Full enzymatic synthesis of a precursor of bioactive pentapeptide OGP(10-14) in organic solvents

Article abstract of DOI:10.1016/S0040-4039(02)00278-2

Full enzymatic synthesis of a fragment of osteogenic growth peptide (OGP), a precursor of bioactive pentapeptide OGP(10-14) (Z-TyrGlyPheGlyGlyOEt), was accomplished by papain, α-chymotrypsin, and thermolysin via 2+3 or 3+2 synthetic routes in organic solvents for the first time. The factors influencing the enzymatic synthesis of fragments of OGP(10-14) were systematically studied.

Full text of DOI:10.1016/S0040-4039(02)00278-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 2423–2425
Full enzymatic synthesis of a precursor of bioactive pentapeptide
OGP(10-14) in organic solvents
a
a
b
b
a,
Ping Liu, Gui-ling Tian, Kin-Sing Lee, Man-Sau Wong and Yun-hua Ye *
a
The Key Laboratory of Bioorganic Chemistry and Molecular Engineering, Ministry of Education, Department of Chemistry,
Peking University, Beijing 100871 China
b
Open Laboratory of Chirotechnology and Department of Applied Biology and Chemical Technology, The Hong Kong
Polytechnic University, Kowloon, Hong Kong, China
Received 14 December 2001; revised 30 January 2002; accepted 6 February 2002
Abstract—Full enzymatic synthesis of a fragment of osteogenic growth peptide (OGP), a precursor of bioactive pentapeptide
OGP(10-14) (Z-TyrGlyPheGlyGlyOEt), was accomplished by papain, a-chymotrypsin, and thermolysin via 2+3 or 3+2 synthetic
routes in organic solvents for the first time. The factors influencing the enzymatic synthesis of fragments of OGP(10-14) were
systematically studied. © 2002 Elsevier Science Ltd. All rights reserved.
7
Enzymatic synthesis of peptides has drawn much atten-
tion because of enzyme stereospecificity, mild reaction
conditions, minimum side-chain protection and avoid-
ance of racemization. Enzymatic synthesis of peptides
in organic solvents offers special advantages. The
enzymes are more stable in organic solvents where the
formation of peptide bonds is more favorable than the
hydrolysis of the peptide. Most organic compounds are
tiation codon. OGP increases bone formation and
5
density when administered to rats. OGP regulates not
only proliferation, alkaline phosphatase activity and
matrix mineralization but also hematopoiesis.
OGP C-terminal pentapeptide, H-Tyr-Gly-Phe-Gly-
Gly-OH [OGP(10-14)], shares the OGP-like in vitro
mitogenic effect and in vivo stimulation of osteogenesis
and hematopoiesis. It is the minimal OGP-derived
sequence that retains the peptides full proliferative
5,8–11
The
1
–4
soluble in organic solvents and so on.
Enzymatic
synthesis of peptides has mostly been focused on the
synthesis of dipeptides and tripeptides. To our knowl-
edge, there are only a few papers dealing with enzy-
matic synthesis of pentapeptides or longer peptides. In
our previous study, we reported the enzymatic synthesis
5
activity.
The synthesis of the precursor of bioactive pentapeptide
OGP(10-14), i.e. Z-TyrGlyPheGlyGlyOEt, by catalysis
using protease has not been reported before. We have
now successfully completed the full enzymatic synthesis
of OGP(10-14) precursor via 2+3 (Scheme 1) or 3+2
2
of a Leu-enkephaline precursor. Herein we describe the
enzymatic synthesis of a precursor of bioactive pen-
tapeptide OGP(10-14) in organic solvents.
(
Scheme 2) synthetic routes in organic solvents for the
first time. In this paper, the factors influencing the
synthesis of the fragments of OGP(10-14) were also
studied.
The osteogenic growth peptide (OGP) is a naturally
occurring tetradecapeptide identical to the C-terminal
amino acid sequence 89-102(H-Ala-Leu-Lys-Arg-Gln-
Gly-Arg-Thr-Leu-Tyr-Gly-Phe-Gly-Gly-OH) of histone
5
,6
H4.
Endogenous OGP is a proteolytic cleavage
product of PreOGP translated from H4 mRNA via
alternative translational initiation at a downstream ini-
Keywords: full enzymatic synthesis; OGP(10-14); protease; organic
solvent.
*
Corresponding author. Fax: 86-10-62751708.; e-mail: yhye@
pku.edu.cn
Scheme 1. (2+3) Synthetic route of the precursor of bioactive
pentapeptide OGP(10-14).
0
040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)00278-2

2
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P. Liu et al. / Tetrahedron Letters 43 (2002) 2423–2425
(
GlyGlyOEt·HCl) in cyclohexane. Therefore, it was
difficult to investigate the effect of pH on the synthesis
of Z-PheGlyGlyOEt. The result indicated that the
amount of Et N had little effect on the synthesis of
3
Z-PheGlyGlyOEt when the amount of Et N was 1ꢀ2
3
times that of the amino component. The yield of Z-
PheGlyGlyOEt was a little higher when the amount of
Et N was 1.5 times the amino component concentration
3
(
Fig. 2).
Scheme 2. (3+2) Synthetic route of the precursor of bioactive
pentapeptide OGP(10-14).
In addition, the effect of Z and Boc protecting groups
on the yield of P-PheGlyGlyOEt (P=Z, Boc) was
ignored (Table 1, entries 4 and 7). In comparison with
N-protected amino acid ethyl ester, the coupling yield
of the corresponding trifluoroethyl ester as the carboxyl
component was considerably improved (Table 1, entries
The yields of the fragments of OGP(10-14) synthesized
by a-chymotrypsin depended on their solubility in
organic solvents. For the synthesis of Z-PheGlyGly-
OEt, the yield in cyclohexane was about double that in
DCM. This could be explained by the fact that the
solubility of the product in cyclohexane was so poor
that the product precipitated from the reaction solu-
tion, which shifted the reaction equilibrium toward
peptide formation. However, the solvents had little
effect on the synthetic yield of Z-TyrGlyOEt because
the solubility of the product was poor in both cyclohex-
ane and DCM.
6
and 7).
Full enzymatic synthesis of the precursor of bioactive
pentapeptide OGP (10-14) (Z-TyrGlyPheGlyGlyOEt)
was achieved using a-chymotrypsin, papain and ther-
molysin via 2+3 or 3+2 synthetic routes in organic
solvents for the first time (Table 1, entries 9 and 10).
The yield of the above precursor obtained by thermo-
dynamically controlled peptide synthesis via the 2+3
synthetic route was higher than that obtained by kinet-
ically controlled peptide synthesis via the 3+2 synthetic
route, but the number of the synthetic steps in the 2+3
route was more than that in the 3+2 route. In the 2+3
route, we also tried to synthesize the precursor of
OGP(10-14) using papain as catalyst in several organic
solvents including DCM, ethyl acetate, methanol, tert-
amyl alcohol. The carboxyl component was Z-TyrGly-
OR(R=H, Et, Me), the amino component was
PheGlyGlyOEt, but the expected product was not
obtained.
The water content of the cyclohexane influenced the
yield of Z-PheGlyGlyOEt synthesized using a-chy-
motrypsin. The expected product was not obtained
when no water was added to the reaction system as
water is essential to make the enzyme active. The yield
of the product increased with the increasing water
content from 0 to 0.5%, but the yield decreased when
the water content was above 0.5% due to the increase of
hydrolysis of the product. So the optimal water content
for the synthesis of Z-PheGlyGlyOEt using a-chy-
motrypsin in cyclohexane was around 0.5% (Fig. 1).
In conclusion, the precursor of bioactive pentapeptide
OGP(10-14) (Z-TyrGlyPheGlyGlyOEt) was synthesized
by a full enzymatic method via two different synthetic
routes. Some important factors, such as organic sol-
The effect of the amount of Et N added to the reaction
3
system on the synthesis of Z-PheGlyGlyOEt was also
studied. The observed pH of the reaction solution
would be much higher than 7 when the equivalent
vents, water content, the amount of Et N added and so
3
amount of Et N was added to the reaction system to
on, were systematically optimized. Our study broadens
the range of the application of proteases in the synthe-
sis of peptide and provided some useful information for
enzymatic synthesis of peptides, especially for longer
peptides.
3
neutralize the hydrochloride salt of the amino compo-
nent. This is because the hydrochloride salt of the
amino component could not be completely neutralized
by Et N due to the poor solubility of amino component
3
Figure 2. The effect of the amount of Et N on the yield of
3
Figure 1. The effect of water content on the yield of Z-Phe-
GlyGlyOEt catalyzed by a-chymotrypsin in cyclohexane.
Z-PheGlyGlyOEt catalyzed by a-chymotrypsin in cyclohex-
ane.

P. Liu et al. / Tetrahedron Letters 43 (2002) 2423–2425
2425
Table 1. Yields of N-protected OGP(10-14) and its fragments synthesized by catalysis using protease in organic solvents
Productb
Yield (%) (solventc)
Mp (°C)
[h] (c, solvent)
20
Entry
Carboxyl
Amino
component
D
componenta
a
d
1
2
3
4
5
6
7
8
9
Z-TyrOEt
Z-TyrOEt
Z-PheOCH CF
Z-PheOCH CF
Z-TyrOEt
Boc-PheOEt
Boc-PheOCH CF
Z-TyrGlyOMe
Z-TyrGlyOH
Z-TyrGlyPheOMe
GlyOEt
GlyOEt
GlyGlyOEt
GlyGlyOEt
GlyOMe
GlyGlyOEt
GlyGlyOEt
PheOMe
PheGlyGlyOEt
GlyGlyOEt
Z-TyrGlyOEt
Z-TyrGlyOEt
54 (D)
63 (C)
32 (D)
75 (C)
58 (C)
37 (C)
75 (C)
61 (C)
168–170
169–171
92–93.5
91–93
138–140
98.5–100
98–100
79–81
−23.5 (1, DMF)
−23.3 (1, DMF)
e
d
h
Z-PheGlyGlyOEt
Z-PheGlyGlyOEt
Z-TyrGlyOMe
Boc-PheGlyGlyOEt
Boc-PheGlyGlyOEt
Z-TyrGlyPheOMe
Z-TyrGlyPheGlyGlyOEt
Z-TyrGlyPheGlyGlyOEt
+4.6
+4.4
2
3
e
h
2
3
e
−24.5 (1, DMF)
+9.0 (1, DMF)
+8.9 (1, DMF)
23.1 (1, CHCl3)
−23.8 (0.5, DMF)
−23.8 (0.5, DMF)
e
e
2
3
f
g
70 (T)
51 (C)
159–161
159–161
e
1
0
a
Carboxyl component:amino component=0.2:0.2 (mmol). As for 8, carboxyl component:amino component=0.2:0.3 (mmol). As for 9, 10,
carboxyl component:amino component=0.1:0.3 (mmol).
All products were confirmed by FAB-MS. Three (product 6 (or 7), 8, 9 (or 10)) are new compounds and their structures were confirmed by H
b
1
NMR, High Resolution-SIMS or elemental analysis. The physical data of other products were consistent with literature values.
D: DCM; C: cyclohexane; T: tert-amyl alcohol.
c
d
e
f
The products were synthesized using 3 mg a-chymotrypsin with 0.25% H O(v/v), pH 10 at 20°C.
2
Et N:amino component=1.25:1. The other reaction conditions were the same as in footnote d.
3
The product was synthesized using 3.5 mg papain with 0.25% H O (v/v), 1.7% (v/v) HSCH CH OH at 37°C, Et N:amino component=1.25:1.
2
2
2
3
g
h
The product was synthesized using 5 mg thermolysin with 8% Ca(OAc)2 buffer (v/v), pH 8.5 at 40°C.
c=1, CHCl :MeOH=4:1.
3
Acknowledgements
6. Bab, I.; Gazit, D.; Chorev, M.; Muhlrad, A.; Shteyer, A.;
Greenberg, Z.; Namdar, M.; Kahn, A. EMBO J. 1992,
1
1, 1867–1873.
The authors thank the Hong Kong Polytechnic Univer-
sity for financial support and the National Natural
Science Foundation of China (NO. 29872002).
7
. Bab, I.; Smith, E.; Gavish, H.; Attar-Namdar, M.;
Chorev, M.; Chen, Y. C.; Muhlrad, A.; Birnbaum, M. J.;
Gary, S.; Frenkel, B. J. Biol. Chem. 1999, 274, 14474–
14481.
8
9
. Greenberg, Z.; Chorev, M.; Muhlrad, A.; Shteyer, A.;
Namdar, M.; Mausm, N.; Bab, I. Biochim. Biophys. Acta
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