Synthesis of a precursor tripeptide Z-Asp-Val-Tyr-OH of thymopentin by chemo-enzymatic method

Article abstract of DOI:10.1080/10826068.2012.660902

The precursor tripeptide of thymopentin was synthesized by a combination of chemical and enzymatic methods. First, Val-Tyr-OH dipeptide was synthesized by a novel chemical method in two steps involving preparation of NCA-Val. Second, the linkage of the third amino acid Z-Asp-OMe to Val-Tyr-OH was completed by an enzymatic method under kinetic control. An industrial alkaline protease alcalase was used in water-organic cosolvent systems. The synthesis reaction conditions were optimized by examining the effects of several factors including organic solvents, water content, temperature, pH, and reaction time on the yield of Z-Asp-Val-Tyr-OH. The optimum condition is of pH 10.0, 35C, acetonitrile/Na ₂CO₃-NaHCO₃ buffer system (85:15, v/v), and reaction time of 2.5hr, which achieves tripeptide yield of more than 70%.

Full text of DOI:10.1080/10826068.2012.660902

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SYNTHESIS OF A PRECURSOR TRIPEPTIDE
Z-Asp-Val-Tyr-OH OF THYMOPENTIN BY
CHEMO-ENZYMATIC METHOD
Kun Zheng a b , Ruiyan Zhan b , Yang Hong b , Jing Li c , Wei Shi a &
Shijun Li b
a Key Laboratory for Molecular Enzymology and Engineering, Ministry
of Education, Jilin University , Changchun , P.R. China
b Jilin Institute of Chemical Technology , Jilin , P. R. China
c Changsheng Gene Pharmaceutical Co., Ltd , Changchun , P. R.
China
Accepted author version posted online: 03 Apr 2012.Published
online: 02 Oct 2012.
To cite this article: Kun Zheng , Ruiyan Zhan , Yang Hong , Jing Li , Wei Shi & Shijun Li (2012)
SYNTHESIS OF A PRECURSOR TRIPEPTIDE Z-Asp-Val-Tyr-OH OF THYMOPENTIN BY CHEMO-
ENZYMATIC METHOD, Preparative Biochemistry and Biotechnology, 42:6, 520-534, DOI:
10.1080/10826068.2012.660902
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Preparative Biochemistry & Biotechnology, 42:520–534, 2012
Copyright # Taylor & Francis Group, LLC
ISSN: 1082-6068 print/1532-2297 online
DOI: 10.1080/10826068.2012.660902
SYNTHESIS OF A PRECURSOR TRIPEPTIDE Z-Asp-Val-Tyr-OH
OF THYMOPENTIN BY CHEMO-ENZYMATIC METHOD
Kun Zheng,1,2 Ruiyan Zhan,2 Yang Hong,2 Jing Li,3 Wei Shi,1 and
Shijun Li2
1Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education,
Jilin University, Changchun, P.R. China
2Jilin Institute of Chemical Technology, Jilin, P. R. China
3Changsheng Gene Pharmaceutical Co., Ltd, Changchun, P. R. China
& The precursor tripeptide of thymopentin was synthesized by a combination of chemical and
enzymatic methods. First, Val-Tyr-OH dipeptide was synthesized by a novel chemical method in
two steps involving preparation of NCA-Val. Second, the linkage of the third amino acid Z-Asp-
OMe to Val-Tyr-OH was completed by an enzymatic method under kinetic control. An industrial
alkaline protease alcalase was used in water–organic cosolvent systems. The synthesis reaction con-
ditions were optimized by examining the effects of several factors including organic solvents, water
content, temperature, pH, and reaction time on the yield of Z-Asp-Val-Tyr-OH. The optimum
condition is of pH 10.0, 35ꢀC, acetonitrile=Na2CO3-NaHCO3 buffer system (85:15, v=v), and
reaction time of 2.5 hr, which achieves tripeptide yield of more than 70%.
Keywords alcalase, organic solvents, peptide synthesis, thymopentin
INTRODUCTION
Thymopoietin (TP) is a polypeptide hormone produced by the thymus,
consisting of 49 amino acids. Thymopentin (TP-5), the pentapepide Arg-
Lys-Asp-Val-Tyr, corresponding to amino acids 32–36 of TP, appears to
represent the active site of TP.[1,2] Thymopentin is pleiotropic in action,
affecting neuromuscular transmission, inducing early T-cell differentiation
and immune regulation, and so on. Thymopentin has been used clinically
for the treatment of rheumatoid arthritis and atopic dermatitis.[3,4] It is also
used for treatment of cancer patients in combination with chemotherapy.[5,6]
Address correspondence to Shijun Li, Environmental and Bioengineering Technology College,
Jilin Institute of Chemical Technology, Jilin, 132022, P. R. China. E-mail: csgp02@126.com; or Wei Shi,
Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education, Jilin University,
Changchun, 130012, P. R. China. E-mail: shiw@jlu.edu.cn

Synthesis of a Precursor Tripeptide
521
At present, the commercially available products of TP-5 are prepared
by the approach of chemical synthesis. However, there is an increasing
commercial
interest
in
enzymatic
preparation
of
small
bioactive peptides.[7] An enzymatic method has great advantages—for
instance, mild conditions of the reaction, the high regiospecificity of the
enzyme allowing the use of minimally protected substrates, and the stereo-
specificity without racemization. A number of hydrophobic small peptides
have been synthesized in high yields using proteases in organic media, as
reported previously.[8–11] However, the formation of peptide bonds involv-
ing hydrophilic amino acids led to many problems, such as the solubility of
a hydrophilic amino acid substrate in hydrophobic organic solvents, an
undesirable hydrolysis of the ester substrate, the growing peptide chain
due to high water content in the reaction system, and deactivation of an
enzyme in polar organic solvents. Therefore, appropriate solvents should
be selected considering the balance between the solubility of substrates
and enzymatic activity. At the same time, when selecting an enzyme, we con-
sider not only its substrate specificity, but also its stability in hydrophilic
organic solvents. Chen et al.[12,13] reported that an industrial alkaline pro-
tease, alcalase (Novo product), prepared from submerged formation of a
selective strain of Bacillus licheniformis, is very stable in ethanol or
2-methyl-2-propanol and is suitable for catalysis of peptide bond formation
via a kinetically controlled approach. In our previous work, several cell
adhesion motif RGD-containing peptides and the precursors were success-
fully synthesized by using this enzyme.
In this report, we discuss the synthesis of a precursor tripeptide of TP-5
in the form of Z-Asp-Val-Tyr-OH by a chemo-enzymatic method. Val-Tyr-OH
dipeptide was synthesized in high yield by a novel chemical method called
the NCA (N-carboxyanhydride) method.[14] The coupling between Z-Asp-
OMe and Val-Tyr-OH was carried out in organic solvents by an enzymatic
method using alcalase as the catalyst in reasonable yields. The reaction con-
ditions for synthesis of Z-Asp-Val-Tyr-OH were optimized by examining the
effects of several factors, including organic solvents, water content, tem-
perature, pH and reaction time, on the tripeptide yield. In addition, the
target peptide is also a good model for the study of the synthesis of
hydrophilic amino acids-containing peptides.
EXPERIMENTAL
Materials
Alcalase was purchased from Novo Industrial (Denmark) as a brown liquid
with a specific activity of 2.5 A.U. (activity unit) mLꢁ1. Z-Asp-OMe, H-Val-OH,
and H-Tyr-OH were purchased from GL Biochem (Shanghai, China).

522
K. Zheng et al.
Trifluoroacetic acid was from Merck (Darmstadt, Germany). Sephadex G-10
was from Pharmacia. Acetonitrile was high-performance liquid chromato-
graphy (HPLC) grade. All other organic solvents were analytical grade.
Chemical Synthesis of Val-Tyr-OH
Synthesis of NCA–Val
H-Val-OH (10 g, 0.85 mol) was mixed with 100 mL of dry THF (tetrahy-
drofuran) in a round-bottomed flask with magnetic stirring and heating.
When the temperature was raised to 60ꢀC, triphosgene (10.12 g, 0.34 mol)
was added to the solution. This mixed solution was stirred for 30–60 min
until it became clear. The solution was evaporated until the volume
reduced to one-third and precipitated in anhydrous hexanes for 24 hr at
4ꢀC. The precipitate was filtered and dried at 35ꢀC for 10–20 min. The
dry product is NCA-Val.
Synthesis of Val-Tyr-OH
One gram Na2CO3 and 10 mL of 1 M NaOH (pH 10.2) was dissolved in
40 mL water in a round-bottomed flask with magnetic stirring. When the sol-
ution became clear, H-Tyr-OH (1 g, 0.006 mol) was added. When the solution
become clear again, 50 mL of acetonitrile was added; then it was cooled to
ꢁ10ꢀC in an ice salt bath.[15] The NCA-Val (0.95 g, 0.006 mol) dissolved in
20 mL acetonitrile was added into the preceding solution. When reaction
proceeded for 3 hr under rapid magnetic stirring at ꢁ10ꢀC, it was stopped
and the upper layer organic phase was removed. The water phase was washed
several times with cold acetonitrile to remove the unreacted NCA-Val. After
adjusting the pH to 5–6, the mixture was evaporated to dryness and redis-
solved in alcohol and filtered to remove the salt. At last, the filtrate was eva-
porated to dryness. The dipeptide product H-Val-Tyr-OH was obtained.
Enzymatic Synthesis of Z-Asp-Val-Tyr-OH
Pretreatment of Alcalase
Alcalase (0.4 mL) and anhydrous ethanol (2 mL) were added to a cen-
trifuge tube, and the mixture was agitated for 5 min. The resulting mixture
was centrifuged at 3000 rpm for 10 min to separate the enzyme from the sol-
vent, and the ethanol was removed by decantation. The procedure was
repeated three times.
Synthesis of Z-Asp-Val-Tyr-OH
The synthesis of Z-Asp-Val-Tyr-OH from Z-Asp-OMe and Val-Tyr-OH was
carried out in a 2-mL volume with magnetic stirring under a series of

Synthesis of a Precursor Tripeptide
523
conditions. For a typical reaction system, Z-Asp-OMe (50 mmol) and Val-
Tyr-OH (150 mmol) and triethylamine (100 mL) were dissolved in 1.7 mL
of acetonitrile; 0.3 mL of Na2CO3-NaHCO3 buffer (0.1 M, pH 10.0) was
added to the pretreated enzyme obtained from 0.4 mL of the untreated
alcalase, incubated for 10 min at 35ꢀC. At a desired time interval, an aliquot
of 0.05 mL was taken from the reaction mixture and centrifuged at
12,000 rpm for 10 min to remove alcalase. Then the supernatant was taken
for HPLC analysis.
Separation and Purification of Peptide Product
The target peptide products Val-Tyr-OH and Z-Asp-Val-Tyr-OH were
further purified on a Sephadex G-10 column (16 ꢂ 1000 mm) equilibrated
and eluted with water at the elution rate of 0.4 mL minꢁ1. The elution pro-
cess was monitored at 220 nm. The collected fractions were lyophilized.
Analytical Control of Peptide Synthesis
Analytical control of the peptide synthesis was carried out by HPLC
(Shimadzu model) with a reverse-phase C18 column (Diamonsil C18,
250 mm ꢂ 4.6 mm). The mobile phase was 30% acetonitrile containing
0.1% TFA and flow rate 1 mL=min. The calibration curves to be used calcu-
lating the yields of the peptide products were constructed from the peak
areas of the purified products at 220 nm.
The reaction products were identified by HPLC–mass spectroscopy
(MS). The chromatographic condition was the same as already described.
The MS condition was as follows: ionization mode: API–ES (atmospheric
pressure ionization-electrospray); polarity: positive; Vcap (capillary volt-
age): 4000 V; nebulizer pressure: 35 psi; drying gas: 8 L minꢁ1; gas tempera-
ture: 350ꢀC; analyzer scan range: 10–800 a.t.u. (atomic mass units).
RESULTS AND DISCUSSION
Synthesis of Val-Tyr-OH
In the reaction of synthesizing the dipeptide Val-Tyr-OH, rapid mixing
enhances the rate of dissolution of the NCA and thereby reduces the possi-
bility for carbamate exchange, which leads to overreaction resulting in for-
mation of tripeptide. The control of pH is important because carbamate
stability increases with increase of pH. However, at a pH of higher than
10.5, hydrolysis of the NCA becomes an important side reaction and the
formation of the NCA anion is increased. Low temperature is in favour

524
K. Zheng et al.
FIGURE 1 HPLC profile of unpurified Val-Tyr-OH.
of the desired reaction over side reactions, partly because of the change in
dissociation constant kw with temperature. With careful control of reaction
conditions, the NCA method permits the rapid synthesis of optically pure
peptides with the use of a minimum of protecting groups.[14,15] For the syn-
thesis of Val-Tyr-OH, the yield was more than 90%. The HPLC profile and
the elution data of the unpurified dipeptide product are given in Figure 1
and Table 1, respectively.
Synthesis of Z-Asp-Val-Tyr-OH
Choice of Enzyme
The synthesis of Z-Asp-Val-Tyr-OH was completed through the linkage
of Z-Asp-OMe to Val-Tyr-OH by an enzymatic method under kinetic control.
For formation of a peptide bond using an enzyme as the catalyst, the first
concern is to choose an appropriate enzyme. Studies on the selectivity of an
alcalase-catalyzed reaction showed that only L-amino acid acyl donors are
suitable substrates at the p-1 subsite of the enzyme, while both D- and
L-amino acid nucleophiles are suitable substrates at the p-10 subsite. Amino
acids with hydrophilic and charged side chains in p-10 position are accepted
better than those with bulky hydrophobic side chains.[16] Therefore, in this
TABLE 1 Data of HPLC Elution for Val-Tyr-OH
Number
Name
Retention Time (min)
Area
Percent Area
Height
1
2
3
4
unknown
Val-Tyr-OH
unknown
unknown
1.826
2.569
3.946
5.455
418185
20411572
488942
1.92
93.96
2.25
12560
820180
8015
405528
1.87
12963

Synthesis of a Precursor Tripeptide
525
case, Z-Asp-OMe as the acyl donor and Val-Tyr-OH as the nucleophile
should be available substrates. The industrial alkaline protease alcalase as
a brown liquid was purified to remove the additives and the other compo-
nents that may affect the experimental results by the combination of preci-
pitating and washing the enzyme with absolute ethanol. The water content
in the pretreated alcalase can be reduced to 0.1%.[12]
Effect of Organic Solvent
The selection of organic solvents is important in the enzyme-catalyzed
synthesis of a peptide bond due to their effects on both the enzyme activity
and the substrate solubility, and further on the yield of a peptide pro-
duct.[17] In this study, seven kinds of organic solvents (methanol, chloro-
form, ethyl acetate, acetonitrile, ethanol, DMF, DMSO) were tested under
the same other experimental conditions. The results for the synthesis of
Z-Asp-Val-Tyr-OH in the different organic solvent systems are shown in
Figure 2, where it can be seen that acetonitrile (85=15, v=v) system is the
best one among the organic solvents tested.
The data in Table 2 show the log P values of some organic solvents. Log
P was generally adopted to describe the hydrophobic property of an
organic solvent in anhydrous or microaqueous media.[18] Apolar solvents
with higher log P values are often less harmful to an enzyme than the sol-
vents with higher polarity. Polar solvents have a greater tendency to strip
the tightly bound essential water from the enzyme molecules. This is a
FIGURE 2 Effects of the organic solvents on the yield of Z-Asp-Val-Tyr-OH. (A) Methanol, (B) chloro-
form, (C) ethyl acetate, (D) acetonitrile, (E) ethanol, (F) DMF, (G) DMSO. Reaction conditions: Z-Asp-
OMe, 50 mM; Val-Tyr-OH, 150 mM; triethylamine, 100 mL; each organic solvent, 85%; reaction tempera-
ture, 35ꢀC; 0.1 M Na2CO3–NaHCO3 buffer (pH 10.0), 15%; alcalase, 0.4 mL; reaction time, 2.5.

526
K. Zheng et al.
TABLE 2 Log P Values of Some Organic Solvents Used
Organic Solvents
Log P
Ethanol
–0.24
0.68
Ethyl acetate
Acetonitrile
Methanol
DMF
DMSO
Chloroform
–0.33
–0.76
–1.00
–1.30
2.0
Note. The values of log P are cited from Laane et al. (1987).
general principle for the selection of organic solvents. In our case, it seems
that we are not able to interpret the result here well by only using log P
value. We should also consider the factors of the substrates solubility and
the enzyme stability in organic solvents. Okazaki et al.[19] reported the syn-
thesis of peptide bonds catalyzed by subtilisin Carlsberg in different hydro-
philic organic solvents with different H2O contents and demonstrated that
the yields of peptide products were higher in most cases when acetonitrile
containing low H2O content was used. On the other hand, we cannot use
hydrophobic organic solvents due to the poor solubility of the hydrophilic
amino acid substrates. Hydrophilic organic solvents are suitable as the reac-
tion media to enhance the solubility of hydrophilic substrates. Z-Asp-OMe
and Val-Tyr-OH have a reasonable solubility in the acetonitrile systems used
in this study. We found that the enzyme alcalase was very stable and active in
acetonitrile.
Effect of Water Content
Water molecules play a key role in the catalytic performance of enzymes
in organic media. The conformation of an enzyme in organic solvents is
very rigid and not favorable for expression of its activity. Small amount of
water is thought to reduce the rigidity by forming multiple hydrogen bonds
with the main chain of the enzyme protein and the conformational flexi-
bility of enzyme molecules rapidly increases. As a result an enzyme becomes
catalytically active in organic media. Excess amount of water, however, leads
an enzyme to a denaturation state because enzyme conformation changes
to a thermodynamically stable state, that is, an inactive state with high con-
formational flexibility. Therefore, the relationship between the catalytic
activity of enzymes and the water content in organic media draws a bell-
shaped profile in many cases.[20] On the other hand, water favors of the
solubility of the hydrophilic substrates. Figure 3 shows the dependence of
water content on the tripeptide yield. For the synthesis of Z-Asp-Val-Tyr-OH
from Z-Asp-OMe and Val-Tyr-OH in acetonitrile, the optimum water content

Synthesis of a Precursor Tripeptide
527
FIGURE 3 Effect of water content on the yield of Z-Asp-Val-Tyr-OH. Except for the change in reaction
water content, the reaction conditions were same as that of D (acetonitrile) in Figure 2.
was about 15% with the best yield after 2.5 hr. When the water content in
the reaction system was higher than the optimum amount, the yield of tri-
peptide product decreased due to the hydrolysis of the ester substrate.
Effect of pH
The effect of pH of the reaction system on the synthesis of the target
tripeptide is shown in Figure 4. The pH values in Figure 4 are those of
the buffer solution contained in organic solvents. The optimum pH value
FIGURE 4 Effect of pH on the yield of Z-Asp-Val-Tyr-OH.

528
K. Zheng et al.
is about 10.0, with the highest yield for the alcalase-catalyzed synthesis of
Z-Asp-Val-Tyr-OH in 85% acetonitrile. It is well known that the pH of reac-
tion media is related to ionization state of the essential groups in the active
site of an enzyme. Therefore, this will affect its catalytic activity. We found
that if the enzyme was dissolved in the buffer before adding it in the reac-
tion system, a higher yield of the target tripeptide would be achieved. In
this way, the enzyme molecules can combine with the essential water layer
around the enzyme molecules to make it maintain a favourable confor-
mation for the catalytic activity. However, in our case it seems that the
effect of the tested pH on the tripeptide synthesis is existent and not
obvious, which may be attribute to the property of alcalase as an alkaline
protease.
Effect of Temperature
Figure 5 shows the effect of reaction temperature on the tripeptide
Z-Asp-Val-Tyr-OH syntheses catalyzed by alcalase in organic solvents. The
optimum reaction temperature is about 35ꢀC as seen in this figure. It is
known that the optimal temperature for alcalase-catalyzed decomposition
reaction in the aqueous phase is 60ꢀC. However, in our study, the alcalase
as a catalyst was employed to catalyze the synthesis of tripeptide in the
organic phase. The experimental result demonstrates that when the reac-
tion temperature was higher than 35ꢀC, the yield of the tripeptide
decreased (Figure 5), which maybe due to the heat denaturation of the
enzyme, especially in organic solvents. The temperature of peptide syn-
thesis mainly depended on two aspects, the enzymatic stability and activity
FIGURE 5 Effect of temperature on the yield of Z-Asp-Val-Tyr-OH.

Synthesis of a Precursor Tripeptide
529
in organic solvents. Generally, higher temperature is unfavorable for
synthesis of the peptide bond because it can cause thermal deactivation
of an enzyme. On the other hand, too low a temperature is also unfavorable
to the rate of enzymatic reaction, as indicated by large amount of the sub-
strate remaining in the reaction system.[21]
Time Course of the Tripeptide Synthesis
Figure 6 shows the time course of the synthesis of the tripeptide Z-Asp-
Val-Tyr-OH catalyzed by alcalase. Generally, the control of the reaction time
is a key point for kinetically controlled peptide synthesis catalyzed by a pro-
tease. If the reaction time is over the optimum one, the yield of a peptide
product would decrease rapidly. However, it was observed in this study that
when the reaction time was over 2.5 hr, the yield of the tripeptide product
could keep relatively constant, indicating that hydrolysis of the peptide
product did not obviously take place. The optimum time was about 2.5 hr
with the best yield.
Effect of Molar Ratio of the Substrates
Figure 7 shows the effect of the molar ratio of the substrates on the tri-
peptide synthesis catalyzed by alcalase. It can be seen that the optimum
molar ratio of the acyl donor to the nucleophile is 1:3, which achieves
the highest yield of the tripeptide. In some reports,[22,23] a high concentra-
tion of nucleophile was adopted in enzymatic synthesis of peptides,
rendering back a high yield. The high concentration of nucleophile is
advantageous for improving productivities of peptides because the deacyla-
tion step is the competitive reaction of the amide component and water.
FIGURE 6 Effect of reaction time on the yield of Z-Asp-Val-Tyr-OH.

530
K. Zheng et al.
FIGURE 7 Effect of molar ratio of substrates on the yield of Z-Asp-Val-Tyr-OH.
Under the optimum conditions of pH 10.0, 35ꢀC, acetonitrile=Na2CO3–
NaHCO3 buffer system (85:15 V=V), reaction time of 2.5 hr, and usage of
alcalase as catalyst, the maximum yield of Z-Asp-Val-Tyr-OH was 71.3%.
The HPLC profile of the unpurified tripeptide product is shown in
Figure 8 and the data on HPLC elution for Z-Asp-Val-Tyr-OH are summar-
ized in Table 3.
Purification of the Target Peptide Products
A Sephadex G-10 column was used to separate the target peptide pro-
ducts of Val-Tyr-OH and Z-Asp-Val-Tyr-OH from the crude products. The
elution profiles are shown in Figure 9 and Figure 10, respectively. The peak
of Z-Asp-Val-Tyr-OH in Figure 10 is lower than that of Val-Tyr-OH. This is
due to the high molar ratio (3:1) of the nucleophile (Val-Tyr-OH) to the
acyl donor (Z-Asp-OMe) in the reaction system.
FIGURE 8 HPLC profile of unpurified Z-Asp-Val-Tyr-OH.

Synthesis of a Precursor Tripeptide
531
TABLE 3 Data of HPLC Elution for Z-Asp-Val-Tyr-OH
Number
Name
Retention Time (min)
Area
Percent Area
Height
1
2
3
4
Val-Tyr-OH
Z-Asp-OH
Z-Asp-Val-Tyr-OH
Z-Asp-OMe
2.569
7.814
14.132
18.213
2733755
9026261
32698914
883573
5.48
18.08
74.67
1.77
289103
416759
848475
22689
FIGURE 9 Elution profile of the crude dipeptide product on a Sephadex G-10 column; 1 mL of the
crude dipeptide product (100 mg=mL) was loaded on the column.
FIGURE 10 Elution profile of the crude tripeptide product on a Sephadex G-10 column; 1 mL of the
tripeptide reaction mixture was loaded on the column.

532
K. Zheng et al.
FIGURE 11 Mass spectrum of the target peptide product Val-Tyr-OH. Ion mass (m=z) of 281.1
corresponds to Val-Tyr-OH.
Identification of the Target Peptide Products
The molecular weights of the peptide products, Val-Tyr-OH and Z-Asp-
Val-Tyr-OH, were confirmed by LC-MS as illustrated in Figure 11 and
Figure 12, respectively. The target product, Z-Asp-Val-Tyr-OH, was further
purified by HPLC. The structure (Scheme 1) was characterized by1H-NMR
(nuclear magnetic resonance) and elemental analysis measurements.
1H-NMR (300 MHz, DMSO-d6): d12.42 (s, 1H), 9.31 (s, 1H), 9.17 (s, 1H),
8.13 (s, 1H), 7.67 (d, J ¼ 9 Hz, 1H), 7.52 (d, J ¼ 9 Hz, 1H), 7.33 (s, 5H),
7.14 (t, J1 ¼ 9 Hz, J2 ¼ 9 Hz, 1H), 6.98 (d, J ¼ 9 Hz, 3H), 6.74 (t, J1 ¼ 6 Hz,
J2 ¼ 6 Hz, 1H), 6.63 (d, J ¼ 6 Hz, 2H), 5.02 (s, 2H), 4.38-4.27 (m, 1H),
FIGURE 12 Mass spectrum of the target peptide product Z-Asp-Val-Tyr-OH. Ion mass (m=z) of 530.2
corresponds to Z-Asp-Val-Tyr-OH.

Synthesis of a Precursor Tripeptide
533
SCHEME 1 The structural formulas of the target peptide product Z-Asp-Val-Tyr-OH.
4.20–4.16 (m, 1H), 1.97–1.88 (m, 1H), 0.99 (d, J ¼ 6 Hz, 1H), 0.78–0.71 (m,
6H) Elemental analysis of Z-Asp-Val-Tyr-OH, Calc.: 58.98% C; 5.86% H;
7.94% N; Found: 53.96% C; 5.688% H; 6.73% N.
CONCLUSIONS
In summary, we succeeded in synthesis of the Z-Asp-Val-Tyr-OH by a
combination of chemical and enzymatic methods. The chemical method
used here provides an opportunity to prepare Val-Tyr-OH in high yield at
a large scale with low cost. The linkage of the third amino acid Z-Asp-
OMe to Val-Tyr-OH was completed by an enzymatic method in organic sol-
vents. The industrial alkaline protease alcalase was successfully used as the
catalyst again for the different target peptide containing hydrophilic amino
acids. The optimum condition for Z-Asp-Val-Tyr-OH synthesis by using alca-
lase as a catalyst is of pH 10.0, 35ꢀC, acetonitrile=Na2CO3-NaHCO3 buffer
system (85:15 v=v), and reaction time of 2.5 hr, which achieves reasonable
yields of more than 70%.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the financial support of the Special
Research Grant from State Administration of Traditional Chinese Medicine
of China (2004ZDZX003).

534
K. Zheng et al.
REFERENCES
1. Audhya, T.; Goldstein, G. Comparative Efficacy of Various Routes of Administration of Thymopen-
tin (TP-5) with Consideration of Degradative Mechanisms. Int. J. Pept. Protein Res. 1983, 22(2),
187–193.
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