DESCRIPTION
Title of the Invention: METHOD FOR REMOVING Fmoc GROUP
Technical Field
[ OOOl]
5 The present invention relates to a method of removing an
F'moc (9-fluorenylmethoxycarbonyl) group, and a production
method of a peptide by using said method.
Background Art
[0002]
10 Frnoc group is an important protecting group of amino acid
and an amino group of a peptide in the peptide synthesis. As a
result of this removal of the Fmoc group, dibenzofulvene (DBF)
or DBF derivative is produced as a byproduct. Fmoc group is
generally removed by using a base. For example, when Fmoc
1s group is removed by using an amine, an adduct of DBF and amine
(hereinafter sometimes to be abbreviated as a "DBF-amine
adduct") is by-produced as a DBF derivative. When the peptide
synthesis is continued while DBF or DBF derivative still
remains, a side reaction such as 9-fluorenylmethylation occurs.
20 Thus, it is desirable to remove DBF or DBF derivative
efficiently. In addition, when amine is used to remove Fmoc
group and DBF in the peptide synthesis, the amine needs to be
removed before the next condensation step.
[0003]
25 Non-patent document 1 describes a method for removal of a
DBF-amine adduct in the liquid phase peptide synthesis, which
includes adding a hydrocarbon solvent such as hexane and the
like for trituration of a residue obtained by concentrating a
reaction extract to dryness, thereby dissolving a DBF-amine
30 adduct in the solvent, and isolating the deprotected peptide as
crystals. However, this method is poor in operability,
sometimes fails to reproduce at a large scale, and is
unsuitable for industrial production. In addition, when a
desired deprotected peptide is an oily substance, this method
35 cannot be used. Furthermore, the method is associated with
problems of low recovery rate and the like due to dissolution
of peptide itself in a hydrocarbon solvent when the peptide
chain is short.
[0004]
5 To solve the problem mentioned above, patent document 1
describes a method including stirring a reaction mixture after
removal of the Fmoc group in a hydrocarbon solvent and a polar
organic solvent, separating the hydrocarbon solvent layer and
the polar organic solvent layer, and removing the hydrocarbon
lo solvent layer in which a DBF and/or DBF-amine adduct is A
dissolved. Patent document 2 describes a method including
contacting a reaction mixture containing a DBF-amine adduct
with carbon dioxide to form a carbonate of a DBF-amine adduct,
and removing the carbonate. However, both patent documents 1
15 and 2 do not describe use of sulfanyl group (SH)-containing
fatty acid for the removal of an F'moc group.
[OOOS]
On the other hand, non-patent document 2 describes a
method including using 1-octanethiol and the like as scavengers
20 of DBF produced during removal of knoc group. In this method,
an adduct of DBF and thiol (hereinafter sometimes to be
abbreviated as a "DBF-thiol adduct") is produced. In an
experiment using 1-octanethiol in non-patent document 2, (a)
trituration or (b) trituration and recrystallization was
2s performed in the same manner as in the method of non-patent
document 1 to remove a byproduced DBF-thiol adduct (Table I),
and the operability is poor.
[0006]
Patent document 3 describes a method including using an
30 amine containing free anion or potential anion, or a thiol
containing free anion or potential anion, as a scavenger to
remove a residual carboxy component in the peptide synthesis
comprising reacting an excess amount of the carboxy component
with an amino component. Patent document 3 further describes,
35 "the aforementioned scavengers are also used for the
deprotection of a peptide under elongation". However, in the
method of patent document 3, the aforementioned scavengers
(thiol containing free anion or potential anion etc.) are used
to deprotect peptide to perform removal of a temporal
5 protecting group contained in the free anion (scavenger), and
deprotection of the N-terminal of peptide under elongation by a
single treatment. Patent document 3 does not describe use of
the aforementioned scavengers for removing Fmoc group or
trapping DBF.
10 [00071
Patent document 4 describes a method of producing a
compound represented by Frn-S-X-COOH (wherein Fm is 9-
fluorenylmethyl), comprising reacting a compound represented by
HS-X-COOH (wherein X is an alkylene chain having 1 - 5 carbon
15 atoms) with a compound represented by Fm-R1 (wherein F'm is 9-
fluorenylmethyl, and R' is a chlorine atom and the like), or
~rnoc-R' (wherein F'moc is 9-f luorenylmethoxycarbonyl, and R* is
succinimidyloxy and the like). However, patent document 4
relates to a production method of Fm-S-X-COOH, and does not
20 relate to the deprotection of an amino group-containing
compound protected by an E'moc group. Patent document 4
proposes using an inorganic base since the above-mentioned
reaction using an organic base affords HS-X-COOH only in a low
yield.
25 [Document List]
[patent documents]
[0008]
patent document 1: WO 2009/014177
patent document 2: WO 2010/016551
30 patent document 3: JP-A-2003-55396
patent document 4: JP-A-2008-303195
[non-patent documents]
[0009]
non-patent document 1: Jikken Kagaku Kouza fifth edition,
35 Maruzen Co., Ltd, published on March 31, 2005, vol. 16, page
272
non-patent document 2: James E. Sheppeck I1 et al., "A
convenient and scaleable procedure for removing the Fmoc group
in solution", Tetrahedron Letter 41 (2000) 5329-5333
5 SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[ 0 0 10 1
The present invention has been made taking note of the
above-mentioned situation, and aims to provide a method of
10 removing an Fmoc group, which can easily remove a byproduct, a
DBF derivative.
Means of Solving the Problems
[OOll]
As mentioned above, since an F'moc group is removed from a
15 protected amino group by using a base, those of ordinary skill
in the art do not expect the presence of an acid in the removal
reaction of an Fmoc group. Nevertheless, the present inventors
have conducted intensive studies in an attempt to achieve the
above-mentioned object and surprisingly found that, when
20 removing an F'moc group by using a base, the removal proceeds
even in the presence of sulfanyl group-containing fatty acid,
and a DBF derivative (i.e., DBF-sulfanyl group-containing fatty
acid adduct) can be successfully removed by subsequently
washing with a basic aqueous solution.
25 [0012]
While non-patent document 2 suggests use of thiosalicylic
acid as a scavenger of DBF, an experiment of thiosalicylic acid
has not been performed, and thiosalicylic acid is a mere
exemplification. When the present inventors performed an
30 experiment using thiosalicylic acid, a DBF derivative (i.e.,
DBF-thiosalicylic acid adduct) was not sufficiently produced
(the following Comparative Example 1). The present invention
based on these findings is as described below.
[ 0 0 13 ]
35 [I] A method of removing an F'moc group, comprising a step of
mixing a compound represented by the formula (I):
HS-L-COOH ( I)
wherein L is a C1+ alkylene group optionally having
substituent(s), an amino group-containing compound protected by
5 an Fmoc group, and a base to give a reaction mixture containing
a compound represented by the formula (11):
m-S-L-COOH (11)
wherein F'm is a 9-fluorenylmethyl group, and L is as mentioned
above, and an amino group-containing compound, and a step of
lo removing the compound represented by the formula (11) by
washing the obtained reaction mixture with a basic aqueous
solution.
[Z] The method of the above-mentioned [I], wherein the base is
an organic base.
15 [3] The method of the above-mentioned [2], wherein the organic
base is 1,8-diazabicyclo[5.4.0]-7-undecene.
[4] The method of any one of the above-mentioned [1] - [3],
wherein the compound represented by the formula (I) is at least
one selected from the group consisting of 3-mercaptopropionic
20 acid, thiomalic acid and cysteine.
[5] The method of any one of the above-mentioned [I] - [4],
wherein the basic aqueous solution is an aqueous solution of at
least one selected from the group consisting of lithium
carbonate, potassium carbonate, sodium carbonate, lithium
25 hydrogen carbonate, potassium hydrogen carbonate, sodium
hydrogen carbonate, lithium hydroxide, potassium hydroxide and
sodium hydroxide.
[6] The method of any one of the above-mentioned [I] - [5],
wherein the amino group-containing compound protected by an
30 Fmoc group is N-Fmoc-C-protected peptide, N-F'moc-C-protected
amino acid or N-F'moc-C-protected amino acid amide, and
the obtained amino group-containing compound is Cprotected
peptide, C-protected amino acid or C-protected amino
acid amide.
35 [7] A method of producing a peptide by a liquid phase synthesis
method, comprising the method of the above-mentioned [6].
[8] The production method of the above-mentioned [7],
comprising (1) a step of condensing C-protected peptide, Cprotected
amino acid or C-protected amino acid amide, and N-
5 Fmoc amino acid or N-Fmoc peptide in the presence of a
condensing agent to give an N-Fmoc-C-protected peptide, and/or
(2) a step of condensing C-protected peptide, C-protected amino
acid or C-protected amino acid amide, and N-F'moc amino acid
active ester or N-Fmoc peptide active ester to give an N-Fmocl
o C-protected peptide.
[9] The production method of the above-mentioned [8], wherein
the aforementioned step (1) is performed in the further
presence of an activator.
[lo] The production method of the above-mentioned [8] or 191,
15 wherein the C-protected peptide, C-protected amino acid or Cprotected
amino acid amide obtained by the method of the abovementioned
[6] is used in the aforementioned step (1) and/or the
aforementioned step (2) without isolating as a solid.
[ll] The production method of the above-mentioned [lo], wherein
20 the peptide is produced by one-pot synthesis.
[0014]
In the following, the "compound represented by the
formula (I)" and the like are sometimes to be abbreviated as
"compound (I) " and the like.
25 Effect of the Invention
[0015]
According to the removal method of the Fmoc group of the
present invention, DBF is trapped by compound (I), compound
(11) as a byproduct can be easily removed by washing with a
30 basic aqueous solution. The removal method of the Fmoc group
of the present invention does not require complicated
operations such as trituration and the like, and can also be
applied easily to large-scale reactions. In addition, by
applying the removal method of the Fmoc group of the present
35 invention to the peptide synthesis, intermediate peptide
obtained after deprotection of Fmoc can be used, without
isolating as a solid, for the next condensation step, which
enables one-pot synthesis of peptide and is particularly
preferable for industrial production.
5 Description of Embodiments
[0016]
1. Symbols
The meanings of the symbols used in the present invention
(that is, DESCRIPTION and Claims) are described below.
10 Ac: acetyl
Alloc: allyloxycarbonyl
At: 7-azabenzotriazol-1-yl
Boc: tert-butoxycarbonyl
BOP: l-benzotriazolyloxy-tris-dimethylamino-phosphonim
15 hexafluorophosphate
Bpr: 1,l-dimethyl-2-phenyl-ethyl
Bsmoc: 1,l-dioxobenzo[b]thiophen-2-ylmethoxycarbonyl
Bt: benzotriazol-1-yl
Bzl: benzyl
2 o Bz1(2,4-OPhy) : 2,4-di (2', 3' -dihydrophytyloxy) benzyl
Bz1(3,4,5-OPhy) : 3,4,5-tri (2' ,3' -dihydrophytyloxy) benzyl
Bzl(2-OBz1(3,4,5-OPhy) -4-OMe) : 2- [3,4,5-tri (2', 3'-
dihydrophytyloxy)benzyloxybenzyloxy]-4-methoxybenzyl
CDI: carbonyldiimidazole
6-C1-HOBt(H0Ct): 6-chloro-1-hydroxybenzotriazole
CPME: cyclopentyl methyl ether
Ct: 6-chlorobenzotriazol-1-yl
DABCO: 1,4-diazabicyclo[2.2.2]octane
DBF: dibenzof ulvene
3 o DBU: 1,8-diazabicyclo[5.4.0]-7-undecene
DCC: dicyclohexylcarbodiimide
Dhbt: 3,4-dihydro-4-oxo-l,2,3-benzotriazin-3-y1
DIPC: diisopropylcarbodiimide
DMAP: N,N-dimethyl-4-aminopyridine
Dmb: 2,4-dimethoxybenzyl
DMF: N,N-dimethylformamide
DMT-MM: 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-
methylmorpholinium chloride
Dpm: diphenylmethyl
5 Dpm(4,4'-OPhy) : 4,4'- (2', 3'-
dihydrophyty1oxy)diphenylmethyl
EDC: 1-ethyl-3-(3-dimethylaminopropy1)-carbodiimide
EDC.HC1: 1-ethyl-3-(3-dimethylaminopropy1)-carbodiimide
hydrochloride
10 Et: ethyl
F'm: 9-fluorenylmethyl
F'moc: 9-fluorenylmethoxycarbonyl
HATU: 0-(7-azabenzotriazol-1-y1)-1,1,3,3-
tetramethyluroniurn hexafluorophosphate
15 HBTU: 0-(benzotriazol-1-y1)-1,1,3,3-tetramethyluronium
hexafluorophosphate
HCTU: 0-( 6-chlorobenzotriazol-1-yl)- 1,1,3,3-
tetramethyluronium hexafluorophosphate
HOAt: 1-hydroxy-7-azabenzotriazole
HOBt: 1-hydroxybenzotriazole
HONb: N-hydroxy-5-norbornane-2,3-dicarboxyimide
HOOBt(H0Dhbt): 3-hydroxy-3,4-dihydro-4-0x0-143-
benzotriazine
HOPht: N-hydroxyphthalimide
HOSu: N-hydroxysuccinimide
iPr: isopropyl
Me: methyl
MsOH: methanesulfonic acid
Nb: 5-norbornane-2,3-dicarboxyimidoyl
NMP: N-methylpyrrolidone
Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
Pht: phthalimidoyl
PyBOP: 1-benzotriazolyloxy-tris-pyrrolidino-phosphonium
hexafluorophosphate
PyBroP: bromo-tris-pyrrolidino-phosphonium
hexafluorophosphate
Su: succinimidoyl
TBTU: 0-benzotriazol-1-yl-1,1,3,3-tetramethyluronium
tetrafluoroborate
5 tBu: tert-butyl
Trt: trityl
THF: tetrahydrofuran
TsOH: p-toluenesulfonic acid
Z: benzyloxycarbonyl
10 [0017]
AA,: amino acid residue (subscript n is any integer of 1
or more, and shows the order of AA, from peptide C-terminal)
PGo: protecting group of C-terminal carboxyl group or Cterminal
amide group of peptide
15 PGn: amino-protecting group (subscript n is any integer
of 1 or more, and PG, is a protecting group of amino group for
AAn )
HOE: activator
E: activated group
Gly: glycine
Ala: alanine
Val: valine
Leu: leucine
Ile: isoleucine
Met: methionine
Phe: phenylalanine
Tyr: tyrosine
Trp: tryptophan
His: histidine
Lys: lysine
Arg: arginine
Ser: serine
Thr: threonine
Asp: aspartic acid
Glu: glutamic acid
Asn: asparagine
Gln: glutamine
Cys: cysteine
Pro: proline
Orn: ornithine
Sar: sarcosine
P-Ala: p-alanine
GABA: y-aminobutyric acid
Dap: 2,3-diaminopropionic acid
10 [00181
Examples of the protecting group of the C-terminal
carboxy group for PGo include alkyl groups such as Me, Et, iPr,
tBu and the like, Z, Fm, Trt, Dpm, Bpr, 1-1-dimethylbenzyl,
dimethylphenyl and the like.
15 Examples of the protecting group of the C-terminal amide
group for PGo include Dmb, bis(4-methoxyphenyl)methyl, trityl
and the like. The amide group optionally has substituent(s)
such as an alkyl group and the like. The amide group is also
referred to as a carbamoyl group.
20 [00191
In addition, as the protecting group of the C-terminal
carboxy group or C-terminal amide group for PGo,
(1) a protecting group using the diphenylmethane compound
described in WO 2010/113939Al as a protecting reagent,
25 ( 2 ) a protecting group using the fluorene compound described in
WO 2010/104169Al as a protecting reagent,
(3) a protecting group using the benzyl compound described in
WO 2011/078295A1 as a protecting reagent,
(4) a protecting group using the branched chain-containing
so aromatic compound described in WO 2012/029794A1 as a protecting
reagent
and the like can be used.
When the C-terminal carboxy group or C-terminal amide
group is protected using such protecting reagents, the
35 liposolubility of the below-mentioned N-F'moc-C-protected
peptide and the like, and C-protected peptide and the like can
be improved. In the production method of peptide by the belowmentioned
liquid phase synthesis method, for example,
impurities can be efficiently removed from the aqueous layer in
5 washing with water in the workup of the coupling step.
[0020]
Examples of the diphenylmethane compound described in WO
2010/113939Al include
2,3,4-trioctadecanoxybenzohydrol;
lo [phenyl(2,3,4-trioctadecanoxyphenyl)methyl]amine;
4,4r-didocosoxybenzohydrol;
di(4-docosoxyphenyl)methylamine;
4,4-di(l2-docosoxydodecyloxy)benzohydrol;
amino-bis[4-(12-docosoxydodecyloxy)phenyl]methane;
15 N-benzyl-[bis(4-docosyloxyphenyl)]methylamine;
(4-methoxy-pheny1)-[4-(3,4,5-tris-octadecyloxycyclohexylmethoxy)-
phenyll-methanol;
{ (4-methoxy-phenyl) - [4- (3,4,5-tris-octadecyloxycyclohexylmethoxy)-
phenyll-methyl}-amine;
20 [bis-(4-docosoxy-pheny1)-methyl]-amine
and the like.
[0021]
Examples of the fluorene compound described in WO
2010/104169Al include
25 2-docosyloxy-9-(4-chlorophenyl)-9-f1uorenol;
2-docosyloxy-9-(4-chlorophenyl)-9-bromofluorene;
2,7-didocosyloxy-9-(4-chlorophenyl)-9-bromo£luorene;
2-(12-docosyloxy-dodecanoxy)-9-(3-f1uorophenyl)-9-
bromof luorene;
30 1,12-bis- [12- (2' -0-9- (4-chlorophenyl) -9-fluorenol) -dodecyloxy] -
dodecane ;
1,12-bis- [12- (2' -0-9- (4-chlorophenyl) -9-bromofluorene) -
dodecyloxy3-dodecane;
2-(3-octadecyloxy-2r2-bi~-octadecyloxymethyl-propoxy)-9-(4-
35 chloropheny1)-9-fluorenol;
2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl-propoxy)-9-(4-
chloropheny1)-9-bromofluorene;
9- (4-chlorophenyl) -2- (3'4'5-
tris(octadecyloxy)cyclohexylmethoxy)-9-fluorenol;
5 9- (4-chlorophenyl) -2- (3'4'5-
tris(octadecyloxy)cyclohexylmethoxy)-9-bromofluorene
and the like.
[0022]
Examples of the benzyl compound described in WO
10 2011/078295Al include
4-(12'-docosyloxy-1'-dodecy1oxy)benzyl alcohol;
4-(12'-docosyloxy-1'-dodecy1oxy)-2-methoxybenzyl alcohol;
4-(12'-docosyloxy-l'-dodecyloxy)-2-methoxybenzylamine;
2-(12'-docosyloxy-1'-dodecy1oxy)-4-methoxybenzyl alcohol;
15 2-(12'-docosyloxy-l'-dodecyloxy)-4-methoxybenzylamine;
4-methoxy-2- [ 3 ' , 4'' 5' -tris (octadecyloxy)b enzyloxy]b enzyl
alcohol;
2- [3', 5' -di (docosyloxy)b enzyloxy] -4-methoxybenzyl alcohol;
2-methoxy-4- [2', 2'' 2' -tris (octadecyloxymethyl) ethoxy] benzyl
20 alcohol;
2-methoxy-4- [2', 2'' 2'-
tris(octadecyloxymethyl)ethoxy]benzylamine;
4-methoxy-2- [ 3 ' , 4'' 5' -
tris(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol;
25 4- [3', 4'' 5' -tris (octadecyloxy)c yclohexylmethyloxy]b enzyl
alcohol;
1'22-bis [12- (4-hydroxymethyl-3-
methoxyphenoxy)dodecyloxy]docosane;
1'22-bis [12- (2-hydroxymethy1-5-
30 methoxyphenoxy) dodecyloxy] docosane;
2-docosyloxy-4-methoxybenzyl alcohol;
2-methoxy-4- [3', 4'' 5' -
tris(octadecyloxy)cyclohexylmethyloxy]benzyl alcohol;
3'5-dimethoxy-4- [3', 4', 5' -
35 tris (octadecyloxy)c yclohexylmethyloxy]b enzyl alcohol;
N-(4-hydroxymethyl-3-methoxypheny1)-3,4,5-
tris(octadecyloxy)cyclohexylcarboxamide;
N- (5-hydroxymethyl-2-methoxyphenyl)- 3,4,5-
tris(octadecyloxy)cyclohexylcarboxamide;
5 N- (4-hydroxymethylphenyl) -3'4'5-
tris(octadecyloxy)cyclohexylcarboxamide
and the like.
[0023]
Examples of the branched chain-containing aromatic
l o compound described in WO 2012/029794Al include
2,4-di(2',3'-dihydrophyty1oxy)benzyl alcohol;
3,5-di (2 ' '3' -dihydrophytyloxy) benzyl alcohol;
4-(2',3r-dihydrophytyloxy)benzyl alcohol;
1- [ (2-chloro-5- (2' '3' -dihydrophytyloxy) phenyl) ] -1-
is phenylmethaneamine;
3,4,5-tri(Zr,3'-dihydrophyty1oxy)benzyl alcohol;
3,4,5-tri (2' ,3' -dihydrophytyloxy) benzylamine;
4- (2'' 3' -dihydrophytyloxy) benzylamine;
2- [ 3 ' , 4'' 5' -tri (2' I 3' I-dihydrophytyloxy) benzyloxy] -4-
20 methoxybenzyl alcohol;
4-(2',3'-dihydrophyty1oxy)-2-methoxybenzyl alcohol;
4-(2',3'-dihydrophyty1oxy)-2-methoxybenzylamine;
4- (2', 3' -dihydrophytyloxy) -2-methylbenzy alcohol;
4-(2',3'-dihydrophyty1oxy)-2-methylbenzylamine;
25 2121418,10,10-hexamethyl-5-dodecanoica cid(4-
hydroxymethy1)phenylamide;
4-(3,7,11-trimethyldodecyloxy)benzyl alcohol;
2-(3,7,11-trimethyldodecyloxy)-9-phenylfluorene-9-ol
and the like.
30 100241
Examples of the amino-protecting group for PG, include
Boc, Z, F'moc, Bsmoc, Alloc, Ac and the like.
The activated group for E means a group which can be
easily dissociated as "EO-" on nucleophilic attack by an amino
35 group and produce an amide bond, and examples thereof include
Bt, Ct, At, OBt, Su, Pht, Nb, pentafluorophenyl and the like.
[0025]
2. Terms
The terms used in the present invention are sequentially
s explained below.
The "amino group-containing compound" in the present
invention means a compound having the primary amino group
and/or the secondary amino group.
The "amino group-containing compound protected by an Fmoc
lo group" means a compound wherein at least one of the primary
amino group and/or the secondary amino group that the amino
group-containing compound has is protected by an Fmoc group.
[0026]
When an amino acid is indicated as "H-aA-OH" in the
1s present invention, it means that the left side is an amino
group, the right side is a carboxy group, and both the amino
group and the carboxy group are not protected.
An amino acid wherein the carboxy group is protected is
indicated as "H-AA-OPGo", and an amino acid wherein the amino
20 group is protected is indicated as "PGn-AA-OH".
An amino acid wherein the amino group is protected and
the carboxy group is active-esterified is indicated as "PG,-AAOE".
A symmetric anhydride of PG,-AA-OH is indicated as "(PG,-
25 AA)2-0N.
[0027]
In the present invention, when amino acid amide is
indicated as "H-AA-NH2", it means that the left side is an
amino group, the right side is an amide group, and both the
so amino group and the amide group are not protected.
An amino acid amide wherein the amide group is protected
is indicated as "H-AA-NHPGoU, and an amino acid amide wherein
the amino group is protected is indicated as "PGn-AA-NH*".
[0028]
35 An amino acid or amino acid amide having a protected side
chain functional group is indicated as "H-AA(PG)-(OH or NH2)"
(PG is a protecting group of the side chain functional group).
[0029]
When peptide is indicated as "H-AA,r -AAn1 -I-. . . -AA1- (OH or
5 NH2)" (subscript n' is an integer of two or more) in the
present invention, it means that the left side is the Nterminal,
the right side is the C-terminal, and the peptide
contains amino acid residues in the number of nf having
unprotected N-terminal and unprotected C-terminal. Here, the
10 N-terminal is not limited to an a-position amino group of the
amino acid residue and, when peptide elongation is performed
via this side chain amino group, it also includes the side
chain amino group (e.g., an €-amino group of Lys), hereinafter
the same.
1s A peptide with protected C-terminal is indicated as "HAAnf-
AAnr-l-**- MI-( OPGo or NHPGo)" , and further, a peptide with
protected N-terminal is indicated as "PGnt -W,I -AA,T-~-. *-AA1-
(OPGo or NHPGo) " .
[0030]
20 The "C-protected amino acid" means an amino acid wherein
the carboxyl group is protected and the amino group is not
protected, and is indicated as "H-AA-OPGo".
The "C-protected amino acid amide" means an amino acid
amide wherein the amide group is protected and the amino group
25 is not protected, and is indicated as "H-AA-NHPGoU.
[0031]
The "N-protected amino acid" means an amino acid wherein
the amino group is protected and the carboxy group is not
protected, and this is indicated as shown by "PGn-AA-OH".
30 The "N-protected amino acid amide" means amino acid amide
wherein the amino group is protected and the amide group is not
protected, and is indicated as "PGn-AA-NH2".
The "N-protected amino acid active ester" means an amino
acid wherein the amino group is protected and the carboxy group
35 is activated by E, and is indicated as "PGn-AA-OE".
The "N-protected peptide active ester" means a peptide
wherein the N-terminal amino group is protected and the Cterminal
carboxy group is activated by E.
N-Protected amino acid active ester or N-protected
5 peptide active ester that can be isolated is one wherein E is
pentafluorophenyl, Su or Nb. Other N-protected amino acid
active ester or N-protected peptide active ester is produced in
the reaction system by reacting N-protected amino acid with a
condensing agent (e . g., EDC) and an activator (e. g . , HOBt ) .
10 [00321
The "N-E'moc amino acid" means an amino acid wherein the
amino group is protected by Fmoc and the carboxy group is not
protected.
The "N-Fmoc amino acid amide" means an amino acid amide
15 wherein the amino group is protected by Fmoc and the amide
group is not protected.
The "N-Fmoc amino acid active ester" means an amino acid
wherein the amino group is protected by Fmoc and the carboxy
group is active-esterified by E.
2 o The "N-Fmoc peptide active ester" means a peptide wherein
the N-terminal amino group is protected by Fmoc and the Cterminal
carboxy group is active-esterified by E.
N-Fmoc amino acid active ester or N-Fmoc peptide active
ester that can be isolated is one wherein E is
25 pentafluorophenyl, Su or Nb. Other N-Fmoc amino acid active
ester or N-Fmoc peptide active ester is produced in the
reaction system by reacting N-Fmoc amino acid with a condensing
agent (e. g., EDC) and an activator (e. g., HOBt) .
[ 0 0 3 3 ]
3 o The "C-protected peptide" is a peptide wherein the Cterminal
carboxy group or C-terminal amide group is protected,
and the N-terminal amino group is not protected, and is
indicated as "H-AA,. -AAnr-l-.o.-A&- (OPGo or NHPGO) " (n' is an
integer of two or more).
The "N-protected-C-protected peptide" means a peptide
wherein all of the N-terminal amino group, and the C-terminal
carboxy group or the C-terminal amide group are protected, and
is indicated as "PG,I-AA,I -AAnt-l-.-*- AAl- (OPGo or NHPGo)" (n' is
an integer of two or more).
5 The "N-Fmoc-C-protected peptide" means a peptide wherein
the N-terminal amino group is protected by Fmoc, and the Cterminal
carboxy group or C-terminal amide group is protected.
[0034]
3. Method of removing Fmoc group
10 The method of removing the E'moc group of the present
invention includes
a step of mixing compound (I) (that is, HS-L-COOH), an
amino group-containing compound protected by an Fmoc group and
a base to give a reaction mixture containing compound (11)
1s (that is, Fm-S-L-COOH) and the amino group-containing compound,
and
a step of washing the obtained reaction mixture with a
basic aqueous solution to remove compound (11)
(in the aforementioned formulas, L is a CI-~al kylene group
20 optionally having substituent(s), and Fm is a 9-fluorenylmethyl
group) .
LO0351
The amino group-containing compound is not particularly
limited as long as it is a compound having the primary amino
25 group and/or the secondary amino group as mentioned above.
Examples of the amino group-containing compound include peptide,
amino acid, amino acid amide and the like. In the method of
removing the Fmoc group of the present invention, only one kind
of the amino group-containing compound protected by the Fmoc
30 group may be used, or two or more kinds thereof may be used in
combination.
[0036]
When the amino group-containing compound is a lowmolecular-
weight compound having a free carboxy group, compound
35 (11) as a byproduct, and the amino group-containing compound
may be dissolved in a basic aqueous solution in the
aforementioned step of washing with the basic aqueous solution,
and the yield of the obtained amino group-containing compound
may decrease. Therefore, the amino group-containing compound
5 preferably does not have a free carboxy group. The amino
group-containing compound protected by the &oc group is
preferably N-Fmoc-C-protected peptide, N-Fmoc-C-protected
peptide, N-Fmoc-C-protected amino acid or N-Fmoc-C-protected
amino acid amide, which does not have a free carboxy group
l o (sometimes to be abbreviated as "N-Fmoc-C-protected peptide
etc." in the present specification). The amino groupcontaining
compound obtained corresponding to the N-Fmoc-Cprotected
peptide and the like is preferably C-protected
peptide, C-protected amino acid or C-protected amino acid amide
15 (sometimes to be abbreviated as "C-protected peptide and the
like" in the present specification).
100371
The amino acid to be the base of the N-Fmoc-C-protected
peptide and the like and C-protected peptide and the like, and
20 the below-mentioned N-Fmoc amino acid, N-Fmoc amino acid active
ester, N-Fmoc peptide and N-Fmoc peptide active ester may be
any of natural amino acid and nonnatural amino acid. In
addition, the amino acid may be any of an L form and a D form.
Moreover, a mixture of racemic amino acids may also be used.
25 Examples of the natural amino acid include Gly, Ala, Val, Leu,
Ile, Ser, Thr, Asn, Gln, Asp, Glu, Lys, Arg, Cys, Met, Phe, Tyr,
Trp, His, Pro, Orn, Sar, P-Ala, GABA and the like. Examples of
the nonnatural amino acid include Dap and the like.
[0038]
30 The N-&oc-C-protected peptide and the like and the Cprotected
peptide and the like may have a side chain functional
group (amino group, carboxy group, sulfanyl group, hydroxy
group, guanidyl group etc.). The side chain amino group may
not be protected, but is preferably protected by a protecting
35 group other than the Fmoc group (e-g., Boc, Z, Bsmoc, Alloc, Ac
etc.). In addition, the side chain carboxy group is preferably
protected by a protecting group like the C-terminal.
[0039]
Examples of the carboxy-protecting group include alkyl
5 having 1 - 6 carbon atoms such as Me, Et, tBu and the like, Bzl,
p-nitrobenzyl, p-methoxybenzyl, Dpm, allyl, Bpr and the like.
Examples of the amide-protecting group include Dmb, bis(4-
methoxypheny1)methyl and the like. In addition, the carboxyprotecting
group and amide-protecting group preferably have a
l o branched chain. Using a protecting group having a branched
chain, the liposolubility of the N-F'moc-C-protected peptide and
the like and the C-protected peptide and the like can be
improved, and a peptide having many amino acid residues is
easily synthesized in the below-mentioned production method of
15 peptide by the liquid phase synthesis method. Examples of the
protecting group having a branched chain include Bz1(2,4-OPhy),
Bz1(3,4,5-OPhy) , Bzl(2-OBz1(3,4,5-OPhy) -4-OMe) , Dpm(4,4'-OPhy)
and the like.
[0040]
20 Examples of the sulfanyl-protecting group include
phenylcarbamoyl, Trt and the like. Examples of the hydroxylprotecting
group include Bzl, tBu and the like. Examples of
the guanidyl-protecting group in the side chain include Pbf and
the like.
25 [0041]
L in the formula (I) shows a C1-8 alkylene group
optionally having substituent (s) . Here, the "C1-811i s the
number of carbons contained in the alkylene group, and does not
contain the number of carbons of the substituent that L has.
30 When the number of carbons of the alkylene group is too large,
the solubility of compound (11) as a byproduct in a basic
aqueous solution decreases, and it cannot be removed
sufficiently in the aforementioned washing step. Therefore,
the number of carbons of the alkylene group is 8 or less,
35 preferably 6 or less, more preferably 2 or less. Examples of
the substituent that L can have include an alkyl group (e.g.,
Me, Et etc.), a carboxy group, an amino group and the like.
Compound (I) is preferably at least one selected from the group
consisting of 3-mercaptopropionic acid, thiomalic acid (also
5 referred to as 2-mercaptosuccinic acid) and cysteine, more
preferably 3-mercaptopropionic acid.
[0042]
The amount of compound (I) to be used is preferably 1.0 -
30 mol, more preferably 3 - 10 mol, per 1 mol of the Fmoc group
10 contained in the amino group-containing compound protected by
the Fmoc group. When the amount of compound (I) to be used is
too small, the Fmoc group cannot be sufficiently removed. On
the other hand, when the amount is too large, removal of
compound (I) itself becomes difficult. When the method of
15 removing the Fmoc group of the present invention is utilized to
a production method of peptide, impurity is sometimes produced
easily due to the condensation reaction of peptide.
[0043]
Compound (I), the amino group-containing compound
20 protected by the Fmoc group and a base are generally reacted in
a solvent. Examples of the solvent include chloroform,
methylene chloride, CPME, DMF, NMP, ethyl acetate, acetonitrile,
THF, a mixed solvent thereof and the like. The amount of the
solvent to be used is generally 3- to 100-fold weight,
25 preferably 5- to 30-fold weight, relative to the amino groupcontaining
compound protected by the F'moc group. While the
reaction temperature varies depending on the amino groupcontaining
compound protected by the Fmoc group, it is
generally 0 to 50°C, preferably 10 to 30°C. The reaction time
30 is generally 0.1 - 24 hr, preferably 1 - 5 hr.
[0044]
Examples of the base to be used for the removal reaction
of the Fmoc group include DBU, DABCO, Et3N, Na2C03, NaOtBu,
KOtBu, iPr2EtN and the like. Only one kind of the base may be
35 used, or two or more kinds thereof may be used in combination.
The base is preferably an organic base, more preferably DBU.
The amount of the base to be used is preferably 0.5 - 5 mol,
more preferably 1 - 3 moll per 1 mol of the carboxy group of
compound (I) to be used. When the amount of the base to be
5 used is too small, the reaction rate of the Fmoc group removal
reaction is not sufficiently improved. On the other hand, when
the amount is too large, removal of the base becomes difficult,
and side reactions such as racemization of peptide and the like
can occur when the method of removing the Fmoc group of the
lo present invention is used for the production method of peptide.
[0045]
One of the characteristics of the method of removing the
Fmoc group of the present invention is removal of compound (11)
as a byproduct by washing the reaction mixture obtained by the
15 aforementioned reaction with a basic aqueous solution.
Generally, washing means removal of a contaminant substance by
dissolving same in a liquid. The washing in the present
invention means removal of compound (11) by dissolving same in
a basic aqueous solution. The washing with a basic aqueous
20 solution is performed by, for example, mixing and stirring a
solution containing the reaction mixture and a basic aqueous
solution, partitioning the organic layer and the aqueous layer,
and removing the aqueous layer. In addition, the washing with
a basic aqueous solution can easily remove not only compound
25 (11) as a byproduct but also the residual compound (I).
[0046]
In the conventional method of removing an Fmoc group
using amine and the like, the reaction solution needs to be
washed with an acidic aqueous solution to remove amine. In
30 this connection, when the reaction solution after removal of
the Fmoc group is washed with an acidic aqueous solution in the
peptide synthesis, the obtained peptide transfers to the acidic
aqueous solution since peptide has an amino group, and the
yield of the peptide problematically decreases. Moreover,
35 removal of the aqueous layer is problematically difficult since
a reaction solution containing peptide (that is, organic layer)
shows poor separation property from an acidic aqueous solution
(that is, aqueous layer). As compared to
conventional methods wherein an Fmoc group is removed by
5 washing with an acidic aqueous solution after using such amine,
the method of removing the Fmoc group of the present invention
including washing with a basic aqueous solution can avoid a
decrease in the yield of the peptide, shows good separation of
the organic layer and the aqueous layer.
10 [00471
The basic aqueous solution is preferably at least one
aqueous solution selected from the group consisting of lithium
carbonate, potassium carbonate, sodium carbonate, lithium
hydrogen carbonate, potassium hydrogen carbonate, sodium
15 hydrogen carbonate, lithium hydroxide, potassium hydroxide and
sodium hydroxide, more preferably an aqueous sodium carbonate
solution. The concentration of the base in the basic aqueous
solution is preferably 1 - 20 wt%, more preferably 5 - 15 wt%.
When the concentration of the base is too low, compound (11)
20 cannot be removed sufficiently. On the other hand, when the
concentration is too high, the base sometimes remains
undissolved in water or a side reaction may occur.
[0048]
A basic aqueous solution is added to the reaction mixture
25 until the pH of the basic aqueous solution after mixing with
the reaction mixture reaches preferably 7 - 14, more preferably
8 - 12. The washing temperature with a basic aqueous solution
is preferably 0 to 50°C, more preferably 10 to 30°C. The
washing with a basic aqueous solution may be repeated.
30 [0049]
The basic aqueous solution may contain a polar solvent.
This polar solvent is preferably at least one selected from the
group consisting of DMF, acetonitrile, methanol, ethanol, THF
and NMP, more preferably DMF. When a polar solvent is used,
35 the content thereof in the basic aqueous solution is preferably
1 - 50% by volume, more preferably 5 - 30% by volume.
[0050]
By concentrating a solution obtained by the method of
removing the F'moc group of the present invention, an amino
5 group-containing compound wherein the Fmoc group has been
removed can be isolated. Where necessary, the amino groupcontaining
compound may be isolated as an acid addition salt
(hydrochloride, toluenesulfonate, methanesulfonate,
hydrobromide, trifluoroacetate etc.) by adding an acid (e.g.,
10 hydrochloric acid, toluenesulfonic acid, methanesulfonic acid,
hydrobromic acid, trifluoroacetic acid etc.) to the solution.
Furthermore, a solution of the obtained amino group-containing
compound can be directly used as a starting material of the
below-mentioned production method of peptide by the liquid
15 phase synthesis method.
4. Production method of peptide by liquid phase synthesis
method
When the amino group-containing compound protected by an
20 Fmoc group is N-Fmoc-C-protected peptide and the like, and the
corresponding amino group-containing compound obtained is Cprotected
peptide and the like, the method of removing the Fmoc
group of the present invention can be preferably used for the
production method of peptide by the liquid phase synthesis
25 method (hereinafter sometimes to be abbreviated as "liquid
phase peptide synthesis method"). The "liquid phase synthesis
method" in the present invention means a synthesis method other
than the solid phase synthesis method, which includes, in
addition to a homogenous reaction wherein all reagents are
30 dissolved in the solvent, a heterogeneous reaction wherein all
or a part of the reagents is not dissolved in the solvent, but
dispersed or suspended therein.
The liquid phase peptide synthesis method of the present
invention including the aforementioned method of removing the
35 Fmoc group is explained below.
[0052]
While the peptide to be the final object product of the
liquid phase peptide synthesis method of the present invention
is not particularly limited, it preferably shows an amino acid
5 residue number of about 2 - 40, which is generally seen in
synthetic peptides. The peptide obtained by the liquid phase
peptide synthesis method of the present invention can be
utilized for, for example, synthetic pharmaceutical peptide,
cosmetic, electronic material (organic EL and the like), food
lo and the like.
[0053]
One embodiment of the liquid phase peptide synthesis
method of the present invention includes
(1) a step of condensing C-protected peptide, C-protected amino
15 acid or C-protected amino acid amide, and N-E'moc amino acid or
N-F'moc peptide in the presence of a condensing agent
(preferably condensing agent and activator) to give an N-E'moc-
C-protected peptide (hereinafter to be abbreviated as "coupling
step (1) " ) , and/or
20 ( 2 ) a step of condensing C-protected peptide, C-protected amino
acid or C-protected amino acid amide, and N-Fmoc amino acid
active ester or N-F'moc peptide active ester to give an N-E'moc-
C-protected peptide (hereinafter to be abbreviated as "coupling
step (2)".). For the liquid phase peptide synthesis method of
25 the present invention, a general method conventionally used for
the peptide synthesis chemistry can be employed without a
particular limitation.
[0054]
By repeating the aforementioned coupling steps (1) and/or
30 (2), and then a step of removing the Fmoc group from the
obtained N-F'moc-C-protected peptide (hereinafter sometimes to
be abbreviated as "N-terminal deprotection step"), C-protected
peptide having a desired number of amino acid residues is
obtained. Finally, a step of removing the C-terminal
35 protecting group of the C-protected peptide, and a step of
removing the protecting group of the side chain functional
group where necessary (hereinafter sometimes to be abbreviated
as the "final deprotection stepN) afford the final object
product peptide.
5 [00551
One embodiment of the aforementioned liquid phase peptide
synthesis method using the N-Fmoc amino acid or N-Fmoc amino
acid active ester can be shown by the following scheme. In the
following scheme, the nth peptide elongation reaction is
l o indicated as "peptide elongation reaction (n)", and the
coupling steps (1) and/or (2) constituting the peptide
elongation reaction (n), and the N-terminal deprotection step
thereafter are indicated as "coupling step (1-n) ", "coupling
step (2-n) " and "N-terminal deprotection step (n) ",
is respectively. A liquid phase peptide synthesis method wherein
the N-Fmoc amino acid or N-Fmoc amino acid active ester in the
scheme below is replaced by N-Fmoc peptide or N-Fmoc peptide
active ester, respectively, is also encompassed in the scope of
the present invention.
20 E00561
I coupling step (1-1) / coupling step (2-1)
N-terminal deprotection step (1) I H-AA2-AA1-(0-PGo or NHPGo) (P2)
PG2-AA2-oH , condensing agent,
(PA21 (HOE)
PG2-AA2-OE
(PAE2)
coupling step (I-n ') I coupling step (2-17')
v
coupling step (1-2)
N-terminal deprotection step (2)
peptide elongation reation (1)
coupling step (2-2) one cycle
v
N-terminal deprotection step (n') 1
PGn~+l-AAn~+l-OH
(PA,,+,) , condensing agent,
peptide elongation reation (n')
one cycle
t
H2N-AA,-AA,-1-AA,-2- ........ -A$-(0-PGo or NHPGo) (P,)
PGn~+l-AAn~tl-OE
(PAEnltl)
final deprotection step
v
H2N-AA,-AA,..1-AA,_2- ........ -MI-(OH or NHPG,) (P)
(HOE) P
In the above-mentioned scheme,
Al is C-protected amino acid or C-protected amino acid
5 amide;
PA2 and are each N-Fmoc amino acid;
PAE2 and PAE,,+l are each N-Fmoc amino acid active ester;
PP2 and PP,I+~ are each N-Frnoc-C-protected peptide;
P2, P,,r, Pnr+] and P, are each C-protected peptide,
subscripts 2, n', nf+l and m are each the number of amino acid
residues of the C-protected peptide, n' is an integer of two or
more, m is an integer of not less than 3, and shows the number
of amino acid residues of the final object product peptide; and
5 P is the final object product peptide (number of amino
acid residues m).
[0058]
In the liquid phase peptide synthesis method of the
present invention, the aforementioned removal method of the
lo Fmoc group may be performed before the start of the elongation
reaction (i-e., before the aforementioned coupling steps (1-1)
and/or (2-2)) to prepare C-protected peptide, C-protected amino
acid or C-protected amino acid amide to be used in these steps.
In the liquid phase peptide synthesis method of the present
15 invention, moreover, the aforementioned removal method of the
Fmoc group may be performed as at least one (preferably all) of
the N-terminal deprotection step to prepare a C-protected
peptide.
Each step is sequentially explained in the following.
20 [00591
4-1. Coupling step (1)
In coupling step (I), for example, an N-F'moc-C-protected
peptide wherein one amino acid residue has elongated is
obtained by mixing C-protected peptide, C-protected amino acid
25 or C-protected amino acid amide, and N-koc amino acid, and a
condensing agent (preferably a condensing agent and an
activator) in a solvent. Using N-F'moc peptide instead of NFmoc
amino acid, moreover, an N-F'moc-C-protected peptide
wherein amino acid residues in the number of the amino acid
30 residues of N-Fmoc peptide have elongated is obtained. The
number of the amino acid residues of N-Fmoc peptide used here
is preferably 2 - 20, more preferably 2 - 10.
[0060]
While the order of addition of the components is not
35 particularly limited, when the C-protected peptide is obtained
by peptide elongation reaction (n-1) before this one, N-Fmoc
amino acid or N-Fmoc peptide and a condensing agent (and
preferably an activator) can be added to a solution of the Cprotected
peptide in a reaction vessel.
5 [00611
The amount of N-F'moc amino acid or N-Fmoc peptide to be
used is generally 0.9 to 4.0 equivalents, preferably 1.0 to 1.5
equivalents, relative to the C-protected peptide and the like.
When the amount is smaller than this range, unreacted Cro
protected peptide and the like tends to remain, and when the
amount is larger, excess N-Fmoc amino acid or N-F'moc peptide
cannot be removed easily.
[0062]
When C-protected peptide and the like is used as acid
1s addition salt, a base is added for neutralization. Examples of
the base include triethylamine, diisopropylethylamine, pyridine,
N-methylmorpholine and the like. The amount of this base to be
used is generally 0.5 to 2.0 equivalents, preferably 1.0 to 1.5
equivalents, relative to the C-protected peptide and the like.
20 When the amount of the base to be used is smaller than this
range, neutralization becomes insufficient and the reaction
does not often proceed smoothly.
[0063]
Examples of the condensing agent include EDC(inc1uding
25 hydrochloride and free form) , DIPC, DCC, BOP, PyBOP, PyBroP,
HBTU, HCTU, TBTU, HATU, CDI, DMT-MM and the like can be
mentioned. Of these, EDC is preferable from the aspects of
residual condensing agent and decomposed condensing agent. The
amount of the condensing agent to be used is generally 0.8 to
30 4.0 equivalents, preferably 1.0 to 1.5 equivalents, relative to
N-Fmoc amino acid.
[0064]
In coupling step (I), an activator is preferably added to
promote the reaction and suppress side reactions such as
35 racemization and the like. Here, the activator is a reagent
that converts, in the presence of a condensing agent, amino
acid to the corresponding active ester, symmetric anhydride and
the like to facilitate formation of a peptide bond (amide bond).
Examples of the activator include HOBt, HOCt, HOAt, HOOBt, HOSu,
s HOPht, HONb, pentafluorophenol and the like. Of these, HOBt,
HOOBt, HOCt, HOAt, HONb, and HOSu are preferable. The amount
of the activator to be used is generally 0 to 4.0 equivalents,
preferably 0.1 to 1.5 equivalents, relative to N-Fmoc amino
acid.
l o [00651
The solvent to be used in coupling step (1) is not
particularly limited as long as it does not inhibit the
reaction. Examples of the solvent include DMF, NMP, ethyl
acetate, THF, acetonitrile, chloroform, methylene chloride, a
is mixed solvent thereof and the like. Of these, ethyl acetate
and DMF are preferable. The amount of the solvent to be used
is generally 3- to 100-fold weight, preferably 5- to 20-fold
weight, relative to the C-protected peptide and the like.
[0066]
2 o The reaction temperature is generally within the range of
-20°C to 40°C, preferably 0°C to 30°C. The reaction time is
generally 0.5 to 30 hr.
[0067]
Workup may be performed after completion of the reaction
2s of coupling step (1). This workup is the same as that after
completion of the reaction of coupling step (2), and will be
explained altogether in "4-3. Workup of coupling steps (1) and
(2)" after "4-2. Coupling step (2) ".
[0068]
30 4-2. Coupling step (2)
In coupling step (2), for example, an N-Fmoc-C-protected
peptide wherein one amino acid residue has elongated is
obtained by mixing C-protected peptide, C-protected amino acid
or C-protected amino acid amide, and N-F'moc amino acid active
35 ester in a solvent. Using N-F'moc peptide active ester instead
of N-Fmoc amino acid active ester, moreover, an N-F'moc-Cprotected
peptide wherein amino acid residues in the number of
the amino acid residues of N-Frnoc peptide active ester have
elongated is obtained. The number of the amino acid residues
5 of N-Fmoc peptide active ester used here is preferably 2 - 10,
more preferably 2 - 5.
[0069]
While the order of addition of the components is not
particularly limited, when the C-protected peptide is obtained
lo by peptide elongation reaction (n-1) before this one, N-F'moc
amino acid active ester or N-F'moc peptide active ester can be
added to a solution of the C-protected peptide in a reaction
vessel.
[0070]
15 The amount of N-F'moc amino acid active ester or N-Frnoc
peptide active ester to be used is the same as that of the NE'rnoc
amino acid or N-Fmoc peptide in coupling step (1). The
base, solvent and the amounts of use thereof in coupling step
(2), the reaction temperature, reaction time and the like and
20 other reaction conditions are also the same as those in
coupling step (1). Workup may be performed after completion of
the reaction of coupling step (2).
[0071]
4-3. Workup of coupling steps (1) and (2)
25 After completion of the reactions of coupling steps (1)
and (2), solid nucleophile removing reagents such as sulfanyl
group-supported silica gel and the like (e.g., SH silica
(manufactured by Fuji Silysia Chemical Ltd.) etc.) may be added,
and the mixture is stirred and filtered to remove residues and
30 byproducts in the reaction mixture that can be condensed with
amine components, such as N-Fmoc amino acid activated ester, NFmoc
peptide activated ester, isourea ester of N-Fmoc amino
acid, symmetric anhydride of N-Fmoc amino acid and the like.
In addition, activated ester may be deactivated in the washing
35 step by washing with weak basic aqueous solution such as sodium
carbonate and the like.
[0072]
In the workup in coupling steps (1) and (2), washing with
acidic aqueous solution and/or washing with basic aqueous
5 solution is preferably performed. The washing with acidic
aqueous solution can remove C-protected peptide, residual
condensing agent and a decomposed product thereof, a base and
the like into the aqueous layer. The washing with basic
aqueous solution can remove additive, residual N-Fmoc amino
l o acid and the like into the aqueous layer.
[0073]
Washing with an acidic aqueous solution is performed, for
example, by mixing and stirring the reaction mixture, a dilute '
aqueous hydrochloric acid solution (e.g., 1N aqueous
15 hydrochloric acid solution), an aqueous solution of sulfuric
acid, formic acid, citric acid, phosphoric acid and the like,
partitioning the organic layer and the aqueous layer, and
removing the aqueous layer.
[0074]
2 o Washing with a basic aqueous solution is performed, for
example, by mixing and stirring the reaction mixture, an
aqueous sodium hydrogen carbonate solution (e-g., 5 wt% aqueous
sodium hydrogen carbonate solution), an aqueous sodium
carbonate solution, an aqueous potassium carbonate solution and
25 the like, partitioning the organic layer and the aqueous layer,
and removing the aqueous layer.
[0075]
Where necessary, washing with water may be further
applied.
30 Particularly, when the above-mentioned (1) protecting
group using the diphenylmethane compound described in WO
2010/113939Al as a protecting reagent, (2) protecting group
using the fluorene compound described in WO 2010/104169Al as a
protecting reagent, (3) protecting group using the benzyl
35 compound described in WO 2011/078295A1 as a protecting reagent,
(4) protecting group using the branched chain-containing
aromatic compound described in WO 2012/029794Al is used as a
protecting group of the C-terminal carboxy group or amide group,
impurities other than the object product can be efficiently
5 removed into the aqueous layer side by washing with an acidic
aqueous solution, washing with a basic aqueous solution and/or
washing with water to be performed as necessary.
[0076]
By concentrating the organic layer, N-E'moc-C-protected
lo peptide can be obtained. A solution of N-E'moc-C-protected
peptide without concentration or a concentrated solution
thereof may be used for the N-terminal deprotection step
thereafter.
[0077]
15 4-4. N-terminal deprotection step
In the liquid phase peptide synthesis method of the
present invention, the aforementioned removal method of the
Fmoc group may be performed to prepare C-protected peptide, Cprotected
amino acid or C-protected amino acid amide to be used
20 when the elongation reaction is started, or may be performed as
at least one (preferably all) of the N-terminal deprotection
step. In the following, the Fmoc group removal method for the
preparation of C-protected peptide, C-protected amino acid or
C-protected amino acid amide to be used when the elongation
25 reaction is started is explained in the N-terminal deprotection
step.
[0078]
When the removal method of the E'moc group of the present
invention is performed as an N-terminal deprotection step,
30 compound (11) as a byproduct can be sufficiently removed.
Therefore, the C-protected peptide, C-protected amino acid or
C-protected amino acid amide obtained after the N-terminal
deprotection step, which is in the form of a solution, can be
used for the next step (i-e., coupling step (1) and/or (2), or
35 final deprotection step) without isolation as a solid. A
solution of the C-protected peptide and the like may be used
for the next step after concentration as necessary.
[0079]
As mentioned above, when the removal method of the Fmoc
5 group of the present invention is performed as an N-terminal
deprotection step, the obtained C-protected peptide and the
like do not need to be isolated as a solid. Therefore, a
peptide as the final object product can be produced by one-pot
synthesis. Here, the one-pot synthesis means a synthesis
lo method wherein, in the liquid phase peptide synthesis method, a
peptide as the final object product is produced without taking
the intermediate peptide obtained in each step (i-e., synthesis
intermediate) from the reaction vessel.
[ 0 0 8 0 1
15 In addition, when the removal method of the Fmoc group of
the present invention is performed as an N-terminal
deprotection step, N-Fmoc amino acid active ester, which is a
byproduct of the coupling steps (1) and/or (2) and the like can
be trapped by compound (I) and removed by washing thereafter
20 with a basic aqueous solution. Therefore, the workup using
sulfanyl group-supported silica gel and the like after the
coupling steps (1) and/or (2) can be omitted.
[0081]
4-5. Final deprotection step
25 In the final deprotection step, by removing the
protecting group of the C-terminal of the C-protected peptide
having the desired number of amino acid residues and, where
necessary, the protecting group of the side chain functional
group thereof, a peptide as the final object product can be
30 obtained.
[0082]
The method for removing the C-terminal protecting group
and the side chain functional group is not particularly limited,
and a deprotection method known per se can be used.
For example, when the protecting group is a lower alkyl
group such as Me, Et and the like, it can be removed by
reacting C-protected peptide with a base such as sodium
hydroxide, potassium hydroxide and the like in a solvent such
as water, aqueous organic solvent and the like at -20 - 40°C
5 for 0.5 - 10 hr.
When the protecting group is tBu, Pbf, Drnb, bis(4-
methoxypheny1)methyl and the like, it can be removed by
reacting C-protected peptide with an acid such as
trifluoroacetic acid, hydrochloric acid, methanesulfonic acid,
10 tosylic acid, formic acid and the like in a solvent such as
chloroform, methylene chloride, ethyl acetate, dioxane and the
like at -20 - 40°C for 0.5 - 10 hr.
When the protecting group is a Z group, it can be removed
by a hydrogenation reaction of C-protected peptide using a
15 catalyst such as palladium carbon and the like in a solvent
such as methanol, DMF, acetic acid and the like at 0 - 40°C for
0.5 - 100 hr, or by reacting C-protected peptide with a strong
acid such as hydrogen fluoride, trifluoromethanesulfonic acid
and the like at -20 to 40°C for 0.5 - 10 hr.
20 When the protecting group is an Alloc group, it can be
removed by a decomposition reaction of C-protected peptide
using a homogeneous zero-valent palladium catalyst such as
tetrakistriphenylphosphine palladium and the like. The amount
of the homogeneous zero-valent palladium catalyst to be used is
25 generally 0.01 - 1.0 equivalent, preferably 0.05 - 0.5
equivalent, relative to the protecting group to be removed.
[0083]
The obtained peptide as the final object product can be
isolated and purified by a method conventionally used in the
30 peptide chemistry. For example, the final object product can
be isolated and purified by extraction and washing,
crystallization, chromatography and the like of the reaction
mixture in the workup after the final deprotection step.
Examples
35 [00841
The present invention is explained in more detail in the
following by referring to Examples, which are not to be
construed as limitative. In the following, "%" showing the
concentration means "wt%", unless specifically indicated.
5 [00851
Example 1
(i) Condensation of 3,4,5-tri(2',3'-dihydrophyty1oxy)benzyl
alcohol and N-Fmoc amino acid, and subsequent removal of Fmoc
group using 3-mercaptopropionic acid
10 3,4,5-Tri (2' ,3' -dihydrophytyloxy) benzyl alcohol (2.0 g,
2.00 mmol) was dissolved in chloroform (20 ml), Fmoc-Leu-OH
(779 mg, 2.20 mmol) was added, EDC.HC1 (465 mg, 2.43 rnrnol) and
DMAP (24 mg, 0.20 mrnol) were added under ice-cooling, and the
mixture was stirred at room temperature overnight. After
15 completion of the reaction, the solvent was evaporated under
reduced pressure, and the residue was dissolved in CPME (20 ml).
To this solution were added 3-mercaptopropionic acid (0.87 mL,
10.02 mmol) and DBU (1.70 mL, 11.39 mmol) under ice-cooling,
and the mixture was stirred at room temperature for 3 hr.
20 After completion of the reaction, IN hydrochloric acid/CPME
(1.40 mL, 1.40 mrnol) , CPME (10 ml) and 20% brine (20 ml) were
added under ice-cooling to give an organic layer and an aqueous
layer. While stirring the mixture at room temperature, 10%
aqueous sodium carbonate solution was added dropwise until the
25 pH of the aqueous layer became 9.0, and the organic layer and
the aqueous layer were partitioned (hereinafter this operation
is sometimes to be abbreviated as "pH=9.0 washing"). The
pH=9.0 washing was repeated one more time. The obtained
organic layer was washed twice with 10% aqueous sodium
30 carbonate solution (20 ml) and once with 20% brine (20 ml) with
stirring at room temperature, and the organic layer and the
aqueous layer were partitioned. The obtained organic layer was
dried over sodium sulfate and filtered to give a CPME solution
(30 ml) containing H-Leu-OBzl(3,4,5-OPhy). This CPME solution
35 was directly used in the next step.
[0086]
A small amount was sampled from the obtained CPME
solution, and TOF-MS was measured. The measurement results and
the measurement conditions thereof are described below. TOF-MS
5 was measured in the same manner in the below-mentioned Example
l(ii) and the following.
TOF-MS: 1110.9[~~+]
measurement device: Waters LCT Premier XE
capillary voltage: 3000V
sample cone voltage: 86V
dissolution temperature: 350°C
source temperature: 120°C
injection volume: 2 pL
[0087]
15 (ii) Condensation of C-protected amino acid and N-F'moc amino
acid, and subsequent removal of Fmoc group using 3-
mercaptopropionic acid
To the CPME solution (30 ml) of H-Leu-OBz1(3,4,5-OPhy)
obtained in the above-mentioned Example l(i) were added HOBt
20 (81 mg, 0.60 rnrnol) and Fhoc-Tyr(tBu)-OH (1.01 g, 2.20 mmol),
EDC.HC1 (465 mg, 2.43 mmol) was added under ice-cooling, and
the mixture was stirred at room temperature overnight. After
completion of the reaction, the solvent was evaporated under
reduced pressure until the mixture became 20 mL, to the
25 concentrated solution were added under ice-cooling 3-
mercaptopropionic acid (0.85 mL, 9.81 rnrnol) and DBU (1.66 mL,
11.18 mrnol), and the mixture was stirred at room temperature
for 3 hr. After completion of the reaction, to this solution
were added under ice-cooling 1N hydrochloric acid/CPME (1.30 mL,
30 1.30 mrnol) , CPME (15 mL) and 20% brine (25 mL) to give an
organic layer and an aqueous layer. Then, the aforementioned
pH=9.0 washing was performed twice. The obtained organic layer
was washed twice with 10% aqueous sodium carbonate solution and
once with 20% brine with stirring at room temperature, and the
35 organic layer and the aqueous layer were partitioned. The
obtained organic layer was dried over sodium sulfate and
filtered to give a CPME solution of H-Tyr(tBu)-Leu-OBzl(3,4,5-
OPhy). This CPME solution was directly used in the next step.
TOF-MS : 132 9.9 [MH']
5 100881
(iii) Condensation of C-protected peptide and N-F'moc amino acid
or N-Fmoc peptide, and subsequent removal of Fmoc group using
3-mercaptopropionic acid
In the same manner as in the above-mentioned Example
10 l(ii), C-protected peptide obtained in the previous step and
the following N-F'moc amino acid or N-E'moc peptide (indicated as
"N-F'moc amino acid etc." in the following Table 1) were
condensed, and then the Fmoc group was removed, whereby the
peptide chain was sequentially elongated.
15 [00891
Table 1
*' organic layer: After completion of Fmoc group removal
reaction, CPME was added to the reaction solution.
3 7
changes from Example 1
(ii)
organic layer*' 40 mL
organic layer 40 mL
amount of DBU 2.1 mL
(13.45 mrnol)
-
organic layer 40 mL
organic layer 40 mL
pH=9.0 washed three
times
organic layer 40 rnL
each washed twice*'
organic layer 50 mL
each washed twice
organic layer 50 mL
each washed twice
organic layer 65 mL
each washed twice
amount of DBU 2.04 mL
(13.67 mrnol)
organic layer 65 mL
each washed twice
peptide
chain
3rd residue
4th residue
5th residue
6th residue
7th residue
8th residue
9th residue
loth residue
11th residue
lzth residue
13th and
14th
residues
N-Fmoc amino
acid etc.
F'moc-
Glu (OtBu) -OH
Fmoc-
Glu (OtBu) -OH
Frnoc-Pro-OH
Fmoc-Ile-OH
Fmoc-
Glu (OtBu)- OH
moc-
Glu (OtBu) -OH
Fmoc-Phe-OH
F'moc-
A~~ (OtBu) -OH
F'moc-Gly-OH
moc-
A~~ ( ~ ~-OtH )
E'moc-Gly-Gly-
OH
TOF-MS
value [MH']
1514.9
1700.0
1797.1
1910.2
2095.2
2280.3
2427.3
2598.3
2655.3
3011.3
3125-2
*2 each washed twice: pH=9.0 washing, after which washing with
10% aqueous sodium carbonate solution, and washing with 20%
brine, each performed twice.
5 [00901
(iv) Condensation of C-protected peptide and N-Frnoc amino acid,
and subsequent removal of Fmoc group using 3-mercaptopropionic
acid
To the CPME solution (65 liiL) of H-Gly-Gly-Asn (Trt) -Glylo
Asp (OtBu) -Phe-Glu (OtBu) -Glu (OtBu) -1le-Pro-Glu (OtBu) -Glu (OtBu) -
Tyr(tBu)-Leu-OBzl(3,4,5-OPhy) obtained in the above-mentioned
Example 1 (iii) were added HOBt (81 mg, 0.60 mmol) , Fmoc-Gly-
Gly-OH (765 mg, 2.16 mmol) and EDC.HC1 (465 mg, 2.43 mmol), and
the mixture was stirred at room temperature overnight. To this
15 solution were added under ice-cooling 3-mercaptopropionic acid
(0.85 mL, 9.81 rnmol) and DBU (2.34 rnL, 15.68 mrnol), and the
mixture was stirred at room temperature for 3 hr. After
completion of the reaction, the reaction solution was
neutralized with 1N hydrochloric acid/CPME (5 mL, 5.00 mrnol),
20 and the solvent was evaporated under reduced pressure. To the
obtained residue was added 80% by volume aqueous acetonitrile
solution (60 ml), and the precipitate was recovered by
filtration. The recovered precipitate was slurry washed with
acetonitrile (50 ml), then dried to give H-Gly-Gly-Gly-Gly-
25 Asn (Trt) -Gly-Asp (OtBu) -Phe-Glu (OtBu)- Glu (OtBu)- 1le-Pro-
Glu (OtBu)- Glu (OtBu)- Tyr (tBu)- Le~-OBzl(3,4~5-OPh()4 .60 g, 1.42
mol) . The yield of the finally obtained C-protected peptide
was calculated from 3,4,5-tri(2',3'-dihydrophyty1oxy)benzyl
alcohol (2.00 mmol), which is the starting material of Example
30 1 (i) , to find 71%.
~0~-~~:323[MH9']. 2
[0091]
Example 2
(i) Condensation of 4,4' - (2', 3' -
35 dihydrophytyloxy)diphenylmethylamine and N-E'moc amino acid, and
subsequent removal of Fmoc group using 3-mercaptopropionic acid
38
4,4' - (2' ,3' -Dihydrophytyloxy) diphenylmethylamine (2.0 g,
2.38 mmol) was dissolved in chloroform (20 ml) , HOBt (32 mg,
0.24 mmol) and F'moc-Leu-OH (1.02g, 2.89 mmol) were added,
EDC.HC1 (607 mg, 3.17 mmol) was added under ice-cooling, and
5 the mixture was stirred at room temperature overnight. After
completion of the reaction, the solvent was evaporated under
reduced pressure, and the residue was dissolved in CPME (20 ml) .
To this solution were added under ice-cooling 3-
mercaptopropionic acid (1.03 mL, 11.89 mmol) and DBU (2.02 mL,
lo 13.55 mmol), and the mixture was stirred at room temperature
for 3 hr. After completion of the reaction, to this solution
were added under ice-cooling IN hydrochloric acid/CPME (1.50 mL,
1.50 mmol) , CPME (10 ml) and 20% brine (20 ml) to give an
organic layer and an aqueous layer. Then, the aforementioned
is pH=9.0 washing was performed twice. The obtained organic layer
was washed once with a mixed solvent of 10% aqueous sodium
carbonate solution (20 ml) and DMF (4 mL), once with 10%
aqueous sodium carbonate solution (20 ml) and twice with 20%
brine (20 ml) with stirring at room temperature, and the
20 organic layer and the aqueous layer were partitioned. The
obtained organic layer was dried over sodium sulfate and
filtered to give a CPME solution (30 ml) of H-Leu-NHDpm(4,4'-
OPhy). This CPME solution was directly used in the next step.
TOF-MS : 8 8 9.6 [MH']
2s COO921
(ii) Condensation of C-protected amino acid amide and N-Fmoc
amino acid, and subsequent removal of F'moc group using 3-
mercaptopropionic acid
To the CPME solution (30 ml) of H-Leu-NHDpm (4,4' -0Phy)
30 were added HOBt (192 mg, 1.43 mmol) and Fmoc-Tyr (tBu) -OH (1.26
g, 2.74 rnrnol), EDC.HC1 (607 mg, 3.17 mmol) was added under icecooling,
and the mixture was stirred at room temperature
overnight. After completion of the reaction, the solvent was
evaporated under reduced pressure until the mixture became 25
35 mL, to the concentrated solution were added under ice-cooling
3-mercaptopropionic acid (1.03 mL, 11.89 mmol) and DBU (2.02 mL,
13.55 mmol), and the mixture was stirred at room temperature
for 3 hr. After completion of the reaction, to this solution
were added under ice-cooling IN hydrochloric acid/CPME (1.50 mL,
5 1.50 mmol) , CPME (5 mL) and 20% brine (25 mL) to give an
organic layer and an aqueous layer. Then, the aforementioned
pH=9.0 washing was performed twice. The obtained organic layer
was washed twice with 10% aqueous sodium carbonate solution and
twice with 20% brine with stirring, and the organic layer and
10 the aqueous layer were partitioned. The obtained organic layer
was dried over sodium sulfate and filtered to give a CPME
solution of H-Tyr (tBu) -Leu-NHDpm (4 , 4' -0Phy) . This CPME
solution was directly used in the next step.
TOF-MS : 1108.7 [MH']
is [00931
(iii) Condensation of C-protected peptide and N-Fmoc amino acid,
and subsequent removal of E'moc group using 3-mercaptopropionic
acid
To the CPME solution (30 ml) of H-Tyr(tBu)-Leu-
20 NHDpm(4,4' -0Phy) were added HOBt (96 mg, 0.71 mmol) and Fmoc-
Glu(0tBu) -OH (1.11 g, 2.61 mrnol) , EDC.HC1 (552 mg, 2.88 mmol)
was added under ice-cooling, and the mixture was stirred at
room temperature overnight. After completion of the reaction,
the solvent was evaporated under reduced pressure until the
25 mixture became 25 mL, to the concentrated solution were added
under ice-cooling 3-mercaptopropionic acid (0.62 mL, 7.13 mmol)
and DBU (1.60 mL, 10.70 mmol), and the mixture was stirred at
room temperature for 3 hr. After completion of the reaction,
to this solution were added under ice-cooling 1N hydrochloric
30 acid/CPME (3 -21 mL, 3.21 mmol) , CPME (10 ml) and 20% brine (25
mL) to give an organic layer and an aqueous layer. Then, the
aforementioned pH=9.0 washing was performed three times. The
obtained organic layer was washed once with a mixed solvent of
10% aqueous sodium carbonate solution (25 mL) and DMF (5 rnL),
35 twice with 10% aqueous sodium carbonate solution (25 rnL) and
twice with 20% brine (25 mL) with stirring at room temperature,
and the organic layer and the aqueous layer were partitioned.
The obtained organic layer was dried over sodium sulfate and
filtered to give a CPME solution of H-Glu(0tBu)-Tyr(tBu)-Leu-
5 NHDpm(4,4' -0Phy) . This CPME solution was directly used in the
next step .
TOF-MS: 1293.8 [MH']
[0094]
(iv) Condensation of C-protected peptide and N-Fmoc amino acid,
10 and subsequent removal of F'moc group using 3-mercaptopropionic
acid
In the same manner as in the above-mentioned Example
2(iii), the C-protected peptide obtained in the previous step
and the following N-F'moc amino acid were condensed, and then
15 the Fmoc group was removed, whereby the, peptide chain was
sequentially elongated.
[0095]
Table 2
peptide
chain
4th
residue
5th
residue
6th
residue
T°F-MS
value [MH']
1478.8
N-Fmoc
amino acid
F'moc-
Glu (OtBu) -
OH
1874.0
Glu (OtBu) -
residue
2059.1
changes from Example 2 (iii)
no washing with mixed solvent
of 10% aqueous sodium
carbonate solution and DMF
orqanic layer*' 40 mL
F'moc-Pro-OH
F'moc-Leu-OH
organic layer 40 mL
organic layer 40 mL
amount of DBU 1.95 mL (13.08
rnrnol)
9th
residue
loth
residue
*' organic layer: After completion of F'moc group removal
'Ith
residue
1575'
1688.9
F'moc-
Lys (Boc) -OH
F'moc-Pro-OH
-
no washing with mixed solvent
of 10% aqueous sodium
carbonate solution and DMF
-
Fmoc-Gly-OH
2287.2
2384.1
organic layer 40 mL
organic layer 40 mL
amount of DBU 1.95 mL (13.08
mrnol)
2441.2
organic layer 45 mL
DMF 9 mL was added in 3rd
pH=9.0 washing
reaction, CPME was added to the reaction solution.
[ 0 0 9 6 I
(v) Condensation of C-protected peptide and N-Fmoc amino acid,
5 and subsequent removal of Fmoc group using 3-mercaptopropionic
acid
To the CPME solution (45 mL) of H-Gly-Pro-Lys (Boc) -
Glu (OtBu) -Glu (OtBu) -Leu-Pro-Glu (OtBu) -Glu (OtBu) -Tyr (tBu) -Leu-
NHDpm(4,4'-OPhy) obtained in the above-mentioned Example 2 (iv)
lo were added HOBt (192 mg, 1.43 mmol), Fmoc-Gly-Gly-OH (765 mg,
2.62 mmol) and EDC. HC1 (552 mg, 2.88 mmol) , and the mixture was
stirred at room temperature overnight. After completion of the
reaction, 3-mercaptopropionic acid (0.62 mL, 7.13 mmol) and DBU
(1.78 mL, 11.89 mmol) were added under ice-cooling, and the
zs mixture was stirred at room temperature for 3 hr. After
completion of the reaction, the reaction solution was
neutralized with 1N hydrochloric acid/CPME (4.28 mL, 4.28 mrnol)
and the solvent was evaporated under reduced pressure. To the
obtained residue was added 80% by volume aqueous acetonitrile
20 solution (70 ml), and the precipitate was recovered by
filtration. The recovered precipitate was slurry washed with
acetonitrile (50 ml), then dried to give H-Gly-Pro-Lys(Boc)-
Glu (OtBu) -Glu (OtBu) -Leu-Pro-Glu (OtBu) -Glu (OtBu) -Tyr (tBu) -Leu-
NHDpm ($,$' -0Phy) (4.40 g, 1.72 mmol) . The yield of the finally
25 obtained C-protected peptide was calculated from 4,4r-(2r,3rdihydrophytyloxy)
diphenylmethylamine (2.38 mmol), which is the
starting material of Example 2(i), to find 72%.
TOF-MS: 2555.1 [MH']
[0097]
30 Example 3
(i) Condensation of 2,4-di (2' ,3' -dihydrophytyloxy) benzyl
alcohol and N-Fmoc amino acid, and subsequent removal of Fmoc
group using 3-mercaptopropionic acid
2,4-Di(2',3'-dihydrophyty10xy)benzyl alcohol (2.0 g, 2.85
35 mmol) was dissolved in chloroform (20 ml) , Fmoc-Gly-OH (1.12 g,
3.77 mmol) was added, EDC.HC1 (794 mg, 4.14 mrnol) and DMAP (42
mg, 0.34 mmol) were added under ice-cooling, and the mixture
was stirred at room temperature overnight. After completion of
the reaction, the solvent was evaporated under reduced pressure,
5 and the residue was dissolved in CPME (20 ml). To this
solution were added under ice-cooling 3-mercaptopropionic acid
(0.74 mL, 8.56 rnrnol) and DBU (1.91 mL, 12.83 mmol) , and the
mixture was stirred at room temperature for 3 hr. After
completion of the reaction, to this solution were added under
lo ice-cooling 1N hydrochloric acid/CPME (3.85 mL, 3.85 mmol),
CPME (15 mL) and 20% brine (25 mL) to give an organic layer and
an aqueous layer. Then, the aforementioned pH=9.0 washing was
performed twice. The obtained organic layer was washed once
with a mixed solvent of 10% aqueous sodium carbonate solution
15 (25 mL) and DMF (2.5 mL) , twice with 10% aqueous sodium
carbonate solution (25 mL) and three times with 20% brine (25
mL) with stirring, and the organic layer and the aqueous layer
were partitioned. The obtained organic layer was dried over
sodium sulfate and filtered to give a CPME solution (35 mL) of
20 H-Gly-OBzl(2,4-OPhy). This CPME solution was directly used in
the next step.
TOF-MS : 758.6 [MH']
[0098]
(ii) Condensation of C-protected amino acid and N-Fmoc amino
25 acid, and subsequent removal of Fmoc group using 3-
mercaptopropionic acid
To the CPME solution (35 mL) of H-Gly-OBzl(2,4-OP~;) were
added HOBt (116 mg, 0.86 mmol) and Fmoc-Glu(0tBu)-OH (1.60 g,
3.76 mmol), EDC.HC1 (794 mg, 4.14 rnmol) was added under ice-
30 cooling, and the mixture was stirred at room temperature
overnight. After completion of the reaction, the solvent was
evaporated under reduced pressure until the mixture became 20
mL, to the concentrated solution was added under ice-cooling
DBU (1.02 mL, 6.84 mmol), and the mixture was stirred at room
35 temperature for 3 hr. After completion of the reaction, to
this solution were added under ice-cooling 1N hydrochloric
acid/CPME (5.48 mL, 5.48 mmol) , CPME (20 ml) and 20% brine (30
ml) to give an organic layer and an aqueous layer. Then, the
aforementioned pH=9.0 washing was performed twice. The
5 obtained organic layer was washed once with a mixed solvent of
10% aqueous sodium carbonate solution (30 ml) and DMF (3 mL),
once with 10% aqueous sodium carbonate solution (30 ml) and
twice with 20% brine (30 ml) with stirring at room temperature,
and the organic layer and the aqueous layer were partitioned.
lo The obtained organic layer was dried over sodium sulfate and
filtered to give a CPME solution (40 ml) of H-Glu(0tBu)-Gly-
OBz1(2,4-OPhy), which was directly transferred to the next step.
TOF-MS : 943.5 [MH']
[0099]
15 (iii) Condensation of C-protected peptide and N-Fmoc amino acid,
and subsequent removal of Fhoc group using 3-mercaptopropionic
acid
To the CPME solution (40 mL) of H-Glu (OtBu) -Gly-OBzl(2,4-
OPhy) were added HOBt (116 mg, 0.86 mmol) and Fhoc-Leu-OH (1.11
20 g, 3.14 mmol) , EDC. HC1 (662 mg, 3.45 mmol) was added under icecooling,
and the mixture was stirred at room temperature
overnight. After completion of the reaction, the solvent was
evaporated under reduced pressure until the mixture became 25
mL, to the concentrated solution were added under ice-cooling
25 3-mercaptopropionic acid (0.74 mL, 8.56 mmol) and DBU (1.91 rnL,
12.83 rnrnol), and the mixture was stirred at room temperature
for 3 hr. After completion of the reaction, to this solution
were added under ice-cooling IN hydrochloric acid/CPME (3.85 rnL,
3.85 mmol) , CPME (15 mL) and 20% brine (25 mL) to give an
30 organic layer and an aqueous layer. Then, the aforementioned
pH=9.0 washing was performed three times. The obtained organic
layer was washed once with a mixed solvent of 10% aqueous
sodium carbonate solution (25 mL) and DMF (2.5 mL), twice with
10% aqueous sodium carbonate solution (25 mL) and three times
35 with 20% brine (25 mL) with stirring at room temperature, and
the organic layer and the aqueous layer were partitioned. The
obtained organic layer was dried over sodium sulfate and
filtered to give a CPME solution of H-Leu-Glu(0tBu)-Gly-
OBz1(2,4-OPhy). This CPME solution was directly used in the
5 next step.
TOF-MS: 1056.7 [MH']
[OlOO]
(iv) Condensation of C-protected peptide and N-Fmoc amino acid,
and subsequent removal of E'moc group using 3-mercaptopropionic
l o acid
In the same manner as in the above-mentioned Example
3(iii), the C-protected peptide obtained in the previous step
and the following N-Fmoc amino acid were condensed, and then
the Fmoc group was removed, whereby the peptide chain was
15 sequentially elongated.
[OlOl]
Table 3
TOF-MS
1275.7
changes from Example 3 (iii)
6th
residue
amount of HOBt 193 mg (1.43
mmol )
amount of HOBt 193 mg (1.43
Fmoc-
Ser (tBu) -OH
mmol )
DMF 3 mL was added in 1st and
1561.9
3rd pH=9.0 washings
amount of HOBt 193 mg (1.43
mmo 1 )
organic layer*' 50 mL
DMF 3 mL was added in 3rd
pH=9.0 washing
amount of DBU 2.12 rnL (14.20
rnrnol)
organic layer 50 mL
DMF 3 mL was added in 1st
~H=9.0 washina
*' organic layer: After completion of Fmoc group removal
reaction, CPME was added to the reaction solution.
20
[0102]
(v) Condensation of C-protected peptide and N-Fmoc amino acid,
and subsequent removal of E'moc group using 3-mercaptopropionic
acid
To the CPME solution (80 ml) of H-Val-Ser (tBu) -Ser (tBu) -
Tyr (tBu) -Leu-Glu (OtBu) -Gly-OBz1 (2, 4-OPhy obtained in the
above-mentioned Example 3(iv) were added HOBt (193 mg,. 1.43
5 mmol), Fmoc-Asp(0tBu)-OH (1.29 g, 3.14 rnmol) and EDC.HC1 (662
mg, 3.45 mrnol), and the mixture was stirred at room temperature
overnight. After completion of the reaction, 3-
mercaptopropionic acid (0.74 mL, 8.56 rnrnol) and DBU (1.91 mL,
12.83 rnmol) were added under ice-cooling, and the mixture was
lo stirred at room temperature for 3 hr. After completion of the
reaction, and the mixture was neutralized with a mixture of
MsOH (0.25 mL, 3.85 mmol) and chloroform (2.5 mL) and the
solvent was evaporated under reduced pressure. To the obtained
residue was added 80% by volume of aqueous acetonitrile
15 solution (60 ml), and the precipitate was recovered by
filtration. The recovered precipitate was slurry washed with
acetonitrile (50 ml), then dried to give H-Asp(0tBu)-Val-
Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-OBzl (2, 4-
OPhy) (3.62 g, 1.97 mmol) . The yield of the finally obtained C-
20 protected peptide was calculated from 2,4-di(2',3'-
dihydrophyty1oxy)benzyl alcohol (2.85 mmol), which is the
starting material of Example 3(i), to find 69%.
TOF-MS: 1832.0 [MH']
[0103]
25 Example 4
(i) Condensation of 2- (3,4,5-tri (2', 3'-
dihydrophytyloxy)benzyloxy)-4-methoxybenzyl alcohol and N-Fmoc
amino acid, and subsequent removal of E'moc group using 3-
mercaptopropionic acid
30 2- (3,4,5-Tri (2' ,3' -dihydrophytyloxy) benzyloxy) -4-
methoxybenzyl alcohol (2.0 g, 1.76 mmol) was dissolved in
chloroform (25 mL) , Fmoc-Ser (tBu) -OH (176 mg, 0.46 mrnol) ,
EDC.HC1 (97 mg, 0.51 mmol) and DMAP (2.2 mg, 0.018 mmol) were
added six times every 30 min under ice-cooling, and the mixture
35 was stirred at room temperature overnight. After completion of
the reaction, the solvent was evaporated under reduced pressure,
and the residue was dissolved in CPME (20 ml). To this
solution were added under ice-cooling 3-mercaptopropionic acid
(0.46 mL, 5.27 mrnol) and DBU (1.34 mL, 9.00 mmol), and the
5 mixture was stirred at room temperature for 3 hr. After
completion of the reaction, to this solution were added
dropwise under ice-cooling a mixture of MsOH (0.22 mL, 3.33
mmol) and CPME (2.2 rnL) , and CPME (15 mL) and 20% brine (30 ml)
were added to give an organic layer and an aqueous layer. Then,
10 the aforementioned pH=9.0 washing was performed three times.
The obtained organic layer was washed once with a mixed solvent
of 10% aqueous sodium carbonate solution (30 ml) and DMF (3 mL),
twice with 10% aqueous sodium carbonate solution (30 ml) and
three times with 20% brine (30 ml) with stirring at room
15 temperature, and the organic layer and the aqueous layer were
partitioned. The obtained organic layer was dried over sodium
sulfate and filtered to give a CPME solution (35 mL) of HSer
(tBu) -OBzl(2-OBzl(3,4,5-OPhy) -4-OMe) . This CPME solution
was directly used in the next step.
[0104]
(ii) Condensation of C-protected amino acid and N-E'moc amino
acid
To the CPME solution (35 mL) of H-Ser(tBu)-OBzl(2-
25 OBz1(3,4,5-0Phy)-4-OMe) was added HOBt (71 mg, 0.43 mmol),
Fmoc-Thr (tBu) -OH (1.00 g, 2.51 mmol) , EDC.HC1 (529 mg, 2.76
mrnol) and DMF (10 ml) were added under ice-cooling, and the
mixture was stirred at room temperature overnight. After
completion of the reaction, to the reaction solution was added
30 20% brine (30 ml) to give an organic layer and an aqueous layer.
While stirring the obtained organic layer and aqueous layer,
10% aqueous sodium carbonate solution was added dropwise until
the pH of the aqueous layer became 6.0, and the organic layer
and the aqueous layer were partitioned (hereinafter this
35 operati-on is sometimes to be abbreviated as "pH=6.0 washing").
The pH=6.0 washing was further repeated twice. The solvent in
the obtained organic layer was evaporated under reduced
pressure, the residue was dissolved in cyclohexane (40 ml), and
the mixture was washed four times with 80% by volume of aqueous
5 acetonitrile solution (30 ml) with stirring, and the organic
layer and the aqueous layer were partitioned. The obtained
organic layer was dried over sodium sulfate and filtered. The
solvent in the obtained filtrate was evaporated under reduced
pressure to give Frnoc-Thr (tBu) -Ser (tBu) -OBz1(2-OBz1(3,4,5-
10 OPhy) -4-OMe) .
TOF-MS : 1655.9 [MH']
[0105]
(iii) Removal of Fmoc group, and subsequent condensation of Cprotected
peptide and N-Fmoc amino acid
15 Fmoc-Thr (tBu) -Ser (tBu) -OBzl(2-OBzl(3,4,5-OPhy) -4-OMe) was
dissolved in CPME (25 mL). To this solution was added DBU
(0.26 mL, 1.76 mmol) under ice-cooling, and the mixture was
stirred at room temperature for 3 hr. Compound (I) was not
used for the removal of the Fmoc group.
20 After completion of the reaction, to this solution were
added under ice-cooling 1N hydrochloric acid/CPME (1.67 mL,
1.67 mmol) , HOBt (237 mg, 1.76 mmol) , Fmoc-Phe-OH (1.16 g, 2.98
mmol), EDC.HC1 (629 mg, 3.28 mmol) and DMF (6 mL), and the
mixture was stirred at room temperature overnight. After
25 completion of the reaction, 20% brine (30 ml) was added to give
an organic layer and an aqueous layer. Then, the
aforementioned pH=6.0 washing was performed three times. The
solvent in the obtained organic layer was evaporated under
reduced pressure, the residue was dissolved in cyclohexane (40
30 ml), and the mixture was washed four times with 80% by volume
of aqueous acetonitrile solution (30 ml) and once with 20%
brine (30 ml) with stirring, and the organic layer and the
aqueous layer were partitioned. The obtained organic layer was
dried over sodium sulfate and filtered. The solvent in the
35 obtained filtrate was evaporated under reduced pressure to give
F'moc-Phe-Thr (tBu) -Ser (tBu) -OBzl(2-OBzl(3,4,5-OPhy) -4-OMe) .
TOF-MS: 1802.9 [MH']
[0106]
(iv) Removal of F'moc group, and subsequent condensation of C-
5 protected peptide and N-Fmoc amino acid
The total amount of F'moc-Phe-Thr (tBu) -Ser (tBu) -OBzl(2-
OBzl(3,4,5-OPhy) -4-0Me) obtained in Example 4 (iii) was
dissolved in CPME (25 mL). To this solution was added under
ice-cooling DBU (0.26 mL, 1.76 mmol) , and the mixture was
lo stirred at room temperature for 3 hr. Compound (I) was not
used for the removal of the F'moc group.
After completion of the reaction, to this solution were
added under ice-cooling 1N hydrochloric acid/CPME (1.67 mL,
1.67 mrnol) , HOBt (237 mg, 1.76 mrnol) , Fmoc-Thr (tBu) -OH (0.91 g,
15 2.29 mmol), EDC.HC1 (629 mg, 3.28 mmol) and DMF (6 mL), and the
mixture was stirred at room temperature overnight. After
completion of the reaction, 20% brine (30 ml) was added to give
an organic layer and an aqueous layer. Then, the
aforementioned pH=6.0 washing was performed three times. The
20 solvent in the obtained organic layer was evaporated under
reduced pressure, the residue was dissolved in cyclohexane (40
ml), and the mixture was washed once with 20% brine (30 ml)
with stirring, and the organic layer and the aqueous layer were
partitioned. The obtained organic layer was dried over sodium
2s sulfate and filtered. The solvent in the obtained filtrate was
evaporated under reduced pressure to give E'moc-Thr(tBu)-Phe-
Thr (tBu) -Ser (tBu) -OBz1(2-OBzl(3 , 4,550Phy) -4-OMe) .
TOF-~~:1959.9[~~+]
[0107]
30 (v) Removal of F'moc group using 3-mercaptopropionic acid, and
subsequent condensation of C-protected peptide and N-Fmoc amino
acid
F'moc-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -OBzl(2-OBzl(3,4,5-
0Phy)-4-OMe) obtained in Example 4(iv) was dissolved in CPME to
35 give a CPME solution (30 ml). To this CPME solution were added
under ice-cooling 3-mercaptopropionic acid (0.46 mL, 5.27 mmol)
and DBU (1.18 mL, 7.90 mmol), and the mixture was stirred at
,room temperature for 3 hr. After completion of the reaction,
to this solution was added under ice-cooling a mixture of MsOH
5 (0.15 mL, 2.37 mrnol) and CPME (1.5 mL) , CPME (20 ml) and 20%
brine (30 ml) to give an organic layer and an aqueous layer.
Then, the aforementioned pH=9.0 washing was performed twice.
The obtained organic layer was washed three times with 10%
aqueous sodium carbonate solution and three times with 20%
lo brine with stirring at room temperature, and the organic layer
and the aqueous layer were partitioned. The obtained organic
layer was dried over sodium sulfate and filtered to give a CPME
solution of H-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -OBzl(2-OBzl(3, 4 , 5-
0Phy)-4-OMe) . To this CPME solution were added under ice-
15 cooling HOBt (71 mg, 0.53 mmol), knoc-Lys(Boc)-OH (994 mg, 2.12
mrnol) and EDC.HC1 (402 mg, 2-01 mmol), and the mixture was
stirred at room temperature overnight. After completion of the
reaction, the solvent in the reaction solution was evaporated
under reduced pressure, 80% by volume of aqueous acetonitrile
20 solution (40 ml) was added, and the precipitate was recovered
by filtration. The recovered precipitate was dried to give
knoc-Lys (Boc) -Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -OBzl(2-OBzl(3,4,5-
OPhy) -4-OMe) (3.56 g, 1.63 mmol) . The yield of the finally
obtained N-Fmoc-C-protected peptide was calculated from 2-
25 (3, 4,5-tri (2', 3' -dihydrophytyloxy) benzyloxy) -4-methoxybenzyl
alcohol (1.76 mmol), which is the starting material of Example
4(i), to find 93%.
TOF-~~:21818 .[MH ']
[0108]
30 Example 5
(i) Condensation of C-protected amino acid salt and N-Fmoc
amino acid, and subsequent removal of Fmoc group using
thiomalic acid
H-Leu-0Bzl.TsOH salt (394 mg, 1.00 mmol) was dissolved in
35 chloroform (10 ml) , triethylamine (0.14 mL, 1.00 mmol) , HOBt
(14 mg, 0.10 mmol) and Ehoc-Tyr (tBu) -OH (506 mg, 1.10 -01)
were added, EDC.HC1 (232 mg, 1.21 mmol) was added under icecooling,
and the mixture was stirred at room temperature
overnight. After completion of the reaction, to this solution
5 were added under ice-cooling thiomalic acid (450 mg, 3.00 mmol)
and DBU (1.49 mL, 10.00 mmol), and the mixture was stirred at
room temperature for 3 hr. After completion of the reaction,
to this solution was added dropwise under ice-cooling a mixture
of MsOH (0 -23 mL, 3.60 mrnol) and chloroform (2.3 mL) ,
10 chloroform (10 ml) and 20% brine (20 ml) were added to give an
organic layer and an aqueous layer. Then, the aforementioned
pH=9.0 washing was performed three times. The obtained organic
layer was washed three times with 10% aqueous sodium carbonate
solution (20 ml) and three times with 20% brine (20 ml) with
15 stirring at room temperature, and the organic layer and the
aqueous layer were partitioned. The obtained organic layer was
dried over sodium sulfate and filtered to give a chloroform
solution (20 ml) of H-Tyr (tBu) -Leu-OBzl. This chloroform
solution was directly used in the next step.
20 TOF-MS : 441.2 [MH']
[0109]
(ii) Condensation of C-protected amino acid and N-Fmoc amino
acid, and subsequent removal of Fmoc group using thiomalic acid
To the chloroform solution (20 ml) of H-Tyr(tBu)-Leu-OBzl
25 was added HOBt (41 mg, 0.30 mmol) , Ehoc-Glu (OtBu) -OH (468 mg,
1.10 mmol) and EDC.HC1 (232 mg, 1.21 mmol) were added under
ice-cooling, and the mixture was stirred at room temperature
overnight. After completion of the reaction, to this solution
were added under ice-cooling thiomalic acid (450 mg, 3.00 mmol)
30 and DBU (1.49 mL, 10.00 mmol), and the mixture was stirred at
room temperature for 3 hr. After completion of the reaction,
to this solution was added dropwise under ice-cooling a mixture
of MsOH (0.23 mL, 3.60 rnrnol) and chloroform (2.3 mL) ,
chloroform (10 ml) and 20% brine (20 ml) were added to give an
35 organic layer and an aqueous layer. Then, the aforementioned
pH=9.0 washing was performed three times. The obtained organic
layer was washed three times with 10% aqueous sodium carbonate
solution (20 ml) and twice with 20% brine (20 ml) with stirring
at room temperature, and the organic layer and the aqueous
5 layer were partitioned. The obtained organic layer was dried
over sodium sulfate and filtered to give a chloroform solution
(30 ml) of H-Glu (OtBu) -Tyr (tBu) -Leu-OBzl. This chloroform
solution was directly used in the next step.
TOF-MS: 626.2 [MH']
lo [0110]
(iii) condensation of C-protected peptide and N-E'moc amino acid,
and subsequent removal of Fmoc group using thiomalic acid
To the chloroform solution (30 ml) of H-Glu (OtBu) -
Tyr(tBu)-Leu-OBzl was added HOBt (68 mg, 0.50 mmol), E'moc-
15 Glu(0tBu)-OH (468 mg, 1.10 mmol) and EDC.HC1 (232 mg, 1.21
mmol) were added under ice-cooling, and the mixture was stirred
at room temperature overnight. After completion of the
reaction, the solvent was evaporated under reduced pressure
until the mixture became 15 mL, to the concentrated solution
20 were added under ice-cooling thiomalic acid (450 mg, 3.00 mmol)
and DBU (1.34 mL, 9.00 mmol), and the mixture was stirred at
room temperature for 3 hr. After completion of the reaction,
to this solution was added dropwise under ice-cooling a mixture
of MsOH (0.17 mL, 2.70 mmol) and chloroform (1.7 mL),
25 chloroform (15 mL) and 20% brine (30 ml) was added to give an
organic layer and an aqueous layer. Then, the aforementioned
pH=9.0 washing was performed three times. The obtained organic
layer was washed three times with 10% aqueous sodium carbonate
solution (30 ml) and twice with 20% brine (30 ml) with stirring
30 at room temperature, and the organic layer and the aqueous
layer were partitioned. The obtained organic layer was dried
over sodium sulfate and filtered. The filtrate was recovered,
and the solvent in the recovered filtrate was evaporated under
reduced pressure. To the obtained residue was added hexane (10
35 ml), and the precipitate was recovered by filtration. The
recovered precipitate was dried to give H-Glu(0tBu)-Glu(0tBu)-
Tyr (tBu) -Leu-OBzl (787 mg, 0.97 mmol) . The yield of the
finally obtained C-protected peptide was calculated from H-Leu-
OBzl-TsOH salt (1.00 mmol), which is the starting material of
5 Example 7 (i) , to find 97%.
TOE-MS: 811.2 [MH']
[Olll]
Example 6: Removal of Fmoc group using thiomalic acid
Fmoc-Leu-OBzl(3,4,5-OPhy) (0.075 mmol) was dissolved in
l o chloroform (1 mL) , thiomalic acid (33.8 mg, 0.23 mmol) and DBU
(0.09 mL, 0.60 mmol) were added under ice-cooling, and the
mixture was stirred at room temperature for 4 hr. The
production rate of compound (11) (i-e., DBF-thiomalic acid
adduct) on completion of the reaction was 91%. The production
15 rate of compound (11) was calculated by quantitative analysis
of DBF by HPLC using synthesized DBF as a standard product,
calculating the production rate of DBF and then calculating the
production rate of compound 2 (100-production rate of DBF).
The calculation method of the following production rates is the
20 same.
HPLC conditions are as described below.
measurement device: Waters AQUITY UPLC BEH C18 50 rnrn x
2.1 mm I.D., 1.7 pm
measurement temperature: 40°C
25 flow rate: 0.4 mL/min
injection volume: 2 pL
measurement wavelength: 220 nm
mobile phase A: 0.05% by volume aqueous trifluoroacetic
acid solution
30 mobile phase B: 0.05% by volume trifluoroacetic acid
acetonitrile solution
gradient time program: the concentration of mobile phase
B was linearly increased from 20% by volume to 90% by volume in
10 min.
35 [01121
After completion of the reaction, to this solution were
added under ice-cooling 1N hydrochloric acid/CPME (0.05 rnL,
0.05 mrnol) , chloroform (5 mL) and 20% brine (5 mL) and, while
stirring the mixture at room temperature, 10% aqueous sodium
5 carbonate solution was added dropwise until the pH of the
mixture became 9.0, and the organic layer and the aqueous layer
were partitioned. The obtained organic layer was washed three
times with 10% aqueous sodium carbonate solution (20 ml) at
room temperature, and the organic layer and the aqueous layer
lo were partitioned. The absence of compound (11) in the organic
layer (i.e., compound (11) was entirely removed into the
aqueous layer) was confirmed by HPLC.
[0113]
Example 7: Removal of Fmoc group using cysteine
15 F'moc-Leu-OBzl(3,4,5-OPhy) (0.075 mmol) was dissolved in
chloroform (1 mL) , cysteine (27 mg, 0.23 mmol) and DBU (0.022
mL, 0.15 mmol) were added under ice-cooling, and the mixture
was stirred at room temperature for 3 hr. The production rate
of compound (11) (i.e., DBF-cysteine adduct) on completion of
20 the reaction was 95%. After completion of the reaction, the
reaction solution was concentrated, to the concentrated
reaction solution was added CPME (5 rnL), and the reaction
solution was washed three times with 10% aqueous sodium
carbonate solution (5 mL) at room temperature to remove
25 compound (11) .
[0114]
Comparative Example 1: Removal of F'moc group using
thiosalicylic acid
F'moc-Leu-OBzl(3,4,5-OPhy) (0.075 mmol) was dissolved in
30 CPME (I mL), thiosalicylic acid (35 mg, 0.23 mmol) and DBU
(0.041 mL, 0.28 mrnol) were added under ice-cooling, and the
mixture was stirred at room temperature for 3.5 hr. The
production rate of DBF-thiosalicylic acid adduct on completion
of the reaction was 21%.
35 Industrial Applicability
[0115]
The removal method of the E'moc group of the present
invention can be utilized for industrial production of peptide
and the like.
5 [01161
This application is based on patent application No. 2011-
274901 filed in Japan, the contents of which are encompassed in
full herein.
Although the present invention have been presented or
l o described by referring to preferred embodiments of this
invention, it will, however, be understood by those of ordinary
skill in the art that various modifications may be made to the
forms and details without departing from the scope of the
invention as set forth in the appended claims. All patents,
15 patent publications and other publications indicated or cited
in the Specification are hereby incorporated in their
entireties by reference.
CLAIMS
1. A method of removing an F'moc group, comprising a step of
mixing a compound represented by the formula (I):
5 HS-L-COOH (I)
wherein L is a CL-* alkylene group optionally having
substituent(s), an amino group-containing compound protected by
an Frnoc group, and a base to give a reaction mixture containing
a compound represented by the formula (11):
10 Fm-S-L-COOH (11)
wherein F'm is a 9-fluorenylmethyl group, and L is as mentioned
above, and an amino group-containing compound, and a step of
removing the compound represented by the formula (11) by
washing the obtained reaction mixture with a basic aqueous
15 solution.
2. The method according to claim 1, wherein the base is an
organic base.
20 3 . The method according to claim 2, wherein the organic base is
1,8-diazabicyclo[5.4.0]-7-undecene.
4. The method according to any one of claims 1 to 3, wherein
the compound represented by the formula (I) is at least one
25 selected from the group consisting of 3-mercaptopropionic acid,
thiomalic acid and cysteine.
5. The method according to any one of claims 1 to 4, wherein
the basic aqueous solution is an aqueous solution of at least
30 one selected from the group consisting of lithium carbonate,
potassium carbonate, sodium carbonate, lithium hydrogen
carbonate, potassium hydrogen carbonate, sodium hydrogen
carbonate, lithium hydroxide, potassium hydroxide and sodium
hydroxide.
6. The method according to any one of claims 1 to 5, wherein
the amino group-containing compound protected by an Fmoc group
is N-Fmoc-C-protected peptide, N-F'moc-C-protected amino acid or
N-Fmoc-C-protected amino acid amide, and
5 the obtained amino group-containing compound is Cprotected
peptide, C-protected amino acid or C-protected amino
acid amide.
7. A method of producing a peptide by a liquid phase synthesis
10 method, comprising the method according to claim 6.
8. The production method according to claim 7, comprising (1) a
step of condensing C-protected peptide, C-protected amino acid
or C-protected amino acid amide, and N-Fmoc amino acid or Nis
F'moc peptide in the presence of a condensing agent to give an
N-Fmoc-C-protected peptide, and/or
(2) a step of condensing C-protected peptide, C-protected amino
acid or C-protected amino acid amide, and N-F'moc amino acid
active ester or N-Fmoc peptide active ester to give an N-F'moc-
20 C-protected peptide.
9. The production method according to claim 8, wherein the step
(1) is performed in the further presence of an activator.
25 10. The production method according to claim 8 or 9, wherein
the C-protected peptide, C-protected amino acid or C-protected
amino acid amide obtained by the method according to claim 6 is
used in the step (1) and/or the step (2) without isolating as a
solid.
30
11. The production method according to claim 10, wherein the
peptide is produced by one-pot synthesis.
Dated this loth day of July 2014
5 7 Of Anand and Anand Advocates
Agent for the Applicant