Synthesis of a precursor of bioactive pentapeptide OGP-(10-14) and the fragment of enkephalin catalyzed by MCM-22 immobilized or free proteases in organic solvents

Article abstract of DOI:10.1055/s-2002-25762

Full enzymatic synthesis of a precursor of osteogenic growth peptide fragment (10-14), Z-Tyr-Gly-Phe-Gly-Gly-OEt, was accomplished using immobilized proteases on molecular sieve MCM-22 by adsorption as catalyst via 3+2 synthetic route for the first rime. The reuse time of immobilized enzyme was dependent on concrete reaction system. Compared with free enzyme, the reaction rate catalyzed by immobilized enzyme was remarkably enhanced and its synthetic yield was also increased in most cases. The effects of water content and the added amount of Et₃N on synthesis of fragments of bioactive peptides including OGP-(10-14) and enkephalin by immobilized enzymes were different from free enzyme.

Full text of DOI:10.1055/s-2002-25762

726
PAPER
Synthesis of a Precursor of Bioactive Pentapeptide OGP-(10–14) and the
Fragment of Enkephalin Catalyzed by MCM-22 Immobilized or Free
Proteases in Organic Solvents
Synthesis of
a
i
Precur
s
or of Bioact
g
ive Pentapeptide
O
L
G
P-(10–14) iu,a Yun-hua Ye,*a Gui-ling Tian,a Kin-Sing Lee,b Man-Sau Wong,b Wai-hung Lob
a
The Key Laboratory of Bioorganic Chemistry and Molecular Engineering, Ministry of Education, Department of Chemistry, Peking
University, Beijing, 100871, PRC
b
Open Laboratory of Chirotechnology and Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic
University, Kowloon, Hong Kong, PRC
Fax +86(10)62751708; E-mail: yhye@pku.edu.cn
Received 16 January 2002; revised 5 March 2002
aware, few papers deal with peptide synthesis by molecu-
lar sieve immobilized enzyme in organic solvents. Our
group was the first to report on the enzymatic peptide syn-
thesis of a tripeptide fragment of enkephalin and aspar-
Abstract: Full enzymatic synthesis of a precursor of osteogenic
growth peptide fragment (10–14), Z-Tyr-Gly-Phe-Gly-Gly-OEt,
was accomplished using immobilized proteases on molecular sieve
MCM-22 by adsorption as catalyst via 3+2 synthetic route for the
first time. The reuse time of immobilized enzyme was dependent on tame precursor in organic solvents with microporous Y
concrete reaction system. Compared with free enzyme, the reaction
zeolites and mesoporous dealuminized DAYzeolites as
immobilization matrixes.8
rate catalyzed by immobilized enzyme was remarkably enhanced
and its synthetic yield was also increased in most cases. The effects
Herein, we describe the synthesis of a precursor of bioac-
tive pentapeptide OGP-(10–14), Z-Tyr-Gly-Phe-Gly-Gly-
OEt, and the fragment of enkephalin using microporous
molecular sieve MCM-22 immobilized proteases by ad-
sorption or free proteases as catalyst in organic solvents.
The osteogenic growth peptide (OGP) is a naturally oc-
curring 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
H4.16,17 Endogenous OGP is a proteolytic cleavage prod-
uct of PreOGP translated from H4 mRNA via alternative
translational initiation at a downstream initiation codon.18
OGP increases bone formation and density when admin-
istered to rats.16 OGP regulates proliferation, alkaline
phosphatase activity, matrix mineralization and hemato-
poiesis.16,19–22 The 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 os-
teogenesis and hematopoiesis. It is the minimal OGP-de-
rived sequence that retains the peptide’s full proliferative
activity.16 In this paper, full enzymatic synthesis of OGP-
(10–14) precursor by MCM-22 immobilized -chymo-
trypsin and papain via 3+2 (Scheme 1) synthetic route in
organic solvents was successfully achieved for the first
time. Synthesis of fragments of bioactive peptides includ-
ing OGP-(10–14) and enkephalin by immobilized enzyme
was compared with free enzyme in regard to water con-
tent, the added amount of Et3N, reaction yield and rate etc.
The reuse time of immobilized enzyme was also investi-
gated.
of water content and the added amount of Et3N on synthesis of frag-
ments of bioactive peptides including OGP-(10–14) and enkephalin
by immobilized enzymes were different from free enzyme.
Key words: immobilized enzyme, MCM-22, OGP-(10–14), enzy-
matic peptide synthesis, organic solvents
Peptide synthesis catalyzed by protease has attracted
much attention in recent years.1 The enzymatic method
presents some noticeable advantages over the chemical
coupling, such as minimal racemization, minimal side
chain protection, high stereospecificity etc. Enzymatic
peptide synthesis in organic media offers special merits.
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
soluble in organic solvents.2,3 However, peptide synthesis
by free enzyme exhibits some drawbacks including the
cost of enzyme, the tendency of the enzyme to denature
and aggregate. In addition to preventing autolysis, immo-
bilized enzymes have more stability and can be reused
more times than free enzymes.4–8 The adsorptive binding
method is a mild coupling and common method for immo-
bilization of enzyme on various supports such as ion-ex-
change polymers, Celite, alumina, glass powder, etc.
Molecular sieves are better inorganic carriers for immobi-
lization of enzyme by adsorption because of its resistance
of biodegradation, high surface areas, hydrophobic or hy-
drophilic behavior, electrostatic interactions, etc.8–10
Some papers reported that molecular sieves were used for
immobilization of enzymes and the catalytic activity of
immobilized enzyme was measured,11–15, but as we are
A series of fragments of OGP-(10–14) were synthesized
by the catalysis of adsorptively immobilized -chymo-
trypsin on MCM-22 in cyclohexane (Table). The results
indicated that the synthetic yield of products by immobi-
lized -chymotrypsin on MCM-22 was higher than those
of free -chymotrypsin in most cases. This could be ex-
Synthesis 2002, No. 6, 29 04 2002. Article Identifier:
1437-210X,E;2002,0,06,0726,0732,ftx,en;F00602SS.pdf.
© Georg Thieme Verlag Stuttgart · New York
ISSN 0039-7881

PAPER
Synthesis of a Precursor of Bioactive Pentapeptide OGP-(10–14)
727
water that made the enzyme inactive; however, the corre-
sponding product was obtained using immobilized en-
zyme as catalyst in 78% yield (Figure 1).
Scheme 1 3+2 Synthetic route to the precursor of bioactive penta-
peptide OGP-(10–14)
plained by the fact that -chymotrypsin molecule was ho-
mogeneously dispersed on the surface of MCM-22, which
increased the contact efficiency between enzymes and
substrates. So the reaction efficiency by immobilized en-
zyme was enhanced and the yield was increased compara-
ble to free -chymotrypsin.2 As for Z-Tyr-Gly-OMe, the
synthetic yield by immobilized enzyme was lower than
that by free enzyme probably because immobilized en-
zymes were easily gathered together and adsorbed on the
bottom of reaction flask under reaction conditions, which
prevented immobilized enzymes from efficiently contact-
Figure 1 The effect of water content on the yield of Z-Phe-Gly-Gly-
OEt catalyzed by free and MCM-22 immobilized -chemotrypsin
Molecular sieves have a strong ability for adsorbing wa-
ter, so the water from the immobilized enzyme could not
be completely removed during the lyophilization for prep-
aration of immobilized enzyme. Therefore, the immobi-
lized enzyme itself still retains a small amount of water
ing with substrates and decreased the yield. Moreover, the which serves as the essential water for the enzyme immo-
effect of water content on the synthetic yield of Z-Phe- bilized on MCM-22 to maintain its catalytic activity in or-
ganic solvents without addition of water.2 The optimal
water content (0.15%) for the reaction by immobilized en-
zyme was lower compared to free enzyme. This may also
result from a small amount of water retained in immobi-
lized enzyme. The synthetic yield by immobilized enzyme
Gly-Gly-OEt by MCM-22 immobilized -chymotrypsin
in cyclohexane was different from that of free -chymot-
rypsin to some extent. When no water was added to the re-
action system, the expected product was not obtained
using free enzyme as catalyst because of lack of essential
Table Yields of OGP-(10–14) Precursor and its Fragments Synthesized by Free Proteases or Immobilized Proteases in Cyclohexane
No
1
Carboxyl Component
Z-Tyr-OEt
Amino Component Product
Enzyme
-CTa
Yield (%)
Gly-OEt
Z-Tyr-Gly-OEt
63
72
75
80
75
82
59
51
62
63
51
71
2
Z-Tyr-OEt
Gly-OEt
Z-Tyr-Gly-OEt
Imm- -CTb
-CT
3
Boc-Phe-OCH2CF3
Boc-Phe-OCH2CF3
Z-Phe-OCH2CF3
Z-Phe-OCH2CF3
Z-Tyr-OEt
Gly-Gly-OEt
Gly-Gly-OEt
Gly-Gly-OEt
Gly-Gly-OEt
Gly-OMe
Boc-Phe-Gly-Gly-OEt
Boc-Phe-Gly-Gly-OEt
Z-Phe-Gly-Gly-OEt
Z-Phe-Gly-Gly-OEt
Z-Tyr-Gly-OMe
4
Imm- -CT
-CT
5
6
Imm- -CT
-CT
7
8
Z-Tyr-OEt
Gly-OMe
Z-Tyr-Gly-OMe
Imm- -CT
Papc
9
Z-Tyr-Gly-OMe
Z-Tyr-Gly-OMe
Z-Tyr-Gly-Phe-OMe
Z-Tyr-Gly-Phe-OMe
Phe-OMe
Z-Tyr-Gly-Phe-OMe
Z-Tyr-Gly-Phe-OMe
Z-Tyr-Gly-Phe-Gly-Gly-OEt
Z-Tyr-Gly-Phe-Gly-Gly-OEt
10
11
Phe-OMe
Imm-Papd
-CT
Gly-Gly-OEt
Gly-Gly-OEt
12
Imm- -CT
a
-CT: -chymotrypsin.
b Imm- -CT: MCM-22 immobilized -chymotrypsin.
c Pap: papain.
d Imm-papain: MCM-22 immobilized papain.
Synthesis 2002, No. 6, 726–732 ISSN 0039-7881 © Thieme Stuttgart · New York

728
P. Liu et al.
PAPER
was obviously decreased as well as that by free enzyme method. Although the precursor of OGP-(10–14) was suc-
when the water content in the reaction system was beyond cessfully obtained by -chymotrypsin and thermolysin via
the optimal water content. In addition, the effect of the 2+3 synthetic route (Scheme 2), we failed to synthesize
amount of Et3N added to the reaction system for the syn- the precursor by immobilized enzyme via 2+3 synthetic
thesis of Z-Phe-Gly-Gly-OEt by MCM-22 immobilized - route because the amount of thermolysin immobilized on
chymotrypsin was also investigated (Figure 2).
MCM-22 was poor.
Scheme 2 2+3 Synthetic route to the precursor of bioactive penta-
peptide OGP-(10–14)
Synthesis of a protected tripeptide fragment of enkepha-
lin, Z-Tyr-Gly-Gly-OEt, by MCM-22 immobilized -chy-
motrypsin was studied. In contrast to free enzyme, water
content of dichloromethane had lower effect on the yield
of Z-Tyr-Gly-Gly-OEt synthesized by immobilized en-
zyme (Figure 3).
Figure 2 The effect of Et3N on the yield of Z-Phe-Gly-Gly-OEt ca-
talyzed by free or MCM-22 immobilized -chymotrypsin
The observed pH value of reaction solution would be
much higher than 7 even if the equivalent amount of Et3N
was added to the reaction system to neutralize the HCl of
amino component. This was caused by the poor solubility
of amino component (Gly-Gly-OEt HCl) in cyclohexane.
Therefore, it was difficult to accurately investigate the in-
fluence of pH value on the synthesis of Z-Phe-Gly-Gly-
OEt. The result indicated that the synthetic yield by im-
mobilized enzyme with a 1:1 molar ratio of Et3N to amino
component was clearly lower than that with a 1.25:1–2:1
molar ratio of Et3N to amino component. However, the
amount of Et3N had little effect on the synthesis of Z-Phe-
Gly-Gly-OEt by free enzyme when the amount of Et3N
was 1–2 times that of the amino component. This may
contribute to the fact that the hydrochloride of amino
component could not be completely neutralized by Et3N Figure 3 The effect of water content on the yield of Z-Tyr-Gly-Gly-
OEt catalyzed by free or MCM-22 immobilized -chymotrypsin
when equimolar amounts of Et3N and amino component
were added, because the buffer solution with weak acidity
(pH 6.0) for the preparation of immobilized enzyme neu-
tralized a part of Et3N.
The expected product was obtained in about 70% yield us-
ing immobilized enzyme as catalyst whereas the corre-
sponding product was not obtained using free enzyme as
catalyst in dichloromethane without addition of water.
The reason was same as mentioned above. The yield of
product by immobilized enzyme increased slightly (70–
72%) when water content varied from 0% to 0.75%. Nev-
ertheless, there was an obvious change for the yield of
product by free enzyme. The yield of product increased
from 0 to 71% with increasing water content from 0 to
0.15% and distinctly decreased (71–54%) when water
content continuously increased from 0.15% to 0.75%.
Full enzymatic synthesis of the precursor of bioactive
pentapeptide OGP-(10–14) was achieved by immobilized
-chymotrypsin and papain via 3+2 synthetic route
(Scheme 1) in cyclohexane for the first time. Z-Tyr-Gly-
OMe synthesized by MCM-22 immobilized -chymot-
rypsin was coupled with Phe-OMe by MCM-22 immobi-
lized papain in cyclohexane to give Z-Tyr-Gly-Phe-OMe.
Then Z-Tyr-Gly-Phe-OMe was condensed with Gly-Gly-
OEt to afford the precursor of OGP-(10–14), Z-Tyr-Gly-
Phe-Gly-Gly-OEt (Table), by the catalysis of MCM-22
immobilized -chymotrypsin via 3+2 synthetic route in
cycohexane with 71% yield. The yield of the precursor by
immobilized enzyme was markedly higher than the free
enzyme. We also attempted to synthesize the precursor of
OGP-(10–14) by thermodynamically controlled peptide
synthesis via 2+3 synthetic route using full enzymatic
The yield of product by immobilized enzyme decreased
only by 5% even if water content increased from 0.75 to
2%. It showed that water content (0–2%) had low effect
on the yield of Z-Tyr-Gly-Gly-OEt by immobilized en-
zyme. However, addition of a little water in reaction sys-
tem obviously enhanced the reaction rate of synthesis of
Synthesis 2002, No. 6, 726–732 ISSN 0039-7881 © Thieme Stuttgart · New York

PAPER
Synthesis of a Precursor of Bioactive Pentapeptide OGP-(10–14)
729
Z-Tyr-Gly-Gly-OEt by immobilized enzyme in dichlo- washing with acetone after the first use. This resulted in
romethane.2
increasing the amount of amino component for the next
use of immobilized enzyme to catalyze the reaction.
The expected peptide quickly precipitated from reaction
solution when a little water was added whereas it took
much more time to observe the precipitate of product
without the addition of water. Additionally, the reaction
rate by immobilized enzyme was much faster than that by
free enzyme. The reaction by immobilized enzyme nearly
reached equilibrium within about one day while it took
2–3 days for the reaction catalyzed by free enzyme to
reach equilibrium because of the increasing contact effi-
ciency between immobilized enzymes and substrates
(Figure 4).
Figure 5 Reusabilitiy of MCM-22 immobilized -chymotrypsin.
Reaction time: 1 day for the first and second use and 2 days for the
others
In conclusion, full enzymatic synthesis of a precursor of
osteogenic growth peptide (10–14), Z-Tyr-Gly-Phe-Gly-
Gly-OEt, was synthesized by adsorptively immobilized
proteases on molecular sieve MCM-22 via 3+2 synthetic
route for the first time. Immobilized enzyme could be
used for several times and its reuse time was relevant to
concrete reaction system. In comparison with free en-
zyme, the reaction rate catalyzed by immobilized enzyme
was remarkably enhanced and its synthetic yield was also
increased in most cases. The effects of water content and
the added amount of Et3N on synthesis of fragments of
bioactive peptides including OGP-(10–14) and enkepha-
lin by immobilized enzymes were different from free en-
zyme. Without the addition of water to reaction system,
immobilized enzyme was still active for catalyzing pep-
tide synthesis while no product was obtained using free
enzyme as catalyst.
Figure 4 The effect of reaction time on the yield of Z-Tyr-Gly-Gly-
OEt catalyzed by free or MCM-22 immobilized -chymotrypsin
Reuse time of immobilized enzyme plays an important
role in its practical application. The more reuse times of
immobilized enzyme are, the less the amount of immobi-
lized enzyme is consumed, and the lower the cost of en-
zyme is. The reuse times of MCM-22 adsorptively
immobilized -chymotrypsin were evaluated by the syn-
thesis of Z-Tyr-Gly-Gly-OEt and Z-Phe-Gly-Gly-OEt in
organic solvents (Figure 5). As for synthesis of Z-Tyr-
Gly-Gly-OEt in dichloromethane, the synthetic yield for
the first use of immobilized enzyme was comparable to
that of the first reuse. Then the yield of product was grad-
ually declined with increasing of the reuse times of immo-
bilized enzyme. The immobilized enzyme could be
-Chymotrypsin (EC 3.4.21.1, from bovine pancreas) and thermol-
ysin (EC 3.4.24.4 from bacillus thermoprotedyticus rokko) were
purchased from Sigma Chemical Company. Papain (EC 3.4.22.2,
2000 units/mg) was prepared by Sino-America Biotech. All amino
acids including N-protected amino acids are L-configuration and
commercial reagents. CH2Cl2 was dried (MgSO4) and redistilled.
Cyclohexane was redistilled from Na/benzophenone. tert-Amyl al-
repeatedly used for five times. As for synthesis of Z-Phe- cohol was dried over K2CO3 and redistilled. Microporous molecular
sieve MCM-22 (pore volume: 0.16 cm3/g; Si/Al = 14; pore size:
Gly-Gly-OEt in cyclohexane, there was obviously no dif-
4.0 5.9Å, 4.0 5.4 Å; specific surface: 400 m2/g) was provided
ference among the synthetic yields for the first use to the
by Research Institute of Petroleum Processing, SINOPEC.
HCl Gly-OMe, HCl GlyOEt, HCl GlyGlyOEt were synthesized by
SOCl2 method. Z-Tyr-OEt, Z-Phe-OCH2CF3, Boc-Phe-OCH2CF3
second reuse of immobilized enzyme. The synthetic yield
was markedly dropped to 23% for the third reuse of im-
mobilized enzyme. These results indicate that the reuse
times of immobilized enzyme were dependent on concrete
reaction probably because some factors influencing the
activity of immobilized enzyme including substrates, or-
ganic solvents and pH value etc., were discrepant in dif-
ferent reactions. Additionally, the yield of product for the
first reuse of immobilized enzyme was slightly higher
than that for the first use of immobilized enzyme possibly
because the immobilized enzyme still retained some ami-
no component that could not be completely eliminated by
were synthesized by DCC/HOBt coupling method with addition of
10% (molar ratio) 4-dimethylaminopyridine (DMAP) as catalyst.23
Petroleum ether used had bp 30–60 °C.
The melting points were determined on aYanaco micromelting
point apparatus and are uncorrected. Mass spectra were recorded on
1
VG-ZAB-HS and Bruker APEXTMII spectrometers. H NMR was
recorded on a Bruker ARX-200 spectrometer. Elemental analysis
was performed on a Elementar Vario EL appartus (Germany). Op-
tical rotations were measured using a Perkin-Elmer 341LC polarim-
eter.
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P. Liu et al.
PAPER
Standard abbreviations for amino acid derivatives and peptides are
according to the suggestions of the IUPAC-IUB Commission on
Biochemical Nomenclature (1984).24 Other abbreviations: Z (ben-
zyloxycarbonyl), Boc (tert-butyloxycarbonyl), PBS (phosphorus
buffer solution).
High Resolution-SIMS: m/z calcd for C29H31O7N3 [M + H]+:
534.2234; found [M + H]+ 534.2238.
1H NMR (200 MHz, CDCl3): = 2.90–3.10 (m, 4 H, 2 CH2) 3.66 (s,
3 H, CH3), 3.84–4.00 (m, 2 H, CH2), 4.31 (m, 1 H, CH), 4.78 (m, 1
H, CH), 5.03 (d, 2 H, J = 2.8 Hz, CH2O), 5.60 (d, 1 H, J = 6.8 Hz,
CONH), 6.66 (d, 2 H, J = 8.2 Hz, ArH), 6.91 (d, 2 H, J = 8.6 Hz,
ArH), 6.75–7.29 (m, 12 H, 10 Harom + 2 CONH).
Synthesis of Z-Tyr-Gly-OEt, Z-Tyr-Gly-OMe, Z-Phe-Gly-Gly-
OEt, Boc-Phe-Gly-Gly-OEt Catalyzed by -Chymotrypsin in
Cyclohexane; General Procedure
HCl Phe-Gly-Gly-OEt
To a suspension of the carboxyl component Z-Tyr-OEt (or Z-Phe-
OCH2CF3, Boc-Phe-OCH2CF3) (0.2 mmol) and the amino compo-
nent HCl Gly-OMe (or HCl Gly-OEt, HCl Gly-Gly-OEt) (0.2
mmol) in cyclohexane (3 mL) were added Et3N (35 L, 0.25 mmol),
H2O (7.5 L, 0.25%, v/v) and -chymotrypsin (3 mg). The reaction
mixture was stirred at 17–20 °C for about 3 d and monitored by
TLC. At the end of the reaction, the solvent was evaporated under
vacuum and the residue was dissolved in EtOAc (30 mL). Then it
was successively washed with aq 1 M HCl (5% citric acid was used
for Boc-protected product) (3 7 mL), H2O (3 7 mL), aq 5%
Na2CO3 (3 7 mL), H2O (3 7 mL) and brine. The organic layer
was dried (MgSO4) and filtered. The solvent was removed and the
crude product was purified by silica gel chromatography (CHCl3–
MeOH, 35:1 60:1) to afford the pure product. As for the amino
component HCl Gly-OEt, the product (Z-Tyr-Gly-OEt) was puri-
fied as follows: at the end of the reaction, the solvent was evaporat-
ed under vacuum. The residue was diluted with CH2Cl2 (4 mL) and
filtered. The precipitate was successively washed by 1 M HCl and
distilled H2O. The crude product was dried at r.t. and recrystallized
from acetone to give the pure product as a white solid.
To a stirred solution of Boc-PheGlyGlyOEt (203.5 mg, 0.5 mmol)
in EtOAc (1.5 mL) was added a 5 M solution of HCl in EtOAc (4
mL) under ice cooling with stirring. After stirring for 2 h under ice
cooling, the solution was stirred overnight at r.t. The resulting pre-
cipitate was filtered and crystallized from MeOH–EtOAc to give
the pure product as a white solid; yield: 149 mg (87%); mp 125–
127 °C.
Z-Tyr-Gly-OH
To a stirred solution of Z-Tyr-Gly-OEt (200 mg, 0.5 mmol) in DMF
(1.5 mL) and MeOH (2 mL) was added dropwise an aq 1 M LiOH
soln (1.5 mL) over ca. 15 min at 30 °C. The solution was continu-
ously stirred for ca. 2–3 h. Then aq 1 M HCl was added to adjust
pH to 7–8. After removal of a part of solvent under vacuum, aq 2 M
HCl was added to bring the pH to 2. The solution was concentrated,
the residue was diluted with brine (10 mL) and extracted with
EtOAc (3 20 mL). After successive washings with aq 1 M HCl,
H2O, aq 5% NaCO3, H2O and brine, the organic layer was dried
(MgSO4) and filtered. The solvent was removed and the crude prod-
uct was recrystallized from CHCl3–petroleum ether to yield the pure
product as a white solid; yield: 173 mg (93%); mp 99–101 °C;
[ ]D20 –13.9 (c = 1, MeOH).
Boc-Phe-Gly-Gly-OEt
Yield: 61 mg (75%); mp 98.5–100 °C; [ ]D20 +8.9 (c = 1, MeOH).
FAB-MS: m/z = 373 (M + H)+.
FAB-MS: m/z = 408 (M + H)+.
Anal. Calcd for C20H29N3O6: C, 58.97; H, 7.12; N, 10.32. Found C,
59.06; H, 7.25; N, 10.27.
Synthesis of Z-Tyr-Gly-Phe-Gly-Gly-OEt by Kinetic Control
via 3+2 Synthetic Route
To a mixture of Z-Tyr-Gly-Phe-OMe (53.3 mg, 0.1 mmol) and
HCl Gly-Gly-OEt (54.2 mg, 0.3 mmol) in cyclohexane (3 mL) were
added Et3N (52 L, 0.37 mmol), H2O (7.5 L, 0.25%, v/v) and -
chymotrypsin (3 mg). The reaction mixture was stirred at 17–20 °C
for about 3 d. Then the solvent was evaporated under vacuum and
the residue was dissolved in EtOAc (70 mL). After successively
washing with aq 1 M HCl, H2O, aq 5% Na2CO3, H2O and brine, the
organic layer was dried (MgSO4) and filtered. The solvent was re-
moved and the crude product was purified by silica gel chromatog-
raphy (gradient elution: CHCl3–MeOH, 30:1 15:1) and
recrystallized from acetone–petroleum ether to give the pure prod-
Z-Phe-Gly-Gly-OEt
Yield: 66 mg (75%); mp 92–93.5 °C (Lit.25 mp 90–92 °C, Lit.26 mp
88–90 °C); [ ]D20 +4.6 (c = 2, CHCl3–MeOH, 4:1) {Lit.26
[ ]D20 +3.0 (c = 2, CHCl3–MeOH, 4:1)}.
FAB-MS: m/z = 442 (M + H)+
Z-Tyr-Gly-OEt
Yield: 50 mg (63%); mp 168–170 °C; [ ]D20 –24.5 (c = 1, DMF);
[Lit. 27 mp 168–170 °C; [ ]D20 –23.5 (c = 5.0, DMF)].
20
uct as a white solid; yield: 33.8 mg (51%); mp 161–163 °C; [ ]D
–23.6 (c = 0.5, DMF).
FAB-MS: m/z = 662 (M + H)+.
Z-Tyr-Gly-OMe
Yield: 46 mg (59%); mp 138–140 °C; [ ]D20 –24.5 (c = 1, DMF).
[Lit.28 mp 141–143 °C; [ ]D20 –22.2 (c = 1.3, DMF)].
Anal. Calcd for C34H39N5O9: C, 61.72; H, 5.90; N, 10.59. Found C,
61.40; H, 5.58; N 10.37.
Synthesis of Z-Tyr-Gly-Gly-OEt Catalyzed by -Chymotrypsin
in CH2Cl2
This compound was prepared according to Ref.2
Synthesis of Z-Tyr-Gly-Phe-Gly-Gly-OEt by Thermodynamic
Control via 2+3 Synthetic Route
Synthesis of Z-Tyr-Gly-Phe-OMe Catalyzed by Papain in Cy-
clohexane
To a suspension of Z-Tyr-Gly-OH (37.2 mg, 0.1 mmol) and
HCl Phe-Gly-Gly-OEt (103.0 mg, 0.3 mmol) in tert-amyl alcohol
(2 mL) were added Et3N (70 L, 0.5 mmol) and 10 mM CaAc2 buff-
er (0.12 mL, 6%, v/v). The substrates dissolved completely on stir-
ring. Then thermolysin (5 mg) was added and the reaction mixture
was stirred at 40 °C for about 3 d. The workup procedure was the
same as described for the 3+2 synthetic route. The resulting crude
product was recrystallized from acetone–petroleum ether to give the
pure product as a white solid; yield: 46 mg (70%); mp 159–161 °C;
[ ]D20 –23.8 (c = 0.5, DMF).
To a mixture of Z-Tyr-Gly-OMe (77.2 mg, 0.2 mmol) and HCl Phe-
OMe (64.8 mg, 0.3 mmol) in cyclohexane (3 mL) were added Et3N
(50 L, 3.6 mmol), H2O (7.5 L, 0.25%, v/v), HOCH2CH2SH (50
L) and papain (3.5 mg). The reaction mixture was stirred at 37 °C
for about 3 d and monitored by TLC. The workup procedure was the
same as described under the general procedure for the purification
of Z-Tyr-Gly-OMe. The resulting crude product was purified by sil-
ica gel chromatograph (EtOAc–petroleum ether, 1.5:1; CHCl3–
MeOH, 30:1) to give the pure product as a white solid; yield: 66 mg
(62%); mp 79–81 °C; [ ]D20 +20.5 (c = 1, CHCl3).
FAB-MS: m/z = 662 (M + H)+.
Synthesis 2002, No. 6, 726–732 ISSN 0039-7881 © Thieme Stuttgart · New York

PAPER
Synthesis of a Precursor of Bioactive Pentapeptide OGP-(10–14)
731
Preparation of Immobilized -Chymotrypsin and Papain on
MCM-22 by Adsorption.
Synthesis of Z-Tyr-Gly-Phe-OMe Catalyzed by MCM-22
Immobilized Papain in Cyclohexane
Immobilized -Chymotrypsin on MCM-22
To a mixture of Z-Tyr-Gly-OMe (77.2 mg, 0.2 mmol) and HCl Phe-
OMe (64.7 mg, 0.3 mmol) in cyclohexane (3 mL) were added Et3N
(50 L, 3.6 mmol), H2O (7.5 L, 0.25%, v/v), HOCH2CH2SH (50
L) and MCM-22 immobilized papain (100 mg). The mixture was
stirred at 37 °C for about 2 d and detected by TLC. Then a larger
amount of EtOAc was added to make the product dissolve com-
pletely. The reaction solution was filtered and washed with several
portions of EtOAc. The immobilized enzyme was dried as de-
scribed above and stored at –2 °C for reuse. The workup and purifi-
cation procedure were the same as for the corresponding product
from free enzyme; yield: 67 mg (63%). Its physical data were iden-
tical to that of the corresponding product from free enzyme.
To a solution of -chymotrypsin (21.8 mg) in PBS (10 mL, pH 6.0,
0.05M) was added MCM-22 (600 mg) under ice-water cooling and
the mixture was stirred for 2 h. The mixture was filtered and washed
with PBS (5 mL). The resulting immobilized enzyme was suspend-
ed in PBS (4 mL) and lyophilized for 8 h to give the expected im-
mobilized enzyme that was stored at –2 °C.
Immobilized Papain on MCM-22
To a stirred solution of papain (18 mg) in PBS (10 mL, pH 6.0, 0.05
M) was added MCM-22 (500 mg) under ice-water cooling and the
mixture was stirred for 2 h. The workup procedure was the same as
above.
Synthesis of Protected Pentapeptide OGP-(10–14) (Z-Tyr-Gly-
Phe-Gly-Gly-OEt) by Kinetic Control via 3+2 Synthetic Route
To a mixture of Z-Tyr-Gly-Phe-OMe (53.3 mg, 0.1 mmol) and
HCl Gly-Gly-OEt (54.2 mg, 0.3 mmol) in cyclohexane (3 mL) were
added Et3N (52 L, 0.37 mmol) and H2O (7.5 L, 0.25%, v/v) and
MCM-22 immobilized -chymotrypsin (100 mg). The reaction
mixture was stirred at 17–20 °C for about 2 d. Then a larger amount
of MeOH was added to make the product dissolve completely. The
mixture was filtered with several washings of MeOH. The solvent
of the filtrate was evaporated under vaccum and the residue was dis-
solved in EtOAc (70 mL). The workup and purification procedure
were the same as for the corresponding product from free enzyme
via the 3+2 synthetic route; yield: 47 mg (71%). The physical data
were identical with that of the corresponding product by free en-
zyme.
Synthesis of Z-Tyr-Gly-Gly-OEt Catalyzed by MCM-22 Immo-
bilized -Chymotrypsin
To a mixture of Z-Tyr-OEt (102.9 mg, 0.3 mmol) and HCl Gly-Gly-
OEt (59.0 mg, 0.3 mmol) in CH2Cl2 (3 mL) was added Et3N (84 L,
0.6 mmol). The mixture was stirred to completely dissolve the sub-
strate. The observed pH value was about 10. Then H2O (7.5 L,
0.25%, v/v) and MCM-22 immobilized -chymotrypsin (100 mg
containing about 2.6 mg of enzyme) were added and the mixture
was continuously stirred for 2 d at 20 °C. The precipitate from the
reaction solution and the immobilized enzyme were filtered and
washed with CH2Cl2 (4 mL). Then the precipitate was thoroughly
dissolved in acetone by several washings. The expected product
was obtained by evaporation of acetone and crystallized from ace-
tone. The immobilized enzyme was stored –2 °C for reuse; yield:
98.7 mg (72%); mp 167–168 °C; [ ]D20 +3.2 (c = 2, AcOH) [Lit.2
mp 164–165 °C; [ ]D20 +2.9 (c = 2, AcOH)].
Acknowledgements
Synthesis of Z-Tyr-Gly-OEt, Z-Tyr-Gly-OMe, Z-Phe-Gly-Gly-
OEt, Boc-Phe-Gly-Gly-OEt Catalyzed by MCM-22 Immobil-
ized -Chymotrypsin in Cyclohexane; General Procedure
To a suspension of Z-Tyr-OEt (or Z-Phe-OCH2CF3, Boc-Phe-
OCH2CF3) (0.2 mmol) and HCl Gly-OMe (or HCl Gly-OEt,
HCl Gly-Gly-OEt) (0.2 mmol) in cyclohexane (3 mL) were added
Et3N (35 L, 0.25 mmol), H2O (7.5 L, 0.25%, V/V) and MCM-22
immobilized -chymotrypsin (100 mg). The mixture was stirred at
17–20 °C for about 2 d and monitored by TLC. Then a larger
amount of EtOAc was added to the solution to make the product dis-
solve completely. The solution was filtered and the residue was
washed several times with EtOAc. The immobilized enzyme was
dried under reduced pressure at r.t. for about 5 min and stored at –2
°C for reuse. The filtrate was evaporated under vacuum and the res-
idue was dissolved in EtOAc (30 mL). The workup procedure was
the same as described for the corresponding product from free en-
zyme. The resulting crude product was purified by silica gel chro-
matography (CHCl3–MeOH, 35:1 60:1) to afford pure products.
As for the amino component HCl Gly-OEt, the product (Z-Tyr-Gly-
OEt) was purified as follows: At the end of reaction, the reaction
mixture was diluted with acetone, filtered and washed with several
portions of acetone to make the product completely dissolve in ac-
etone. The immobilized enzyme was dried as described above and
stored at –2 °C for reuse. The solvent was removed and the residue
was diluted with CH2Cl2 (4 mL) and filtered. The precipitate was
successively washed by aq 1 M HCl (3 7 mL) and distilled H2O
(3 7 mL). The crude product was dried at r.t. and recrystallized
from acetone to yield the pure product as a white solid. The yields
of Boc-Phe-Gly-Gly-OEt, Z-Phe-Gly-Gly-OEt, Z-Tyr-Gly-OEt and
Z-Tyr-Gly-OMe were 65 mg (80%), 72 mg (82%), 58 mg (72%)
and 39 mg (51%), respectively. Their physical data were identical
with those of corresponding products by free enzyme.
The authors thank the Research Institute of Petroleum Processing
for providing MCM-22. The financial support of the Hong Kong
Polytechnic University and the National Natural Science Foundati-
on of China (2982002) for this project is gratefully acknowledged.
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