Atlantic cod trypsin-catalyzed peptide synthesis with inverse substrates as acyl donor components

Article abstract of DOI:10.1248/cpb.58.484

Atlantic cod trypsin-catalyzed peptide synthesis has been studied by using p-amidino- and p-guanidinophenyl esters of N-(tert-butyloxycarbonyl)amino acid as acyl donor components. The reaction temperature was optimized at 0 °C. The method was shown to be successful as effectively for synthesizing the peptide and useful for preparing dipeptide between D-amino acid with D-amino acid and β-amino acid with β-amino acid, respectively. The enzymatic hydrolysis of the resulting products was negligible.

Full text of DOI:10.1248/cpb.58.484

484
Chem. Pharm. Bull. 58(4) 484—487 (2010)
Vol. 58, No. 4
Atlantic Cod Trypsin-Catalyzed Peptide Synthesis with Inverse
Substrates as Acyl Donor Components
Tomoyoshi FUCHISE,a Hideki KISHIMURA,b Zhi-hong YANG,c Mareshige KOJOMA,a Eiko TOYOTA,a and
,a
*
Haruo SEKIZAKI
a Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido; Ishikari-Tobetsu, Hokkaido 061–0293,
b
Japan: Laboratory of Marine Products and Food Science, Research Faculty of Fisheries Sciences, Hokkaido University;
c
Hakodate, Hokkaido 041–8611, Japan: and Pharmacy Department, Medical College of Qingdao University; Qingdao
266021, Shandong, China. Received November 9, 2009; accepted January 19, 2010; published online January 20, 2010
Atlantic cod trypsin-catalyzed peptide synthesis has been studied by using p-amidino- and p-guanidino-
phenyl esters of N-(tert-butyloxycarbonyl)amino acid as acyl donor components. The reaction temperature was
optimized at 0 °C. The method was shown to be successful as effectively for synthesizing the peptide and useful
for preparing dipeptide between D-amino acid with D-amino acid and b-amino acid with b-amino acid, respec-
tively. The enzymatic hydrolysis of the resulting products was negligible.
Key words Atlantic cod trypsin; enzymatic peptide synthesis; inverse substrate; cold-adapted trypsin; N-(tert-butyloxycar-
bonyl)amino acid p-amidinophenyl ester; N-(tert-butyloxycarbonyl)amino acid p-guanidinophenyl ester
A large number of biologically active peptides have been cod trypsin with chum salmon trypsin. In the present work,
isolated recently from bacterial, fungal, plant and animal we investigated Atlantic cod trypsin-catalyzed peptide syn-
sources and characterized in some detail. In particular, short- thesis using p-amidino- and p-guanidinophenyl esters of Na-
sequence peptides play important roles in the sensory appre- tert-butyloxycarbonyl (Boc)-amino acid as acyl donor com-
ciation of food toward four basic taste sensations (sweet, bit- ponents with two types of acyl acceptors. We have found that
ter, sour and salty).1) Such peptides sometimes contain D- Atlantic cod trypsin-catalyzed peptide synthesis of the D-
amino acid and other unusual amino acids. Synthetic chem- amino acid and b-amino acid-containing products is more ef-
istry has witnessed remarkable progress with the develop- ficient than chum salmon trypsin-catalyzed synthesis.
ment of novel biologically active peptides. Enzymatic pep-
tide synthesis has emerged as a powerful approach to the Results and Discussion
preparation of short sequences. Especially, peptide synthesis
The kinetic constants for the trypsin-catalyzed hydrolysis
using protease-catalyzed reverse reaction has been exten- of synthetic inverse substrates were analyzed on the basis of
sively studied with a variety of amino acids and peptide the following scheme.
derivatives as coupling components.2—8) It has been reported
that the protease-catalyzed peptide synthesis is superior to
the chemical coupling method because it is highly stereose-
lective, racemization-free, and requires less side-chain pro-
tection. The major drawback of the enzymatic method, how-
ever, is the respective substrate specificity. Thus, the applica- Where: Eꢀenzyme; Sꢀsubstrate; ESꢀenzyme–substrate
tion of proteases for peptide synthesis has not been fully in- complex; EAꢀacyl enzyme; P1ꢀalcohol component of the
vestigated synthetic possiblity of a number of biologically substrate; P2ꢀacid component of the substrate; Ksꢀdissocia-
significant peptides containing D-amino acid or other unusual tion constant of enzyme-substrate complex; k2ꢀrate constant
amino acids. A few previous reports have shown the enzy- of acylation step; and k3ꢀrate constant of deacylation step.
matic condensation of noncorded amino acids,9,10) peptide The kinetic parameters Ks and k2 are useful for the evaluation
mimetics,11) peptide conjugates,12) and D-amino acid.13)
of substrates. The former can provide information on the
Previously, we reported that the inverse substrates such as strength of the bond between the substrate and the enzyme,
p-amidino-14) and p-guanidinophenyl esters15—17) behave as which is a characteristic of the enzymatic process, while the
specific substrates for trypsin and trypsin-like enzymes and latter directly reflects the accessibility of the carbonyl func-
allow the specific introduction of an acyl group carrying a tion of the substrate molecule to the catalytic residue of the
non-specific residue into the enzymatic active site. The char- enzyme in the ES complex. The acetic acid and Na-Boc-
acteristics of inverse substrates suggested that they are useful amino acid p-amidinophenyl esters were subjected to kinetic
for enzymatic peptide synthesis.18—24)
analysis.
The kinetic parameters were determined as previously de-
Many studies on the characterization of trypsins from
cold-adapted species have been reported.25) These trypsins scribed,16) and the values obtained are listed in Table 1. The
display substantially higher catalytic efficiency than their parameters were compared with those of chum salmon
mammalian counterparts.25—33) We previously reported the trypsin. p-Amidinophenyl esters behave as specific substrates
chum salmon trypsin-catalyzed synthesis of peptides using for Atlantic cod trypsin based on their k2/Ks values. The pa-
inverse substrates.23,24) We had obtained commercially avail- rameter k2/Ks has been introduced by Brot and Bender34) to
able Atlantic cod trypsin at almost the same time. We are in- evaluate of the specificity of substrates. The observed k2/Ks
terested in comparing of the catalytic efficiency of Atlantic values for Atlantic cod trypsin, 105—106 (Mꢁ1 sꢁ1), are mod-
© 2010 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: sekizaki@hoku-iryo-u.ac

April 2010
485
Table 1. Kinetic Parameters for the Trypsin-Catalyzed Hydrolysis of In-
verse Substrates
Ac-OAm
Ks
(M)
k2
k3
k2/Ks
Enzyme
(sꢁ1
)
(sꢁ1
)
(Mꢁ1 sꢁ1
)
Fig. 2. Structures of Acyl Acceptors
Atlantic cod trypsin
1.92ꢄ10ꢁ5 1.35ꢄ10 5.66ꢄ10ꢁ3 7.02ꢄ105
Chum salmon trypsina) 3.87ꢄ10ꢁ5 1.70ꢄ10 9.26ꢄ10ꢁ3 4.39ꢄ105
Na-Boc-L-Ala-OAm (2)
Ks
(M)
k2
k3
k2/Ks
Enzyme
(sꢁ1
)
(sꢁ1
)
(Mꢁ1 sꢁ1
)
Atlantic cod trypsin
3.52ꢄ10ꢁ6 1.93ꢄ10
1.39
1.14
5.49ꢄ105
1.28ꢄ107
Chum salmon trypsina) 1.00ꢄ10ꢁ6 1.28ꢄ10
Na-Boc-D-Ala-OAm (5)
Ks
(M)
k2
k3
k2/Ks
Enzyme
(sꢁ1
)
(sꢁ1
)
(Mꢁ1 sꢁ1
)
Atlantic cod trypsin
8.37ꢄ10ꢁ6 1.17ꢄ10 6.84ꢄ10ꢁ3 1.40ꢄ106
Chum salmon trypsina) 4.00ꢄ10ꢁ6
9.01
2.20ꢄ10ꢁ3 2.25ꢄ106
Fig. 3. Atlantic Cod Trypsin-Catalyzed Peptide Synthesis at Various Tem-
peratures of Na-Boc-L-Ala-L-Ala-pNA (2a) (ꢀ), Na-Boc-D-Ala-L-Ala-pNA
(5a) (ꢁ), Na-Boc-L-Ala-D-Ala-pNA (2b) (ꢂ), and Na-Boc-D-Ala-D-Ala-
pNA (2a) (ꢄ)
a) See ref. 33.
Conditions: acyl donor, 1 mM; acyl acceptor, 20 mM; Atlantic cod trypsin, 5 mM; 50%
DMSO-GTA (50 mM, pH 8.5, containing 20 mM CaCl2); 0 °C.
and 113 h at 25 °C and 0 °C, respectively.23) Analysis of the
temperature dependence of Atlantic cod trypsin-catalyzed
reactions is in progress. The Atlantic cod trypsin-catalyzed
coupling reaction of Na-Boc-L-Ala-OAm (2) and Na-Boc-D-
Ala-OAm (5) with L-Ala-pNA (a) and D-Ala-pNA (b) to give
corresponding Na-Boc-Ala-Ala-pNA (2a, 5a, 2b, 5b) was
examined under various reaction temperatures. The highest
yield was obtained at 0 °C in all tested reactions as shown in
Fig. 3. Therefore, we were also investigated enzymatic pep-
tide synthesis using another type of inverse substrate, N-Boc-
Fig. 1. Structures of Inverse Substrates
erately large for trypsin substrates. All compounds have suf- AA-OGu (9—16), which were not previously studied on en-
ficient affinity and susceptibility to Atlantic cod trypsin. In zymatic peptide synthesis at low temperature. The other reac-
particular, the observed k2/Ks value of Na-Boc-D-alanine p- tion conditions, such as organic solvent, pH, and the acyl ac-
amidinophenyl esters was larger than that of Na-Boc-L-ala- ceptor concentration, were used according to the same condi-
nine p-amidinophenyl esters. p-Guanidinophenyl esters show tions of chum salmon trypsin.23) Consequently, the standard
similar behavior to the trypsin-catalyzed hydrolysis of p- procedure for the coupling reaction was fixed as follows: acyl
amidinophenyl esters as previously described.16) Conse- donor (inverse substrate), 1 mM; acyl acceptor, 20 mM; At-
quently, two series of inverse substrates were tested against lantic cod trypsin, 5 mM; 50% dimethyl sulfoxide (DMSO)-
cold-adapted trypsins (chum salmon and Atlantic cod) for GTA (50 mM, pH 8.5, containing 20 mM CaCl2); 0 °C.
trypsin-catalyzed peptide synthesis.
The results of the Atlantic cod trypsin-catalyzed coupling
The structures of the inverse substrates (1—16) used in reaction were compared with those of the chum salmon
this study are shown in Fig. 1. These inverse substrates were trypsin-catalyzed coupling reaction previously reported,23)
prepared by condensation of the appropriate Na-Boc-pro- and they are summarized in Table 2. In general, toward all
tected amino acid and p-[Nꢂ,Nꢃ-bis(Z)guanidine]phenol, fol- inverse substrates (1—16), both cold-adapted trypsins (At-
lowed by deprotection by catalytic hydrogenation.16—18) The lantic cod and chum salmon) behaved as a moderately effec-
structures of acyl acceptors (a—c) are also shown in Fig. 2.
tive catalyst for the synthesis of the peptides (Entries 1—19
Because the hydrolysis of reactant components is a serious in Table 2). As shown in Table 2, Atlantic cod trypsin can be
problem in the enzyme-catalyzed synthetic method, our inter- more utilized for the synthesis of peptides containing D-
est is centered on the temperature dependence of Atlantic amino acid and b-amino acid than those of the chum salmon
cod trypsin-catalyzed hydrolysis of inverse substrates in con- trypsin (Entries 6, 9, 10, 15, 18). Atlantic cod trypsin can be
nection with enzymatic peptide synthesis. We previously in- also utilized for the synthesis of some D-amino acid and b-
vestigated the possibility of the nonenzymatic reaction. At amino acid peptide (Entries 7, 19).
50% solvent content, Na-Boc-L-Ala-OAm (2) underwent
In conclusion, the utility of Atlantic cod trypsin can be
some spontaneous hydrolysis with a half-life of about 15 h proposed as a catalyst for the synthesis of peptides by the use

486
Vol. 58, No. 4
Table 2. Yield of Atlantic Cod Trypsin-Catalyzed Peptide Synthesisa)
Entry
No.
Acyl donor
(No.)
Reaction
Product
(No.)
Yieldc)
(%)
Acyl acceptor
timeb) (min)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Na-Boc-Gly-OAm (1)
Na-Boc-L-Ala-OAm (2)
Na-Boc-L-Ala-OAm (2)
Na-Boc-L-Leu-OAm (3)
Na-Boc-L-Phe-OAm (4)
Na-Boc-D-Ala-OAm (5)
Na-Boc-D-Ala-OAm (5)
Na-Boc-D-Leu-OAm (6)
Na-Boc-D-Phe-OAm (7)
Nb-Boc-b-Ala-OAm (8)
Na-Boc-Gly-OGu (9)
L-Ala-pNA (a)
L-Ala-pNA (a)
D-Ala-pNA (b)
L-Ala-pNA (a)
L-Ala-pNA (a)
L-Ala-pNA (a)
D-Ala-pNA (b)
L-Ala-pNA (a)
L-Ala-pNA (a)
L-Ala-pNA (a)
L-Ala-pNA (a)
L-Ala-pNA (a)
L-Ala-pNA (a)
L-Ala-pNA (a)
L-Ala-pNA (a)
L-Ala-pNA (a)
L-Ala-pNA (a)
L-Ala-pNA (a)
b-Ala-b-NA (c)
15 [15]d)
30 [30]d)
60 [60]e)
30 [45]d)
30 [30]d)
90 [60]d)
180 [180]e)
90 [120]d)
90 [60]d)
60 [90]d)
20 [20]e)
45 [30]e)
45 [30]e)
45 [30]e)
90 [90]e)
90 [90]e)
90 [90]e)
60 [60]e)
300 [300]e)
Na-Boc-Gly-L-Ala-pNA (1a)
Na-Boc-L-Ala-L-Ala-pNA (2a)
Na-Boc-L-Ala-D-Ala-pNA (2b)
Na-Boc-L-Leu-L-Ala-pNA (3a)
Na-Boc-L-Phe-L-Ala-pNA (4a)
Na-Boc-D-Ala-L-Ala-pNA (5a)
Na-Boc-D-Ala-D-Ala-pNA (5b)
Na-Boc-D-Leu-L-Ala-pNA (6a)
Na-Boc-D-Phe-L-Ala-pNA (7a)
Nb-Boc-b-Ala-L-Ala-pNA (8a)
Na-Boc-Gly-L-Ala-pNA (1a)
Na-Boc-L-Ala-L-Ala-pNA (2a)
Na-Boc-L-Leu-L-Ala-pNA (3a)
Na-Boc-L-Phe-L-Ala-pNA (4a)
Na-Boc-D-Ala-L-Ala-pNA (5a)
Na-Boc-D-Leu-L-Ala-pNA (6a)
Na-Boc-D-Phe-L-Ala-pNA (7a)
Nb-Boc-b-Ala-L-Ala-pNA (8a)
Nb-Boc-b-Ala-b-Ala-b-NA (9c)
53 [56]d)
83 [82]d)
37 [5]e)
90 [85]d)
73 [85]d)
83 [77]d)
38 [4]e)
87 [90]d)
84 [59]d)
25 [4]d)
52 [51]e)
74 [76]e)
84 [77]e)
85 [80]e)
68 [66]e)
69 [63]e)
65 [66]e)
66 [37]e)
45 [26]e)
Na-Boc-L-Ala-OGu (10)
Na-Boc-L-Leu-OGu (11)
Na-Boc-L-Phe-OGu (12)
Na-Boc-D-Ala-OGu (13)
Na-Boc-D-Leu-OGu (14)
Na-Boc-D-Phe-OGu (15)
Nb-Boc-b-Ala-OGu (16)
Nb-Boc-b-Ala-OGu (16)
a) Conditions: acyl donor, 1 mM; acyl acceptor, 20 mM; Atlantic cod trypsin, 5 mM; 50% DMSO-GTA (50 mM, pH 8.5, containing 20 mM CaCl2); 0 °C. b) The values in
brackets are reaction times (min) of chum salmon trypsin-catalyzed peptide synthesis. c) The values in brackets are yields (%) of chum salmon trypsin-catalyed peptide synthesis.
d) See ref. 23. e) This work.
br s), 6.39 (1H, br s), 7.39—7.47 (4H, m), 7.75—7.76 (2H, m), 8.11 (1H,
br s), 8.23 (1H, s). Anal. Calcd for C21H27N3O4: C, 65.44; H, 7.06; N, 10.90.
Found: C, 64.78; H, 7.20; N, 10.78. HR-MS m/z: 408.1898 (Calcd for
C21H27N3O4Na: 408.1899).
of two series of inverse substrates. The utility of chum
salmon trypsin can also be proposed as a catalyst for the syn-
thesis of peptide by the use of p-guanidinophenyl esters. This
method, which is operative at low temperature, is advanta-
Kinetic Parameters for Atlantic Cod Trypsin-Catalyzed Hydrolysis
geous since the spontaneous hydrolysis of the acyl donor is Active titration revealed that the concentration of Atlantic cod trypsin was
retarded as previously described.18,23) It must be emphasized
that a longer incubation period did not decrease the coupling
yields in the present method. This result suggested that sec-
ondary hydrolysis of the resulting products by enzyme is
negligible.
55% using p-methylumbelliferyl pꢂ-guanidinobenzoate according to the lit-
erature.38) The kinetic parameters, Ks, k2 and k3 for Atlantic cod trypsin-cat-
alyzed hydrolysis, were determined by means of the thionine displacement
method using a stopped-flow technique.15,34) The reaction was carried out in
0.05 M Tris–HCl buffer, pH 8.0, containing 0.02 M CaCl2 at 25 °C. In these
experiments, the concentrations were: enzyme, 3.31ꢄ10ꢁ6—7.83ꢄ10ꢁ6 M;
substrate, 2.40ꢄ10ꢁ5—1.01ꢄ10ꢁ3 M; thionine; 2.50ꢄ10ꢁ5 M, respectively.
Enzymatic Peptide Coupling Reaction The peptide coupling reaction
was carried out at 0 °C in 50% DMSO solution, which was mixed with
50 mM solution of 3,3-dimethylglutaric acid (G), tris(hydroxymethyl)amino-
methane (T), and 2-amino-2-methyl-1,3-propanediol (A) (GTA buffer) (pH
8.5, containing 20 mM CaCl2). The concentrations of acyl donors (1—16),
acyl acceptors (a—c), and enzymes were 1 mM, 20 mM, and 5 mM, respec-
tively. The progress of the peptide coupling reaction was monitored by
HPLC under the following conditions: isocratic elution at 1 ml/min, 0.1%
trifluoroacetic acid/acetonitrile. An aliquot of the reaction mixture was in-
jected, and the eluate was monitored at 310 nm (chromophore due to p-ni-
troanilide moiety) and 240 nm (chromophore due to b-naphthylamido moi-
ety). Peak identification was made by correlating the retention time with that
of an authentic sample that was chemically synthesized.39—41) The observed
peak areas were used to estimate sample concentration. In Fig. 3, yield of
the same product was same reaction time in each reaction temperature.
Experimental
HPLC analysis was performed by using a reversed column (Shim-pack,
CLC-ODS (M), 4.6ꢄ250 mm) on a Shimadzu LC-10AD pump system
equipped with a Shimadzu SPD-10A UV–VIS spectrophotometric detector.
1H-NMR spectra were recorded on a JEOL ECA-500 spectrometer. Kinetic
parameters were determined with a Union Giken RA-410 stopped-flow spec-
trometer. All inverse substrates, N-(tert-butyloxycarbonyl)amino acid p-
amidino-(N-Boc-AA1-OAm) and N-(tert-butyloxycarbonyl)amino acid p-
guanidinophenyl ester (N-Boc-AA1-OGu), were prepared according to our
previous papers.16—18) L-Alanine-p-nitroanilide (L-Ala-pNA) was purchased
from Peptide Institute, Inc. D-Alanine-p-nitroanilide (D-Ala-pNA) was pre-
pared following the reported procedure.35—37) b-Alanine-b-naphthylamide
hydrobromide (b-Ala-b-NA·HBr) was purchased from Bachem. Atlantic
cod trypsin [EC 3.4.21.4] (lyophilized) (Lot 24H7150) was purchased from
Sigma Chemical Co. HPLC grade DMSO from Kanto Chemical Co., Inc.
was used. 3,3-Dimethylglutaric acid and 2-amino-2-methyl-1,3-propanediol,
and tris(hydroxymethyl)aminomethane were obtained from Tokyo Chemical
Industry Co., Ltd. and ICN Biomedicals, Inc., respectively. p-Methylumbel-
liferyl pꢂ-guanidinobenzoate was purchased from Merk and Co., Inc.
Nb-(tert-Butyloxycarbonyl)-b-alanyl-b-alanine b-Naphthylamide (8c)
A solution of Nb-Boc-Ala-OH (189 mg, 1 mmol), b-alanine-b-naphthyl-
amide hydrochloride (250 mg, 1 mmol), N,N-diisopropylethylamine (129
mg, 1 mmol) and 1-hydroxybenzotriazole (135 mg, 1 mmol) in N,N-di-
methylformamide (DMF) (1 ml) was treated with dicyclohexylcarbodiimide
(DCC) (226 mg, 1.1 mmol) at 0 °C. The reaction mixture was stirred for 1 h
at the same temperature, then warmed to room temperature, and stirring was
continued for 20 h. The resulting precipitate of DCUrea was filtered off, and
the filtrate was evaporated to dryness in vacuo. The residue was diluted with
benzene–AcOEt (1 : 5) and purified on a silica gel column. The pure com-
pound 8c was obtained by recrystallization from AcOEt (77.3%). Colorless
Acknowledgments This work was supported in part by a Grant-in-Aid
for High Technology Research Programs and Strategic Projects to Support
the Formation of Research Bases at Private Universities from the Ministry of
Education, Culture, Sports, Science and Technology of Japan, and by a grant
from the Japan Private School Promotion Foundation.
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