Superiority of the carbamoylmethyl ester as an acyl donor for the kinetically controlled amide-bond formation mediated by α-chymotrypsin

Article abstract of DOI:10.1039/b108735p

The superiority of the carbamoylmethyl ester as an acyl donor for the α-chymotrypsin-catalysed kinetically controlled peptide-bond formation is demonstrated in the couplings of an inherently poor amino acid substrate, Ala, with various amino acid residues as amino components and in the couplings of non-protein amino acids such as halogenophenylalanines as carboxylic components. Furthermore, this approach is applied to the amide-bond formation between an amino acid residue and a chiral amine, which is highly diastereoselective.

Full text of DOI:10.1039/b108735p

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Superiority of the carbamoylmethyl ester as an acyl donor for
the kinetically controlled amide-bond formation mediated by
ꢀ-chymotrypsin†
1
Toshifumi Miyazawa,* Eiichi Ensatsu, Nobuhiro Yabuuchi, Ryoji Yanagihara and
Takashi Yamada
Department of Chemistry, Faculty of Science and Engineering, Konan University,
Higashinada-ku, Kobe 658-8501, Japan
Received (in Cambridge, UK) 25th September 2001, Accepted 27th November 2001
First published as an Advance Article on the web 8th January 2002
The superiority of the carbamoylmethyl ester as an acyl donor for the α-chymotrypsin-catalysed kinetically
controlled peptide-bond formation is demonstrated in the couplings of an inherently poor amino acid substrate, Ala,
with various amino acid residues as amino components and in the couplings of non-protein amino acids such as
halogenophenylalanines as carboxylic components. Furthermore, this approach is applied to the amide-bond
formation between an amino acid residue and a chiral amine, which is highly diastereoselective.
Introduction
Protease-catalysed peptide synthesis has been intensively
investigated during the last few decades,1 because it has a
potential advantage in circumventing the major problems
associated with the chemical synthesis of peptides such as the
risk of racemisation during the activation step and other
undesirable side reactions. However, proteases’ narrow sub-
Scheme 1 Kinetically controlled peptide synthesis: H–E, α-chymo-
trypsin; R1CO2R, carboxylic component in the form of an ester;
R2NH2, amino component.
strate specificity is often considered as a main drawback from a
synthetic standpoint which emphasises the general applicability
of the reaction. In connection with this unsolved problem, we
have recently reported on the broadening of substrate tolerance
of α-chymotrypsin (EC 3.4.21.1) by taking advantage of such
activated esters as the 2,2,2-trifluoroethyl (Tfe) or carbamoyl-
methyl (Cam) ester as acyl donors in kinetically controlled
peptide synthesis.2,3 Thus, as a typical example, the extremely
low coupling efficiency obtained by employing the Me ester of
an inherently poor amino acid substrate, Ala, was significantly
improved by the use of the Cam ester.3 The ameliorating effect
of this ester was observed also in the couplings of other amino
acid residues such as Gly and Ser as carboxylic components.
In the present work, first the effectiveness of the Cam ester
was examined in the α-chymotrypsin-catalysed couplings of
Ala with different amino acid amides as amino components.
Next this approach was applied to the incorporation of non-
protein amino acids such as halogenophenylalanines into
peptides which had previously been attempted with only partial
success by employing the Tfe ester.2 Furthermore, the α-chymo-
trypsin-catalysed amide-bond formation between an amino
acid residue and a chiral amine, not an amino acid derivative,
was attempted by means of the Cam ester method.4
converted to the acyl-enzyme intermediate, which then reacts
with an amino acid nucleophile in competition with water to
afford the peptide product and the hydrolysis product of the
donor ester, respectively.1b Even in a water-miscible organic
solvent with low water content where the synthetic reaction
is favoured due to the reduced water activity, the coupling
efficiency must be affected by the amine nucleophile used. As
the amino acid residue employed as the amino component
had been restricted solely to (S)-Leu in the previous works,2,3
we first intended to investigate the influence of the amino
component in the α-chymotrypsin-catalysed couplings of Ala.
Thus, the reactions of Z-(S)-Ala esters with several amino acid
amides (Xbb-NH2) were conducted in acetonitrile containing
4% (v/v) Tris buffer (pH 7.8) in the presence of the immobilised
enzyme on Celite (Scheme 2).3 The yields of the targeted peptide
Scheme 2
Results and discussion
Effectiveness of the carbamoylmethyl ester in the couplings of
Ala with different amino acid nucleophiles
[Z-(S)-Ala-Xbb-NH2] and hydrolysis product of the donor
ester [Z-(S)-Ala] were quantified by HPLC analysis, and the
results obtained after 48 h of incubation are compiled in Table
1. The yield of the desired peptide, as well as that of Z-Ala was
extremely poor even after 48 h when the Me ester was used as
the acyl donor, irrespective of the amine nucleophile. With
the Tfe ester, the coupling efficiencies were generally improved,
but the extents differed depending on the amine nucleophile
In the kinetically controlled peptide-bond formation mediated
by α-chymotrypsin (Scheme 1), the ester substrate used is
† Electronic supplementary information (ESI) available: elemental
analyses and HPLC separation data. See http://www.rsc.org/suppdata/
p1/b1/b108735p/
390
J. Chem. Soc., Perkin Trans. 1, 2002, 390–395
This journal is © The Royal Society of Chemistry 2002
DOI: 10.1039/b108735p

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Table 1 Effect of donor esters on the α-chymotrypsin-catalysed coup-
lings of Z-(S)-Ala-OR with various amino acid amides (Xbb-NH2)a
Table 2 α-Chymotrypsin-catalysed couplings of Z-(RS)-Phe(X)-OR
a
with (S)-Leu-NH2
Yield (%)c
Yield (%)b
Peptidec
Xbb
Gly
Rb
Peptide
Z-(S)-Ala
X
R
Z-Phe(X)
Me
Tfe
Cam
Me
Tfe
Cam
Me
Tfe
Cam
Me
Tfe
3
44
95.6
3.2
20
96.8
2.9
17
73.6
6.7
82.4
88.4
89.6
4.3
0
3
4.4
0
4
2.5
0.9
7
22.2
1.0
8.6e
10.9e
10.4
0.6
8
2Br
Med
CH2CH2Cl
CH2CCl3
Tfed
0.8
2.3
7.7
13.9
7.0
0.5
0.9
1.7
3.4
1.3
2.1
8.8
4.8
2.4
2.9
3.1
1.9
6.7
4.7
3.0
(S)-Ala
(S)-Val
(S)-Leud
CH2CF2CF3
N᎐CMe2
5.4e
33.0
45.6
46.2
47.5
47.6
49.2
39.4
47.1
48.8
᎐
Cam
Cam
Cam
Cam
Cam
Cam
Cam
Cam
Cam
3Br
4Br
2F
3F
4F
2Cl
3Cl
4Cl
Cam
Cam
Me
(S)-Met
(S)-Phe
Tfe
55
Cam
87.8
12.2
a Reactions were conducted using 0.1 mmol of Z-(RS)-Phe(X)-OR and
0.2 mmol of (S)-Leu-NH2ؒHCl together with 0.2 mmol of TEA in the
same manner as described in Table 1. b After 1 h of incubation. c S-S
Isomer. d Ref. 2. e S-S Isomer, 5.2%; R-S isomer, 0.2%.
a Reactions were conducted using 0.05 mmol of Z-(S)-Ala-OR, 0.2
mmol of Xbb-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 Tfe, 2,2,2-trifluoroethyl;
Cam, carbamoylmethyl. c After 48 h of incubation. d Ref. 3. e Corrected
for non-enzymatic hydrolysis.
was gratifying to find that the peptide yield obtained using the
Cam ester was 2.4-times greater than that with the Tfe ester. As
the reaction profile in Fig. 1 shows, the amount of the donor
employed: the peptide yield was only 20% at best when the
amino component was (S)-Ala or (S)-Val. In this respect, (S)-
Leu proved to be rather exceptional (82% peptide yield) among
the amino acid residues examined as amino components.
When the Cam ester was employed, the peptide yields were
profoundly improved, reaching almost 90% in most cases. With
(S)-Val as the amino component, the peptide yield was just over
70% and the production of Z-Ala was exceptionally large,
suggesting the deteriorating effect of the bulky side chain
of this amino acid residue on its nucleophilic attack toward
the acyl-enzyme intermediate. Thus, the superiority of the
Cam ester over other esters was once again ascertained in the
couplings using several amino acids as amino components.
Incorporation of halogenophenylalanines into peptides using the
carbamoylmethyl ester as an acyl donor
We reported in previous papers2 that in the α-chymotrypsin-
catalysed couplings of N-Z-(RS)-halogenophenylalanines [Z-
(RS)-Phe(X)] with (S)-Leu-NH2 the coupling yields were
generally improved by the use of the Tfe esters as acyl donors
instead of the conventional Me esters. However, with the amino
acid residues carrying bulky substituents on the ortho-position
of the benzene ring as the carboxylic components, the coupling
efficiencies were still low [Phe(2Cl), 19% yield; Phe(2Br), 14%
yield, after 1 h of incubation], though they themselves were
prominent compared with the almost negligible yields obtained
using the Me esters. As the deleterious effect of the o-Br sub-
stituent was especially noteworthy, we examined in the present
study the couplings of Z-(RS)-Phe(2Br) by employing a series
of its esters (Scheme 3). Table 2 summarises the yields of the
Fig. 1 Reaction profile in the α-chymotrypsin-catalysed coupling of
Z-(RS)-Phe(2Br)-OCam with (S)-Leu-NH2. Symbols: ᮀ, Z-(RS)-
Phe(2Br)-OCam; ᭺, Z-(S)-Phe(2Br)-(S)-Leu-NH2; ᭝, Z-Phe(2Br).
ester decreased gradually, reaching nearly 50% of starting
quantity after 2 h, and then remained constant afterwards. The
yield of the S-S peptide reached a maximum (39.2%) after 5 h,
while production of the R-S peptide was not detected. At
this point in time the hydrolysis product of the donor ester
[Z-Phe(2Br)] was formed in 10.8% yield and was found to be
enantiomerically pure (S), which was confirmed by converting
it to the Me ester followed by HPLC analysis on a chiral
column.5 These results indicate that the deacylation of the
acyl-enzyme intermediate by the nucleophilic amino compon-
ent or by water proceeded enantiospecifically within at least
5 h. Taking into consideration our previous report that in the
coupling of Z-(R)-Ala-OCam with (S)-Leu-NH2 the R-S
peptide was produced in moderate yield (41%) after 48 h of
incubation,3 it follows that the (S)-Phe(2Br) residue is a better
amino acid substrate than is the (S)-Ala residue and that the
difference in reactivity of the enantiomers of Phe(2Br) is much
greater than that of the enantiomers of Ala.
Scheme 3
desired peptide and the hydrolysis product of the donor ester
after 1 h of incubation. When halogenated alkyl esters other
than the Tfe ester were employed, the improvement in the
coupling efficiency was not very significant. Once again it
J. Chem. Soc., Perkin Trans. 1, 2002, 390–395
391

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Table 3 Effect of water content of the solvent on the α-chymotrypsin-
catalysed acylation of (S)-1-phenylethylamine with Z-(S)-Phe-OTfea
The couplings of other N-Z-halogenophenylalanines were
next examined using their Cam esters as acyl donors. The
results obtained after 1 h of incubation are compiled in Table 2.
The behaviour of Phe(2Cl) resembled that of Phe(2Br): the
peptide yield was moderate (39%), but still doubled that
obtained using the Tfe ester. In all other cases, the yields of
the S-S peptides were above 45% after 1 h, which were better
than those obtained using the Tfe esters. Moreover, in some
cases the reaction proceeded almost to completion within 1 h.
Thus, incorporation of the non-protein amino acid residues
was found to be best achieved by employing the Cam esters as
acyl donors.
Yield (%)c
Tris buffer (%)b
Amide
Z-(S)-Phe
0
1
1.5
2
4
11.0
70.3
92.6
89.0
71.5
0.7
4.4
7.1
9.5
27.0
a Reactions were conducted using 0.05 mmol of Z-(S)-Phe-OTfe,
0.2 mmol of (S)-1-phenylethylamine and 150 mg of the immobilised
α-chymotrypsin in a solvent composed of 2 ml of acetonitrile and
0 µl, 20 µl (water content, 1%), 31 µl (1.5%), 41 µl (2%), or 83 µl (4%)
of 0.05 M Tris buffer (pH 7.8) at 30 ЊC. b By volume. c After 5 h of
incubation.
Enantioselective acylation of chiral amines with an N-protected
amino acid ester as an acyl donor
If an amine is used as a nucleophile instead of an amino acid
derivative in the kinetically controlled approach of protease-
catalysed peptide synthesis as depicted in Scheme 1, the for-
mation of an amide can be expected. To our best knowledge,
however, there have been few reports concerning the protease-
catalysed formation of an amide bond between an amino
acid and an amine. Accordingly, the nucleophilicity or steric
demand of amines during the reaction with the acyl-enzyme
intermediate is not fully understood. Chinsky et al. reported on
the chemoselective acylation of 6-aminohexan-1-ol with the
2-chloroethyl ester of N-Ac-(S)-Phe mediated by subtilisin
Carlsberg in 2-methylbutan-2-ol.6 On the other hand, in the
case of α-chymotrypsin there had been no relevant reports until
we reported preliminarily on the results of the first attempt to
achieve the enantioselective acylation of chiral amines using an
amino acid Cam ester as an acyl donor (Scheme 4).4 Here we
describe the relevant details.
Table 4 α-Chymotrypsin-catalysed acylation of homochiral amines
with Z-(S)-Phe-OCama
Yield (%)b
Amine
Amide
Z-(S)-Phe
(S)-1-Phenylethylamine
(R)-1-Phenylethylamine
(S)-1-(1-Naphthyl)ethylamine
(R)-1-(1-Naphthyl)ethylamine
92.5
58.1
89.8
8.2
6.8
16.5
8.7
48.8
a Reactions were conducted in a solvent containing 1.5% water in the
same manner as described in Table 3. b After 2 h of incubation.
6.3%]. (S)-1-Phenylethylamine was found to be a poor acyl
acceptor compared with the amino acid amides used previously
[peptide yields (%) after 1 h during the couplings of Z-(S)-Phe-
OTfe: Gly-NH2, 99; (S)-Leu-NH2, 98; (S)-Val-NH2, 97].2 Even
with this amine, however, the Cam ester was a much better acyl
donor than the halogenated alkyl ester, resulting in a marked
increase of the amide yield (78.6% even after 15 min).
Accordingly, employing Z-(S)-Phe-OCam as the acyl donor
the acylation of each enantiomer of 1-phenylethylamine and
1-(1-naphthyl)ethylamine was examined. The results obtained
after 2 h of incubation are compiled in Table 4. The (S)-
enantiomer of 1-phenylethylamine was a more reactive nucleo-
phile than the (R)-counterpart and the acyl donor was
consumed almost completely after 2 h together with the con-
comitant reaction with water (6.8%). The proportion of the
hydrolysis product of the donor ester increased when the less
reactive (R)-enantiomer was employed as the nucleophile. In
the case of 1-(1-naphthyl)ethylamine the difference in reactivity
of the two enantiomers was much larger: the (S)-enantiomer
was as reactive as (S)-1-phenylethylamine, while the (R)-
counterpart was far less reactive as a nucleophile, resulting in
the formation of a large quantity of the hydrolysis product of
the donor ester.
Based on the above results, the enantioselective acylation of
some racemic amines was examined using Z-(S)-Phe-OCam as
the acyl donor (Table 5). The amount of the immobilised
enzyme was reduced to one-fifth of that used in the above-
mentioned investigations. The diastereomers of the resulting
amides were separated well by normal-phase HPLC on a chiral
column, which allowed the accurate determination of dia-
stereomeric excess (d.e.)-values. The d.e.-value obtained was
used together with the yield of the amide product to estimate the
enantiomeric ratio E,7 the discrimination between the two com-
peting enantiomers of the amine nucleophile. The acylation of
1-phenylethylamine or 3-amino-1-phenylbutane was somewhat
faster than that of 1-(1-naphthyl)ethylamine; the competing
hydrolysis of the donor ester was at a low level (<6% yield). The
E-values were good to excellent in all the cases examined and,
Scheme 4
As shown below, the amines examined were found to be poor
acyl acceptors compared with the amino acid amides employed
previously2,3 Consequently, the water content of the solvent
must affect the amide-bond formation more severely and the
minimal amount of water necessary for guaranteeing reason-
able enzyme activity should be determined. Thus, the α-
chymotrypsin-catalysed acylation of (S)-1-phenylethylamine
with Z-(S)-Phe-OTfe was chosen as a model system and the
influence of buffer content in acetonitrile on the yields of the
desired amide and hydrolysis product of the donor ester was
investigated. As shown in Table 3, even in the absence of water
the enzyme showed some activity, but it was too low to give a
practical yield of the amide within a reasonable time. Water
content of 1.5% by volume was found to be the best, and
further experiments were conducted using the solvent of this
composition. When the amount of water was increased beyond
this value, the yield of the product amide decreased due to
competing hydrolysis of the donor ester.
Fig. 2 shows the comparison of reaction profiles of the α-
chymotrypsin-catalysed acylations of (S)-1-phenylethylamine
when different esters of Z-(S)-Phe were employed as acyl
donors. The yield of the desired amide was extremely low when
the Me ester was used. In contrast, the coupling efficiency was
profoundly improved with the Tfe ester, while the competing
hydrolysis of the donor ester was little accelerated [yield of
Z-(S)-Phe after 1 h: Me ester, 0.5%; Tfe ester, 4.6%; Cam ester,
392
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Fig. 2 Reaction profiles in the α-chymotrypsin-catalysed acylation of (S)-1-phenylethylamine with different N-Z-(S)-phenylalanine esters [Z-(S)-
Phe-OR]. Symbols: ᭺, R = Cam; ᭝, R = Tfe; ᮀ, R = Me.
Table 5 α-Chymotrypsin-catalysed acylation of racemic amines with Z-(S)-Phe-OCama
Amide
Z-(S)-Phe
Yield (%)
Amine
Time/min
Yield (%)b
% d.e.c
Ed
42
660
25
1-Phenylethylamine
1-(1-Naphthyl)ethylamine
3-Amino-1-phenylbutane
30
60
30
45.8
40.0
44.5
89.6
99.4
85.1
4.5
5.7
5.0
a Reactions were conducted in a solvent containing 1.5% water in the same manner as described in Table 3. b Total yield of the S-S and S-R amides.
c Diastereomeric excess of the S-S amide. d Enantiomeric ratio (Ref. 7). In this case E = ln [1 Ϫ c(1 ϩ d.e.)]/ln [1 Ϫ c(1 Ϫ d.e.)], where c is the total
yield of the S-S and S-R amides.
among others, the enantiodiscrimination of 1-(1-naphthyl)-
ethylamine proved to be extremely efficient. It is anticipated
from the E-values obtained that if a racemic amine is used in
an amount equivalent to the acyl donor, the enantiomeric
excess of the untouched (R)-isomer will be >99% when the
formation of amide is allowed to reach 50% yield with 1-(1-
naphthyl)ethylamine, and 60% with 1-phenylethylamine or
3-amino-1-phenylbutane, respectively.
In summary, the results reported here together with those
obtained previously2,3 demonstrate the superiority of the
Cam ester as an acyl donor in the α-chymotrypsin-catalysed
formation of amide bonds via the kinetically controlled
approach.
Elemental analysis data for all new compounds are available as
Supplementary material.†
Preparation of N-Z-amino acid carbamoylmethyl esters
The following Cam esters of N-Z-amino acids were prepared
via the reaction of their Cs salts with 2-chloroacetamide in
DMF at 60 ЊC overnight as described previously for the prep-
aration of Z-(S)-Ala-OCam.3b The crude products were
purified by recrystallisation from EtOAc–petroleum spirit
(60–86% yield).
Z-(RS)-Phe(2F)-OCam. Mp 121.5–122.5 ЊC; δH (DMSO-d6)
2.84–3.27 (2H, d of AB q, J 14.1, 10.8 and 4.2), 4.38–4.44 (1H,
m), 4.45 (2H, s), 4.95 (2H, s), 7.07–7.32 (10H, m), 7.39 (1H, br),
7.90 (1H, d, J 8.7).
Experimental
General
Z-(RS)-Phe(3F)-OCam. Mp 107.5–108.5 ЊC; δH (DMSO-d6)
2.83–3.22 (2H, d of AB q, J 14.1, 10.8 and 4.2), 4.39–4.45 (1H,
m), 4.46 (2H, s), 4.96 (2H, s), 7.02–7.37 (10H, m), 7.43 (1H, br),
7.90 (1H, d, J 8.7).
1H NMR spectra (300 MHz) were obtained on a Varian Unity
300 spectrometer using CDCl3 or DMSO-d6 as a solvent
with TMS as an internal standard. Mps were determined
on a Yamato MP-21 apparatus and are uncorrected. Optical
rotations were measured using a JASCO DIP-4 digital polar-
imeter. [α]D-Values are given in units of 10Ϫ1 deg cm2 gϪ1.
All solvents were distilled prior to use following standard
protocols and dried over molecular sieves. Petroleum spirit
refers to the fraction with distillation range 30–70 ЊC. Amino
acid amide hydrochlorides were purchased from Kokusan
Chemical Works. α-Chymotrypsin (type II, ex bovine pancreas)
was purchased from Sigma and had a specific activity of 48
units per mg solid with Bz-Tyr-OEt. It was immobilised by dis-
solution in pH 7.8 Tris buffer, mixing with Celite No. 535
(Johns-Mansville Co.), and lyophilising as described before.2
Z-(RS)-Phe(4F)-OCam. Mp 119–120.5 ЊC; δH (DMSO-d6)
2.80–3.18 (2H, d of AB q, J 13.8, 10.8 and 4.2), 4.34–4.44 (1H,
m), 4.46 (2H, s), 4.96 (2H, s), 7.05–7.36 (10H, m), 7.42 (1H, br),
7.88 (1H, d, J 8.4).
Z-(RS)-Phe(2Cl)-OCam. Mp 148.5–149 ЊC; δH (DMSO-d6)
2.90–3.37 (2H, d of AB q, J 13.8, 10.8 and 4.2), 4.43–4.51 (1H,
m), 4.47 (2H, s), 4.94 (2H, s), 7.20–7.43 (11H, m), 7.94 (1H, d, J
8.7).
Z-(RS)-Phe(3Cl)-OCam. Mp 83.5–84.5 ЊC; δH (DMSO-d6)
J. Chem. Soc., Perkin Trans. 1, 2002, 390–395
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2.79–3.21 (2H, d of AB q, J 13.8, 11.1 and 4.2), 4.37–4.44 (1H,
m), 4.46 (2H, s), 4.95 (2H, s), 7.20–7.36 (10H, m), 7.43 (1H, br),
7.89 (1H, d, J 8.7).
described previously for the preparation of Z-(S)-Ala-(S)-Leu-
NH2.3b As Z-(S)-Ala-Gly-NH2 and Z-(S)-Ala-(S)-Ala-NH2
were highly water-soluble, the washings were reduced to 1/5
of the usual quantity during the work-up. Z-(S)-Ala-Gly-
NH2: mp 118–119 ЊC (from aq. MeOH); [α]2D5 ϩ7.5 (c 1.0,
DMF) {lit.,10 mp 119–121 ЊC; [α]2D3 ϩ4.8 (c 2, DMF)}. Z-(S)-
Ala-(S)-Val-NH2: mp 243–246 ЊC (from MeOH); [α]2D5 ϩ12.8
(c 1.0, DMF) (lit.,11 mp 239–241 ЊC). Z-(S)-Ala-(S)-Phe-NH2:
mp 209–210 ЊC (from aq. MeOH); [α]2D5 Ϫ19.9 (c 1.0, DMF)
(lit.,11 mp 199–201 ЊC).
Z-(RS)-Phe(4Cl)-OCam. Mp 118.5–119.5 ЊC; δH (DMSO-d6)
2.80–3.19 (2H, d of AB q, J 13.8, 11.1 and 4.2), 4.38–4.44 (1H,
m), 4.47 (2H, s), 4.96 (2H, s), 7.22–7.34 (10H, m), 7.42 (1H, br),
7.87 (1H, d, J 8.4).
Z-(RS)-Phe(2Br)-OCam. Mp 164–165 ЊC; δH (DMSO-d6)
2.92–3.33 (2H, d of AB q, J 14.1, 11.1 and 4.2), 4.44–4.53 (1H,
m), 4.47 (2H, s), 4.95 (2H, s), 7.15–7.36 (10H, m), 7.58 (1H, d,
J 8.1), 7.94 (1H, d, J 8.7).
Z-(S)-Ala-(S)-Ala-NH2. 50% yield; mp 224–225 ЊC (from
EtOAc); [α]2D5 Ϫ5.4 (c 1.0, DMF); δH (DMSO-d6) 1.18 (6H, d,
J 7.2), 4.02 (1H, apparent quintet, J ≈7), 4.17 (1H, apparent
quintet, J ≈7), 4.96–5.05 (2H, AB q, J 12.6), 7.00 (1H, s), 7.28
(1H, s), 7.29–7.38 (5H, m), 7.48 (1H, d, J 7.2), 7.84 (1H, d,
J 7.2).
Z-(RS)-Phe(3Br)-OCam. Mp 92.5–93 ЊC; δH (DMSO-d6)
2.78–3.19 (2H, d of AB q, J 13.8, 11.1 and 4.2), 4.37–4.43 (1H,
m), 4.46 (2H, s), 4.96 (2H, s), 7.20–7.43 (10H, m), 7.50 (1H, s),
7.89 (1H, d, J 8.4).
Z-(S)-Ala-(S)-Met-NH2. 53% yield; mp 195.5–196 ЊC (from
MeOH); [α]2D5 Ϫ7.9 (c 1.0, DMF); δH (DMSO-d6) 1.19 (3H, d,
J 7.2), 1.70–2.01 (2H, m), 2.01 (3H, s), 2.37–2.44 (2H, m),
3.98–4.08 (1H, quintet-like), 4.22–4.29 (1H, m), 5.00 (2H, s),
7.07 (1H, s), 7.30–7.34 (6H, m), 7.50 (1H, d, J 7.5), 7.88 (1H, d,
J 7.8).
Z-(RS)-Phe(4Br)-OCam. Mp 122–123 ЊC; δH (DMSO-d6)
2.76–3.17 (2H, d of AB q, J 13.8, 10.8 and 4.2), 4.34–4.42 (1H,
m), 4.45 (2H, s), 4.95 (2H, s), 7.19–7.48 (11H, m), 7.87 (1H, d,
J 8.4).
Z-(S)-Phe-OCam. Mp 88–89 ЊC; [α]2D5 Ϫ40.0 (c 1.0, DMF)
{lit.,8 mp 88.5–89.5 ЊC; [α]2D1.5 Ϫ38.5 (c 1.0, DMF)}; δH (DMSO-
d6) 2.80–3.18 (2H, d of AB q, J 13.8, 11.1 and 4.5), 4.34–4.42
(1H, m), 4.45 (2H, s), 4.95 (2H, s), 7.18–7.35 (11H, m), 7.39
(1H, br), 7.87 (1H, d, J 8.4).
Peptide synthesis mediated by immobilised ꢀ-chymotrypsin
A mixture of an N-Z-amino acid ester [0.05 mmol for the (S)-
amino acid derivatives or 0.1 mmol for the (RS)-amino acid
derivatives], an amino acid amide hydrochloride (0.2 mmol),
TEA (28 µl, 0.2 mmol) and the immobilized enzyme (150 mg,
corresponding to 4.7 mg of α-chymotrypsin) was incubated
with shaking (180 strokes minϪ1) in a solvent composed 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, desired peptide
and hydrolysis product of the donor ester were determined by
HPLC analysis on a GL Sciences PU-610 liquid chromatograph
under the following conditions: column, Inertsil ODS 3 (5 µm;
1.5 mm id × 150 mm, GL Sciences); mobile phase, 30 or
35% aq. acetonitrile containing H3PO4 (0.01 M) or 55–65% aq.
MeOH containing H3PO4 (0.01 M); flow rate, 0.1 ml minϪ1;
column temperature, 30 or 40 ЊC; detection, at 254 nm on a
GL Sciences UV-620 variable-wavelength UV monitor; data
acquisition and processing, Shimadzu C-R6A data processor.
The diastereomers (S-S and R-S) of the resulting peptides
were also separated on the same column by regulating the
fraction of MeOH in the mobile phase (aq. MeOH). The
HPLC separations of compounds relevant to some α-chymo-
trypsin-catalysed couplings are available as Supplementary
material.†
Preparation of N-Z-(RS)-2-bromophenylalanine esters
The following esters were prepared through the reaction of
Z-(RS)-Phe(2Br) (830 mg, 2.2 mmol) with the corresponding
alcohol (2.1 mmol) in the presence of DMAP (135 mg, 1.1
mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDCؒHCl) (460 mg, 2.4 mmol) in dichloro-
methane (DCM) (8 ml) according to the procedure of Dhaon
et al.9 The crude products were purified by recrystallisation
from EtOAc–petroleum spirit (60–72% yield). The physical
properties of Z-(RS)-Phe(2Br)-OTfe were described
previously.2b
Z-(RS)-Phe(2Br)-OCH2CF2CF3. Mp 82.5–83.5 ЊC; δH
(CDCl3) 3.11–3.40 (2H, d of AB q, J 14.1, 9.0 and 5.7), 4.58
(2H, t, J 13.2), 4.75–4.83 (1H, m), 5.06 (2H, s), 5.24 (1H, br d,
J 8.4), 7.10–7.57 (9H, m).
Z-(RS)-Phe(2Br)-OCH2CH2Cl. Mp 54.5–56 ЊC; δH (CDCl3)
3.14–3.39 (2H, d of AB q, J 13.8, 8.1 and 5.7), 3.61 (2H, t,
J 5.7), 4.36 (2H, t, J 5.7), 4.71–4.78 (1H, q-like), 5.06 (2H, s),
5.30 (1H, br d, J ≈8), 7.08–7.56 (9H, m).
An aliquot of the reaction mixture from Z-(RS)-Phe(2Br)-
OCam ϩ (S)-Leu-NH2 was treated with an ethereal solution
of diazomethane and the resulting ester Z-Phe(2Br)-OMe was
analysed on a Chiralcel OD column (4.6 mm id × 250 mm,
Daicel Chemical Industries; eluent, hexane–propan-2-ol) to
determine its enantiomeric purity as described before.5
Z-(RS)-Phe(2Br)-OCH2CCl3. Mp 53–55 ЊC; δH (CDCl3)
3.11–3.49 (2H, d of AB q, J 13.8, 8.7 and 5.1), 4.72–4.84 (2H,
AB q, J 11.7), 4.78–4.89 (1H, m), 5.05 (2H, s), 5.28 (1H, br d,
J ≈7), 7.09–7.57 (9H, m).
Preparation of authentic N-[N-Z-(S)-phenylalanyl]amines
Z-(RS)-Phe(2Br)-ON᎐C(CH ) . Mp 71.5–73 ЊC; δ (CDCl3)
᎐
3
2
H
The preparation of N-[Z-(S)-Phe]-(S)-1-(1-naphthyl)ethyl-
amine is described as a typical example. To a stirred solution of
Z-(S)-Phe (600 mg, 2.0 mmol), (S)-1-(1-naphthyl)ethylamine
(340 mg, 2.0 mmol) and HOBT (270 mg, 2.0 mmol) in DCM
(3 ml) was added EDCؒHCl (380 mg, 2.0 mmol) under ice-
cooling. After stirring of the mixture at this temperature for 3 h
and then at room temperature for 3 days, the solvent was
removed in vacuo. The residue 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 afforded crystals,
1.81 (3H, s), 2.02 (3H, s), 3.15–3.37 (2H, d of AB q, J 13.8, 8.1
and 6.6), 4.83–4.90 (1H, q-like), 5.06 (2H, s), 5.42 (1H, br d,
J 8.4), 7.07–7.56 (9H, m).
Preparation of authentic dipeptides
The authentic samples of N-protected dipeptide amides, Z-(S)-
Ala-Xbb-NH2 [Xbb = Gly, (S)-Ala, (S)-Val, (S)-Met or (S)-
Phe], were prepared through the coupling of Z-(S)-Ala and
Xbb-NH2ؒHCl [in the presence of an equimolar amount of
triethylamine (TEA)] by the EDC–HOBT method in DMF as
394
J. Chem. Soc., Perkin Trans. 1, 2002, 390–395

View Article Online
which were recrystallised from EtOAc–hexane; 77% yield;
mp 183–185 ЊC; [α]2D5 ϩ6.7 (c 1.0, DMF); δH (CDCl3) 1.55
(3H, d, J 6.9), 2.89–3.09 (2H, d of AB q, J 13.8, 8.4 and 6.0),
4.31–4.38 (1H, q-like), 5.04 (2H, apparent s), 5.47 (1H, br d),
5.81–5.91 (1H, quintet-like, J 7.5), 6.03 (1H, br d), 6.97–8.05
(17H, m).
Reactions of N-Z-(S)-phenylalanine carbamoylmethyl ester with
racemic amines mediated by ꢀ-chymotrypsin
A mixture of Z-(S)-Phe-OCam (0.05 mmol), a racemic amine
(0.2 mmol) and the immobilised α-chymotrypsin (30 mg) was
incubated, with shaking, in the same solvent as above at 30 ЊC.
The progress of the reaction was monitored by reversed-phase
HPLC analysis, and the diastereomers of the resulting amide
were quantified by normal-phase HPLC on a Chiralpak AD
column (4.6 mm id × 250 mm, Daicel Chemical Industries)
using a Shimadzu LC-5A liquid chromatograph equipped
with an SPD-2A UV monitor. The retention times (min) are as
follows [mobile phase, hexane–propan-2-ol (9 : 1, v/v); flow rate,
1.0 ml minϪ1; column temperature, 40 ЊC]: N-[Z-(S)-Phe]-(R)-1-
phenylethylamine, 10.8; N-[Z-(S)-Phe]-(S)-1-phenylethylamine,
12.3; N-[Z-(S)-Phe]-(R)-1-(1-naphthyl)ethylamine, 13.0; N-[Z-
(S)-Phe]-(S)-1-(1-naphthyl)ethylamine, 14.4; N-[Z-(S)-Phe]-
(R)-3-amino-1-phenylbutane, 7.9; N-[Z-(S)-Phe]-(S)-3-amino-
1-phenylbutane, 9.4.
N-[Z-(S)-Phe]-(S)-1-phenylethylamine. 79% yield; mp 186–
188 ЊC; [α]2D5 Ϫ18.7 (c 1.0, DMF); δH (CDCl3) 1.38 (3H, d, J 6.9),
2.93–3.11 (2H, d of AB q, J 13.6, 8.0 and 6.0), 4.31–4.40
(1H, q-like), 4.97–5.07 (1H, quintet-like), 5.08 (2H, apparent s),
5.40 (1H, br d), 5.88 (1H, br d), 7.07–7.33 (15H, m).
N-[Z-(S)-Phe]-(R)-1-methyl-3-phenylpropylamine. 83% yield;
mp 162–163 ЊC; [α]2D5 Ϫ14.8 (c 1.0, DMF); δH (CDCl3) 0.95
(3H, d, J 6.9), 1.57–1.69 (2H, m), 2.53 (2H, t, J 7.8), 2.93–3.15
(2H, d of AB q, J 13.5, 8.1 and 6.0), 3.94 (1H, septet-like, J ≈7),
4.26 (1H, q-like, J ≈7), 5.09 (2H, s), 5.24 (1H, d, J 8.7), 5.29 (1H,
br), 7.09–7.37 (15H, m).
The mixtures of diastereomers (S-S ϩ S-R) of N-[Z-(S)-
Phe]-amines were also prepared through the coupling of Z-(S)-
Phe with(RS)-1-phenylethylamine, (RS)-1-(1-naphthyl)ethyl-
amine or (RS)-1-methyl-3-phenylpropylamine by the EDC–
HOBT method in DCM in the same manner as above and
purified by recrystallisation from EtOAc–hexane. Each sample
thus prepared showed only two peaks corresponding to both
the diastereomers on normal-phase HPLC using the chiral
column mentioned below. The slower eluting diastereomer
proved to be S-S by comparison with the authentic sample.
Acknowledgements
This work was supported in part by a grant from the Hirao
Taro Foundation of the Konan University Association for
Academic Research.
References
1 For reviews, see: (a) K. Faber, Biotransformations in Organic
Chemistry, Springer-Verlag, Berlin, 4th edn., 2000, p. 370; (b) H.-D.
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Reactions of N-Z-(S)-phenylalanine esters with homochiral
amines mediated by ꢀ-chymotrypsin
The reaction of Z-(S)-Phe-OTfe with (S)-1-phenylethylamine
is described as a typical example. To a solution of Z-(S)-Phe-
OTfe (0.05 mmol) and (S)-1-phenylethylamine (0.2 mmol) in
acetonitrile (2 ml) was added the immobilised α-chymotrypsin
on Celite (150 mg), followed by 31 µl of pH 7.8 Tris buffer
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10AS liquid chromatograph under the following conditions:
column, Cosmosil 5C18 (4.6 mm id × 150 mm, Nacalai Tesque);
mobile phase, 60% aq. MeOH containing H3PO4 (0.01 M); flow
rate, 1.0 ml minϪ1; column temperature, 40 ЊC; detection, at 254
nm on a Shimadzu SPD-10A UV monitor. Aliquots (10 µl
each) of the reaction mixture were withdrawn at frequent inter-
vals, diluted with AcOH (100 µl), and then injected onto the
column. The retention times (min) of the relevant compounds
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J. Chem. Soc., Perkin Trans. 1, 2002, 390–395
395