Broadening of the substrate tolerance of α-chymotrypsin by using the carbamoylmethyl ester as an acyl donor in kinetically controlled peptide synthesis

Article abstract of DOI:10.1039/b004180g

In the kinetically controlled approach of peptide synthesis mediated by α-chymotrypsin, the broadening of the protease's substrate tolerance is achieved by switching the acyl donor from the conventional methyl ester to the carbamoylmethyl ester. Thus, as a typical example, the extremely low coupling efficiency obtained by employing the methyl ester of an inherently poor amino acid substrate, Ala, is significantly improved by the use of this particular ester. Its ameliorating effect is observed also in the couplings of other amino acid residues such as Gly and Ser as carboxy components.

Full text of DOI:10.1039/b004180g

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Broadening of the substrate tolerance of ꢀ-chymotrypsin by using
the carbamoylmethyl ester as an acyl donor in kinetically
controlled peptide synthesis
1
Toshifumi Miyazawa,* Kayoko Tanaka, 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
In the kinetically controlled approach of peptide synthesis mediated by α-chymotrypsin, the broadening of the
protease’s substrate tolerance is achieved by switching the acyl donor from the conventional methyl ester to the
carbamoylmethyl ester. Thus, as a typical example, the extremely low coupling efficiency obtained by employing
the methyl ester of an inherently poor amino acid substrate, Ala, is significantly improved by the use of this
particular ester. Its ameliorating effect is observed also in the couplings of other amino acid residues such as
Gly and Ser as carboxy components.
either by a nucleophilic amine component to give a peptide
Introduction
product or by water to form the hydrolysis product of the donor
ester. Such protease-catalysed peptide-bond formation can be
carried out in organic solvents with only a small amount of
water as well as in aqueous media.4 In the former the water
activity is reduced, which favours the synthetic reaction and
virtually eliminates the secondary hydrolysis of the formed
peptide. Along these lines, we have investigated the α-chymo-
trypsin-catalysed peptide synthesis using esters of N-protected
amino acids (or peptides) as acyl donors in hydrophilic organic
solvents containing only a few percent of water and we have
found that such activated esters as the 2,2,2-trifluoroethyl ester
are useful for the incorporation of non-protein amino acids
such as halogenophenylalanines into peptides.2 As an extension
of this study, we have examined the couplings of α-chymo-
trypsin’s inherently poor amino acid substrates, e.g., Ala, by
employing a series of its esters. Table 1 summarises the yields
of the desired peptide and the hydrolysis product of the donor
ester in the α-chymotrypsin-catalysed coupling of an ester of
Z--Ala with -Leu-NH2 in acetonitrile containing 4% (v/v)
Tris buffer (pH 7.8) after 48 h of incubation (Scheme 2),
Although enzymic methods for peptide synthesis1 have several
advantages over conventional chemical methodologies, a
narrow substrate specificity is often regarded as a major draw-
back from a synthetic standpoint: only limited amino acid
residues are acceptable as substrates. Accordingly, developing
a means to broaden the applicability of proteases for peptide
synthesis remains a challenging task. In the preceding paper,2
we have reported that the coupling efficiency in the kinetically
controlled peptide-bond formation catalysed by α-chymo-
trypsin is profoundly 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 in an organic solvent such as
acetonitrile with low water content and that this approach is
useful for the incorporation of non-protein amino acids such
as halogenophenylalanines into peptides. The success of this
approach prompted us to attempt to overcome the narrow sub-
strate tolerance of the protease by modulating the ester moiety
of an acyl donor. Thus, we have examined a series of activated
esters for the couplings of α-chymotrypsin’s inherently poor
substrates such as Ala lacking a large hydrophobic side chain.
We have at last found that the carbamoylmethyl ester is superior
even to the halogenated alkyl esters,3 and that this particular
ester is able to broaden the protease’s substrate tolerance. This
paper describes the details of the relevant works.
Scheme 2
Results and discussion
together with the relative initial rate (νrel) of consumption of the
substrate ester. The reaction conditions adopted here were the
same as those determined as optimal ones in the previous
investigation.2 As was expected, the yield of the desired peptide,
as well as that of the hydrolysed donor ester, was extremely low
even after 48 h when the methyl ester (Entry 1) was used as
an acyl donor. This was also the case with other alkyl esters
carrying a longer chain (Entries 2, 3, 5–11). By contrast, when
halogenated alkyl esters (Entries 12–15) were employed, the
coupling efficiency was improved profoundly in accordance
with our previous results.2 As shown in Scheme 1, the acyl-
enzyme intermediate is partitioned between aminolysis and
hydrolysis, affording the peptide product and the hydrolysis
product of the donor ester. Once the acyl-enzyme intermediate
is formed, its competitive partitioning is supposed to be
unaffected by the ester moiety of the acyl donor, according to
In the kinetically controlled peptide synthesis catalysed by a
serine or cysteine protease1b,e (Scheme 1), an ester substrate is
used as an acyl donor, which facilitates the formation of the
acyl-enzyme intermediate. This intermediate is then deacylated
Scheme 1 Kinetically controlled peptide synthesis: H-E, serine or
cysteine protease; R1CO2R, carboxy component in the form of an
ester; R2NH2, amino component.
DOI: 10.1039/b004180g
J. Chem. Soc., Perkin Trans. 1, 2001, 87–93
This journal is © The Royal Society of Chemistry 2001
87

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a
Table 1 α-Chymotrypsin-catalysed couplings of Z--Ala-OR with -Leu-NH2
Yield (%)d
Peptide
b
Entry
R
νrel
σ*c
Z--Ala-OH
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
CH3
CH2CH3
1e
0.54
0.39
0
6.7
4.2
2.5
1.0
3.4
10.2
3.0
2.0
3.4
6.3
1.0
0.7
0.5
0
0.6
1.0
0.4
0.3
0.6
Ϫ0.100
Ϫ0.115
(CH2)2CH3
CH(CH3)2
(CH2)3CH3
CH2CH(CH3)2
(CH2)4CH3
(CH2)5CH3
C6H11-c
CH2C5H9-c
CH2C6H11-c
CH2CF3
0.53
0.76
0.34
0.24
0.41
0.74
0.68
17.5
8.8
3.4
14.1
4.2
18.7
11.4
5.3
2.8
29.1
0.61
0.42
29.6
6.7
6.9
0.51
3.0
133
110
4.8
Ϫ0.130
Ϫ0.125
0.9
Ϫ0.06
ϩ0.92
4.9
0.7
82.4
63.2
30.5
87.8
28.6
79.3
72.7
33.6
19.2
89.9
9.1
8.6g
6.8g
3.8g
6.9g
4.2
CH2CF2CF3
CH2CH2Cl
CH2CCl3
ϩ0.385
ϩ0.215
CH2Ph
CH2C6H4NO2-4
CH2C6H4CN-4
CH2C6H4Cl-4
CH2C6H4OMe-4
CH2C5H4N
(CH2)2Ph
9.2
9.6
4.0
2.2
10.0
0.7
ϩ0.080
ϩ0.02
ϩ1.30
ϩ0.64 f
ϩ0.60
(CH2)3Ph
CH2CN
6.8
0.5
88.3
45.6
43.0
7.5
24.0
88.4
89.1
34.4
6.4g
5.6g
7.0g
4.1
CH2OCH3
CH2COCH3
CH2COPh
CH2CO2Et
CH2CONH2
CH2CONHCH3
CH2CON(CH3)2
4.1
10.9g
10.8g
5.2
a Coupling conditions: Z--Ala-OR (0.05 mmol), -Leu-NH2ؒHCl (0.2 mmol), TEA (0.2 mmol), immobilised α-chymotrypsin on Celite (150 mg),
acetonitrile (2 ml), 0.05 M Tris buffer (pH 7.8) (80 µl), 30 ЊC. b The initial rate of consumption of the substrate ester was determined through
the periodical assay of the reaction mixture over 8 h (4 h and 1 h in the case of moderately fast reacting esters and fast reacting esters, respectively).
c Ref. 11. d After 48 h of incubation. e 2.79 × 10Ϫ2 mM hϪ1 mgϪ1. The value in Table 1 of ref. 3b was misprinted and should be corrected. f Ref. 12.
g Corrected for non-enzymic hydrolysis (see Table 2).
the simplified reaction scheme. It may have some effect in the
case when the leaving alkoxy group (OR) is not completely
detached from the acyl-enzyme intermediate before the
deacylation by nucleophiles occurs. In practice, the competing
hydrolysis of acyl donors was little accelerated when the
halogenated alkyl esters were used. The use of the 2,2,2-
trifluoroethyl and 2,2,2-trichloroethyl esters (Entries 12 and 15,
respectively) was especially effective.
alkylestersbearingelectron-withdrawinggroupsotherthanhalo-
gens and phenyl rings were examined next. The cyanomethyl
ester8 (Entry 24) increased the peptide yield significantly. In
this case, no peptide product was obtained non-enzymically,
while spontaneous hydrolysis of the donor ester occurred
to some extent (see Table 2).† The methoxymethyl and
acetonyl esters (Entries 25 and 26, respectively) had a con-
siderable ameliorating effect. Finally, it was gratifying to find
that the carbamoylmethyl ester (Entry 29) was superior
to the halogenated alkyl esters. The use of this ester as an
enzyme substrate was reported some decades ago.9 It was later
examined as a donor ester in the α-chymotrypsin-catalysed
coupling of Z- or Boc-Phe conducted in aqueous organic
media.10 The chief concern at that time was to take advantage
of the better solubility of this ester in the aqueous phase for the
coupling of the α-chymotrypsin’s good amino acid substrates.
The superiority of the carbamoylmethyl ester can be seen from
its reaction profile as compared with those of the other esters,
e.g., the methyl and trifluoroethyl esters (Fig. 1): even after
2 h of incubation the peptide yield reached to 77% and the
maximum yield (88%) was attained after ca. 6 h.
With the benzyl ester5 (Entry 16) the peptide yield was
improved to a considerable extent, and its p-nitro or p-cyano
derivative (Entries 17 and 18, respectively) had a further
ameliorating effect, which was comparable to that shown by
the above mentioned halogenated alkyl esters. The effect of
substituents on the initial rate of consumption of the sub-
strate ester resembled that on the rates of alkaline hydrolysis
of substituted-benzyl acetates6 or benzoates.7 Thus, when the
p-methoxybenzyl ester (Entry 20) was employed as an acyl
donor, the peptide yield was lower than that obtained using the
benzyl ester itself. It is interesting to note that the 4-pyridyl-
methyl ester (Entry 21) afforded a result similar to that given by
the p-nitrobenzyl ester. The insertion of a methylene group
between the phenyl ring and the ester oxygen atom resulted in
a substantial decrease in the peptide yield (Entries 22 and 23),
indicating the inductive (electron-withdrawing) effect of the
phenyl ring. The importance of the electronic effect, over the
steric effect which must be responsible mainly for the stability
of the enzyme–substrate (ES) complex, was supported by the
fact that the cyclohexylmethyl or cyclopentylmethyl ester
(Entry 11 or 10, respectively), which had a steric demand more
similar to that of the benzyl ester, exerted no marked influence
compared with the other alkyl esters. Accordingly, substituted
When the log vrel-value for each ester substrate in Table 1
was plotted against the polar substituent constant, σ*,11 for
R, which is a measure of its polar or inductive effect, a rough
proportionality was found between them (Fig. 2): the higher
† Non-enzymic peptide synthesis was not detected when the other
activated esters were employed, while a small amount of the donor
ester was hydrolysed non-enzymically in some cases, as shown
in Table 2. In Table 1, corrections were made for non-enzymic
hydrolysis.
88
J. Chem. Soc., Perkin Trans. 1, 2001, 87–93

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a
Table 2 Non-enzymic reactions of Z--Ala-OR with -Leu-NH2
Table
3
α-Chymotrypsin-catalysed couplings of Z-Xaa-OR with
a
-Leu-NH2
Yield (%)b
Yield (%)b
Peptide
R
Peptide
Z--Ala-OH
Xaa
Gly
R
Z-Xaa-OH
CH2CF3
0
0
0
0
0
0
0
0
0
1.9
2.3
0.2
2.0
5.3
2.9
1.6
0.7
0.1
CH2CF2CF3
CH2CH2Cl
CH2CCl3
CH3
CH2CF3
CH2Ph
CH2C6H4NO2-4
CH2CONH2
CH3
CH2CF3
CH2Ph
CH2C6H4NO2-4
CH2CONH2
CH3
CH2CF3
CH2CONH2
CH3
CH2CONH2
CH2CF3
CH2CONH2
CH2CONH2
0.4
74.3
3.9
23.4
90.9
8.7
87.9
69.7
92.4
91.3
0
0.9
13.6
0
40.6
0.6
0.6
0.6
9.1c
1.3
3.3
6.0c
1.2
0.5c
4.6
4.6
6.3c
0.3
0.8
6.8
0.8
7.0
0.5
0.3
1.4
CH2CN
CH2OCH3
CH2COCH3
CH2CONH2
CH2CONHCH3
-Ser
-Val
a Reactions were conducted using Z--Ala-OR (0.05 mmol), -Leu-
NH2ؒHCl (0.2 mmol), and TEA (0.2 mmol) in a solvent composed
of acetonitrile (2 ml) and 0.05 M Tris buffer (pH 7.8) (80 µl) at 30 ЊC.
b After 48 h.
-Ala
-Pro
Aib
6.4
a The coupling conditions were the same as described in Table 1,
using 0.05 mmol of acyl donor. b After 48 h. c Corrected for non-
enzymic hydrolysis. The yields (%) of Z-Xaa-OH formed after
48 h of incubation under the same conditions as described in
Table
2 were as follows: Z-Gly-OCH2CF3, 1.8; Z-Gly-OCH2-
CONH2, 2.1; Z--Ser-OCH2CF3, 10.2; Z--Ser-OCH2CONH2, 2.2.
The corresponding values in Table 2 of ref. 3b were uncorrected
ones.
esters whose amide hydrogens were substituted by methyl
group(s) were examined next (Entries 30 and 31 in Table 1).
The monomethylated ester gave almost the same result as
the parent ester, while with the dimethylated ester the peptide
yield was diminished to a great extent. This result implies the
necessity of at least one amide proton of the carbamoyl-
methyl ester for stabilisation through hydrogen bonding of
the ES-complex.
Fig. 1 Reaction profiles in the α-chymotrypsin-catalysed couplings
of Z--Ala-OR with -Leu-NH2. Symbols: ᭺, R = CH2CONH2;
᭝, R = CH2CF3; ᮀ, R = CH3.
The ameliorating effect of the carbamoylmethyl ester was
observed also in the couplings of other amino acid residues as
carboxy components. As shown in Table 3, Gly behaved as a
poorer amino acid substrate for α-chymotrypsin than did -Ala,
the yield of the desired peptide being negligible even after 48 h
of incubation when the methyl ester was used as the acyl donor.
In this case also, a marked enhancement of peptide yield
by the use of the trifluoroethyl ester and the superiority of
the carbamoylmethyl ester over other activated esters were
demonstrated. The same trend was observed also among the
methyl, trifluoroethyl and carbamoylmethyl esters of -Ser.
But a feature observed only in this particular case was that
the p-nitrobenzyl ester was as good a donor ester as the carb-
amoylmethyl ester. The methyl ester of Z--Val failed in the
production of the desired peptide, probably due to the steric
hindrance caused by the β-branching in the side chain, while the
carbamoylmethyl ester managed to give the peptide product,
albeit in low yield. This was also the case with another bulky
amino acid, α-aminoisobutyric acid (Aib, 2-amino-2-methyl-
propanoic acid),15 the peptide yield being even lower. Further-
more, the use of the carbamoylmethyl ester allowed even the
coupling of a -amino acid as the carboxy component.16 Thus,
the Z--Ala ester gave the desired --peptide in moderate
yield, although the νrel-value was less than one sixth of that for
the -counterpart. On the other hand, it proved to be quite
difficult to obtain the peptide of -Pro even by the use of the
carbamoylmethyl ester.
Fig. 2 Plot of log νrel against σ* of R for the α-chymotrypsin-catalysed
coupling of Z--Ala-OR with -Leu-NH2 (see Table 1).
the electron-withdrawing ability of the R group, the higher
the νrel-value. The log νrel-value corresponds to the relative
activation free energy which is dependent both on the stability
of the ES complex and on the rate of acyl-enzyme form-
ation. The correlation between log νrel and σ* is indicative
of the predominance of the effect of the R group on the
acyl-enzyme formation over the binding of the substrate ester
onto the enzyme. In the case of the carbamoylmethyl ester,
however, its effect on the binding stage must also be relatively
important, because the electron-withdrawing ability of this
group should not be markedly large compared with other
groups, e.g., the acetonyl group (σ* = ϩ0.60), though its σ*-
value is not reported in the literature.‡ Carbamoylmethyl
In summary, α-chymotrypsin’s narrow substrate specificity
was broadened by the use of the carbamoylmethyl ester as the
acyl donor in the kinetically controlled approach of peptide
synthesis mediated by this protease.
‡ The σ*-value for the carbamoylmethyl group is estimated to be ϩ0.59
according to the following equation,13 pKa(RCO2H) = Ϫ1.700σ*(R) ϩ
4.644, by employing the reported Ka-value for propanedioic acid
monoamide (2.284 × 10Ϫ4 at 25 ЊC).14
J. Chem. Soc., Perkin Trans. 1, 2001, 87–93
89

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Table 4 Elemental analyses of new compounds
C (%)
Found
(required)
H (%)
Found
(required)
Molecular
formula
Compound
Z--Ala-OCH2CH3
C13H17NO4
C14H19NO4
C14H19NO4
C15H21NO4
C15H21NO4
C16H23NO4
C17H25NO4
C17H23NO4
C17H23NO4
C18H25NO4
C13H14F3NO4
C14H14F5NO4
C13H16ClNO4
C13H14Cl3NO4
C18H19NO4
C18H18N2O6
C19H18N2O4
C18H18ClNO4
C19H21NO5
C19H21NO4
C20H23NO4
C13H14N2O4
C13H17NO5
C14H17NO5
C15H19NO6
C14H18N2O5
C15H20N2O5
C13H16N2O6
C15H20N2O5
C15H18N2O5
C14H18N2O5
C17H25N3O4
C16H23N3O4
C17H25N3O5ؒ1/2H2O
C19H29N3O4
C19H27N3O4
C18H27N3O4
62.21 (62.14)
63.11 (63.38)
63.39 (63.38)
64.22 (64.50)
64.33 (64.50)
65.33 (65.51)
66.29 (66.43)
66.61 (66.86)
66.71 (66.86)
67.23 (67.69)
51.46 (51.15)
47.51 (47.33)
54.70 (54.65)
44.30 (44.03)
68.81 (69.00)
60.41 (60.33)
67.14 (67.45)
62.17 (62.16)
66.29 (66.46)
69.66 (69.71)
70.25 (70.36)
59.41 (59.54)
58.21 (58.42)
60.24 (60.21)
58.05 (58.25)
57.03 (57.14)
58.08 (58.43)
52.64 (52.70)
58.10 (58.43)
58.63 (58.82)
56.87 (57.14)
61.08 (60.88)
59.72 (59.80)
56.61 (56.65)
62.69 (62.79)
62.84 (63.14)
61.61 (61.87)
6.87 (6.82)
7.28 (7.21)
7.23 (7.21)
7.60 (7.58)
7.62 (7.58)
7.98 (7.90)
8.23 (8.20)
7.61 (7.59)
7.75 (7.59)
7.82 (7.89)
4.63 (4.62)
3.92 (3.97)
5.63 (5.64)
4.06 (3.98)
6.19 (6.11)
5.03 (5.06)
5.24 (5.36)
5.19 (5.22)
6.14 (6.16)
6.48 (6.47)
6.78 (6.79)
5.54 (5.38)
6.47 (6.41)
6.19 (6.13)
6.16 (6.19)
6.24 (6.16)
6.64 (6.54)
5.54 (5.44)
6.73 (6.54)
5.91 (5.92)
6.02 (6.16)
7.36 (7.51)
7.17 (7.21)
7.18 (7.27)
8.07 (8.04)
7.53 (7.53)
8.01 (7.79)
Z--Ala-O(CH2)2CH3
Z--Ala-OCH(CH3)2
Z--Ala-O(CH2)3CH3
Z--Ala-OCH2CH(CH3)2
Z--Ala-O(CH2)4CH3
Z--Ala-O(CH2)5CH3
Z--Ala-OC6H11-c
Z--Ala-OCH2C5H9-c
Z--Ala-OCH2C6H11-c
Z--Ala-OCH2CF3
Z--Ala-OCH2CF2CF3
Z--Ala-OCH2CH2Cl
Z--Ala-OCH2CCl3
Z--Ala-OCH2Ph
Z--Ala-OCH2C6H4NO2-4
Z--Ala-OCH2C6H4CN-4
Z--Ala-OCH2C6H4Cl-4
Z--Ala-OCH2C6H4OMe-4
Z--Ala-O(CH2)2Ph
Z--Ala-O(CH2)3Ph
Z--Ala-OCH2CN
Z--Ala-OCH2OCH3
Z--Ala-OCH2COCH3
Z--Ala-OCH2CO2Et
Z--Ala-OCH2CONHCH3
Z--Ala-OCH2CON(CH3)2
Z--Ser-OCH2CONH2
Z--Val-OCH2CONH2
Z--Pro-OCH2CONH2
Z-Aib-OCH2CONH2
Z--Ala--Leu-NH2
Z-Gly--Leu-NH2
Z--Ser--Leu-NH2
Z--Val--Leu-NH2
Z--Pro--Leu-NH2
Z-Aib--Leu-NH2
(c 1.0, MeOH) {lit.,18 mp 54–55 ЊC; [α]D20 Ϫ18.9 (c 1, MeOH)};
Z--Ala-OMe: mp 47–48.5 ЊC; [α]D25 ϩ33.8 (c 1.0, MeOH).
(ii) The following esters were prepared through the reaction
of an N-Z-amino acid (2.2 mmol) with the corresponding
alcohol (2.5 mmol for the group A esters or 2.1 mmol for the
group B esters) in the presence of DMAP (135 mg, 1.1 mmol)
and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (EDCؒHCl) (460 mg, 2.4 mmol) in DCM (8 ml) accord-
ing to the procedure of Dhaon et al.:19 (A) ethyl, n-propyl,
isopropyl, n-butyl, isobutyl, cyclohexyl, cyclopentylmethyl,
cyclohexylmethyl, 2,2,2-trifluoroethyl, 2,2,3,3,3-pentafluoro-
propyl, 2-chloroethyl, or 2,2,2-trichloroethyl ester; (B) n-pentyl,
n-hexyl, benzyl, 4-chlorobenzyl, 4-methoxybenzyl, 2-phenyl-
ethyl, 3-phenylpropyl, or ethoxycarbonylmethyl ester.
Z-Gly-OCH2CF3: mp 73–73.5 ЊC (Found: C, 49.56; H, 4.10;
N, 4.87. C12H12F3NO4 requires C, 49.49; H, 4.15; N, 4.81%);
Z-Gly-OCH2Ph: mp 71–72 ЊC (Found: C, 68.26; H, 5.84;
N, 4.70. C17H17NO4 requires C, 68.22; H, 5.72; N, 4.68%);
Z--Ser-OCH2CF3: mp 73–77 ЊC; [α]D25 Ϫ14.2 (c 1.0, MeOH)
(Found: C, 48.87; H, 4.36; N, 4.48. C13H14F3NO5 requires C,
48.60; H, 4.39; N, 4.36%); Z--Val-OCH2CF3: mp 58 ЊC;
[α]D25 Ϫ17.8 (c 1.0, MeOH) (Found: C, 53.97; H, 5.42; N, 4.24.
C15H18F3NO4 requires C, 54.05; H, 5.44; N, 4.20%); Z--Pro-
OCH2CF3: oil; [α]D25 Ϫ48.8 (c 1.0, MeOH) (Found: C, 54.17;
H, 4.87; N, 4.23. C15H16F3NO4 requires C, 54.38; H, 4.87;
N, 4.23%).
Experimental
All solvents were distilled, and dried over molecular sieves
prior to use. -Leu-NH2ؒHCl was purchased from Kokusan
Chemical Works (Japan). α-Chymotrypsin (type II, ex bovine
pancreas) was purchased from Sigma and had a specific activity
of 48 units per mg solid with N-Bz-Tyr ethyl ester. It was
immobilised on Celite using a pH 7.8 buffer as described
before.2 IR spectra were recorded on a Nicolet N-750B
FT-IR spectrometer using attenuated total reflectance
(ATR). 1H NMR spectra (300 MHz) were recorded with a
Varian Unity 300 spectrometer using CDCl3 as solvent with
TMS as internal standard unless otherwise stated. Mps were
determined on a Yamato MP-21 apparatus and are un-
corrected. Optical rotations were measured with a JASCO
DIP-4 digital polarimeter. [α]D-Values are given in units of
10Ϫ1 deg cm2 gϪ1. Elemental analyses of new compounds are
compiled in Table 4. Petroleum spirit refers to the fraction with
distillation range 30–70 ЊC.
Preparation of N-Z-amino acid esters
The physical and 1H NMR data of the esters of Z--Ala
prepared as described below are compiled in Table 5, and those
of the carbamoylmethyl esters of other N-Z-amino acids are
in Table 6.
(i) The methyl esters were prepared through treatment of
the corresponding N-Z-amino acids with an ethereal solution
of diazomethane in a nearly quantitative yield. Z-Gly-OMe:
oil; Z--Ser-OMe: oil; [α]D25 Ϫ14.0 (c 1.0, MeOH) {lit.,17 mp 45–
46 ЊC; [α]D22 Ϫ12.5 (c 1, MeOH)}; Z--Val-OMe: oil; [α]D25 Ϫ19.4
(iii) The following esters were prepared via reaction of the Ce
salt of an N-Z-amino acid with the corresponding chloride:20
the carbamoylmethyl, cyanomethyl, methoxymethyl, acetonyl,
benzoylmethyl, N-methylcarbamoylmethyl, or N,N-dimethyl-
90
J. Chem. Soc., Perkin Trans. 1, 2001, 87–93

View Article Online
Table 5 Physical and 1H NMR data of Z--Ala-OR
[α]D25 (10Ϫ1
IR (ATR)
c
b
R
Mp (T/ЊC)a deg cm2 gϪ1
)
(νmax/cmϪ1
)
1H NMR δH (CDCl3)
d
CH3
46–47.5
(A)
oil
Ϫ33.8
Ϫ31.1
Ϫ32.0
Ϫ28.4
3339, 1752, 1691
3334, 1720
1.41 (3H, d, J 6.9), 3.75 (3H, s), 4.39 (1H, quint, J 6.9), 5.11 (2H, s), 5.24–5.40
(1H, br d), 7.26–7.40 (5H, m)
1.27 (3H, t, J 7.2), 1.41 (3H, d, J 6.9), 4.20 (2H, q, J 7.2), 4.36 (1H, quint,
J 6.9), 5.11 (2H, s), 5.26–5.44 (1H, br), 7.25–7.40 (5H, m)
0.94 (3H, t, J 7.5), 1.42 (3H, d, J 7.2), 1.57–1.74 (2H, m), 4.10 (2H, t, J 6.5),
4.39 (1H, quint, J 7.2), 5.11 (2H, s), 5.28–5.42 (1H, br d), 7.26–7.41 (5H, m)
1.24 (3H, d, J 6.1), 1.26 (3H, d, J 6.1), 1.40 (3H, d, J 6.9), 4.33 (1H, quint,
J 6.9), 4.97–5.05 (1H, sept, J 6.1), 5.11 (2H, s), 5.28–5.42 (1H, br d), 7.26–7.41
(5H, m)
0.93 (3H, t, J 7.5), 1.27–1.43 (2H, m), 1.41 (3H, d, J 7.2), 1.54–1.69 (2H, m),
4.14 (2H, t, J 6.6), 4.38 (1H, quint, J 7.2), 5.11 (2H, s), 5.26–5.41 (1H, br d),
7.27–7.42 (5H, m)
CH2CH3
(CH2)2CH3
CH(CH3)2
oil
oil
3347, 1720
3349, 1721
(CH2)3CH3
oil
Ϫ29.0
CH2CH(CH3)2
(CH2)4CH3
oil
oil
Ϫ31.3
Ϫ29.1
3349, 1722
3342, 1723
0.93 (6H, d, J 6.6), 1.43 (3H, d, J 7.2), 1.86–2.02 (1H, m), 3.82–4.00 (2H, m),
4.40 (1H, quint, J 7.2), 5.11 (2H, s), 5.28–5.42 (1H, br d), 7.26–7.40 (5H, m)
0.90 (3H, t, J 6.9), 1.23–1.37 (4H, m), 1.41 (3H, d, J 7.2), 1.59–1.69 (2H, m),
4.13 (2H, t, J 6.8), 4.38 (1H, quint, J 7.2), 5.11 (2H, s), 5.27–5.41 (1H, br d),
7.26–7.40 (5H, m)
(CH2)5CH3
oil
Ϫ27.6
3355, 1723
0.89 (3H, t, J 6.9), 1.19–1.40 (6H, m), 1.42 (3H, d, J 7.2), 1.57–1.68 (2H, m),
4.13 (2H, t, J 6.5), 4.38 (1H, quint, J 7.2), 5.11 (2H, s), 5.28–5.40 (1H, br d),
7.26–7.38 (5H, m)
C6H11-c
57.5–58.5 Ϫ31.1
3363, 1751, 1694
3335, 1722
1.17–1.90 (10H, m), 1.41 (3H, d, J 7.2), 4.35 (1H, quint, J 7.2), 4.74–4.86
(1H, m), 5.11 (2H, s), 5.28–5.41 (1H, br d), 7.26–7.40 (5H, m)
1.10–1.82 (8H, m), 1.42 (3H, d, J 7.2), 2.11–2.37 (1H, m), 3.93–4.10 (2H, m),
4.38 (1H, quint, J 7.2), 5.11 (2H, s), 5.28–5.42 (1H, br d), 7.27–7.40 (5H, m)
0.84–1.82 (11H, m), 1.42 (3H, d, J 7.2), 3.85–4.30 (2H, m), 4.39 (1H, quint,
J 7.2), 5.11 (2H, s), 5.37 (1H, br d, J 6.9), 7.26–7.42 (5H, m)
1.46 (3H, d, J 7.2), 4.34–4.54 (2H, m), 4.54–4.70 (1H, m), 5.12 (2H, AB q, J 13),
5.16–5.29 (1H, br), 7.26–7.41 (5H, m)
1.45 (3H, d, J 7.5), 4.40–4.57 (2H, m), 4.72 (1H, AB q, J 13), 5.12 (2H, AB q,
J 12), 5.18–5.29 (1H, br d), 7.27–7.41 (5H, m)
1.44 (3H, d, J 7.2), 3.67 (2H, t, J 5.6), 4.23–4.51 (3H, m), 5.11 (2H, s),
5.21–5.36 (1H, br), 7.25–7.45 (5H, m)
1.51 (3H, d, J 7.2), 4.54 (1H, quint, J 7.2), 4.65 and 4.93 (2H, AB q, J 12), 5.12
(2H, AB q, J 12), 5.23–5.36 (1H, br d), 7.27–7.40 (5H, m)
1.42 (3H, d, J 7.2), 4.44 (1H, quint, J 7.2), 5.11 (2H, s), 5.17 (2H, AB q, J 12),
5.29–5.40 (1H, br d), 7.24–7.43 (10H, m)
1.46 (3H, d, J 7.2), 4.47 (1H, quint, J 7.2), 5.12 (2H, s), 5.15–5.34 (1H, br),
5.27 (2H, AB q, J 9.3), 7.24–7.40 (5H, m), 7.55 (2H, d, J 8.4), 8.22 (2H, d,
J 8.4)
(B)
oil
CH2C5H9-c
CH2C6H11-c
CH2CF3
Ϫ30.5
Ϫ25.7
Ϫ28.6
Ϫ27.6
Ϫ30.3
Ϫ31.7
Ϫ31.4
oil
3357, 1735, 1709
3331, 1773, 1683
3351, 1756, 1687
3330, 1752, 1691
3327, 1763, 1705
3338, 1747, 1688
3324, 1744, 1688
59
(B)
56–56.5
(A)
53–54
(B)
CH2CF2CF3
CH2CH2Cl
CH2CCl3
CH2Ph
oil
38–39
(B)
CH2C6H4NO2-4 100.5–101 Ϫ17.2
(C)
CH2C6H4CN-4
CH2C6H4Cl-4
3333, 1743, 1685
3300, 1736, 1689
3336, 1735, 1686
3215, 1747, 1709
1.44 (3 H, d, J 7.2), 4.46 (1H, quint, J 7.2), 5.11 (2H, s), 5.22 (2H, AB q, J 15),
5.24–5.34 (1H, br d), 7.23–7.40 (5H, m), 7.43 and 7.62 (4H, AB q, J 8.0)
1.41 (3H, d, J 7.2), 4.43 (1H, quint, J 7.2), 5.10 (2H, s), 5.13 (2H, AB q, J 12),
5.21–5.35 (1H, br d), 7.15–7.40 (9H, m)
1.40 (3H, d, J 7.2), 3.81 (3H, s), 4.40 (1H, quint, J 7.2), 5.10 (4H, s), 5.27–5.37
(1H, br d), 6.88 and 7.28 (4H, AB q, J 8.4), 7.30–7.40 (5H, m)
1.47 (3H, d, J 7.2), 4.49 (1H, quint, J 7.2), 5.12 (2H, s), 5.19 (2H, AB q,
J 13), 5.32–5.45 (1H, br d), 7.23 (2H, d, J 5.4), 7.27–7.40 (5H, m), 8.60 (2H,
d, J 5.4)
CH2C6H4OMe-4 61
CH2C5H4Ne
(CH2)2Ph
(CH2)3Ph
CH2CN
oil
Ϫ32.6
Ϫ24.7
Ϫ45.3
Ϫ36.6
Ϫ43.0
Ϫ24.8
3307, 1734, 1682
3304, 1738, 1686
3329, 1762, 1705
3352, 1801, 1713
1.34 (3H, d, J 7.2), 2.95 (2H, t, J 6.9), 4.27–4.43 (3H, m), 5.10 (2H, s), 5.24–
5.36 (1H, br d), 7.16–7.41 (10H, m)
1.42 (3H, d, J 7.7), 1.95–2.04 (2H, m), 2.68 (2H, t, J 12), 4.16 (2H, t, J 6.6),
4.39 (1H, quint, J 7.7), 5.11 (2H, s), 5.24–5.37 (1H, br d), 7.11–7.40 (10H, m)
1.46 (3H, d, J 7.2), 4.46 (1H, quint, J 7.2), 4.76 (2H, AB q, J 17), 5.11 (2H,
AB q, J 14), 5.21–5.34 (1H, br d), 7.26–7.42 (5H, m)
1.45 (3H, d, J 6.9), 3.46 (3H, s), 4.42 (1H, quint, J 6.9), 5.11 (2H, AB q, J 12),
5.25 and 5.33 (2H, AB q, J 6.0), 5.28–5.40 (1H, br), 7.27–7.40 (5H, m)
1.51 (3H, d, J 7.2), 2.16 (3H, s), 4.50 (1H, quint, J 7.2), 4.64 and 4.78 (2H,
AB q, J 17), 5.11 (2H, AB q, J 13), 5.23–5.38 (1H, br d), 7.27–7.41 (5H, m)
1.58 (3H, d, J 7.2), 4.58 (1H, quint, J 7.2), 5.12 (2H, AB q, J 12), 5.29 and 5.51
(2H, AB q, J 17), 5.34 (1H, br d, J 8.1), 7.26–7.40 (5H, m), 7.44–7.97 (5H, m)
1.28 (3H, t, J 7.2), 1.50 (3H, d, J 7.2), 4.22 (2H, q, J 7.2), 4.50 (1H, quint,
J 7.2), 4.56 and 4.76 (1H, AB q, J 16), 5.12 (2H, AB q, J 13), 5.23–5.38 (1H, br
d), 7.26–7.41 (5H, m)
1.46 (3H, d, J 7.2), 4.33 (1H, quint, J 7.2), 4.63 (2H, AB q, J 16), 5.10 (2H,
AB q, J 11), 5.44 (1H, br), 5.73 (1H, br), 6.78 (1H, br), 7.28–7.41 (5H, m)
1.46 (3H, d, J 7.2), 2.78 (3H, d, J 4.8), 4.31 (1H, quint, J 7.2), 4.64 (2H, AB q,
J 16), 5.12 (2H, AB q, J 12), 5.31–5.42 (1H, br d), 6.80 (1H, br), 7.28–7.41
(5H, m)
49–50
(D)
oil
CH2OCH3
CH2COCH3
CH2COPh f
CH2CO2Et
oil
68–68.5
(D)
156–157
(E)
50–52.5
(A)
3333, 1756, 1725,
1692
3367, 1754, 1687
(CHCl3)
Ϫ46.6
3329, 1761, 1744,
1686
g
CH2CONH2
71.5–72.5 Ϫ16.3
(F) (DMF)
3421, 3328, 1747,
1711, 1686
CH2CONHCH3 98.5–99
Ϫ21.8
(E)
CH2CON(CH3)2 oil
Ϫ40.7
3308, 1753, 1716,
1663
1.53 (3H, d, J 7.2), 2.95 (6H, s), 4.51 (1H, quint, J 7.2), 4.67 and 4.87 (2H,
AB q, J 14), 5.11 (2H, AB q, J 11), 5.44 (1H, br d), 7.27–7.39 (5H, m)
a Recrystallisation solvent: A, cyclohexane; B, CCl4–petroleum spirit; C, benzene–petroleum spirit; D, CCl4; E, EtOAc; F, EtOAc–petroleum spirit.
b c 1.0 in MeOH unless otherwise noted. c Wavenumbers of only the peaks ascribed to the N–H and C᎐O stretching vibrations are quoted.
᎐
d Lit.,19 mp 45–46 ЊC; [α]D22 Ϫ33.9 (c 2, MeOH). e 4-Picolyl ester. Lit.,24 mp 111–112.5 ЊC; [α]D20 Ϫ20.2 (c 1.0, DMF). f Lit.,25 mp 154–155 ЊC; [α]D18 Ϫ26.5
(c 2.0, CHCl3). g Lit.,26 mp 74–75 ЊC; [α]D24 Ϫ16.8 (c 2.0, DMF).
J. Chem. Soc., Perkin Trans. 1, 2001, 87–93
91

View Article Online
Table 6 Physical and 1H NMR data of Z-Xaa-OCH2CONH2
[α]D25 (10Ϫ1
IR (ATR)
b
c
Xaa
Mp (T/ЊC)a
deg cm2 gϪ1
)
(νmax/cmϪ1
)
1H NMR
Glyd
-Ser
-Val
104–105.5
122.5–124
89–89.5
3446, 3328, 1770,
1745, 1687
3308, 1766, 1684
δH (DMSO-d6) 3.89 (2H, d, J 6.0), 4.45 (2H, s), 5.04 (2H, s), 7.22–7.37 (6H, m),
7.44 (1H, br), 7.75 (1H, t, J 6.0)
Ϫ16.6
δH (DMSO-d6) 3.61–3.79 (2H, m), 4.24–4.31 (1H, m), 4.47 (2H, AB q, J 15),
5.04 (2H, s), 5.14 (1H, t, J 5.7), 7.24–7.46 (7H, m), 7.68 (1H, d, J 7.8)
δH (CDCl3) 0.98 (3H, d, J 6.9), 1.02 (3H, d, J 6.9), 2.05–2.24 (1H, m), 4.16
(1H, dd, J 7.1 and 6.3), 4.55 and 4.73 (2H, AB q, J 16), 5.10 (2H, s), 5.36
(1H, br d, J 7.1), 5.73 (1H, br), 6.70 (1H, br), 7.28–7.42 (5H, m)
δH (CDCl3) 1.87–2.36 (4H, m), 3.50–3.69 (2H, m), 4.37–4.47 (1H, m), 4.65
(2H, AB q, J 16), 5.14 (2H, s), 5.49 (1H, br), 7.21 (1H, br), 7.28–7.43 (5H, m)
δH (DMSO-d6) 1.39 (6H, s), 4.40 (2H, s), 5.01 (2H, s), 7.18 (1H, br), 7.30–7.40
(6H, m), 7.94 (1H, br)
Ϫ17.5
3445, 3317, 1750,
1707, 1666
-Pro
oil
Ϫ53.8
3339, 1754, 1678
Aib
153.5–154.5
3373, 3284, 1745,
1675
a From EtOAc. b c 1.0 in MeOH. c Wavenumbers of only the peaks ascribed to the N–H and C᎐O stretching vibrations are quoted. d Lit.,26 mp 105–
᎐
108 ЊC.
carbamoylmethyl ester. The preparation of the carbamoyl-
methyl ester of Z--Ala is described as a typical example. To
a solution of Z--Ala (447 mg, 2 mmol) in MeOH (4 ml) was
added a solution of Cs2CO3 (326 mg, 1 mmol) in water (1.5 ml),
and the mixture was evaporated under reduced pressure. After
repeated evaporation to dryness with toluene, the residue was
stored over P4O10 in a vacuum desiccator. The Ce salt of Z--
Ala thus obtained was mixed with 2-chloroacetamide (187 mg,
2 mmol) in DMF (8 ml) and the mixture was stirred at 60 ЊC
overnight. The mixture was distributed between EtOAc (40 ml)
and water (10 ml), and the aqueous phase was extracted further
with EtOAc (2 × 10 ml), and the combined organic extracts
were washed successively with 1 M aq. NaHCO3 and water and
dried over Na2SO4. Evaporation of the solvent afforded white
crystals, which were recrystallised from EtOAc–petroleum
spirit; yield 485 mg (87%).
afforded white crystals, which were recrystallised from
benzene–petroleum spirit; yield 4.9 g (21%); mp 37–38.5 ЊC
(lit.,27 45–46 ЊC) (Found: C, 33.77; H, 5.50; N, 13.36. C3H6ClNO
requires C, 33.51; H, 5.62; N, 13.62%); δH (CDCl3) 2.90 (3H, d,
J 5.1 Hz), 4.06 (2H, s), 6.70 (1H, br).
N,N-Dimethyl-2-chloroacetamide. This compound was pre-
pared from dimethylamine hydrochloride and chloroacetyl
chloride in the same manner as above. An oil obtained after
washings was distilled under reduced pressure; yield 33%;
bp 100 ЊC at 14 mmHg (lit.,27 98.5–99.5 ЊC at 11 mmHg)
(Found: C, 39.71; H, 6.75; N, 11.46. C4H8ClNO requires C,
39.52; H, 6.63; N, 11.52%); δH (CDCl3) 2.99 (3H, s), 3.06 (3H,
s), 4.09 (2H, s).
Preparation of authentic dipeptides
The benzyl ester of Z--Ser was also prepared by the
same route: mp 83–83.5 ЊC; [α]D25 ϩ3.8 (c 1.0, CHCl3) {lit.,21
mp 83–84 ЊC; [α]D20 ϩ5.1 (c 2.95, CHCl3)}.
The authentic samples of N-protected dipeptide amides,
Z-Xaa--Leu-NH2, were prepared as illustrated below for the
preparation of Z--Ala--Leu-NH2. To a stirred solution of
Z--Ala (447 mg, 2.0 mmol), -Leu-NH2ؒHCl (333 mg, 2.0
mmol), TEA (203 mg, 2.0 mmol) and HOBT (270 mg,
2.0 mmol) in DMF (8 ml) was added EDCؒHCl (384 mg,
2.0 mmol) under ice-cooling. After stirring at this temperature
for 2 h and then at ambient temperature overnight, the reac-
tion 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 white
crystals, which were recrystallised from aq. EtOH; yield 532 mg
(94%). Physical and 1H NMR data of Z-Xaa--Leu-NH2
thus prepared are compiled in Table 7.
(iv) The 4-nitrobenzyl and 4-cyanobenzyl esters were pre-
pared via reaction of the triethylamine (TEA) salt of an N-Z-
amino acid with the corresponding bromide.22 The preparation
of the 4-nitrobenzyl ester of Z--Ala is described as a typical
example. A mixture of Z--Ala (447 mg, 2 mmol), 4-nitrobenzyl
bromide (432 mg, 2 mmol), and TEA (202 mg, 2 mmol) in
EtOAc (7 ml) was refluxed for 8.5 h. The white precipitate was
filtered off and the filtrate was washed successively with 2 M
HCl, water, 1 M aq. NaHCO3 and brine, and dried over
Na2SO4. Evaporation of the solvent in vacuo afforded white
crystals, which were recrystallised from benzene–petroleum
spirit; yield 464 mg (65%).
Z-Gly-OCH2C6H4(4NO2): mp 111 ЊC (lit.,23 107–109.5 ЊC);
Z--Ser-OCH2C6H4(4NO2): mp 117.5–118 ЊC; [α]D25 Ϫ10.8
(c 1.0, MeOH) {lit.,21 mp 116–117 ЊC; [α]D20 Ϫ11.0 (c 1, MeOH)}.
(v) The 4-picolyl ester24 of Z--Ala was prepared by
stirring a mixture of Z--Ala (447 mg, 2 mmol), 4-picolyl
chloride hydrochloride (392 mg, 2 mmol), and 1,1,3,3-tetra-
methylguanidine (461 mg, 4 mmol) in DMF (7 ml) at
90 ЊC for 9 h. Similar work-up as above yielded pale yellow
crystals, which were recrystallised from EtOAc; yield 392 mg
(62%).
HPLC analyses
The liquid chromatograph employed was a Shimadzu (Japan)
LC-10AS instrument, equipped with a Rheodyne 7725i sample
injector and a Shimadzu SPD-10A variable-wavelength UV
monitor. A Shimadzu 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, Cosmosil 5C18 (4.6 mm id ×
150 mm, Nacalai Tesque, Japan); mobile phase, 30–55% aq.
MeOH containing H3PO4 (0.01 M); flow rate, 1.0 ml minϪ1
column temperature, 30 ЊC; detection, UV at 254 nm.
;
Preparation of N-substituted 2-chloroacetamides
N-Methyl-2-chloroacetamide. Methylamine hydrochloride
(14.9 g, 0.22 mol) was dissolved in 24% (w/v) aq. NaOH (100
ml) and 1,2-dichloroethane (100 ml) was added. To the stirred
mixture was added a solution of chloroacetyl chloride (24.9 g,
0.22 mol) in 1,2-dichloroethane (25 ml) below Ϫ5 ЊC over a
period of 40 min. After stirring for 30 min, the organic layer
was separated, washed successively with 0.5 M HCl and water,
and dried over Na2SO4. Evaporation of the solvent in vacuo
Peptide synthesis mediated by immobilised ꢀ-chymotrypsin in
acetonitrile with low water content
The preparation of Z--Ala--Leu-NH2 is described as a
typical example. A mixture of Z--Ala-OCH2CONH2 (14 mg,
0.05 mmol), -Leu-NH2ؒHCl (33 mg, 0.2 mmol), TEA (28 µl,
0.2 mmol) and the immobilised enzyme on Celite (150 mg,
92
J. Chem. Soc., Perkin Trans. 1, 2001, 87–93

View Article Online
Table 7 Physical and 1H NMR data of Z-Xaa--Leu-NH2
[α]D25 (10Ϫ1
b
Xaa
Mp (T/ЊC)a
191–191.5
deg cm2 gϪ1
)
1H NMR δH (DMSO-d6)
-Alac
Ϫ18.3
0.82 (3H, d, J 6.5), 0.85 (3H, d, J 6.5), 1.17 (3H, d, J 7.2), 1.40–1.46 (2H, t-like), 1.48–1.65 (1H,
m), 3.93–4.11 (1H, quint-like), 4.16–4.24 (1H, q-like), 5.00 (2H, s), 6.97 (1H, s), 7.28 (1H, s),
7.19–7.40 (5H, m), 7.46 (1H, br d, J 8.4), 7.77 (1H, br d, J 8.4)
(DMF)d
-Ala
189.5–191
Ϫ2.8
0.81 (3H, d, J 6.0), 0.85 (3H, d, J 6.0), 1.18 (3H, d, J 6.9), 1.25–1.61 (3H, m), 3.99–4.10 (1H,
quint-like), 4.14–4.23 (1H, m), 5.00 (2H, s), 7.04 (1H, s), 7.25 (1H, s), 7.21–7.39 (5H, m), 7.47
(1H, d, J 7.2), 8.01 (1H, d, J 8.4)
Gly
124–126
Ϫ19.1
Ϫ19.6
0.82 (3H, d, J 6.5), 0.86 (3H, d, J 6.5), 1.36–1.64 (3H, m), 3.55–3.72 (2H, m), 4.16–4.28 (1H,
q-like), 5.02 (2H, s), 7.00 (1H, s), 7.24–7.40 (6H, m), 7.44 (1H, t, J 5.7), 7.88 (1H, d, J 8.1)
0.82 (3H, d, J 6.3), 0.86 (3H, d, J 6.3), 1.40–1.50 (2H, m), 1.50–1.67 (1H, m), 3.56 (2H, t, J 5.9),
4.04–4.11 (1H, q-like), 4.16–4.24 (1H, m), 5.02 (2H, s), 5.04 (1H, t, J 5.4), 7.04 (1H, s), 7.27–7.40
(7H, m), 7.96 (1H, d, J 8.7)
-Ser
184–184.5
-Val
-Pro
260–261
189–194
Ϫ43.7
Ϫ78.2
0.80–0.88 (12H, m), 1.32–1.48 (2H, m), 1.48–1.66 (1H, m), 1.87–2.04 (1H, m), 3.84 (1H, dd, J 8.7
and 6.9), 4.21–4.29 (1H, m), 5.02 (2H, s), 6.96 (1H, s), 7.30–7.40 (7H, m), 7.80 (1H, d, J 8.4)
0.67–0.95 (6H, m), 1.29–1.64 (3H, m), 1.71–1.89 (3H, m), 2.01–2.21 (3H, m), 3.27–3.52 (2H, m),
4.13–4.32 (2H, m), 4.78–5.13 (2H, m), 6.94 and 6.99 (1H, s ϩ s), 7.16–7.41 (6H, m), 7.95 and 7.98
(1H, d ϩ d, J 7.5)
Aib
137.5–139
Ϫ28.4
0.79 (3H, d, J 6.0), 0.84 (3H, d, J 6.0), 1.31 (6H, s), 1.44–1.60 (3H, m), 4.08–4.18 (1H, m), 4.99
(2H, AB q, J 13), 7.00 (1H, s), 7.11 (1H, s), 7.26–7.38 (5H, m), 7.57 (1H, s), 7.65 (1H, br d, J 8.4)
a From aq. EtOH. b c 1.0 in MeOH unless otherwise noted. c Lit.,10 mp 187–189 ЊC; [α]D22 Ϫ17.1 (c 1, DMF). d Ϫ43.7 in MeOH.
6 E. Tommila and C. N. Hinshelwood, J. Chem. Soc., 1938, 1801.
7 Y. Yukawa, Y. Tsuno and M. Sawada, Bull. Chem. Soc. Jpn., 1966,
39, 2274.
corresponding to 4.7 mg of α-chymotrypsin) was incubated
with shaking (180 strokes minϪ1) in a solvent composed of
acetonitrile (2 ml) and 0.05 M Tris buffer (pH 7.8) (83 µl) at
30 ЊC. An aliquot (10 µl) of the reaction mixture was withdrawn
periodically, diluted with AcOH (100 µl) and injected onto the
HPLC column.
8 This ester was proposed as an active ester for conventional peptide
synthesis a long time ago, see: R. Schwyzer, B. Iselin, W. Rittel and
P. Sieber, Helv. Chim. Acta, 1956, 39, 872.
9 R. J. Kerr and C. Niemann, J. Org. Chem., 1958, 23, 304. See also:
S. G. Cohen, V. M. Vaidya and R. M. Schultz, Proc. Natl. Acad. Sci.
USA, 1970, 66, 249. This ester was also recommended as a carboxy-
protecting group for peptide synthesis by conventional chemical
methods: J. Martinez, J. Laur and B. Castro, Tetrahedron, 1985,
41, 739.
Acknowledgements
This work was supported in part by a grant from the Hirao
Taro Foundation of the Konan University Association for
Academic Research.
10 P. Kuhl, U. Zacharias, H. Burckhardt and H.-D. Jakubke, Monatsh.
Chem., 1986, 117, 1195.
11 R. W. Taft, Jr., in Steric Effects in Organic Chemistry, ed.
M. S. Newman, Wiley, New York, 1956, ch. 13.
12 P. Ballinger and F. A. Long, J. Am. Chem. Soc., 1960, 82, 795.
13 S. Takahashi, L. A. Cohen, H. K. Miller and E. G. Peake, J. Org.
Chem., 1971, 36, 1205.
References
1 For reviews, see: (a) K. Faber, Biotransformations in Organic
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14 G. H. Jeffery and A. I. Vogel, J. Chem. Soc., 1934, 1101.
15 A method has been proposed for very bulky couplings including
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16 Incorporation of -amino acids into peptides via subtilisin-
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93