α-chymotrypsin-catalysed peptide synthesis via the kinetically controlled approach using activated esters as acyl donors in organic solvents with low water content: Incorporation of non-protein amino acids into peptides

Article abstract of DOI:10.1039/b004183l

The α-chymotrypsin-catalyzed peptide synthesis via the kinetically controlled approach using activated esters as acyl donors in orgnanic solvents with low water content was presented. The methyl esters of N-Z derivatives of racemic non-protein amino acids were chosen as carboxy components. They allowed the peptide-bond formation and optical resolution simultaneously to yield homochiral peptides. This method is useful for the incorporation of non-protein amino acids into peptides.

Full text of DOI:10.1039/b004183l

View Article Online / Journal Homepage / Table of Contents for this issue
ꢀ-Chymotrypsin-catalysed peptide synthesis via the kinetically
controlled approach using activated esters as acyl donors in
organic solvents with low water content: incorporation of
non-protein amino acids into peptides
1
Toshifumi Miyazawa,* Shin’ichi Nakajo, Miyako Nishikawa, Kazumi Hamahara,
Kiwamu Imagawa, Eiichi Ensatsu, Ryoji Yanagihara and Takashi Yamada
Department of Chemistry, Faculty of Science, Konan University, Higashinada-ku,
Kobe 658-8501, Japan. Fax: ϩ81-78-435-2539; E-mail: miyazawa@base2.ipc.konan-u.ac.jp
Received (in Cambridge, UK) 25th May 2000, Accepted 30th October 2000
First published as an Advance Article on the web 11th December 2000
The coupling efficiency in the α-chymotrypsin-catalysed peptide synthesis via the kinetically controlled approach
is greatly improved by the use of activated esters such as the 2,2,2-trifluoroethyl ester as acyl donors instead of the
conventional methyl ester in organic solvents such as acetonitrile with low water content. This approach is useful
for the incorporation of non-protein amino acids such as halogenophenylalanines into peptides.
approach where carboxy components were used in the form of
Introduction
esters at pH-values greater than 8, as introduced by Mitin et al.8
as opposed to the equilibrium-controlled approach. The methyl
esters of N-Z derivatives of racemic non-protein amino acids
were chosen as carboxy (C) components, because papain is
known to exhibit a preference for bulky hydrophobic residues
in the P2-position9 of the substrate, and they were allowed to
react with -Leu anilide used as an amino (N) component
(Scheme 1). The use of racemic C-components would, if
The use of proteases for peptide synthesis has attracted grow-
ing attention in recent years.1 Enzymic peptide synthesis has
several advantages over conventional chemical methodologies:
enzyme specificity suppresses side reactions and ensures the
production of chemically and chirally pure peptides. Thus,
peptide synthesis using enzymes has come to constitute an
important complement to chemical methods and even to
recombinant DNA techniques. However, most of the relevant
works deal with peptides consisting of only proteinogenic
amino acids which are universally distributed as protein con-
stituents in all living organisms. Of hundreds of known amino
acids, other naturally occurring ones are of non-protein origin,2
and there are also a number of purely synthetic (or unnatural)
ones. Non-protein amino acids are important as building
blocks for the synthesis of analogues of biologically active
peptides necessary for structure–activity studies.3 The synthesis
of peptides containing non-protein amino acids has usually
been carried out by chemical methods, and moreover,
recombinant DNA techniques are not easily applicable to the
synthesis of peptides containing these non-coded amino acids.
Therefore, the enzymic incorporation of non-protein amino
acids into peptides remains a challenge.
We have recently investigated the optical resolution of non-
protein amino acids4 via their ester hydrolysis using lipases5
and proteases6 from various sources, and consequently we have
been interested in incorporating these amino acids into peptides
using easily available proteases via the kinetically controlled
approach.1a,b,e During the course of our investigation, we have
found that the coupling efficiency can greatly be improved by
the use of activated esters such as the 2,2,2-trifluoroethyl ester
as an acyl donor instead of the conventional methyl ester.7 In
this paper, we describe the details of our relevant works.
Scheme 1 Protease-catalysed couplings of non-protein -amino
acids (-Xaa) as C-components. Enzyme and conditions: (a) papain,
Tris–maleate buffer (pH 8.0), 35 ЊC; (b) α-chymotrypsin, carbonate–
bicarbonate buffer (pH 10.0) containing 20% (v/v) DMF, 35 ЊC.
fortunate, allow peptide-bond formation and optical resolution
simultaneously to yield homochiral peptides. The couplings
were performed in Tris–maleate buffer (pH 8.0) at 35 ЊC with
an N/C molar quotient of 1.5 which was determined through
preliminary experiments. The progress of reaction was followed
by reversed-phase HPLC analysis. In the couplings of amino
acids carrying aliphatic side-chains as C-components, the
targeted peptides were obtained generally in high yields, the
coupling efficiency being reduced by branching in the side-
chain and especially by a long side-chain of 7 or more carbon
atoms.10 Fortunately, the diastereoselectivities toward the -
peptides were sufficiently high. In sharp contrast, the yields and
diastereoselectivities were low in the couplings of amino acids
bearing aromatic side-chains as C-components (the first three
entries in Table 1). Consequently, we turned our attention to the
use of another protease, α-chymotrypsin (EC 3.4.21.1), which
is known to hydrolyse preferentially a peptide bond adjacent
to the carbonyl group of aromatic amino acid residues.1d
Adopting the kinetically controlled approach in this case also,
the methyl esters of N-Z--halogenophenylalanines were
treated with -Leu anilide (N/C molar quotient 1.5) in
carbonate–bicarbonate buffer (pH 10.0) containing 20% (v/v)
DMF after some preliminary experiments searching for the
optimum coupling conditions. As shown in Table 1, the yields
Results and discussion
Initially, we intended to use papain (EC 3.4.22.2), a sulfydryl
protease from papaya latex which is known to possess a wide
substrate specificity,1d for the purpose. Our recent study of the
papain-catalysed enantioselective hydrolysis of non-protein
amino acid esters also illustrated a rather broad substrate toler-
ance of this enzyme.6b We adopted the kinetically controlled
82
J. Chem. Soc., Perkin Trans. 1, 2001, 82–86
This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b004183l

View Article Online
Table 1 Protease-catalysed coupling of Z--Phe(X)-OMea with
-Leu-NHPhb
Table 2 Effect of donor esters and water content of the solvent on the
a
α-chymotrypsin-catalysed coupling of Z--Phe-OR with -Leu-NH2
X
Protease
Yield (%)c
% -
Yield (%)b
Water content
R
(%, v/v)
Peptide
Z--Phe-OH
H
Papain
Papain
Papain
α-CT
α-CT
α-CT
8.1
6.3
5.9
39.2
29.1
25.8
35.0
15.2
57
55
54
93
86
99
97
98
4F
4Cl
H
4F
2Cl
4Cl
4Br
Me
2
3
4
5
2
3
4
5
4
4
4
20.2
26.9
31.0
31.3
87.1
93.7
98.2
98.1
56.8
96.3
70.2
0.7
1.0
1.1
1.3
1.8
2.2
2.3
2.2
1.4
2.5
1.9
α-CT
α-CT
CH2CF3
a Phe(X) stands for a Phe analogue bearing a substituent X on the
benzene ring; for example, Phe(4F) represents 4-fluorophenylalanine.
b Coupling conditions with α-chymotrypsin (α-CT): see the Experi-
mental section. With papain: Z--Phe(X)-OMe (0.5 mmol) and
-Leu-NHPhؒHCl (182 mg, 0.75 mmol) were treated in the presence
of papain (75 mg) and 2-mercaptoethanol (50 µl) in 7 ml of 0.05 M
Tris–maleate buffer (pH 8.0) at 35 ЊC for 24 h. c Total yield of peptide
diastereomers (- ϩ -).
CH2CH2Cl
CH2CCl3
CH(CF3)2
a A mixture of Z--Phe-OR (0.05 mmol), -Leu-NH2ؒHCl (0.2 mmol),
TEA (0.2 mmol), and the immobilised enzyme (150 mg) was incubated
with shaking in a solvent composed of 2 ml of acetonitrile and 41 µl
(water content: 2%), 62 µl (3%), 83 µl (4%), or 105 µl (5%) of 0.05 M
Tris buffer (pH 7.8) at 30 ЊC. b After 1 h of incubation.
of the targeted peptides were much higher than those obtained
using papain as a catalyst. The replacement of a hydrogen by a
halogen atom on the benzene ring caused a decrease in the
coupling efficiency. This was especially serious with the coup-
ling of the bromophenylalanine derivative. ortho-Substitution
also caused a deterioration in the coupling yield. On the other
hand, the diastereoselectivities toward the - peptides were
high in these cases, with the exception of the p-fluorophenyl-
alanine derivative.
The protease-catalysed peptide-bond formation based on the
kinetically controlled approach can be carried out in organic
solvents with low water content as well as in aqueous media.11
It is expected that in the former the water activity is reduced,
which favours the synthetic reaction and virtually eliminates the
secondary hydrolysis of the peptide formed. In addition, our
recent experiences with the successful use of 2-chloroethyl
and 2,2,2-trifluoroethyl esters in the lipase-catalysed enantio-
selective hydrolysis5a,b or transesterification5c of amino ester
substrates prompted us to try such activated esters as acyl
donors instead of the conventional methyl ester.7 Along these
lines, we chose the α-chymotrypsin-catalysed synthesis of
Z--Phe--Leu-NH2 as a model system and several kinds of
activated esters were evaluated as donor esters (Scheme 2).
Table 3 Effect of donor esters on the α-chymotrypsin-catalysed coup-
ling of Z--Phe(X)-OR with -Leu-NH2 in acetonitrile containing 4%
(v/v) Tris buffera
Yield (%)b
X
R
Peptidec
Z-Phe(X)-OH
2F
Me
CH2CF3
Me
CH2CF3
Me
CH2CF3
Me
CH2CF3
Me
CH2CF3
Me
CH2CF3
Me
CH2CF3
Me
15.9
45.6
15.3
47.5
19.2
48.7
3.2
19.2
21.3
45.7
3.9
48.3
0.8
13.9
18.8
42.4
1.4
3.9
2.2
1.6
2.3
1.5
1.2
1.0
3.2
2.8
3.2
1.2
1.6
0.5
3.4
3.0
3.6
0.2
2.2
3F
4F
2Cl
3Cl
4Cl
2Br
3Br
4Br
CH2CF3
Me
CH2CF3
40.4
a Coupling conditions as described in the Experimental section, using
0.1 mmol of Z--Phe(X)-OR, 0.2 mmol of -Leu-NH2ؒHCl, 0.2 mmol
of TEA, and 150 mg of the immobilised enzyme in a solvent composed
of 2 ml of acetonitrile and 83 µl of 0.05 M Tris buffer (pH 7.8) at 30 ЊC.
b After 1 h of incubation. c Only the - peptide was detected in each
case.
Scheme 2
Hydrophilic organic solvents such as acetonitrile, propan-1-ol,
2-methylbutan-2-ol, acetone, 1,4-dioxane, THF, etc. containing
a small amount of water were tested, and acetonitrile was found
to be the best. Table 2 shows the results (after 1 h of incubation)
of couplings conducted in acetonitrile containing different
amounts [2–5% (v/v)] of Tris buffer (pH 7.8)12 in the presence
of the enzyme adsorbed on Celite. In this case, not only the
yield of the desired peptide but also that of the hydrolysis
product of the donor ester was determined by HPLC analysis.
Under the present reaction conditions, non-enzymic reactions
(peptide-bond formation and hydrolysis of the donor ester)
were not detected even when the activated esters were
employed. The optimal water content was found to be 4%, and
the solvent of this composition was used throughout further
experiments. The coupling yield was almost doubled when
the acyl donor was switched from the methyl ester to the
2-chloroethyl ester,13 and the use of the 2,2,2-trifluoroethyl
ester14 was much more effective, forcing the reaction to comple-
tion, while the competing hydrolysis of the donor ester was
little accelerated compared with the case of the methyl ester.
Such a significant increase in the coupling efficiency on
replacing the methyl ester with the trifluoroethyl ester was also
observed in the couplings with several other amino acid amides
as nucleophiles.7
This promising method employing the trifluoroethyl ester
was next applied to the couplings of non-protein amino acids
as C-components in acetonitrile containing 4% Tris buffer
(Scheme 3). Table 3 shows a comparison between the methyl
Scheme 3 Immobilised α-chymotrypsin-catalysed couplings of -
halogenophenylalanines as C-components. Conditions: acetonitrile
containing 4% (v/v) Tris buffer (pH 7.8), 30 ЊC.
and trifluoroethyl esters in the couplings of N-Z--halogeno-
phenylalanines with -Leu-NH2. The peptide yields obtained
using methyl esters were rather low; especially noteworthy was
J. Chem. Soc., Perkin Trans. 1, 2001, 82–86
83

View Article Online
the deleterious effect of bulky substituents on the aromatic ring
(o-Cl or o-Br). On the other hand, the yields were greatly
improved by the use of trifluoroethyl esters without a large
increase in the yields of hydrolysis products of the donor esters.
In some cases, the reaction proceeded almost to completion.
Moreover, it is worth noting that the reactions proceeded
diastereospecifically to produce only the - peptides even when
the trifluoroethyl esters were employed.
solvent [e.g., EtOAc–petroleum spirit (bp 30–70 ЊC)]. The phys-
ical properties of Z--Phe(X) bearing X = 2F, 3F, 4F and 4Cl
were reported elsewhere.5d The mps (ЊC) of the other Z--
Phe(X) employed in this study are as follows: X = 2Cl, 124–125;
X = 3Cl, 92.5–93; X = 2Br, 126–127; X = 3Br, 99–99.5; X = 4Br,
166–168.
Preparation of Z-L- or Z-DL-Phe(X)-OMe
The reaction mechanism of the α-chymotrypsin-catalysed
peptide-bond formation via the kinetically controlled approach
studied here is thought to be the one described for the synthesis
of Z--Phe--Leu-NH2 as an example in Scheme 4.1b,e The ester
Treatment of Z-- or Z--Phe(X) with an ethereal solution of
diazomethane afforded their methyl esters in a nearly quantit-
ative yield. Z--Phe-OMe, oil; [α]D25 Ϫ14.4 (c 1.3, MeOH) {lit.,16
[α]D19 Ϫ15.6 (c 1.02, MeOH)}. The mps (ЊC) of Z--Phe(X)-
OMe obtained as crystals are as follows: X = 3F, 56–58;
X = 2Cl, 66.5–68; X = 4Cl, 68–70; X = 4Br, 86–88. The methyl
esters bearing X = 2F, 4F and 2Br were obtained as oils.
Preparation of the halogenated alkyl esters of Z-L- or
Z-DL-Phe(X)
Z-- or Z--Phe(X) was converted to its halogenated alkyl
esters by treatment with the corresponding halogenated alcohol
in the presence of DMAP and 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDCؒHCl) in DCM accord-
ing to the procedure of Dhaon et al.17 The preparation of Z--
Phe-OCH2CF3 is described as a typical example. To a stirred
solution of Z--Phe (660 mg, 2.2 mmol), DMAP (135 mg, 1.1
mmol) and 2,2,2-trifluoroethanol (250 mg, 2.5 mmol) in dry
DCM (8 ml) was added EDCؒHCl (460 mg, 2.4 mmol) under
ice-cooling, and the mixture was stirred at this temperature for
2 h and then at ambient temperature overnight. The reaction
mixture was partitioned between EtOAc and water, and the
organic layer was washed successively with 1 M HCl, water, 1 M
aq. NaHCO3 and brine, and dried over Na2SO4. Evaporation of
the solvent in vacuo gave crystals in 96% yield; mp 80–80.5 ЊC;
[α]D25 Ϫ20.8 (c 1.0, MeOH) (Found: C, 59.90; H, 4.73; N, 3.79.
C19H18F3NO4 requires C, 59.84; H, 4.76; N, 3.67%); νmax/cmϪ1
3324, 1764, 1686; δH 3.06–3.22 (2H, d of AB q, J 15.3 and 6.6),
4.37–4.61 (2H, m), 4.76 (1H, m), 5.10 (2H, apparent s), 5.16
(1H, br d, J 7), 7.10–7.36 (5H, m), 7.34 (5H, apparent s).
Scheme 4
substrate acting as an acyl donor first forms the enzyme–
substrate (ES) complex with the enzyme, and then it affords the
acyl-enzyme intermediate with the liberation of the leaving
group (OR). This intermediate is then deacylated either by a
nucleophilic amino component to yield the peptide product or
by water to form the hydrolysis product of the donor ester. The
results obtained in this study indicate the usefulness of the
activated esters such as the 2,2,2-trifluoroethyl ester as acyl
donors. Since there seems to be no significant difference in the
binding of the donor ester onto the enzyme between the methyl
and trifluoroethyl esters, for example, the acylation rates of the
enzyme should be very different between them: the activated
ester must accelerate the acylation step compared with the
conventional methyl ester. The successful incorporation of
non-protein amino acids such as halogenophenylalanines into
peptides illustrated in this paper suggests that the present
approach will be promising for broadening the narrow substrate
tolerance of enzymes, which will be the subject of the following
paper.
Z-L-Phe-OCH2CH2Cl. 83% yield; mp 43–44 ЊC; [α]D25 Ϫ20.3
(c 1.0, MeOH) (Found: C, 63.17; H, 5.52; N, 3.98. C19H20ClNO4
requires C, 63.07; H, 5.57; N, 3.87%); νmax/cmϪ1 3346, 1752,
1683; δH 3.13 (2H, m), 3.61 (2H, t, J 5.7), 4.34 (2H, m), 4.69
(1H, m), 5.00 (1H, apparent s), 5.23 (1H, br d, J 8), 7.11–7.38
(5H, m), 7.33 (5H, apparent s).
Experimental
All solvents were distilled and dried over molecular sieves prior
to use. Amino acid amide hydrochlorides were purchased from
Kokusan Chemical Works (Japan). Papain (twice crystallised,
lyophilised powder, ex papaya latex) was purchased from Sigma
and had a specific activity of 2.8 units per mg solid with Bz--
Arg-OEt. α-Chymotrypsin (type II, ex bovine pancreas) was
also from Sigma and had a specific activity of 48 units per mg
solid with Bz--Tyr-OEt. IR spectra were recorded on a Nicolet
N-750B FT-IR spectrometer using attenuated total reflectance
Z-L-Phe-OCH2CCl3. 92% yield; oil; [α]D25 ϩ6.9 (c 1.3, CHCl3)
(Found: C, 53.11; H, 4.20; N, 3.24. C19H18Cl3NO4 requires C,
52.98; H, 4.21; N, 3.25%); νmax/cmϪ1 3333, 1704; δH 3.09–3.28
(2H, d of AB q, J 14.1 and 6.9), 4.75 (2H, AB q, J 12.0), 4.81
(1H, m), 5.00 (1H, apparent s), 5.18 (1H, br d, J 8), 7.14–7.38
(5H, m), 7.33 (5H, apparent s).
Z-L-Phe-OCH(CF3)2. 95% yield; mp 73.5–75 ЊC; [α]D25 ϩ3.2
(c 1.1, CHCl3) (Found: C, 53.78; H, 3.78; N, 3.26. C20H17F6NO4
requires C, 53.46; H, 3.81; N, 3.12%); νmax/cmϪ1 3327, 1782,
1686; δH 3.05–3.26 (2H, d of AB q, J 14.1 and 6.5), 4.83 (1H,
m), 5.09 (2H, apparent s), 5.12 (1H, br d, J 7), 5.77 (1H, septet,
J 6.0), 7.11–7.39 (10H, m).
(ATR). Wavenumbers of only the peaks ascribed to the N–H
and C᎐O stretching vibrations are quoted. H NMR spectra
1
᎐
(300 MHz) were recorded with a Varian Unity 300 spectrometer
using CDCl3 as solvent with TMS as internal standard unless
otherwise stated. J-Values are given in Hz. Mps were deter-
mined on a Yamato (Japan) MP-21 apparatus and are uncor-
rected. Optical rotations were measured with a JASCO (Japan)
DIP-4 digital polarimeter, with [α]D-values given in units of
10Ϫ1 deg cm2 gϪ1.
Z-DL-Phe(2F)-OCH2CF3. 84% yield; mp 39–40 ЊC (Found:
C, 56.97; H, 4.21; N, 3.27. C19H17F4NO4 requires C, 57.15; H,
4.29; N, 3.51%); νmax/cmϪ1 3331, 1772, 1696; δH 3.09–3.31 (2H,
m), 4.49 (2H, q, J 8.4), 4.75 (1H, m), 5.09 (2H, AB q, J 12.3),
5.25 (1H, br d, J 9), 7.00–7.39 (9H, m).
Preparation of Z-L- or Z-DL-Phe(X)
- or -Phe(X) (X = H, 2F, 3F, 4F, 2Cl, 3Cl, 4Cl, 2Br, 3Br or
4Br) was benzyloxycarbonylated using Z-Cl under the usual
Schotten–Baumann conditions15 to give Z-- or Z--Phe(X),
which was purified by recrystallisation from an appropriate
Z-DL-Phe(3F)-OCH2CF3. 94% yield; mp 68–69 ЊC (Found:
C, 57.31; H, 4.02; N, 3.56%); νmax/cmϪ1 3318, 1762, 1685; δH
3.05–3.22 (2H, d of AB q, J 14.1 and 5.4), 4.40–4.60 (2H, m),
84
J. Chem. Soc., Perkin Trans. 1, 2001, 82–86

View Article Online
Table 4 HPLC separation of the diastereomers (- and -) of
4.73 (1H, m), 5.10 (2H, AB q, J 6.2), 5.21 (1H, br d, J 8), 6.80–
7.39 (4H, m), 7.35 (5H, apparent s).
a
Z-Phe(X)-Leu-NH2
c
c
X
Mobile phaseb
kЈ
kЈ
αd
L-L
D-L
Z-DL-Phe(4F)-OCH2CF3. 91% yield; mp 79–81 ЊC (Found:
C, 57.32; H, 4.37; N, 3.30%); νmax/cmϪ1 3328, 1761, 1687;
δH 3.03–3.20 (2H, d of AB q, J 14.1 and 6.6), 4.37–4.62 (2H, m),
4.75 (1H, m), 5.13 (2H, AB q, J 14.1), 5.19 (1H, br d, J 8),
6.97–7.40 (9H, m).
2F
3F
4F
2Cl
3Cl
4Cl
2Br
3Br
4Br
A
A
A
B
B
B
B
B
B
16.5
18.8
18.8
12.9
15.0
16.3
14.1
17.9
19.5
20.1
22.4
21.5
16.2
18.1
18.3
17.9
21.7
21.5
1.22
1.19
1.14
1.26
1.21
1.12
1.27
1.21
1.10
Z-DL-Phe(2Cl)-OCH2CF3. 91% yield; mp 79–80 ЊC (Found:
C, 55.03; H, 4.09; N, 3.62. C19H17ClF3NO4 requires C, 54.88; H,
4.12; N, 3.37%); νmax/cmϪ1 3352, 1766, 1696; δH 3.15–3.38 (2H,
d of AB q, J 14.1 and 6.3), 4.49 (2H, q, J 8.4), 4.79 (1H, m), 5.07
(2H, AB q, J 12.9), 5.26 (1H, br d, J 8), 7.16–7.39 (9H, m).
a HPLC conditions: see the Experimental section. b A, 55% aq. MeOH
containing H3PO4 (0.01 M); B, 60% aq. MeOH containing H3PO4
(0.01 M). c Capacity factor: kЈ = (tR Ϫ t0)/t0, where t0 = 1.50 min. d Sep-
Z-DL-Phe(3Cl)-OCH2CF3. 86% yield; mp 66–67 ЊC (Found:
C, 54.69; H, 4.36; N, 3.09%); νmax/cmϪ1 3321, 1766, 1689; δH
(DMSO-d6) 2.92 (1H, dd-like, J 13.8 and 5.4), 3.07 (1H, dd-like,
J 13.8 and 5.1), 4.41 (1H, m), 4.78 (2H, q, J 9.0), 4.99 (2H, AB
q, J 12.6), 7.21–7.37 (9H, m), 7.99 (1H, d, J 7.8).
c
aration factor: α = kЈD-L/kЈ
.
L-L
was added EDCؒHCl (190 mg, 1.0 mmol) under ice-cooling.
After stirring at this temperature for 2 h and then at ambient
temperature overnight, the reaction mixture was diluted with
EtOAc, washed successively with 1 M HCl, water, 1 M aq.
NaHCO3 and brine, and dried over Na2SO4. Evaporation of the
solvent in vacuo afforded crystals, which were recrystallised
from aq. MeOH (370 mg, 90%); mp 192.5–193 ЊC; [α]D25 Ϫ18.3
(c 1.0, DMF), Ϫ21.7 (c 1.0, MeOH) {lit.,18 mp 195–196 ЊC;
[α]D25 Ϫ19.7 (c 1, DMF)}; δH (DMSO-d6) 0.83 (3H, d, J 6.3),
0.87 (3H, d, J 6.6), 1.45 (2H, apparent t, J 7), 1.48–1.66
(1H, m), 2.72 (1H, dd, J 13.8 and 11.1), 3.00 (1H, dd, J 13.8
and 3.9), 4.16–4.33 (2H, m), 4.93 (2H, AB q, J 13.2), 6.99
(1H, s), 7.11–7.35 (11H, m), 7.51 (1H, d, J 8.4), 7.97 (1H, d,
J 8.4).
Z-DL-Phe(4Cl)-OCH2CF3. 85% yield; mp 95–101 ЊC (Found:
C, 55.16; H, 4.27; N, 3.39%); νmax/cmϪ1 3343, 1766, 1697;
δH 3.02–3.20 (2H, d of AB q, J 14.1 and 6.0), 4.37–4.63 (2H, m),
4.74 (1H, m), 5.09 (2H, AB q, J 12.3), 5.16 (1H, br d, J 8), 7.02–
7.40 (9H, m).
Z-DL-Phe(2Br)-OCH2CF3. 95% yield; mp 83.5–84 ЊC
(Found: C, 49.50; H, 4.01; N, 3.27. C19H17BrF3NO4 requires C,
49.58; H, 3.72; N, 3.04%); νmax/cmϪ1 3320, 1770, 1697; δH 3.14–
3.41 (2H, m), 4.45–4.55 (2H, m), 4.80 (1H, m), 5.06 (2H, s), 5.27
(1H, br d, J 9), 7.09–7.57 (9H, m).
The mixtures of diastereomers (- and -) of Z-Phe(X)-
Leu-NHPh or Z-Phe(X)-Leu-NH2 were prepared through the
coupling of Z--Phe(X) with -Leu-NHPh or -Leu-NH2 (in
the form of the hydrochloride with an equimolar amount of
TEA) by the EDC–HOBT method in DMF in the same manner
as above and purified by recrystallisation from aq. MeOH.
Each sample thus prepared showed only two peaks, corre-
sponding to both the diastereomers on reversed-phase HPLC.
The faster eluting diastereomer proved to be - by comparison
with authentic samples prepared through the coupling of a very
small amount of Z--Phe(X) (X = 2F, 3F, 4F, 4Cl and 4Br),
which was obtained by enzymic resolution,5,6 with -Leu-NHPh
or -Leu-NH2 salt by the EDC–HOBT method.
Z-DL-Phe(3Br)-OCH2CF3. 88% yield; mp 76–77 ЊC (Found:
C, 49.81; H, 3.67; N, 3.24%); νmax/cmϪ1 3319, 1766, 1689;
δH (DMSO-d6) 2.90 (1H, dd-like, J 12.0 and 10.5), 3.07 (1H,
dd-like, J 12.0 and 4.8), 4.41 (1H, m), 4.77 (2H, q, J 9.0), 4.98
(2H, AB q, J 12.3), 7.20–7.50 (9H, m), 7.98 (1H, d, J 7.8).
Z-DL-Phe(4Br)-OCH2CF3. 85% yield; mp 111–113 ЊC
(Found: C, 49.44; H, 3.59; N, 3.25%); νmax/cmϪ1 3322, 1761,
1687; δH 3.00–3.19 (2H, d of AB q, J 14.1 and 6.3), 4.37–4.63
(2H, m), 4.74 (1H, m), 5.09 (2H, AB q, J 12.3), 5.16 (1H, br d,
J 8), 6.97–7.43 (9H, m).
Preparation of L-Leu-NHPhؒHCl
Preparation of immobilised ꢀ-chymotrypsin
Boc--Leuؒ1/2H2O (2.4 g, 10 mmol), aniline (1.4 g, 15 mmol),
EDTA (60 mg) and -Cys (200 mg) were dissolved in a mixture
of pH 4.8 acetate buffer (140 ml) and DMF (60 ml). After the
pH had been adjusted to 4.8, papain (400 mg) was added and
the mixture was stirred at 38 ЊC for 4 days. The precipitate
was filtered off, washed with the buffer, and dried in a vacuum
desiccator over P4O10. Materials insoluble in EtOAc were fil-
tered off and the filtrate was concentrated to give white crystals
(2.6 g; mp 136–143 ЊC), which were recrystallised from EtOAc–
petroleum spirit to give Boc--Leu-NHPh (1.7 g, 56%); mp 148–
150 ЊC; [α]D25 Ϫ34.8 (c 1.0, MeOH) (Found: C, 70.12; H, 9.28; N,
9.63. C17H26N2O2 requires C, 70.31; H, 9.02; N, 9.65%). Boc--
Leu-NHPh (1.5 g, 5 mmol) thus obtained was stirred in 3.7 M
HCl in 1,4-dioxane (15 ml) for 1.5 h. Evaporation of the solvent
in vacuo afforded white crystals, which were recrystallised from
propan-2-ol–diisopropyl ether to give -Leu-NHPhؒHCl (0.9 g,
74%); mp 218–222 ЊC.
α-Chymotrypsin (32 mg) was dissolved in 4 ml of 0.05 M Tris
buffer (pH 7.8) and mixed with Celite No. 535 (Johns-Mansville
Co., Denver, CO; 1 g), and the mixture was lyophilised using an
Eyela (Japan) freeze-dryer FDU-830 for 20 h.
HPLC analyses
The liquid chromatograph employed was a GL Sciences (Japan)
PU-610 instrument, equipped with a Rheodyne 8125 sample
injector and a GL Sciences UV-620 variable-wavelength UV
monitor. A Shimadzu (Japan) C-R6A data processor was
used for data acquisition and processing. The amounts of the
donor ester, peptide and hydrolysis product of the donor
ester were determined by HPLC analysis on an ODS column
under the following conditions: column, Inertsil ODS 3 (5 µm;
1.5 mm id × 150 mm, GL Sciences); mobile phase, 55–60%
aq. MeOH containing H3PO4 (0.01 M); flow rate, 0.1 ml
minϪ1; column temperature, 40 ЊC; detection, UV at 254 nm.
The diastereomers (- and -) of the resulting peptides were
also separated well on the same column by decreasing the
amount of MeOH in the mobile phase. The separation of the
diastereomers (- and -) of Z-Phe(X)-Leu-NH2 is compiled
in Table 4.
Preparation of authentic dipeptides
An authentic sample of Z--Phe--Leu-NH2 was prepared as
follows. To a stirred solution of Z--Phe (300 mg, 1.0 mmol),
-Leu-NH2ؒHCl (130 mg, 1.0 mmol), triethylamine (TEA) (100
mg, 1.0 mmol) and HOBT (135 mg, 1.0 mmol) in DMF (10 ml)
J. Chem. Soc., Perkin Trans. 1, 2001, 82–86
85

View Article Online
(e) H.-D. Jakubke, in The Peptides, ed. S. Udenfriend and J.
ꢀ-Chymotrypsin-catalysed coupling of Z-DL-Phe(X)-OMe with
L-Leu-NHPh in aqueous media
Meienhofer, Academic Press, San Diego, 1987, vol. 9, ch. 3.
2 S. Hunt, in Chemistry and Biochemistry of the Amino Acids, ed. G. C.
Barrett, Chapman and Hall, London, 1985, p. 55.
3 D. C. Roberts and F. Vellaccio, in The Peptides, ed. E. Gross and
J. Meienhofer, Academic Press, New York, 1983, vol. 5, ch. 6.
4 For reviews, see: (a) T. Miyazawa, Amino Acids, 1999, 16, 191; (b)
T. Miyazawa, in Fluorine-containing Amino Acids: Synthesis and
Properties, ed. V. P. Kukhar’ and V. A. Soloshonok, Wiley,
Chichester, 1995, p. 267.
5 (a) T. Miyazawa, T. Takitani, S. Ueji, T. Yamada and S. Kuwata,
J. Chem. Soc., Chem. Commun., 1988, 1214; (b) T. Miyazawa,
H. Iwanaga, S. Ueji, T. Yamada and S. Kuwata, Chem. Lett., 1989,
2219; (c) T. Miyazawa, M. Mio, Y. Watanabe, T. Yamada and
S. Kuwata, Biotechnol. Lett., 1992, 14, 789; (d) T. Miyazawa,
H. Iwanaga, S. Ueji and T. Yamada, Biocatal. Biotransform., 2000,
17, 445.
6 (a) T. Miyazawa, H. Iwanaga, T. Yamada and S. Kuwata, Chirality,
1992, 4, 427; (b) T. Miyazawa, H. Iwanaga, T. Yamada and
S. Kuwata, Biotechnol. Lett., 1994, 16, 373; (c) T. Miyazawa,
H. Minowa, T. Miyamoto, K. Imagawa, R. Yanagihara and
T. Yamada, Tetrahedron: Asymmetry, 1997, 8, 367; (d) T. Miyazawa,
K. Imagawa, R. Yanagihara and T. Yamada, Biotechnol. Tech.,
1997, 11, 931.
Z--Phe(X)-OMe (0.25 mmol) and -Leu-NHPhؒHCl (92 mg,
0.38 mmol) were added to a mixture of 5 ml of carbonate–
bicarbonate buffer (pH 10.0) and 1.2 ml of DMF, followed by
α-chymotrypsin (32 mg). The reaction mixture was stirred at
35 ЊC for 24 h. After addition of 10% (w/v) aq. citric acid
(10 ml), the mixture was extracted with EtOAc. After addition
of p-nitrotoluene (13.7 mg, 0.1 mmol) as internal standard, the
combined EtOAc extracts were washed successively with 1 M
HCl, 5% aq. NaHCO3, and brine, and dried over Na2SO4. After
evaporation of the solvent, a part of the residue was subjected
to reversed-phase HPLC analysis using aq. MeOH as the
mobile phase to obtain the coupling yield. Calibration plots
of the ratio of peak areas on reversed-phase HPLC between
each targeted peptide (synthesised chemically as a mixture of
diastereomers) and p-nitrotoluene against their molar ratio
gave straight lines going through the origin, which were used
for estimating the coupling yield.
7 Preliminary communication, T. Miyazawa, S. Nakajo, M.
Nishikawa, K. Imagawa, R. Yanagihara and T. Yamada, J. Chem.
Soc., Perkin Trans. 1, 1996, 2867.
Peptide synthesis mediated by immobilised ꢀ-chymotrypsin in
acetonitrile with low water content
8 Yu. V. Mitin, N. P. Zapevalova and E. Yu. Gorbunova, Int. J. Pept.
Protein Res., 1984, 23, 528.
9 I. Schechter and A. Berger, Biochem. Biophys. Res. Commun., 1967,
27, 157.
10 Preliminary communication, T. Miyazawa, K. Hamahara, M.
Matsuoka, Y. Shindo and T. Yamada, Biotechnol. Lett., 1998, 20,
389.
11 cf. P. Clapés, P. Adlercreutz and B. Mattiasson, Biotechnol. Appl.
Biochem., 1990, 12, 376.
12 The optimal pH for amide-bond hydrolysis mediated by α-chymo-
trypsin: see ref. 1(d).
13 This ester was used as an acyl donor in the incorporation of
-amino acids into peptides mediated by subtilisin in 2-methyl-
butan-2-ol: A. L. Margolin, D.-F. Tai and A. M. Klibanov, J. Am.
Chem. Soc., 1987, 109, 7885.
The coupling of Z--Phe(2F)-OCH2CF3 with -Leu-NH2 is
described as a typical example. A mixture of Z--Phe(2F)-
OCH2CF3 (40 mg, 0.1 mmol), -Leu-NH2ؒHCl (33 mg, 0.2
mmol), TEA (28 µl, 0.2 mmol) and the immobilised enzyme
(150 mg, corresponding to 4.7 mg of α-chymotrypsin) was
incubated with shaking (180 strokes minϪ1) in a solvent com-
posed of 2 ml of acetonitrile and 83 µl of 0.05 M Tris buffer
(pH 7.8) at 30 ЊC. The amounts of the donor ester, peptide
and hydrolysis product were determined as described in ‘HPLC
analyses,’ above.
Acknowledgements
14 2,2,2-Trifluoroethyl esters were employed in the protease-catalysed
transesterifications, see: B. Danieli, P. Bellis, G. Carrea and S. Riva,
Gazz. Chim. Ital., 1991, 121, 123; J. Broos, J. F. J. Engbersen, I. K.
Sakodinskaya, W. Verboom and D. N. Reinhoudt, J. Chem. Soc.,
Perkin Trans. 1, 1995, 2899; H. G. Park, H. N. Chang and J. S.
Dordick, Biotechnol. Lett., 1995, 17, 1085; K. Kawashiro, H.
Sugahara, S. Sugiyama and H. Hayashi, Biotechnol. Bioeng., 1997,
53, 26.
15 M. Bodanszky and A. Bodanszky, The Practice of Peptide Synthesis,
Springer-Verlag, Berlin, 2nd edn., 1994, p. 11.
16 A. Ito, R. Takahashi, C. Miura and Y. Baba, Chem. Pharm. Bull.,
1975, 23, 3106.
This work was supported in part by grants from the Science
Research Promotion Fund of the Japan Private School Promo-
tion Foundation and from the Ministry of Education, Science
and Culture, Japan (to T. M.).
References
1 For reviews, see: (a) K. Faber, Biotransformations in Organic
Chemistry, Springer-Verlag, Berlin, 3rd edn., 1997, p. 331; (b) H.-D.
Jakubke, in Enzyme Catalysis in Organic Synthesis, ed. K. Drauz
and H. Waldmann, VCH, Weinheim, 1995, B.2.5, p. 431; (c) C.-H.
Wong and G. M. Whitesides, Enzymes in Synthetic Organic
Chemistry, Pergamon Press, Oxford, 1994, p. 46; (d) W. Kullmann,
Enzymatic Peptide Synthesis, CRC Press, Boca Raton, FL, 1987;
17 M. K. Dhaon, R. K. Olsen and K. Ramasamy, J. Org. Chem., 1982,
47, 1962.
18 P. Kuhl, U. Zacharias, H. Burckhardt and H.-D. Jakubke, Monatsh.
Chem., 1986, 117, 1195.
86
J. Chem. Soc., Perkin Trans. 1, 2001, 82–86