DESCRIPTION
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
^%. each disclose a method of using 3, 4, 5-tri (n-octadecyloxy)benzyl
alcohol as a protective reagent 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 document 6: WO2010/104169
20 [non-patent document]
[0006]
non-patent dociiment 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
tf*| reactions 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.
[0008]
5 When an anchor other than the above-mentioned trityl type
compound (patent document 4) and fluorene compound (patent
document 6) is used, the same problem of by-production of
diketopiperazine as in the liquid phase reaction occurs in the
peptide synthesis when an isolation and purification operation
10 including concentration, precipitation, filtration, washing and
drying is performed after removal of the N-terminal temporary
protecting group. Particularly, when a sequence having proline
at the C-terminal or the second residue is contained or when
two residues on the C-terminal. are different optically active
15 amino acids, for example, D-form and L-form amino acid and the
like, by-production of diketopiperazine is remarkable.
Furthermore, the present inventors have found that, in the
anchor method, different from the liquid phase reaction,
purification of peptide is difficult since the anchor itself,
20 which was eliminated along with the by-production of
diketopiperazine, also precipitates along with the object
product in an isolation step of peptide protected by an anchor
group, and the yield and purity of the object product decrease
since an amino acid sequence derived from the eliminated anchor
25 and a peptide having a different chain length are also byproduced.
[0009]
The present invention has been made in view of the abovementioned
problems specific to the peptide synthesis by
30 conventional anchor methods, and aims to provide an
industrially useful production method of peptide, which
suppresses conventionally problematic by-production of
diketopiperazine, and incorporates a step that can be performed
successively in one pot by omitting isolation and purification
35 operations of an intermediate as much as possible.
Means of Solving the Problems
[0010]
The present inventors have found that, in an elongation
step of a peptide chain having a 9-fluorenylmethyloxycarbonyl
5 group (hereinafter to be also referred to as Fmoc group) as a
temporary protecting group of the N-terminal amino group of an
amino acid or peptide, and an anchor group derived from an
anchor as a protecting group of the C-terminal carboxy group,
the steps up to the condensation reaction with N-Fmoc amino
10 acid or N-Fmoc peptide can be performed successively in one pot
by omitting a set of isolation and purification operations of
concentration, precipitation, filtration, washing and drying
from a temporary protecting group removal step by merely
performing neutralization with an acid after removal of the
15 Fmoc group with a non-nucleophilic organic base. In addition,
they have found that said production method can suppress
elimination of the anchor group by suppressing the byproduction
of diketopiperazine problematic in the peptide
synthetic reaction by the conventional anchor method, which
20 resulted in the completion of the present invention. The
present invention is as follows.
[0011]
[1] A production method of a peptide, comprising the following
step (1);
25 (1) removing N-terminal Fmoc group of N-Fmoc C-protected amino
acid or N-Fmoc 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
30 300, with a non-nucleophilic organic base in a halogenated
solvent or ether solvent to give a C-protected amino acid or Cprotected
peptide, neutralizing with an acid, adding N-Fmoc
amino acid or N-Fmoc peptide, a condensing agent and a
condensation accelerator to the reaction solution after
35 neutralization, and condensing the N-terminal of the Cprotected
amino acid or C-protected peptide with N-Fmoc amino
acid or N-Fmoc peptide to give an N-Fmoc C-protected peptide.
[2] The method of the above-mentioned [1], further comprising
step (2) precipitating N-Fmoc C-protected peptide with a polar
5 solvent, and obtaining same by solid-liquid separation, after
step (1) .
[3] The method of the above-mentioned [2], further comprising
step (3) removing N-terminal Fmoc group and/or C-terminal
anchor group of the N-Fmoc C-protected peptide, after step (2).
10 [4] The method of any one of the above-mentioned [1] - [3],
wherein the anchor is a compound represented by the following
formula (I):
[0012]
Y
0R2)p (')
15 [0013]
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
20 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
25 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
30 by a halogen atom; and
R*^ is a hydrogen atom, or a group represented by the formula
^ (a):
[0014]
(OR^)r
(a)
[0015]
5 wherein * is a binding site;
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
10 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
halogen atom, a Ci_6 alkyl group optionally substituted by a
15 halogen atom, and a Ci-e 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.
[5] The method of the above-mentioned [4], wherein the compound
20 represented by the formula (I) is a compound selected from the
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,
25 4-methoxy-2-[3',4',5'-
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,
30 3,5-dimethoxy-4-[3',4',5'-
tri(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol,
2,4-di(dodecyloxy)benzyl alcohol.
^D 3,4,5-tri(octadecyloxy)benzylamine,
bis (4-docosyloxyphenyl)methanol,
bis(4-docosyloxyphenyl)methylamine, and
2- (12-docosyloxy-dodecyloxy)-9- (3-fluorophenyl)-9-bromofluorene.
5 [6] The method of any one of the above-mentioned [1] - [5],
wherein the non-nucleophilic organic base is selected from the
group consisting of 1,8-diazabicyclo[5.4.0]-7-undecene, 1,4-
diazabicyclo[2.2.2]octane and 1,5-diazabicyclo[4.3.0]-5-nonene.
[7] The method of any one of the above-mentioned [1] - [5],
10 wherein the non-nucleophilic organic base is 1,8-
diazabicyclo[5.4.0]-7-undecene.
[8] The method of any one of the above-mentioned [1] - [7],
wherein the acid is selected from the group consisting of
methanesulfonic acid, trifluoromethanesulfonic acid,
15 benzenesulfonic acid, p-toluenesulfonic acid anhydride,
sulfuric acid and hydrogen chloride/ether solution.
[9] The method of any one of the above-mentioned [1] - [7],
wherein the acid is selected from the group consisting of
methanesulfonic acid, trifluoromethanesulfonic acid, hydrogen
20 chloride/diethyl ether, and hydrogen chloride/cyclopentyl
methyl ether.
[10] The method of any one of the above-mentioned [1] - [9],
wherein the condensation accelerator is 1-hydroxybenzotriazole,
ethyl 1-hydroxy-lH-l,2,3-triazole-5-carboxylate, l-hydroxy-7-
25 azabenzotriazole, 0-(benzotriazol-1-yl)-N,N,N',N'-
tetramethyluronium hexafluorophosphate, or 0-(6-
chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate.
[11] A production method of peptide by improved successive
30 immobilization-protection of C-te2nninal and elongation of Nterminal,
comprising a deprotection step by a heterocyclic
compound having an amidine structure.
[12] The production method of peptide by improved successive
immobilization-protection of C-terminal and elongation of N-
35 terminal of the above-mentioned [11], wherein the protecting
^y group is an Fmoc group.
Effect of the•Invention
[0016]
According to the production method of peptide of the
5 present invention, a step of removing an Fmoc group which is a
temporary protecting group of the N-terminal amino group of an
amino acid or peptide, a neutralization step, and a subsequent
condensation step with N-Fmoc amino acid or N-Etooc peptide can
be successively performed in one pot without isolation and
10 purification operations of an intermediate. Therefore, a
convenient and efficient production method of peptide, which is
suitable for industrial production, can be provided. According
to the present invention, moreover, in an elongation step of a
peptide chain containing a particular sequence conventionally
15 difficult to synthesize in a good yield due to the byproduction
of diketopiperazine, the by-production of an
impurity peptide compound is suppressed and an object peptide
compound can be produced with high purity and high yield.
Description of Embodiments
20 [0017]
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
25 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 present
30 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.
[0018]
35 An amino acid which is a constitutional unit of a peptide
produced by the method of the present invention is a compound
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
5 repeating a dehydration condensation step (condensation step)
of an amino group of an amino acid component and a carboxy
group of other amino acid component, according to the amino
acid sequence of the peptide. Of the two amino acid components
involved in the fojrmation of the peptide bond, a component
10 providing an amino group is hereinafter sometimes referred to
as an amine component, and a component providing a carboxy
group as an acid component.
[0019]
The protecting group of the N-terminal amino group of an
15 acid component to be used for the condensation step in the
present invention is an Fmoc group.
[0020]
In the present specification, the "N-Fmoc amino acid" or
"N-Fmoc peptide" means an amino acid or peptide wherein an N-
20 terminal amino group thereof is protected by an Fmoc group and
a carboxy group is not protected.
[0021]
In the present specification, the "C-protected amino
acid" or "C-protected peptide" means an amino acid or peptide
25 wherein the C-terminal carboxy group thereof is protected by
forming an anchor group by condensing with an anchor soluble in
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
30 compound), and the N-terminal amino group is not protected.
[0022]
In the present specification, the "N-Fmoc C-protected
amino acid" or "N-Fmoc C-protected peptide" means the abovementioned
"C-protected amino acid" or "C-protected peptide"
35 wherein the N-terminal amino acid is protected by an Fmoc group
^u of a temporary protecting group.
[0023]
Examples of the halogenated solvent in the present
invention include halogenated hydrocarbons such as chloroform,
5 dichloromethane, 1,2-dichloroethane, chlorobenzene and the like.
The halogenated solvent may be a mixed solvent of two or more
kinds. Among the halogenated solvents, chloroform and
dichloromethane are particularly preferable.
[0024]
10 Examples of the ether solvent in the present invention
include 1,4-dioxane, cyclopentyl methyl ether (hereinafter
sometimes to be referred to as CPME), tetrahydrofuran
(hereinafter sometimes to be referred to as THE) and the like.
The ether solvent may be a mixed solvent of two or more kinds.
15 Among the ether solvents, CPME and THE are particularly
preferable.
[0025]
Examples of the polar solvent in the present invention
include methanol, ethanol, isopropanol, acetonitrile,
20 propionitrile, dimethylformamide, dimethylacetamide, dimethyl
sulfoxide, water and the like, and a mixed solvent of two or
more kinds of these. Of these, methanol or acetonitrile is
preferably used. As the polar solvent in the present invention,
methanol is preferable particularly from the aspect of
25 practical utility.
[0026]
One embodiment 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
30 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.
[0027]
The formula (I):
35 [0028]
10
^
[0029]
wherein
R''" is a hydrogen atom or, when R'^ is a group represented by the
5 following formula (a), optionally shows a single bond together
with R^ to form a fluorene ring together with ring A and ring
B;
R^ in the number of p is each independently an organic group
having an aliphatic hydrocarbon group;
10 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
halogen atom, a Ci-e alkyl group optionally substituted by a
halogen atom, and a Ci-e alkoxy group optionally substituted by
15 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
(a) :
20 [0030]
* ^
(a)
[0031]
wherein * is a binding site;
r is an integer of 0 to 4;
25 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
11
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
5 halogen atom, and a Ci-g 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.
[0032]
10 The anchor represented by the above-mentioned formula (I)
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.
15 [0033]
In the present specification, as the "alkyl group" for R,
a straight or branched Ci_3o alkyl group can be mentioned. It
is preferably a Ci-io alkyl group, more preferably a Ci-e alkyl
group. Specific preferable examples include methyl, ethyl,
20 propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl and
the like, and methyl and ethyl are particularly preferable.
[0034]
In the present specification, as the "aralkyl group" for
R, a C7-30 aralkyl group can be mentioned. It is preferably a
25 C7-20 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,
naphthylmethyl, 1-naphthylethyl, 1-naphthylpropyl and the like,
and benzyl is particularly preferable.
30 [0035]
As R, a hydrogen atom, a Ci-e alkyl group or a C7-16
aralkyl group is preferable, a hydrogen atom, methyl, ethyl or
benzyl is more preferable, and a hydrogen atom is particularly
preferable.
35 [0036]
12
^ In the present specification, the "halogen atom" is a
fluorine atom, a chlorine atom, a bromine atom or an iodine
atom. In the present specification, as the "halogen atom" for
Y, a chlorine atom, a bromine atom or an iodine atom is
5 preferable, and a bromine atom is more preferable.
[0037]
In the present specification, the "organic group having
an aliphatic hydrocarbon group" for R^ or R'' is a monovalent
organic group having an aliphatic hydrocarbon group in a
10 molecule structure thereof.
[0038]
The "aliphatic hydrocarbon group" in the "organic group
having an aliphatic hydrocarbon group" is a straight or
branched saturated or unsaturated aliphatic hydrocarbon group,
15 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
hydrocarbon group having 5 to 30 carbon atoms, particularly
preferably an aliphatic hydrocarbon group 10 to 30 carbon atoms.
20 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
(monovalent group), or other site (for example, divalent group).
[0039]
25 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
the like, and divalent groups derived therefrom, preferably
monovalent groups such as a methyl group, an ethyl group, a
30 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
lauryl group, a tridecyl group, a myristyl group, a cetyl group,
a stearyl group, an arachyl group, a behenyl group, an oleyl
35 group, an isostearyl group and the like, and divalent groups
13
derived therefrom.
[0040]
The moiety other than the "aliphatic hydrocarbon group"
of the "organic group having an aliphatic hydrocarbon group"
5 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.
Examples of the "hydrocarbon group" include an aliphatic
hydrocarbon group, an aromatic-aliphatic hydrocarbon group, a
10 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
group, a cycloalkyl group, an aryl group, an aralkyl group and
the like, and divalent groups derived therefrom are used. As
15 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
like can be mentioned. As the "alkenyl group", a C2-6 alkenyl
group and the like are preferable and, for example, vinyl, 1-
20 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,
propargyl, 1-propynyl and the like can be mentioned. As the
"cycloalkyl group", a C3-6 cycloalkyl group and the like are
25 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
the like and, for example, phenyl, 1-naphthyl, 2-naphthyl,
biphenylyl, 2-anthryl and the like can be mentioned. Of these,
30 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-
phenylethyl, 2-phenylethyl, 1-phenylpropyl, naphthylmethyl, 1-
naphthylethyl, 1-naphthylpropyl and the like can be mentioned.
35 Of these, a C7-16 aralkyl group (Ce-io aryl-Ci-e alkyl group) is
14
more preferable, and benzyl is particularly preferable. The
"hydrocarbon group" may be substituted by a substituent
selected from a halogen atom (a chlorine atom, a bromine atom,
a fluorine atom, an iodine atom), an alkyl group having 1 to 6
5 carbon atoms and optionally substituted by one or more halogen
atoms, an oxo group and the like.
[0041]
In the "organic group having an aliphatic hydrocarbon
group" constituting the OR^ group or OR^ group in the above-
10 mentioned formula (I), plural "aliphatic hydrocarbon groups"
may be present by branching and the like. When plural
"aliphatic hydrocarbon groups" are present in the "organic
group having an aliphatic hydrocarbon group", they may be the
same or different.
15 [0042]
In the "organic group having an aliphatic hydrocarbon
group" for R^ or R^ in the above-mentioned formula (I), the
lower limit of the total carbon number is preferably 5, more
preferably 10, further preferably 12, still more preferably 14,
20 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
carbon number is preferably 200, more preferably 150, further
preferably 120, still more preferably 100, especially
25 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
represented by the formula (I) in a polar solvent becomes, even
when the peptide chain is a long chain.
30 [0043]
Specific preferable examples of the "OR^" group or "OR^"
group include dodecyloxy, cetyl oxy, octadecyloxy, docosyloxy,
docosyloxy-dodecyloxy, triacontyloxy and the like. The "OR^"
group or "OR^" group is present in a total number of p or r (p
35 is an integer of 1 to 4 and r is an integer of 0 to 4), p is
15
preferably 2 or 3, and r is preferably an integer of 0 to 2.
[0044]
Specific preferable examples of the substituent
optionally present in ring A or ring B in the above-mentioned
5 formula (I) include a Ci-g 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
group optionally substituted by one or more halogens (e.g., a
Ci-6 alkyl group such as methyl, ethyl, propyl, isopropyl, butyl,
10 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
Ci-6 alkoxy group is preferable.
[0045]
15 A 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;
R^ is a hydrogen atom;
20 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
r is an integer of 0 to 2.
[0046]
25 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;
R^, R'', and R''' are each a hydrogen atom;
30 R^ is an aliphatic hydrocarbon group having 5 to 60 carbon
atoms; and
p is an integer of 1 to 3.
[0047]
Another preferable embodiment of the anchor represented
35 by the above-mentioned formula (I) is a compound of the formula
16
^ (I), wherein
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
5 p is 2 or 3.
[0048]
Another preferable embodiment of the anchor represented
by the above-mentioned formula (I) is a compound of the formula
(I), wherein
10 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.
[0049]
15 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;
R^, R'^, and R'"' are each a hydrogen atom;
20 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.
[0050]
Another preferable embodiment of the anchor represented
25 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 a cyclohexylmethyl group having 1 to 3 alkoxy groups
30 having 12 to 30 carbon atoms; and
p is an integer of 1 to 3.
[0051]
Preferable examples of the anchor soluble in halogenated
solvents or ether solvents, insoluble in polar solvents and
35 having a molecular weight of not less than 300 in the present
17
^ invention include the following anchors.
3,4,5-tri(octadecyloxy)benzyl alcohol,
2,4-di(docosyloxy)benzyl alcohol,
4-methoxy-2-[3' ,4',5'-tri(octadecyloxy)benzyloxy]benzyl alcohol,
5 4-methoxy-2-[3',4',5'-
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,
10 3,5-dimethoxy-4-[3',4',5'-
tri(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol,
2,4-di(dodecyloxy)benzyl alcohol,
3,4,5-tri(octadecyloxy)benzylamine,
bis(4-docosyloxyphenyl)methanol,
15 bis(4-docosyloxyphenyl)methylamine, and
2-(12-docosyloxy-dodecyloxy)-9-(3-fluorophenyl)-9-bromofluorene.
[0052]
While the production method of the aforementioned anchor
is not particularly limited, it can be produced from a starting
20 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 compound, for example,
halide corresponding to the group R^ or R'' in the formula (I)
and the like can be obtained as a commercially available
25 product or can be produced by a method known per se or a method
analogous thereto.
[0053]
An amino acid or peptide which is an acid component or an
amine component to be used in the present invention often has,
30 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
carboxy group, a hydroxy group and the like. Such functional
group is distinguished from an amino group and a carboxy group
35 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
not impair the gist of the present invention, it is preferably
protected by an appropriate protecting group to prevent an
5 undesirable side reaction during peptide bond formation by a
dehydration condensation reaction and deprotection of an Nterminal
amino groups.
[0054]
The protecting group of the side chain functional group
10 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
is, the protecting group of the side chain functional group
needs to be maintained until the completion of the desired
15 amino acid sequence, without being removed even under the
removing conditions of an Fmoc group which is the protecting
group of the N-terminal amino group. The protecting group is
not particularly limited as long as the side chain functional
group does not cause an undesirable side reaction during
20 formation of the peptide bond by a dehydration condensation
reaction and deprotection of the N-terminal amino group.
[0055]
The protecting group of the side chain functional group
is not particularly limited as long as it is stable under the
25 deprotection conditions of an Fmoc group which is the
protecting group of the N-terminal amino group (temporary
protecting group). For example, the protecting groups
described in PEPTIDE GOUSEI NO KISO TO JIKKENN (basis and
experiment of peptide synthesis), published by Maruzen Co., Ltd.
30 (1985), PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, the third
edition, published by JOHN WILLY&SONS (1999) and the like can
be mentioned.
[0056]
When the side chain functional group is a carboxy group,
35 an ester-type protecting group, an amide-type protecting group,
19
^
a hydrazide-type protecting group and the like can be mentioned.
[0057]
As the ester-type protecting group, substituted or
unsubstituted alkyl ester, and substituted or unsubstituted
5 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
unsubstituted aralkyl ester, benzyl ester, p-nitrobenzyl ester,
10 p-methoxybenzyl ester, diphenylmethyl ester, 9-fluorenylmethyl
(Fm) ester, 4-picolyl (Pic) ester and the like are preferably
used.
[0058]
As the amide-type protecting group, unsubstituted amide,
15 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.
[0059]
20 As the hydrazide-type protecting group, unsubstituted
hydrazide, N-phenylhydrazide, N,N'-diisopropylhydrazide and the
like are preferably used.
[0060]
Of these, ester-type protecting groups which are stable
25 under the deprotection conditions of an Fmoc group, such as tbutyl
ester, substituted or unsubstituted benzyl ester and the
like are preferably used, and substituted or unsubstituted
benzyl ester is particularly preferably used since synthesis
thereof is comparatively easy.
30 [0061]
When the side chain functional group is an amino group, a
urethane-type protecting group, an acyl-type protecting group,
a sulfonyl-type protecting group and the like can be mentioned.
[0062]
35 As the urethane-type protecting group, for example, a
20
^
methoxycarbonyl group, an ethoxycarbonyl group, a tertbutoxycarbonyl
(Boc) group, a benzyloxycarbonyl (Z) group and
the like are used, and a methoxycarbonyl group, an
ethoxycarbonyl group, a Boc group and the like are preferable.
5 Of these, a Boc group is particularly preferably used since
selective deprotection thereof is possible under mild acidic
conditions.
[0063]
As the acyl-type protecting group, for example, a formyl
10 group, an acetyl group, a trifluoroacetyl group and the like
are preferably used.
[0064]
As the sulfonyl-type protecting group, for example, a ptoluenesulfonyl
(Ts) group, a p-tolylmethanesulfonyl group, a
15 4-methoxy-2,3,6-trimethylbenzenesulfonyl group and the like are
preferably used.
[0065]
As for side chain functional groups other than those
mentioned above, a protecting group stable under the
20 deprotection conditions of an Fmoc group which is the
protecting group (temporary protecting group) of the N-terminal
amino group can be selected and used.
[0066]
The side chain functional group can be deprotected as
25 necessary after forming the object peptide bond.
[0067]
Next, the production method of the present invention is
explained. The production method of the present invention is a
production method of peptide, which characteristically includes
30 the following step (1).
(1) removing N-terminal Fmoc group of N-Fmoc C-protected amino
acid or N-Fmoc 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
35 polar solvents and having a molecular weight of not less than
21
300, with a non-nucleophilic organic base in a halogenated
solvent or ether solvent to give a C-protected amino acid or Cprotected
peptide, neutralizing with an acid, adding N-Fmoc
amino acid or N-Fmoc peptide, a condensing agent and a
5 condensation accelerator to the reaction solution after
neutralization, and condensing the N-terminal of the Cprotected
amino acid or C-protected peptide with N-Fmoc amino
acid or N-Fmoc peptide to give an N-Fmoc C-protected peptide
(N-terminal deprotection step and subsequent condensation step)
10 [0068]
The N-Fmoc C-protected amino acid or N-Fmoc C-protected
peptide to be used in step (1) in the production method of the
present invention can be produced by the following step (a).
In the following, step (a) is first explained before
15 explanation on step (1).
[0069]
Step (a) (C-terminal protection step)
In this step, an anchor soluble in halogenated solvents
or ether solvents, insoluble in polar solvents and having a
20 molecular weight of not less than 300 is condensed with a Cterminal
of N-Fmoc amino acid or N-Fmoc peptide to give N-Ftaoc
C-protected amino acid or N-Fmoc C-protected peptide.
[0070]
While the upper limit of the number of amino acid
25 residues of the N-Ftaoc peptide is not particularly limited as
long as the N-Fmoc 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 N-Fmoc peptide is preferably not more than 100,
more preferably not more than 50, still more preferably not
30 more than 30.
[0071]
The condensation reaction is preferably performed by
dissolving anchor, N-Fmoc amino acid or N-Fmoc peptide and a
catalytic amount of dimethylaminopyridine in a solvent, adding
35 a condensing agent (and a condensation accelerator where
22
^
necessary) and stirring the mixture.
[0072]
This step is performed in a solvent that does not
influence the reaction. The higher the solubility in the
5 solvent becomes, the more superior the reactivity is expected
to be. Therefore, a solvent showing high solubility of the
aforementioned N-Fmoc amino acid or N-Fmoc peptide is
preferably selected. Specifically, halogenated solvents such
as chloroform, dichloromethane, 1,2-dichloroethane and the
10 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
an appropriate ratio. In addition, aromatic hydrocarbons such
as benzene, toluene, xylene and the like; nitriles such as
15 acetonitrile, 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
sulfoxide and the like may be mixed at an appropriate
proportion with the above-mentioned halogenated solvent and
20 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
or tetrahydrofuran is preferable, and chloroform is
particularly preferable.
25 [0073]
While the concentration of the N-Fmoc amino acid or NFmoc
peptide in a solution in this step is not particularly
limited as long as it is dissolved, it is preferably 1 - 3 0 wt%.
[0074]
30 The amount of the N-Fmoc amino acid or N-Fmoc peptide to
be used in this step can be 1 - 10 mol, preferably 1 - 5 mol,
per 1 mol of the aforementioned anchor.
[0075]
When Y is a hydroxy group, an ester bond is formed by
35 adding a condensing agent and, where necessary, a condensation
23
accelerator in a solvent that does not influence the reaction
in the presence of a dimethylaitiinopyridine catalyst.
[0076]
When Y is an -NHR group, an amide bond is fo2nned by
5 adding a condensing agent in the presence of a condensation
accelerator.
[0077]
When Y is a halogen atom, an ester bond -is formed by
adding a base such as diisopropylethylamine and the like in a
10 solvent that does not influence the reaction.
[0078]
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)-
15 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.
[0079]
20 The amount of the condensation accelerator to be used is
preferably 0.05 - 1.5 mol per 1 mol of the aforementioned
anchor.
[0080]
As a condensing agent, dicyclohexylcarbodiimide (DCC),
25 diisopropylcarbodiimide (DIG), 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-
30 [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.
[0081]
35 The amount of the condensing agent to be used is, for
24
^
example, 1 - 1 0 mol, preferably 1 - 5 mol, per 1 mol of the
aforementioned anchor.
[0082]
While the reaction temperature is not particularly
5 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 - 70 hr.
[0083]
10 The N-Fmoc C-protected amino acid or N-Fmoc C-protected
peptide contained in the thus-obtained reaction solution can be
isolated by concentrating the reaction solvent under reduced
pressure, adding the aforementioned polar solvent to allow for
precipitation, solid-liquid separation (filtration) of the
15 precipitate, and washing same with acetonitrile. Examples of
the polar solvent to be used in this step include methanol,
acetonitrile and the like, preferably methanol.
[0084]
Step (1) (N-terminal deprotection step and subsequent
20 condensation step)
In this step, an N-terminal Fmoc group of N-Fmoc Cprotected
amino acid or N-Fmoc C-protected peptide is removed
by a treatment with a non-nucleophilic organic base to give a
C-protected amino acid or C-protected peptide, which is then
25 neutralized and, without an isolation and purification
operation, C-protected amino acid or C-protected peptide and NFmoc
amino acid or N-Fmoc peptide are subjected to dehydrating
condensation.
[0085]
30 While the upper limit of the number of the amino acid
residues of the N-Fmoc C-protected peptide is not particularly
limited as long as the N-Fmoc C-protected peptide to be used in
this step is soluble in a solvent to be used in this step, the
number of the amino acid residues of the N-Fmoc C-protected
35 peptide is preferably not more than 100, more preferably not
25
^
more than 50, further preferably not more than 30. While the
upper limit of the number of amino acid residues of the Cprotected
peptide is not particularly limited as long as the Cprotected
peptide to be used in this step is soluble in a
5 solvent to be used in this step, the number of amino acid
residues of C-protected peptide is preferably not more than 100,
more preferably not more than 50, still more preferably not
more than 30.
[0086]
10 In the following, step (1) is explained by dividing into
step (1-1) (N-terminal deprotection step) and step (1-2)
(condensation step).
[0087]
Step (1-1) (N-terminal deprotection step)
15 The Fmoc group is removed (deprotected) by treating same
with a non-nucleophilic organic base in a halogenated solvent
or ether solvent. The deprotection is performed in a solvent
that does not influence the reaction.
[0088]
20 As the non-nucleophilic base, 1,8-diazabicyclo[5.4.0]-7-
undecene (DBU), 1,4-diazabicyclo[2.2.2]octane (DABCO), and 1,5-
diazabicyclo[4.3.0]-5-nonene (DBN) and the like can be
mentioned, DBU and DBN are preferable, and DBU is more
preferable.
25 [0089]
The halogenated solvent' or ether solvents may be a mixed
solvent of two or more kinds thereof. The halogenated solvent
or ether solvent is preferably chloroform, dichloromethane, THF
or CPME.
30 [0090]
In this deprotection step, when nucleophilic organic base
(secondary amine) such as dimethylamine, diethylamine,
piperidine, morpholine and the like, which are widely used for
removal of Fmoc group, are used in a nonpolar organic solvent
35 such as chloroform and the like, the organic base in large
26
excess is necessary to complete the reaction. When a
nucleophilic base is used, a side reaction wherein an acid
component is amidated occurs in the next condensation step.
When the aforementioned non-nucleophilic base is used in this
5 step, deprotection reaction is completed both in a halogenated
solvent and an ether solvent. The amount of the nonnucleophilic
base to be used is preferably not less than 0.8
equivalent, more preferably not less than 1 equivalent,
preferably not more than 5 equivalents, more preferably not
10 more than 3 equivalents, relative to the reactive substrate (NFmoc
C-protected amino acid or N-Fmoc C-protected peptide).
[0091]
To achieve one pot in this step, an organic base that
exerts an adverse influence (side reaction such as deprotection
15 of N-terminal of N-Fmoc amino acid or N-Fmoc peptide itself,
which is a newly added acid component, and the like) on the
next condensation reaction of C-protected amino acid or Cprotected
peptide and N-Fmoc amino acid or N-Fmoc peptide needs
to be removed. In this step, therefore, it is essential to
20 incorporate a neutralization step for addition of an acid to
the reaction solution after the deprotection step.
[0092]
Examples of the acid to be used for the neutralization
step include methanesulfonic acid, trifluoromethanesulfonic
25 acid, benzenesulfonic acid, p-toluenesulfonic acid anhydride,
sulfuric acid, hydrogen chloride/ether solution and the like.
Of these, methanesulfonic acid, trifluoromethanesulfonic acid,
hydrogen chloride/diethyl ether and hydrogen
chloride/cyclopentyl methyl ether are preferable.
30 [0093]
The amount of the acid to be used is preferably not less
than 0.5 mol, more preferably not less than 0.9 mol, preferably
not more than 1.1 mol, more preferably not more than 1.0 mol,
per 1 mol of the non-nucleophilic organic base.
35 [0094]
27
While the reaction temperature of step (1-1) is not
particularly lim.ited 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
5 than 30°C. The reaction time of step (1-1) is, for example, 1
- 70 hr.
[0095]
Step (1-2) (condensation step)
In this step, N-Fmoc amino acid or N-Fmoc peptide, a
10 condensing agent and a condensation accelerator are directly
added to the reaction solution after the neutralization step in
step (1-1) to perform condensation of the N-terminal of the Cprotected
amino acid or C-protected peptide with N-Fmoc amino
acid or N-E^oc peptide to give N-Fmoc C-protected peptide.
15 [0096]
This step is performed using the condensing agent,
condensation accelerator and the like described in the
aforementioned step (a) and under the peptide synthesis
conditions generally used in the field of peptide chemistry. A
20 particularly preferable combination of a condensing agent and a
condensation accelerator in this step is that of N-ethyl-N'-3-
dimethylaminopropylcarbodiimide hydrochloride (EDC HCl) and 1-
hydroxybenzotriazole (HOBt).
[0097]
25 The amount of the condensation accelerator to be used is
preferably not less than 0.05 mol, more preferably not less
than 0.9 mol, preferably not more than 1.5 mol, more preferably
not more than 1.1 mol, per 1 mol of the aforementioned Cprotected
amino acid or C-protected peptide.
30 [0098]
The production method of the present invention may
further contain step (2) (precipitation step) wherein N-Fmoc Cprotected
peptide is precipitated in a polar solvent, and
obtained by solid-liquid separation, after step (1). Step (2)
35 is explained in the following.
28
[0099]
Step (2) (precipitation step)
In this step, the condensate (N-Fmoc C-protected peptide)
obtained in the above-mentioned step (1) is isolated by
5 changing the solvent, in which the condensate is dissolved
(e.g., change of solvent composition, change of solvent kind),
to allow for precipitation. That is, the reaction is performed
under the conditions allowing dissolution of the condensate,
the solvent is exchanged to cause precipitation of the
10 condensate, followed by solid-liquid separation, and slurry
washing to remove impurity. Evaporation of the solvent and the
like may be performed before solvent exchange. As the solvent
for exchange, a polar solvent such as methanol, acetonitrile
and the like is used. That is, the reaction is performed under
15 the conditions allowing dissolution of the compound and, after
the reaction, as the solvent for exchange, for example, a
halogenated solvent and the like are used for dissolution and a
polar solvent such as methanol, acetonitrile and the like are
used for precipitation.
20 [0100]
The above-mentioned steps (1) and (2) may be repeated two
times or more to obtain an N-Fmoc C-protected peptide having an
elongated peptide chain. While the upper limit of the number
of amino acid residues of the N-Fmoc C-protected peptide having
25 an elongated peptide chain is not particularly limited as long
as the N-Fmoc C-protected peptide is soluble in the solvent to
be used in step (1), 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.
30 [0101]
The production method of the peptide of the present
invention can further contain step (3) for removing the Nterminal
Fmoc group and/or the C-terminal anchor group of the
N-Fmoc C-protected peptide after the precipitation step (2).
35 The N-terminal Fmoc group is removed according to, for example,
29
^
the above-mentioned step (1).
[0102]
The C-terminal anchor group is removed after the abovementioned
precipitation step (2) and after removal of the N-
5 terminal Fmoc group, or before removal of the Fmoc group. As a
result, 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
10 group) can be obtained.
[0103]
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
15 performed by an acid treatment. As an acid to be used for the
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
20 deprotection, for example, chloroform, dichloromethane, 1,2-
dichloroethane or a mixed solvent thereof and the like can be
mentioned. The concentration of an acid to be used for the
deprotection is, for example, 0.lw/v% - 5w/v%.
[0104]
25 It is also possible to remove an anchor group derived
from an aromatic compound (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
30 used in the field, particularly peptide synthesis, is used, and
a method including 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
35 particularly preferable. The amount of the acid to be used is
30
appropriately set according to the kind 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
5 than 100 mol, more preferably not more than 50 mol, per 1 mol
of the N-Fmoc C-protected peptide. Along with such use,
trifluoromethanesulfonic acid, trimethylsilyl
trifluoromethanesulfonate, BFa-etherate and the like can also
be added as a further source of strong acid.
10 [0105]
While the reaction temperature is not particularly
limited as long as the reaction proceeds, for example, it is
preferably 0°C - 50°C, more preferably 0°C - 30°C. The reaction
time is, for example, 0.5 - 24 hr.
15 [0106]
For confirmation of the progress of the reaction in the
above-mentioned step (a), step (1) and step (3), a method
similar to general liquid phase organic synthetic reaction can
be applied. That is, thin layer silica gel chromatography,
20 high performance liquid chromatography and the like can be used
to trace the reaction.
[Examples]
[0107]
The present invention is explained in more detail in the
25 following by referring to Examples, which are not to be
construed as limiting the scope 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
30 the like are indicated by abbreviations, each indication is
based on the abbreviation of the lUPAC-IUB Commission on
Biochemical Nomenclature or conventional abbreviations in the
art.
[0108]
35 The anchor used in the Example can be produced by a
31
^
method known per se (see the aforementioned 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
5 product).
[0109]
Reference Example 1: synthesis of 4-methoxy-2-[3', 4', 5'-
tri(octadecyloxy)benzyloxy]benzyl alcohol
(i) 3,4,5-Tri(octadecyloxy)benzyl alcohol (83.0 g, 90.8 mmol)
10 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
3,4,5-tri(octadecyloxy)benzyl chloride as wet crystals (93.6 g).
15 [0110]
(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
(830 ml), and the mixture was stirred at 80°C overnight. The
20 reaction solution 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
evaporated, and the residue was crystallized from methanol (800
25 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%) .
[0111]
(iii) 4-Methoxy-2-[3',4',5'-
30 tri(octadecyloxy)benzyloxy]benzaldehyde (93.5 g, 8 9.2 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
(100 ml) was added at 0°C to quench the reaction. About half
35 the solvent was evaporated, the residue was dissolved in
32
^
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
(1200 ml). The solvent was evaporated, and the residue was
5 crystallized from methanol (900 ml), and washed with
acetonitrile to give 4-methoxy-2-[3',4',5'-
tri(octadecyloxy)benzyloxy]benzyl alcohol (92.4 g, 88.0 mmol,
yield 97% (vs 3,4,5-tri(octadecyloxy)benzyl alcohol)).
^H-NMR (300MHz, CDCl3):5 0 . 88 (9H, t, J=6. 3Hz,Ci7H34-Me) , 1.15-
10 1.40(84H,br,C3',4',5'-OC3H6-Ci4H28-CH3), 1.40-
1 . 5 5 ( 6 H , b r , C 3 ' , 4 ' , 5'-OC2H4-CH2-C15H31) , 1. 7 0 - 1 . 85 (6H,m, C 3 ' , 4 ' , 5 ' -
OCH2-CH2-C16H33) , 2 . 1 8 ( l H , t , J=6.3Hz,OH) , 3 . 79 (3H, s , C4-0Me) , 3 . 9 0 -
4 . 0 3 ( 6 H , m , C 3 ' , 4 ' , 5'-O-CH2-C17H35) , 4 . 65 (2H,d, J = 6 . 6Hz, Ar-CHg-OH) ,
4.97(2H,s,Ar-0-CH2-Ar) , 6. 4 7 ( I H , d d , J = 2 . 1 , 8 . l H z , C 5 - H ) ,
15 6 . 5 3 ( l H , d , J = 2 . 4 H z , C 3 - H ) , 6 . 6 0 ( 2 H , s , C 2 ' , 6 ' - H ),
7 . 1 9 ( I H , d , J = 8 . i H z , C6-H)
[0112]
Example 1: Synthesis of dipeptide (Fmoc-Tyr(t-Bu)-Leu-
OBzl (3,4,5-OCi8H37) ) from Fmoc-Leu-OBzl (3, 4, 5-OC18H37) wherein
20 deprotection step (removal step of Fmoc group), neutralization
step and dehydrating condensation step with Fmoc-Tyr(t-Bu)-OH
are successively performed in one pot
3,4,5-tri(Octadecyloxy)benzyl alcohol (hereinafter
sometimes indicated as Bzl (3,4,5-OC18H37)-OH) (3.0 g, 3.28
25 mmol) and Fmoc-Leu-OH (1.3 equivalents) were dissolved in
chloroform, EDC HCl (1.4 equivalents) and dimethylaminopyridine
(0.1 equivalent) were added under ice-cooling and the mixture
was stirred. After completion of the reaction, the solvent was
evaporated, MeOH (45 ml) was added and the mixture was stirred.
30 The precipitate was collected by filtration, washed with
acetonitrile and dried.
The obtained Fmoc-Leu-OBzl (3,4,5-OC18H37) was dissolved
in chloroform, DBU (1.0 equivalent) was added under ice-cooling
and the mixture was stirred. After completion of the
35 deprotection, HCl/CPME solution (0.95 equivalent relative to
33
^
DBU) was added, HOBt (1.0 equivalent) and Fmoc-Tyr(tBu)-OH (1.1
equivalents) were added, EDCHCl (1.2 equivalents) was added
and the mixture was stirred. After completion of the
condensation reaction, the solvent was evaporated, MeOH (45 ml)
5 was added and the mixture was stirred. The precipitate was
collected by filtration, washed with acetonitrile, and dried
under reduced pressure to give Fmoc-Tyr(tBu)-Leu-OBzl (3,4,5-
OC18H37) (4.04 g, 3.19 mmol, yield 97%).
TOF-MS(+)=1468.1
10 [0113]
The measurement device and conditions thereof used in
Example 1 are as described below.
Measurement device: LCT Premier XE manufactured by Waters
Capillary voltage: 3000V
15 Sample cone voltage: 8 6V
Dissolution temperature: 350°C
Source part temperature: 120°C
LC part
Solvent: aqueous trifluoroacetic acid solution (trifluoroacetic
20 acid concentration: 0.05v/v%) - mixed solvent of MeCN and THE
(MeCN concentration: 0.05v/v%)
Column: AQUITY BEH C18 1.7 ym 2.1x50 mm
Temperature: 4 0°C
Flow rate: 0.7 ml/min
25 Wavelength: 220 nm
Injection volume: 2 ]ii
[0114]
Example 2: Synthesis of tripeptide (Fmoc-Ala-Pro-Gly-OBzl
(3,4,5-OCi8H37) ) from Fmoc-Pro-Gly-OBzl (3, 4, 5-OC18H37) wherein
30 deprotection step (removal step of Fmoc group), neutralization
step and dehydrating condensation step with Fmoc-Ala-OH are
successively performed in one pot
Fmoc-Pro-Gly-OBzl (3,4,5-OC18H37) (400 mg) obtained in the
same manner as in Example 1 was dissolved in chloroform (5 ml) ,
35 DBU (1.0 equivalent) was added under ice-cooling and the
34
^
mixture was stirred. After completion of the deprotection,
HCl/CPME solution (0.97 equivalent relative to DBU) was added,
HOBt (1.0 equivalent) and Fmoc-Ala-OH (1.1 equivalents) and EDC
HCl (1.2 equivalents) were added and the mixture was stirred.
5 After completion of the condensation reaction, the solvent was
evaporated, MeOH (5 ml) was added and the mixture was stirred.
The precipitate was collected by filtration, washed with
acetonitrile, and dried under reduced pressure to give Fmoc-
Ala-Pro-Gly-OBzl (3,4,5-OC18H37) (415 mg) . The content of Bzl
10 (3,4,5-OC18H37)-OH in the obtained solid was measured under the
same conditions as in Example 1 to find 0.9 mol% of the object
product.
TOF-MS(+)=1361.5
[0115]
15 Example 3: Synthesis of tripeptide (Fmoc-Gly-Cys(Trt)-Tyr(t-
Bu)-0-anchor group) from Fmoc-Cys(Trt)-Tyr(t-Bu)-0-anchor group
wherein deprotection step (removal step of Emoc group),
neutralization step and dehydrating condensation step with
Fmoc-Gly-OH are successively performed in one pot
20 Fmoc-Cys(Trt)-Tyr(tBu)-OBzl(2-TOB-4-OMe) (9.0 g (4.90
mmol)) obtained in the same manner as in Example 1 except that
4-methoxy-2-[3',4',5'-tri(octadecyloxy)benzyloxy]benzyl alcohol
(hereinafter sometimes to be indicated as HO-Bzl(2-TOB-4-OMe))
was used as a starting material was dissolved in chloroform (90
25 ml), DBU (870 mg, 1.2 equivalents) was added under ice-cooling
and the mixture was stirred. After completion of the
deprotection, methanesulfonic acid (522 mg, 0.95 equivalent
relative to DBU). was added, HOBt (640 mg, 1.0 equivalent),
Fmoc-Gly-OH (1.56 g, 1.1 equivalents), and EDC HCl (1.11 g, 1.2
30 equivalents) were added and the mixture was stirred. After
completion of the condensation reaction, the solvent was
evaporated, MeOH (90 ml) was added and the mixture was stirred.
The precipitate was collected by filtration, washed with
acetonitrile, and dried under reduced pressure to give Fmoc-
35 Gly-Cys(Trt)-Tyr(tBu)-OBzl(2-TOB-4-OMe) (9.04 g, 4.77 mmol,
• 35
^
yield 99%). The content of the decomposed Bzl(2-TOB-4-OMe)-OH
in the obtained solid was measured under the same conditions as
in Example 1 to find 1 mol% of the object product.
TOF-MS(+)=1893.2
5 [0116]
Example 4: Synthesis of tripeptide(Fmoc-Ala-Pro-Gly-0-anchor
group) from Fmoc-Gly-0-anchor group wherein deprotect'ion step
(removal step of Fmoc group), neutralization step and
dehydrating condensation step with Fmoc-Pro-OH are successively
10 performed in one pot, and further, peptide chain elongation
step using Fmoc-Ala-OH is also performed in one pot
2-(12-Docosyloxy-dodecyloxy)-9-(3-fluorophenyl)-9-
bromofluorene (1.0 g) and Fmoc-Gly-OH (2 equivalents) were
dissolved in chloroform, diisopropylethylamine (2 equivalents)
15 was added and the mixture was stirred at 60°C. After
completion of the reaction, the solvent was evaporated,
methanol (5 ml) was added and the mixture was stirred. The
precipitate was collected by filtration, washed with
acetonitrile, and dried under reduced pressure. The solid was
20 dissolved in chloroform, and DBU (1.5 equivalents) was added
under ice-cooling to remove Fmoc. After completion of the
reaction, HCl/CPME solution (0.95 equivalent relative to DBU)
was added, HOBt (1.0 equivalent) and Fmoc-Pro-OH (1.1
equivalents) were added, EDC HCl (1.2 equivalents) was added,
25 and the mixture was stirred. After completion of the
condensation reaction, the solvent was evaporated, methanol (5
ml) was added and the mixture was stirred. The precipitate was
collected by filtration, washed with acetonitrile, and dried
under reduced pressure to give Fmoc-Pro-Gly-0-anchor group
30 (1.28 g) . The solid (1 g) was further subjected to the removal
of Fmoc and subsequent condensation reaction by operations
similar to those in the above to give Fmoc-Ala-Pro-GlyO-anchor
group (1.04 g) . The content of the decomposed 2-(12-
docosyloxy-dodecyloxy)-9-(3-fluorophenyl)-9-fluorenol in the
35 obtained solid was measured under the samie conditions as in
36
^
Example 1 to find 0.2 area % of the object product.
TOF-MS(+)=1232.5
[0117]
Experimental Example 1: Influence of various organic bases and
5 solvents on by-production of diketopiperazine in deprotection
reaction of Fmoc-Tyr (tBu)-Leu-OBzl (3, 4, 5-OC18H37) (removal of
Fmoc group)
[0118]
Experimental method
10 The dipeptide (Fmoc-Tyr (tBu)-Leu-OBzl (3, 4, 5-OC18H37) )
obtained in Example 1 was dissolved in chloroform or CPME,
subjected to deprotection reaction using various bases for 2.5
hr, and the content of 3,4,5-tri(octadecyloxy)benzyl alcohol
formed as a result of decomposition in conjunction with the by-
15 production of diketopiperazine was measured by HPLC as a ratio
relative to the yield of the object product.
[0119]
Experimental results
When non-nucleophilic DBU was used as an organic base,
20 elimination of the anchor group was scarcely found in both an
ether solvent THE and a halogenated solvent chloroform, and byproduction
of diketopiperazine could be suppressed. On the
other hand, when nucleophilic diethylamine or piperidine was
used, the amount of the eliminated anchor group increased
25 remarkably, which indicates by-production of a large amount of
diketopiperazine.
[0120]
Table 1
Solvent
THE
THE
THE
chloroform
Organic base for
deprotection
diethylamine (15
equivalents)/DBU (1
equivalent)
piperidine (15 equivalents)
DBU (1 equivalent)
DBU (1 equivalent)
2.5 hr later
(mol%)
18
80
0.5
0.2
37
^
[0121]
Experimental Example 2: Comparison of one pot synthetic method
of the present invention (method A: Example 2) and conventional
sequential synthetic method (method B, method C) for by-
5 production of diketopiperazine in Fmoc-Ala-Pro-Gly-OBzl (3,4,5-
OC18H37) synthetic reaction
[0122]
Experimental method
The by-production rate of diketopiperazine when
10 tripeptide (Fmoc-Ala-Pro-Gly-OBzl (3,4,5-OC18H37) ) was produced
by the following methods B, C was calculated from the
measurement of the amount of the eliminated anchor, and
compared.
Method B: Under the same conditions as in method A, the N-
15 terminal Fmoc group of Ftaoc-Pro-Gly-0-anchor group was removed
using DBU (1 equivalent)/diethylamine (15 equivalents). After
completion of the reaction, the mixture was neutralized with
0.95 equivalent of hydrogen chloride/CPME relative to DBU,
concentrated and precipitated. Thereafter, the precipitate was
20 dissolved and subjected to a condensation step with Fmoc-Ala to
isolate tripeptide.
Method C: Under the same conditions as in method A, the Nterminal
Fmoc group of Fmoc-Pro-Gly-0-anchor group was removed
using diethylamine (50 equivalents). After completion of the
25 reaction, and the mixture was subjected to concentration and
precipitation. Thereafter, the precipitate was dissolved and
subjected to a condensation step with Fmoc-Ala to isolate
tripeptide.
[0123]
30 Experimental results
As compared to method A using DBU for removal of Ftaoc
group, method C using diethylamine required an excess amount of
a base to complete the reaction in a halogenated solvent. Not
only in the above-mentioned method C but also in method B using
35 diethylamine to trap dibenzofulvene, elimination of the anchor
38
^
group caused by the by-production of diketopiperazine could not
be suppressed as shown in Table 2, even though the
concentration and precipitation step was performed after
removal of the Fmoc group.
5 [0124]
Table 2
Production method of
tripeptide
Method A
Method B
Method C
Content of eliminated
anchor in tripeptide (mol%)
0.9
19
38
[0125]
As shown above, it was found that, when diethylamine or
10 piperidine, which is a nucleophilic organic base widely used as
a deprotecting agent for Fmoc group, is used, the deprotection
speed is slow in halogenated solvents or ether solvents as
compared to DBU, which is a non-nucleophilic organic base, even
when an excess amount of the organic base is used, and a side
15 reaction of a reaction with an acid component to form amide
occurs in the subsequent condensation step, thus decreasing the
yield of the object product. Furthermore, when diethylamine or
piperidine is used, the by-production of diketopiperazine
during the deprotection cannot be suppressed. These results
20 have clarified that use of about 0.8-5 equivalents of a nonnucleophilic
and highly basic organic base such as DBU and the
like is suitable for removing the Fmoc group, and further
incorporation of a neutralization step enables steps up to the
subsequent condensation step to be performed successively in
25 one pot and suppression of the by-production of
diketopiperazine.
[INDUSTRIAL APPLICABILITY]
[0126]
According to the production method of peptide of the
30 present invention, a step of removing an Fmoc group which is a
39
^fh ^H^^
temporary protecting group of the N-terminal amino group of an
amino acid or peptide, a neutralization step, and a subsequent
condensation step with N-E^oc amino acid or N-Fmoc peptide can
be successively performed in one pot without isolation and
5 purification operations during the process. Therefore, a
convenient and efficient production method of peptide, which is
suitable for industrial production, can be provided. According
to the present invention, moreover, since by-production of
diketopiperazine can be suppressed, an object peptide compound
10 can be produced with good purity for any sequence.
[0127]
A differently expression method showing the
characteristics of the production method of peptide of the
present invention is described below.
15 An "immobilizing-protecting group" is defined as a
"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
20 deprotected in a (deprotection step)".
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
25 repeating a step of N-terminal deprotection step of "N-terminal
protected amino acid or peptide having immobilized and
protected C-terminal (deprotection step) and a step of
condensing with a novel N-terminal protected amino acid or
peptide (condensation step)", Fmoc method and Boc method are
30 included therein, and the Fmoc method is preferable.
[0128]
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
35 and elongating N-terminal characterized by successively
40
performing a (deprotection step) and a (condensation 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 (condensation
5 step)", the production method of peptide of the present
invention can also be expressed as follows.
[1] A production method of peptide by improved successive
immobilization-protection of C-terminal and elongation of Nterminal,
comprising a deprotection step by a heterocyclic
10 compound having an amidine structure.
[2] The production method of peptide of [1], wherein the
heterocyclic compound having the amidine structure is at least
one kind selected from the group consisting of 1,8-
diazabicyclo[5.4.0]-7-undecene, 1,4-diazabicyclo[2.2.2]octane,
15 1,5-diazabicyclo[4.3.0]-5-nonene.
[3] The production method of peptide of [1] or [2],
further comprising a neutralization step with an acid after the
deprotection step.
[4] The production method of peptide of [3], comprising a
20 precipitation step after the condensation step.
[5] The production method of peptide of any one of [1] -
[4], wherein the protecting group is an Fmoc group.
[6] The production method of peptide of any one of [1] -
[4], comprising a final deprotection step.
25 [0129]
The production method of peptide of the present invention
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
30 peptide with immobilized and protected C-terminal by reacting
C-terminal of an N-terminal protected amino acid or peptide
with an immobilizing-protecting group,
(the second step: deprotection step)
A step of producing amino acid or peptide With
35 immobilized and protected C-terminal by removing the N-terminal
41
^ protecting group of the N-terminal protected amino acid or
peptide with immobilized and protected C-terminal.
(the third step: condensation step)
A step of producing elongated N-terminal protected
5 peptide with immobilized and protected C-terminal by condensing
amino acid or peptide with immobilized and protected C-terminal
and a novel N-terminal protected amino acid or peptide,
(the fourth step: precipitation step)
A step of purifying the elongated N-terminal protected
10 peptide with immobilized and protected C-terrtiirial by
precipitating the elongated N-terminal protected peptide with
immobilized and protected C-terminal in a polar solvent,
(the fifth step: final deprotection step)
A step of producing an elongated peptide by removing the
15 immobilized-protected C-terminal from the elongated N-terminal
protected peptide with immobilized and protected C-terminal or
elongated peptide with immobilized and protected C-terminal.
[0130]
Since the above-mentioned second step, third step and
20 fourth step are repeated as one set, the production method of
peptide of the present invention specifically includes two
cases as follows, m is a natural number, and "(...) x m times"
means that the step in the parenthesis is repeated m times.
(Case 1) the first step -^ (the second step —> the third step ->
25 the fourth step) x m times —>• the fifth step
(Case 2) the first step ->• (the second step -^ the third step ->
the fourth step) x m times —> the second step —> the fifth step
[0131]
This application is based on a patent application No.
30 2011-122798 filed in Japan, the contents of which are
incorporated in full herein.
42

W CLAIMS AMENDED UNDER PCT ARTICLE 19
1. A production method of a peptide, comprising the following
step (1);
5 (1) removing N-terminal Fmoc group of N-E^oc C-protected amino
acid or N-Fmoc 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
10 300, with a non-nucleophilic organic base in a halogenated
solvent or ether solvent to give a C-protected amino acid or Cprotected
peptide, neutralizing with an acid, adding N-Fmoc
amino acid or N-Fmoc peptide, a condensing agent and a
condensation accelerator to the reaction solution after
15 neutralization, and condensing the N-terminal of the Cprotected
amino acid or C-protected peptide with N-Fmoc amino
acid or N-Fmoc peptide to give an N-Fmoc C-protected peptide.
2. The method according to claim 1, further comprising step (2)
20 precipitating N-Fmoc C-protected peptide with a polar solvent,
and obtaining same by solid-liquid separation, after step (1).
3. The method according to claim 2, further comprising step (3)
removing N-terminal Fmoc group and/or C-terminal anchor group
25 of the N-Fmoc C-protected peptide, after step (2).
4. The method according to any one of claims 1 - 3 , wherein the
anchor is a compound represented by the following formula (I):
Y
R^-l—R''
0R2)p (')
30 wherein
R'"' is a hydrogen atom or, when R'^ is a group represented by the
H'i
im
* ' l - *• *^ 1?
following formula (a), optionally shows a single bond together -
with R to form a fluorene ring together with ring A and ring
B; f) R^ in the number of p is each independently an organic group
5 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
halogen atom, a Ci-6 alkyl group optionally substituted by a
10 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
15 (a) :
iOR%
(a)
wherein * is a binding site;
r is an integer of 0 to 4;
R'' in the number of r is each independently an organic group
20 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
and ring B; and
ring B optionally further has, in addition to OR* in the number
25 of r, a substituent selected from the group consisting of a
halogen atom, a Ci_6 alkyl group optionally substituted by a
halogen atom, and a Ci-g alkoxy group optionally substituted by
a halogen atom; and
Y is a hydroxy group, NHR (R is a hydrogen atom, an alkyl group
30 or an aralkyl group) or a halogen atom.
5. The method according to claim 4, wherein the compound
M4
A represented by the formula (I) is a compound.selected from the
group consisting of ^^
3, 4, 5-tri (octadecyloxy) benzyl alcohol, •-•'*
2,4-di(docosyloxy)benzyl alcohol,
5 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' -
tri(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol,
10 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,
3,4,5-tri (octadecyloxy)benzylamine,
15 bis(4-docosyloxyphenyl)methanol,
bis(4-docosyloxyphenyl)methylamine, and
2-(12-docosyloxy-dodecyloxy)-9-(3-fluorophenyl)-9-
bromofluorene.
20 6. The method according to any one of claims 1- 5, wherein the
non-nucleophilic organic base is selected from the group
consisting of 1,8-diazabicyclo[5.4.0]-7-undecene, 1,4-
diazabicyclo[2.2.2]octane and 1,5-diazabicyclo[4.3.0]-5-nonene.
25 7. The method according to any one of claims 1 - 5 , wherein the
non-nucleophilic organic base is 1,8-diazabicyclo[5.4.0]-7-
undecene.
8. The method according to any one of claims 1 - 7 , wherein the
30 acid is selected from the group consisting of methanesulfonic
acid, trifluoromethanesulfonic acid, benzenesulfonic acid, ptoluenesulfonic
acid anhydride, sulfuric acid and hydrogen
chloride/ether solution.
35 9. The method according to any one of claims 1 - 7 , wherein the
MS
n r . •-..''' •" -% -f i r'^ '<• -^ .• acid is selected from the group consisting of methanesulfonic
acid, trifluoromethanesulfonic acid, hydrogen chloride/diethyl
ether, and hydrogen chloride/cyclopentyl methyl ether.
5 10. The method according to any one of claims 1 - 9 , wherein
the condensation accelerator is 1-hydroxybenzotriazole, ethyl
l-hydroxy-lH-1,2,3-triazole-5-carboxylate, l-hydroxy-7-
azabenzotriazole, 0-(benzotriazol-1-yl)-N,N,N',N'-
tetramethyluronium hexafluorophosphate, or 0-(6-
10 chlorobenzotriazol-1-yl) -N,N,N' ,N' -tetramethyluroniiom
hexafluorophosphate.
11. A production method of peptide, comprising a step
of deprotecting an N-terminal of an amino acid or peptide
15 having protected N-terminal and protected C-terminal by a
heterocyclic compound having an amidine structure, a step of
neutralizing same with an acid, and a step of condensing the
amino acid or peptide having the N-terminal deprotected in the
deprotection step with an N-protected amino acid or N-protected
20 peptide.
12. The production method of peptide according to
claim 11, wherein the protecting group removed in the
deprotection step is an Fmoc group.