Kinetically controlled peptide synthesis mediated by papain using the carbamoylmethyl ester as an acyl donor

Article abstract of DOI:10.1007/s10989-014-9393-0

A series of dipeptides were synthesized generally in good yields with carbamoylmethyl (Cam) esters as acyl donors in the presence of a cysteine protease, papain, immobilized on Celite. Several segment condensations were also achieved generally in high yields without danger of racemization and formation of the secondary-hydrolysis product. Moreover, partial sequences of some bioactive peptides were prepared through segment condensations, and aimed-at peptides were obtained generally in high yields without the racemization of C-terminal residues of the carboxyl components. Thus, the superiority of the Cam ester in the kinetically controlled peptide synthesis was once again ascertained in couplings mediated by the cysteine protease as in those catalyzed by the serine proteases reported earlier.

Full text of DOI:10.1007/s10989-014-9393-0

Int J Pept Res Ther
DOI 10.1007/s10989-014-9393-0
Kinetically Controlled Peptide Synthesis Mediated by Papain
Using the Carbamoylmethyl Ester as an Acyl Donor
•
Toshifumi Miyazawa Takao Horimoto
•
Kayoko Tanaka
Accepted: 7 January 2014
Ó Springer Science+Business Media New York 2014
Abstract A series of dipeptides were synthesized gen-
erally in good yields with carbamoylmethyl (Cam) esters as
acyl donors in the presence of a cysteine protease, papain,
immobilized on Celite. Several segment condensations
were also achieved generally in high yields without danger
of racemization and formation of the secondary-hydrolysis
product. Moreover, partial sequences of some bioactive
peptides were prepared through segment condensations,
and aimed-at peptides were obtained generally in high
yields without the racemization of C-terminal residues of
the carboxyl components. Thus, the superiority of the Cam
ester in the kinetically controlled peptide synthesis was
once again ascertained in couplings mediated by the cys-
teine protease as in those catalyzed by the serine proteases
reported earlier.
et al. 2012). This enzymatic methodology has a lot of
advantages compared to the conventional chemical
approaches: freedom from racemization, high regio- and
stereoselectivity, and minimal side-chain protection. On
the other hand, narrow substrate specificity and the sec-
ondary hydrolysis of the growing peptides are counted as
major disadvantages. We previously demonstrated the
superiority of the carbamoylmethyl (Cam) ester as an acyl
donor in the kinetically controlled peptide-bond formation
mediated by the mammalian serine protease a-chymo-
trypsin: the above-mentioned drawbacks were overcome by
employing this particular ester (Miyazawa et al. 2001a, b,
2002a, b). The effectiveness of the Cam ester was ascer-
tained also in peptide syntheses mediated by some micro-
bial serine proteases (Miyazawa et al. 2002c, 2003, 2008).
In peptide synthesis mediated by a serine or cysteine
protease, the kinetically controlled approach employing
ester substrates has often been chosen, because compared
with the thermodynamically controlled approach which
represents the direct reversal of proteolysis it generally
gives a higher yield of the targeted peptide in a shorter
reaction time and it requires a lower concentration of the
Keywords Carbamoylmethyl ester Á Cysteine protease Á
Kinetically controlled peptide synthesis Á Papain Á Segment
condensation
Introduction
¨
enzyme employed (Nuijens et al. 2012). Doring et al.
The specificities of enzymes, especially proteases, have
been increasingly exploited for peptide-bond formation. As
the consequence of intensive investigations during the last
few decades, protease-catalyzed peptide synthesis is now
becoming popular as an alternative or complement to
chemical synthesis of biologically active peptides (Nuijens
(1981) first observed that papain-mediated peptide syn-
theses could proceed when esterified acyl group donors
were employed. These syntheses were performed in a
biphasic aqueous-organic system at pH 5.5. Mitin et al.
(1984) reported later on peptide syntheses at pH values
ranging from 8 to 9.5 where the peptidase activity of the
protease was largely reduced.
In the present paper, using the Cam ester as the acyl
donor and the immobilized papain (EC 3.4.22.2) in an
organic solvent with a minimum amount of water, we have
examined a series of dipeptide syntheses and several model
segment condensations, and the synthesis of partial
T. Miyazawa (&) Á T. Horimoto Á K. Tanaka
Department of Chemistry, Faculty of Science and Engineering,
Konan University, 8-9-1 Okamoto, Higashinada-ku,
Kobe 658-8501, Japan
e-mail: miyazawa@konan-u.ac.jp
123

Int J Pept Res Ther
sequences of some bioactive peptides, in order to demon-
strate the superiority of the Cam ester in the kinetically
controlled peptide synthesis mediated by the cysteine pro-
tease (Wang et al. 2011; de Beer et al. 2011, 2012).
data processor. HPLC analyses were undertaken under the
following conditions: column, YMC ODS (4.6 mm
i.d. 9 250 mm); mobile phase, 50–65 % MeOH aq. con-
taining H3PO4 (0.01 M); flow rate, 1.0 ml min-1; column
temperature, 30 °C; detection, UV at 254 nm.
Materials and Methods
Papain-Catalyzed Peptide Synthesis
Materials
In a typical coupling experiment, a mixture of the Cam
ester of an N-Z-amino acid or N-protected peptide
(0.05 mmol), an amino acid amide hydrochloride or a
peptide amide hydrobromide (0.2 mmol), triethylamine
(28 ll, 0.2 mmol) and the immobilized enzyme (150 mg)
was incubated with shaking in a solvent composed of 2 ml
of acetonitrile and 42 ll of 0.1 M phosphate buffer
(pH 8.0) at 30 °C. The yields of the desired peptide, its
epimer which might occur as the result of the racemization
of the C-terminal residue, the hydrolysis product of the
donor ester and possible by-products were determined by
HPLC analysis.
Papain (twice-crystallized, lyophilized powder from
papaya latex) was purchased from Sigma Chemical Co. and
had a specific activity of 2.8 U (mg solid)-1 with N-ben-
zoyl-L-Arg-OEt. a-Chymotrypsin (type II, from bovine
pancreas) and Bacillus licheniformis protease (subtilisin
Carlsberg) were also from Sigma. The Cam esters of N-Z-
amino acids (Z = benzyloxycarbonyl) or N-protected
peptides were prepared through the reaction of their Cs
salts with 2-chloroacetamide in DMF at 60 °C overnight
(Miyazawa et al. 2001b, 2002b). Amino acid amide
hydrochlorides were purchased from Kokusan Chemical
Works. Gly-L-Leu-L-Met-NH2ÁHBr and L-Phe-Gly-NH2Á
HBr were prepared from their corresponding N-Z-peptide
amides through debenzyloxycabonylation with HBr in
acetic acid. The authentic samples of N-protected di- and
tripeptide amides were prepared by the conventional
chemical methods. The racemization-free authentic sam-
ples of partial sequences of bioactive peptides were pre-
pared by the EDC [1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide]–HOBt (1-hydroxybenzotriazole)–CuCl2 method
(Miyazawa et al. 1992), while a mixture of epimeric pep-
tides was prepared through the same segment condensa-
tions by the EDC method which usually results in a large
amount of racemization of the C-terminal residue. All
organic solvents were distilled and dried over molecular
sieves prior to use.
Results and Discussion
In the kinetically controlled peptide bond formation med-
iated by a serine or cysteine protease, the ester substrate
acting as an acyl group donor first forms the enzyme–
substrate (ES) complex, and then it affords the acyl-
enzyme intermediate (Nuijens et al. 2012). This interme-
diate is then deacylated either by a nucleophilic amino
component to yield the peptide product or by water to form
the hydrolysis product of the donor ester. Accordingly, an
anhydrous or nearly anhydrous organic solvent should be
an ideal reaction medium if the enzyme of question is
active enough in that environment, because any undesirable
hydrolytic side reactions are, in principle, avoidable. Thus
far, papain-catalyzed kinetically controlled peptide syn-
theses have been conducted mainly in aqueous-organic
solvent mixtures employing conventional esters such as the
methyl ester as the acyl donor. We intended to use the Cam
ester as the acyl donor in an organic solvent with low water
content to minimize the secondary hydrolysis of the
growing peptide. The minimal amount of water necessary
for guaranteeing reasonable activity varies with each
enzyme, and the kind of buffer solution which contains the
water might have some effect on the enzyme’s activity.
Moreover, we found previously that the coupling efficiency
was largely dependent on the pH of the buffer solution
from which the immobilized protease was prepared
(Miyazawa et al. 2002c).
Preparation of Immobilized Proteases
The immobilization procedure was essentially the same as
that adopted previously for a-chymotrypsin (Miyazawa
et al. 2001a). Papain (150 mg) was dissolved in 15 ml of
phosphate buffer (pH 8.0), and mixed up with cysteine
hydrochloride (25 mg) and Celite No. 535 (1 g), and the
mixture was lyophilized using an Eyela freeze-dryer FDU-
830 for 24 h. B. licheniformis protease was immobilized in
a similar way as shown in the footnote f of Table 3.
HPLC Analyses
The liquid chromatograph employed was a Shimadzu LC-
10AS instrument equipped with a Shimadzu SPD-10A
variable wavelength UV monitor and a Shimadzu C-R6A
Based on the preliminary results obtained by employing
model couplings, e.g., Z-L-Phe-OCam ? L-Leu-NH2 and
Z-L-Ala-OCam ? L-Ile-NH2, we decided to conduct
123

Int J Pept Res Ther
Table 1 Papain-catalyzed couplings of Z-Xbb-OCam with Xcc-NH2
in aqueous acetonitrile
Table 2 Papain-catalyzed couplings of Z-Xaa-Xbb-OCam with L-
Leu-NH2 in aqueous acetonitrile
Xbb
Xcc
Yield (%)
Peptide
Yield (%)
Z-Xbb-OH
Xbb = L-Phe
Xbb = L-Ala
Xaa
Peptide
Z-Xaa-L-Phe-OH
Peptide
Z-Xaa-L-Ala-OH
Gly
L-Leu
L-Leu
L-Leu
L-Leu
L-Leu
Gly
87.9
87.2
82.4
83.9
85.1
67.4
57.5
86.1
77.4
81.2
75.7
87.5
0.6
L-Ala
L-Leu
L-Ser
L-Met
L-Phe
L-Phe
L-Phe
L-Phe
L-Phe
L-Phe
L-Phe
2.8
Gly
62.7
89.3
46.9
7.8
4.8
5.0
89.4
97.2
79.9
4.2
1.8
1.5
3.3
L-Ala
L-Phe
3.1
7.7
Reactions were conducted using 0.05 mmol of Z-Xaa-Xbb-OCam,
0.2 mmol of L-Leu-NH2ÁHCl, 0.2 mmol of TEA and 150 mg of the
immobilized papain (prepared at pH 8.0) in a solvent composed of the
specified amount of acetonitrile and 2 % (v/v) of phosphate buffer
(pH 8.0) at 30 °C for 24 h
19.3
15.1
8.4
L-Val
L-Leu
L-Ile
12.4
8.1
L-Ser
L-Met
L-Phe
other hand, Val was a poor amine nucleophile, probably
because of its bulky side chain which might interfere with
the nucleophilic attack toward the acyl-enzyme interme-
diate. Gly, Ile and Met occupied the intermediate position
between the two groups of residues. Data on the specificity
for the S1’-site of papain have been less documented thus
far. In the papain-catalyzed coupling (via the thermody-
13.6
8.6
Reactions were conducted using 0.05 mmol of Z-Xbb-OCam,
0.2 mmol of Xcc-NH2ÁHCl, 0.2 mmol of TEA and 150 mg of the
immobilized papain (prepared at pH 8.0) in 2 mL of acetonitrile and
2 % (v/v) of phosphate buffer (pH 8.0) at 30 °C for 24 h
papain-catalyzed couplings in acetonitrile containing 2 %
(v/v) phosphate buffer (pH 8.0) at 30 °C in the presence of
immobilized papain prepared at pH8.0. Thus, a series of
Z-Xbb-OCam (Xbb = Gly, L-Ala, L-Leu, L-Ser, L-Met or L-
Phe) were allowed to react with Xcc-NH2 (Xcc = Gly, L-
Val, L-Leu, L-Ile, L-Ser, L-Met or L-Phe). The yields of the
targeted peptide (Z-Xbb-Xcc-NH2) and the hydrolysis
product of the donor ester (Z-Xaa) after 24 h of incubation,
quantified by HPLC analysis, are compiled in Table 1. The
coupling efficiencies were generally low compared with
those obtained using a-chymotrypsin as a biocatalyst
(Miyazawa et al. 2001b). First, the influence of the amino
acid residue as the carboxyl component (Xbb) was exam-
ined when the amino acid residue as the amino component
(Xcc) was fixed to L-Leu. The differences among the res-
idues were rather small: the peptide yields were more than
80 % in all the cases. This is in accordance with the well-
known fact that the S1-site of papain can be occupied by a
variety of amino acid residues (Kullmann 1987). The pri-
mary specificity of papain is for a bulky aliphatic or aro-
matic residue in the P2-position and this requirement is met
with the use of the N-Z-protecting group. The influence of
the amino acid residue as the amino component (Xcc) was
examined next when the amino acid residue as the carboxyl
component (Xbb) was fixed to L-Phe. Since an amino acid
nucleophile competes with water in the reaction with the
acyl-enzyme intermediate, the coupling efficiency must be
affected by the amino component employed. Leu, Phe and
Ser proved to be rather good residues: the peptide yields
were more than 80 % and the production of Z-Phe was
comparatively small among the residues examined. On the
namically controlled approach) of Z-DL-Gla-OH (Gla = c-
ˇ
carboxyglutamic acid) with L-Xcc-NHNHPh, Cerovsky
ˇ
´
ˇ
and Jost (1985) reported that the yields of the homochiral
dipeptides Z-L-Gla-L-Xcc-NHNHPh were dependent on the
chemical nature of Xcc, following the order: Leu [
Phe [ Met [ Ala [ Val [ Asn. Thus, the two different
types of approaches, i.e., kinetically-controlled or thermo-
dynamically-controlled, showed almost the same tendency
toward the preference for the S1’-site of papain.
We examined next some model segment condensations,
Z-Xaa-L-Phe-OCam ? L-Leu-NH2 or Z-Xaa-L-Ala-OCam ?
L-Leu-NH2 (Xaa = Gly, L-Ala, or L-Phe), under the same
reaction conditions as above. The results obtained after 24 h
of incubation are compiled in Table 2. The peptide yields
were generally much higher and the amounts of the hydro-
lysis products of the donor esters were lower with Z-Xaa-L-
Ala-OCam than with Z-Xaa-L-Phe-OCam. As stated above,
the S1-site of papain can be occupied by a variety of amino
acid residues. However, in the couplings (via the thermo-
dynamically controlled approach) of N-Z-protected amino
acids or peptides, it was reported that hydrophilic or small
hydrophobic amino acids were more appropriate as acyl
group donors (Isowa et al. 1977). Our present result is in
accordance with the reported specificity for the P1-position.
As far as the specificity for the P2-position is concerned, the
less hydrophobic Ala residue gave better peptide yield
than the more hydrophobic Phe residue. This is not in
accordance with what was observed by Isowa et al.: with
some remarkable exceptions, improved peptide yields
resulted if bulky hydrophobic amino acid residues occupied
the P2-position of the substrate. In the present segment
123

Int J Pept Res Ther
Table 3 Protease-catalyzed syntheses of substance
pentapeptide
P
(7-11)-
Table 4 Protease-catalyzed syntheses of eledoisin (6-11)-
hexapeptide
Protease
2 ? 3 Couplinga
4 ? 1 Couplingb
Proteasea
Yield (%)
Peptide
Yield (%)
Yield (%)
Boc-L-Ala-L-Phe-L-Ile-OH
Peptide
Boc-L-Phe-
L-Phe-OH
Peptide
Boc-L-Phe-
L-Phe-Gly-
L-Leu-OH
Papain
90.3
88.0
86.8
4.9
1.9
4.5
a-Chymotrypsin
B. licheniformis protease
Papainc
a-Chymotrypsind
82.8
83.9
84.1
4.8
5.1
1.7
78.3
75.2
73.5
7.2
6.9
9.9
Boc-L-Ala-L-Phe-L-Ile-Gly-L-Leu-L-Met-NH2: mp 257–258 °C; [a]D25
-
29.6° (c 1.0, DMF). Reactions were conducted using 0.05 mmol of
Boc-L-Ala-L-Phe-L-Ile-OCam, 0.2 mmol of Gly-L-Leu-L-Met-
NH2ÁHBr, and 0.2 mmol of TEA at 30 °C for 48 h
B. licheniformis
proteasee
a
See footnotes d–f of Table 3
Boc-L-Phe-L-Phe-Gly-L-Leu-L-Met-NH2: mp 166–168 °C; [a]2D5-23.8°
(c 1.0, DMF)
a
Reactions were conducted using 0.05 mmol of Boc-L-Phe-L-Phe-
OCam, 0.2 mmol of Gly-L-Leu-L-Met-NH2ÁHBr, and 0.2 mmol of
peptide was obtained in ca. 80 % yield after 48 h of incu-
bation. Although the concomitant formation of a small
amount of the hydrolysis product of the donor esters was
inevitable, no epimeric peptides were detected on HPLC.
Moreover, no significant formation of defective peptides
was observed from the HPLC profile. The two serine pro-
teases yielded almost similar results to those obtained using
papain in both of these segment couplings. Eledoisin is an
undecapeptide of mollusk origin and also belongs to the
tachykinin family of neuropeptides. Its minimum sequence
is also the C-terminal (8-11)-tetrapeptide, i.e., L-Ile-Gly-L-
Leu-L-Met-NH2 (Broccardo et al. 2003). We tried the prep-
aration of the (6-11)-hexapeptide sequence through a 3 ? 3
(Boc-L-Ala-L-Phe-L-Ile-OCam ? Gly-L-Leu-L-Met-NH2)
segment condensation (Table 4). The desired peptide was
obtained in ca. 90 % yield after 48 h of incubation. No
indications of the formation of the epimeric peptide and
defective peptides were detected on HPLC. In this seg-
ment coupling also the two serine proteases yielded
almost similar results to those obtained using papain.
Dermorphin is a heptapeptide having potent analgesic
activity isolated from the skin of a South American frog. The
N-terminal (1-4)-tetrapeptide, i.e., L-Tyr-D-Ala-L-Phe-Gly,
is the minimum sequence that retains dermorphin activity
(Montecucchi 1981). The synthesis of the tetrapeptide
sequence, which contains a D-amino acid residue, was
examined through both 2 ? 2 (Boc-L-Tyr-D-Ala-OCam ?
L-Phe-Gly-NH2) and 3 ? 1 (Boc-L-Tyr-D-Ala-L-Phe-OCam ?
Gly-NH2) segment condensations (Table 5). As previously
reported (Miyazawa et al. 2001b), the use of the Cam ester
allowed even the coupling of a D-amino acid as the carboxyl
component mediated by a-chymotrypsin. Therefore, we
thought it worthwhile to examine the 2 ? 2 segment conden-
sation in which the C-terminal residue was a D-amino acid,
considering also the fact that the specificity of papain for the
P1-position is rather broad. In fact, the aimed-at peptide
was obtained in a moderate yield (57 %) after 48 h of incu-
bation in this case. No racemization of the D-Ala residue at the
P1-position was detected. The 3 ? 1 segment condensation
TEA at 30 °C for 48 h
b
Reactions were conducted using 0.05 mmol of Boc-L-Phe-L-Phe-
Gly-L-Leu-OCam, 0.2 mmol of L-Met-NH2ÁHCl, and 0.2 mmol of
TEA at 30 °C for 48 h
c
Using 150 mg of the immobilized papain (prepared at pH 8.0) in
2 mL of acetonitrile and 2 % (v/v) of phosphate buffer (pH 8.0)
d
Using 150 mg of the immobilized a-chymotrypsin (prepared at pH
8.0) in 2 mL of acetonitrile and 2 % (v/v) of phosphate buffer (pH
8.0)
e
Using 150 mg of the immobilized subtilisin Carlsberg (prepared at
pH 10.7) in 2 mL of acetonitrile and 2 % (v/v) of carbonate buffer
(pH 10.7)
condensations, the formation of the defective peptides and
the racemization of the C-terminal residues were not
detected on HPLC, as clarified by preparing the epimeric
authentic samples chemically.
Furthermore, the synthesis of partial sequences of some
bioactive peptides was examined through segment con-
densations employing Cam esters as acyl donors. These
peptides are the partial sequences containing the minimum
sequences which retain their bioactivities. For comparison,
the same couplings were also conducted by using a-chy-
motripsin and B. licheniformis protease as biocatalysts.
These proteases were also employed in immobilized form.
In these couplings the quantification of the aimed-at peptide,
its epimer which might occur as the result of the racemi-
zation of the C-terminal residue, and the hydrolysis product
of the donor ester were done by HPLC analysis. Substance P
is an undecapeptide functioning as a neurotransmitter and
neuromodulator which belongs to the tachykinin neuro-
peptide family. The minimum sequence was found to be the
C-terminal (8-11)-tetrapeptide, i.e., L-Phe-Gly-L-Leu-L-
Met-NH2 (Nakamura et al. 1999). We examined the syn-
thesis of the (7-11)-pentapeptide sequence through both
2 ? 3 (Boc-L-Phe-L-Phe-OCam ? Gly-L-Leu-L-Met-NH2)
(Boc = t-butoxycarbonyl) and 4 ? 1 (Boc-L-Phe-L-Phe-
Gly-L-Leu-OCam ? L-Met-NH2) segment condensations
(Table 3). In both the segment condensations the desired
123

Int J Pept Res Ther
the cysteine protease-catalyzed kinetically controlled pep-
Table 5 Protease-catalyzed syntheses of dermorphin (1-4)-
tetrapeptide
tide synthesis.
Proteasea
2 ? 2 Couplingb
3 ? 1 Couplingc
Yield (%)
Yield (%)
References
Peptide Boc-L-Tyr- Peptide Boc-L-Tyr-
D-Ala-L-Phe-
OH
Broccardo M, Erspamer V, Falconieri G, Improta G, Linari G,
Melchiorri P, Grace RCR, Chandrashekar IR, Cowsik SM (2003)
Solution structure of the tachykinin peptide eledoisin. Biophys J
84:655–664
D-Ala-OH
Papain
60.2
7.2
5.6
4.0
57.2
41.1
37.0
5.8
3.2
2.8
ˇ
ˇ
´
ˇ
Cerovsky V, Jost K (1985) Enzymatically catalyzed synthesis of
dipeptides of c-carboxy-L-glutamic acid from benzyloxycar-
bonyl-c-carboxy-DL-glutamic acid. Coll Czech Chem Commun
50:878–884
a-Chymotrypsin 33.3
B. licheniformis
protease
34.5
Boc-L-Tyr-D-Ala-L-Phe-Gly-NH2: mp 130–130.5 °C; [a]2D5 ? 29.2°
(c 1.0, DMF)
de Beer RJAC, Zarzycka B, Amatdjais-Groenen HIV, Jans SCB,
Nuijens T, Quaedflieg PJL, van Delft FL, Nabuurs SB, Rutjes
FPJT (2011) Papain-catalyzed peptide bond formation: enzyme-
specific activation with guanidinophenyl esters. ChemBioChem
12:2201–2207
de Beer RJAC, Zarzycka B, Mariman M, Amatdjais-Groenen HIV,
Mulders MJ, Quaedflieg PJL, van Delft FL, Nabuurs SB, Rutjes
FPJT (2012) Papain-specific activating esters in aqueous dipep-
tide synthesis. ChemBioChem 13:1319–1326
a
See footnotes d–f of Table 3
b
Reactions were conducted using 0.05 mmol of Boc-L-Tyr-D-Ala-
OCam, 0.2 mmol of L-Phe-Gly-NH2ÁHBr, and 0.2 mmol of TEA at
30 °C for 48 h
c
Reactions were conducted using 0.05 mmol of Boc-L-Tyr-D-Ala-L-
Phe-OCam, 0.2 mmol of Gly-NH2ÁHCl, and 0.2 mmol of TEA at
¨
Doring G, Kuhl P, Jakubke H-D (1981) Modelluntersuchungen zur
¨
30 °C for 48 h
papainkatalysierten Peptidsynthese im waßrig-organischen
Zweiphasensystem. Monatsh Chem 112:1165–1173
Isowa Y, Ohmori M, Ichikawa T, Kurita H, Sato M, Mori K (1977)
The synthesis of peptides by means of proteolytic enzymes. Bull
Chem Soc Jpn 50:2762–2765
Kullmann W (1987) Enzymatic peptide synthesis. CRC Press, Boca
Raton, pp 50–52
Mitin YV, Zapevalova NP, Gorbunova EY (1984) Peptide synthesis
catalyzed by papain at alkaline pH values. Int J Pept Protein Res
23:528–534
Miyazawa T, Otomatsu T, Fukui Y, Yamada T, Kuwata S (1992)
Simultaneous use of 1-hydroxybenzotriazole and copper(II)
chloride as additives for racemization-free and efficient peptide
synthesis by the carbodiimide method. Int J Pept Protein Res
39:308–314
also afforded the desired peptide in almost a similar yield as the
2 ? 2 condensation. In this case also no racemization of the
Phe residue at the P1 position was detected. Both the segment
condensations no indications of the formation of defective
peptides were detected on HPLC. The two serine proteases also
gave the desired peptide in lower yields in both the segment
condensations. In particular, the yields were about half of that
obtained by the catalysis of papain in the 3 ? 1 segment
condensation.
Miyazawa T, Nakajo S, Nishikawa M, Hamahara K, Imagawa K,
Ensatsu E, Yanagihara R, Yamada T (2001a) a-Chymotrypsin-
catalysed peptide synthesis via the kinetically controlled
approach using activated esters as acyl donors in organic
solvents with low water content: incorporation of non-protein
amino acids into peptides. J Chem Soc, Perkin Trans 1:82–86
Miyazawa T, Tanaka K, Ensatsu E, Yanagihara R, Yamada T (2001b)
Broadening of the substrate tolerance of a-chymotrypsin by
using the carbamoylmethyl ester as an acyl donor in kinetically
controlled peptide synthesis. J Chem Soc, Perkin Trans 1:87–93
Miyazawa T, Ensatsu E, Yabuuchi N, Yanagihara R, Yamada T
(2002a) Superiority of the carbamoylmethyl ester as an acyl
donor for the kinetically controlled amide-bond formation
Conclusions
A series of dipeptides were synthesized generally in good
yields by employing Cam esters as acyl donors in the
presence of immobilized papain. In these couplings the
differences among the amino acid residues as the carboxyl
components were rather small and Leu and Phe were good
amino acid residues as the amino components. Several
segment condensations were achieved generally in high
yields without danger of racemization and formation of the
secondary-hydrolysis product. Partial sequences of some
bioactive peptides were prepared through segment con-
densations, and aimed-at peptides were obtained generally
in high yields without the racemization of C-terminal res-
idues of the carboxyl components. In the synthesis of
partial sequences of bioactive peptides through segment
condensations, aimed-at peptides were obtained generally
in high yields without the racemization of C-terminal res-
idues of the carboxyl components. Thus, the present results
indicate the usefulness of the Cam ester as the acyl donor in
mediated by a-chymotrypsin.
1:390–395
J Chem Soc, Perkin Trans
Miyazawa T, Ensatsu E, Hiramatsu M, Yanagihara R, Yamada T
(2002b) a-Chymotrypsin-catalysed segment condensations via
the kinetically controlled approach using carbamoylmethyl
esters as acyl donors in organic media. J Chem Soc, Perkin
Trans 1:396–401
Miyazawa T, Hiramatsu M, Murashima T, Yamada T (2002c)
Bacillus licheniformis protease-catalyzed peptide synthesis via
the kinetically controlled approach using the carbamoylmethyl
ester as an acyl donor in anhydrous acetonitrile. Lett Pept Sci
9:173–177
123

Int J Pept Res Ther
Miyazawa T, Hiramatsu M, Murashima T, Yamada T (2003)
Utilization of proteases from Aspergillus species for peptide
synthesis via the kinetically controlled approach. Biocatal
Biotrans 21:93–100
Nakamura M, Chikama T, Nishida T (1999) Synergistic effect with
Phe-Gly-Leu-Met-NH2 of the C-terminal of substance P and
insulin-like growth factor-1 on epithelial wound healing of rabbit
cornea. Br J Pharmacol 127:489–497
Miyazawa T, Shindo H, Murashima T, Yamada T (2008) Peptide
synthesis using carbamoylmethyl esters as acyl donors mediated
by Bacillus amyloliquefaciens protease. Protein Pept Lett
15:1050–1054
Nuijens T, Quaedflieg PJLM, Jakubke H-D (2012) Synthesis of
¨
peptides. In: Drauz K, Groger H, May O (eds) Enzyme catalysis
in organic synthesis, 3rd edn. Wiley-VCH, Weinheim,
pp 692–738
Montecucchi PC (1981) Pharmacological data on dermorphins, a new
class of potent opioid peptides from amphibian skin. Br J
Pharmacol 73:625–631
Wang M, Qi W, Yu Q, Su R, He Z (2011) Kinetically controlled
enzymatic synthesis of dipeptide precursor of L-alanyl–L-gluta-
mine. Biotechnol Appl Biochem 58:449–455
123