Methyltrypsin-catalyzed peptide coupling: Comparison of alkyl ester and guanidinophenyl ester derivatives as acyl donor component

Article abstract of DOI:10.1006/bioo.1997.1076

Methyltrypsin-catalyzed peptide synthesis has been studied by using conventional alkyl ester and p-guanidinophenyl ester derivatives of α-amino acid as the acyl donor component. They were found to be coupled with α- amino acid derivatives (acyl acceptor component) to produce dipeptide. The behavior of methyltrypsin toward both the substrates has been studied.

Full text of DOI:10.1006/bioo.1997.1076

BIOORGANIC CHEMISTRY 25, 307–319 (1997)
ARTICLE NO. BH971076
Methyltrypsin-Catalyzed Peptide Coupling: Comparison of
Alkyl Ester and Guanidinophenyl Ester Derivatives as
Acyl Donor Component1
Kunihiko Itoh, Haruo Sekizaki, Eiko Toyota, and Kazutaka Tanizawa2
Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu,
Hokkaido 061–02, Japan
Received August 15, 1997
Methyltrypsin-catalyzed peptide synthesis has been studied by using conventional alkyl
ester and p-guanidinophenyl ester derivatives of Ͱ-amino acid as the acyl donor component.
They were found to be coupled with Ͱ-amino acid derivatives (acyl acceptor component)
to produce dipeptide. The behavior of methyltrypsin toward both the substrates has been
studied.
1997 Academic Press
INTRODUCTION
It is well known that arginine- or lysine-containing peptides interact specifically
with trypsin-like enzymes and hence possess a variety of pharmacological activities.
However, chemical synthesis of these peptides is tedious. Synthesis of arginyl pep-
tides, for example, requires the extensive protection of the guanidino side-chain.
Enzymatic peptide synthesis is more advantageous than chemical synthesis in many
respects since it is highly stereoselective and racemization-free and requires minimal
side-chain protection (1, 2). Enzymatic peptide synthesis, however, has two serious
drawbacks. One of them is the potential loss of the product due to the hydrolysis
of the resulting peptide bonds by the enzyme. Another serious defect is the discrimi-
nation of amino acid residues by the enzyme being employed in the enzymatic
synthesis. In the case of trypsin-catalyzed coupling, for example, the acyl donor is
limited to peptides containing a positively charged arginine or lysine residue at the
carboxyl end position. As a solution to the former problem, application of chemically
modified enzymes such as methylchymotrypsin (3, 4) and methylsubtilisin (5) has
been developed by C.-H. Wong et al. The latter problem could be solved by use
of inverse substrates, as reported in our previous papers (6–8).
It has been demonstrated that methyltrypsin can be easily prepared from the
inexpensive commercial bovine trypsin (9–11). Methyltrypsin is known to lose the
amidase activity of the original enzyme whereas the esterase activity is retained (3,
4). We wish now to report the application of methyltrypsin as a catalyst for peptide
1 Dedicated to Dr. Yuichi Kanaoka (Editorial Board of this Journal, Emeritus Professor of Hokkaido
University) on the occasion of his 70th birthday.
2 To whom correspondence should be addressed.
307
0045-2068/97 $25.00
Copyright 1997 by Academic Press
All rights of reproduction in any form reserved.

308
ITOH ET AL.
synthesis by using conventional substrates (Z-
L
-Lys-OR and Bz- -Arg-OEt) and
L
inverse substrates for trypsin (Boc-amino acid p-guanidinophenyl esters) (12, 13)
as the acyl donor component.
EXPERIMENTAL PROCEDURES
Melting points were determined on a Yanaco MP-500 melting point apparatus
and were uncorrected. IR spectra were recorded on a JASCO VALOR-III FT-IR
1
spectrometer. H-NMR spectra were recorded on a JEOL JNM-FX-400 FT NMR
spectrometer. Optical rotations were measured with a JASCO DIP-360 digital
polarimeter. Kinetic parameters were determined with a Union Giken RA-401
stopped-flow spectrometer, a Hitachi U-2,000 UV spectrophotometer, and a Radi-
ometer TTT-80 pH-stat. HPLC was performed on a Shimadzu LC-6A pump system
equipped with a Shimadzu SPD-6AV UV–VIS spectrophotometric detector.
Materials
Bovine pancreatic trypsin (EC 3.4.21.4) was purchased from Worthington Bio-
chemical Co. (twice recrystallized, lot TRL). It was further purified by the treatment
with
L
-1-p-tosylamino-2-phenylethyl chloromethyl ketone (TPCK) and subse-
quently by affinity chromatography (14) using Benzamidine Sepharose 6B (Phar-
macia). m-Aminobenzenesulfonic acid (metanilic acid) and cyanamide were pur-
chased from Kanto Chemical Co., Inc. Trimethyloxonium tetrafluoroborate and
TPCK were products of Aldrich Chemical Co., Inc. p-Nitrophenyl-pЈ-guanidinoben-
zoate hydrochloride (NPGB) was obtained from Merck Co., Inc. (p-Amidino-
phenyl)methanesulfonyl fluoride hydrochloride was purchased from Wako Pure
␧
Chemical Industries, Ltd. NͰ-(benzyloxycarbonyl)-N -(tert-butyloxycarbonyl)-
L
-
lysine [Z-
chloride (Z-
national, Inc.
and NͰ-(benzoyl)-
supplied from Peptide Institute, Inc. NͰ-(benzoyl)-
p-nitroanilide (Bz- -Phe- -Val- -Arg-pNA) was purchased from Sigma Chemical
L
-Lys(Boc)-OH] and NͰ-(benzyloxycarbonyl)-
-Lys-OMe · HCl) were obtained from Calbiochem-Novabiochem Inter-
-Alanine p-nitroanilide ( -Ala-pNA), -leucine amide ( -Leu-NH2),
-arginine ethyl ester hydrochloride (Bz- -Arg-OEt · HCl) were
-phenylalanyl- -valyl- -arginine
L
-lysine methyl ester hydro-
L
L
L
L
L
L
L
L
L
L
L
L
L
Co. Inverse substrates [NͰ-(tert-butyloxycarbonyl)amino acid p-guanidinophenyl
ester p-toluenesulfonate (Boc-amino acid-OGp · TsOH)] were prepared following
reported procedures (12, 13).
Preparation of Methyltrypsin
Methylation of the enzyme was carried out by a modification of literature proce-
dure (9–11). A solution of bovine trypsin (350 mg, 60% purity, determined from
active site titration using NPGB (15)) in 100 ml of 50 mM Tris–HCl buffer (pH
7.4, containing 20 mM CaCl2) was treated with 10 ml of 0.1 M solution of methyl
m-guanidinobenzenesulfonate tetrafluoroborate in acetonitrile. The reaction mix-
ture was incubated at 25ЊC for 3 h. During the incubation the pH was kept at 7.4
by occasional addition of 50 mM NaOH. After the pH of the reaction mixture was

METHYLTRYPSIN-CATALYZED PEPTIDE COUPLING
309
lowered to 7.0 by the addition of 0.1 M HCl, (p-amidinophenyl)methanesulfonyl
fluoride hydrochloride (5 mg) was added to the reaction mixture and incubation
was continued for 10 min at 25ЊC. During the incubation the pH was kept at 7.0
by occasional addition of 50 mM NaOH. After the resulting precipitate was removed
by centrifugation, the pH of the supernatant was adjusted to 3.0 by addition of
1 M HCl. The solution was dialyzed against 1 mM HCl (pH 3.0) at 4ЊC, and then
pH and salt concentration of the dialysate was adjusted to those of eluate buffer.
The solution was loaded on a column of Benzamidine Sepharose 6B (2.0 ϫ 7.0
cm), which was equilibrated with 0.05 M sodium acetate buffer (pH 5.0, containing
0.02 M CaCl2, and 0.1 M KCl). The column was washed with the same buffer until
the effluent exhibited negligible absorbance at 280 nm. The bound methyltrypsin
was eluted with 5 mM HCl (pH 2.3, containing 0.1 M KCl). The collected fractions
were dialyzed against 1 mM HCl (pH 3.0). Methyltrypsin (90 mg) was obtained as
a colorless powder after lyophilization.
The molar concentration of methyltrypsin was estimated spectrophotometrically
from its absorbance at 280 nm assuming E1 cm ϭ 15.4 and a molecular weight of
23,800 (16). The purity of the preparation was determined by NPGB titration
(15). The amidase activity was determined using Bz-
L
-Phe-
L
-Val- -Arg-pNA as a
L
substrate according to the reported method (17–19).
Synthesis of Substrate
Synthesis of NͰ-(tert-Butyloxycarbonyl)-
tion of Boc- -tyrosine p-nitrophenyl ester (20) (443 mg, 1.1 mmol) in EtOH (30
L-tyrosine p-aminophenyl ester. A solu-
L
ml) containing 10% Pd-C (10 mg) was vigorously stirred in an atmosphere of
hydrogen at room temperature for 20 h. The catalyst was filtered off and the filtrate
was evaporated to dryness in vacuo. The crude residue was subjected to the next
reaction without purification.
Synthesis of NͰ-(tert-butyloxycarbonyl)-
carbonyl)guanidino]phenyl ester (Boc-
L
-tyrosine p-[NЈ,NЉ-bis(benzyloxy-
L
-Tyr-OGp(Z2)]. A solution of the crude
aminophenyl ester (described above) and 1-[N,NЈ-bis(benzyloxycarbonyl)amidino]-
pyrazole (21, 22) (378 mg, 1.0 mmol) in absolute THF (0.5 ml) was stirred for 3 h
at room temperature in an atmosphere of nitrogen. The reaction mixture was diluted
with benzene-ethyl acetate (5 : 1) and passed through a short silica gel column
(2.5 ϫ 30 cm). The eluate was evaporated to dryness in vacuo and the solid residue
was recrystallized from MeOH. Boc- -Tyr-OGp(Z2) (478 mg, 70%) was obtained
L
as colorless solid. mp 131–132ЊC. [Ͱ]2D5 Ϫ6.9Њ (c ϭ 1.1, CH3CN). Anal. Calcd. for
C37H38N4O9 · H2O: C, 63.42; H, 5.75; N, 8.00. Found: C, 63.52; H, 5.66; N, 7.99.
Synthesis of NͰ-(tert-Butyloxycarbonyl)-
L-tyrosine p-guanidinophenyl ester p-tolu-
enesulfonate (Boc-
L
-Tyr-OGp · TsOH). A solution of Boc- -Tyr-OGp(Z2) (341 mg,
L
0.5 mmol) and TsOH · H2O (95 mg, 0.5 mmol) in MeOH (30 ml) containing 10%
Pd-C (5 mg) was vigorously stirred overnight in an atmosphere of hydrogen at
room temperature. The catalyst was filtered off, and the filtrate was evaporated to
dryness in vacuo. The residue was washed with dry diethyl ether. The procedure
gave Boc-
L
-Tyr-OGp · TsOH (282 mg, 96%) as a colorless amorphous material.
[Ͱ]2D5 ϩ 1.6Њ(c ϭ 1.0, MeOH). FAB-MS m/z: 415 (M ϩ H)ϩ.

310
ITOH ET AL.
Synthesis of NͰ-(tert-butyloxycarbonyl)-
D-tyrosine p-guanidinophenyl ester p-tolu-
enesulfonate (Boc-D-Tyr-OGp · TsOH). This series of compounds was obtained from
Boc- -tyrosine p-nitrophenyl ester by a procedure similar to that described for the
D
L
-tyrosine derivatives. Boc- -Tyr-OGp(Z2) was obtained in 62% yield as a
D
colorless solid. mp 130–131ЊC. [Ͱ]2D5 ϩ 6.4Њ(c ϭ 1.1, CH3CN). Anal. Calcd for
C37H38N4O9 · H2O: C, 63.42; H, 5.75; N, 8.00. Found: C, 63.71; H, 5.77; N, 8.10. Boc-
D
-Tyr-OGp · TsOH was obtained in 94% yield as a colorless amorphous material.
[Ͱ]D Ϫ 1.6Њ(c ϭ 1.0, MeOH). FAB-MS m/z: 415 (M ϩ H)ϩ.
␧
Synthesis of NͰ-(benzyloxycarbonyl)-N -(tert-butyloxycarbonyl)-
L-lysine p-chlo-
rophenyl ester (Z-
L
-Lys(Boc)-OCp). A solution of Z- -Lys(Boc)-OH (1.141 g, 3.0
L
mmol) and p-chlorophenol (424 mg, 3.3 mmol) in a mixture of dimethylformamide
(5 ml) and ethyl acetate (10 ml) was treated with dicyclohexylcarbodiimide (681
mg, 3.3 mmol) at 0ЊC. The reaction mixture was stirred for 1 h at 0ЊC and for 12
h at room temperature. The resulting precipitate of dicyclohexylurea was filtered
off and the filtrate was concentrated to dryness in vacuo. Recrystallization from
diethyl ether-hexane afforded a colorless needle (930 mg, 63%). mp 80–81ЊC.
[Ͱ]2D5 Ϫ 6.7Њ(c ϭ 1.0, CHCl3). Anal. Calcd for C25H31N2O6Cl: C, 61.16; H, 6.36; N,
5.71; Cl, 7.22. Found: C, 61.16; H, 6.41; N, 5.69; Cl, 7.18.
Synthesis of NͰ-(benzyloxycarbonyl)-
OCp). Z- -Lys(Boc)-OCp (490 mg, 1.0 mmol) was treated with 2 M HCl in dioxane
L
-lysine p-chlorophenyl ester (Z-
L
-Lys-
L
(6 ml) for 2 h at room temperature. The resulting precipitate was filtrated and
washed with dry diethyl ether. Recrystallization from diethyl ether-ethanol gave a
colorless solid. mp 162–164ЊC, 385 mg (90% yield). [Ͱ]2D5 Ϫ 30.6Њ(c ϭ 1.1, MeOH).
Anal. Calcd for C20H23N2O4Cl · HCl: C, 56.21; H, 5.66; N, 6.56; Cl, 16.48. Found:
C, 55.70; H, 5.61; N, 6.48; Cl, 16.59.
Measurements of Enzyme Activity
The kinetic parameters, Ks, k2, and k3, for the hydrolysis of Bz-L-Arg-OEt, were
determined by the thionine displacement method using stopped-flow techniques.
The reaction was carried out in 50 mM Tris–HCl (pH 8.0, containing 20 mM CaCl2)
according to the reported method (17–19). The kinetic parameters, Km and kcat,
for Boc- -Phe-OGp · TsOH, were determined with a pH-stat device. The reaction
L
was carried out in 0.1 M KCl (containing 20 mM CaCl2), with 10 mM NaOH as
the titrant.
Enzymatic Peptide Coupling Reactions
Peptide coupling reactions were carried out at 25ЊC in 50 mM 4-morpholinepro-
panesulfonate (MOPS) buffer, pH 8.0, containing dimethylsulfoxide (DMSO). The
concentrations of acyl donor (conventional substrate), acyl acceptor (
ide, - or -alanine p-nitroanilide), and methyltrypsin were 1 mM, 100 mM (or 20
mM), and 10 ȐM, respectively. In the case of inverse substrate, the concentrations
of acyl donor (inverse substrate), acyl acceptor ( -arginine amide, -lysine amide,
or -alanine p-nitroanilide), and methyltrypsin were 1 mM, 40 mM, and 20 ȐM,
L-leucine am-
L
D
L
L
L
respectively. The time course of the peptide coupling reaction was determined
aliquots of the reaction mixture by HPLC. HPLC conditions were as follows: column

METHYLTRYPSIN-CATALYZED PEPTIDE COUPLING
311
FIG. 1. Time course of methyltrypsin-catalyzed hydrolysis of NPGB. Reaction was carried out in
0.1 M Veronal buffer (pH 8.3, containing 20 mM CaCl2) at 25ЊC. Concentration of methyltrypsin and
NPGB were 8.45 and 98 ȐM, respectively.
TABLE 1
Comparison of Kinetic Parameters for Native Trypsin and Methyltrypsin-Catalyzed Hydrolysis of
Conventional and Inverse Substrate
Ks(Km)
(M)
k2
k3(kcat
)
k2/Ks (kcat/Km)
1
1
1
1
(sϪ
)
(sϪ
)
(sϪ MϪ
)
Reference
NPGBa
Native trypsin
Methyltrypsin
Bz-
-Arg-OEtb
5
4
5
3.65 ϫ 10Ϫ
2.69 ϫ 102
4.06 ϫ 10Ϫ
4.10 ϫ 10Ϫ
7.37 ϫ 106
1.21 ϫ 102
23
2
1.84 ϫ 10Ϫ
Not determined
This work
L
6
3
Native trypsin
Methyltrypsin
4.26 ϫ 10Ϫ
1.10 ϫ 102
5.01 ϫ 10Ϫ
2.04 ϫ 100
2.58 ϫ 105
2.96 ϫ 102
This work
This work
1
3
1.69 ϫ 10Ϫ
4.02 ϫ 10Ϫ
Boc-L
-Phe-OGpc
5
5
Native trypsin
Methyltrypsin
(2.25 ϫ 10Ϫ
)
)
Not observed
Not observed
(2.68 ϫ 10)
(1.19 ϫ 106)
(3.03 ϫ 10)
12
3
(3.73 ϫ 10Ϫ
(1.13 ϫ 10Ϫ
)
This work
a Reaction was carried out in 0.1 M Veronal (pH 8.3, containing 20 mM CaCl2) at 25ЊC.
b Reaction was carried out in 50 mM Tris–HCl (pH 8.0, containing 20 mM CaCl2) at 25ЊC and
parameters were estimated by thionine displacement method using stopped-flow technique.
c Reaction was carried out in 0.1 M KCl (pH 8.0, containing 20 mM CaCl2) at 25ЊC and parameters
were estimated using pH-stat technique.

312
ITOH ET AL.
TABLE 2
Methyltrypsin-Catalyzed Peptide Coupling by Use of Conventional Substrate as
Acyl Donor Componenta
Entry
no.
Reaction
time (h)
Yieldb
(%)
Acyl donor
Product
1
2
3
4
5
6
7
Z-
Z-
Bz-
Z-
Z-
Z-
L
-Lys-OMe
3
0.5
4
24
3
Z-
Z-
Bz-
Z-
Z-
Z-
L
L
-Lys-
-Lys-
-Arg-
-Lys-
-Lys-
-Lys-
L
-Leu-NH2
-Leu-NH2
-Leu-NH2
-Leu-NH2
83 (53)
28 (7)
66 (35)
L-Lys-OCp
L
L
-Arg-OEt
L
L
L-Lys-OMe
L-Lys-OMe
L-Lys-OCp
L
L
L
D
Not detected
80 (48)
L
-Ala-pNA
-Ala-pNA
0.5
1
L
55 (24)
86 (76)
Bz-
L
-Arg-OEt
Bz-
L
-Arg-
L-Ala-pNA
a Condition: acyl donor, 1 mM; actyl acceptor, 100 mM; methyltrypsin, 10 ȐM; 20% DMSO–MOPS
(50 mM, pH 8.0, containing 10 mM CaCl2); 25ЊC.
b The values in parentheses are yields by using 20 mM acyl acceptor.
(4.6 ϫ 250 mm, Wakosil 5C18–200), isocratic elution at 1 ml/min, 0.1% trifluoroacetic
acid/acetonitrile. Elution peaks were detected at 310 nm for the p-nitroanilide
moiety, at 254 nm for the benzyloxycarbonyl moiety, and at 260 nm for the
p-hydroxybenzyl moiety, respectively.
RESULTS AND DISCUSSION
Methyltrypsin
The purity of the commercial preparation of bovine pancreatic trypsin was verified
by active site titration using NPGB according to the reported method (15), and
the preparation used was determined to be 60% pure. Methyltrypsin was prepared
using methyl m-guanidinobenzenesulfonate tetrafluoroborate as described in the
experimental procedure. The pH was kept at 7.4 by occasional addition of NaOH.
It was found that strict control of pH during the methylation step is crucial for the
purity of the modified enzyme. The amidase activity of methyltrypsin was measured
with Bz-
L
-Phe-
L
-Val- -Arg-pNA and no appreciable activity was detected. The
L
purity of methyltrypsin (95%) was determined by active site titration according to
a method similar to that described for bovine trypsin using NPGB.
The time course of methyltrypsin-catalyzed hydrolysis of NPGB is shown in Fig.
1. After mixing enzyme and substrate, a very slow but clear acylation reaction was
observed but no distinct steady-state turnover process. As shown in Table 1, the
affinity of methyltrypsin toward NPGB is weaker than that of native trypsin (fivefold
larger Ks value) (23). In contrast, the k2 value for methyltrypsin is approximately
four orders of magnitude smaller than that of native trypsin. The parameter, k2/Ks
(or kcat/Km), introduced by Brot and Bender (24) was used for the evalution of the
specificity of the substrates. In terms of these k2/Ks (or kcat/Km) values, the specificity
of methyltrypsin is four orders of magnitude lower than those of native trypsin.

METHYLTRYPSIN-CATALYZED PEPTIDE COUPLING
313
TABLE 3
Methyltrypsin-Catalyzed Peptide Coupling by Use of Inverse Substrate as
Acyl Donor Componenta
Entry
no.
Reaction
time (h)
Yield
(%)
Acyl donor
Product
8
9
Boc-Gly-OGp
4
4
2
0.5
4
3
Boc-Gly-
L
-Ala-pNA
38
42
59
44
62
35
45
2
31
39
21
20
13
13
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
L
L
L
L
L
L
L
-Als-OGp
-Leu-OGp
-Phe-OGp
-Tyr-OGp
-Tyr-OGp
-Tyr-OGp
-Tyr-OGp
-Ala-OGp
-Leu-OGp
-Phe-OGp
-Tyr-OGp
-Tyr-OGp
-Tyr-OGp
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
L
L
L
L
L
L
L
-Ala-L-Ala-pNA
-Leu-
-Phe-
-Tyr-
-Tyr-
-Tyr-
-Tyr-
-Ala-
-Leu-
-Phe-
10
11
12
13
14
15
16
17
18
19
20
21
L
-Ala-pNA
-Ala-pNA
-Ala-pNA
-Leu-NH2
-Arg-NH2
L
L
L
L
8
12
10
6
D-Ala-pNA
D
D
D
D
D
D
D
D
D
D
D
D
L-Ala-pNA
L
-Ala-pNA
-Ala-pNA
-Ala-pNA
-Leu-NH2
-Arg-NH2
8
L
20
48
48
-Tyr-
-Tyr-
-Tyr-
L
L
L
a Condition: acyl donor, 1 mM; acyl acceptor, 40 mM; methyltrypsin, 20 ȐM; 20% DMSO–MOPS
(50 mM, ph 8.0, containing 10 mM CaCl2); 25ЊC.
These diminished values were approximately the same order of magnitude as those
reported for methylchymotrypsin, which was used previously as a catalyst for enzy-
matic peptide synthesis (3).
Kinetic parameters for the modified enzyme were obtained with two comparable
types of esters, and the results are shown in Table 1. In the hydrolysis of Bz- -Arg-
L
OEt, which is a typical specific substrate for trypsin and trypsin-like enzymes,
methyltrypsin was three orders of magnitude less active than the native trypsin in
terms of k2/Ks value. The k2 and k3 values were three orders of magnitude smaller
than those for native trypsin.
In a previous paper (7), we reported that Boc-amino acid p-guanidinophenyl ester
such as Boc-
L
-phenylalanine p-guanidinophenyl ester (Boc- -Phe-OGp) behaves as
L
a specific substrate for trypsin and trypsin-like enzymes, in spite of the fact that
the site-specific group (a charged guanidinium) for the enzyme is not included in
the acyl moiety but in the leaving group portion. Such a substrate was termed an
‘‘inverse substrate’’ for trypsin and trypsin-like enzymes (25). In comparing kinetic
parameters for methyltrypsin and native trypsin-catalyzed hydrolyses of the ‘‘inverse
substrate’’ (Boc- -Phe-OGp), both enzymes showed almost the same affinity,
L
though their total catalytic efficiencies (kcat/Km) are markedly different.
Methyltrypsin-Catalyzed Peptide Coupling Reaction
Methyltrypsin-catalyzed peptide coupling reactions were carried out and moni-
tored as described under Experimental Procedures. In the analysis of the reaction
product by HPLC, eluate was identified by comparison with the authentic sample
which was chemically synthesized according to literature procedures (7, 26, 27).

314
ITOH ET AL.
FIG. 2. Effect of organic solvent on methyltrypsin-catalyzed condensation of Z-
-Leu-NH2 and that of Boc- -Tye-OGp with -Ala-pNA. Reaction was carried out in 50 mM MOPS
buffer (pH 8.0) containing DMSO (᭿) and DMF (᭹) at 25ЊC. Product yield was analyzed after a reaction
period of 4 h in which the coupling was completed. (—) Z- -Lys-OMe, 1 mM; -Leu-NH2 , 100 mM;
methyltrypsin, 10 ȐM. (----) Boc- -Tyr-OGp, 1 mM; -Ala-pNA, 40 mM; methyltrypsin, 20 ȐM.
L-Lys-OMe with
L
L
L
L
L
L
L
The coupling yields reached 66–86% within 4 h (entries 1, 3, 5, and 7 in Table
2). This result indicated that methyltrypsin could be a valuable catalyst in peptide
synthesis with conventional acyl donors. The enantiomeric preference of the acyl
acceptor is rather strict (entry 4 in Table 2). This is in contrast to the results obtained
with the native enzyme. It has been reported that the enantiomeric preference
for the acyl acceptor in trypsin- and Ͱ-chymotrypsin-catalyzed peptide coupling
reactions is not very strict with substantial reaction occurring with -acyl acceptors
D
(26, 27).
Activated esters such as cyanomethyl and p-chlorophenyl ester have often been
used in peptide couplings catalyzed by modified enzymes, since the catalytic activity
of modified enzymes is usually less than native enzymes and reactive substrates are
preferred (3, 28). A rapid coupling reaction was observed when the Z-
L
-lysine
p-chlorophenyl ester was used as acyl donor, but the yield was low (entries 2 and

METHYLTRYPSIN-CATALYZED PEPTIDE COUPLING
315
FIG. 3. pH dependency of methyltrypsin-catalyzed condensation. Reactions were carried out in
50 mM 4-morpholinoethanesulfonic acid (MES) (᭿), 50 mM MOPS (᭹), and 0.1 M carbonate buffers
(᭡) containing 20% DMSO at 25ЊC (—) Z-
L-Lys-OMe, 1 mM; L-Leu-NH2 , 100 mM; methyltrypsin,
10 ȐM. (----) Boc- -Tyr-OGp, 1 mM; -Ala-pNA, 40 mM; methyltrypsin, 20 ȐM.
L
L
6 in Table 2). It was determined that Z- -lysine p-chlorophenyl ester is so reactive
L
that the ester is consumed by nonspecific hydrolysis within 0.5 h of reaction. For
the methyltrypsin-catalyzed reaction the reactive ester is thus less suited than methyl
and ethyl esters. This contrasts with results obtained in methylchymotrypsin-cata-
lyzed and thiolsubtilisin-catalyzed peptide coupling reactions (3, 28).
The methyltrypsin-catalyzed coupling reaction was also applied to inverse sub-
strates. The results are summarized in Table 3 together with the coupling conditions.
It was noticed that the reaction involving
15 in Table 3). In contrast, the structure of the acyl donor does not markedly affect
the reaction yield. Reactions with acyl donors derived from -amino acids are more
or less slower than those with the corresponding -enantiomers and this is reflected
D-acyl acceptors is not favorable (entry
D
L
in the final reaction yields. The reaction conditions were further investigated with
regard to reaction media, pH, acyl acceptor concentration, and reaction time. The
effect of DMSO and DMF concentration on the coupling yields is shown in Fig.
2. The coupling reaction of inverse substrates depends on the organic solvent

316
ITOH ET AL.
FIG. 4. (A) Effect of acyl acceptor concentration on methyltrypsin-catalyzed condensation. Reactions
were carried out in 50 mM MOPS (pH 8.0) containing 20% DMSO at 25ЊC. (—) Z- -Lys-OMe, 1 mM;
-Tyr-OGp, 1 mM;
-Ala-pNA (᭿), 1–60 mM. (B) Double reciprocal plots. Dissociation constants,
-Tyr-OGp (᭿), were determined from the plot.
L
methyltrypsin, 10 ȐM;
methyltrypsin, 20 ȐM;
L-Leu-NH2 (᭹) and L-Ala-pNA (᭡), 1–200 mM. (----) Boc-L
L
10.5 mM for Z-L-Lys-OMe (᭡) and 7.0 mM for Boc-L

METHYLTRYPSIN-CATALYZED PEPTIDE COUPLING
317
FIG. 5. Time course of the peptide coupling reaction. Reaction was carried out in 50 mM MOPS
(pH 8.0) containing 20% DMSO at 25ЊC. (—) Z- -Lys-OMe, 1 mM; -Leu-NH2 (᭹) or -Ala-pNA (᭿),
-Tyr-OGp, 1 mM; -Ala-pNA (᭡), 40 mM; methyltrypsin,
L
L
L
100 mM; methyltrypsin, 10 ȐM. (----) Boc-
L
L
20 ȐM.
concentration and the best coupling yield for the reaction with Boc- -Tyr-OGp was
L
resulted at 10% DMF. However, every inverse substrate was not always soluble in
10% DMF. Therefore, the medium containing 20% DMSO was selected as a standard
condition for the coupling reaction. It is noticeable that the concentration of organic
solvent does not affect so much the reaction of conventional esters. The effect of
pH of the reaction medium on the coupling yields was analyzed. The maximum
yield was obtained at pH 8.0 for both inverse substrates and conventional esters,
as shown in Fig. 3. Acyl acceptor concentration influenced the coupling yield. Wong
et al. reported that the yield of methylchymotrypsin-catalyzed peptide coupling
was dependent on the acceptor concentration (3). Methyltrypsin-catalyzed peptide
coupling with conventional substrates showed the same dependence, as shown in
Fig. 4A. In the case of the inverse substrate, the coupling yield reached a plateau
at the concentration of 40 mM of the acceptor, suggesting the saturation of the
enzyme active site with the acceptor. Double reciprocal plots of the coupling yield

318
ITOH ET AL.
vs acceptor concentration may afford the equilibrium constant for the dissociation
of the acyl acceptor from the ternary complex (enzyme–acyl donor–acyl acceptor).
Dissociation constants, 10.5 and 7.0 mM, were analyzed for both the conventional
and the inverse substrates, respectively (Fig. 4B). The time course of the coupling
of Z-
L-Lys-OMe with
L-Leu-NH2 and
L
-Ala-pNA, and Boc-
L
-Tyr-OGp with -Ala-
L
pNA, are shown in Fig. 5. The coupling yields were not changed after a long
period of incubation. This result indicated that enzymatic hydrolysis of the products
is negligible.
Enzymatic synthesis of arginine- or lysine-containing peptides has been investi-
gated by several groups. Oka and Morihara carried out trypsin-catalyzed syntheses
using high concentrations of substrate (27). Wong et al. reported that the amidase
activity of trypsin, papain, and chymotrypsin decreased by the addition of DMF
and DMSO, whereas esterase activity remained essentially unchanged. Therefore
these enzymes have proved useful for peptide bond formation in the presence of
DMF and DMSO (29). Meanwhile, Jakubke et al. proposed a new concept called
the ‘‘freeze-concentration model,’’ which involves the concentration of reactants
under freezing conditions, and they succeeded in the preparation of peptides in
good yields with chymotrypsin (30). These methods were advantageous for the
avoidance of the secondary hydrolysis of the products.
In summary, this paper provides a simple chemical procedure for the preparation
of methyltrypsin in which the amidase activity was greatly decreased but the esterase
activity retained. The utility of methyltrypsin as a catalyst for the synthesis of
peptides by use of conventional and inverse substrates was studied. The method
has been shown to be useful for the formation of peptides, which are not subject
to secondary hydrolysis by the enzyme.
ACKNOWLEDGMENTS
This work was supported in part by a Grant-in-Aid for Encouragement of Young Scientists (No.
08772030) from the Ministry of Education, Science, Sports, and Culture of Japan and by a grant from
the Japan Private School Promotion Foundation.
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