz, H^C-octadecyloxy) ,
30 1.26-1.81 (m, 102H, -CH2-octadecyloxy), 3.77-3.86 (m, 2H, -0-
CHg-octadecyloxy), 3.91-3.95 (m, 4H, -0-CH2-octadecyloxy), 5.40
(s, 1H, H2N-CH-benzhydryl), 6.58 (d, 1H, J=8.7Hz, -benzhydryl),
6.88 (d, 1H, J=8.7Hz, -benzhydryl), 7.20-7.37 (m, 5H, -
benzhydryl)
35 [0163]
60
Reference Example 7: Synthesis of 3,4,5-tris[3,4,5-
tris(octadecyloxy)benzyloxy]benzyl alcohol
[0164]
pC-|8H37
OC18H37
OCi8H37
18H37
OC18H37
OC18H37
'18n37 "OC18H37
OC18H37
5 [0165]
(1) Synthesis of methyl {3,4,5-tris[3,4,5-
tris(octadecyloxy)benzyloxy]}benzoate
Under an argon atmosphere, methyl gallate (182 mg, 1.00
mmol), 3,4,5-tris(octadecyloxy)benzyl chloride (2.81 g, 3.01
10 mmol) and potassium carbonate (1.39 g, 10.1 mmol) were added
to dehydrating N,N-dimethylformamide (20 mL) , and the mixture
was stirred at 70°C overnight. The reaction mixture was
allowed to cool to room temperature, purified water (50 mL)
was added, and the precipitated solid was collected by
15 filtration. The obtained solid was slurry-washed in
acetonitrile and filtered, and the obtained solid was purified
by silica gel column chromatography (dichloromethane-hexane)
to give the title compound (2.31 g, 80.5%) as a white solid.
(2) Synthesis of 3,4,5-tris[3,4,5-
20 tris(octadecyloxy)benzyloxy]benzyl alcohol
Methyl {3,4,5-tris[3,4,5-
tris(octadecyloxy)benzyloxy]}benzoate (1.01 g, 350 umol) was
dissolved in dehydrating cyclopentyl methyl ether (20 mL) and,
61
under ice-cooling, lithium aluminum hydride (31.8 mg, 840 jjmol)
was added, and the mixture was stirred at room temperature for
2 hr. After completion of the reaction, 0.1 mol/L aqueous
hydrochloric acid was added dropwise to decompose unreacted
5 lithium aluminum hydride and the mixture was washed with 1.0
mol/L aqueous hydrochloric acid. The organic layer was dried
over sodium sulfate and filtered. The obtained filtrate was
concentrated under reduced pressure, methanol was added, and
the precipitated solid was collected by filtration and dried
io under reduced pressure to give the title compound (940 mg,
94.6%) as a white solid.
TLC: Rf=0.80 (dichloromethane:hexane=2:1)
1H-NMR(400MHz): 5 0.88 (t, 27H, J=7.0Hz, HaC-octadecyloxy),
1.26-1.75 (m, 306H, -CH2-octadecyloxy), 3.77 (t, 4H, J=6.6Hz, -
15 O-CHs-octadecyloxy), 3.87 (t, 10H, J=6.6Hz, -0-CHro
c t a d e c y l o x y ) , 3.92 (t, 4H, J=6.6Hz, -O-CHz-octadecyloxy) , 4.57
(d, 2H, J=6.0Hz, HO-CHr-benzyl) , 4.97 (s, 2H, HO-benzyl-O-CHbp
h e n y l ) , 5.01 (s, 4H, HO-benzyl-O-CHz-phenyl) , 6.61 (s, 4H, H0-
b e n z y l - O - b e n z y l ) , 6.63 (s, 2H, HO-benzyl-O-benzyl), 6.66 (s,
20 2H, HO-benzyl-O-benzyl).
[0166]
Reference Example 8: Synthesis of 4 , 4 ' -
bis(docosyloxy)benzhydrol
[0167]
OC22H45
25
OC22H45
[0168]
(1) Synthesis of 4,4'-didocosyloxy-benzophenone
To 4,4'-dihydroxy-benzophenone (8.2 g, 38.3 mmol) and 1-
bromodocosane (31.3 g, 80.4 mmol) were added DMF (300 mL) and
62
potassium carbonate (15.9 g, 115 mmol), and the mixture was
stirred at 80°C for 6.5 hr. After confirmation of the
disappearance of the monoalkylated compound, the reaction
mixture was ice-cooled, and IN hydrochloric acid (300 mL) and
5 water (150 mL) were slowly added to the thoroughly-stirred
mixture. The slurry was filtered, and the obtained crystals
were washed with water and methanol to give the title compound
(28.3 g, 34.1 mmol, 89%).
1H-NMR(CDCl3/300MHz) 5=0.88 (6H, t, J=6.6Hz, OC22H45 C22-H) , 1.1-
io 1.6 (76H, br, OC22H45 C3-21-H) , 1.81 (4H, m, OC22H45C2-H) , 2.04
(1H, s, -OH), 4.03 (4H, t, J=6.5Hz, OC22H45CI-H) , 6.94 (4H, d,
J=8.8Hz, Ph C3,3',5,5'-H), 7.77 (4H, d, J=8.7Hz, Ph
C2,2',6,6'-H)
[0169]
15 (2) Synthesis of 4,4'-bis(docosyloxy)benzhydrol
To 4,4'-didocosyloxy-benzophenone (28.3 g, 34.1 mmol)
were added THF (300 mL) and methanol (15 mL), and the mixture
was heated to 60°C. Sodium borohydride (6.10 g, 161 mmol) was
slowly added, and the mixture was stirred at the same
20 temperature for 4 hr. The reaction mixture was ice-cooled, and
IN hydrochloric acid (80 mL) was added dropwise. THF was
evaporated, water (450 mL) was added, and IN hydrochloric acid
was added to adjust the pH to 5 - 7. The slurry was filtered,
and the obtained crystals were washed with water and methanol
25 to give the title compound (28.5 g, 34.1 mmol, 99%).
1H-NMR(CDCl3/300MHz) 6 0.88 (6H, t, J=6.6Hz, OC22H45 C22-H) , 1.1-
1.6 (76H, br, OC22H45 C3-21-H) , 1.73 (4H, m, OC22H45C2-H) , 2.04
(1H, s, -OH), 3.93 (4H, t, J=6.6Hz, OC22H45 Cl-H) , 5.76 (1H, s,
HO-CHPh2), 6.85 (4H, d, J=8.7Hz, Ph C3,3',5,5'-H), 7.25 (4H, d,
30 J=8.6Hz, Ph C2,2',6,6'-H)
[0170]
Reference Example 9: Synthesis of 4,4'-
bis (docosyloxy)benzhydryl amine
[0171]
63
OC22H45
[0172]
(1) Synthesis of N-(9-fluorenylmethoxycarbonyl)-bis(4-
docosyloxyphenyl)methyl amine
5 To 4,4'-bis(docosyloxy)benzhydrol (713 mg, 856 umol) were
added toluene (15 ml), 9-fluorenylmethylcarbamate (246 mg,
1.03 mmol) and methanesulfonic acid (8.3 u.1, 128 umol), and the
mixture was stirred at 100°C for 3 hr. The disappearance of
4,4'-bis(docosyloxy)benzhydrol was confirmed, and the mixture
10 was cooled to room temperature. 2.5% Aqueous sodium
bicarbonate (10 ml) was added, and the mixture was stirred.
The mixture was partitioned, and the organic layer was further
washed with water (10 ml*2). The organic layer was evaporated
under reduced pressure, and the residue was washed with
15 methanol (10 ml) to give the title compound (540 mg, 512 umol,
yield 60%).
^-NMR (CDCl3/300MHz) 5 0.88 (9H, t, J=6.6, C21H42-CH3) , 1.10-
1.50 (82H, br, Alkyl-H) , 1.77 (4H, m, -O-CH2-CH2-C20H41) , 3.93
(4H, d, J=6.6, -O-CH2-C21H43) , 4.21 (IH, br, s, fluorene C9-H),
20 4.43 (2H, br, d, J=6.6, fluorene-CH^-O) , 5.23 (IH, br, s, Fmoc-
NH- or Fmoc-NH-CH), 5.85 (IH, br, s, Fmoc-NH- or Fmoc-NH-CH),
6.84 (4H, d, J=8.7, PhC3,5-H), 7.11(4H, d, J=8.7, PhC2,6-H),
7.25-7.35 (2H, br, m, fluorene C2,7-H), 7.39 (2H, br, t, J=6.9
fluorene C3,6-H), 7.59 (2H, br, s, fluorene Cl,8-H), 7.75 (2H,
25 br, d, J=6.6, fluorene C4,5-H)
(2) Synthesis of 4,4'-bis(docosyloxy)benzhydryl amine
To dichloromethane (10 ml) was added DBU (1,8-
diazabicyclo [5, 4, 0]-7-undecene, 200 |_il) , the compound (500 mg,
64
474 umol) obtained in Reference Example 9(1) was added thereto,
and the mixture was stirred at room temperature for 5 hr. IN
Hydrochloric acid (1.3 ml) was added dropwise, and the mixture
was stirred. The solvent was evaporated, and the residue was
5 washed with acetonitrile (10 ml) to give the title compound
(340 mg, 408 umol, yield 86%).
1H-NMR(CDCl3/300MHz) 5 0.88 (6H, t , J=6.6Hz, OC22H45 C22-H) , 1 . 1 -
1.6 (78H, b r , OC22H45C3-21-H, -NH2) , 1.75 (4H, m, OC22H45C2-H) ,
3 . 9 2 (4H, t , J=6.6Hz, OC22H45 Cl-H) , 5.12 (1H, s, H2N-CHPh2) ,
10 6.83 (4H, d, J=8.6Hz, Ph C3, 3 ' , 5 , 5 ' - H ) , 7.24 (4H, d, J=8.6Hz,
Ph C 2 , 2 ' , 6 , 6 ' - H)
[0173]
Example 1 (synthesis of nucleoside): Synthesis of 5'-0-(4,4'-
dimethoxytrityl) deoxythymidin-3' -yl- [3,4,5-
15 tris(octadecyloxy)benzyl]succinate
[0174]
DMTrO
OC18H37
OCi8H37
OC18H37
[0175]
(1) Synthesis of 5'-0-(4,4'-dimethoxytrityl)deoxythymidine-3'-
20 O-succinate
Under an argon atmosphere, 5'-0-(4,4'-
dimethoxytrityl)deoxythymidine (5.00 g, 9.18 mmol), succinic
anhydride (1.38 g, 13.8 mmol) and triethylamine (3.85 mL, 27.5
mmol) were dissolved in dichloromethane (95 mL), and the
25 mixture was stirred at room temperature for 8 hr. The
completion of the reaction was confirmed by thin layer
65
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 triethylamine salt (7.02 g, 98%) of
5 5'-0-(4,4'-dimethoxytrityl)deoxythymidine-3' -O-succinate as a
colorless frothy solid.
(2) Synthesis of 5' -0-(4, 4'-dimethoxytrityl)deoxythymidin-3'-
yl- [3,4,5-tris(octadecyloxy)benzyl]succinate
3,4,5-Tris(octadecyloxy)benzyl alcohol (3.06 g, 3.35
10 mmol) was dissolved in dichloromethane (35 mL), a
triethylamine salt (3.00 g, 4.02 mmol) of 5'-0-(4,4'-
dimethoxytrityl)deoxythymidine-3'-O-succinate, 2-(1Hbenzotriazol-
1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate [HBTU] (1.98 g, 5.23 mmol),
15 diisopropylethylamine (925 ^L, 5.23 mmol) and
dimethylaminopyridine (639 mg, 5.23 mmol) were added, and the
mixture was stirred. The completion of the reaction was
confirmed by thin layer chromatography, methanol was added,
and the mixture was concentrated and suction-filtered to give
20 5'-0- (4,4'-dimethoxytrityl)deoxythymidin-3' -yl-[3,4,5-
tris (octadecyloxy)benzyl]succinate (4.77 g, yield 93%) as a
white solid.
Rf=0.78 (CH2Cl2/MeOH,10:l).
^-NMR (400MHz) : 5=0.88 (t, 9H, Ar-CHj) , 1.29 (br, s, 90H,
25 (CHzhs), 1.36 (s, 3H, N5-CH3), 1.77 (m, 6H, Ar-OCH2CH2) , 2 . 4 5 (m,
2H, 2 ' - H ) , 2,67 (m, 4H, succinyl-CHgCH^) , 3.48 (m, 2H, 5 ' - H ),
3 . 7 9 (s, 6H, DMTr-OCHs) , 3.95 (m, 6H, Ar-OCHs) , 4.12 (m, 1H,
4 ' - H ) , 5.01 (s, 2H, A r - C H 2 - s u c c i n y l ) , 5.50 (m, 1H, 3 ' - H ) , 6.42
( t , 1H, l ' - H ) , 6.53 (s, 2H, Ar-H), 6 . 8 4 - 7 . 2 6 (m, 13H, DMTr-Ar-
30 H) , 7.60 (s, 1H, N6-H) .
[0176]
Example 2 (continuous synthesis 1 of dinucleotide in
solution): Synthesis of 5'-0-(4,4'-
dimethoxytrityl)deoxythymidine-3'-[0-(2-
35 cyanoethyl)]phosphoryl-deoxythymidin-3' -yl-[3,4,5-
66
tris(octadecyloxy)benzyl]succinate
[0177]
O
NH
DMTrO^ ~N O
1 A,
yj
o
I °
„ Y^
c 18H37° y^Y^ o'J l s^^vY °
C18H37° IT
OC18H37
[0178]
5 Under an argon atmosphere, 5'-0-(4,4' -
dimethoxytrityl)deoxythymidin-3'-yl-[3,4,5-
tris (octadecyloxy) benzyl] succinate (511 mg, 332 jjmol) was
dissolved in dichloromethane (5.11 mL) , pyrrole (230 \iL, 3.32
mmol) and trifluoroacetic acid (296 jo.L, 3.98 mmol) were added,
10 and the mixture was stirred at room temperature for 15 min.
The completion of the reaction was confirmed by thin layer
chromatography. The reaction mixture was neutralized with
pyridine (322 (J.L, 3.98 mmol), a solution of N-methylimidazole
(158 ]iL, 1.99 mmol) and 5'-0-(4,4'-
15 dimethoxytrityl)deoxythymidine-3'-[0-(2-cyanoethyl)-(N,Ndiisopropyl)
] -phosphoramidite (494 mg, 664 jjmol) in
acetonitrile were added, and the mixture was stirred at room
temperature for 60 min. The completion of the reaction was
confirmed by thin layer chromatography. Furthermore, 0.2M
20 iodine pyridine/THF/H20 solution (3.32 mL) was added, and the
mixture was stirred at room temperature for 10 min. A methanol
67
solution saturated with sodium thiosulfate was poured into a
reaction vessel, and the mixture was stirred at 0°C for 10 min,
suction-filtered using Kiriyama funnel, and dried to give 5'-0-
(4,4' -dimethoxytrityl) deoxythymidine-3'- [0- (2-
5 cyanoethyl)]phosphoryl deoxythymidin-3'-yl-[3,4,5-
tris(octadecyloxy)benzyl]succinate (59 7 mg, 99.2%) as a white
solid.
^-NMR (4 00MHz) : 5=0.88 (t, 9H, J=7.04Hz, Ar-CHj) , 1.26 (br, s,
90H, (CH2)i5), 1-30 (s, 3H, Ni5-CH3) , 1.40 (s, 3H ,N2
5-CH3) , 1.77
10 (m, 6H, Ar-OCH2CH2_) , 2.30 (m, 2H, 2'i-H), 2.40 (m, 2H, 2'2-H),
2.68 (m, 4H, succinyl-CHsCHg) , 2.75 (m, 2H, 5'X-H), 3.39 (m, 2H,
5'2-H), 3.79 (s, 6H, DMTr-OCHa) , 3.94 (m, 6H, Ar-OCHz) , 4.11 (m,
IH, 4'i-H), 4.25 (m, 4H, cyanoethyl-CH2CH2 and m, IH, 4'2-H),
5.00 (s, 2H, Ar-CHg-succinyl) , 5.18 (m, IH, 3'2-H), 5.27 (m, IH,
15 3'i-H), 6.24 (m, IH, l'i-H), 6.42 (m, IH, 1'2-H), 6.53 (s, 2H,
Ar-H), 6.83-7.38 (m, 13H, DMTr-Ar-H), 7.38 (m, IH, N6-H), 7.53
(m, IH, N6-H)
^-NMR (400MHz) : 6=0.88 (t, 9H, J=7.04HZ, Ar-CH3) , 1.26 (br, s,
90H, (CHzhs), 1.30 (s, 3H, Ni5-CH3) , 1.40 (s, 3H, N2
5-CH3) , 1.77
20 (m, 6H, Ar-OCH2CH2) , 2.30 (m, 2H, 2'i-H), 2.68 (m, 2H, 2'2-H and
m, 4H, succinyl-CHgCH^) , 2.75 (m, 2H, 5'i-H), 3.53 (m, 2H, 5'2-
H) , 3.79 (s, 6H, DMTr-0CH3) , 3.94 (m, 6H, Ar-OCH^) , 4.11 (m, IH,
4'i-H), 4.25 (m, 4H, cyanoethyl-CH2CH2 and m, IH, 4'2-H), 5.01
(s, 2H, Ar-CH2-succinyl), 5.18 (m, IH, 3'2-H), 5.31 (m, IH, 3'i-
25 H), 6.24 (m, IH, l'i-H), 6.42 (m, IH, 1'2-H), 6.53 (s, 2H, Ar-
H), 6.83-7.38 (m, 13H, DMTr-Ar-H), 7.38 (m, IH, Ni6-H), 7.53 (m,
IH, N2
6-H)
[0179]
Example 3 (continuous synthesis of phosphorthioate dimer in
30 solution): Synthesis of 5'-0-(4,4'-
dimethoxytrityl)deoxythymidine-3' -[0-(2-
cyanoethyl)]phosphorthionyl deoxythymidin-3'-yl-[3,4,5-
tris(octadecyloxy)benzyl]succinate
[0180]
68
c 18H37° Y ^ Y ^ o ^ ^ ^ Y °
C18H37° 1f^
06-18^37
[0181]
Under an argon atmosphere, 5'-0-(4,4'-
dimethoxytrityl)deoxythymidin-3'-yl-[3,4,5-
5 tris (octadecyloxy)benzyl] succinate (200 mg, 130 |amol) was
dissolved in dichloromethane (3.00 mL), pyrrole (89.8 uL, 1.30
mmol) and trifluoroacetic acid (116 uL, 1.56 mmol) were added,
and the mixture was stirred at room temperature for 15 min.
The completion of the reaction was confirmed by thin layer
10 chromatography. The reaction mixture was neutralized with
pyridine (126 \xL, 1.56 mmol), a solution of N-methylimidazole
(61.8 \xL, 0.779 mmol) and 5'-0-(4,4'-
dimethoxytrityl)deoxythymidine-3'-[0-(2-cyanoethyl)-(N,Ndiisopropyl)
]-phosphoramidite (194 mg, 260 jjmol) in
15 acetonitrile was added, and the mixture was stirred at room
temperature for 60 min. The completion of the reaction was
confirmed by thin layer chromatography. Furthermore, 0.05M 3-
( (N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-
thione pyridine/acetonitrile solution (7.79 mL) was added, and
20 the mixture was stirred at room temperature for 30 min. A
methanol solution saturated with sodium thiosulfate was poured
69
into a reaction vessel, suction-filtered using Kiriyama funnel,
and dried to give 5'-0-(4,4'-dimethoxytrityl)-deoxythymidine-
3'-[0-(2-cyanoethyl)]phosphorthionyl deoxythymidin-3'-yl-
[3,4,5-tris(octadecyloxy)benzyl]succinate (247 mg, 99.5%) as a
5 white solid.
1H-NMR(400MHz) : 5=0.88 (t, 9H, J=6.8Hz, Ar-CHs) , 1.28 (br, s,
90H, (CH2)i5), 1.30 (s, 3H, N^-CHs) , 1.46 (s, 3H, N2
5-CH3) , 1.73
(m, 6H, Ar-OCH2CH2) , 2.28 (m, 2H, 2'i-H), 2.41 (m, 2H, 2'2-H),
2.68 (m, 2H, 5'i-H and m, 4H, succinyl-CH^CHz) , 3.44 (m, 2H,
10 5'2-H), 3.79 (s, 6H, DMTr-OCHa) , 3.95 (m, 6H, Ar-OCH^) , 4.10 (m,
IH, 4'i-H), 4.31 (m, 4H, cyanoethyl-CHzCH^ and m, IH, 4'2-H),
5.01 (s, 2H, Ar-CH2-succinyl) , 5.26 (m, IH, 3'i-H), 5.32 (m, IH,
3'2-H), 6.27 (m, IH, l'i-H), 6.38 (m, IH, 1'2-H), 6.53 (s, 2H,
Ar-H), 6.84-7.29(m, 13H, DMTr-Ar-H), 7.29 (m, IH, Ni6-H), 7.56
15 (m, IH, N2
6-H)
1H-NMR(400MHz) : 6=0.88 ( t , 9H, Ar-CH3) , 1.28 (br, s, 90H,
(CHg)^), 1.30 (s, 3H, Ni5-CH3) , 1.46 (s, 3H, N2
5-CH3) , 1.73 (m,
6H, Ar-OCH2CH2) , 2 . 4 1 (m, 2H, 2 ' 2 - H ) , 2.68 (m, 2H, 2 ' i -H and m,
4H, succinyl-CHgCHs) , 2.77 (m, 2H, 5 ' i - H ) , 3.44 (m, 2H, 5'2 - H ),
20 3.79 (s, 6H, DMTr-OCH3) , 3.95 (m, 6H, Ar-OCH^) , 4.17 (m, IH,
4 ' i - H ) , 4.31 (m, 4H, cyanoethyl-CH^CH^ and m, IH, 4 ' 2 - H ) , 5.01
( s , 2H, Ar-CHg-succinyl) , 5.33 (m, IH, 3 ' r H and m, IH, 3'2 - H ),
6 . 2 7 (m, IH, l ' i - H ) , 6.38 (m, IH, 1'2 - H ) , 6.53 (s, 2H, Ar-H),
6 . 8 4 - 7 . 2 9 (m, 13H, DMTr-Ar-H), 7.29 (m, IH, Nx
6-H), 7.56 (m, IH,
25 N2
6-H)
[0182]
Example 4 (continuous synthesis 2 of dinucleotide in
solution): Synthesis of 5'-0-(4,4'-dimethoxytrityl)-2'-0-
(tert-butyldimethylsilyl)uridine-3'-[0-(2-
30 cyanoethyl)]phosphoryl deoxythymidin-3'-yl-[3,4,5-
tris (octadecyloxy)benzyl]succinate
[0183]
70
o
DMTrCL N O
O OTBS
P-P-O Y -
NC O^ N^O
C18H37° l^
OC18H37
[0184]
Under an argon atmosphere, 5'-0-(4,4'-
dimethoxytrityl)deoxythymidin-3'-yl-[3,4,5-
5 tris(octadecyloxy)benzyl]succinate (200 mg, 130 umol) was
dissolved in dichloromethane (3.00 mL) , pyrrole (89.8 |j,L, 1.30
mmol) and trifluoroacetic acid (116 \ih, 1.56 mmol) were added,
and the mixture was stirred at room temperature for 15 min.
The completion of the reaction was confirmed by thin layer
10 chromatography. The reaction mixture was neutralized with
pyridine (126 |i,L, 1.56 mmol), a solution of N-methylimidazole
(61.8 uL, 0.779 mmol) and 5'-0-(4,4'-dimethoxytrityl)-2'-0-
(tert-butyldimethylsilyl)uridine-3'-[0-(2-cyanoethyl)-(N,Ndiisopropyl)
]-phosphoramidite (224 mg, 260 fimol) in
is acetonitrile was added, and the mixture was stirred at room
temperature for 60 min. The completion of the reaction was
confirmed by thin layer chromatography. Furthermore, 0.2M
iodine pyridine/THF/H20 solution (1.33 ml) was added, and the
mixture was stirred at room temperature for 10 min. A methanol
20 solution saturated with sodium thiosulfate was poured into a
reaction vessel, suction-filtered using Kiriyama funnel, and
dried to give 5'-0-(4,4'-dimethoxytrityl)-2'-0-(tert-
71
butyldimethylsilyl)uridine-3'-[0-(2-cyanoethyl)]phosphoryl
deoxythymidin-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate
(257 mg, 98.3%) as a white solid.
1H-NMR(4 00MHz) : 5=0.13 ( t , 6H, J=3.8Hz, -OSi (Meg) tBu) , 0.88 (t,
5 9H, J = 6 . 4 , -0Si(Me2 ) t B u and t , 9H, J = 6 . 3 6 , Ar-CHg) , 1.28 (br, s,
90H, (CHghs), 1-30 (s, 3H, N5-CH3) , 1.75 (m, 6H, Ar-OCHzCHg) ,
2 . 2 6 (m, 2H, 2 ' 2 - H ) , 2.38 (m, IH, 2 ' i - H ) , 2.66 (m, 4H,
succinyl-CHzCHz) , 2.73 (m, 2H, 5 ' r - H ) , 3.47 (m, 2H, 5 ' 2 - H ) , 3.79
( s , 6H, DMTr-OCH3) , 3.94 (m, 6H, Ar-OCHg) , 4 . 2 5 (m, 4H,
io cyanoethyl-CH2CH2 and m, l H , 4 ' i - H ) , 4 . 5 1 (m, IH, 4 ' 2 - H ) , 4.93 (m,
IH, 3 ' 2 - H ) , 5.00 (s, 2H, A r - C H g - s u c c i n y l ) , 5.20 (m, 1 H , 3 ' I - H ),
5 . 2 5 (m, IH, Ni5-H) , 5.97 (m, IH, l ' i - H ) , 6.21 (m, IH, 1'2 - H ),
6 . 5 3 (s, 2H, Ar-H), 6 . 8 5 - 7 . 2 8 (m, 13H, DMTr-Ar-H), 7.29 (m, IH,
Ni6-H) , 7.83 (m, IH, N2
6-H)
15 1H-NMR(400MHz) : 5=0.13 ( t , 6H, J=3.8Hz, -OSi(Meg) tBu) , 0.88 (t,
9H, J = 6 . 4 , -OSi(Me2)tBu and t , 9H, J = 6 . 3 6 , Ar-CHg) , 1.28 (br, s,
90H, (CHghs), 1.30 (s, 3H, N5-CH3) , 1.75 (m, 6H, Ar-OCHsCHg) ,
2 . 3 8 (m, IH, 2 ' i - H ) , 2.58 (m, 2H, 2 ' 2 - H ) , 2.68 (m, 4H,
succinyl-CHgCHg) , 2.73 (m, 2H, 5 ' i - H ) , 3.62 (m, 2H, 5 ' 2 - H ) , 3.80
20 (s, 6H, DMTr-OCH3) , 3.94 (m, 6H, Ar-OCHg) , 4.25 (m, 4H,
cyanoethyl-CHgCHg and m, 1 H , 4 ' I - H ) , 4 . 5 1 (m, IH, 4 ' 2 - H ) , 4.93 (m,
IH, 3 ' 2 - H ) , 5.01 (s, 2H, Ar-CHg-succinyl) , 5.25 (m, IH, Ni5-H) ,
5 . 3 0 (m, IH, 3 ' i - H ) , 5.97 (m, IH, l ' i - H ) , 6.21 (m, IH, 1'2 - H ),
6 . 5 3 (s, 2H, Ar-H), 6 . 8 5 - 7 . 2 8 ( m , 13H, DMTr-Ar-H), 7.29 (m, IH,
25 Nx
6-H) , 7.83 (m, IH, N2
6-H)
[0185]
Example 5 (continuous synthesis of 20mer oligonucleotide in
solution): Synthesis of deoxythymidinyl-[3'—>5' ] -
deoxycytidinyl- [3' —>5' ] -deoxycytidinyl-[3'->5' ] -deoxycytidinyl-
30 [3'-»5' ] -deoxyguanidyl- [3f->5/" ] -deoxycytidinyl- [3' —>5' ] -
deoxycytidinyl-[3f—>5f ] -deoxythymidinyl-[3'-»5' ]-deoxyguanidyl-
[ 3'->5' ] -deoxythymidinyl- [ 3' ->5' ] -deoxyguanidyl- [ 3'->5' ]-
deoxyadenylyl-[3'—>5' ] -deoxycytidinyl-[3'—>5' ] -deoxyadenylyl-
[3'-»5' ] -deoxythymidinyl- {?>'-+b' ] -deoxyguanidyl- [3'->5' ] -
35 deoxycytidinyl-[3'->5' ]-deoxyadenylyl-[3'—>5' ] -deoxythymidinyl-
72
[3' —>5r]-deoxythymidine
(1) Synthesis of 5'-0-(4, 4'-dimethoxytrityl)-deoxythymidine
3' -[0-(2-cyanoethyl)]phosphoryl-N4-benzoyl-deoxycytidine 3'-[0-
(2-cyanoethyl)]phosphoryl-N4-benzoyl-deoxycytidine 3'-[0-(2-
cyanoethyl)
cyanoethyl)
cyanoethyl)
cyanoethyl)
cyanoethyl)
cyanoethyl)
cyanoethyl)
cyanoethyl)
cyanoethyl)
cyanoethyl)
cyanoethyl)
cyanoethyl)
cyanoethyl)
cyanoethyl)
cyanoethyl)
cyanoethyl)
cyanoethyl)
] phosphoryl-N4-benzoyl-deoxycytidine 3' -
]phosphoryl-N2-isobutyryl-deoxyguanosine
] phosphoryl-N4-benzoyl-deoxycytidine 3' -
] phosphoryl-N4-benzoyl-deoxycytidine 3' -
]phosphoryl-deoxythymidine 3'-[0-(2-
]phosphoryl-N2-isobutyryl-deoxyguanosine
]phosphoryl-deoxythymidine 3'-[0-(2-
] phosphoryl-N2-isobutyryl-deoxyguanosine
] phosphoryl-N6-benzoyl-deoxyadenosine 3'
] phosphoryl-N4-benzoyl-deoxycytidine 3' -
]phosphoryl-N6-benzoyl-deoxyadenosine 3'
]phosphoryl-deoxythymidine 3'-[0- (2-
]phosphoryl-N2-isobutyryl-deoxyguanosine
] phosphoryl-N4-benzoyl-deoxycytidine 3' -
] phosphoryl-N6-benzoyl-deoxyadenosine 3'
]phosphoryl-deoxythymidine 3'-[0-(2-
]phosphoryl-deoxythymidin-3' -yl- [3,4,5-
[0-
3'
[0-
[0-
3'
3'
-[o
[0-
-[o
3'
[0-
-[0
(2-
-[0-
(2-
(2-
-[0-
-[0-
-(2-
(2-
-(2-
-[0-
(2-
-(2-
(2-
(2-
(2-
(2-
tris(octadecyloxy)benzyl]succinate
The operations in the same manner as in Example 2 were
repeated 19 times to give the above-mentioned compound (2.55
25 g) .
(2) Deprotection and purification step
The compound (100 mg, 10.3 jxmol) synthesized in Example
5(1) and 30% aqueous ammonia:ethanol=3:1 solution (5.00 mL)
were placed in an autoclave, and the mixture was heated at 80°C
30 for 2 hr, and freeze-dried. The freeze-dried product was
diluted with 0.1M aqueous ammonium acetate solution, the
mixture was applied to C-18 cartridge purification, and the
obtained eluate was freeze-dried to give the object product,
deoxythymidinyl- [3'-»5' ] -deoxycytidinyl- [3'—>5' ]-
35 deoxycytidinyl- [3'->5' ] -deoxycytidinyl-[3' —>5' ]-deoxyguanidyl-
73
[3'-»5' ] -deoxycytidinyl-[3'-»5' ] -deoxycytidinyl- [3'->5' ]-
deoxythymidinyl-[3'->5' ] -deoxyguanidyl-[3'—>5' ] -
deoxythymidinyl- [3'—»5' ]-deoxyguanidyl-[3'—>5' ] -deoxyadenylyl-
[3'->5' ] -deoxycytidinyl- [3'-»5' ] -deoxyadenylyl- [3'^-5' ] -
5 deoxythymidinyl- [3'—»5' ] -deoxyguanidyl-[3'—»5' ] -deoxycytidinyl-
[3'->5' ]-deoxyadenylyl-[3'->5' ] -deoxythymidinyl- [3'->5' ]-
deoxythymidine.
HPLC (shodex ODP (4.6 cp*150 mm), flow rate 1.0 mL/min, MeCN,
H20 gradient: 0-15 min; 2 to 98%, 15 to 25 min; 98%, A=254 nm):
10 Rt=5.61 min (96.3%);
MALDI-TOF/MS: 6043.66[M-H]"
[0186]
Experimental Example 1 (study of cation scavenger)
Using the compound of Example 1 wherein the 5'-hydroxyl
15 group is protected by 4,4'-dimethoxytrityl (hereinafter
sometimes to be abbreviated as DMTr) (hereinafter sometimes to
be abbreviated as a DMTr-protected compound) as a test
compound, a DMTr cation scavenger candidate substance was
tested and evaluated.
20 The DMTr-protected compound (50.0 mg, 32.5 umol) was
dissolved in dichloromethane (350 uL) to give a starting
material solution, and a cation scavenger candidate substance
(162 umol) described in Table 1 was added. Trifluoroacetic
acid (29.2 uL, 394 umol) was added, and the mixture was stirred
25 at room temperature for about 30 min until the disappearance
of the spot of the DMTr-protected compound in the reaction
mixture could be confirmed by both UV and color reaction under
the following TLC measurement conditions.
Furthermore, the reaction mixture was neutralized with
30 pyridine (31.8 uL, 394 umol) and evaluation was conducted
according to the following evaluation criteria under the
following TLC measurement conditions.
O (effective): when spot of DMTr-protected compound was not
confirmed by both UV and color reaction
35 x (ineffective): when spot of DMTr-protected compound was
74
confirmed by any or both of UV and color reaction
The results are shown in Table 1.
[thin layer chromatography (TLC) measurement conditions]
The reaction mixture was spotted near the point of origin
5 of a TLC plate (2 cmx5 cm rectangle, manufactured by Merch) by
using a TLC spotting capillary tube (5 |iL) (manufactured by
Hirschmann Laborgeraete), developed with a developing solvent
(dichloromethane/methanol=10/l), and confirmed by visual
observation by UV (254 nm) and color reaction (after immersion
o in phosphomolybdic acid-ethanol solution, on a hot plate
(about 300°C) for 10 sec or above).
(DMTr-protected compound Rf value = 0.70, DMTr-deprotected
compound Rf value = 0.40)
[0187]
75
Table 1
Cation scavengers
methanol
dimethylsulfide
anisole
thioanisole
m-cresol
1,3,5-
trimethylbenzene
1,3-dimethoxybenzene,
1,3,5-
trimethoxybenzene
triisopropylsilane
triethylsilane
pyrrole
3-methylpyrrole
2,4-dimethylpyrrole
indole
succinimide
phthalimide
evaluation of scavenging of DMTr cation after
neutralization
(effective (o); ineffective (x))
O
X
X
X
X
X
X
X
X
X
o
O
O
O
X
X
non-protection state of 5'-hydroxyl
group was maintained even after
neutralization
5'-hydroxyl group was DMTr-ized after
neutralization
5'-hydroxyl group was DMTr-ized after
neutralization
5'-hydroxyl group was DMTr-ized after
neutralization
5'-hydroxyl group was DMTr-ized after
neutralization
5'-hydroxyl group was DMTr-ized after
neutralization
5'-hydroxyl group was DMTr-ized after
neutralization
5'-hydroxyl group was DMTr-ized after
neutralization
5'-hydroxyl group was DMTr-ized after
neutralization
5'-hydroxyl group was triethylsilylated
after neutralization
non-protection state of 5'-hydroxyl
group was maintained even after
neutralization
non-protection state of 5'-hydroxyl
group was maintained even after
neutralization
non-protection state of 5'-hydroxyl
group was maintained even after
neutralization
non-protection state of 5'-hydroxyl
group was maintained even after
neutralization
5'-hydroxyl group was DMTr-ized after
neutralization
5'-hydroxyl group was DMTr-ized after
neutralization
[0188]
When dimethylsulfide, anisole, thioanisole, cresol,
1,3,5-trimethylbenzene, 1,3-dimethoxybenzene, 1,3,5-
trimethoxybenzene, succinimide and phthalimide widely used as
a cation scavenger were used, the 5'-terminal hydroxyl group
was protected again by protecting group Q after neutralization,
which clarifies that they do not sufficiently function as a
76
cation scavenger. When trialkylsilane (e.g.,
triisopropylsilane, triethylsilane etc.), which is a
conventionally-used cation scavenger, was used, trialkylsilane
could not be used for the subsequent condensation step since
5 the 5'-terminal hydroxyl group was trialkylsilylated after
neutralization. Furthermore, it was found that use of methanol
as a cation scavenger prevents the condensation reaction in
the next step, though it irreversibly traps cation, and
therefore, methanol cannot be used.
10 On the other hand, it was found that effective scavengers
capable of maintaining the scavenging state of DMTr cation
even after neutralization reaction were pyrrole derivatives
such as pyrrole, 3-methylpyrrole, 2,4-dimethylpyrrole, and the
like, and indole derivatives such as indole and the like.
15 [0189]
Experimental Example 2 (when methanol was used as cation
scavenger)
Using the compound of Example 1, wherein the 5'-hydroxylprotecting
group is 4,4'-dimethoxytrityl, as a test compound,
20 whether or not methanol confirmed to be effective as a DMTr
cation scavenger in Experimental Example 1 permits continuous
synthesis of deprotection and condensation in solution was
studied by the following method.
[0190]
25 Under an argon atmosphere, 5'-0-(4,4'-
dimethoxytrityl)deoxythymidin-3' -yl-[3,4,5-
tris(octadecyloxy)benzyl]succinate (100 mg, 65.0 umol) was
dissolved in dichloromethane (1.5 mL) , methanol (13.2 uL, 325
umol) and trifluoroacetic acid (57.9 uL, 779 umol) were added,
30 and the mixture was stirred at room temperature for 15 min.
The completion of the reaction was confirmed by HPLC. The
reaction mixture was neutralized with pyridine (63.0 uL, 779
umol), a solution of N-methylimidazole (30.9 uL, 390 umol) and
5'-0-(4,4'-dimethoxytrityl)deoxythymidine-3'-[0-(2-
35 cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (96.8 mg, 130
77
umol) in acetonitrile was added, and the mixture was stirred at
room temperature for 60 min and analyzed by HPLC. HPLC peak
area values of the reaction system after deprotection reaction
and after condensation reaction are shown in Table 2.
5 [0191]
Table 2
after deprotection
after
neutralization
after condensation
reaction
HPLC peak area value (%)
test compound with
deprotected DMTr
96.9
97.1
92.6
condensation
product (same as
Example 3 compound)
" • — _ _ _ _ _
^ ^ ^ — _ _ _ _ _
3.2
[0192]
The above-mentioned results have revealed that methanol
io maintained scavenging of DMTr cation even after neutralization
but continuous condensation reactions in the same system do
not advance condensation reaction.
[0193]
Experimental Example 3 (when triethylsilane was used as cation
15 scavenger)
Using the compound of Example 1, wherein the 5'-hydroxylprotecting
group is 4,4'-dimethoxytrityl, as a test compound
and triethylsilane as a DMTr cation scavenger, deprotection
was studied by the following method.
20 [0194]
Under an argon atmosphere, 5'-0-(4,4'-
dimethoxytrityl)deoxythymidin-3'-yl-[3,4,5-
tris(octadecyloxy)benzyl]succinate (100 mg, 65.0 umol) was
dissolved in dichloromethane (1.5 mL) , triethylsilane (51.7 uL,
25 325 umol) and trifluoroacetic acid (57.9 uL, 779 jjitiol) were
added, and the mixture was stirred at room temperature for 15
min. The completion of the reaction was confirmed by thin
layer chromatography. The reaction mixture was neutralized
78
with pyridine (63.0 U.L, 779 umol), and a methanol solution was
supplied into a reaction vessel, suction-filtered using
Kiriyama funnel, and dried. The solid obtained by drying was
measured for 1H-NMR (below). As a result, a mixture of 5'-0-
5 (triethylsilyl)deoxythymidin-3'-yl-[3,4,5-
tris(octadecyloxy)benzyl]succinate wherein the 5'-hydroxyl
group was triethylsilylated, and 5'-OH-deoxythymidin-3'-yl-
[3,4,5-tris(octadecyloxy)benzyl]succinate was obtained at a
proton intensity ratio at the 1'-position of 5'-triethylsilyl
io compound:5'-OH compound=0.26:1.00.
1H-NMR(400MHz) : 6=0.68 (dd, 6H, Si(CH2CH3) 3), 0.86 (t, 9H, Ar-
CH3), 0.99 (t, 9H, (CH2CH3)3), 1.26 (br, s, 90H, (CH2) 15) , 1.30
(s, 3H, N5-CH3_) , 1.75 (m, 6H, Ar-OCH2CH2) , 2.12 (m, 2H, 2'-H),
2.68 (m, 6H, succinyl-CH^CH^) , 3.88 (m, 2H, 5'-H), 3.98 (m, 6H,
15 Ar-OCH2) , 4.07 (m, 1H, 4'-H), 5.02 (s, 2H, Ar-CHg-succinyl),
5.28 (m, 1H, 3'-H), 6.37 (t, 1H, l'-H), 6.54 (s, 2H, Ar-H),
7.62 (s, 1H, N6-H)
[0195]
The above-mentioned results have revealed that
20 triethylsilane is effective for DMTr cation scavenging during
deprotection, but unsuitable as a DMTr cation scavenger, since
it partly triethylsilylates the 5'-hydroxyl group of the
deprotected compound after neutralization, and adversely
affects continuous condensation steps.
25 [0196]
Experimental Example 4 (when step of precipitation and
isolation after continuous solution reactions was changed)
Using 5'-OH-deoxythymidin-3'-yl-[3,4,5-
tris(octadecyloxy)benzyl]succinate as a test compound and
30 condensation, oxidation and deprotection were continuously
performed in this order in the same reaction system by the
following method.
[0197]
Under an argon atmosphere, 5'-OH-deoxythymidin-3'-yl-
35 [3,4,5-tris(octadecyloxy)benzyl]succinate (79.3 mg, 52.3 umol)
79
was dissolved in dichloromethane (1.5 mL), a solution of 5'-0-
(4,4'-dimethoxytrityl)deoxythymidine-3'-[0-(2-cyanoethyl)-
(N,N-diisopropyl)]-phosphoramidite (77.9 mg, 105 jamol) and 1Htetrazole
(36.6 mg, 523 (jitiol) in acetonitrile was added, and
5 the mixture was stirred at room temperature for 60 min. The
completion of the reaction was confirmed by thin layer
chromatography. Furthermore, 0.2M iodine pyridine/THF/H20
solution (1.1 mL) was added to the reaction mixture, and the
mixture was stirred at room temperature for 10 min. After
10 stirring, TFA (46.6 uL, 628 |jmol) was added and the reaction
was traced by HPLC analysis. The HPLC peak area values of the
reaction system after condensation reaction and before
addition of TFA or after stirring for 30 min at room
temperature after addition of TFA are shown in Table 3.
is [0198]
Table 3
before addition of
TFA
after addition of
TFA
HPLC peak area value (%)
condensation product
wherein 5'-hydroxy1
group is protected by
DMTr group
92.3
91.0
condensation
product wherein
DMTr group is
deprotected
^ ^ ^ ^ ^
N.D.
N.D.: not detected
[0199]
20 The above-mentioned results have revealed that when a
continuous solution synthesis method using iodine/pyridine as
an oxidizing agent is started from the condensation reaction,
the reaction system after the condensation reaction added with
2% TFA does not achieve deprotection of the DMTr group of the
25 5'-hydroxy1 group. Therefrom it was found that a preferable
order of reactions in the continuous solution synthesis of
oligonucleotide in the present invention is removal
(deprotection) of DMTr group, which is a temporary protecting
group of 5'-hydroxyl group, condensation and oxidation,
80
followed by precipitation by addition, after oxidation, of
methanol and the like saturated with hypo, and isolation
operation for solid-liquid separation.
[0200]
5 Example 6: Synthesis of 5'-0-(4,4'-
dimethoxytrityl) deoxythymidin-3' -yl-N- [3, 4, 5-
tris(octadecyloxy)benzyl]succinamate '
[0201]
DMTrO
OC18H37
0C18H37
OC18H37
io [0202]
A triethylamine salt (1.45 g, 1.94 mmol) of 5'-0-(4,4'-
dimethoxytrityl)deoxythymidine-3' -O-succinate and 3,4,5-
tris(octadecyloxy)benzyl amine (1.02 g, 1.10 mmol) were
dissolved in dehydrating dichloromethane (15 mL), 2-(lH-
15 benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
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
20 layer chromatography, and the mixture was washed with
saturated 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
25 solution, the mixture was filtered, and the obtained solid was
purified by silica gel column chromatography
(dichloromethane/methanol, 1% v/v triethylamine) to give the
81
title compound (1.22 g, 72.4%) as a white solid.
TLC: Rf=0.50 (dichloromethane:methanol=4:1)
^-NMR (4 00MHz) : 5 0.89 (t, 9H, J=7.0Hz, H3C(octadecyloxy)),
1.25-1.79 (m, 102H, -CH2- (octadecyloxy) ) , 1.35 (s, 3H, N5-CH3-
5 thymidine), 2.45 (m, 2H, 2' -thymidine) , 2.51 (m, 2H, succinyl),
2.70 (m, 2H, succinyl), 3.46 (m, 2H, 5'-thymidine), 3.79 (s,
6H, H3CO-DMT1:) , 3.79-3.95 (m, 6H, Bn-O-CH^-) , 4.15 (m, IH, 4'-
thymidine), 4.32 (d, 2H, J=5.5Hz, -NH-CH2-benzyl), 5.47 (m, IH,
3'-thymidine), 5.72 (d, 2H, J=5.5Hz, -NH-CH^-benzyl) , 6.41 (m,
10 IH, 1'-Thymidine), 6.45 (s, 2H,-benzyl), 6.83 (d, 4H, J=9.0Hz,
DMTr), 7.24-7.38 (m, 9H, DMTr),.7.61 (s, IH, N6-thymidine),
7.95 (br s, N3-NH-thymidine)
[0203]
Example 7: Synthesis of 5'-0-(4,4'-
15 dimethoxytrityl) deoxythymidin-3' -yl- [3, 5-
bis(docosyloxy)benzyl]succinate
[0204]
DMTrO
22H45
20
OC22H45
[0205]
A triethylamine salt (837 mg, 1.12 mmol) of 5'-0-(4,4'-
dimethoxytrityl)deoxythymidine-3'-O-succinate and 3,5-
bis(docosyloxy)benzyl alcohol (500 mg, 660 umol) were dissolved
in a mixed solvent of dehydrating dichloromethane (10 mL) and
dehydrating diethyl ether (10 mL), 2-(lH-benzotriazol-1-yl)-
25 1,1,3,3-tetramethyluronium hexafluorophosphate [HBTU] (3.00 g,
7.82 mmol) and N,N-diisopropylethylamine (1.40 mL, 7.82 mmol)
82
were added, and the mixture was stirred at 30°C for 4 hr. The
disappearance of the starting material was confirmed by thin
layer chromatography, and the mixture was washed with
saturated aqueous sodium hydrogen carbonate solution and
5 saturated brine. The obtained organic layer was dried over
sodium sulfate and filtered. The filtrate was concentrated
under reduced pressure, and methanol was added. The
precipitated solid was collected by filtration and purified by
silica gel column chromatography (dichloromethane/methanol, 1%
10 v/v triethylamine) to give the title compound (702 mg, 76.9%)
as a white solid.
TLC: Rf=0.60 (dichloromethane:methanol=9:1)
1H-NMR(400MHz): 5 0.88 (t, 6H, J=7.0, KbC-docosyloxy), 1.25-
1.76 (m, 84H, -CHr-docosyloxy) , 1.35 (s, 3H, N5-CH3-thymidine) ,
15 2.45 (m, 2H, 2'-thymidine), 2.67 (m, 4H, succinyl), 3.46 (m,
2H, 5'-thymidine), 3.79 (s, 6H, CH30-DMTr), 3.90 (t, 4H,
J=6.6Hz, Bn-0-CH2-), 4.11 (m, IH, 4'-thymidine), 5.03 (s, 2H,
NH-CH2-benzyl), 5.47 (m, IH, 3'-thymidine), 6.38 (m, IH, 3'-
thymidine), 6.85 (s, 2H, -benzyl), 6.83 (d, 4H, J=9.0, DMTr),
20 7.24-7.38 (m, 9H, DMTr), 7.60 (m, IH, N6-thymidine), 7.97 (br s,
IH, N3-NH-thymidine)
[0206]
Example 8: Synthesis of 5'-0-(4,4'-
dimethoxytrityl)deoxythymidin-3'-yl-N-[3,5-
25 bis(docosyloxy)benzyl]succinamate
[0207]
83
DMTrO
22H45
OC22H45
[0208]
A triethylamine salt (223 mg, 299 (jmol) of 5'-0-(4,4'-
dimethoxytrityl)deoxythymidine-3'-O-succinate and 3,5-
5 bis (docosyloxy)benzyl amine (133 mg, 175 |imol) were dissolved
in dehydrating dichloromethane (5 mL), 2-(lH-benzotriazol-lyl)
-1, 1, 3, 3-tetramethyluronium hexafluorophosphate [HBTU] (375
mg, 954 (imol) and N,N-diisopropylethylamine (170 uL, 954 (jmol)
were added, and the mixture was stirred at room temperature
10 for 2 hr. The disappearance of the starting material was
confirmed by thin layer chromatography, and the mixture was
washed with saturated aqueous sodium hydrogen carbonate
solution and saturated brine. The obtained organic layer was
dried over sodium sulfate, filtered and concentrated under
15 reduced pressure. Methanol was added to the filtrate, and the
precipitated solid was collected by filtration. The obtained
solid was dried under reduced pressure to give the title
compound (205 mg, 84.6%) as a white solid.
TLC: Rf=0.40 (dichloromethane:methanol=9:1)
20 ^-NMR (400MHz) : 5 0.88 (t, 6H, J=7.0, HaC-docosyloxy), 1.25-
1.75 (m, 84H, -CHa-docosyloxy), 1.35 (s, 3H, N5-CH3-thymidine) ,
2.45 (m, 2H, 2'-thymidine), 2.52 (m, 2H, succinyl), 2.69 (m,
2H, succinyl), 3.4 6 (m, 2H, 5'-thymidine), 3.7 9 (s, 6H, CH3ODMTr),
3.89 (t, 4H, J=6.6Hz, -O-CHg-docosyloxy), 4.14 (m, 1H,
25 4'-thymidine), 4.34 (d, 2H, J=5.6Hz, -NH-CHg-benzyl) , 5.47 (m,
1H, 3'-thymidine), 5.74 (t, 1H, J=5.6Hz, -NH-CH2-benzyl) , 6.35
84
(m, 1H, 1' -thymidine), 6.38 (s, 2H,-benzyl), 6.83 (d, 4H,
J=9.0Hz, -DMTr), 7.24-7.38 (m, 9H, -DMTr) , 7.61 (s, 1H, N6-
thymidine), 7.93 (br s, 1H, N3-NH-thymidine)
[0209]
5 Example 9: Synthesis of 5'-0-(4,4'-
dimethoxytrityl)deoxythymidin-3'-yl-[2,4-
bis(docosyloxy)benzyl]succinate
[0210]
DMTrO
OC22H45
OC22H45
10 [0211]
A triethylamine salt (1.69 g, 2.24 mmol) of 5'-0-(4,4'-
dimethoxytrityl)deoxythymidine-3'-O-succinate and 2,4-
bis(docosyloxy)benzyl alcohol (990 mg, 1.32 mmol) were
dissolved in dehydrating dichloromethane (15 mL), 2-(lH-
15 benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate [HBTU] (2.77 g, 7.20 mmol) and N,Ndiisopropylethylamine
(1.28 mL, 7.20 mmol) were added, and the
mixture was stirred at room temperature for 1 hr. The
disappearance of the starting material was confirmed by thin
20 layer chromatography, and the mixture was washed with
saturated aqueous sodium hydrogen carbonate solution and
saturated brine. The obtained organic layer was dried over
sodium sulfate, filtered and concentrated under reduced
pressure. Methanol was added to the filtrate, and the
25 precipitated solid was collected by filtration and dried to
give the title compound (1.68 g, 92.0%) as a white solid.
85
TLC: Rf=0.70 (dichloromethane:methanol=9:1)
^-NMR (4 00MHz) : 5 0.88 (t, 6H, J=7.,0Hz, HsC-docosyloxy) , 1.25-
1.75 (m, 84H, -CH2-docosyloxy) , 1.35 (s, 3H, N5-CH3-thymidine) ,
2.44 (m, 2H, 2'-thymidine), 2.64 (m, 4H, succinyl), 3.45 (m,
5 2H, 5'-thymidine), 3.79 (s, 6H, H3C-DMTr), 3.92 (m, 4H, -O-CH2-
docosyloxy), 4.10 (m, 1H, 4'-thymidine), 5.11 (s, 2H, -O-CHgbenzyl),
5.47 (m, 1H, 3'-thymidine), 6.39 (m, 1H, 1'-
thymidine), 6.42 (s, 2H, -benzyl), 6.83 (d, 4H, cJ=8.9Hz, -
DMTr) , 7.17-7.39 (m, 9H, -DMTr) , 7.60 (s, 1H, N6-thymidine) ,
10 7.97 (br s, 1H, N3-NH-thymidine)
[0212]
Example 10: Synthesis of 5'-0-(4,4'-
dimethoxytrityl)deoxythymidin-3/ -yl-N-[2,4-
bis(docosyloxy)benzyl]succinamate
15 [0213]
DMTrO
OC22H45
OC22H45
[0214]
A triethylamine salt (1.70 g, 2.28 mmol) of 5'-0-(4,4'-
dimethoxytrityl)deoxythymidine-3'-O-succinate and 2,4-
20 bis(docosyloxy)benzyl amine (1.01 g, 1.32 mmol) were dissolved
in dehydrating dichloromethane (15 mL), 2-(lH-benzotriazol-1-
yl)-1,1,3,3-tetramethyluronium hexafluorophosphate [HBTU]
(2.73 g, 7.20 mmol) and N,N-diisopropylethylamine (1.28 mL,
7.20 mmol) were added, and the mixture was stirred at room
25 temperature for 1 hr. The disappearance of the starting
material was confirmed by thin layer chromatography, and the
86
mixture was washed with saturated aqueous sodium hydrogen
carbonate solution and saturated brine. The obtained organic
layer was dried over sodium sulfate, filtered and concentrated
under reduced pressure. Methanol was added to the filtrate,
5 and the precipitated solid was collected by filtration and
dried under reduced pressure to give the title compound (1.75
g, 96.0%) as a white solid.
TLC: Rf=0.60 (dichloromethane:methanol=9:1)
XH-NMR(400MHz): 5 0.88 (t, 6H, J=7.0Hz, HsC-docosyloxy), 1.25-
10 1.74 (m, 84H, -CHg-docosyloxy) , 1.34 (s, 3H, N5-CH3-thymidine) ,
2.45 (m, 2H, 2'-thymidine), 2.46 (m, 2H, succinyl), 2.66 (m,
2H, succinyl), 3.45 (m, 2H, 5'-thymidine), 3.79 (s, 6H, H3CODMTr),
3.89 (t, 2H, J=6.6Hz, -O-CHz-docosyloxy), 3.94 (t, 2H,
J=6.6Hz, -0-CH2-docosyloxy) , 4.11 (m, IH, 4'-thymidine) , 4.35
15 (d, 2H, jr=5.7Hz, -NH-CHg-benzyl) , 5.47 (m, IH, 3'-thymidine) ,
5.93 (t, IH, J=5.7Hz, -NH-CH2-benzyl), 6.38 (m, IH, 1'-
thymidine), 6.42 (s, 2H, -benzyl), 6.83 (d, 4H, J=9.0Hz, -
DMTr), 7.11-7.38 (m, 9H, -DMTr), 7.60 (s, IH, N6-thymidine),
7.96 (br s, IH, N3-NH-thymidine)
20 [0215]
Example 11: Synthesis of 5 ' - 0 - ( 4 , 4 ' -
dimethoxytrityl)deoxythymidin-3'-yl-N-[4,4'-
bis(docosyloxy)benzhydryl]succinamate
[0216]
OC22H45
DMTrO
OC22H45
25
[0217]
87
A triethylamine salt (2.02 g, 2.71 mmol) of 5'-0-(4,4'-
dimethoxytrityl)deoxythymidine-3'-O-succinate and 4,4'-
bis(docosyloxy)benzhydryl amine (1.25 g, 1.50 mmol) were
dissolved in dehydrating dichloromethane (15 mL), 2-(lH-
5 benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate [HBTU] (3.40 g, 8.93 mmol) and N,Ndiisopropylethylamine
(1.56 mL, 9.00 mmol) were added, and the
mixture was stirred at 40°C overnight. The disappearance of
the starting material was confirmed by thin layer
10 chromatography, and the reaction mixture was concentrated
under reduced pressure. Methanol was added to the concentrated
solution, and the precipitated solid was collected by
filtration. The obtained solid was purified by silica gel
column chromatography (dichloromethane/methanol,1% v/v
15 triethylamine) to give the title compound (1.65 g, 75.3%) as a
white solid.
TLC: Rf=0.70 (dichloromethane:methanol=10:1)
XH-NMR(400MHz): 5 0.88 (t, 6H, Jfc7.0Hz, HaC-docosyloxy), 1.25-
1.76 (m, 84H, -CH2-docosyloxy), 1.36 (s, 3H, N5-CH3-thymidine),
20 2.42 (m, 2H, 2'-thymidine), 2.55 (m, 2H, succinyl), 2.70 (m,
2H, succinyl), 3.45 (m, 2H, 5'-thymidine), 3.78 (s, 6H, H3CODMTr)
, 3.89 (dt, 4H, J=6.7, 13.6Hz, -O-CHg-docosyloxy) , 4.11 (m,
1H, 4'-thymidine) , 5.48 (m, 1H, 3'-thymidine) , 6.03 (d, 1H,
J=7.8Hz, -NH-CH-benzhydryl), 6.10 (d, 1H, J=7.8Hz, -NH-CH-
25 benzhydryl), 6.39 (m, 1H, 1' -thymidine), 6.81 (m, 4H, -
benzhydryl), 6.83 (d, 4H, J=9.0Hz, -DMTr), 7.08-7.39 (m, 9H+4H,
-DMTrt-benzhydryl), 7.59 (s, 1H, N6-thymidine), 7.89 (br s, 1H,
N3-NH-thymidine)
[0218]
30 Example 12: Synthesis of 5'-0-(4,4'-
dimethoxytrityl)deoxythymidin-3'-yl-N-[2,3,4-
tris (octadecyloxy)benzhydryl]succinamate
[0219]
88
DMTrO
OC18H37
OCi8H37
OC18H37
[0220]
A triethylamine salt (1.29 g, 1.70 mmol) of 5'-0-(4,4'-
dimethoxytrityl)deoxythymidine-3'-O-succinate and 2,3,4-
5 tris (octadecyloxy)benzhydryl amine (1.01 g, 1.02 mmol) were
dissolved in dehydrating dichloromethane (15 mL), 2-(lHbenzotriazol-
1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate [HBTU] (2.31 g, 6.09 mmol) and N,Ndiisopropylethylamine
(1.07 mL, 6.06 mmol) were added, and the
10 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 aqueous sodium hydrogen carbonate solution and
saturated brine. The obtained organic layer was dried over
15 sodium sulfate, filtered and concentrated under reduced
pressure. Methanol was added to the filtrate, and the
precipitated solid was collected by filtration and dried under
reduced pressure to give the title compound (1.14 g, 68.8%) as
a white solid.
20 TLC: Rf=0.50 (dichloromethane:methanol=9:1)
XH-NMR(400MHz) spectra of diastereomer 1: 5 0.88 (t, 6H,
J=7.0Hz, H3C-octadecyloxy), 1.16-1.80 (m, 3H+102H, N5-CH3-
thymidine+-CH2-octadecyloxy), 2.42-2.80 (m, 1H+4H, 2'-
thymidine+succinyl), 3.26 (m, 2H, 2' -thymidine), 3.45 (m, 2H,
25 5'-thymidine), 3.78 (s, 6H, HjCO-DMTr), 3.94 (m, 6H, -0-CH2-
octadecyloxy), 4.12 (m, 1H, 4'-thymidine), 5.50 (m, 1H, 3'-
thymidine), 6.25 (d, 1H, J=8.6Hz, -NH-CH-benzhydryl), 6.41 (m,
89
IH, 1' -thymidine) , 6.59 (d, IH, j=8.6Hz, -benzhydryl) , 6.74 (d,
IH, J=8.6Hz, -NH-CH-benzhydryl), 6.83 (d, 4H, J=9.0Hz, -DMTr),
6.90 (d, IH, J=8.6Hz, -benzhydryl), 7.11-7.39 (m, 9H+5H, -
DMTr+-benzhydryl), 7.60 (s, IH, N6-thymidine), 8.00 (br s, IH,
5 N3-NH-thymidine)
1H-NMR(400MHz) spectra of diastereomer 2: 5 0.88 (t, 6H,
J=7.0Hz, H3C-octadecyloxy), 1.16-1.80 (m, 3H+102H, N5-CH3~
thymidine+-CH2-octadecyloxy), 2.42-2.80 (m, 1H+4H, 2'-
thymidine+succinyl), 3.26 (m, 2H, 2'-thymidine), 3.4 5 (m, 2H,
10 5'-thymidine), 3.78 (s, 6H, HaCO-DMTr), 3.94 (m, 6H, -O-CHgoctadecyloxy),
4.12 (m, IH, 4'-thymidine), 5.50 (m, IH, 3'-
thymidine), 6.26 (d, IH, J=8.6Hz, -NH-CH-benzhydryl), 6.41 (m,
IH, V -thymidine) , 6.61 (d, IH, J=8.6Hz, -benzhydryl), 6.76 (d,
IH, J=8.6Hz, -NH-CH-benzhydryl), 6.83 (d, 4H, J=9.0Hz, -DMTr),
15 6.92 (d, IH, J=8.6Hz, -benzhydryl), 7.11-7.39 (m, 9H+5H, -
DMTr+-benzhydryl), 7.60 (s, IH, N6-thymidine), 8.00 (br s, IH,
N3-NH-thymidine)
[0221]
Example 13: Synthesis of 5'-0-(4,4'-
20 dimethoxytrityl)deoxythymidin-3'-yl-{3,4,5-tris [3,4,5-
tris(octadecyloxy)benzyloxy]benzyl}succinate
[0222]
90
C18H37O |^ OC18H37
OCi8H37
[0223]
A triethylamine salt (408 mg, 548 jjmol) of 5'-0-(4,4'-
dimethoxytrityl)deoxythymidine-3'-O-succinate and 3,4,5-
5 tris[3,4,5-tris(octadecyloxy)benzyloxy]benzyl alcohol (904 mg,
318 |jmol) were dissolved in dehydrating dichloromethane (10 mL) ,
2- (lH-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate [HBTU] (1.09 g, 2.87 mmol) and N,Ndiisopropylethylamine
(507 \iL, 2.85 mmol) were added, and the
10 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 aqueous sodium hydrogen carbonate solution and
saturated brine. The obtained organic layer was dried over
15 sodium sulfate, filtered and concentrated under reduced
pressure. Methanol was added to the filtrate, and the
precipitated solid was collected by filtration and purified by
silica gel column chromatography
(dichloromethane/methanol=99/2, 1% v/v triethylamine) to give
20 the title compound (207 mg, 65.2%) as a white solid.
TLC: Rf=0.50 (dichloromethane:methanol=19:1)
^-NMR(4 00MHz): 5 0.88 (t, 27H, J=6.9, HsC-octadecyloxy), 1.25-
91
1.75 (m, 306H, -CHg-octadecyloxy) , 1.35 (s, 3H, N5-CH3-
thymidine) , 2.47 (m, 2H, 2'-thymidine) , 2.66 (m, 4H, succinyl) ,
3.47 (m, 2H, 5'-thymidine), 3.74 (t, 4H, J=6.3Hz, -benzyl-OCH^-
octadecyloxy), 3.78 (s, 6H, CH30-DMTr), 3.86 (t, 10H,
5 J=6.3Hz, -benzyl-O-CH^-octadecyloxy) , 3.92 (t, 4H, J=6.3Hz, -
benzyl-O-CHg-octadecyloxy) , 4.15 (m, IH, 4'-thymidine) , 4.99 (m,
2H+6H, -0-CH2-benzyl-0-CH2-benzyl+-0-CH2-benzyl-0-CH2-benzyl) ,
5.48 (m, IH, 3'-thymidine), 6.42 (m, IH, 3'-thymidine), 6.61
(s, 6H, -O-benzyl-0-benzyl), 6.66 (s, 2H, -0-benzyl-O-benzyl),
io 6.82 (d, 4H, J=8.9Hz, DMTr), 7.23-7.38 (m, 9H, DMTr), 7.60 (m,
IH, N6-thymidine), 7.93 (br s, IH, N3-NH-thymidine)
[0224]
Example 14: Synthesis of 5'-0-(4,4'-dimethoxytrityl)-2'-
methoxyuridin-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate
15 [0225]
O
NH
DMTrO^I o . i
C I 8H3 7 0 ^ Y ^ 0 A ^ Y O OMe
C18H37O J
OCi8H37
[0226]
(1) Synthesis of 5'-0-(4, 4'-dimethoxytrityl)-2'-
methoxyuridine-3/' -O-succinate
20 Under an argon atmosphere, 5'-0-(4,4'-dimethoxytrityl)-
2'-methoxyuridine (1.98 g, 3.57 mmol), succinic anhydride (631
mg, 6.30 mmol) and triethylamine (1.49 mL, 10.7 mmol) were
dissolved in dichloromethane (30 mL), and the mixture was
stirred at room temperature for 3 hr. The completion of the
25 reaction was confirmed by thin layer chromatography, and the
mixture was partition-washed three times with 2.OM phosphoric
acid-triethylamine buffer (pH 7.50). The organic layer was
92
evaporated under reduced pressure to quantitatively give a
triethylamine salt (2.80 g) of the title compound as a
colorless frothy solid.
(2) Synthesis of 5' -0- (4, 4' -dimethoxytrityl) -2''-methoxyuridin-
5 3' -yl-[3, 4, 5-tris(octadecyloxy)benzyl]succinate
The compound (1.40 g, 1.84 mmol) synthesized in Example
14-(1) and 3, 4, 5-tris(octadecyloxy)benzyl alcohol (989 mg,
1.08 mmol) were dissolved in dehydrating dichloromethane (40
mL), 2-(lH-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
10 hexafluorophosphate [HBTU] (2.46 g, 6.48 mmol) and N,Ndiisopropylethylamine
(1.13 mL, 6.4 8 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, methanol was added to the reaction
15 mixture, and the mixture was filtered. The obtained solid was
purified by silica gel column chromatography (hexane/ethyl
acetate, 1% v/v triethylamine) to give the title compound
(1.41 g, 84.0%) as a white solid.
TLC: Rf=0.72 (dichloromethane:methanol=9:1)
20 ^-NMR (400MHz) : 5 0.88 (t, 9H, J=6.6Hz, H3C (octadecyloxy) ) ,
1.18-1.80 (m, 102H, -CH2-(octadecyloxy)), 2.70 (m, 4H,
succinyl), 3.42-3.50 (m, IH, 5' -thymidine), 3.46 (s, 3H, 2'-
OMe), 3.56-3.62 (m, IH, 5'-thymidine), 3.79 (s, 6H, HgCO-DMTr),
3.90-4.00 (m, 6H, Bn-O-CH^-) , 4.08 (m, IH, 2'-H), 4.24 (m, IH,
25 4'-H), 5.01 (s, 2H, -0-CH2-benzyl), 5.28-5.33 (m, 2H, 3'-H and
N5-H), 6.02 (m, IH, l'-H), 6.53 (s, 2H, -benzyl), 6.84 (m, 4H,
DMTr), 7.24-7.38 (m, 9H, DMTr), 7.86 (d, IH, J=8.20Hz, N6-H),
8.10 (br s, N3-NH)
[0227]
30 Example 15: Synthesis of deoxycytidinyl- [3' —>5' ]-deoxyadenylyl-
[3' ->5']-deoxythymidinyl- [3'-»5' ] -deoxythymidine (5'-d[CATT]-
3')
Synthesis of 5'-d[CATT]-3' using
[0228]
93
DMTrO
18H37
O C 1 8 H 37
O C 1 8 H 37
[0229]
(1) Synthesis of 5' - 0 - (4, 4' -dimethoxytrityl) deoxythymidine-3' -
[0-(2-cyanoethyl)]phosphoryl-deoxythymidin-3'-yl-N-[3,4,5-
5 tris(octadecyloxy)benzyl]succinamate
The compound (302 mg, 195 umol) synthesized in Example 6
was dissolved in dichloromethane (4.5 mL), trifluoroacetic
acid (72.2 (j,L, 975 |jmol) and pyrrole (66.9 p.L, 975 fxmol) were
added, and the mixture was stirred for 5 min. The completion
10 of the deprotection was confirmed by thin layer chromatography,
pyridine (78.9 (J.L, 975 jumol) and N-methylimidazole (38.7 pL,
488 umol) were added, and the mixture was stirred for 10 min.
After neutralization, a dT-CE phosphoramidite reagent (5'-0-
(4,4'-dimethoxytrityl)deoxythymidine-3'-[0-(2-cyanoethyl)-
15 (N,N-diisopropyl) ]-phosphoramidite) (435 mg, 585 jjmol)
dissolved in 0.25 mol/L 5-(benzylthio)-1Htetrazole/
acetonitrile solution (1.5 mL) was added to the
reaction mixture, and the mixture was stirred for 10 min. 0.2
mol/L Iodine pyridine/tetrahydrofuran/water =49/49/2 solution
20 (5.9 mL) was added, and the mixture was stirred for 10 min.
After completion of the reaction, a methanol solution
saturated with sodium thiosulfate was added to the reaction
mixture, and the resultant solid was collected by'filtration
and dried to quantitatively give the title compound (385 mg)
25 as a white solid.
(2) Synthesis of 5'-0-(4,4'-dimethoxytrityl)-N6-benzoyl-2' -
94
deoxyadenosine-3' -[0-(2-cyanoethyl)]phosphoryl-deoxythymidine-
3' - [0- (2-cyanoethyl) Jphosphoryl-deoxythymidin-3' -yl-N- [3, 4, 5-
tris(octadecyloxy)benzyl]succinamate
The compound (383 mg, 200 umol) synthesized in Example
5 15-(1) was dissolved in dichloromethane (4.5 mL),
trifluoroacetic acid (72.2 uL, 975 umol) and pyrrole (66.9 uL,
975 (jmol) were added, and the mixture was stirred for 5 min.
The completion of the deprotection was confirmed by thin layer
chromatography, pyridine (78.9 uL, 975 (jmol) and N-
10 methylimidazole (38.7 uL, 488 umol) were added, and the mixture
was stirred for 10 min. After neutralization, a dA-CE
phosphoramidite reagent (5'-0-(4,A'-dimethoxytrityl)-N6-
benzoyl-2'-deoxyadenosine-3'-[0-(2-cyanoethyl)-(N,Ndiisopropyl)
]-phosphoramidite) (514 mg, 600 umol) dissolved in
15 0.25 mol/L 5-(benzylthio)-lH-tetrazole/acetonitrile solution
(1.5 mL) was added to the reaction mixture, and the mixture
was stirred for 10 min. 0.2 mol/L Iodine
pyridine/tetrahydrofuran/water = 49/49/2 solution (5.9 mL) was
added, and the mixture was stirred for 10 min. After
20 completion of the reaction, a methanol solution saturated with
sodium thiosulfate was added to the reaction mixture, and the
resultant solid was collected by filtration and dried to give
the title compound (479 mg, 99.8%) as a white solid.
(3) Synthesis of 5'-0-(4,A'-dimethoxytrityl)-N4-benzoyl-2' -
25 deoxycytidine-3' - [0- (2-cyanoethyl) ]phosphoryl-N6-benzoyl-2' -
deoxyadenosine-3'-[0-(2-cyanoethyl)]phosphoryl-deoxythymidine-
3'-[0-(2-cyanoethyl)]phosphoryl-deoxythymidin-3'-yl-N-[3,4,5-
tris(octadecyloxy)benzyl]succinamate
The compound (476 mg, 198 umol) synthesized in Example
30 15-(2) was dissolved in dichloromethane (4.5 mL),
trifluoroacetic acid (72.2 uL, 975 umol) and pyrrole (66.9 uL,
975 (jmol) were added, and the mixture was stirred for 5 min.
The completion of the deprotection was confirmed by thin layer
chromatography, pyridine (78.9 uL, 975 umol) and N-
35 methylimidazole (38.7 uL, 488 umol) were added, and the mixture
95
was stirred for 10 min. After neutralization, a dC-CE
phosphoramidite reagent (5'-0-(4,4'-dimethoxytrityl)-N4-
benzoyl-2'-deoxycytidine-3'-[0-(2-cyanoethyl)-(N,Ndiisopropyl)
]-phosphoramidite) (500 mg, 600 umol) dissolved in
5 0.25 mol/L 5-(benzylthio)-lH-tetrazole/acetonitrile solution
(1.5 inL) was added to the reaction mixture, and the mixture
was stirred for 10 min. 0.2 mol/L Iodine
pyridine/tetrahydrofuran/water =4 9/4 9/2 solution (5.9 inL) was
added, and the mixture was stirred for 10 min. After
10 completion of the reaction, a methanol solution saturated with
sodium thiosulfate was added to the reaction mixture, and the
resultant solid was collected by filtration and dried to give
the title compound (522 mg, 91.9%) as a white solid.
(4) Synthesis of deoxycytidinyl- [3' ->5' ]-deoxyadenylyl-
15 [3'->5' ] -deoxythymidinyl- [3'->5' ] -deoxythymidine (5' -d[CATT] -
3')
The compound synthesized in Example 15-(3) and a solution
(4.0 mL) of 28% aqueous ammonia solution:ethanol=3:1 were
placed in an autoclave, and the mixture was heated at 65°C for
20 16 hr. The reaction mixture was concentrated by a centrifugal
evaporator under reduced pressure. The mixture 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
25 deprotected with 2% aqueous trifluoroacetic acid solution and
eluted with 20% aqueous acetonitrile solution to give the
title compound.
m/z(MALDI TOF) : Anal. Calc. for C39H51N12O23P3: 1148.2. Found
1147.0(M-H)~
30 [0230]
Example 16: Synthesis of deoxycytidinyl- [3'—>5' ]-deoxyadenylyl-
[ 3' -»5' ] -deoxythymidinyl-[3'->5' ] -deoxythymidine (5' -d[CATT]-
3')
S y n t h e s i s of 5'-d[CATT]-3' using
35 [0231]
96
DMTrO
22H45
OC22H45
[0232]
(1) Synthesis of 5'-0-(4,4'-dimethoxytrityl)-deoxythymidine-
3'- [0-(2-cyanoethyl)]phosphoryl-deoxythymidin-3'-yl-[3,5-
5 bis(docosyloxy)benzyl]succinate
The compound (202 mg, 146 pinol) synthesized in Example 7
was dissolved in dichloromethane (3.0 mL), trifluoroacetic
acid (53.5 uL, 723 umol) and pyrrole (50.0 \XL, 723 umol) were
added, and the mixture was stirred for 5 min. The completion
10 of the deprotection was confirmed by thin layer chromatography,
pyridine (58.4 |iL, 723 (jmol) and N-methylimidazole (28.6 uL,
361 jjmol) were added, and the mixture was stirred for 10 min.
After neutralization, a dT-CE phosphoramidite reagent (208 mg,
289 \mol) dissolved in 0.25 mol/L 5-(benzylthio)-1H-
15 tetrazole/acetonitrile solution (1.0 mL) was added to the
reaction mixture, and the mixture was stirred for 10 min. 0.2
mol/L Iodine pyridine/tetrahydrofuran/water=49/49/2 solution
(2.9 mL) was added, and the mixture was stirred for 10 min.
After completion of the reaction, a methanol solution
20 saturated with sodium thiosulfate was added to the reaction
mixture, and the resultant solid was collected by filtration
and dried to give the title compound (226 mg, 89.0%) as a
white solid.
(2) Synthesis of 5'-0-(4,4'-dimethoxytrityl)-N6-benzoyl-2'-
25 deoxyadenosine-3' -[0-(2-cyanoethyl)]phosphoryl-deoxythymidine-
3'-[0-(2-cyanoethyl)]phosphoryl-deoxythymidin-3'-yl-[3,5-
97
bis(docosyloxy)benzyl]succinate
The compound (225 mg, 129 umol) synthesized in Example
16-(1) was dissolved in dichloromethane (3.0 mL),
trifluoroacetic acid (47.8 uL, 645 (amol) and pyrrole (44.6 jiL,
5 645 pmol) were added, and the mixture was stirred for 5 min.
The completion of the deprotection was confirmed by thin layer
chromatography, pyridine (52.1 ^L, 645 pmol) and Nmethylimidazole
(25.6 fiL, 323 pmol) were added, and the mixture
was stirred for 10 min. After neutralization, a dA-CE
10 phosphoramidite reagent (334 mg, 387 jxmol) dissolved in 0.25
mol/L 5- (benzylthio)-lH-tetrazole/acetonitrile solution (1.0
mL) was added to the reaction mixture, and the mixture was
stirred for 10 min. 0.2 mol/L Iodine
pyridine/tetrahydrofuran/water=49/49/2 solution (3.9 mL) was
15 added, and the mixture was stirred for 10 min. After
completion of the reaction, a methanol solution saturated with
sodium thiosulfate was added to the reaction mixture, and the
resultant solid was collected by filtration and dried to
quantitatively give the title compound (288 mg) as a white
20 solid.
(3) Synthesis of 5' -0- (4, 4' -dimethoxytrityl) -N4-benzoyl-2' -
deoxycytidine-3' - [0- (2-cyanoethyl) ] phosphoryl -N6-benzoyl-2' -
deoxyadenosine-3' - [0- (2-cyanoethyl) jphosphoryl-deoxythymidine-
3'-[0-(2-cyanoethyl)]-phosphoryl-deoxythymidin-3'-yl-[3,5-
25 bis(docosyloxy)benzyl]succinate
The compound (287 mg, 129 umol) synthesized in Example
16-(2) was dissolved in dichloromethane (3.0 mL),
trifluoroacetic acid (47.8 JJ.1, 645 jxmol) and pyrrole (44.6 (j.1,
645 (jmol) were added, and the mixture was stirred for 5 min.
30 The completion of the deprotection was confirmed by thin layer
chromatography, pyridine (52.1 uL, 645 (jmol) and Nmethylimidazole
(25.6 |j.L, 323 umol) were added, and the mixture
was stirred for 10 min. After neutralization, a dC-CE
phosphoramidite reagent (325 mg, 387 )amol) dissolved in 0.25
35 mol/L 5-(benzylthio)-lH-tetrazole/acetonitrile solution (1.0
98
mL) was added to the reaction mixture, and the mixture was
stirred for 10 min. 0.2 mol/L Iodine
pyridine/tetrahydrofuran/water=49/49/2 solution (3.9 mL) was
added, and the mixture was stirred for 10 min. After
5 completion of the reaction, a methanol solution saturated with
sodium thiosulfate was added to the reaction mixture, and the
resultant solid was collected by filtration and dried to give
the title compound (316 mg, 91.2%) as a white solid.
(4) Synthesis of deoxycytidinyl-[3' —>5r ] -deoxyadenylyl-
10 [3'-»5' ] -deoxythymidinyl-[3'->5' ] -deoxythymidine (5' -d[CATT]-
3')
The compound synthesized in Example 16-(3) and a solution
(4.0 mL) of 28% aqueous ammonia solution:ethanol=3:1 were
placed in an autoclave, the mixture was heated at 65°C for 16
15 hr, and concentrated by a centrifugal evaporator under reduced
pressure. The concentrated 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
20 deprotected with 2% aqueous trifluoroacetic acid solution and
eluted with 20% aqueous acetonitrile solution to give the
title compound.
m/z(MALDI TOF) : Anal. Calc. for C39H51N12O23P3: 1148.2. Found
1147.0 (M-H)~
25 [0233]
Example 17: Synthesis of d e o x y c y t i d i n y l - [ 3 ' —>5' ] -deoxyadenylyl-
[ 3 ' - > 5 ' ] - d e o x y t h y m i d i n y l - [ 3 ' - > 5 ' ] -deoxythymidine (5'-d[CATT]-
3')
Synthesis of 5'-d[CATT]-3' using
30 [0234]
99
DMTrO
OC22H45
OC22H45
[0235]
(1) Synthesis of 5'-0-(4,4'-dimethoxytrityl)-deoxythymidine-
3'~[0-(2-cyanoethyl)]phosphoryl-deoxythymidin-3'-yl-N-[2,4-
5 bis(docosyloxy)benzyl]succinamate
The compound (197 mg, 143 umol) synthesized in Example 10
was dissolved in dichloromethane (3.0 mL) under an argon
atmosphere, trifluoroacetic acid (53.5 uL, 720 umol) and
pyrrole (50.0 uL, 720 umol) were added, and the mixture was
10 stirred for 5 min. The completion of the deprotection was
confirmed by thin layer chromatography, pyridine (58.4 uL, 720
umol) and N-methylimidazole (28.6 uL, 360 umol) were added, and
the mixture was stirred for 10 min. After neutralization, a
dT-CE phosphoramidite reagent (201 mg, 289 umol) dissolved in
15 0.25 mol/L 5-(benzylthio)-IH-tetrazole/acetonitrile solution
(1.0 mL) was added to the reaction mixture, and the mixture
was stirred for 10 min. 0.2 mol/L Iodine
pyridine/tetrahydrofuran/water=4 9/4 9/2 solution (2.9 mL) was
added, and the mixture was stirred for 10 min. After
20 completion of the reaction, a methanol solution saturated with
sodium thiosulfate was added to the reaction mixture, and the
resultant solid was collected by filtration and dried to give
the title compound (248 mg, 99.9%) as a white solid.
(2) Synthesis of 5' -0- (4, 4' -dimethoxytrityl) -N6-benzoyl-2' -
25 deoxyadenosine-3' - [0- (2-cyanoethyl) jphosphoryl-deoxythymidine-
3'-[0- (2-cyanoethyl)]phosphoryl-deoxythymidin-3,-yl-N-[2, 4-
100
bis(docosyloxy)benzyl]succinamate
The compound (235 mg, 135 umol) synthesized in Example
17-(1) was dissolved in dichloromethane (3.0 mL) under an
argon atmosphere, trifluoroacetic acid (53.5 uL, 720 umol) and
5 pyrrole (50.0 uL, 720 umol) were added, and the mixture was
stirred for 5 min. The completion of the deprotection was
confirmed by thin layer chromatography, pyridine (58.4 uL, 720
umol) and N-methylimidazole (28.6 uL, 360 umol) were added, and
the mixture was stirred for 10 min. After neutralization, a
10 dA-CE phosphoramidite reagent (347 mg, 405 umol) dissolved in
0.25 mol/L 5-(benzylthio)-lH-tetrazole/acetonitrile solution
(1.0 mL) was added to the reaction mixture, and the mixture
was stirred for 10 min. 0.2 mol/L Iodine
pyridine/tetrahydrofuran/water=49/4 9/2 solution (3.9 mL) was
15 added, and the mixture was stirred for 10 min. After
completion of the reaction, a methanol solution saturated with
sodium thiosulfate was added to the reaction mixture, and the
resultant solid was collected by filtration and dried to give
the title compound (276 mg, 91.9%) as a white solid.
20 (3) Synthesis of 5' -0- (4, 4' -dimethoxytrityl) -N4-benzoyl-2' -
deoxycytidine-3' - [0- (2-cyanoethyl) ] phosphoryl-N6-benzoyl-2' -
deoxyadenosine-3'-[0-(2-cyanoethyl)]phosphoryl-deoxythymidine-
3'-[0-(2-cyanoethyl)]phosphoryl-deoxythymidin-3' -yl-N-[2,4-
bis(docosyloxy)benzyl]succinamate
25 The compound (253 mg, 94.2 umol) synthesized in Example
17-(2) was dissolved in dichloromethane (3.0 mL) under an
argon atmosphere, - trifluoroacetic acid (34.9 uL, 471 umol) and
pyrrole (32.6 uL, 471 umol) were added, and the mixture was
stirred for 5 min. The completion of the deprotection was
30 confirmed by thin layer chromatography, pyridine (38.1 uL, 471
umol) and N-methylimidazole (18.7 uL, 235 umol) were added, and
the mixture was stirred for 10 min. After neutralization, a
dC-CE phosphoramidite reagent (236 mg, 282 umol) dissolved in
0.25 mol/L 5-(benzylthio)-lH-tetrazole/acetonitrile solution
35 (1.0 mL) was added to the reaction mixture, and the mixture
101
was stirred for 10 min. 0.2 mol/L Iodine
pyridine/tetrahydrofuran/water=4 9/4 9/2 solution (3.9 mL) was
added, and the mixture was stirred for 10 min. After
completion of the reaction, a methanol solution saturated with
5 sodium thiosulfate was added to the reaction mixture, and the
resultant solid was collected by filtration and dried to give
the title compound (252 mg, 99.5%) as a white solid.
(4) Synthesis of deoxycytidinyl- [3'—>5' ] -deoxyadenylyl-
[3'-»5' ] -deoxythymidinyl- [3'->5' ] -deoxythymidine (5'-d[CATT]-
10 y±
The compound synthesized in Example 17-(3) and a solution
(4.0 mL) of 28% aqueous ammonia solution:ethanol=3:1 were
placed in an autoclave, the mixture was heated at 65°C for 16
hr, and concentrated by a centrifugal evaporator under reduced
15 pressure. The concentrated 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
20 eluted with 20% aqueous acetonitrile solution to give the
title compound.
m/z(MALDI TOF) : Anal. Calc. for C39H51N12O23P3: 1148.24. Found
1149.63(M+H)+
[0236]
25 Example 18: Synthesis of deoxycytidinyl- [3'—>5' ]-deoxyadenylyl-
[3' ->5']-deoxythymidinyl- [3'->5' ]-deoxythymidine(5'-d [CATT]-3')
Synthesis of 5'-d[CATT]-3' using
[0237]
102
DMTrO
9^18^37
0C1 8H37
OC1 8H37
[0238]
(1) Synthesis of 5'-0-(4,4'-dimethoxytrityl)-deoxythymidine-
3'-[0-(2-cyanoethyl)]phosphoryl-deoxythymidin-3'-yl-N-[2,3,4-
5 tris(octadecyloxy)benzhydryl]succinamate
The compound (401 mg, 246 umol) synthesized in Example 12
was dissolved in dichloromethane (3.0 mL) under an argon
atmosphere, trifluoroacetic acid (91.0 uL, 1.23 mmol) and
pyrrole (85.0 uL, 1.23 mmol) were added, and the mixture was
io stirred for 5 min. The completion of the deprotection was
confirmed by thin layer chromatography, pyridine (99.5 uL, 1.23
mmol) and N-methylimidazole (48.8 ul, 615 umol) were added, and
the mixture was stirred for 10 min. After neutralization, a
dT-CE phosphoramidite reagent (373 mg, 492 umol) dissolved in
is 0.25 mol/L 5-(benzylthio)-IH-tetrazole/acetonitrile solution
(1.0 mL) was added to the reaction mixture, and the mixture
was stirred for 10 min. 0.2 mol/L Iodine
pyridine/tetrahydrofuran/water=49/49/2 solution (4.9 mL) was
added, and the mixture was stirred for 10 min. After
20 completion of the reaction, a methanol solution saturated with
sodium thiosulfate was added to the reaction mixture, and the
resultant solid was collected by filtration and dried to give
the title compound (491 mg, 95.7%) as a white solid.
(2) Synthesis of 5' -0-(4,4'-dimethoxytrityl)-N6-benzoyl-2' -
25 deoxyadenosine-3' -[0-(2-cyanoethyl)]phosphoryl-deoxythymidine-
3'- [0-(2-cyanoethyl)j phosphoryl-deoxythymidin-3'-yl-N-[2,3, 4-
tris(octadecyloxy)benzhydryl]succinamate
103
The compound (228 mg, 115 umol) synthesized in Example
18-(1) was dissolved in dichloromethane (3.0 mL) under an
argon atmosphere, trifluoroacetic acid (42.6 uL, 575 umol) and
pyrrole (39.8 uL, 575 umol) were added, and the mixture was
5 stirred for 5 min. The completion of the deprotection was
confirmed by thin layer chromatography, pyridine (46.5 uL, 575
umol) and N-methylimidazole (22.9 uL, 288 umol) were added, and
the mixture was stirred for 10 min. After neutralization, a
dA-CE phosphoramidite reagent (296 mg, 345 umol) dissolved in
10 0.25 mol/L 5-(benzylthio)-IH-tetrazole/acetonitrile solution
(1.0 mL) was added to the reaction mixture, and the mixture
was stirred for 10 min. 0.2 mol/L Iodine
pyridine/tetrahydrofuran/water =49/49/2 solution (2.9 mL) was
added, and the mixture was stirred for 10 min. After
15 completion of the reaction, a methanol solution saturated with
sodium thiosulfate was added to the reaction mixture, and the
resultant solid was collected by filtration and dried to give
the title compound (269 mg, 94.1%) as a white solid.
(3) Synthesis of 5'-0-(4,4'-dimethoxytrityl)-N4-benzoyl-2' -
20 deoxycytidine-3' -[0-(2-cyanoethyl)]phosphoryl-N6-benzoyl-2' -
deoxyadenosine-3'-[0-(2-cyanoethyl)]phosphoryl-deoxythymidine-
3' - [0- (2-cyanoethyl) ]phosphoryl-deoxythymidin-3' -yl-N- [2, 3, 4-
tris(octadecyloxy)benzhydryl]succinamate
The compound (254 mg, 102 umol) synthesized in Example
25 18-(2) was dissolved in dichloromethane (3.0 mL) under an
argon atmosphere, trifluoroacetic acid (37.8 uL, 510 umol) and
pyrrole (35.3 uL, 510 umol) were added, and the mixture was
stirred for 5 min. The completion of the deprotection was
confirmed by thin layer chromatography, pyridine (41.2 uL, 510
30 umol) and N-methylimidazole (20.2 uL, 255 umol) were added, and
the mixture was stirred for 10 min. After neutralization, a
dC-CE phosphoramidite reagent (254 mg, 306 umol) dissolved in
0.25 mol/L 5-(benzylthio)-IH-tetrazole/acetonitrile solution
(1.0 mL) was added to the reaction mixture, and the mixture
35 was stirred for 10 min. 0.2 mol/L Iodine
104
pyridine/tetrahydrofuran/water=49/49/2 solution (2.1 mL) was
added, and the mixture was stirred for 10 min. After
completion of the reaction, a methanol solution saturated with
sodium thiosulfate was added to the reaction mixture, and the
5 resultant solid was collected by filtration and dried to give
the title compound (293 mg, 97.3%) as a white solid.
(4) Synthesis of deoxycytidinyl-[3' ->5' ] -deoxyadenylyl-
[ 3'->5' ] -deoxythymidinyl-[3' ->5' ] -deoxythymidine (5'-d[CATT]-
3')
io The compound synthesized in Example 18-(3) and a solution
(4.0 mL) of 28% aqueous ammonia solution:ethanol=3:1 were
placed in an autoclave, the mixture was heated at 65°C for 16
hr, and concentrated by a centrifugal evaporator under reduced
pressure. The concentrated solution was adsorbed to C-18
15 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 give the
20 title compound.
m/z(MALDI TOF) : Anal. Calc. for C39H51Ni2023P3: 1148.24. Found
1146.8(M-H)"
[0239]
Example 19: Synthesis of 5'-0-(4,4'-dimethoxytrityl)-2'-
25 fluorouridine-3'-[0-(2-cyanoethyl)]phosphoryl-deoxythymidin-
3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate
[0240]
105
DMTrO
O
C18H37O y^Y^Q^^^Y °
>V ° O C 1 8 H 3 7
C18H37O
[0241]
5'-0-(4,4'-dimethoxytrityl)-deoxythymidin-3' -yl-[3,4, 5-
tris(octadecyloxy)benzyl]succinate (214 mg, 138 umol) was
5 dissolved in dichloromethane (3.0 mL) under an argon
atmosphere, trifluoroacetic acid (96.3 p.L, 1.38 mmol) and
pyrrole (90.0 (J.L, 1.38 mmol) were added, and the mixture was
stirred for 5 min. The completion of the deprotection was
confirmed by thin layer chromatography, pyridine (105 |u.L, 1.38
10 mmol) and N-methylimidazole (51.6 \xL, 690 |jinol) were added, and
the mixture was stirred for 10 min. After neutralization, a
2'-F-U-CE phosphoramidite reagent (5'-0-(4,4'-
dimethoxytrityl)-2'-fluorouridine-3'-[0-(2-cyanoethyl)-(N,Ndiisopropyl)
]-phosphoramidite) (390 mg, 520 jimol) dissolved in
15 0.25 mol/L 5-(benzylthio)-IH-tetrazole/acetonitrile solution
(1.5 mL) was added to the reaction mixture, and the mixture
was stirred for 10 min. 0.2 mol/L Iodine
pyridine/tetrahydrofuran/water=49/49/2 solution (3.9 mL) was
added, and the mixture was stirred for 10 min. After
20 completion of the reaction, a methanol solution saturated with
sodium thiosulfate was added to the reaction mixture, and the
resultant solid was collected by filtration and dried to give
106
the title compound (243 mg, 93.2%) as a white solid.
m/z(ESI-MS): Anal. Calc. for C108Hi63FN502oP: 1900.16. Found
1919.19(M+NH4) +
[0242]
5 Example 20: Synthesis of 5'-0-(4,4'-dimethoxytrityl)-2'-
methoxyuridine-3' - [0- (2-cyanoethyl) ] phosphoryl-2' -
methoxyuridin-3' -yl-[3,4,5-tris(octadecyloxy)benzyl]succinate
[0243]
O
NH
DMTrCL ~N °
^
O OMe
O
NC O. NA0
C i s H s / O y ^ s ^ o ^ ^ y O OMe
C18H37° |^
OC18H37
10 [0244]
5'-0-(4,4'-dimethoxytrityl)-2'-methoxyuridin-3'-yl-
[3, 4,5-tris(octadecyloxy)benzyl]succinate (202 mg, 129 umol)
was dissolved in dichloromethane (3.0 mL) under an argon
atmosphere, trifluoroacetic acid (96.3 uL, 1.38 mmol) and
15 pyrrole (90.0 u.L, 1.38 mmol) were added, and the mixture was
stirred for 5 min. The completion of the deprotection was
confirmed by thin layer chromatography, pyridine (105 u.L, 1.38
mmol) and N-methylimidazole (51.6 u.L, 690 umol) were added, and
the mixture was stirred for 10 min. After neutralization, a
20 2'-OMe-U-CE phosphoramidite reagent (5'-0-(4,4'-
dimethoxytrityl)-2'-methoxyuridine-3'-[0-(2-cyanoethyl)-(N,Ndiisopropyl)]-
phosphoramidite) (402 mg, 528 umol) dissolved in
107
0.25 mol/L 5-(benzylthio)-lH-tetrazole/acetonitrile solution
(1.5 mL) was added to the reaction mixture, and the mixture
was stirred for 10 min. 0.2 mol/L Iodine
pyridine/tetrahydrofuran/water=49/49/2 solution (3.9 mL) was
5 added, and the mixture was stirred for 10 min. After
completion of the reaction, a methanol solution saturated with
sodium thiosulfate was added to the reaction mixture, and the
resultant solid was collected by filtration and dried to give
the title compound (237 mg, 95.7 %) as a white solid.
io m/z(ESI-MS): Anal. C a l c . for C109H166N5O22P: 1 9 2 9 . 4 8 . Found
1 9 4 7 . 2 1 (M+NH4) +
[0245]
Example 21: S y n t h e s i s of 5 ' - 0 - ( 4 , 4 ' - d i m e t h o x y t r i t y l ) - 2 ' -
f l u o r o u r i d i n e - 3 ' - [ 0 - ( 2 - c y a n o e t h y l ) ] p h o s p h o r y l - 2 ' -
15 methoxyuridin-3' - y l - [ 3 , 4 , 5 - t r i s ( o c t a d e c y l o x y ) b e n z y l ] s u c c i n a te
[0246]
CisHsrOy^Y^O^^^Y0 °Me >v ° OC18H37
C-18H37O
[0247]
5'-0-(4,4'-Dimethoxytrityl)-2'-methoxyuridin-3'-yl-
20 [3,4,5-tris(octadecyloxy)benzyl]succinate (197 mg, 127 umol)
was dissolved in dichloromethane (3.0 mL) under an argon
atmosphere, trifluoroacetic acid (96.3 uL, 1.38 mmol) and
108
pyrrole (90.0 pL, 1.38 mmol) were added, and the mixture was
stirred for 5 min. The completion of the deprotection was
confirmed by thin layer chromatography, pyridine (105 jaL, 1.38
mmol) and N-methylimidazole (51.6 JJ.L, 690 pmol) were added, and
5 the mixture was stirred for 10 min. After neutralization, a
2'-F-U-CE phosphoramidite reagent (404 mg, 539 umol) dissolved
in 0.25 mol/L 5-(benzylthio)-lH-tetrazole/acetonitrile
solution (1.5 mL) was added to the reaction mixture, and the
mixture was stirred for 10 min. 0.2 mol/L Iodine
10 pyridine/tetrahydrofuran/water =49/49/2 solution (3.9 mL) was
added, and the mixture was stirred for 10 min. After
completion of the reaction, a methanol solution saturated with
sodium thiosulfate was added to the reaction mixture, and the
resultant solid was collected by filtration and dried to give
15 the title compound (228 mg, 94.7 %) as a white solid.
m/z(ESI-MS) : Anal. Calc. for C108H163FN5O21P: 1917.44. Found
1935.18(M+NH4)+
[0248]
Example 22: Synthesis of 5 ' - 0 - ( 4 , 4 ' - d i m e t h o x y t r i t y l ) - 2 ' -
20 methoxyuridine-3'-[0-(2-cyanoethyl)jphosphoryl-deoxythymidin-
3 ' - y l - [ 3 , 4 , 5 - t r i s ( o c t a d e c y l o x y ) b e n z y l ] s u c c i n a te
[0249]
O
NH
DMTrCL NO
O OMe
I
O —P = 0 - N ^ M H
NA0
C18H37O y*^Y^oyi^^y0
Ci8H370 y"^
O C 1 8 H 37
109
[0250]
5'-0-(4,4'-Dimethoxytrityl)-deoxythymidin-3'-yl-[3,4,5-
tris (octadecyloxy) benzyl] succinate (214 mg, 138 umol) was
dissolved in dichloromethane (3.0 mL) under an argon
5 atmosphere, trifluoroacetic acid (96.3 uL, 1.38 mmol) and
pyrrole (90.0 uL, 1.38 mmol) were added, and the mixture was
stirred for 5 min. The completion of the deprotection was
confirmed by thin layer chromatography, pyridine (105 uL, 1.38
mmol) and N-methylimidazole (51.6 uL, 690 umol) were added, and
10 the mixture was stirred for 10 min. After neutralization, a
2'-OMe-U-CE phosphoramidite reagent (396 mg, 520 |jmol)
dissolved in 0.25 mol/L 5-(benzylthio)-1Htetrazole/
acetonitrile solution (1.5 mL) was added to the
reaction mixture, and the mixture was stirred for 10 min. 0.2
15 mol/L Iodine pyridine/tetrahydrofuran/water=49/49/2 solution
(3.9 mL) was added, and the mixture was stirred for 10 min.
After completion of the reaction, a methanol solution
saturated with sodium thiosulfate was added to the reaction
mixture, and the resultant solid was collected by filtration
20 and dried to give the title compound (246 mg, 94.0%) as a
white solid.
m/z(ESI-MS): Anal. Calc. for Ci09Hi66N5O2iP: 1912.18. Found
1931.21(M+NH4)+
[0251]
25 Example 23: One pot reaction using 2,4,6-trimethylpyridine as
neutralization base
Synthesis of 5'-0-(4,4'-dimethoxytrityl)-deoxythymidine-3'-[0-
(2-cyanoethyl)]phosphoryl-deoxythymidin-3'-yl-[3,4,5-
tris(octadecyloxy)benzyl]succinate
30 Under an argon atmosphere, 5'-0-(4,4'-dimethoxytrityl)-
deoxythymidin-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate
(200 mg, 130 umol) was dissolved in dichloromethane (3.0 mL),
trifluoroacetic acid (96.3 uL, 1.30 mmol) and pyrrole (90.0 uL,
1.30 mmol) were added, and the mixture was stirred for 5 min.
35 The completion of the deprotection was confirmed by thin layer
110
chromatography, 2,4,6-trimethylpyridine (171 uL, 1.30 mmol) and
N-methylimidazole (48.6 uL, 650 umol) were added, and the
mixture was stirred for 10 min. After neutralization, a dT-CE
phosphoramidite reagent (193 mg, 260 umol) dissolved in 0.25
5 mol/L 5-(benzylthio)-lH-tetrazole/acetonitrile solution (1.0
mL) was added to the reaction mixture, and the mixture was
stirred for 10 min. 0.2 mol/L Iodine
pyridine/tetrahydrofuran/water=4 9/4 9/2 solution (2.6 mL) was
added, and the mixture was stirred for 10 min. After
io completion of the reaction, a methanol solution saturated with
sodium thiosulfate was added to the reaction mixture, and the
resultant solid was collected by filtration and dried to give
the title compound (241 mg, 96.9%) as a white solid.
m/z(ESI-MS): Anal. Calc. for C109Hi66N502oP: 1896.19. Found
15 1915.23 (M+NH4) +
[0252]
Example 24: One pot reaction using trifluoromethanesulfonic
acid as deprotection reagent and benzimidazole as
neutralization reagent
20 Synthesis of 5'-0-(4,4'-dimethoxytrityl)-deoxythymidine-3'-[0-
(2-cyanoethyl)]phosphoryl-deoxythymidin-3'-yl-[3,4,5-
tris(octadecyloxy)benzyl]succinate
Under an argon atmosphere, 5'-0-(4,4'-dimethoxytrityl)-
deoxythymidin-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate
25 (200 mg, 130 umol) was dissolved in dichloromethane (3.0 mL) ,
trifluoromethanesulfonic acid (5.75 uL, 65.0 umol) and pyrrole
(90.0 uL, 1.30 mmol) were added, and the mixture was stirred
for 5 min. The completion of the deprotection was confirmed by
thin layer chromatography, benzimidazole (7.68 mg, 65.0 umol)
30 was added, and the mixture was stirred for 10 min. After
neutralization, a dT-CE phosphoramidite reagent (193 mg, 260
umol) dissolved in 0.25 mol/L 5-(benzylthio)-1Htetrazole/
acetonitrile solution (1.0 mL) was added to the
reaction mixture, and the mixture was stirred for 10 min. 0.2
35 mol/L Iodine pyridine/tetrahydrofuran/water=49/49/2 solution
111
(2.6 mL) was added, and the mixture was stirred for 10 min.
After completion of the reaction, a methanol solution
saturated with sodium thiosulfate was added to the reaction
mixture, and the resultant solid was collected by filtration
5 and dried to give the title compound (248 mg, 98.6%) as a
white solid.
m/z(ESI-MS): Anal. Calc. for C109H166N5O20P: 1896.19. Found
1915.23(M+NH4) +
[0253]
io Example 25 One pot reaction using 90% aqueous acetonitrile
solution as precipitation solvent
Synthesis of 5' -0-(4, 4'-dimethoxytrityl)-deoxythymidine-3'-[0-
(2-cyanoethyl)]phosphoryl-deoxythyinidin-3' -yl-[3,4,5-
tris(octadecyloxy)benzyl]succinate
15 Under an argon atmosphere, 5'-0-(4,4'-dimethoxytrityl)-
deoxythymidin-3'-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate
(200 mg,130 pmol) was dissolved in dichloromethane (3.0 mL),
trifluoroacetic acid (96.3 p,L, 1.30 mmol) and pyrrole (90.0 jiL,
1.30 mmol) were added, and the mixture was stirred for 5 min.
20 The completion of the deprotection was confirmed by thin layer
chromatography, pyridine (105 \iL, 1.30 mmol) and Nmethylimidazole
(51.6 \iL, 650 pmol) were added, and the mixture
was stirred for 10 min. After neutralization, a dT-CE
phosphoramidite reagent (193 mg, 260 pmol) dissolved in 0.25
25 mol/L 5-(benzylthio)-IH-tetrazole/acetonitrile solution (1.5
mL) was added to the reaction mixture, and the mixture was
stirred for 10 min. 0.2 mol/L Iodine
pyridine/tetrahydrofuran/water=49/49/2 solution (2.6 mL) was
added, and the mixture was stirred for 10 min. After
30 completion of the reaction, a 90% aqueous acetonitrile
solution saturated with sodium thiosulfate was added to the
reaction mixture, and the resultant solid was collected by
filtration and dried to give the title compound (248 mg,
99.6%) as a white solid.
35 m/z(ESI-MS): Anal. Calc. for Cio9Hi66N502oP: 1896.19. Found
112
1915.22(M+NH4)+
INDUSTRIAL APPLICABILITY
[0254]
The present invention provides a method of producing an
5 n+p-mer oligonucleotide efficiently in a high yield, which
includes use of, as a starting material, an n-mer
oligonucleotide wherein the 3'-terminal hydroxyl group is
protected by a pseudo solid phase protecting group, and the
5'-terminal hydroxyl group is protected by a temporary
10 protecting group, and (1) a deprotection step of the 5'-
terminal hydroxyl group protected by a temporary protecting
group, (2) a 5'-terminal elongation step by the addition of a
p-mer oligonucleotide wherein the 3'-position is
phosphoramidited, and (3) an oxidation step or a sulfurization
15 step of a phosphite triester moiety, by adding a particular
cation scavenger during the deprotection step of a 5'-terminal
hydroxyl group protected by a temporary protecting group,
applying a neutralization treatment after completion of the
deprotection reaction, and using a particular oxidizing agent
20 or sulfurizing agent in the oxidation step or sulfurization
step, and the present invention further provides RNA, DNA,
nucleic acid medicine and the like. In addition, an
oligonucleotide automatic synthesis program and an
oligonucleotide automatic synthesis apparatus, which utilize
25 the production method of oligonucleotide of the present
invention can also be developed.
[0255]
According to "GENOME CHEMISTRY" SEKINE, Mitsuo and SAITO,
Isao ed., Kodansha Scientific, 1-3(2003)", a general
30 phosphoramidite method is known to include the following as a
basic unit.
STEP1: deprotection step (removal of 5'hydroxylprotecting
group of chain elongation resultant product),
STEP2: condensation step (step of condensing nucleoside-
35 3'-phosphoramidite wherein various functional groups are
113
protected and protected nucleotide),
STEP3: capping step (step of capping unreacted 5'-
hydroxyl group), and
STEP4: oxidation step (step of obtaining nucleoside
5 phosphate triester compound by oxidation).
[0256]
When the "improved continuous phosphoramidite method"
defined as "a method comprising (STEP 1) deprotection step,
(STEP 2) condensation step and (STEP 4) oxidation step as a
10 basic unit, which continuously performs STEPs 1, 2, 4 in a
solution, without an isolation and purification step after
STEPs 1, 2", the production method of oligonucleotide of the
present invention can also be defined as follows.
[1] A production method of oligonucleotide by an improved
15 continuous phosphoramidite method, comprising at least one
kind of cation scavenger selected from a pyrrole derivative
and an indole derivative in a deprotection step.
[2] The production method of [1], further comprising an
organic base after the deprotection step and before a
20 condensation step.
[3] The production method of [1] or [2], further comprising
using an oxidizing agent or a sulfurizing agent in the
oxidation step.
[4] The production method of any one of [1] to [3], further
25 comprising crystallization and isolation of the oxidized
compound or sulfurated compound in a polar solvent after the
oxidation step.
[5] The production method of [4], further comprising
deprotection of all protecting groups.
30 [0257]
Thus, it is highly significant that the co-presence of
least one kind of cation scavenger selected from a pyrrole
derivative and an indole derivative affords an improved
continuous phosphoramidite method that does not require an
35 isolation and purification step after STEPs 1 and 2, or a
114
^3 capping step.
[0258]
This application is based on patent application No. 2011-
110872 filed in Japan, the contents of which are incorporated
5 in full herein.
115

\ CLAIMS ©!**'*«« lAJp
1. A method of producing an oligonucleotide comprising the
following steps (1) to (3) : ^J' '•-''11v i; ^J*^
5 (1) a step of reacting, in a non-polar solvent, an n-mer
oligonucleotide (n is an integer of one or more) wherein the
3'-hydroxy1 group is protected by a pseudo solid phase
protecting group, and the 5'-hydroxyl group is protected by a
temporary protecting group removable under acidic conditions,
10 an acid, and.at least one kind of cation scavenger selected
from a pyrrole derivative and an indole derivative to remove
the temporary protecting group of the 5'-hydroxyl group, and
neutralizing the reaction mixture with an organic base,
(2) a step of adding, to the reaction mixture after the
15 neutralization in step (1), a p-mer oligonucleotide (p is an
integer of one or more) wherein the 3'-hydroxy1 group is
phosphoramidited, and the 5'-hydroxyl group is protected by a
temporary protecting group removable under acidic conditions
to allow condensation with the n-mer oligonucleotide obtained
20 in step (1), wherein the temporary protecting group of the 5'-
hydroxyl group is removed, by forming a phosphite triester
bond via the 5'-hydroxyl group thereof, and
(3) a step of adding an oxidizing agent or a sulfurizing agent
to the reaction mixture obtained in step (2) to convert the
25 phosphite triester bond of the n+p-mer oligonucleotide
obtained in step (2) to a phosphate triester bond or a
thiophosphate triester bond.
2. The method according to claim 1, wherein p is 1.
30
3. The method according to claim 1 or 2, further comprising
the following step (4):
(4) a step of adding a polar solvent to the reaction mixture
obtained in step (3) to precipitate the n+p-mer
35 oligonucleotide and obtaining same by solid-liquid separation.
116
4. The method according to claim 3, further comprising the
following step (5):
(5) a step of removing all the protecting groups of the n+p-
5 mer oligonucleotide obtained in step (4) . fj> ,< \ .-,,-v,'.-.
5. The method according to any one of claims 1 to 4, wherein
the temporary protecting group removable under acidic
conditions is a dimethoxytrityl group or a monomethoxytrityl
io group.
ORIGINAL
6. The method according to any one of claims 1 to 5, wherein
the non-polar solvent is a solvent selected from the group
consisting of a halogenated solvent, an aromatic solvent, an
15 ester solvent, an aliphatic solvent, a non-polar ether solvent,
and a combination thereof.
7. The method according to any one of claims 1 to 5, wherein
the non-polar solvent is a solvent selected from the group
20 consisting of dichloromethane, chloroform, 1,2-dichloroethane,
benzene, toluene, xylene, mesitylene, hexane, pentane, heptane,
nonane, cyclohexane, ethyl acetate, isopropyl acetate, tertbutyl
methyl ether, cyclopentyl methyl ether, and a
combination thereof.
25
8. The method according to any one of claims 3 to 7, wherein
the polar solvent is an alcohol solvent or a nitrile solvent.
9. The method according to any one of claims 3 to 7, wherein
30 the polar solvent is methanol or acetonitrile.
10. The method according to any one of claims 1 to 9, wherein
the pyrrole derivative or the indole derivative is at least
one kind selected from the group consisting of pyrrole, 3-
35 methylpyrrole, 2,4-dimethylpyrrole, indole, 4-methylindole, 5-
117
; 95 74^1 a.
methylindole, 6-methylindole, 7-methylindole, 5,6-
dimethylindole and 6,7-dimethylindole. ORIGINAL
11. The method according to any one of claims 1 to 10, wherein
5 the oxidizing agent is iodine, (IS) - ( + ) - (10- A .< • :/ ««,
camphorsulfonyl)oxaziridine, tert-butyl hydroperoxide, 2-
butanone peroxide, 1,1-dihydroperoxycyclododecane,
bis(trimethylsilyl)peroxide or m-chloroperbenzoic acid.
10 12. The method according to any one of claims 1 to 10, wherein
the sulfurizing agent is 3-((N,Ndimethylaminomethylidene)
amino)-3H-1,2,4-dithiazole-5-thione,
3H-1,2-benzodithiol-3-one-l,1-dioxide, 3H-1,2-benzodithiol-3-
one, phenylacetyl disulfide, tetraethylthiuram disulfide, 3-
15 amino-1,2,4-dithiazole-5-thione or sulfur.
13. The method according to any one of claims 1 to 12, wherein
the acid is trifluoroacetic acid, dichloroacetic acid,
trifluoromethanesulfonic acid, trichloroacetic acid,
20 methanesulfonic acid, hydrochloric acid, acetic acid or ptoluenesulfonic
acid.
14. The method according to any one of claims 1 to 13, wherein
the organic base is at least one kind selected from the group
25 consisting of pyridine, 2,4,6-trimethylpyridine, benzimidazole,
1,2,4-triazole, N-phenylimidazole, 2-amino-4,6-
dimethylpyrimidine, 1,10-phenanthroline, imidazole, Nmethylimidazole,
2-chlorobenzimidazole, 2-bromobenzimidazole,
2-methylimidazole, 2-phenylbenzimidazole, N-
30 phenylbenzimidazole and 5-nitrobenzimidazole.
15. A method of producing an oligonucleotide by an improved
continuous phosphoramidite method, comprising using at least
one kind of cation scavenger selected from a pyrrole
35 derivative and an indole derivative in a deprotection step.
118
£
10
15
DELNP \ J ;
16. A pseudo solid phase protecting group represented by the
formula (I):
-L-Y-Z (I)
5 wherein
L is a group represented by the formula (al) : P 0 h'OV 2015
o
**
o
(a1) °RIGINAL
wherein shows the bonding position to Y;
** indicates the bonding position to a group to be
protected;
Li 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 Li,
shows the bonding position to C=0, 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):
R'
OR5)k (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
119
£
10
15
20
25
: 95 7 4™ 13;
xanthenyl group together with ring B; 0 6 NOV 2011
R in the number of k are each independently an organic
group having an aliphatic hydrocarbon group having 10 or
more carbon atoms;
k is an integer of 1 to 4;
ring A optionally further has, in addition to OR5 in the
number of k, substituent(s) selected from the group
consisting of a halogen atom, a C±-e alkyl group optionally
substituted by a halogen atom, and a Ci_6 alkoxy group
optionally substituted by a halogen atom;
Ra is a hydrogen atom; and
Rb is a hydrogen atom, or a group represented by the formula
(a3) :
ORIGINAL
wherein * shows a bonding position;
j is an integer of 0 to 4;
R7 in the number of j are each independently an organic
group having an aliphatic hydrocarbon group having 10 or
more carbon atoms;
R6 is a hydrogen atom, or optionally a single bond or -Clin
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 OR7 in the
number of j, substituent(s) selected from the group
consisting of a halogen atom, a Ci_6 alkyl group
optionally substituted by a halogen atom, and a Ci_6
alkoxy group optionally substituted by a halogen atom.
17. A nucleotide represented by the formula (II):
120
95 74^13)
QWG.
0 6NOV20H
Base
wherein ORIGINAL
m is an integer of 0 or more,
Base in the number of m+1 are each independently an optionally
5 protected nucleic acid base,
Q is a hydrogen atom, or a temporary protecting group
removable under acidic conditions,
X is a hydrogen atom, a halogen atom, or an optionally
protected hydroxyl group,
10 X' in the number of m are each independently a hydrogen atom,
a halogen atom, or an optionally protected hydroxyl group,
R8 in the number of m are each independently an oxygen atom or
a sulfur atom,
WG in the number of m are each independently an electron-
15 withdrawing group,
L is a group represented by the formula (al):
o
**
*
o (a1)
20
wherein shows the bonding position to Y;
indicates the bonding position to a 3'-hydroxy group of
the nucleotide;
Li is an optionally substituted divalent Ci_22 hydrocarbon
121
7 4M 13'
group; and ~ * ~ * 0 6 NOV 2013 L2 is a single bond, or a group represented by C(=0)N(R 2)N -3
R1-N(R3)*** wherein ** shows the bonding position to Llf
shows the bonding position to C=0, R1 is an optionally
5 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
10 alkyl group or an aralkyl group, and
Z is a group represented by the formula (a2):
Ra \ * /R ^b
ORIGINAL
wherein* shows the bonding position to Y;
R4 is a hydrogen atom, or when Rb is a group represented by
15 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;
R5 in the number of k are each independently is an organic
group having an aliphatic hydrocarbon group having 10 or
20 more carbon atoms;
k is an integer of 1 to 4;
ring A optionally further has, in addition to OR5 in the
number of k, substituent(s) selected from the group
consisting of a halogen atom, a Ci_6 alkyl group optionally
25 substituted by a halogen atom, and a Ci_6 alkoxy group
optionally substituted by halogen atom;
Ra is a hydrogen atom; and
Rb is a hydrogen atom, or a group represented by the formula
(a3) :
122
£
10
15
574^1$
0 6NQV2OB
ORIGINAL
OR7)j
(a3)
wherein * shows a bonding position;
j is an integer of 0 to 4;
R7 in the number of j are each independently is an
organic group having an aliphatic hydrocarbon group
having 10 or more carbon atoms;
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 OR7 in the
number of j, substituent(s) selected from the group
consisting of a halogen atom, a Ci_6 alkyl group
optionally substituted by a halogen atom, and a Ci_6
alkoxy group optionally substituted by halogen atom.
18. The nucleotide according to claim 17, wherein m is 0.
19. The nucleotide according to claim 17 or 18, wherein
L in the formula (II) is a succinyl group, and
20 R5 and/or R7 are/is an alkyl group having 10 to 40 carbon atoms.
20. The nucleotide according to claim 17 or 18, wherein
L in the formula (II) is a succinyl group, and
Ra and Rb are both hydrogen atoms, and
25 R5 is an alkyl group having 10 to 40 carbon atoms.
21. The nucleotide according to claim 17 or 18, wherein
L in the formula (II) is a succinyl group, and
R5 and/or R7 are/is an alkyl group having 12 to 30 carbon atoms.
30
22. The nucleotide according to claim 17 or 18, wherein
L in the formula (II) is a succinyl group, and
Y-Z is a group selected from the group consisting of
123
ORIGINS 5 74DBffl3i 44
a 3,4,5-tri(octadecyloxy)benzyloxy group,
a 3,5-di(docosyloxy)benzyloxy group, 0 6 NOV 2 08
a 3,5-di[3',4',5'-tri(octadecyloxy)benzyloxy]benzyloxy group,
a 3,4,5-tri[3',4',5'-tri(octadecyloxy)benzyloxy]benzyloxy
5 group,
a 3,4,5-tri(octadecyloxy)benzylamino group,
a 2,4-di(docosyloxy)benzylamino group,
a 3,5-di(docosyloxy)benzylamino group,
a di(4-docosyloxyphenyl)methylamino group,
10 a 4-methoxy-2-[3',4',5'-tri(octadecyloxy)benzyloxy]benzylamino
group,
a 4-methoxy-2-[3',4',5'-
tri(octadecyloxy)cyclohexylmethyloxy]benzylamino group,
a 2,4-di(dodecyloxy)benzylamino group,
15 a phenyl(2,3, 4-tri(octadecyloxy)phenyl)methylamino group,
a di[4-(12-docosyloxydodecyloxy)phenyl]methylamino group,
a 3,5-di[3',4',5'-tri(octadecyloxy)benzyloxy]benzylamino group,
and
a 3,4, 5-tri[3', 4', 5' -tri(octadecyloxy)benzyloxy]benzylamino
20 group.
23. The nucleotide of according to any one of claims 17 to 22,
wherein Q is a monomethoxytrityl group or a dimethoxytrityl
group.