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

Abstract

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.

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Introduction

Although enzymic methods for peptide synthesis ¹ have several advantages over conventional chemical methodologies, a narrow substrate specificity is often regarded as a major drawback 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-chymotrypsin 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 substrate 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.

Results and discussion

In the kinetically controlled peptide synthesis catalysed by a serine or cysteine protease ^1b,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 either by a nucleophilic amine component to give a peptide 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-chymotrypsin-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-chymotrypsin’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-L-Ala with L-Leu-NH₂ in acetonitrile containing 4% (v/v) Tris buffer (pH 7.8) after 48 h of incubation (Scheme 2), together with the relative initial rate (ν_rel) 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 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.

Table 1 α-Chymotrypsin-catalysed couplings of Z-L-Ala-OR with L-Leu-NH₂ ^a

				Yield (%) ^d
Entry	R	ν rel ^b	σ* ^c	Peptide	Z-L-Ala-OH
a Coupling conditions: Z-L-Ala-OR (0.05 mmol), L-Leu-NH2·HCl (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 °C. 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 × 10^−2 mM h^−1 mg^−1. 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).
1	CH3	1 ^e	0	6.7	1.0
2	CH2CH3	0.54	−0.100	4.2	0.7
3	(CH2)2CH3	0.39	−0.115	2.5	0.5
4	CH(CH3)2			1.0	0
5	(CH2)3CH3	0.53	−0.130	3.4	0.6
6	CH2CH(CH3)2	0.76	−0.125	10.2	1.0
7	(CH2)4CH3	0.34		3.0	0.4
8	(CH2)5CH3	0.24		2.0	0.3
9	C6H11-c	0.41		3.4	0.6
10	CH2C5H9-c	0.74		6.3	0.9
11	CH2C6H11-c	0.68	−0.06	4.9	0.7
12	CH2CF3	17.5	+0.92	82.4	8.6 ^g
13	CH2CF2CF3	8.8		63.2	6.8 ^g
14	CH2CH2Cl	3.4	+0.385	30.5	3.8 ^g
15	CH2CCl3	14.1		87.8	6.9 ^g
16	CH2Ph	4.2	+0.215	28.6	4.2
17	CH2C6H4NO2-4	18.7		79.3	9.2
18	CH2C6H4CN-4	11.4		72.7	9.6
19	CH2C6H4Cl-4	5.3		33.6	4.0
20	CH2C6H4OMe-4	2.8		19.2	2.2
21	CH2C5H4N	29.1		89.9	10.0
22	(CH2)2Ph	0.61	+0.080	9.1	0.7
23	(CH2)3Ph	0.42	+0.02	6.8	0.5
24	CH2CN	29.6	+1.30	88.3	6.4 ^g
25	CH2OCH3	6.7	+0.64  ^f	45.6	5.6 ^g
26	CH2COCH3	6.9	+0.60	43.0	7.0 ^g
27	CH2COPh	0.51		7.5	4.1
28	CH2CO2Et	3.0		24.0	4.1
29	CH2CONH2	133		88.4	10.9 ^g
30	CH2CONHCH3	110		89.1	10.8 ^g
31	CH2CON(CH3)2	4.8		34.4	5.2

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Scheme 1 Kinetically controlled peptide synthesis: H-E, serine or cysteine protease; R¹CO₂R, carboxy component in the form of an ester; R²NH₂, amino component.

Scheme 2

With the benzyl ester ⁵ (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 substrate ester resembled that on the rates of alkaline hydrolysis of substituted-benzyl acetates ⁶ 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-pyridylmethyl 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 alkyl esters bearing electron-withdrawing groups other than halogens and phenyl rings were examined next. The cyanomethyl ester ⁸ (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 considerable 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.

Table 2 Non-enzymic reactions of Z-L-Ala-OR with L-Leu-NH₂ ^a

	Yield (%) ^b
R	Peptide	Z-L-Ala-OH
a Reactions were conducted using Z-L-Ala-OR (0.05 mmol), L-Leu-NH2·HCl (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 °C. b After 48 h.
CH2CF3	0	1.9
CH2CF2CF3	0	2.3
CH2CH2Cl	0	0.2
CH2CCl3	0	2.0
CH2CN	0	5.3
CH2OCH3	0	2.9
CH2COCH3	0	1.6
CH2CONH2	0	0.7
CH2CONHCH3	0	0.1

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Fig. 1 Reaction profiles in the α-chymotrypsin-catalysed couplings of Z-L-Ala-OR with L-Leu-NH₂. Symbols: ○, R = CH₂CONH₂; △, R = CH₂CF₃; □, R = CH₃.

When the log v_rel-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 the electron-withdrawing ability of the R group, the higher the ν_rel-value. The log ν_rel-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 formation. The correlation between log ν_rel 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* = +0.60), though its σ*-value is not reported in the literature.‡ Carbamoylmethyl 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 carbamoylmethyl ester for stabilisation through hydrogen bonding of the ES-complex.

Fig. 2 Plot of log ν_rel against σ* of R for the α-chymotrypsin-catalysed coupling of Z-L-Ala-OR with L-Leu-NH₂ (see Table 1).

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 L-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 L-Ser. But a feature observed only in this particular case was that the p-nitrobenzyl ester was as good a donor ester as the carbamoylmethyl ester. The methyl ester of Z-L-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-methylpropanoic acid),¹⁵ the peptide yield being even lower. Furthermore, the use of the carbamoylmethyl ester allowed even the coupling of a D-amino acid as the carboxy component.¹⁶ Thus, the Z-D-Ala ester gave the desired D-L-peptide in moderate yield, although the ν_rel-value was less than one sixth of that for the L-counterpart. On the other hand, it proved to be quite difficult to obtain the peptide of L-Pro even by the use of the carbamoylmethyl ester.

Table 3 α-Chymotrypsin-catalysed couplings of Z-Xaa-OR with L-Leu-NH₂^a

		Yield (%) ^b
Xaa	R	Peptide	Z-Xaa-OH
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-OCH2CONH2, 2.1; Z-L-Ser-OCH2CF3, 10.2; Z-L-Ser-OCH2CONH2, 2.2. The corresponding values in Table 2 of ref. 3b were uncorrected ones.
Gly	CH3	0.4	0.6
	CH2CF3	74.3	9.1 ^c
	CH2Ph	3.9	1.3
	CH2C6H4NO2-4	23.4	3.3
	CH2CONH2	90.9	6.0 ^c
L-Ser	CH3	8.7	1.2
	CH2CF3	87.9	0.5 ^c
	CH2Ph	69.7	4.6
	CH2C6H4NO2-4	92.4	4.6
	CH2CONH2	91.3	6.3 ^c
L-Val	CH3	0	0.3
	CH2CF3	0.9	0.8
	CH2CONH2	13.6	6.8
D-Ala	CH3	0	0.8
	CH2CONH2	40.6	7.0
L-Pro	CH2CF3	0.6	0.5
	CH2CONH2	0.6	0.3
Aib	CH2CONH2	6.4	1.4

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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.

Experimental

All solvents were distilled, and dried over molecular sieves prior to use. L-Leu-NH₂·HCl 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 uncorrected. Optical rotations were measured with a JASCO DIP-4 digital polarimeter. [α]_D-Values are given in units of 10⁻¹ deg cm² g⁻¹. Elemental analyses of new compounds are compiled in Table 4. Petroleum spirit refers to the fraction with distillation range 30–70 °C.

Table 4 Elemental analyses of new compounds

Compound	Molecular formula	C (%) Found (required)	H (%) Found (required)
Z-L-Ala-OCH2CH3	C13H17NO4	62.21 (62.14)	6.87 (6.82)
Z-L-Ala-O(CH2)2CH3	C14H19NO4	63.11 (63.38)	7.28 (7.21)
Z-L-Ala-OCH(CH3)2	C14H19NO4	63.39 (63.38)	7.23 (7.21)
Z-L-Ala-O(CH2)3CH3	C15H21NO4	64.22 (64.50)	7.60 (7.58)
Z-L-Ala-OCH2CH(CH3)2	C15H21NO4	64.33 (64.50)	7.62 (7.58)
Z-L-Ala-O(CH2)4CH3	C16H23NO4	65.33 (65.51)	7.98 (7.90)
Z-L-Ala-O(CH2)5CH3	C17H25NO4	66.29 (66.43)	8.23 (8.20)
Z-L-Ala-OC6H11-c	C17H23NO4	66.61 (66.86)	7.61 (7.59)
Z-L-Ala-OCH2C5H9-c	C17H23NO4	66.71 (66.86)	7.75 (7.59)
Z-L-Ala-OCH2C6H11-c	C18H25NO4	67.23 (67.69)	7.82 (7.89)
Z-L-Ala-OCH2CF3	C13H14F3NO4	51.46 (51.15)	4.63 (4.62)
Z-L-Ala-OCH2CF2CF3	C14H14F5NO4	47.51 (47.33)	3.92 (3.97)
Z-L-Ala-OCH2CH2Cl	C13H16ClNO4	54.70 (54.65)	5.63 (5.64)
Z-L-Ala-OCH2CCl3	C13H14Cl3NO4	44.30 (44.03)	4.06 (3.98)
Z-L-Ala-OCH2Ph	C18H19NO4	68.81 (69.00)	6.19 (6.11)
Z-L-Ala-OCH2C6H4NO2-4	C18H18N2O6	60.41 (60.33)	5.03 (5.06)
Z-L-Ala-OCH2C6H4CN-4	C19H18N2O4	67.14 (67.45)	5.24 (5.36)
Z-L-Ala-OCH2C6H4Cl-4	C18H18ClNO4	62.17 (62.16)	5.19 (5.22)
Z-L-Ala-OCH2C6H4OMe-4	C19H21NO5	66.29 (66.46)	6.14 (6.16)
Z-L-Ala-O(CH2)2Ph	C19H21NO4	69.66 (69.71)	6.48 (6.47)
Z-L-Ala-O(CH2)3Ph	C20H23NO4	70.25 (70.36)	6.78 (6.79)
Z-L-Ala-OCH2CN	C13H14N2O4	59.41 (59.54)	5.54 (5.38)
Z-L-Ala-OCH2OCH3	C13H17NO5	58.21 (58.42)	6.47 (6.41)
Z-L-Ala-OCH2COCH3	C14H17NO5	60.24 (60.21)	6.19 (6.13)
Z-L-Ala-OCH2CO2Et	C15H19NO6	58.05 (58.25)	6.16 (6.19)
Z-L-Ala-OCH2CONHCH3	C14H18N2O5	57.03 (57.14)	6.24 (6.16)
Z-L-Ala-OCH2CON(CH3)2	C15H20N2O5	58.08 (58.43)	6.64 (6.54)
Z-L-Ser-OCH2CONH2	C13H16N2O6	52.64 (52.70)	5.54 (5.44)
Z-L-Val-OCH2CONH2	C15H20N2O5	58.10 (58.43)	6.73 (6.54)
Z-L-Pro-OCH2CONH2	C15H18N2O5	58.63 (58.82)	5.91 (5.92)
Z-Aib-OCH2CONH2	C14H18N2O5	56.87 (57.14)	6.02 (6.16)
Z-D-Ala-L-Leu-NH2	C17H25N3O4	61.08 (60.88)	7.36 (7.51)
Z-Gly-L-Leu-NH2	C16H23N3O4	59.72 (59.80)	7.17 (7.21)
Z-L-Ser-L-Leu-NH2	C17H25N3O5·1/2H2O	56.61 (56.65)	7.18 (7.27)
Z-L-Val-L-Leu-NH2	C19H29N3O4	62.69 (62.79)	8.07 (8.04)
Z-L-Pro-L-Leu-NH2	C19H27N3O4	62.84 (63.14)	7.53 (7.53)
Z-Aib-L-Leu-NH2	C18H27N3O4	61.61 (61.87)	8.01 (7.79)

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Preparation of N-Z-amino acid esters

The physical and ¹H NMR data of the esters of Z-L-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.

Table 5 Physical and ¹H NMR data of Z-L-Ala-OR

R	Mp (T/°C)^a	[α]^25D (10^−1 deg cm^2 g^−1)^b	IR (ATR) (νmax/cm^−1)^c	^1H NMR δH (CDCl3)
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[double bond, length half m-dash]O stretching vibrations are quoted. d Lit.,^19 mp 45–46 °C; [α]^22D −33.9 (c 2, MeOH). e 4-Picolyl ester. Lit.,^24 mp 111–112.5 °C; [α]^20D −20.2 (c 1.0, DMF). f Lit.,^25 mp 154–155 °C; [α]^18D −26.5 (c 2.0, CHCl3). g Lit.,^26 mp 74–75 °C; [α]^24D −16.8 (c 2.0, DMF).
CH3 ^d	46–47.5 (A)	−33.8	3339, 1752, 1691	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)
CH2CH3	oil	−31.1	3334, 1720	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)
(CH2)2CH3	oil	−32.0		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)
CH(CH3)2	oil	−28.4	3347, 1720	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)
(CH2)3CH3	oil	−29.0	3349, 1721	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)
CH2CH(CH3)2	oil	−31.3	3349, 1722	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)
(CH2)4CH3	oil	−29.1	3342, 1723	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	−27.6	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 (B)	−31.1	3363, 1751, 1694	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)
CH2C5H9-c	oil	−30.5	3335, 1722	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)
CH2C6H11-c	oil	−25.7	3357, 1735, 1709	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)
CH2CF3	59 (B)	−28.6	3331, 1773, 1683	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)
CH2CF2CF3	56–56.5 (A)	−27.6	3351, 1756, 1687	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)
CH2CH2Cl	53–54 (B)	−30.3	3330, 1752, 1691	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)
CH2CCl3	oil	−31.7	3327, 1763, 1705	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)
CH2Ph	38–39 (B)	−31.4	3338, 1747, 1688	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)
CH2C6H4NO2-4	100.5–101 (C)	−17.2	3324, 1744, 1688	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)
CH2C6H4CN-4	91.5–92.5 (D)	−20.0	3333, 1743, 1685	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)
CH2C6H4Cl-4	88.5–89.5 (A)	−25.4	3300, 1736, 1689	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)
CH2C6H4OMe-4	61 (D)	−29.8	3336, 1735, 1686	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)
CH2C5H4N ^e	113–114 (E)	−18.8 (DMF)	3215, 1747, 1709	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)
(CH2)2Ph	oil	−32.6	3307, 1734, 1682	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)
(CH2)3Ph	49–50 (D)	−24.7	3304, 1738, 1686	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)
CH2CN	oil	−45.3	3329, 1762, 1705	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)
CH2OCH3	oil	−36.6	3352, 1801, 1713	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)
CH2COCH3	68–68.5 (D)	−43.0	3333, 1756, 1725, 1692	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)
CH2COPh  ^f	156–157 (E)	−24.8 (CHCl3)	3367, 1754, 1687	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)
CH2CO2Et	50–52.5 (A)	−46.6	3329, 1761, 1744, 1686	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)
CH2CONH2 ^g	71.5–72.5 (F)	−16.3 (DMF)	3421, 3328, 1747, 1711, 1686	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)
CH2CONHCH3	98.5–99 (E)	−21.8		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)
CH2CON(CH3)2	oil	−40.7	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)

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Table 6 Physical and ¹H NMR data of Z-Xaa-OCH₂CONH₂

Xaa	Mp (T/°C) ^a	[α]^25D (10^−1 deg cm^2 g^−1) ^b	IR (ATR) (νmax/cm^−1) ^c	^1H NMR
a From EtOAc. b c 1.0 in MeOH. c Wavenumbers of only the peaks ascribed to the N–H and C[double bond, length half m-dash]O stretching vibrations are quoted. d Lit.,^26 mp 105–108 °C.
Gly ^d	104–105.5		3446, 3328, 1770, 1745, 1687	δ H (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)
L-Ser	122.5–124	−16.6	3308, 1766, 1684	δ H (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)
L-Val	89–89.5	−17.5	3445, 3317, 1750, 1707, 1666	δ H (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)
L-Pro	oil	−53.8	3339, 1754, 1678	δ H (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)
Aib	153.5–154.5		3373, 3284, 1745, 1675	δ H (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)

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(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-L-Ser-OMe: oil; [α]²⁵_D −14.0 (c 1.0, MeOH) {lit.,¹⁷ mp 45–46 °C; [α]²²_D −12.5 (c 1, MeOH)}; Z-L-Val-OMe: oil; [α]²⁵_D −19.4 (c 1.0, MeOH) {lit.,¹⁸ mp 54–55 °C; [α]²⁰_D −18.9 (c 1, MeOH)}; Z-D-Ala-OMe: mp 47–48.5 °C; [α]²⁵_D +33.8 (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 hydrochloride (EDC·HCl) (460 mg, 2.4 mmol) in DCM (8 ml) according 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-pentafluoropropyl, 2-chloroethyl, or 2,2,2-trichloroethyl ester; (B) n-pentyl, n-hexyl, benzyl, 4-chlorobenzyl, 4-methoxybenzyl, 2-phenylethyl, 3-phenylpropyl, or ethoxycarbonylmethyl ester.

Z-Gly-OCH₂CF₃: mp 73–73.5 °C (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 °C (Found: C, 68.26; H, 5.84; N, 4.70. C₁₇H₁₇NO₄ requires C, 68.22; H, 5.72; N, 4.68%); Z-L-Ser-OCH₂CF₃: mp 73–77 °C; [α]²⁵_D −14.2 (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-L-Val-OCH₂CF₃: mp 58 °C; [α]²⁵_D −17.8 (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-L-Pro-OCH₂CF₃: oil; [α]²⁵_D −48.8 (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%).

(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-dimethylcarbamoylmethyl ester. The preparation of the carbamoylmethyl ester of Z-L-Ala is described as a typical example. To a solution of Z-L-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-L-Ala thus obtained was mixed with 2-chloroacetamide (187 mg, 2 mmol) in DMF (8 ml) and the mixture was stirred at 60 °C 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%).

The benzyl ester of Z-L-Ser was also prepared by the same route: mp 83–83.5 °C; [α]²⁵_D +3.8 (c 1.0, CHCl₃) {lit.,²¹ mp 83–84 °C; [α]²⁰_D +5.1 (c 2.95, CHCl₃)}.

(iv) The 4-nitrobenzyl and 4-cyanobenzyl esters were prepared 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-L-Ala is described as a typical example. A mixture of Z-L-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 °C (lit.,²³ 107–109.5 °C); Z-L-Ser-OCH₂C₆H₄(4NO₂): mp 117.5–118 °C; [α]²⁵_D −10.8 (c 1.0, MeOH) {lit.,²¹ mp 116–117 °C; [α]²⁰_D −11.0 (c 1, MeOH)}.

(v) The 4-picolyl ester ²⁴ of Z-L-Ala was prepared by stirring a mixture of Z-L-Ala (447 mg, 2 mmol), 4-picolyl chloride hydrochloride (392 mg, 2 mmol), and 1,1,3,3-tetramethylguanidine (461 mg, 4 mmol) in DMF (7 ml) at 90 °C for 9 h. Similar work-up as above yielded pale yellow crystals, which were recrystallised from EtOAc; yield 392 mg (62%).

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 −5 °C 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 afforded white crystals, which were recrystallised from benzene–petroleum spirit; yield 4.9 g (21%); mp 37–38.5 °C (lit.,²⁷ 45–46 °C) (Found: C, 33.77; H, 5.50; N, 13.36. C₃H₆ClNO requires C, 33.51; H, 5.62; N, 13.62%); δ_H (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 prepared 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 °C at 14 mmHg (lit.,²⁷ 98.5–99.5 °C at 11 mmHg) (Found: C, 39.71; H, 6.75; N, 11.46. C₄H₈ClNO requires C, 39.52; H, 6.63; N, 11.52%); δ_H (CDCl₃) 2.99 (3H, s), 3.06 (3H, s), 4.09 (2H, s).

Preparation of authentic dipeptides

The authentic samples of N-protected dipeptide amides, Z-Xaa-L-Leu-NH₂, were prepared as illustrated below for the preparation of Z-L-Ala-L-Leu-NH₂. To a stirred solution of Z-L-Ala (447 mg, 2.0 mmol), L-Leu-NH₂·HCl (333 mg, 2.0 mmol), TEA (203 mg, 2.0 mmol) and HOBT (270 mg, 2.0 mmol) in DMF (8 ml) was added EDC·HCl (384 mg, 2.0 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 white crystals, which were recrystallised from aq. EtOH; yield 532 mg (94%). Physical and ¹H NMR data of Z-Xaa-L-Leu-NH₂ thus prepared are compiled in Table 7.

Table 7 Physical and ¹H NMR data of Z-Xaa-L-Leu-NH₂

Xaa	Mp (T/°C) ^a	[α]^25D (10^−1 deg cm^2 g^−1) ^b	^1H NMR δH (DMSO-d6)
a From aq. EtOH. b c 1.0 in MeOH unless otherwise noted. c Lit.,^10 mp 187–189 °C; [α]^22D −17.1 (c 1, DMF). d −43.7 in MeOH.
L-Ala ^c	191–191.5	−18.3 (DMF) ^d	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)
D-Ala	189.5–191	−2.8	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	−19.1	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)
L-Ser	184–184.5	−19.6	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)
L-Val	260–261	−43.7	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)
L-Pro	189–194	−78.2	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 + s), 7.16–7.41 (6H, m), 7.95 and 7.98 (1H, d + d, J 7.5)
Aib	137.5–139	−28.4	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)

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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 min⁻¹; column temperature, 30 °C; detection, UV at 254 nm.

Peptide synthesis mediated by immobilised α-chymotrypsin in acetonitrile with low water content

The preparation of Z-L-Ala-L-Leu-NH₂ is described as a typical example. A mixture of Z-L-Ala-OCH₂CONH₂ (14 mg, 0.05 mmol), L-Leu-NH₂·HCl (33 mg, 0.2 mmol), TEA (28 μl, 0.2 mmol) and the immobilised enzyme on Celite (150 mg, corresponding to 4.7 mg of α-chymotrypsin) was incubated with shaking (180 strokes min⁻¹) in a solvent composed of acetonitrile (2 ml) and 0.05 M Tris buffer (pH 7.8) (83 μl) at 30 °C. An aliquot (10 μl) of the reaction mixture was withdrawn periodically, diluted with AcOH (100 μl) and injected onto the HPLC column.

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, 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, Florida, 1987;; (e) H.-D. Jakubke , in The Peptides, ed. S. Udenfriend and J. Meienhofer, Academic Press, San Diego, 1987, vol. 9, ch. 3..
2. Preceding paper: T. Miyazawa, S. Nakajo, M. Nishikawa, K. Hamahara, K. Imagawa, E. Ensatsu, R. Yanagihara and T. Yamada, J. Chem. Soc., Perkin Trans. 1DOI: 10.1039/b004183l;preliminary communication, T. Miyazawa, S. Nakajo, M. Nishikawa, K. Imagawa, R. Yanagihara and T. Yamada, J. Chem. Soc., Perkin Trans. 1, 1996, 1214.
3. Preliminary communications, (a) T. Miyazawa , K. Tanaka , R. Yanagihara and T. Yamada , in Peptide Chemistry 1996, ed. C. Kitada, Protein Research Foundation, Osaka, 1997, p. 33;; (b) T. Miyazawa, K. Tanaka, E. Ensatsu, R. Yanagihara and T. Yamada, Tetrahedron Lett., 1998, 39, 997.
4. cf. P. Clapés, P. Adlercreutz and B. Mattiasson, Biotechnol. Appl. Biochem., 1990, 12, 376.
5. The benzyl and p-nitrobenzyl esters were employed as donor esters in the α-chymotrypsin-catalysed coupling of N-maleyl- or N-glutaryl-L-Leu in an aqueous organic medium, see: V. Schellenberger, A. Görner, A. Könnecke and H.-D. Jakubke, Pept. Res., 1991, 4, 265.
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.
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.
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.
14. G. H. Jeffery and A. I. Vogel, J. Chem. Soc., 1934, 1101.
15. A method has been proposed for very bulky couplings including Aib: H. Wenschuh, M. Beyermann, R. Winter, M. Bienert, D. Ionescu and L. A. Carpino, Tetrahedron Lett., 1996, 37, 5483.
16. Incorporation of D-amino acids into peptides via subtilisin-catalysed condensation using the 2-chloroethyl esters as acyl donors in tert-amyl alcohol has been reported: A. L. Margolin, D.-F. Tai and A. M. Klibanov, J. Am. Chem. Soc., 1987, 109, 7885.
17. C. H. Hassall and J. O. Thomas, J. Chem. Soc. C, 1968, 1495.
18. M. Miyoshi, Bull. Chem. Soc. Jpn., 1973, 46, 1489.
19. M. K. Dhaon, R. K. Olsen and K. Ramasamy, J. Org. Chem., 1982, 47, 1962.
20. S. S. Wang, B. F. Gisin, D. P. Winter, R. Makofske, D. Kulesha, C. Tzougraki and J. Meienhofer, J. Org. Chem., 1977, 42, 1286.
21. N. Ono, T. Yamada, T. Saito, K. Tanaka and A. Kaji, Bull. Chem. Soc. Jpn., 1978, 51, 2401.
22. R. Schwyzer and P. Sieber, Helv. Chim. Acta, 1959, 42, 972.
23. H. Schesrz and K. Arakawa, J. Am. Chem. Soc., 1959, 81, 5691.
24. R. Camble, R. Garner and G. T. Young, J. Chem. Soc. C, 1969, 1911.
25. G. C. Stelakatos, A. Paganou and L. Zervas, J. Chem. Soc. C, 1966, 1191.
26. F. H. C. Stewart, Aust. J. Chem., 1965, 18, 1089.
27. W. A. Jacobs and M. Heidelberger, J. Biol. Chem., 1915, 21, 145.

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Footnotes

† 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.

‡ The σ*-value for the carbamoylmethyl group is estimated to be +0.59 according to the following equation,¹³ pK_a(RCO₂H) = −1.700σ*(R) + 4.644, by employing the reported K_a-value for propanedioic acid monoamide (2.284 × 10⁻⁴ at 25 °C).¹⁴

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