DESCRIPTION
W Title of the Invention: METHOD FOR PRODUCING PEPTIDE
Technical Field
[0001]
5 The present invention relates to a method capable of
obtaining an object peptide with a high purity and a high yield
by a convenient operation omitting an isolation and
purification operation of an intermediate as much as possible,
which is suitable for industrial production.
10 Background Art
[0002]
As a production method of peptide besides a solid phase
method and a liquid phase method, a production method using a
protecting group (hereinafter to be also referred to as an
15 anchor group) permitting a reaction in a homogeneous liquid
phase, and, after changing the solvent composition after the
reaction, performing isolation and purification merely by
filtration and washing (hereinafter to be also referred to as
an anchor method) has recently been proposed. The anchor
20 method is a production method of peptide, wherein, in peptide
synthesis and the like, a particular compound that shows
reversible changes between a dissolved state and an undissolved
(precipitated) state according to the changes of the solvent
composition is used as a compound (anchor) that forms an anchor
25 group for protecting the C-terminal and/or a side chain
functional group of amino acid or peptide. Here, the anchor
group means a protecting group that binds to a reactive
substrate to make the substrate soluble in nonpolar solvents
and capable of reaction in a liquid phase, and that
30 precipitates on addition of a polar solvent to enable solidliquid
separation, thus showing both reactivity and convenience
of working up. The anchor means a compound for forming an
anchor group.
[0003]
35 For example, patent document 1 and non-patent document 1
^^^
^ ^ ^ t
each disclose a method of using 3,4,5-tri(n-octadecyloxy)benzyl
alcohol as an anchor for carboxy group and the like. In
addition, patent documents 2 - 4 each disclose anchors such as
3, 5-di(docosyloxy)benzyl alcohol, 2,4-di(docosyloxy)benzyl
5 alcohol, trityl type compound and the like.
[0004]
The present inventors have also developed a particular
diphenylmethane compound (patent document 5) and a fluorene
compound (patent document 6) as an anchor usable for the anchor
10 method.
[Document List]
[patent documents]
[0005]
patent document 1: JP-A-2000-44493
15 patent document 2: WO2006/104166
patent document 3: WO2007/034812
patent document 4: WO2007/122847
patent document 5: WO2010/113939
patent dociiment 6: WO2010/104169
20 [non-patent document]
[0006]
non-patent document 1: Bull. Chem. Soc. Jpn 74, 733-738 (2001)
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
25 [0007]
The above-mentioned anchor method is a useful method in
the organic synthetic methods such as peptide synthesis and the
like, which shows advantages of both the solid phase reaction
and the liquid phase reaction, such as convenient working up
30 and possible scaling up, and draws attention from an industrial
aspect. However, when sequential multistep synthetic reactions
are needed such as in the peptide synthesis and the like,
isolation and purification operations of concentration,
precipitation by poor solvent, filtration, washing and drying
35 need to be repeated in each step, so that undesirable side
^
reactions such as by-production of a double-hit compound due to
the residual amino acid and the like will not occur in the next
step. Since the high number of operation steps requires a
large amount of time and cost, industrialization is prevented.
5 Here, the double-hit compound means a peptide wherein one extra
amino acid residue has been further inserted into the object
peptide.
[0008],
The present invention has been made in view of the above-
10 mentioned problems specific to the peptide synthesis by
conventional anchor methods, and aims to provide a convenient
and industrially useful production method of peptide, which
incorporates a step that can be performed successively in one
pot by omitting isolation and purification operations of an
15 intermediate as much as possible.
Means of Solving the Problems
[0009]
The present inventors have found that, after condensation
of an amino acid or peptide having an anchor group as a C-
20 teintiinal carboxy-protecting group and an amino acid or peptide
having an N-terminal amino group temporarily protected by a
protecting group (hereinafter to be also referred to as a
temporary protecting group), the steps up to the removing step
of the temporary protecting group can be successively performed
25 in one pot by omitting a set of isolation and purification
operations of concentration, precipitation, filtration, washing
and drying. In the production method of a peptide of the
present invention, a free N-terminal amino group is formed by
removing the temporary protecting group rather than an anchor
30 group, and the C-terminal carboxy group of a new amino acid or
peptide is reacted with the free N-terminal amino group to
elongate the peptide chain. To distinguish from an anchor
group, therefore, a protecting group that temporarily protects
the N-te2nninal amino group is called a "temporary protecting
35 group" in the present specification.
^
[0010]
To perform the above-mentioned one-pot step repeatedly
and efficiently, complete removal of residual amino acid that
causes, side reactions is necessary. The present inventors have
5 found that precipitation and solid-liquid separation by the
addition of a solvent containing water-containing acetonitrile
after removing the above-mentioned temporary protecting group
can completely remove of the residual amino acid from the
condensation product, which resulted in the completion of the
10 present invention. The present invention is as follows.
[0011]
[1] A production method of a protected amino acid or protected
peptide, comprising the following steps (1) and (2);
(1) obtaining a C-protected amino acid or C-protected peptide
15 by removing a N-terminal protecting group from an N-protected
C-protected amino acid or N-protected C-protected peptide
wherein a C-terminal carboxy group is protected by an anchor
group derived from an anchor soluble in halogenated solvents or
ether solvents, insoluble in polar solvents and having a
20 molecular weight of not less than 300, without isolating from a
reaction solution of the aforementioned anchor and an Nprotected
amino acid or N-protected peptide, and
(2) precipitating the C-protected amino acid or C-protected
. peptide in a solvent containing 60 - 95% water-containing
25 acetonitrile after step (1), and obtaining same by solid-liquid
separation.
[2] A production method of peptide, comprising the following
steps (3) to (5) ;
(3) condensing an N-terminal amino group of a C-protected amino
30 acid or C-protected peptide wherein a C-terminal carboxy group
is protected by an anchor group derived from an anchor soluble
in halogenated solvents or ether solvents, insoluble in polar
solvents and having a molecular weight of not less than 300,
and a C-terminal carboxy group of an N-protected amino acid or
35 N-protected peptide in a solvent to give an N-protected C^
^
protected peptide,
(4) removing the N-terminal protecting group of the N-protected
C-protected peptide obtained in step (3) in the reaction
solution of step (3), without isolating the N-protected C-
5 protected peptide, to give a C-protected peptide, and
(5) precipitating C-protected peptide in a solvent containing
60 - 95% water-containing acetonitrile after step (4), and
obtaining same by solid-liquid separation.
[3] The method of the above-mentioned [2], further comprising
10 step (6) removing the C-terminal anchor group of the Cprotected
peptide after step (5).
[4] The method of the above-mentioned [2] or [3], wherein the
anchor soluble in halogenated solvents or ether solvents,
insoluble in polar solvents and having a molecular weight of
15 not less than 300 is a compound represented by the following
formula (I):
[0012]
[0013]
20 wherein
R'"' is a hydrogen atom or, when R"^ is a group represented by the
following formula (a), optionally shows a single bond together
with R^ to form a fluorene ring together with ring A and ring
B;
25 R^ in the number of p is each independently an organic group
having an aliphatic hydrocarbon group;
p is an integer of 1 to 4;
ring A optionally further has, in addition to OR^ in the number
of p, a substituent selected from the group consisting of a
30 halogen atom, a Ci-e alkyl group optionally substituted by a
halogen atom, and a Ci-e alkoxy group optionally substituted by
^
a halogen atom;
R^ is a hydrogen atom, or a phenyl group optionally substituted
by a halogen atom; and
R'' is a hydrogen atom, or a group represented by the formula
5 (a) :
[0014]
[0015]
wherein * is a binding site;
10 r is an integer of 0 to 4;
R^ in the number of r is each independently an organic group
having an aliphatic hydrocarbon group;
R^ is a hydrogen atom, or optionally shows a single bond
together with R''' to form a fluorene ring together with ring A
15 and ring B; and
ring B optionally further has, in addition to OR^ in the number
of r, a substituent selected from the group consisting of a
halogen atom, a Ci-e alkyl group optionally substituted by a
halogen atom, and a Ci-e alkoxy group optionally substituted by
20 a halogen atom; and
Y is a hydroxy group, NHR (R is a hydrogen atom, an alkyl group
or an aralkyl group) or a halogen atom.
[5] The method of the above-mentioned [4], wherein, in the
formula (I) , Y is a hydroxy group, R''' is a hydrogen atom, R^
25 and/or R^ are/is aliphatic hydrocarbon group(s) having 5 to 60
carbon atoms, p is an integer of 1 to 3, and r is an integer of
0 - 2.
[6] The method of the above-mentioned [4], wherein, in the
formula (I), Y is a hydroxy group, R^, R^^, and R'"' are each a
30 hydrogen atom, R^ is an aliphatic hydrocarbon group having 5 to
60 carbon atoms, and p is an integer of 1 to 3.
[7] The method of the above-mentioned [4], wherein, in the
^
formula (I), Y is a hydroxy group, R^, R , and R are each a
hydrogen atom, R^ is an alkyl group having 10 to 40 carbon
atoms, and p is 2 or 3.
[8] The method of the above-mentioned [4], wherein, in the
5 formula (I), Y is a hydroxy group, R^, R'^, and R'"' are each a
hydrogen atom, R^ is an alkyl group having 12 to 30 carbon
atoms, and p is 2 or 3.
[9] The method of the above-mentioned [4], wherein the compound
represented by the formula (I) is a compound selected from the
10 group consisting of
3,4,5-tri(octadecyloxy)benzyl alcohol,
2,4-di(docosyloxy)benzyl alcohol,
4-methoxy-2-[3',4',5'-tri(octadecyloxy)benzyloxy]benzyl alcohol,
4-methoxy-2-[3', 4' , 5' -
15 tri(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol,
2-methoxy-4-[3',4' ,5'-
tri(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol,
4-[3',4',5'-tri(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol,
3,5-dimethoxy-4-[3',4', 5'-
20 tri(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol,
2,4-di(dodecyloxy)benzyl alcohol,
3,4,5-tri (octadecyloxy)benzylamine,
bis(4-docosyloxyphenyl)methanol, and
bis(4-docosyloxyphenyl)methylamine.
25 [10] The method of any one of the above-mentioned [2] to [9],
wherein the water-containing acetonitrile has an acetonitrile
content of 70v/v% - 90v/v%.
[11] The method of any one of the above-mentioned [2] to [9],
wherein the water-containing acetonitrile has an acetonitrile
30 content of 75v/v% - 85v/v%.
[12] The method of any one of the above-mentioned [2] to [9],
wherein the water-containing acetonitrile has an acetonitrile
content of 80v/v%.
[13] The method of any one of the above-mentioned [2] to [12],
35 wherein the solvent containing water-containing acetonitrile is
^u
a mixed solvent of water-containing acetonitrile, and at least
one kind of solvent selected from the group consisting of
methanol, ethanol, dimethylformamide, propionitrile, dimethyl
sulfoxide, acetone, dichloromethane, chloroform,
5 tetrahydrofuran, cyclopentyl methyl ether and ethyl acetate.
[14] The method of any one of the above-mentioned [2] to [12],
wherein the solvent containing water-containing acetonitrile is
water-containing acetonitrile.
[15] The method of any one of the above-mentioned [2] to [14],
10 wherein the amino-protecting group of the N-protected amino
acid or N-protected peptide is a 9-fluorenylmethyloxycarbonyl
group, a tert-butoxycarbonyl group or a benzyloxycarbonyl group.
[16] A production method of peptide by an improved successive
method of immobilizing-protecting C-terminal and elongating N-
15 terminal, comprising, after a deprotection step, a
precipitation step in a polar solvent containing acetonitrile
and water.
Effect of the Invention
[0016]
20 According to the production method of peptide of the
present invention, residual amino acid and the like can be
effectively removed by merely performing precipitating by a
solvent containing water-containing acetonitrile and solidliquid
separation, after removing a temporary N-terminal amino
25 group-protecting group of an amine component used for a
condensation step (amino acid or peptide having an anchor as a
C-terminal carboxy group-protecting group). As a result, for
example, a condensation step of the amine component and Nterminal
amino group with an amino acid or peptide protected by
30 a temporary protecting group, and the subsequent' step for
removing the temporary protecting group can be successively
performed in one pot with a high yield and a high purity,
without isolation and purification operations of an
intermediate. Therefore, a convenient and efficient production
35 method of peptide, which is suitable for industrial production.
^
can be provided.
Description of Embodiments
[0017]
Unless otherwise specified in the sentences, any
5 technical terms and scientific terms used in the present
specification, have the same meaning as those generally
understood by those of ordinary skill in the art the present
invention belongs to. Any methods and materials similar or
equivalent to those described in the present specification can
10 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 present
specification are hereby incorporated by reference so as to
describe and disclose constructed products and methodology
15_ described in, for example, publications usable in relation to
the described invention.
[0018]
An amino acid which is a constitutional unit, of a peptide
produced by the method of the present invention is a compound
20 having an amino group and a carboxy group in the same molecule,
and may be a natural amino acid or non-natural amino acid, and
an L form, a D form or a racemate. A peptide is synthesized by
repeating a dehydration condensation step (condensation step)
of an amino group of an amino acid component and a carboxy
25 group of other amino acid component, according to the amino
acid sequence of the peptide. Of the two amino acid components
involved in the formation of the peptide bond, a component
providing an amino group is hereinafter sometimes referred to
as an amine component, and a component providing a carboxy
30 group as an acid component.
[0019]
The temporary protecting group of the N-terminal amino
group of an acid component to be used for the condensation step
in the present invention is, for example, a 9-
35 fluorenylmethyloxycarbonyl group (hereinafter to be also
referred to as Fmoc group), a tert-butoxycarbonyl group
(hereinafter to be also referred to as Boc group) or a
benzyloxycarbonyl group (hereinafter to be also referred to as
Cbz group), and it is preferably an Fmoc group or a Boc group.
5 [0020]
In the present specification, the "N-protected amino
acid" or "N-protected peptide" means an amino acid or peptide
wherein an N-terminal amino group thereof is protected by a
temporary protecting group and a carboxy group is not protected.
10 [0021]
In the present specification, the "C-protected amino
acid" or "C-protected peptide" means an amino acid or peptide
wherein the C-terminal carboxy group thereof is protected by
forming an anchor group by condensing with an anchor soluble in
15 halogenated solvents or ether solvents, insoluble in polar
solvents and having a molecular weight of not less than 300
(e.g., benzyl compound, diphenylmethane compound or fluorene
compound), and the N-terminal amino group is not protected.
[0022]
20 In the present specification, the "N-protected Cprotected
amino acid" or "N-protected C-protected peptide"
means the above-mentioned "C-protected amino acid" or "Cprotected
peptide" wherein the N-terminal amino acid is
protected by a temporary protecting group.
25 [0023]
Examples of the halogenated solvent in the present
invention include halogenated hydrocarbons such as chloroform,
dichloromethane, 1,2-dichloroethane, chlorobenzene and the like.
The halogenated solvent may be a mixed solvent of two or more
30 kinds.' Among the halogenated solvents, chlorofo2nii and
dichloromethane are particularly preferable.
[0024]
Examples of the ether solvent in the present invention
include 1,4-dioxane, cyclopentyl methyl ether (hereinafter
35 sometimes to be referred to as CPME), tetrahydrofuran
10
(hereinafter sometimes to be referred to as THF) and the like.
The ether solvent may be a mixed solvent of two or more kinds.
Among the ether solvents, CPME and THF are particularly
preferable.
5 [0025]
Examples of the polar solvent in the present invention
include methanol, ethanol, isopropanol, acetonitrile,
propionitrile, dimethylformamide (hereinafter sometimes to be
referred to as DMF), dimethylacetamide, dimethyl sulfoxide,
10 water and the like, and a mixed solvent of two or more kinds of
these.
[0026]
The "water-containing acetonitrile" in the "solvent
containing water-containing acetonitrile" means a mixed solvent
15 of acetonitrile and water. The lower limit of the content of
acetonitrile in the water-containing acetonitrile is preferably
60v/v%, more preferably 70v/v%, and further preferably 75v/v%.
On the other hand, the upper limit of the content of
acetonitrile in the water-containing acetonitrile is preferably
20 95v/v%, more preferably 90v/v%, and further preferably 85v/v%.
A particularly preferable content of acetonitrile in the watercontaining
acetonitrile is 80v/v%. In the present
specification, for example, water-containing acetonitrile
having an acetonitrile content of 80v/v% is sometimes indicated
25 as "80% water-containing acetonitrile".
[0027]
The "solvent containing water-containing acetonitrile"
may be the above-mentioned "water-containing acetonitrile"
alone, or a mixed solvent with other organic solvent. While
30 such other organic solvent is not particularly limited as long
as it can remove the residual amino acid and the like by mixing
with water-containing acetonitrile, alcohol solvents such as
methanol, ethanol and the like, amide solvents such as DMF and
the like, nitrile solvents (excluding acetonitrile) such as
35 propionitrile and the like, halogenated solvents, ether
11
^ •
solvents, dimethyl sulfoxide, acetone, ethyl acetate and the
like can be mentioned.
[0028]
One embodiment of the anchor soluble in halogenated
5 solvents or ether solvents, insoluble in polar solvents and
having a molecular weight of not less than 300 in the present
invention is a compound represented by the following formula
(I) . Among such compounds, one having a molecular weight of
not less than 400 is preferable.
10 [0029]
The formula (I):
[0030]
Y
0R2)P C)
[0031]
15 wherein
R"*^ is a hydrogen atom or, when R^ is a group represented by the
following formula (a), optionally shows a single bond together
with R'^ to form a fluorene ring together with ring A and ring
B;
20 R^ in the number of p is each independently an organic group
having an aliphatic hydrocarbon group;
p is an integer of 1 to 4;
ring A optionally further has, in addition to OR^ in the number
of p, a substituent selected from the group consisting of a
25 halogen atom, a Ci-e alkyl group optionally substituted by a
halogen atom, and a Ci_6 alkoxy group optionally substituted by
a halogen atom;
R^ is a hydrogen atom, or a phenyl group optionally substituted
by a halogen atom; and
30 ^ is a hydrogen atom, or a group represented by the formula
(a):
12
[0032]
[0033]
wherein * is a binding site;
5 r is an integer of 0 to 4;
R^ in the number of r is each independently an organic group
having an aliphatic hydrocarbon group;
R"^ is a hydrogen atom, or optionally shows a single bond
together with R"'' to form a fluorene ring together with ring A
10 and ring B; and
ring B optionally further has, in addition to OR^ in the number
of r, a substituent selected from the group consisting of a
halogen atom, a Ci-e alkyl group optionally substituted by a
halogen atom, and a Ci_6 alkoxy group optionally substituted by
15 a halogen atom; and
Y is a hydroxy group, NHR (R is a hydrogen atom, an alkyl group
or an aralkyl group) or a halogen atom.
[0034]
The anchor represented by the above-mentioned formula (I)
20 is bound to a compound intended to be protected. That is, an
anchor wherein Y is a hydroxy group, an -NHR group or a halogen
atom protects a compound by condensing with a carboxy group on
the C-terminal of amino acid or peptide and the like.
[0035]
25 In the present specification, as the "alkyl group" for R,
a straight or branched Ci-30 alkyl group can be mentioned. It
is preferably a Ci-10 alkyl group, more preferably a Ci-e alkyl
group. Specific preferable examples include methyl, ethyl,
propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl and
30 the like, and methyl and ethyl are particularly preferable.
[0036]
In the present specification, as the "aralkyl group" for
13
^
R, a C7-30 aralkyl group can be mentioned. It is preferably a
C-j-20 aralkyl group, more preferably a C7-16 aralkyl group (Ce-io
aryl-Ci-6 alkyl group). Specific preferable examples include
benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl,
5 naphthylmethyl, 1-naphthylethyl, 1-naphthylpropyl and the like,
and benzyl is particularly preferable.
[0037]
As R, a hydrogen atom, a Ci-e alkyl group or a C7-16
aralkyl group is preferable, a hydrogen atom, methyl, ethyl or
10 benzyl is more preferable, and a hydrogen atom is particularly
preferable.
[0038]
In the present specification, the "halogen atom" is a
fluorine atom, a chlorine atom, a bromine atom or an iodine
15 atom. In the present specification, as the "halogen atom" for
Y, a chlorine atom, a bromine atom or an iodine atom is
preferable, and a bromine atom is more preferable.
[0039]
In the present specification, the "organic group having
20 an aliphatic hydrocarbon group" for R^ or R^ is a monovalent
organic group having an aliphatic hydrocarbon group in a
molecule structure thereof.
[0040]
The "aliphatic hydrocarbon group" in the "organic group
25 having an aliphatic hydrocarbon group" is a straight or
branched saturated or unsaturated aliphatic hydrocarbon group,
preferably an aliphatic hydrocarbon group having 5 or more
carbon atoms, more preferably an aliphatic hydrocarbon group
having 5 to 60 carbon atoms, further preferably an aliphatic
30 hydrocarbon group having 5 to 30 carbon atoms, particularly
preferably an aliphatic hydrocarbon group 10 to 30 carbon atoms.
The moiety of the "aliphatic hydrocarbon group" in the
"organic group having an aliphatic hydrocarbon group" is not
particularly limited, and may be present at the terminal
35 (monovalent group), or other site (for example, divalent group).
14
Q [0041]
Examples of the "aliphatic hydrocarbon group" include
monovalent groups such as an alkyl group, a cycloalkyl group,
an alkenyl group, a cycloalkenyl group, an alkynyl group and
5 the like, and divalent groups derived therefrom, preferably
monovalent groups such as a methyl group, an ethyl group, a
propyl group, an isopropyl group, a butyl group, an isobutyl
group, a sec-butyl group, a tert-butyl group, a pentyl group, a
hexyl group, an octyl group, a decyl group, a dodecyl group, a
10 lauryl group, a tridecyl group, a myristyl group, a cetyl group,
a stearyl group, an arachyl group, a behenyl group, an oleyl
group, an isostearyl group and the like, and divalent groups
derived therefrom.
[0042]
15 The moiety other than the "aliphatic hydrocarbon group"
of the "organic group having an aliphatic hydrocarbon group"
can be set freely. For example, it may have a moiety such as -
0-, -S-, -C00-, -OCONH-, -CONH-, a hydrocarbon group
(monovalent group or divalent group) and the like as a linker.
20 Examples of the "hydrocarbon group" include an aliphatic
hydrocarbon group, an aromatic-aliphatic hydrocarbon group, a
monocyclic saturated hydrocarbon group, an aromatic hydrocarbon
group and the like. Specifically, for example, monovalent
groups such as an alkyl group, an alkenyl group, an alkynyl
25 group, a cycloalkyl group, an aryl group, an aralkyl group and
the like, and divalent groups derived therefrom are used. As
the "alkyl group", a Ci-e alkyl group and the like are
preferable and, for example, methyl, ethyl, propyl, isopropyl,
butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl and the
30 like can be mentioned. As the "alkenyl group", a C2-6 alkenyl
group and the like are preferable and, for example, vinyl, 1-
propenyl, allyl, isopropenyl, butenyl, isobutenyl and the like
can be mentioned. As the "alkynyl group", a C2-6 alkynyl group
and the like are preferable and, for example, ethynyl,
35 propargyl, 1-propynyl and the like can be mentioned. As the
15
"cycloalkyl group", a C3-6 cycloalkyl group and the like are
preferable and, for example, cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl and the like can be mentioned. For
example, the "aryl group" is preferably a C6-14 aryl group and
5 the like and, for example, phenyl, 1-naphthyl, 2-naphthyl,
biphenylyl, 2-anthryl and the like can be mentioned. Of these,
a Ce-io aryl group is more preferable, and phenyl is
particularly preferable. As the "aralkyl group", a C7-20
aralkyl group is preferable and, for example, benzyl, 1-
10 phenylethyl, 2-phenylethyl, 1-phenylpropyl, naphthylmethyl, 1-
naphthylethyl, 1-naphthylpropyl and the like can be mentioned.
Of these, a C7-16 aralkyl group (Ce-io aryl-Ci-e alkyl group) is
more preferable, and benzyl is particularly preferable. The
"hydrocarbon group" may be substituted by a substituent
15 selected from a halogen atom (a chlorine atom, a bromine atom,
a fluorine atom, an iodine atom), an alkyl group having 1 to 6
carbon atoms and optionally substituted by one or more halogen
atoms, an 0x0 group and the like.
[0043]
20 In the "organic group having an aliphatic hydrocarbon
group" constituting the OR^ group or OR* group in the abovementioned
formula (I), plural "aliphatic hydrocarbon groups"
may be present by branching and the like. When plural
"aliphatic hydrocarbon groups" are present in the "organic
25 group having an aliphatic hydrocarbon group", they may be the
same or different.
[0044]
In the "organic group having an aliphatic hydrocarbon
group" for R^ or R* in the above-mentioned formula (I), the
30 lower limit of the total carbon number is preferably 5, more
preferably 10, further preferably 12, still more preferably 14,
• especially preferably 16, and particularly preferably 20. On
the other hand, in the "organic group having an aliphatic
hydrocarbon group" for R^ or R*, the upper limit of the total
35 carbon number is preferably 200, more preferably 150, further
16
^ '
preferably 120, still more preferably 100, especially
preferably 80, particularly preferably 60, particularly further
preferably 40, and most preferably 30. The higher the carbon
number, the better the crystallinity of the compound
5 represented by the formula (I) in a polar solvent becomes, even
when the peptide chain is a long chain.
[0045]
Specific preferable examples of the "OR^" group or '''OR''"
group include dodecyloxy, cetyl oxy, octadecyloxy, docosyloxy,
10 docosyloxy-dodecyloxy, triacontyloxy and the like. The "OR^"
group or "OR^" group is present in a total number of p or r (p
is an integer of 1 to 4 and r is an integer of 0 to 4), p is
preferably 2 or 3, and r is preferably an integer of 0 to 2.
[0046]
15 Specific preferable examples of the substituent
optionally present in ring A or ring B in the above-mentioned
formula (I) include a Ci-e alkoxy group (e.g., a C1-4 alkoxy
group such as methoxy, ethoxy, propoxy, isopropoxy, butoxy,
isobutoxy, sec-butoxy, tert-butoxy and the like), a Ci-e alkyl
20 group optionally substituted by one or more halogens (e.g., a
Ci-6 alkyl group such as methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, sec-butyl, tert-butyl, pentyl, hexyl and the like, a
halogen-substituted Ci-e alkyl group such as trifluoromethyl,
trichloromethyl and the like), and a halogen atom. Of these, a
25 Ci-6 alkoxy group is preferable.
[0047]
A preferable embodiment of the anchor represented by the
above-mentioned formula (I) is a compound of the formula (I),
wherein
30 y is a hydroxy group;
R""" is a hydrogen atom;
R^ and/or R'' are/is an aliphatic hydrocarbon group having 5 to
60 carbon atoms;
p is an integer of 1 to 3; and
35 r is an integer of 0 to 2.
17
^p^
[0048]
Another preferable embodiment of the anchor represented
by the above-mentioned formula (I) is a compound of the formula
(I), wherein .
5 Y is a hydroxy group;
R^, R^, and R^ are each a hydrogen atom;
R^ is an aliphatic hydrocarbon group having 5 to 60 carbon
atoms; and
p is an integer of 1 to 3.
10 [0049]
Another preferable embodiment of the anchor represented
by the above-mentioned formula (I) is a compound of the formula
(I), wherein
y is a hydroxy group;
15 R^, R'^, and R''' are each a hydrogen atom;
R^ is an alkyl group having 10 to 40 carbon atoms; and
p is 2 or 3.
[0050]
Another preferable embodiment of the anchor represented
20 by the above-mentioned formula (I) is a compound of the formula
(I), wherein
y is a hydroxy group;
R^, R'^, and R''' are each a hydrogen atom;
R^ is an alkyl group having 12 to 30 carbon atoms; and
25 p is 2 or 3.
[0051]
Another preferable embodiment of the anchor represented
by the above-mentioned formula (I) is a compound of the formula
(I), wherein
30 Y is a hydroxy group;
R^, R'', and R"'" are each a hydrogen atom;
R^ is a benzyl group having 1 to 3 alkoxy groups having 12 to
30 carbon atoms; and
p is an integer of 1 to 3.
35 [0052]
€ Another preferable embodiment of the anchor represented
by the above-mentioned formula (I) is a compound of the formula
(I), wherein
Y is a hydroxy group;
5 R^, R^^, and R'"' are each a hydrogen atom;
R^ is a cyclohexylmethyl group having 1 to 3 alkoxy groups
having 12 to 30 carbon atoms; and
p is an integer of 1 to 3.
[0053]
10 Preferable examples of the anchor soluble in halogenated
solvents or ether solvents, insoluble in polar solvents and
having a molecular weight of not less than 300 in the present
invention include the following anchors.
3,4,5-tri (octadecyloxy)benzyl alcohol,
15 2,4-di(docosyloxy)benzyl alcohol,
4-methoxy-2-[3',4',5'-tri(octadecyloxy)benzyloxy]benzyl alcohol,
4-methoxy-2-[3',4',5'-
tri(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol,
2-methoxy-4-[3',4',5'-
20 tri(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol,
4-[3',4',5'-tri(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol,
3,5-dimethoxy-4-[3',4',5'-
tri(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol,
2,4-di(dodecyloxy)benzyl alcohol,
25 3,4,5-tri(octadecyloxy)benzylamine,
bis(4-docosyloxyphenyl)methanol,
bis(4-docosyloxyphenyl)methylamine, and
2-(12-docosyloxy-dodecyloxy)-9-(3-fluorophenyl)-9-bromofluorene.
[0054]
30 While the production method of the aforementioned anchor
is not particularly limited, it can be produced from a starting
material compound according to a method known per se (patent
documents 1 - 6 , non-patent document 1) or a method analogous
thereto. A compound used as a starting material, for example,
35 halide corresponding to the group R^ or R'' in the formula (I)
19
^
and the like can be obtained as a commercially available
product or can be produced by a method known per se or a method
analogous thereto.
[0055]
5 An amino acid or peptide which is an acid component or an
amine component to be used in the present invention often has,
in addition to an amino group or carboxy group involved in the
formation of a peptide bond, a functional, group subjected to a
dehydration condensation reaction, such as an amino group, a
10 carboxy group, a hydroxy group and the like. Such functional
group is distinguished from an amino group and a carboxy group
forming a peptide bond of the main chain, and referred to as a
side chain functional group. While the side chain functional
group does not need to be always protected as long as it does
15 not impair the gist of the present invention, it is preferably
protected by an appropriate protecting group to prevent an
undesirable side reaction during peptide bond formation by a
dehydration condensation reaction and deprotection of an Nteanninal
amino groups.
20 [0056]
The protecting group of the side chain functional group
is subject to a certain limitation on the combination with the
N-terminal amino-protecting group, like the C-terminal carboxyprotecting
group of the aforementioned amine component. That
25 is, the protecting group of the side chain functional group
needs to be maintained until the completion of the desired
amino acid sequence, without being removed even under the
removing conditions of the temporary protecting group of the Nterminal
amino group. The protecting group is not particularly
30 limited as long as the side chain functional group does not
cause an undesirable side reaction during formation of the
peptide bond by a dehydration condensation reaction and
deprotection of the N-terminal amino group.
[0057]
35 The protecting group of the side chain functional group
20
is not particularly limited as long as it is stable under the
deprotection conditions of the temporary protecting group of
the N-terminal amino group. For example, the protecting groups
described in PEPTIDE GOUSEI NO KISO TO JIKKENN (basis and
5 experiment of peptide synthesis), published by Maruzen Co., Ltd.
(1985), PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, the third
edition, published by JOHN WILLY&SONS (1999) and the like can
be mentioned.
[0058]
10 When the side chain functional group is a carboxy group,
an ester-type protecting group, an amide-type protecting group,
a hydrazide-type protecting group and the like can be mentioned.
[0059]
As the ester-type protecting group, substituted or
15 unsubstituted alkyl ester, and substituted or unsubstituted
aralkyl ester are preferably used. As the substituted or
unsubstituted alkyl ester, methyl ester, ethyl ester, tertbutyl
ester, cyclohexyl ester, trichloroethyl ester, phenacyl
ester and the like are preferably used. As the substituted or
20 unsubstituted aralkyl ester, benzyl ester, p-nitrobenzyl ester,
p-methoxybenzyl ester, diphenylmethyl ester, 9-fluorenylmethyl
(Fm) ester, 4-picolyl (Pic) ester and the like are preferably
used.
[0060]
25 As the amide-type protecting group, unsubstituted amide,
primary amide such as N-methylamide, N-ethylamide, Nbenzylamide
and the like, secondary amide such as N,Ndimethylamide,
pyrrolidinylamide, piperidinylamide and the like,
and the like are preferably used.
30 [0061]
As the hydrazide-type protecting group, unsubstituted
hydrazide, N-phenylhydrazide, N,N'-diisopropylhydrazide and the
like are preferably used.
[0062]
35 Of these, when the N-terminal temporary protecting group
21
^
is an Fmoc group, ester-type protecting groups which are stable
under the deprotection conditions, such as t-butyl ester,
substituted or unsubstituted benzyl ester and the like are
preferably used, and substituted or unsubstituted benzyl ester
5 is particularly preferably used since synthesis thereof is
comparatively easy.
[0063]
When the side chain functional group is an amino group, a
urethane-type protecting group, an acyl-type protecting group,
10 a sulfonyl-type protecting group and the like can be mentioned.
[0064]
As the urethane-type protecting group, for example, a
methoxycarbonyl group, an ethoxycarbonyl group, a tertbutoxycarbonyl
(Boc) group, a benzyloxycarbonyl (Cbz) group and
15 the like are used, and a methoxycarbonyl group, an
ethoxycarbonyl group, a Boc group and the like are preferable.
Of these, when the N-terminal temporary protecting group is an
Fmoc group, a Boc group is particularly preferably used since
selective deprotection thereof is possible under mild acidic
20 conditions. When the N-terminal temporary protecting group is
a Boc group, a Cbz group and a bromobenzyloxycarbonyl group are
particularly preferably used.
[0065]
As the acyl-type protecting group, for example, a formyl
25 group, an acetyl group, a trifluoroacetyl group and the like
are preferably used.
[0066]
As the sulfonyl-type protecting group> for example, a ptoluenesulfonyl
(Ts) group, a p-tolylmethanesulfonyl group, a
30 4-methoxy-2,3,6-trimethylbenzenesulfonyl group and the like are
preferably used.
[0067]
As for side chain functional groups other than those
mentioned above, a protecting group stable under the
35 deprotection conditions of the temporary protecting group of
22
^
the N-terminal amino group can be selected and used.
[0068]
The side chain functional group can be deprotected as
necessary after forming the object peptide bond.
5 [0069]
Next, the production method of the present invention is
explained. The production method of the present invention is a
production method of protected amino acid, protected peptide or
peptide, which includes a precipitation step and a solid-liquid
10 separation step by the addition of water-containing
acetonitrile after removing the N-terminal temporary protecting
group.
[0070]
The first embodiment of the production method of the
15 present invention is a production method of a protected amino
acid or protected peptide, comprising the following steps (1)
and (2);
(1) obtaining a C-protected amino acid or C-protected peptide
by removing a N-terminal protecting group from an N-protected
20 C-protected amino acid or N-protected C-protected peptide
wherein a C-teirminal carboxy group is protected by an anchor
group derived from an anchor soluble in halogenated solvents or
ether solvents, insoluble in polar solvents and having a
molecular weight of not less than 300, without isolating from a
25 reaction solution of the aforementioned anchor and an Nprotected
amino acid or N-protected peptide (N-terminal
deprotection step), and
(2) precipitating the C-protected amino acid or C-protected
peptide in a solvent containing 60 - 95% water-containing
30 acetonitrile after step (1), and obtaining same by solid-liquid
separation (precipitation and solid-liquid separation step).
[0071]
The N-protected C-protected amino acid or N-protected Cprotected
peptide to be used in the above-mentioned step (1)
35 can be produced by the following step (a). In the following,
23
step (a) is first explained before explanation on step (1).
[0072]
Step (a) (condensation step)
In this step, an anchor soluble in halogenated solvents
5 or ether solvents, insoluble in polar solvents and having a
molecular weight of not less than 300 is condensed with a Cterminal
carboxy group of N-protected amino acid or N-protected
peptide in a solvent to give N-protected C-protected amino acid
or N-protected C-protected peptide.
10 [0073]
While the upper limit of the number of amino acid
residues of the N-protected peptide is not particularly limited
as long as the N-protected peptide to be used in this step is
soluble in a solvent to be used in this step, the number of
15 amino acid residues of N-protected peptide is preferably not
more than 100, more preferably not more than 50, still more
preferably not more than 30, especially preferably not more
than 10, particularly preferably not more than 5.
[0074]
20 The condensation reaction is preferably performed by
dissolving anchor, N-protected amino acid or N-protected
peptide and a catalytic amount of dimethylaminopyridine in a
solvent, adding a condensing agent and stirring the mixture.
[0075]
25 This step is performed in a solvent that does not
influence the reaction. The higher the solubility in the
solvent becomes, the more superior the reactivity is expected
to be. Therefore, a solvent showing high solubility of the
aforementioned N-protected amino acid or N-protected peptide is
30 preferably selected. Specifically, halogenated solvents such
as chloroform, dichloromethane, 1,2-dichloroethane and the
like; and ether solvents such as 1,4-dioxane, cyclopentyl
methyl ether, tetrahydrofuran and the like can be mentioned.
Two or more kinds of these solvents may be used in a mixture in
35 an appropriate ratio. In addition, aromatic hydrocarbons such
24
^
as benzene, toluene, xylene and the like; nitriles (excluding
acetonitrile) such as propionitrile and the like; ketones such
as acetone, 2-butanone and the like; amides such as N,Ndimethylformamide
and the like; sulfoxides such as dimethyl
5 sulfoxide and the like may be mixed at an appropriate
proportion with the above-mentioned halogenated solvent and
ether solvents as long as the compound to be used for the
production method of the present invention can be dissolved.
Of these, chloroform, dichloromethane, cyclopentyl methyl ether
10 or tetrahydrofuran is preferable, and chloroform is
particularly preferable.
[0076]
While the concentration of the N-protected amino apid or
N-protected peptide in a solution in this step is not
15 particularly limited as long as it is dissolved, it is
preferably 1 - 3 0 wt%.
[0077]
The amount of the N-protected amino acid or N-protected
peptide to be used in this step can be 1 - 10 mol, preferably 1
2 0 - 5 mol, per 1 mol of the aforementioned anchor.
[0078]
When Y in the aforementioned formula (I) is a hydroxy
group, an ester bond is formed by adding a condensing agent and,
where necessary, a condensation accelerator in a solvent that
25 does not influence the reaction in the presence of a
dimethylaminopyridine catalyst.
[0079]
When Y in the aforementioned formula (I) is ah -NHR group,
an amide bond is formed by adding a condensing agent in the
30 presence of a condensation accelerator.
[0080]
When Y in the aforementioned fo2nnaula (I) is a halogen
atom, an ester bond is formed by adding a base such as
diisopropylethylamine and the like in a solvent that does not
35 influence the reaction.
25
[0081]
As a condensation accelerator, 1-hydroxybenzotriazole
(HOBt), ethyl 1-hydroxy-lH-l,2,3-triazole-5-carboxylate (HOCt),
l-hydroxy-7-azabenzotriazole (HOAt), 0-(benzotriazol-1-yl)-
5 N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 0-(6-
chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HCTU) and the like can be mentioned, with
preference given to HOBt.
[0082]
10 The amount of the condensation accelerator to be used is
preferably 0.05 - 1.5 mol per 1 mol of the aforementioned
anchor.
[0083]
As a condensing agent, dicyclohexylcarbodiimide (DCC),
15 diisopropylcarbodiimide (DIC) ,. N-ethyl-N'-3-
dimethylaminopropylcarbodiimide and hydrochloride thereof (EDC
HCl), (benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate (PyBop), 0-(benzotriazol-1-yl)-N,N,N',N'-
tetramethyluronium tetrafluoroborate (TBTU), 1-
20 [bis(dimethylamino)methylene]-5-chloro-lH-benzotriazolium 3-
oxide hexafluorophosphate (HCTU), 0-benzotriazole-N,N,N',N'-
tetramethyluronium hexafluorophosphate (HBTU) and the like can
be mentioned.
[0084]
25 The amount of the condensing agent to be used is, for
example, 1 - 1 0 mol, preferably 1 - 5 mol, per 1 mol of the
aforementioned anchor.
[0085]
While the reaction temperature is not particularly
30 limited as long as the reaction proceeds, it is preferably not
less than -10°C, more preferably not less than 0°C, preferably
not more than 50°C, more preferably not more than 30°C. The
reaction time is, for example, 1 --7 0 hr.
[0086]
35 Step (1) (deprotection step of N-terminal)
26
In this step, the N-terminal temporary protecting group
is removed from N-protected C-protected amino acid or Nprotected
C-protected peptide, without isolating from the
reaction solution of the anchor and N-protected amino acid or
5 N-protected peptide. This step may contain a neutralization
step after the removing step.
' [0087]
Removal (deprotection) of the N-terminal temporary
protecting group of the N-protected C-protected amino acid or
10 N-protected C-protected peptide can be performed by a known
method. For example, when the temporary protecting group is an
Fmoc group, the deprotection is performed by treating with an
organic base, when the temporary protecting group is a Boc
group, it is performed by treating with an acid, and when the
15 temporary protecting group is a Cbz group, it is performed by a
catalytic reduction and the like. The deprotection is
performed in a solvent that does not influence the reaction.
[0088]
While the organic base usable for the removal of an Fmoc
20 group is not particularly limited, secondary amines such as
diethylamine, piperidine, morpholine and the like, tertiary
amines such as diisopropylethylamine, dimethylaminopyridine,
l,8-diazabicyclo[5.4.0]-7-undecene (DBU), 1,4-
diazabicyclo[2.2.2]octane (DABCO), 1,5-diazabicyclo[4.3.0]-5-
25 nonene (DBN) and the like can be mentioned. Of these,
deprotection is performed in a halogenated solvent or ether
solvent, DBU and the like are preferable. The amount of the
organic base to be used is, for example, 1 - 100 mol,
preferably 1 - 1 0 mol, per 1 mol of the aforementioned N-
30 protected C-protected amino acid or N-protected C-protected
peptide.
[0089]
While the acid usable for the removal of a Boc group is
not particularly limited, mineral acids such as hydrogen
35 chloride, sulfuric acid, nitric acid and the like, carboxylic
27
^
acids such as formic acid, trifluoroacetic acid (TFA) and the
like, sulfonic acids such as methanesulfonic acid, ptoluenesulfonic
acid and the like, or a mixture thereof can be
used. As the mixture, for example, hydrogen bromide/acetic
5 acid, hydrogen chloride/dioxane, hydrogen chloride/acetic acid
and the like can be mentioned. When an acid other than an
aqueous solution is used, for example, when formic acid,
methanesulfonic acid and the like are used in a non-aqueous
system, for example, it is possible to selectively remove the
10- Boc group while retaining an anchor group, which is a
protecting group of carboxy group subject to hydrolysis under
acidic conditions. Particularly, water-soluble sulfonic acids
which are liquid at ambient temperature such as methanesulfonic
acid and the like are preferable since, when they are used in a
15 non-aqueous system, they can quickly progress the reaction at
room temperature with a comparatively small amount of use. The
amount of the acid to be used is, for example, 1 - 100 mol,
preferably 1 - 1 0 mol, per 1 mol of the aforementioned Nprotected
C-protected amino acid or N-protected C-protected
20 peptide.
[0090]
While the catalyst usable for the removal of the Cbz
• group is not particularly limited, for example, palladium and
the like can be mentioned. The amount of the catalyst to be
25 used is, for example, not less than 1 part by weight,
preferably not less than 5 parts by weight, for example, not
more than 50 parts by weight, preferably 30 parts by weight,
per 100 parts by weight of the aforementioned N-protected Cprotected
amino acid or N-protected C-protected peptide.
30 [0091]
Examples of the solvent that does not influence the
reaction include halogenated solvents such as chloroform,
dichloromethane, 1,2-dichloroethane and the like; aromatic
hydrocarbons such as toluene, xylene and the like; ether
35 solvents such as diethyl ether, CPME, THF, 1,4-dioxane and the
28
^
like; and the like, or a mixture thereof, preferably chloroform,
dichloromethane and THF.
[0092]
When the temporary protecting group of the aforementioned
5 N-terminal is an Fmoc group, a neutralization step by the
addition of an acid may be incorporated before the next
precipitation, solid-liquid separation step, since an excess
amount of the organic base used for the deprotection may
exhibit an adverse influence on the reaction product during
10 working up such as solvent evaporation and the like.
[0093]
As an acid to be used for the neutralization step, for
example, methanesulfonic acid, trifluoromethanesulfonic acid,
benzenesulfonic acid, p-toluenesulfonic anhydride, sulfuric
15 acid, hydrogen chloride and the like can be mentioned. Of
these, methanesulfonic acid, trifluoromethanesulfonic acid, and
hydrogen chloride are preferable.
[0094]
The amount of the acid to be used for the neutralization
20 step is, for example, not less than 0.5 mol, preferably not
less than 0.9 mol, for example, not more than 10 mol,
preferably not more than 5 mol, per 1 mol of the organic base.
[0095]
While the temperature of the reaction of step (1) is not
25 particularly limited as long as the reaction proceeds, it is
preferably not less than -10°C, more preferably not less than
0°C, preferably not more than 50°C, more preferably not more
than 30°C. The reaction time of step (1) is, for example, 1 -
70 hr.
30 [0096]
Step (2) (precipitation and solid-liquid separation step)
In this step, C-protected amino acid or C-protected
peptide obtained in the above-mentioned step (1) is isolated by
changing the solvent dissolving the C-protected amino acid or
35 C-protected peptide (e.g., change of solvent composition,
29
^
change of solvent kind) to allow for precipitation. That is,
the deprotection of step (1) is performed under the conditions
under which C-protected amino acid or C-protected peptide is
dissolved, solvent exchange is performed to precipitate C-
5 protected amino acid or C-protected peptide, and the impurity
(free amino acid, free peptide etc.) is removed by solid-liquid
separation. Solvent evaporation and the like may be perfoinnied
before solvent exchange.
[0097]
10 As a solvent for exchange, polar solvents such as
methanol, acetonitrile and the like can be used. When methanol
is used as a solvent for exchange and removal of the solvent is
insufficient though residual amino acid can be removed,
problems such as transesterification reaction with an anchor
15 group and the like occur during the isolation operation or the
following condensation step. Therefore, a drying step needs to
be formed to sufficiently remove methanol, which
problematically requires much time for industrialization. In
addition, it was found that when acetonitrile is used as a
20 solvent for exchange as mentioned below, residual amino acid
cannot be sufficiently removed and, when subjected to the next
condensation step, a double-hit compound derived from the
residual amino acid is by-produced. Moreover, when water is
used as a solvent for exchange, the object peptide compound
25 coagulates in a state containing impurities, which makes it
difficult to efficiently remove the residual amino acid or
peptide. Therefore, to isolate C-protected amino acid or Cprotected
peptide from the reaction mixture with high purity,
or obtain C-protected amino acid or C-protected peptide having
30 a purity sufficient for the successive one-pot reaction
consisting of a condensation step and a deprotection step, a
solvent containing water-containing acetonitrile needs to be
used as a solvent for exchange.
[0098]
35 In this step, the lower limit of the content of
30
^
acetonitrile in water-containing acetonitrile is preferably
60v/v%, more preferably ipv/v%, still more preferably 75v/v%.
On the other hand, the upper limit of the content of
acetonitrile in water-containing acetonitrile is preferably
5 95v/v%, more preferably 90v/v%, still more preferably 85v/v%.
A particularly preferable content of acetonitrile in watercontaining
acetonitrile is 80v/v%.
[0099]
In this step, the "solvent containing water-containing
10 acetonitrile" may be the above-mentioned "water-containing
acetonitrile" alone or a mixed solvent with other organic
solvent mentioned above. As other organic solvent, at least
one kind of solvent selected from the group consisting of
methanol, ethanol, dimethylformamide, propionitrile, dimethyl
15 sulfoxide, acetone, dichloromethane, chloroform,
tetrahydrofuran, cyclopentyl methyl ether and ethyl acetate is
preferable. The content of the "water-containing acetonitrile"
in the "solvent containing water-containing acetonitrile" is
not par1;icularly limited as long as amino acids can be removed,
20 and 60v/v% - 100v/v% is preferable.
[0100]
The second embodiment of the production method of the
present invention is a production method of peptide, comprising
the following steps (3) to (5);
25 (3) condensing an N-terminal amino group of a C-protected amino
acid or C-protected peptide wherein a C-tejnninal carboxy group
is protected by an anchor group derived from an anchor soluble
in halogenated solvents or ether solvents, insoluble in polar
solvents and having a molecular weight of not less than 300,
30 and a C-terminal carboxy group of an N-protected amino acid or
N-protected peptide in a solvent to give an N-protected Cprotected
peptide,
(4) removing the N-terminal protecting group of the N-protected
C-protected peptide obtained in step (3) in the reaction
35 solution of step (3), without isolating the N-protected C-
31
^
protected peptide, to give a C-protected peptide (N-terminal
deprotection step), and
(5) precipitating C-protected peptide in a solvent containing
60 - 95% water-containing acetonitrile after step. (4), and
5 obtaining same by solid-liquid separation (precipitation and
solid-liquid separation step),
[0101]
Step (3) (condensation step)
In this step, an N-terminal amino group of C-protected
10 amino acid or C-protected peptide and a C-terminal carboxy
group of N-protected amino acid or N-protected peptide are
condensed under the conditions similar to those in step (a)
wherein Y in the aforementioned formula (I) is NHR.
[0102]
15 While the upper limit of the number of amino acid
residues of the C-protected peptide is not particularly limited
as long as the C-protected peptide to be used in this step is
soluble in a solvent to be used in this step, the number of
amino acid residues of C-protected peptide is preferably not
20 more than 100, more preferably not more than 50, still more
preferably not more than 30. While the upper limit of the
number of amino acid residues of the N-protected peptide is not
particularly limited as long as the N-protected peptide to be
used in this step is soluble in a solvent to be used in this
25 step, the number of amino acid residues of N-protected peptide
is preferably not more than 100, more preferably not more than
50, still more preferably not more than 30, especially
preferably not more than 10, particularly preferably not more
than 5.
30 [0103]
Step (4) (N-terminal deprotection step)
This step is performed in the same manner as in the
deprotection step of N-terminal in step (1).
[0104]
35 Step (5) (precipitation and solid-liquid separation step)
32
^
This step is performed in the same manner as in the
precipitation and solid-liquid separation step in step (2).
[0105]
The above-mentioned steps (3) - (5) may be repeated two
5 times or more to obtain a C-protected peptide having an
elongated peptide chain. While the upper limit of the number
of amino acid residues of the C-protected peptide having an
elongated peptide chain is not particularly limited as long as
the C-protected peptide is soluble in the solvent to be used in
10 step (3), the number of the amino acid residues is preferably
not more than 200, more preferably not more than 100, more
preferably not more than 50.
[0106]
The production method of the peptide of the present
15 invention can further contain step (6) for removing the Cterminal
anchor group of the C-protected peptide after the
precipitation step (5).
[0107]
By the removal (deprotection) of the C-terminal anchor
20 group, the final object product peptide wherein the C-terminal
of the peptide is -COOH (e.g., the aforementioned formula (I)
wherein Y is a hydroxy group or a halogen atom), or -CONHR
(e.g., the aforementioned formula (I) wherein Y is an NHR
group) can be obtained.
25 [0108]
When an anchor group derived from an anchor of the
aforementioned formula (I) wherein Y is a hydroxy group or a
halogen atom is selectively removed, deprotection is preferably
performed by an acid treatment. As an acid to be used for the
30 deprotection, trifluoroacetic acid (hereinafter to be referred
to as TFA), hydrochloric acid, sulfuric acid, methanesulfonic
acid, p-toluenesulfonic acid and the like can be mentioned,
with preference given to TFA. As a solvent to be used for the
deprotection, for example, chloroform, dichloromethane, 1,2-
35 dichloroethane or a mixed solvent thereof and the like can be
33
© mentioned. The concentration of an acid to be used for the
deprotection is, for example, 0.lw/v% - 5w/v%.
[0109]
It is also possible to remove an anchor group derived
5 from an anchor of the aforementioned formula (I) wherein Y is a
hydroxy group, an -NHR group, or a halogen atom, simultaneously
with the protecting group of other side chain in a peptide. In
this case, a conventional method used in the field,
particularly peptide synthesis, is used, and a method including
10 adding an acid and the like is preferably used. As the acid,
TFA, hydrochloric acid, sulfuric acid, mesylic acid, tosylic
acid, trifluoroethanol, hexafluoroisopropanol and the like are
used. Of these, TFA is particularly preferable. The amount of
the acid to be used is appropriately set according to the kind
15 of the acid to be used, and an amount suitable for removing the
anchor group is used. The amount of the acid to be used is
preferably not less than 3 mol, more preferably not less than 5
mol, preferably not more than 100 mol, more preferably not more
than 50 mol, per 1 mol of the C-protected peptide. Along with
20 such use, trifluoromethanesulfonic acid, trimethylsilyl
trifluoromethanesulfonate, BFa-etherate and the like can also
be added as a further source of strong acid.
[QUO]
While the reaction temperature is not particularly
25 limited as long as the reaction proceeds, for example, it is
0°C - 50°C, preferably 0°C - 30°C. The reaction time is, for
example, 0.5 - 24 hr.
[0111]
For confirmation of the progress of the reaction in the
30 above-mentioned steps (a), (2), (4) and (5), a method similar
to general liquid phase organic synthetic reaction can be
applied. That is, thin layer silica gel chromatography, high
performance liquid chromatography and the like can be used to
trace the reaction.
35 [Examples]
34
^ [0112]
The present invention is explained in more detail in the
following by referring to Reference Example and Experimental
Examples, which are not to be construed as limiting the scope
5 of the present invention in any way. The reagents, apparatuses
and materials used in the present invention are commercially
available unless otherwise specified. In the present
specification, when amino acid and the like are indicated by
abbreviations, each indication is based on the abbreviation of
10 the lUPAC-IUB Commission on Biochemical Nomenclature or
conventional abbreviations in the art.
[0113]
The anchor used in the Experimental Example can be
produced by a method known per se (see the aforementioned
15 patent documents 1 - 6 , non-patent document 1) or a method
analogous thereto, or method according to the following
Reference Example 1, from a known starting material compound
(or commercially available product).
[0114]
20 Reference Example 1: synthesis of 4-methoxy-2-[3',4',5'-
tri(octadecyloxy)benzyloxy]benzyl alcohol (anchor (A))
[0115]
9^18^37
'OC18H37
CH2OH
OC18H37
(A)
OMe
[0116]
25 (i) 3,4,5-Tri(octadecyloxy)benzyl alcohol (83.0 g, 90.8 mmol)
was dissolved in chloroform (830 ml), thionyl chloride (21.6 g,
0 .182 mol) was added at 0°C and the mixture was stirred for
1.5 hr at room temperature. The solvent was evaporated, and
the residue was crystallized from acetonitrile (800 ml) to give
30 3,4,5-tri(octadecyloxy)benzyl chloride as wet crystals (93.6 g),
35
^ [0117]
(ii) 3,4,5-Tri(octadecyloxy)benzyl chloride (93.6 g, wet, <90.8
mmol), 2-hydroxy-4-methoxybenzaldehyde (15.2 g, 0.10 mol),
potassium carbonate (31.4 g, 0.23 mol) were suspended in DMF
5 (830 ml), and the mixture was stirred at 80°C overnight. The
reaction mixture was dissolved in chloroform (1600 ml), and
washed three times with IN hydrochloric acid (800 ml), once
with 5 wt% aqueous sodium hydrogen carbonate solution (800 ml)
and once with 20 wt% brine (800 ml). The solvent was
10 evaporated, and the residue was crystallized from methanol (800
ml), and washed with acetonitrile (800 ml) to give 4-methoxy-2-
[3',4',5'-tri(octadecyloxy)benzyloxy]benzaldehyde (93.5 g, 89.2
mmol, yield 98%).
[0118]
15 (iii) 4-Methoxy-2-[3',4',5'-
tri (octadecyloxy)benzyloxy]benzaldehyde (93.5 g, 89.2 mmol) was
dissolved in THF-methanol (1870 ml+94 ml), and sodium
borohydride (4.05 g, 107 mmol) was added at 0°C. After
stirring at room temperature for 1.5 hr, 0.2N hydrochloric acid
20 (100 ml) was added at 0°C to quench the reaction. About half
the solvent was evaporated, the residue was dissolved in
chloroform (2400 ml) and washed two times with O.IN
hydrochloric acid (1200 ml), once with 5wt% aqueous sodium
hydrogen carbonate solution (1200 ml) and once with 20wt% brine
25 (1200 ml). The solvent was evaporated, and the residue was
crystallized from methanol (900 ml), and washed with
acetonitrile to give 4-methoxy-2-[3',4',5'-
tri(octadecyloxy)benzyloxy]benzyl alcohol (anchor (A), 92.4 g,
88.0 mmol, yield 97% (vs 3,4,5-tri(octadecyloxy)benzyl
30 alcohol)).
^H-NMR (300MHz, CDCl3):5 0. 88 (9H, t, J=6. 3Hz, Ci7H34-Me) , 1.15-
1.40(84H,br,C3',4' , 5'-OC3H6-C14H28-CH3) , 1.40-
1.55(6H,br,C3' ,4' ,5'-OC2H4-CH2-Ci5H3i) , 1. 70-1. 85 (6H,m, C3' , 4' , 5'-
OCH2-CH2-C16H33) , 2.18(lH,t, J=6.3Hz,OH) , 3 . 79 (3H, s, C4-0Me) , 3.90-
35 4.03(6H,m,C3',4',5'-O-CH2-Ci7H35) , 4 . 65 (2H, d, J=6. 6Hz,Ar-CH2-OH) ,
36
4.97(2H,s,Ar-0-CH2-Ar) , 6.47(IH, dd, J=2.1,8.lHz,C5-H),
6.53(lH,d,J=2.4Hz,C3-H), 6.60(2H,s,C2',6'-H),
7.19(lH,d,J=8.1Hz,C6-H)
[0119]
5 Experimental Example 1: comparison of impurity contents due to
difference of precipitation solvent
test method:
Anchor (A) (500 mg) was dissolved in chloroform, and
Fmoc-Phe-OH (1.3 equivalents), dimethylaminopyridine (0.15
10 equivalent), and EDC HCl (1.4 equivalents) were added to allow
reaction. After completion of the reaction,, diethylamine (15
equivalents) and DBU (2.5 equivalents) were directly added to
the reaction solution to remove the Fmoc group, and the
solution was neutralized with 4M HCl/CPME (2.5 equivalents).
15 The solvent was evaporated under reduced pressure, and the
residue was precipitated with acetonitrile alone or 80% watercontaining
acetonitrile, and the precipitate was filtered and
slurry washed with acetonitrile. Wet crystals were dissolved
in chloroform, condensed by adding Fmoc-His(Trt)-OH (1.1
20 equivalents), EDC HCl (1.2 equivalents) and HOBt (0.1
equivalents) . In the same manner as above, the Fmoc group was
removed, and precipitation was performed with acetonitrile
alone (or 80% water-containing acetonitrile). The same
operation was repeated and, after condensation with Ftaoc-Gly,
25 the Fmoc group was removed to give a tripeptide-protected
product. The thus-obtained tripeptide-protected product was
treated with TEA to remove the anchor group and the side chain
functional group-protecting group. The content of impurity
(e.g., double-hit compound etc.) formed other than the object
30 product H-Gly-His-Phe-OH was compared between precipitation
with acetonitrile alone and precipitation with 80% watercontaining
acetonitrile.
[0120]
HPLC measurement condition
35 Measurement device: LC-20A manufactured by SHIMADZU CORPORATION
37
^ Column: YMC-Pack ODS-A 4.6x150 ram (5 \m)
Temperature: 40°C
Wavelength: 220 nm
Flow rate: 1.0 ml/min
5 Eluent: SOLUTION A 0.05v/v% TFA-water
SOLUTION B 0.05v/v% TFA-MeCN
Time program: SOLUTION B concentration 0 min 1 v/v%
20 min 50 v/v%
25 min 50 v/v%
10 [0121]
Experimental results: As shown in Table 1, when precipitation
was repeated with acetonitrile alone, impurity was by-produced
in a large amount (45 area % (area ratio relative to peak of
object tripeptide in HPLC analysis data)), whereas when
15 precipitation was repeated with 80% water-containing
acetonitrile, impurity was scarcely formed.
[0122]
Table 1
Quantitative ratio of impurity to final deprotected
product (peak area ratio: area %)
acetonitrile
80% water-containing
acetonitrile
+Phe
20
0.3
+His
17
0
+PheHis
3
0
+PhePhe
5
0
+Phe:H-Gly-His-Phe-Phe-OH
20 +His:H-Gly-His-His-Phe-OH
+PheHis:H-Gly-His-Phe-His-Phe-OH
+PhePhe:H-Gly-His-Phe-Phe-Phe-OH
[0123]
Experimental Example 2: comparison of amino acid removal
25 performance by precipitation solvent
Experimental method:
Anchor (A), anchor
(B) or anchor (C) shown below was dissolved in chloroform, and
Fmoc-amino acid (hereinafter sometimes to be also indicated as
"Fmoc-Xaa-OH") (1.2 equivalents), dimethylaminopyridine (0.1
equivalent), and EDC HCl (1.3 equivalents) were added to
38
30
perform dehydration condensation. After the completion of the
reaction, diethylamine (15 equivalents) and DBU (2 equivalents)
were directly added to the reaction solution without isolation
to remove Fmoc group, and the solution was neutralized by the
5 addition of 4M HCl/CPME (2 equivalents). The solvent was
evaporated under reduced pressure. Various solvents for
exchange were added to the residue and the mixture was stirred,
and the precipitate was collected by filtration, washed with
the same solvent for exchange added to the residue and dried.
10 The contents of the residual amino acid and protected amino
acid (H-His(Trt)-OH) in the crude crystals of Xaa-0-anchor
group (derived from anchor (A)) were measured by amino acid
analysis and HPLC (Table 2).
[0124]
15 The measurement conditions of HPLC are the same as those
in Experimental Example 1. The measurement condition of amino
acid analysis is as follows.
Measurement device: AAA (HITACHI L-8900)
Column: HITACHI #2622PH 4.6 mmx60 mm
20 HITACHI #2650L 4.6 mmx40 mm
Solvent: L-8500PH Kit
Temperature: 57°C
[0125]
In addition, the precipitates obtained by adding various
25 solvents for exchange were dissolved in chloroform, Finoc-Leu
was added, and a condensation reaction was performed using EDC*
HCl/HOBt. A small amount of the reaction solution was taken,
and the content of a double-hit compound (Fmoc-Leu-Xaa-Xaa-OH)
in crude Fmoc-Leu-Xaa-OH obtained by removing the anchor group
30 (derived from the following anchor (A), (B) or (C)) in TFA was
measured by HPLC under the same measurement conditions as in
Experimental Example 1 (Table 3).
39
^ ^ t ^ ^
[0126]
Table 2
Contents of residual amino acid and protected amino acid (H-His(Trt)-OH) in crude crystals of Xaa-0-anchor
group (derived from anchor (A)
Xaa
acetonitrile
80% water-containing acetonitrile
Phe
0.06 wt%
0.001 wt%
Gly
0.04 wt%
0.01 wt%
He
0.02 wt%
0.004 wt%
His(Trt)
1.6 wt%
0
5 [0127]
Table 3
Anchor
(A)
(B)
(A)
(B)
(C)
(A)
(B)
(C)
Xaa
Phe
Gly
His
(Trt)
Lys
(Boc)
First residue amino
acid (equivalents)
1.4
1.4
1.2
1.2
1.4
1.2
1.4
1.2
Content (area %) of double-hit compound (Fmoc-Leu-Xaa-Xaa-OH) in
crude Fmoc-Leu-Xaa-OH
acetonitrile
4.8
6.7
4.5
2.5
4.7
2.0
1.5
1.6
90% watercontaining
acetonitrile
1.1
-
-
-
-
-
-
-
80% watercontaining
acetonitrile
0
0.1
0.7
0.7
0.5
0.1
0.1
0.2
60% watercontaining
acetonitrile
0.9
-
-
-
-
-
-
-
40
IWiwgfflwppjPljiwsyw wmm^i^^i^>m^-
u [0128]
The anchors used in Experimental Example 2 were anchor
(A) shown in Reference Example 1, anchor (B) shown by the
formula:
5 [0129]
OCiaH37
OC18H37
OC18H37
(B)
[0130]
and anchor (C) shown by the formula:
[0131]
^;f-v^OC22H45
OC22H45
10
[0132]
[0133]
Experimental results:
As shown in Table 2, it was found that the precipitation
15 with 80% water-containing acetonitrile can efficiently remove
residual amino acid as compared to the precipitation with
acetonitrile alone. As shown in Table 3, moreover, it was
found that the precipitation with water-containing acetonitrile
within the range defined in the present invention (particularly,
20 80% water-containing acetonitrile) remarkably inhibits
problematic by-production of a double-hit compound during
peptide synthesis.
[0134]
Experimental Example 3: comparison of amino acid removal
25 performance in dipeptide synthesis in co-presence of various
Fmoc-amino acids
Experimental method:
A synthesized and isolated Fmoc-Phe-0-anchor group
(derived from the above-mentioned anchor (B) or (C)) was
41
^^f^^-^
dissolved in chloroform, various Fmoc-amino acids (0.2
equivalents) were added to be mixed in the solution. To this
solution were added DBU and diethylamine to remove the Fmoc
group, a crude product of an H-Phe-0-anchor group was obtained
5 by precipitation by the addition of various solvents for
exchange, and condensed with Fmoc-Leu using EDC/HOBt. A small
amount of the condensation reaction mixture was taken, and the
anchor group was removed in TFA and the mixture was analyzed by
HPLC, and a quantitative ratio of a double-hit compound of the
10 amino acid mixed (Ftaoc-Leu-Xaa-Phe) relative to the object
dipeptide Fmoc-Leu-Phe was measured. The results are shown in
Tables 4 and 5.
42
o
[0135]
Table 4
Anchor
(B)
(C)
Xaa
Phe
Gly
His(Trt)
He
Lys{Boc)
Gly-Gly
Arg-Pro
Phe
Gly
His(Trt)
Quantitative ratio (area %) of double-hit compound (Fmoc-Leu-Xaa-Phe)
90% watercontaining
acetonitrile
2.1
-
-
-
-
-
-
0.3
1.4
1.7
85% watercontaining
acetonitrile
0.7
-
-
-
-
-
-
0.2
-
-
80% watercontaining
acetonitrile
0.4
0
0
0.7
0.1
0.2
0
0.6
0
0.1
75% watercontaining
acetonitrile
1.7
-
-
-
-
-
-
1.0
-
-
70% watercontaining
acetonitrile
1.9
-
-
-
-
-
-
1.0
-
-
80% water-containing
acetonitrile +
methanol
-
1.6
-
-
-
-
-
-
-
Content of 80% water-containing acetonitrile in 80% water-containing acetonitrile + methanol: 95 v/v%
43
wm^mm?iss^mimmmms>fm mm^^mimmmv^mmm m^m^mmmi^ mm^m'imrm^f^^m^W'm^m^mmi'sf wmm^'^s^mmmmmi^imw^^sfsm
[0136]
Table 5
©
Anchor
(B)
(C)
Xaa
Phe
Gly
His(Trt)
He
Lys(Boc)
Gly-Gly
Arg-Pro
Phe
Gly
His(Trt)
Quantitative ratio (area %) of double-hit compound (Etnoc-Leu-Xaa-Phe)
acetonitrile
3.1
2.0
1.8
1.9
1.1
1.7
1.5
4.1
2.0
6.9
20% water-containing acetonitrile
4.1
2.5
2.7
4.9
2.2
-
-
4.3
2.5
9.7
44
M^^^^SRWSWPSWW!^ !WlWWS»W'^»f^SWW!|!^^ mmf^i^mm^f?^ m!mw^mamsmmm?m>^f m^i^fim^mmmm^mf'mmHmmimmm, ijwmiBiirw^gifSfffligpjyjtggm^^ mmm>w^mfWlWW^^
1#
[0137]
Experimental results:
As shown in Tables 4 and 5, it was found that the
precipitation with water-containing acetonitrile within the
5 range defined in the present invention (particularly, 80%
water-containing acetonitrile) remarkably inhibits by- .
production of a double-hit compound, as compared to the
precipitation with acetonitrile alone or precipitation with 20%
water-containing acetonitrile. It was also confirmed that 80%
10 water-containing acetonitrile shows less by-production of a
double-hit compound as compared to water-containing acetone,
water-containing DMF, water-containing methanol and watercontaining
ethanol, each having an acetonitrile content of
80v/v%.
15 [INDUSTRIAL APPLICABILITY]
[0138]
According to the production method of peptide of the
present invention, by performing precipitation and solid-liquid
separation by the addition of particular water-containing
20 acetonitrile after dehydration condensation of an N-terminal
amino group of an amino acid or peptide having a C-terminal
protected by an anchor group and a C-terminal carboxy group of
an N-protected amino acid or N-protected peptide, and a
subsequent removing step of an N-terminal temporary protecting
25 group, a condensation step and an N-tejrminal deprotection step
can be successively performed in one pot in a good yield.
According to the present invention, complicated isolation and
purification operations such as solvent evaporation after a
condensation step, precipitation by solvent exchange,
30 filtration, washing, drying and the like can be omitted, and
by-production of a double-hit compound and the like is not
found. Therefore, a convenient and efficient production method
of peptide, which enables scaling up and is suitable for
industrial production can be provided.
35 [0139]
45
^
A differently expression method showing the
characteristics of the production method of peptide of the
present invention is described below.
An "immobilizing-protecting group" is defined as a
5 "protecting group uninfluenced in a (deprotection step) and a
(condensation step), and deprotected in a (final deprotection
step)", and.a general "protecting group" is defined as a
"protecting group uninfluenced in a (condensation step), and
deprotected in a (deprotection step)".
10 When a "production method of peptide by a method of
immobilizing-protecting C-terminal and elongating N-terminal"
is defined as a "production method of peptide comprising
elongating a peptide chain in the N-terminal direction by
repeating a step of condensing an amino acid or peptide having
15 immobilized and protected C-terminal and a novel N-terminal
protected amino acid or peptide (condensation step), and a step
of N-terminal deprotection of the elongated N-terminal
protected peptide having iiranobilized and protected C-terminal
(deprotection step)", Fmoc method and Boc method are included
20 therein, and the Fmoc method is preferable.
[0140]
Here, when an "improved successive method of
immobilizing-protecting C-terminal and elongating N-terminal"
is defined as a "method of immobilizing-protecting C-terminal
25 and elongating N-terminal characterized by successively
performing a (condensation step) and a (deprotection step) in a
solution without purification by extraction, precipitation and
the like on the way between these steps and purifying by a
(precipitation step) after the completion of the (deprotection
30 step)", the production method of peptide of the present
invention can also be expressed as follows.
[1] A production method of peptide by an improved successive
method of immobilizing-protecting C-terminal and elongating Nterminal,
comprising, after a deprotection step, a
35 precipitation step in a polar solvent containing acetonitrile
46
© and water.
[2] The production method of peptide of [1], wherein the polar
solvent is 60 - 95% water-containing acetonitrile.
[3] The production method of peptide of [1]] or [2], wherein
5 the protecting group is an Fmoc group.
[4] The production method of peptide of' any one of [1]- [3],
further comprising a final deprotection step.
[0141]
The production method of peptide of the present invention
10 can be further expressed in detail as follows.
(The first step: C-terminal immobilizing-protecting step)
A step of producing N-terminal protected amino acid or
peptide with immobilized and protected C-terminal by reacting
C-terminal of an N-terminal protected amino acid or peptide
15 with an immobilizing-protecting group.
(The second step: deprotection step)
A step of producing amino acid or peptide with
immobilized and protected C-terminal by removing the N-terminal
protecting group of the N-terminal protected amino acid or
20 peptide with immobilized and protected C-terminal.
(The third step: condensation step)
A step of producing elongated N-terminal protected
peptide with immobilized and protected C-terminal by condensing
amino acid or peptide with immobilized and protected C-terminal
25 and a novel N-tearminal protected amino acid or peptide.
(The fourth step: deprotection step)
A step of producing amino acid or peptide with
immobilized and protected C-terminal by removing the N-terminal
protecting group of the N-terminal protected amino acid or
30 peptide with immobilized and protected C-terminal.
(The fifth step: precipitation step)
A step of purifying the elongated N-terminal protected
peptide with immobilized and protected C-terminal by
precipitating the elongated N-terminal protected peptide with
35 immobilized and protected C-terminal in a polar solvent.
47
^^^
^K^
(The sixth step: final deprotection step)
A step of producing an elongated peptide by removing the
immobilized-protected C-terminal from the elongated N-terminal
protected peptide with immobilized and protected C-terminal or
5 elongated peptide with immobilized and protected C-terminal.
[0142]
Since the above-mentioned third step, fourth step, and
fifth step are repeated as one set, the production method of
peptide of the present invention specifically includes the
10 following case.
The first step -> the second step -> (the third step ->
the fourth step -^ the fifth step) x m times -> the sixth step
wherein m is a natural number, and "(...) x m times" means that
the step in the parenthesis is repeated m times.
15 [0143]
This application is based on a patent application No.
2011-122943 filed in Japan, the contents of which are
incorporated in full herein.
48

-11G1NAL ^ ^^^ ^^
^
1
Claims 1 1 2 ' ^ « - ^^
1. A production method of a protected amino acid or protected
peptide, comprising the following steps (1) and (2);
5 (1) obtaining a C-protected amino acid or C-protected peptide
by removing a N-terminal protecting group from an N-protected
C-protected amino acid or N-protected C-protected peptide
wherein a C-terminal carboxy group is protected by an anchor
group derived from an anchor soluble in halogenated solvents or
10 ether solvents, insoluble in polar solvents and having a
molecular weight of not less than 300, without isolating from a
reaction solution of the anchor and an N-protected amino acid
or N-protected peptide, and
(2) precipitating the C-protected amino acid or C-protected
15 peptide in a solvent containing 60 - 95% water-containing
acetonitrile after step (1), and obtaining same by solid-liquid
separation.
2. A production method of peptide, comprising the following
20 steps (3) to (5);
(3) condensing an N-terminal amino group of a C-protected amino
acid or C-protected peptide wherein a C-terminal carboxy group
is protected by an anchor group derived from an anchor soluble
in halogenated solvents or ether solvents, insoluble in polar
25 solvents and having a molecular weight of not less than 300,
and a C-terminal carboxy group of an N-protected amino acid or
N-protected peptide in a solvent to give an N-protected Cprotected
peptide,
(4) removing the N-terminal protecting group of the N-protected
30 C-protected peptide obtained in step (3) in the reaction
solution of step (3), without isolating the N-protected Cprotected
peptide, to give a C-protected peptide, and
(5) precipitating C-protected peptide in a solvent containing
60 - 95% water-containing acetonitrile after step (4), and
35 obtaining same by solid-liquid separation.
49
3. The method according to claim 2, further comprising step (6)
removing the C-terminal anchor group of the C-protected peptide
after step (5).
4. The method according to claim 2 or 3, wherein the anchor
soluble in halogenated solvents or ether solvents, insoluble in
polar solvents and having a molecular weight of not less than
300 is a compound represented by the following formula (I):
10
:OR2)P ('>
wherein
R'"' is a hydrogen atom or, when R'^ is a group represented by the
following formula (a), optionally shows a single bond together
with R"^ to form a fluorene ring together with ring A and ring
15 B;
R^ in the number of p is each independently an organic group
having an aliphatic hydrocarbon group;
p is an integer of 1 to 4;
ring A optionally further has, in addition to OR^ in the number
20 of p, a substituent selected from the group consisting of a
halogen atom, a Ci-e alkyl group optionally substituted by a
halogen atom, and a Ci-e alkoxy group optionally substituted by
a halogen atom;
R^ is a hydrogen atom, or a phenyl group optionally substituted
25 by a halogen atom; and
R^ is a hydrogen atom, or a group represented by the formula
(a):
50
wherein is a binding site; ^^ h^ ,.'> i\nt-?^l| >Li
r is an integer of 0 to 4; | | /, ,• J \J ' ' -»• ^-^
R^ in the number of r is each independently an organic group
having an aliphatic hydrocarbon group;
5 R^ is a hydrogen atom, or optionally shows a single bond
together with R''" to form a fluorene ring together with ring A
and ring B; and
ring B optionally further has, in addition to OR'' in the number
of r, a substituent selected from the group consisting of a
10 halogen atom, a Ci-e alkyl group optionally substituted by a
halogen atom, and a Ci_6 alkoxy group optionally substituted by
a halogen atom; and
Y is a hydroxy group, NHR (R is a hydrogen atom, an alkyl group
or an aralkyl group) or a halogen atom.
15
20
25
30
35
5. The method according to claim 4, wherein, in the formula (I),
Y is a hydroxy group, R''' is a hydrogen atom, R^ and/or R^ are/is
aliphatic hydrocarbon group(s) having 5 to 60 carbon atoms, p
is an integer of 1 to 3, and r is an integer of 0 - 2.
6. The method according to claim 4, wherein, in the formula (I),
Y is a hydroxy group, R^, R"^, and R''' are each a hydrogen atom,
R^ is an aliphatic hydrocarbon group having 5 to 60 carbon
atoms, and p is an integer of 1 to 3.
7. The method according to claim 4, wherein, in the formula (I),
Y is a hydroxy group, R^, R'', and R'"' are each a hydrogen atom,
R^ is an alkyl group having 10 to 40 carbon atoms, and p is 2
or 3.
8. The method according to claim 4, wherein, in the formula (I),
Y is a hydroxy group, R^, R*', and R'"' are each a hydrogen atom,
R^ is an alkyl group having 12 to 30 carbon atoms, and p is 2
or 3.
51
ORIGINAL 1^2^551115
jp^ 9. The method according to claim 4, wherein the compound ^ T H C P
represented by the formula (I) is a compound selected from the
group consisting of
3,4,5-tri(octadecyloxy)benzyl alcohol,
5 2,4-di(docosyloxy)benzyl alcohol,
4-methoxy-2-[3',4',5'-tri(octadecyloxy)benzyloxy]benzyl alcohol,
4-methoxy-2-[3',4', 5' -
tri(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol,
2-methoxy-4-[3',4', 5' -
10 tri(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol,
4-[3', 4' ,5'-tri(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol,
3,5-dimethoxy-4-[3', 4',5' -
tri(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol,
2,4-di(dodecyloxy)benzyl alcohol,
15 3,4,5-tri(octadecyloxy)benzylamine,
bis(4-docosyloxyphenyl)methanol, and
bis(4-docosyloxyphenyl)methylamine.
10. The method according to any one of claims 2 to 9, wherein
20 the water-containing acetonitrile has an acetonitrile content
of.70v/v% - 90v/v%.
11. The method according to any one of claims 2 to 9, wherein
the water-containing acetonitrile has an acetonitrile content
25 of 75v/v% - 85v/v%.
12. The method according to any one of claims 2 to 9, wherein
the water-containing acetonitrile has an acetonitrile content
of 80v/v%.
30
13. The method according to any one of claims 2 to 12, wherein
the solvent containing water-containing acetonitrile is a mixed
solvent of water-containing acetonitrile, and at least one kind
of solvent selected from the group consisting of methanol,
35 ethanol, dimethylformamide, propionitrile, dimethyl sulfoxide,
52
€» le, acetone, dichloromethane, chloroform, ter-raftvydrofuran.,
cyclopentyl methyl ether and ethyl acetate. , 0 1
14. The method according to any one of claims 2 to 12, wherein
5 the solvent containing water-containing acetonitrile is watercontaining
acetonitrile.
15. The method according to any one of claims 2 to 14, wherein
the amino-protecting group of the N-protected amino acid or N-
10 protected peptide is a 9-fluorenylmethyloxycarbonyl group, a
tert-butoxycarbonyl group or a benzyloxycarbonyl group.
16. A production method of peptide by an improved successive
method of immobilizing-protecting C-terminal and elongating N-
15 terminal, comprising, after a deprotection step, a
precipitation step in a polar solvent containing acetonitrile
and water.