Optimal Synthesis of N-Ac-Phe-Gly-NH2
J. Agric. Food Chem., Vol. 57, No. 2, 2009 407
(4) Brandsch, M.; Brandsch, C.; Prasad, P. D.; Ganapathy, V.; Hopfer,
U.; Leibach, F. H. Identification of a renal cell line that
constitutively expresses the kidney-specific high-affinity H+/
peptide co-transporter. FASEB J. 1995, 9, 1489–1496.
(5) Ganapathy, M. E.; Brandsch, M.; Prasad, P. D.; Ganapathy, V.;
Leibach, F. H. Differential recognition of ꢀ-lactam antibiotics by
intestinal and renal peptide transporters, PEPT1 and PEPT2.
J. Biol. Chem. 1995, 270, 25672–25677.
(6) Ganapathy, M. E.; Prasad, P. D.; Mackenzie, B.; Ganapathy, V.;
Leibach, F. H. Interaction of anionic cephalosporins with the
intestinal and renal peptide transporters PEPT1 and PEPT2.
Biochim. Biophys. Acta 1997, 1324, 296–308.
a pH of 9.0, for which the dipeptide derivative yield could reach
95% or higher; however, the yield could be as low as 20% if
the enzyme activity was 50 U and the pH 7.0. A maximum
yield of 98.02% was predicted at this optimum condition, in
which the incubation time was 30.88 min; the reaction temper-
ature, 35.77 °C; the enzyme activity, 159.16 U; and the pH,
8.98. Extra experimental treatments at the optimum condition
were conducted, for which an average yield of 95.50 ( 0.58%
was obtained. The actual experimental result (95.50%) was in
satisfactory agreement with the predicted value (98.02%).
In the preliminary study, the independent variables were
considered individually. As the reaction time increased, the
dipeptide derivative yield also increased. The results of the
R-chymotrypsin-catalyzed N-Ac-Phe-Gly-NH2 synthesis at 30
°C obtained in this study were consistent with those obtained
by Abdus Salam et al. (12). Mishima et al. (14) recommended
that the optimum water content for dipeptide synthesis by
R-chymotrypsin be less than 10%; however, in this study, higher
yields of the product were obtained in a biphasic system
containing 96% buffer and 4% ethyl acetate. The experimental
results also revealed that R-chymotrypsin functioned as a catalyst
for N-Ac-Phe-Gly-NH2 synthesis under an alkaline condition
(pH 9.0-10.0) much better than under an acidic condition. The
results are similar to those observed in previous studies (23, 24).
However, fewer reports have focused on the optimization of
dipeptide synthesis by an experimental design. Therefore, in
our research, the optimization was obtained via CCRD for the
dipeptide derivative synthesis from N-acetyl-Phe-OEt and Gly-
NH2. The contour plots clearly indicate the relationships among
the manipulated parameters and the yield.
(7) Ganapathy, M. E.; Huang, W.; Wang, H.; Ganapathy, V.; Leibach,
F. H. Valacyclovir: A substrate for the intestinal and renal peptide
transporters PEPT1 and PEPT2. Biochem. Biophys. Res. Commun.
1998, 246, 470–475.
(8) Va´benø, J.; Lejon, T.; Nielsen, C. U.; Steffansen, B.; Chen, W.;
Ouyang, H.; Borchadt, R. T.; Luthman, K. Phe-Gly dipeptido-
mimetics designed for the di/tripeptide transporters PEPT1 and
PEPT2: Synthesis and biological investigations. J. Med. Chem.
2004, 47, 1060–1069.
(9) Hou, R. Z.; Zhang, N.; Li, G.; Huang, Y. B.; Wang, H.; Xiao,
Y. P.; Liu, Y. J.; Yang, Y.; Zhao, L.; Zhang, X. Z. Synthesis of
tripeptide RGD amide by a combination of chemical and
enzymatic methods. J. Mol. Catal. B: Enzym. 2005, 37, 9–15.
(10) Ogino, H.; Yamada, M.; Watanabe, F.; Ichinose, H.; Yasuda, M.;
Ishikawa, H. Peptide synthesis catalyzed by organic solvent-stable
protease from Pseudomonas aeruginosa PST-01 in monophasic
aqueous-organic solvent systems. J. Biosci. Bioeng. 1999, 88, 513–
518.
(11) Shen, H. Y.; Tian, G. L.; Ye, Y. H.; Wang, J. Non-coded amino
acids as acyl donor substrates for peptide bond formation catalyzed
by Thermoase in toluene. J. Mol. Catal. B: Enzym. 2005, 37, 26–
29.
Furthermore, the modeling of R-chymotrypsin-catalyzed
dipeptide synthesis was successfully developed by RSM. The
optimum condition for this synthesis in a buffer-ethyl acetate
(96:4, v/v) system was as follows: incubation time, 30.88 min;
reaction temperature, 35.77 °C: pH, 8.98; and enzyme activity,
159.16 U. The predicted highest dipeptide derivative yield was
98.02%. Two experimental runs at the optimum condition were
conducted, for which an average yield of 95.50 ( 0.58% was
obtained. This study provided a useful technique for finding
optimal operating conditions for N-Ac-Phe-Gly-NH2 synthesis.
Moreover, the technique could be applied to large-scale produc-
tion of the product in a bioreactor. We believe that the findings
in this study can assist further research in dipeptide synthesis.
(12) Abdus Salam, S. M.; Kagawa, K. I.; Kawashiro, K. R-Chymot-
rypsin-catalyzed peptide synthesis in frozen aqueous solution using
N-protected amino acid carbamoylmethyl esters as acyl donors.
Tetrahedron: Asymmetry 2006, 17, 22–29.
(13) Feliciano, A. S.; Dias, A. I.; Prazeres, D. M. F. The effect of
stirring and seeding on the Ac-Phe-Leu-NH2 synthesis and
crystallization in a reversed micellar system. Enzyme Microb.
Technol. 2000, 27, 264–269.
(14) Mishima, E.; Matsuyama, K.; Baba, M.; Chidori, M. Enzymatic
dipeptide synthesis by surfactant-coated R-chymotrypsin com-
plexes in supercritical carbon dioxide. Biotechnol. Prog. 2003,
19, 281–284.
(15) Miyazawa, T.; Nakajo, S. I.; Nishikawa, M.; Hamahara, K.;
Imagawa, K.; Ensatsu, E.; Yanagihara, R.; Yamada, T. R-Chy-
motrypsin-catalyzed peptide synthesis via the kinetically controlled
approach using activated esters as acyl donors in organic solvents
with low water content: Incorporation of non-protein amino acids
into peptides. J. Chem. Soc., Perkin Trans. 2001, 1, 82–86.
(16) Xing, G. W.; Li, F. Y.; Ming, C.; Ran, L. N. Peptide bond
formation catalyzed by R-chymotrypsin in ionic liquids. Tetra-
hedron Lett. 2007, 48, 4271–4274.
ACKNOWLEDGMENT
We express our appreciation to Dr. Cheryl Rutledge, Depart-
ment of English, Da-Yeh University, for her editorial assistance.
(17) Thust, S.; Koksch, B. Protease-catalyzed peptide synthesis for the
site-specific incorporation of R-fluoroalkyl amino acids into
peptides. J. Org. Chem. 2003, 68, 2290–2296.
(18) So, J. E.; Kang, S. H.; Kim, B. G. Lipase-catalyzed synthesis of
peptides containing D-amino acid. Enzyme Microb. Technol. 1998,
23, 211–215.
(19) Zhang, X. Z.; Wang, X.; Chen, S.; Fu, X.; Wu, X.; Li, C. Protease-
catalyzed small peptide synthesis in organic media. Enzyme
Microb. Technol. 1996, 19, 538–544.
(20) Yokozeki, K.; Hara, S. A novel and efficient enzymatic method
for the production of peptides from unprotected starting materials.
J. Biotechnol. 2005, 115, 211–220.
LITERATURE CITED
(1) Calvet, S.; Clape´s, P.; Torres, J. L.; Valencia, G.; Feixas, J.;
Adlercreutz, P. Enzymatic synthesis of X-Phe-Leu-NH2 in low
water content systems: Influence of the N-R protecting group and
the reaction medium composition. Biochim. Biophys. Acta 1993,
1164, 189–196.
(2) Pan, D.; Luo, Y.; Tanokura, M. Antihypertensive peptides from
skimmed milk hydrolysate digested by cell-free extract of Lac-
tobacillus helVeticus JCM1004. Food Chem. 2004, 91, 123–129.
(3) Fazzi, R.; Galimberti, S.; Pacini, S.; Testi, R.; Azzara`, A.;
Orciuolo, E.; Trombi, L.; Metelli, M. R.; Petrini, M. Bone and
bone marrow interactions: Hematological activity of osteoblastic
growth peptide (OGP)-derived carboxy-terminal pentapeptide Ø.
Action on human megakaryocytopoieses: focus on essential
thrombocythemia. Leuk. Res. 2004, 28, 1097–1105.
(21) Huang, Y. B.; Xiao, Y. P.; Wang, H.; Hou, R. Z.; Zhang, N.;
Wu, X. X.; Xu, L.; Zhang, X. Z. Chemo-enzymatic synthesis of
tripeptide RGD diamide in organic solvents. J. Biotechnol. 2005,
116, 51–59.