Enzymatic peptide synthesis in organic solvent with different zeolites as immobilization matrixes

Article abstract of DOI:10.1016/S0040-4020(00)00261-1

A series of zeolite immobilized α-chymotrypsin and thermolysin with microporous Y zeolites (HY, NH₄, NaY) and mesoporous dealuminized DAY zeolites (HDAY, HNH₄DAY) as matrixes have been prepared to catalyze peptide bond formation in organic solvents for the first time. The results indicated that most zeolite immobilized enzymes were active for peptide syntheses in organic media, and still had catalytic activity to some extent after being reused five times. According to the results, the immobilization effect of microporous Y zeolite was better than that of mesoporous DAY zeolite, suggesting that microporous Y zeolite can form more powerful hydrogen bonds with enzyme molecules since there are more hydroxyl groups on the Y zeolite than on the DAY zeolite. In addition, the influences of some reaction conditions such as reaction time and water content of the solvent on the enzymatic peptide synthesis were also studied and optimized. For the two kinds of proteases, NH₄Y zeolite did not show its advantages for thermolysin, but was more suitable for α-chymotrypsin as an immobilization matrix. 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00261-1

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 3517±3522
Enzymatic Peptide Synthesis in Organic Solvent with Different
Zeolites as Immobilization Matrixes
²
Guo-Wen Xing, Xuan-Wen Li, Gui-Ling Tian and Yun-Hua Ye
*
Key Laboratory of Bioorganic Chemistry and Molecular Engineering, Department of Chemistry, Peking University, Beijing 100871,
People's Republic of China
Received 11 January 2000; revised 6 March 2000; accepted 23 March 2000
AbstractÐA series of zeolite immobilized a-chymotrypsin and thermolysin with microporous Y zeolites (HY, NH4Y, NaY) and meso-
porous dealuminized DAY zeolites (HDAY, HNH4DAY) as matrixes have been prepared to catalyze peptide bond formation in organic
solvents for the ®rst time. The results indicated that most zeolite immobilized enzymes were active for peptide syntheses in organic media,
and still had catalytic activity to some extent after being reused ®ve times. According to the results, the immobilization effect of microporous
Y zeolite was better than that of mesoporous DAY zeolite, suggesting that microporous Y zeolite can form more powerful hydrogen bonds
with enzyme molecules since there are more hydroxyl groups on the Y zeolite than on the DAY zeolite. In addition, the in¯uences of some
reaction conditions such as reaction time and water content of the solvent on the enzymatic peptide synthesis were also studied and
optimized. For the two kinds of proteases, NH4Y zeolite did not show its advantages for thermolysin, but was more suitable for a-chymo-
trypsin as an immobilization matrix. q 2000 Elsevier Science Ltd. All rights reserved.
³
Ê
from micropore (,20 A) to mesopore (20±500 A) accord-
Ê
Introduction
ing to the control of preparation technology.
An immobilized enzyme has more stability and can be
reused more times than a free enzyme during the enzymatic
process. Utilizing a chemical or physical method, enzyme
molecules are bound on the surface of an immobilization
matrix. This usually allows the enzyme to maintain its
catalytic activity while inhibiting other processes such as
autolysis. The adsorptive binding method is a common
method that is used in the preparation of immobilized
enzymes on various supports such as ion-exchange poly-
mers, celite, alumina, glass powder, etc.1,2 In the pursuit
of better carriers, zeolites have attracted much attention in
recent years,3±6 since they: (i) have unique structural
characteristics and are resistant to biodegradation; (ii)
possess novel properties such as high surface areas, hydro-
phobic or hydrophilic behavior and electrostatic inter-
actions; (iii) can be readily prepared with cavities ranging
To our knowledge, only a few papers4±6 have reported that
some enzymes such as papain, trypsin or cutinase, etc. have
been trapped on zeolites and their catalytic activities
measured. However, zeolite immobilized enzymes as cata-
lysts for peptide synthesis have not been reported. The
research on enzymatic peptide synthesis in organic solvents
has made great progress since 1980s.7±9 Based on our
studies in this ®eld,10±13 in this article we report a new
type of immobilized a-chymotrypsin and thermolysin with
different kinds of zeolites as matrixes to catalyze peptide
bond formation in organic solvents. The enzymatic peptide
synthesis was performed according to Scheme 1. A tri-
peptide ZTyrGlyGlyOEt, the protected fragment of Leu-
enkephalin, was synthesized by immobilized a-chymotryp-
sin from ZTyrOEt and GlyGlyOEt in dichloromethane. The
other model peptide ZAspPheOMe,
a precursor of
aspartame, was prepared using immobilized thermolysin
as catalyst in tert-amyl alcohol with ZAspOH and PheOMe
as substrates. The preliminary results have previously been
brie¯y reported.14
Keywords: enzymatic peptide synthesis; immobilization; organic solvent;
zeolites.
* Corresponding author. Fax: 186-10-6275-1708; e-mail: yhye@pku.edu.cn
²
Present address: P.O. Box 27, Shanghai Institute of Organic Chemistry,
The purpose of the present study is to investigate the in¯u-
ence of zeolite properties such as acidity and pore diameter
on the enzymatic peptide synthesis. In the previous
papers,10±13 the syntheses of these two model peptides by
free enzymes have been reported. Herein, some immobi-
lized enzyme-catalyzed reaction conditions including
reaction time and optimum water content of the solvent
Chinese Academy of Sciences, 354 Fenglin Road, Shanghai 200032,
People's Republic of China.
Abbreviations: The amino acid residues which were used in this paper
are of l-con®guration. Standard abbreviations for amino acid derivatives
and peptides are according to the suggestions of the IUPAC-IUB Commis-
sion on Biochemical Nomenclature Eur. J. Biochem. 1984, 138, 9±37.
Other abbreviations: Z, benzyloxycarbonyl; MOPS, 3-(N-morpholino)pro-
panesulfonic acid.
³
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00261-1

3518
G.-W. Xing et al. / Tetrahedron 56 (2000) 3517±3522
Scheme 1. Enzymatic strategy for syntheses of two model peptides by zeolite immobilized enzymes.
Table 1. Physical data of ®ve kinds of zeolites
Speci®c surface (m2/g)
Si/Al
Ê
Pore size (A)
Zeolite
Pore volume (ml/g)
HY, NH4Y, NaY
HDAY, HNH4DAY
7.3
40
0.330±0.340
0.460
930±940
840
2.40±2.45
6.10
were studied and compared with those of free enzyme-
catalyzed reaction. In this work, zeolite immobilized
a-chymotrypsin and thermolysin were successfully applied
to peptide syntheses in organic solvents for the ®rst time.
The experiments indicated that most zeolite immobilized
enzymes were active for the model peptides syntheses.
Both the zeolite pore size and its acidity played important
roles in enzyme adsorption and enzymatic reaction. The
preference of immobilization support was distinguishable
between the two enzymes used. Additionally, the reusability
of zeolite immobilized enzyme was studied.
The results indicated that the immobilization effect of
microporous Y zeolites was better than that of mesoporous
DAY zeolites; that is to say, Y zeolite was a more favorable
type of immobilization matrix for a-chymotrypsin than
DAY zeolite. Though the pore size of DAY zeolite (40 A)
Ê
is much larger than that of Y zeolite (7.3 A), the enzyme
Ê
cannot totally enter the inner channels or cages of
DAY zeolite due to the large volume of the a-chymotrypsin
Ê 3
molecule (50£40£40 A ). Accordingly, it was perhaps
partly taken up or adsorbed on the surface of molecular
sieves similarly to the case of Y zeolite. The interaction
between enzyme molecules and zeolite is complicated.
Apart from the electrostatic action among the charged
groups on the surface of the enzyme and the cations from
the framework of molecular sieves as well as the negatively
charged oxygen atoms in the zeolite framework, there are a
lot of hydrogen bonds between the H-bond acceptors on the
enzyme and the hydroxyl groups on the zeolite. The cations
and OH groups in the zeolite decrease with increasing the
Si/Al ratio. After the Y zeolite was dealuminized to enlarge
its pore diameter, the Si/Al ratio of DAY zeolite obviously
increased. Therefore, there are more cations and OH groups
in Y zeolite which can form more powerful hydrogen bonds
with enzyme molecules to provide more stable zeolite±
enzyme complexes. As a result, Y zeolite immobilized
a-chymotrypsin possessed higher catalytic activity than
Results and Discussion
Five kinds of zeolites were used for immobilization and
some of their physical data are listed in Table 1. HY,
NH4Y and NaY zeolites were three kinds of microporous
Y zeolites and HDAY, HNH4DAY zeolites were meso-
porous ones. DAY refers to dealuminized Y zeolite that
was prepared by chemical and hydrothermal dealumination,
and its Si/Al ratio was higher than that of Y zeolite.15
Firstly, we prepared ®ve kinds of zeolite immobilized
a-chymotrypsins by physical adsorption and studied their
utilization in the synthesis of ZTyrGlyGlyOEt with
dichloromethane as solvent.
Reusability of zeolite immobilized a-chymotrypsin and
effect of support properties
Fig. 1 illustrates the relationship between peptide yield and
the reuse time of zeolite immobilized a-chymotrypsin. The
results showed that all the immobilized a-chymotrypsins on
different zeolites were active for peptide synthesis, and the
catalytic yield declined to a varying extent after being
reused 4±5 times. On the whole, the product yield using
Y zeolites as matrixes dropped more slowly than using
DAY zeolites with increasing the reuse time of immobilized
enzyme. Moreover, there was no obvious difference in the
reusability of the three kinds of Y zeolite immobilized
a-chymotrypsins. When judging from the best yield that
each immobilized enzyme obtained during the ®ve times
of reuse, HY zeolite was the best matrix since the yield of
the second reuse reached the highest (78%) of all the
immobilized a-chymotrypsin-catalyzed reactions.
Figure 1. Reusability of immobilized a-chymotrypsins in the synthesis of
ZTyrGlyGlyOEt. Reaction time: 1 day for the ®rst use and 2 days for the
®rst to ®fth reuse.

G.-W. Xing et al. / Tetrahedron 56 (2000) 3517±3522
3519
Table 2. Comparison of the one-day yields of ZTyrGlyGlyOEt synthesized
by different zeolite immobilized a-chymotrypsins and free a-chymotrypsin
Zeolite
HY NaY NH4Y HDAY HNH4DAY Free enzyme
60 58 68 60 53
Yield (%) 69
DAY zeolite immobilized enzyme when reused several
times for the enzymatic reaction.
Table 2 describes the one-day yields of the model reaction
catalyzed by different zeolite immobilized a-chymotrypsins
and free a-chymotrypsin. After the free a-chymotrypsin
was immobilized on zeolites, the yield increased to a
varying degree. The increasing activity of HY zeolite
immobilized enzyme was the highest among the ®ve kinds
of immobilized enzymes used. This showed that it was
bene®cial to have a-chymotrypsin immobilized using
molecular sieves as matrix.
Figure 3. In¯uence of water content on the yield of ZTyrGlyGlyOEt cata-
lyzed by HY zeolite immobilized or free a-chymotrypsin.
a-chymotrypsin-catalyzed peptide synthesis the optimum
water content range was broader (0.25±1.0% (v/v)) than
in the reaction using free enzyme. Even if no water was
injected into the solvent, the model peptide was still
obtained in 30% yield. While, in the free a-chymotrypsin-
catalyzed model peptide synthesis there was no product
obtained without water added, indicating that enzyme
activity could not be shown in the dry solvent.
Effect of reaction time on the zeolite immobilized
a-chymotrypsin-catalyzed reaction
From Fig. 2, it can be seen that for the free a-chymotrypsin-
catalyzed reaction, a good yield was obtained after carrying
out the reaction for 2 days, while for the NH4Y zeolite
immobilized a-chymotrypsin, the 1 day yield was nearly
the same as the 3 day yield. Owing to the increase in contact
ef®ciency between enzymes and substrates when a-chymo-
trypsin molecules were well dispersed on the surface of
zeolite, the reaction ef®ciency was enhanced and therefore
the good yield can be reached in less time in the immobi-
lized enzyme-catalyzed reaction than in the free enzyme-
catalyzed reaction.
Because molecular sieve itself can easily adsorb water
molecules that cannot be completely eliminated during the
lyophilization for preparing the immobilized enzyme, the
obtained zeolite immobilized enzyme still retains a small
amount of water which serves as the essential water for the
enzyme in the organic solvent to maintain its catalytic
activity in the enzymatic reaction. As a result, the sensitivity
of immobilized a-chymotrypsin to the water content
decreased. The zeolite immobilized a-chymotrypsin
extended the range for the optimum water content of the
solvent.
Effect of water content of dichloromethane on the zeolite
immobilized a-chymotrypsin-catalyzed reaction
Although the HY zeolite immobilized a-chymotrypsin
exhibited high catalytic activity as the water content varied
from 0.25 to 1.0% (v/v), when more water was added into
the solvent its reusability went down remarkably as Table 3
indicated. In the case of the water content being 0.25%, the
HY zeolite immobilized a-chymotrypsin can be ef®ciently
used several times and 58% yield obtained for the third
reuse. Compared with this, when 1.0% water content was
chosen in the reaction, the activity of immobilized enzyme
decreased obviously from the second reuse and the tri-
peptide was synthesized in low yield (38%). The results
showed that high water content can lead to a good yield in
the ®rst use, but at the same time the enzyme activity was
lost rapidly. Taking both product yield and enzyme reusa-
bility into consideration, 0.25% water content of the
dichloromethane was selected for the reaction.
The water content of the organic solvent greatly in¯uenced
the activity of the immobilized enzyme and the product
yield. With HY zeolite immobilized a-chymotrypsin as an
example, different amounts of water were added to the
reaction. Fig. 3 shows the changes that took place when
free a-chymotrypsin was immobilized on HY zeolite. The
effect of water content of dichloromethane on the free
a-chymotrypsin-catalyzed reaction has been studied in our
previous works,10,13 and the optimized water content was
0.15% (v/v). Interestingly, in the HY zeolite immobilized
Table 3. Reusability of HY immobilized a-chymotrypsin under different
water contents
Water content (%, v/v)
Product yield for different reuses of the
catalyst (%)
0
1
2
3
0.25
1.00
69
78
74
77
78
38
58
11
Figure 2. Relationship between reaction time and the product yield cata-
lyzed by NH4Y zeolite immobilized or free a-chymotrypsin.

3520
G.-W. Xing et al. / Tetrahedron 56 (2000) 3517±3522
Figure 4. Reusability of immobilized thermolysin in the synthesis of ZAspPheOMe. Reaction conditions: ZAspOH was 100 mM and PheOMe was 150 mM in
the reactions; water content was 6% (v/v); reaction time was 3 days in all cases; 100 mg immobilized thermolysin was added in the ®rst use.
Reusability of zeolite immobilized thermolysin and effect
of support properties
consequence, the activity of HY zeolite immobilized
thermolysin became very low and the yield of dipeptide
ZAspPheOMe was poor. HY zeolite immobilized a-chymo-
trypsin had a high catalytic activity resulting from the
broader stable pH range (pH 3±10) of a-chymotrypsin
compared with that of thermolysin. With this provision,
the acidity of HY zeolite had no noticeable in¯uence on
the enzyme activity. The above results indicated that
different physical properties of enzymes can lead to them
being more effectively immobilized on different matrixes.
The exact pH value changes during the enzyme immobili-
zation are shown in Tables 4 and 5.
In order to study the in¯uence of different zeolites as
matrixes on the activity of different enzymes, four kinds
of zeolite immobilized thermolysins have also been
prepared. They were microporous HY, NH4Y, NaY and
mesoporous HNH4DAY zeolite immobilized thermolysins.
Their reusability was investigated with enzymatic synthesis
of ZAspPheOMe as model reaction.
Unlike immobilized a-chymotrypsin, the catalytic effect of
HY immobilized thermolysin was the worst of the four
kinds of zeolite immobilized thermolysins used (Fig. 4).
Thermolysin is a neutral protease which is stable at pH
6.0±9.0. However, owing to the strong acidity of HY zeolite
the pH value of the solution decreased to 3.22 after the
thermolysin buffer solution (pH 6.98) was stirred for
immobilization over HY zeolite for an hour. As a
In the case of the other three zeolites NH4Y, NaY and
HNH4DAY, their pH values all approached neutral and
were located in the stable pH scope of thermolysin. Thus,
with them as immobilization matrixes, thermolysin
presented catalytic activity to a certain extent in the
reaction. However, it was worth noting that the activity of
dealuminized mesoporous HNH4DAY zeolite immobilized
thermolysin was lower than that of NH4Y or NaY zeolite
immobilized enzyme. This observation is similar to that
using different zeolite immobilized a-chymotrypsins as
catalysts which has been analyzed and described above.
Table 4. The pH value changes during a-chymotrypsin immobilization on
different zeolites
Zeolite
HY NaY NH4Y HDAY HNH4DAY
Starting pH value of the
enzyme solution
pH value of the water phase 4.07 7.74 7.55
after adsorption on zeolite
for 1 h
7.95 7.95 7.95
7.95
6.69
7.95
7.31
Effect of reaction time on the zeolite immobilized
thermolysin-catalyzed reaction
The in¯uence of reaction time on the synthesis of ZAsp-
PheOMe catalyzed by NH4Y zeolite immobilized thermo-
lysin is the same as free thermolysin (Fig. 5). In both cases,
the model peptide yields increased with increasing the reac-
tion time, and during the same period, the corresponding
yields were close to each other. When compared with the
in¯uence of reaction time on the zeolite immobilized
a-chymotrypsin-catalyzed reaction, the results suggest that
for the two kinds of proteases, the zeolite material did not
show its advantages for thermolysin, but was more suitable
for a-chymotrypsin as an immobilization matrix.
Table 5. The pH value changes during thermolysin immobilization on
different zeolites
Zeolite
HY
6.98
3.22
NaY
6.98
7.10
NH4Y
6.98
HNH4DAY
6.98
Starting pH value of the
enzyme solution
pH value of the water phase
after adsorption on zeolite
for 1 h
6.76
6.84

G.-W. Xing et al. / Tetrahedron 56 (2000) 3517±3522
3521
trypsins and four kinds of zeolite immobilized thermolysins
were prepared, and the peptide formation reactions cata-
lyzed by them in organic solvents were investigated and
compared. The results demonstrate that it is feasible to
use zeolites as supports for enzymatic peptide synthesis.
The effects of immobilization matrix and the reaction con-
ditions on the model reactions are complicated. Some
problems such as the identi®cation of hydrogen bonds
formed between enzyme and zeolite are currently being
investigated. The study presented here will be helpful for
the investigation of this type of zeolite immobilized enzyme
and in ®nding more valuable and promising immobilization
matrixes.
Figure 5. Relationship between reaction time and the product yield cata-
lyzed by NH4Y zeolite immobilized or free thermolysin.
Experimental
Effect of water content of tert-amyl alcohol on the zeolite
immobilized thermolysin-catalyzed reaction
a-Chymotrypsin (EC 3.4.21.1 Type II, from bovine
pancreas) and thermolysin (EC 3.4.24.4 from bacillus
thermoprotedyticus rokko) were purchased from Sigma
Chemical Company. All the zeolites (HY, NH4Y, NaY,
HDAY, HNH4DAY) were prepared by our laboratory as
described in the literature.15 ZAspOH, HCl´PheOMe, ZTyr-
OEt and HCl´GlyGlyOEt were prepared according to the
standard method.17 Dichloromethane and tert-amyl alcohol
were from the local chemical factory, they were dried over
K2CO3 and distilled before use.
Fig. 6 illustrates the in¯uence of water content of tert-amyl
alcohol on the peptide yield when NH4Y zeolite immobi-
lized thermolysin was used. The optimum water content in
the NH4Y zeolite immobilized thermolysin-catalyzed reac-
tion was about 6% (v/v), and it was the same as that (6,8%)
in the free thermolysin-catalyzed model peptide synthesis
which was reported in the previous articles.10,11 This result
differed from the situation using immobilized and free
a-chymotrypsin as catalysts. Compared with tert-amyl alco-
hol (log P 0.89),16 dichloromethane (log P 1.25)16 is a more
hydrophobic organic solvent. Thus, adding only a small
amount of water into dichloromethane (the optimum water
content ,1% (v/v)) can provide enough essential water to
maintain the activity of a-chymotrypsin. In contrast, more
water must be added to tert-amyl alcohol (the optimum
water content .1% (v/v)) for thermolysin to show its cata-
lytic activity. In addition, due to the optimum water content
being low (0.25%) in the immobilized a-chymotrypsin-
catalyzed synthesis, a little residual water in the zeolite
carriers can maintain the activity of enzyme even when no
water was added into dichloromethane. In the case of immo-
bilized thermolysin-catalyzed reaction, when no water was
injected, no product was obtained, since the optimum water
content of tert-amyl alcohol system was high (6%) and the
water remaining in the immobilization support was not
enough to enable the thermolysin-catalyzed peptide
synthesis.
Preparation of immobilized a-chymotrypsin and
thermolysin on different zeolites
a-Chymotrypsin (20 mg) was dissolved in phosphate buffer
(4 ml, pH 7.95, 50 mM), or [thermolysin (20 mg) was
dissolved in MOPS(Na1) buffer (4 ml, pH 6.98, 50 mM)],
then zeolite (200 mg) was added at room temperature under
stirring. After about an hour, the pH value of the mixture
was measured by pH meter and the suspension was lyophi-
lized overnight to obtain the zeolite immobilized a-chymo-
trypsin or thermolysin.
Synthesis of ZTyrGlyGlyOEt by different zeolite
immobilized a-chymotrypsins (general procedure)
ZTyrOEt (0.5 mmol) and HCl´GlyGlyOEt (0.5 mmol) were
suspended in dichloromethane (5 ml), then triethylamine
(140 ml) was added and the mixture was shaken thoroughly
to make the substrates dissolve completely. After zeolite
immobilized a-chymotrypsin (50 mg), water (12.5 ml,
H2O/CH2Cl2ˆ0.25% (v/v)) were added, the mixture was
stirred at room temperature for 1±3 days. At the end of
the reaction, the precipitate from the reaction solution and
the immobilized enzyme were ®ltered and washed
thoroughly with acetone. Then the immobilized a-chymo-
trypsin was dried for storage or reuse, and the acetone
solution was evaporated to obtain ZTyrGlyGlyOEt.
In summary, ®ve kinds of zeolite immobilized a-chymo-
Synthesis of ZAspPheOMe by different zeolite
immobilized thermolysins (general procedure)
ZAspOH (0.5 mmol) and HCl´PheOMe (0.5±0.75 mmol)
were suspended in tert-amyl alcohol (5 ml), then triethyl-
amine (220±255 ml) was added and the mixture was shaken
thoroughly. After zeolite immobilized thermolysin (50±
Figure 6. In¯uence of water content on the yield of ZAspPheOMe cata-
lyzed by NH4Y zeolite immobilized or free thermolysin.

3522
G.-W. Xing et al. / Tetrahedron 56 (2000) 3517±3522
F.; Prazeres, D. M. F.; Cabral, J. M. S.; Aires-Barros, M. R. J. Mol.
Catal. B: Enzymatic 1996, 1, 53±60.
100 mg) and 10 mM calcium acetate (0.3±0.4 ml) were
added, the solution was stirred at 408C for 1±3 days. At
the end of the reaction, the immobilized enzyme were
®ltered and washed thoroughly with ethyl acetate. Then
the immobilized thermolysin was dried for storage or
reuse, and the ethyl acetate solution as well as the ®ltrate
were evaporated under reduced pressure. The oily residue
was diluted by ethyl acetate (50 ml), and then washed with
1 M HCl and saturated NaCl, dried over anhydrous Na2SO4.
After removal of solvent, the crude product was recrys-
tallized from methanol±water to give pure ZAspPheOMe.
6. Liu, B.; Hu, R.; Deng, J. Anal. Chem. 1997, 69, 2343±2348.
7. Enzymatic Reactions in Organic Media, Koskinen, A. M. P.,
Klibanov, A. M., Eds.; Blackie Academic and Professional:
London, 1996.
8. Tian, G.-L.; Xing, G.-W.; Ye, Y.-H. Chinese J. Org. Chem.
1998, 18, 11±19.
9. Xing, G.-W.; Ye, Y.-H.; Li, C. X. Chem. J. Chin. Univ. 1998,
19, 1602±1607.
10. Ye, Y.-H.; Tian, G.-L.; Xing, G.-W.; Dai, D. C.; Chen, G.; Li,
C. X. Tetrahedron 1998, 54, 12585±12596.
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Acknowledgements
12. Xing, G.-W.; Liu, D. G.; Ye, Y.-H.; Ma, J. M. Tetrahedron
Lett. 1999, 40, 1971±1974.
This project was supported by the Doctoral Program
Foundation of Institution of the Higher Education and the
Hong Kong Polytechnic University.
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logy and Chemistry (Proceedings of the 1996 Chinese Peptide
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