Enzymatic synthesis of the precursor of Leu-enkephalin in water-immiscible organic solvent systems Yukitaka Kimura, Kazuhiro Nakanishi,* and Ryuichi Matsuno D e p a r t m e n t o f F o o d Science and Technology, Faculty o f Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan

The precursor of Leu-enkephalin, Z-L-TyrGlyGly-L-Phe-L-LeuOEt, was synthesized from amino acid derivatives with three proteinases without the protection of the side chain of L-Tyr. First, Z-GlyGlyOBu t and Z-L-TyrGlyGlyOBu t were synthesized in quite a high yield, 83% and 99%, in an aqueous~organic biphasic system by papain and a-chymotrypsin, respectively. Then, Z-L-Phe-L-LeuOEt was synthesized by thermolysin from Z-L-Phe and L-LeuOEt either in buffer or in a biphasic system; the yields were 95% and 100%, respectively. The synthesis of Z-L-TyrGlyGly-L-Phe-L-LeuOEt from Z-L-TyrGlyGly and LPhe-L-LeuOEt was performed effectively by thermolysin immobilized on Amberlite XAD-7 in a buffer and in an aqueous~organic biphasic system, as well as in saturated ethyl acetate, while the yield was low in reactions by free thermolysin. In the reaction with the immobilized enzyme (IME) in saturated ethyl acetate, the maximum yield of the precursor of Leu-enkephalin was 68%. The reasons for effective synthesis with IME are: (1) higher concentration of L-Phe-L-LeuOEt inside support, which resulted in rising the rate of the synthesis reaction and protecting the competitive hydrolysis of Z-c-TyrGlyGly by thermolysin, (2) entrapment of the product inside the support where thermolysin could not act in the case of reaction in buffer, and (3) extraction of the product with the organic solvent in the case of reaction in a biphasic system or in saturated organic solvent.

Keywords:Enzymatic peptide synthesis; aqueous/organic biphasic systems; immobilized enzymes; Leu-enkephalin; papain; a-chymotrypsin; thermolysin; reactions in organic solvent

Introduction Peptides can be synthesized from amino acids with suitable blocking groups either chemically or enzymatically. The e n z y m a t i c method was first used by Bergmann and Fraenkel-Conrat in 1937. I E n z y m a t i c peptide synthesis has some merits o v e r chemical ones. F o r example, reaction can occur stereospecifically in mild conditions without the protection of side chains. One of the serious problems with the enzymatic method is the low equilibrium yield of the synthesis. There are Abbreviations: Z, N-benzyloxycarbonyl-; IME, immobilized enzyme; OBu t, tert-butyl ester; OEt, ethyl ester; Nps-, o-Nitrophenylsulfenyl-; Boc-, t-Butoxycarbonyl-; Bzl, O-Benzyl-; OTMB, trimethylbenzyl ester * Present address: Department of Biotechnology, Faculty of Engineering, Okayama University, Tsushimanaka, Okayama 700, Japan Address reprint requests to Dr. Ryuichi Matsuno, Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606, Japan Received 10 November 1988; revised 10 March 1989

272

Enzyme Microb. Technol., 1990, vol. 12, April

several ways to o v e r c o m e this. Precipitation of the condensation product in the reaction mixture gives a high yield. The addition of water-miscible organic cosolvent to the reaction mixture 2 is another way. Reaction in an aqueous/organic biphasic s y s t e m reported by Klibanov et al. 3 in 1977 shifts the equilibrium toward the product. This principle has been applied to the production of some useful peptides such as the precursor of aspartame. 4 Martinek et al. 5'6 theoretically evaluated the effects o f the use of biphasic system on the equilibrium yield. One of them is that increasing the volume ratio of the organic to the aqueous phase increases the equilibrium yield. Reaction in a large volume ratio of organic to aqueous phases is similar to reaction in an immiscible organic solvent. The kinetics of the enzymatic synthesis of dipeptides in an aqueous/organic biphasic system were also studied. 7,8 The results were used in the design of effective continuous reactions in organic solvent by I M E 4 and of batch enzymatic synthesis of oligopeptide, des-Tyr~-Leu-enkephalin, in a biphasic system. 9 © 1990 Butterworth

Publishers

Enzymatic synthesis of Leu-enkephalin: Y. Kimura et al. L-Tyr(Y)

GIy(G)

Gly(G) L-Phe(F) L-Leu(L)

IZ

-~- 4-OBut /

z

H2/Pd

Z--

1

~'~°

OBut

OBut Z-

Z ,-Chyr~o~p~

!OBut Z

Z

~ CH3COOH~'

Z

Th~ma~in

-OEt Thern'oly~n

OEt OEt OEt

Figure 1

Outline of the synthesis of the precursor of Leu-enkephalin. Z is a protecting group for the amino terminal. OBu t and OEt are protecting groups for the carboxyl terminal. The peptide bonds between L-Tyr and Gly, and Gly and Gly w e r e synthesized with cx-chymotrypsin and papain, respectively. The bonds between L-Phe and L-LeuOEt, and Gly and L-Phe w e r e formed by thermolysin. Arrows show the removal of protecting groups as described in Materials and methods

Here, the proteinase-catalysed synthesis of the precursor of Leu-enkephalin, j° an opioid peptide, was studied as another model reaction of oligopeptide synthesis. The brief scheme of synthesis of this pentapeptide adopted in this study is shown in Figure 1. Although Leu-enkephalin has been synthesized enzymatically by Kullmann j~ and Wong e t a l . , j2 by their methods the yields were negligible or low unless the side chain of L-tyrosine was blocked. Here, we attempted to synthesize the Leu-enkephalin precursor originally from Z-L-Tyr, Z-Gly, GlyOBut, Z-L-Phe, LLeuOEt, by using reaction in an aqueous/organic biphasic system or reaction in a saturated organic solvent for each step in order to increase the yield. Especially, in the synthesis of Z-L-TyrGlyGIy-L-Phe-L-LeuOEt from Z-L-TyrGIyGIy and L-Phe-L-LeuOEt, attention was paid to the effects of the support used for immobilization of the enzyme, and the partition of reactants among three phases on the yield of Z-L-TyrGlyGly-LPhe-L-LeuOEt.

Materials and methods

Materials Papain (recrystallized twice) and a-chymotrypsin (recrystallized three times) from Sigma Chemical Co. (USA) were used without further purification. Crystalline thermolysin (crystallized once) was supplied by Daiwa Kasei KK. (Osaka, Japan). The enzyme was used without further purification except when the hydrolytic activity of free thermolysin towards Z-L-TyrGlyGly was examined. Z-Gly, Z-L-Tyr, Z-e-Phe, L-LeuOEt • HCI, authentic Leu-enkephalin, and a 25% HBr solution in acetic acid were obtained from the

Peptide Institute, Inc. (Osaka, Japan). GlyO-Bu t • HCI was the product of Kokusan Chemical Works (Tokyo, Japan). As a reagent in the buffer, 2-(N-Morpholino)ethanesulfonic acid (Mes) from Dojin Chemical Ltd. (Kumamoto, Japan) was used. All other chemicals were of analytical grade and were purchased either from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) or Nacalai Tesque, Inc. (Kyoto, Japan).

Analytical methods Substrates and products were analyzed by HPLC (LC6A; Shimadzu Corp., Kyoto, Japan) with an ODS column (4.6 x 150 mm Cosmosil 5C18-P packed column; Nacalai Tesque) with use of a mixture of acetonitrile/ water (60/40, v/v) adjusted to pH 2.5 with phosphoric acid, as the eluent with the flow rate of 0.8 mi mlEluted reactants were detected by a UV detector at 254 nm. Thus, reactants were detected as N-benzyloxycarbonyl, tyrosine, or phenylalanine residues.

Synthesis of Z-GlyGlyOButfrom Z-Gly and GlyOBu t in buffer The papain-catalysed condensation reaction between Z-Gly and GlyOBu t • HCI was carried out at 40°C in 0.05 M Mes/NaOH buffer, pH 6, containing 0.1% 2mercaptoethanol. The mixture was kept at pH 6 with 1 M NaOH under vigorous magnetic stirring for 20 h. The enzyme concentration was 0.43 mM. The final concentrations of the substrates, Z-GIy and GlyOBu t • HCI, were 20 mM. Portions (0.5 ml) taken from the reaction mixture were dissolved in 0.025 ml of 6 M HC1, diluted with an appropriate amount of acetonitrile/water (70/30, v/v), adjusted to pH 2.5 with phosphoric acid, and analyzed by HPLC. The acetonitrile/water solution at pH 2.5 in this composition was used for dilution of reactants in a buffer, for dissolution of dried reactants from organic phase, or for extraction of reactants from supports of IME before analysis by HPLC throughout the experiments.

Synthesis of Z-GlyGlyOBu t from Z-Gly and GlyOBu t in an aqueous~organic biphasic system Throughout the experiments in an aqueous/organic biphasic system, we used ethyl acetate saturated with 0.05 M Mes solution or 0.25 M Tris solution at 40°C for a solvent of organic phase, and 0.05 M Mes/NaOH buffer or 0.25 M Tris/HCl buffer saturated with ethyl acetate at 40°C for a buffer of aqueous phase. Z-GIyGlyOBu ~was synthesized in a biphasic system via the papain-catalysed condensation of Z-GIy and GlyOBu t. The reaction was started by the mixing of 15 ml of saturated ethyl acetate containing 0.2 M Z-Gly and 0.05% 2-mercaptoethanol with 3 ml of saturated 0.05 M Mes/NaOH buffer containing 1.5 M GlyOBu t • HCI, 0.05% 2-mercaptoethanol, 2.55 mM papain, and 5 mM CaCI 2. The reaction mixture was kept at pH 6 with 1 M NaOH and vigorously stirred magnetically for 20 h. Portions 0.5 ml of each of the aqueous and organic phases were sampled. The former was analyzed as

Enzyme Microb. Technol., 1990, vol. 12, April

273

Papers described above. The latter was first e v a p o r a t e d to dryness under reduced pressure, dissolved in an appropriate amount of acetonitrile/water (70/30, v/v), and analyzed by H P L C .

Preparation of GlyGlyOBu t • CH3COOH The ethyl acetate solution containing Z - G l y G l y O B u t was e v a p o r a t e d under reduced pressure. The residue was dissolved in an appropriate amount of acetonitrile/ water (60/40, v/v) adjusted to p H 2.5 with phosphoric acid. Z - G l y G l y O B u t was separated from the other components (2-mercaptoethanol in particular) by passage through an ODS column (25 x 120 mm) by use of the same solution as before as eluent at the flow rate of 2 ml m i n - J. F r o m the fractions, only Z - G i y G l y O B u t was extracted into an appropriate amount of ethyl acetate and e v a p o r a t e d under reduced pressure. The protective group Z was r e m o v e d from Z - G l y G I y O B u t by catalytic hydrogenation with palladium black and H2 gas in the solution (water/acetic acid/methanol = 5/2/3 (v/v/ v)). ]3 The resultant solution was e v a p o r a t e d to produce G I y G l y O B u t • C H 3 C O O H as syrup.

Synthesis of Z-L-TyrGlyGlyOBu t from Z-L-Tyr and GlyGlyOBu t in buffer Z - k - T y r G l y G l y O B u t was synthesized from Z-L-Tyr and G l y G l y O B u t . C H 3 C O O H via a condensation reaction catalysed by c~-chymotrypsin at 40°C in 0.25 M Tris/HCl buffer, pH 7, under magnetic stirring, kept at pH 7 with 1 M N a O H . The final concentrations of Z-LTyr, G l y G l y O B u t • C H 3 C O O H , and ~x-chymotrypsin in the mixture were 5, 50, and 0.08 raM, respectively. After 20 h of reaction, a 0.5-ml portion of the mixture was sampled and analyzed by H P L C as before.

Synthesis of Z-L-TyrGlyGlyOBu t from Z-L-Tyr and GlyGlyOBu t in an aqueous~organic biphasic system Z - L - T y r G I y G l y O B ut was synthesized in a biphasic system via a condensation reaction catalysed by c~-chymotrypsin from Z-L-Tyr and G l y G l y O B u t • C H 3 C O O H at 40°C. The reaction was started by the mixing of 10 ml of saturated ethyl acetate containing 40 mM Z-L-Tyr and 2 ml of saturated 0.25 M Tris/HC! buffer containing both 400 mM G l y G l y O B u t • C H 3 C O O H and 0.48 mM c~-chymotrypsin. The volume ratio of the organic to aqueous phases was 5. The reaction mixture was vigorously stirred with a magnet for 20 hours. A I-ml portion of the organic phase and a 0.2-ml portion of the aqueous phase were sampled and analyzed by H P L C as before.

Synthesis of Z-L-Phe-L-LeuOEt from Z-L-Phe and L-LeuOEt in buffer Z-k-Phe-L-LeuOEt was synthesized via a condensation reaction catalysed by thermolysin from Z-e-Phe and L-LeuOEt • HCI at 40°C in 0.25 M Tris/HCI buffer, p H 7.0, containing 5 mM CaCI 2. The final concentrations of Z-L-Phe, L-LeuOEt • HCI, and thermolysin in the mixture were 80, 120, and 0.053 raM, respectively. Immediately, the reaction mixture was divided into 0.5-

274

Enzyme Microb. Technol., 1990, vol. 12, April

mi portions. Each portion was incubated at 40°C. At appropriate times, 0.025 ml of 6 M HCI was added to stop the reaction; the mixture was diluted with an appropriate amount of acetonitrile/water (70/30, v/v), and analyzed by H P L C .

Synthesis of Z-L-Phe-L-LeuOEt from Z-Phe and L-LeuOEt in an aqueous~organic biphasic system Z-L-Phe-L-LeuOEt was synthesized from Z-L-Phe and L-LeuOEt • HC1 via a condensation reaction catalysed by thermolysin in a biphasic system at 40°C. The reaction was started by the mixing of 5 ml of saturated ethyl acetate containing 80 mM Z-L-Phe and 5 ml of saturated 0.25 M Tris/HCl buffer, p H 7.0, containing 120 mM L-LeuOEt • HCI, 5 mM CaCl 2, and 0.11 mM thermolysin. Then, 0.5-ml portions of the aqueous and organic phases were sampled and analyzed by H P L C as before.

Preparation of Z-L-TyrGlyGly The organic phase of the solution of Z-L-TyrGlyGiyOBu t produced was washed twice with 5% citrate solution, water, 5% liquid a m m o n i a , and water in a separatory funnel. The ethyl acetate containing Z-L-TyrGIyG l y O B u t was dehydrated with MgSO 4 and e v a p o r a t e d under reduced pressure. The powder Z-L-TyrGlyGlyOBu t was dissolved in formic acid at 50 mM and left for 8 h to r e m o v e tert-butanol from the Z - L - T y r G I y G l y O B ut. The mixture was e v a p o r a t e d twice in water under reduced pressure to obtain Z-L-TyrGlyGly.

Preparation of L-Phe-L-LeuOEt • HBr The organic phase of the solution of Z-L-Phe-L-LeuOEt produced was washed in the same way as for Z-LT y r G l y G l y O B u t. The Z-group was r e m o v e d from Z-LPhe-L-LeuOEt by the H B r / A c O H method j4,15 to obtain L-Phe-L-LeuOEt • HBr.

Synthesis of Z-L-TyrGlyGly-L-Phe-L-LeuOEt from Z-t.-TyrGlyGly and L-Phe-L-LeuOEt in buffer Z-L-TyrGlyGIy-L-Phe-L-LeuOEt was synthesized via a condensation reaction catalysed by thermolysin from Z-L-TyrGiyGly and L-Phe-L-Leu-OEt • H B r at 40°C in 0.05 M M e s / N a O H buffer, pH 4.0-7.0, containing 5 mM CaCI 2. The final concentrations of Z-L-TyrGIyGly, L-Phe-L-LeuOEt • HBr, and thermolysin in the reaction mixture were I0, 10, and 0.053 mM, respectively. Immediately,the reaction mixture was divided into 0.5ml portions. Each portion was incubated at 40°C. At appropriate times, 0.025 ml of 6 M HCI was added to stop the reaction; the mixture was diluted with an appropriate amount of acetonitrile/water (70/30, v/v), and analyzed by H P L C .

Synthesis of Z-L-TyrGlyGly-L-Phe-L-LeuOEt from Z-L-TyrGlyGly and L-Phe-L-LeuOEt with IME in buffer Thermolysin was immobilized onto Amberlite XAD-7 (Rohm & H a a s Co., USA) by cross-linking after ad-

Enzymatic synthesis of Leu-enkephalin: Y. Kimura et al. sorption as described previously. 4 One-fifteenth of a gram wet IME, which had been incubated in 0.05 M M e s / N a O H buffer, pH 4.5-7.0, containing 5 mM CaCI2, was put in each vial with 1 ml of the same buffer containing 10 mM Z-L-TyrGlyGly and 10 to 20 mM LPhe-L-LeuOEt • HBr, and the mixtures in several vials were incubated at 40°C with shaking. At appropriate times, 0.02 ml of 6 M HC1 was added to stop the reaction and the mixture was diluted with 2 ml of acetonitrile/ water (70/30, v/v), shaken for 20 min, and assayed by H P L C . By this procedure, almost all of the products that had accumulated in the support were extracted.

Synthesis of Z-L-TyrGlyGly-L-Phe-L-LeuOEt from Z-LTyrGlyGly and L-Phe-L-LeuOEt in an aqueous~organic biphasic system Z-L-TyrGlyGly-L-Phe-L-LeuOEt was synthesized from Z-L-TyrGlyGly and L-Phe-L-LeuOEt via a condensation reaction catalysed by thermolysin in a biphasic system at 40°C. When the effect of the aqueous phase pH on the synthetic reaction was studied, the volume ratio of an organic to aqueous phase was set to one. Six milliliters of saturated ethyl acetate containing 10 or 100 mM L-Phe-L-LeuOEt and saturated 0.05 M M e s / N a O H buffer of various pHs (4-7) containing 10 mM Z-L-TyrGIyGly, 5 mM CaCI 2, and 0.11 mM thermolysin were mixed under vigorous magnetic stirring, incubated for 24 h. The pH of the reaction mixture was controlled at the prescribed value with 1 M NaOH. Then 0.5-ml portions of the aqueous and organic phases were sampled and analyzed by H P L C as before. The synthetic reaction was also performed changing the volume ratio between 1 and 15 as follows. Saturated ethyl acetate containing 5 mM Z-L-TyrGIyGly and 5 mM L-Phe-L-LeuOEt and saturated 0.05 M M e s / N a O H buffer containing 5 mM CaC12 and thermolysin were mixed in the volume ratio of organic to aqueous phase of 1 to 15. Here, the overall e n z y m e concentration of the biphasic system was set at 0.053 mM. The reaction mixture was incubated at 40°C with the starting pH of 4.5 with vigorous magnetic stirring. At appropriate times, portions of the organic and aqueous phases were sampled in an amount proportional to the volume ratio, and assayed by H P L C as before.

Synthesis of Z-L- TyrGlyGly-L-Phe-L-LeuOEt from Z-L-TyrGlyGly and L-Phe-L-LeuOEt with IME in an aqueous~organic biphasic system Two-fifteenths of a gram of IME were preincubated in a 0.05 M M e s / N a O H buffer saturated with ethyl acetate, pH 4.5, containing 5 mM CaC12, and added to a mixture of 1 ml of saturated ethyl acetate and 1 ml of saturated 0.05 M M e s / N a O H buffer at pH 4.5 containing 10 mM Z-L-TyrGlyGly, 10 mM L-Phe-L-LeuOEt • HBr, and 5 mM CaCl 2 with shaking at 40°C. At appropriate times, the phases were filtered on a G-2 glass filter, and 0.2-ml portions of the organic and aqueous phases were sampled and analyzed by H P L C as before. The IME was put in 1 ml of acetonitrile/water (70/30, v/v), shaken for 20 min, and analyzed by H P L C to

assay the products, substrates, and other reactants in the supports.

Synthesis of Z-L-TyrGlyGly-L-Phe-L-LeuOEt from Z-L-TyrGlyGly and L-Phe-L-LeuOEt with IME in an organic solvent One-fifteenth of a gram of IME preincubated in 0.05 M M e s / N a O H buffer saturated with ethyl acetate, pH 4.5-7.0, containing 5 mM CaCI 2 was brought into contact with 3 ml of saturated ethyl acetate containing 5.5 mM Z-L-TyrGlyGIy and 5.5 to 55 mM L-Phe-L-LeuOEt with shaking at 40°C. The resulting concentration of ZL-TyrGlyGly was near the solubility limit in saturated ethyl acetate at 40°C. At appropriate times, 0.2-ml portions of the organic phase were sampled and analyzed by H P L C as before.

Hydrolysis of Z-L-TyrGlyGly-L-Phe-L-LeuOEt by free thermolysin One milliliter of 0.05 M M e s / N a O H buffer, pH 4.5, containing 33% acetonitrile, 4.3 mM Z-L-TyrGlyGly-LPhe-L-LeuOEt, 5 mM CaCI 2, and 0.053 mM thermolysin were divided into 0.2-ml aliquots that were incubated at 40°C. At appropriate times, 0.02 ml of 6 M HCI and 0.5 ml of acetonitrile/water (70/30, v/v) were added, and the concentrations of reactants were measured by HPLC.

Measurement of the hydrolytic activity of free thermolysin towards Z-L-TyrGlyGly To measure the hydrolytic activity of thermolysin towards Z-L-TyrGIyGIy at the peptide bond between Tyr and Gly, 0.8 ml of 0.05 M M e s / N a O H buffer (pH 7.0) containing 5 mM CaCI 2 and 12.5 mM Z-L-TyrGlyGIy was mixed with 0.2 ml of the enzyme solution (0.080 mM, pH 7.0). The reaction mixture was divided into 0.2-mi aliquots which were incubated at 40°C. At appropriate times, 0.02 ml of 6 M HCI and 0.5 ml of acetonitrile/water (70/30, v/v) were added and the concentrations of Z-L-TyrGlyGly and Z-L-Tyr were measured. To confirm further the hydrolytic activity of thermolysin towards Z-L-TyrGIyGly, the same experiment was performed with the enzyme solution (100/zg ml J, pH 6.0) purified electrophoretically as described in the next paragraph.

Purification of thermolysin One milliliter of sample solution containing 40% glycerol, 4% 2-mercaptoethanoi, 0.005% bromophenol blue, and 2 mg thermolysin was separated by electrophoresis with 7.5% acrylamide gel in the refrigerator for 8 h. Three parts of the gel, each containing a protein band, were cut out and put in different cellophane tubes with 1 ml of 0.05 M M e s / N a O H buffer (pH 6.0) containing 5 mM CaCI 2. The tubes were dialyzed against the same 1 1 of buffer at 4°C overnight. The suspensions in the tubes were filtered with a G-2 glass filter. The filtrate sample that caused the synthesis of Z-L-Phe-L-PheOMe

Enzyme Microb. Technol., 1990, vol. 12, April

275

Papers from Z-L-Phe and L-PheOMe • HCI 4 was used as the purified enzyme solution.

Measurement of the partition of the reactants between the support phase (solid) and the aqueous or organic phases Partition ratios of the reactants were measured as follows, with an assumption of negligible diffusion resistance in the support phase. When IME was used in a buffer, the partition ratio of reactants was calculated during the course of the synthesis of Z-e-TyrGlyGlyL-Phe-e-LeuOEt with IME because of the solubility limit of the product in the buffer. At appropriate times, 0.2-mi portions were taken from the reaction solution and analyzed by HPLC to measure the concentrations of substrate and product in the aqueous phase (Cw). Then, to measure the concentrations of substrate and product inside the support (Ci.), immediately after sampling, 0.025 ml of 6 M HCI and 2 ml of acetonitrile/ water (70/30, v/v), pH 2.5, were added to stop the reaction and to extract material in the solid phase. After being shaken for 20 rain, the concentrations of substrates and product were measured (C,op). The concentration, Ci,, was calculated as (Capo " (Vw + Vi, + 2.025) - Cw • Vw)/Vin, where V~, and Vw are the volumes of the solid and buffer phases, respectively. In an aqueous/organic biphasic system, the partition ratio of the reactants in the solid phase to those in the aqueous or organic phases was measured as follows. First, IME was inactivated by incubation in 0.5 M acetate buffer, pH 4.0, for 30 rain at 65°C. One milliliter of saturated ethyl acetate was mixed with 1 ml of saturated 0.05 M Mes/NaOH buffer containing 10 mM Z-LTyrGlyGly, 10 mM L-Phe-e-LeuOEt • HBr, and 5 mM CaCI z. Then inactivated IME (wet 0.134 g) preincubated with a saturated buffer, pH 4.5, was added and the suspension was shaken for 30 min at 40°C and filtered with a G-2 glass filter. The concentrations of Z-L-TyrGlyGly and L-Phe-L-LeuOEt of both phases (Cw and Corg) of the filtrate were measured. An appropriate amount of inactivated IME was suspended in 1 ml of acetonitrile/water (70/30, v/v), and analyzed by HPLC to measure C~, as before. To measure the partition of the product, inactivated IME (0.134 g) was put into a mixture of I ml of saturated ethyl acetate containing 5.5 mM Z-L-TyrGlyGly-L-PheL-LeuOEt and l ml of saturated 0.05 M Mes/NaOH buffer containing 5 mM CaC12, pH 4.5. As described above, the concentrations of Z-L-TyrGlyGIy-L-Phe-LLeuOEt were measured (Corg, Cw, Cin). In the organic solvent system, the partition ratio of the reactants in the solid phase to those in the organic phase was measured as follows. One-fifteenth of a gram of inactivated IME was incubated with 0.05 M Mes/ NaOH buffer saturated with ethyl acetate, pH 4.5, containing 5 mM CaCl 2 for 30 min at 40°C, and put in 1 ml of saturated ethyl acetate containing 5.5 mM Z-LTyrGlyGly and 5.5 mM L-Phe-L-Leu-OEt or 5.5 mM Z-L-TyrGlyGIy-L-Phe-L-LeuOEtwith shaking at 40°C. After 30 min, IME was filtered with a G-2 glass filter 276

Enzyme Microb. Technol., 1990, vol. 12, April

and then put in 1 ml of acetonitrile/water (70/30, v/v) to extract the reactants in the supports. The filtered organic solution was evaporated in reduced pressure. Dried reactants were dissolved in the appropriate amount of acetonitrile/water (70/30, v/v). Both acetonitrile/water solutions were analyzed by HPLC to determine the concentrations of reactants.

Measurement of physical data of the reaction intermediates and products The reaction intermediates and products were purified as described in Preparation of GlyGlyOBut • CH3COOH. The following spectroscopic and analytical instruments were used: EI-MS, JEOL JMS-DX 300 (70 eV, 300/zA); SIMS, HITACHI M-80; ORD, Jasco Model J-5; IH-NMR, JEOL GX 400 (400 MHz, ref. TMS)

Results and discussion

Synthesis of Z-GlyGlyOBu t, Z-L-TyrGlyGlyOBu t, and Z-L-Phe-L-LeuOEt The yield of Z-GIyGIyOBut synthesized from Z-GIy and GlyOBu t with papain and that of Z-L-TyrGlyGlyOBu t synthesized from Z-L-Tyr and GlyGlyOBut with ~chymotrypsin was 9% and 2% in buffer, respectively. It increased to 83% and 99%, respectively, in an aqueous/ organic biphasic system. Thus, the organic solvent was effective in sifting the equilibrium in favor of the products. The yield of Z-L-Phe-e-LeuOEt synthesized from Z-e-Phe and e-LeuOEt with thermolysin was as high as 95% even in buffer, because the product precipitates. Almost 100% of Z-L-Phe was converted to Z-LPhe-e-LeuOEt in an aqueous/organic biphasic system. With condensation catalysed by papain or ~-chymotrypsin, tert-butyi ester was used for the amine substrate instead of ethyl ester. One reason was that ethyl ester is easily fiydrolysed by papain and ~-chymotrypsin, j6,t7 but tert-butyl ester is not easily. The other was that the partition of the product with tert-butyl ester is higher towards the organic phase compared to that with ethyl ester, resulting in a higher yield of the product. On the other hand, thermolysin does not hydrolyse ethyl ester, is With e-Phe-L-LeuOBut as the amino component, the synthetic rate of Z-e-TyrGlyGly-e-Phe-LLeuOBu t was too low in a biphasic system, probably due to a low partition of L-Phe-L-LeuOBut towards the aqueous phase. Thus, ethyl ester substrate was used for this thermolysin-catalysed reaction.

Hydrolysis of Z-L-TyrGlyGly and Z-L-TryGlyGly-L-Phe-L-LeuOEt by thermolysin The hydrolysis of Z-L-TyrGlyGly and Z-L-TyrGlyGlye-Phe-e-LeuOEt by thermolysin significantly affects the yield of the product. Considering the reported substrate specificity of thermolysin, it was not expected that Z-e-TyrGlyGly would be hydrolysed at a bond beween L-Tyr and Gly. However, preliminary experiments suggested the bond between Tyr and Gly was

Enzymatic synthesis of Leu-enkephalin: Y. Kimura et al. I

I

I

r

,-,4

~2 -

~1 ~

0

I

I

I

1 rime (h)

.m

2

Figure 2 Course of hydrolysis of Z-L-TyrGlyGly-L-Phe-L-LeuOEt by thermolysin in acetonitrile/water (1/2, v/v), pH 4.5. (O) Z-LTyrGlyGly-L-Phe-L-LeuOEt, (/k) Z-L-Tyr, (A) Z-L-TyrGlyGly; (.) Z-L-TyrGlyGly-L-Phe; (I') mixture of GlyGly-L-Phe-L-LeuOEtand

L-Phe-L-LeuOEt. Initial concentration of Z-L-TyrGlyGly-L-PheL-LeuOEt, 4.3 mM

cleaved. Thermolysin hydrolysed I 1% Z-L-TyrGIyGly to Z-L-Tyr in 10 rain at 40°C. Since this was unexpected, this finding was confirmed by the use of the purified enzyme obtained by electrophoresis (data not shown). Furthermore, the hydrolysis of a bond between LTyr and Gly of the desired condensation product, ZL-TyrGlyGly-L-Phe-L-LeuOEt, which might strongly affect the yield of the condensation reaction, was examined by the use of free thermolysin in a buffer, pH 4.5, containing 33% acetonitrile to dissolve the substrate. As shown in Figure 2, thermolysin produced Z-L-Tyr, Z-L-TyrGIyGly, probably a mixture of GlyGly-L-PheL-LeuOEt and L-Phe-L-LeuOEt, and less Z-L-TyrGlyGly-L-Phe.

Synthesis of Z-L-TyrGlyGly-L-Phe-L-LeuOEt from Z-I.-TyrGlyGly and L-Phe-L-LeuOEt by free thermolysin Figure 3A shows the course of the condensation reaction in a buffer at pH 4.5. As the substrates decreased, four main products were produced: Z-L-TyrGlyGlyL-Phe-L-LeuOEt, Z-L-Tyr, Z-L-Tyr-L-Phe-L-LeuOEt, and Z-L-Tyr-L-Phe-L-Phe-L-LeuOEt. The concentration of the desired product, Z-L-TyrGIyGIy-L-Phe-LLeuOEt, was low. The maximum yield was only 2.7%. Z-L-TyrGlyGly was hydrolysed by thermolysin to yield Z-L-Tyr, as mentioned above, which is one reason for the low yield of the product. Another reason was the formation of byproduct via complex reactions. Z-LTyr-L-Phe-L-LeuOEt condensed from Z-L-Tyr and LPhe-L-LeuOEt was hydrolysed into Z-L-Tyr-L-Phe and L-LeuOEt. From Z-L-Tyr-L-Phe and L-Phe-L-LeuOEt, Z-L-Tyr-L-Phe-L-Phe-L-LueOEt was produced via a successive condensation reaction. At 24 h, there accumulated a large amount of a mixture of byproducts containing L-phenylalanine residue such as L-Phe-L-

Phe and GIyGly-L-Phe produced by complex multiple reactions including condensation and hydrolysis. There also appeared a small amount of Z-L-Tyr-L-LeuOEt and Z-L-Tyr-L-Phe during the reaction (data not shown in Figure 3A). In the synthetic reaction at pH 6-7, more Z-L-Tyr was produced than at lower pH, and therefore the desired product decreased (data not shown). In Figure 3B, the course of the synthesis of Z-LTyrGlyGly-L-Phe-L-LeuOEtin an aqueous/organic biphasic system (pH 4.5) is shown. The synthetic rate of the desired product was as low as the synthesis in a buffer. Compared with synthesis in a buffer (Figure 3A), Z-L-Tyr was produced quickly as the substrates decreased. An undesired product, Z-L-Tyr-L-Phe-LLeuOEt, was produced in large amounts at the later stage of the synthesis because of the increase of Z-LTyr. At 24 h a large amount of mixture of byproducts containing L-phenylalanine residue such as GlyGly-LPhe and L-Phe-L-Phe was produced. Other products were few. The yield of the desired product did not increase as the volume ratio of the organic to the aqueous phase was increased to 15 (data not shown). In this biphasic system, a high hydrolytic rate of Z-LTyrGlyGly into Z-L-Tyr might be the main reason for the low product yield, and the rate might be ascribed to the partition of the two substrates between the two phases. Column 2 in Table l shows the partition of substrates and product between the two phases. The aqueous phase, where the enzyme reaction proceeded, contained much less of the reactant L-Phe-L-LeuOEt than Z-L-TyrGlyGly. Because of the low concentration of L-Phe-L-LeuOEt in the aqueous phase, the amount of Z-L-TyrGlyGly-enzymecomplex which proceeds to hydrolysis was larger than that of the substrate-enzyme complex which produces the desired product. Consequently, the rate of formation of Z-L-Tyr was high. Z-L-Tyr was combined with L-Phe-L-LeuOEt, producing Z-L-Tyr-L-Phe-L-LeuOEt. Since this compound partitioned in favor of the organic phase, further 10

0

,

]

3

5

240 1 Time (h)

3

5

Figure 3 Courses of the synthesis of Z-L-TyrGlyGly-L-Phe-L-LeuOEt from Z-L-TyrGlyGly and L-Phe-L-LeuOEt by free thermolysin in a buffer solution at initial pH 4.5 (A) and in an aqueous/organic biphasic system at pH 4.5 (B). (0) L-Phe-L-LeuOEt; (&) Z-L-TyrGlyGly; (©) Z-L-TyrGlyGly-L-Phe-L-LeuOEt; (A) Z-L-Tyr; (1-])Z-L-

Tyr-L-Phe-L-LeuOEt; (V) Z-L-Tyr-L-Phe-L-Phe-L-LeuOEt;(V) mixture of byproducts containing L-phenylalanine residue such as GlyGly-L-Phe and L-Phe-L-Phe

Enzyme Microb. Technol., 1990, vol. 12, April

277

Papers Table 1 Ratios of the concentrations of the substrates and the desired product in the aqueous phase, the organic phase, and the phase inside the support With support Without support Biphasic system a

Buffer solution b system

Cw/ Corg

Cin/Cw

Reactants Z-L-TyrGlyGly L-Phe-L-LeuOEt Z-L-TyrGlyGly-L-Phe-L-LeuOEt

1.2 5.7 x 10 1 1.2 × 10 3

Organic solvent d system

Biphasic system c

3.5 x 10 1.6 x 10 >8.1 × 10 3

Cin/Cw

Cin/Corg

Cin/Corg

3.1 8.8 >7.2 × 10 2

3.4 4.2 9.0 × 10 1

2.1 7.5 1.0

a pH of the aqueous phase was 4.5 b Initial pH was 5.0 c Initial pH was 4.5 d Support was preincubated at pH 4.5. The concentrations of the reactants inside the support were calculated on the basis of the w h o l e v o l u m e of the support

multiple reactions, including hydrolysis and condensation which occurred in synthesis in a buffer, were less. Thus, the mechanisms of synthesis in the biphasic system were different from those in the buffer. It was expected that the yield of product in synthesis would increase with increased concentration of amine substrate, L-Phe-L-LeuOEt7 because the concentration of L-Phe-L-LeuOEt in an aqueous phase increases even if the partition coefficient is kept constant. When the concentration of L-Phe-L-LeuOEt was increased to 100 mM, the yield of the desired product increased to 25% because the concentration of L-Phe-L-LeuOEt in an aqueous phase was also high, though the partition was constant.

Synthesis of Z-L-TyrGlyGly-L-Phe-L-LeuOEt from Z-L-TyrGlyGly and L-Phe-L-LeuOEt by IME The reaction with IME was done in an effort to increase the yield. As seen from column 3 of Table 1, the partition of substrates and product between buffer and the solid phase was in favor of the solid phase, which might be expected to increase the yield of product. Figure 4A shows the synthesis of Z-L-TyrGIyGly-L-Phe-L-LeuOEt from Z-L-TyrGlyGly and L-Phe-L-LeuOEt by IME in a buffer. The optimum pH for the yield of desired product was 5.0. The yield of Z-L-TyrGlyGly-L-Phe-LLeuOEt was much higher than with free enzyme. In this case, the desired product and Z-L-Tyr-L-Phe-LLeuOEt were accumulated only in the support. Thus, the nature of the support was important for improvement of the yield. This behavior may be due to two reasons. First, the high partition of L-Phe-L-LeuOEt to the inside of the support, where the enzyme was concentrated, increased the rate of synthesis, even though the rate of hydrolysis of Z-L-TyrGlyGly was the same as with free enzyme. It is the common observation in thermolysincatalysed synthesis that the rate is proportional to the concentration of the amine components. 7 Second, precipitation of Z-L-TyrGlyG|y-L-Phe-L-LeuOEt inside the support would prevent the product from the attack 278

Enzyme

Microb.

Technol.,

1990, v o l . 12, A p r i l

of enzyme and thus shift the equilibrium toward the product. In another view, this second reason may be equivalent to the following. Since the support is not homogeneous, there may be regions where the enzyme is absent. Strong adsorption of the desired product in such regions would also prevent enzyme attack. The second reason is also supported by the experimental observation that the reaction in the presence of free enzyme and support, not carrying the enzyme, produced the product in a higher yield (I 1%) than that in the absence of the support. Figure 4B shows the synthesis of Z-L-TyrGlyGiy-LPhe-L-LeuOEt from Z-L-TyrGlyGly and L-Phe-L-LeuOEt with 1ME in an aqueous/organic biphasic system at initial pH 4.5. The maximum yield of the desired product (23%) was higher than with the free enzyme (Figure 3B). This was the result of partitions of substrates and product among the three phases, that is, 1ME (solid), buffer, and organic phases, shown in col-

10 ~ k '

'

'

'A. . . .

~ '

' c

--o-

0

1

3

5

240 1

3 Time (h)

5

240 1

3

5

24

Figure 4 Courses of the synthesis of Z-L-TyrGlyGly-L-Phe-L-LeuOEt from Z-L-TyrGlyGly and L-Phe-L-LeuOEt by immobilized thermolysin in a buffer at initial pH 5.0 (A), in an aqueous/organic biphasic system at initial pH 4.5 (B), and in organic solvent (C). In the case of synthesis in a saturated organic solvent, IME was preincubated at pH 4.5. (0) L-Phe-L-LeuOEt; (A) Z-L-TyrGlyGiy; (O) Z-L-TyrGlyGly-L-Phe-L-LeuOEt; (A) Z-L-Tyr; (7]) Z-L-Tyr-L-PheL-LeuOEt; (V) Z-L-Tyr-L-LeuOEt; ( × ) Z-L-Tyr-L-Phe-L-Phe-L-LeuOEt; (V) mixture of byproducts containing L-phenylalanine residue such as GlyGly-L-Phe and L-Phe-L-Phe

Enzymatic synthesis of Leu-enkephalin: Y. Kimura et al. 100

I

I

,

I I rll,l

;e v

... 80 6O

N

~6 40 2o 0

y I 1

~

,

I I, 5

,ill

10 r FLOEt] / [Z-YGG] ( - )

Figure 5 Effect of L-Phe-L-LeuOEt (FLOEt) concentration on the yield of Z-L-TyrGlyGly-L-Phe-L-LeuOEt (Z-YGGFLOEt) in the synthesis with the immobilized thermolysin. (0) Reaction in a buffer at initial pH 5.0; (A) reaction in aqueous/organic biphasic system at initial pH 4.5; (©) reaction in saturated ethyl acetate. The initial concentration of Z-L-TyrGlyGly (Z-YGG) was 10 mM in the buffer for • and G, and 5.5 mM in the organic phase for ©. In the case of synthesis in saturated ethyl acetate, IME was preincubated at pH 4.5

umns 4 and 5 of Table 1. The ratio of substrates and product between the solid and buffer phases, Ci,/Cw, in a biphasic system was lower than in a buffer system. However, considering that there are both buffer and organic phases, the concentration of the two substrates, especially L-Phe-L-LeuOEt, is still high in the solid phase. The product, Z-L-TyrGiyGly-L-Phe-LLeuOEt, is not found in the buffer phase but is distributed almost equally in concentration between the solid and organic phases. Because of the low volume ratio of the solid phase to the organic phase, about 0.13, almost of all the product is in the organic phase. High concentrations of substrates, especially L-Phe-L-LeuOEt, in the solid phase make the rate of synthesis of product high and the rate of hydrolysis of Z-L-TyrGlyGly low; extraction of the product mainly into the organic phase shifts the equilibrium of the reaction to

Table 2

the desired product. For these reasons, IME caused the yield to increase in the biphasic system. Although the absolute solubility of the product was low, it was more soluble in a buffer saturated with ethyl acetate than in one without ethyl acetate. This might be a cause of the lower yield in a biphasic system than in a buffer system, as the product is more easily hydrolysed in a biphasic system than in a buffer system. Figure 4C shows the synthesis of Z-L-TyrGlyGly-LPhe-k-LeuOEt from Z-L-TyrGlyGiy and L-Phe-L-LeuOEt by IME in a saturated ethyl acetate. The maximum yield was obtained with IME preincubated at pH 4.5. The yield of the desired product was 20%, the same as in the biphasic system. The high yield is explained by the partitions of substrates and product between the solid and organic phases shown in column 6 of Table 1. Particularly, high concentrations of substrates, especially L-Phe-L-LeuOEt, might increase the rate of synthesis as before. To achieve a higher yield, we examined synthesis in an organic solvent with various concentrations of L-Phe-L-LeuOEt (Figure 5). The yield became higher as the concentration of L-Phe-LLeuOEt increased and reached 68% when the concentrations of Z-L-TyrGIyGIy and e-Phe-L-LeuOEt were 5.5 and 55 mM, respectively. Under this condition, ZL-Tyr-L-Phe-L-LeuOEt was 20% and Z-L-Tyr was not detected. Finally, Table 2 shows the physical data of the reaction intermediates and products obtained by the mass spectra and optical rotation measurements. The values of m/z coincided with the molecular weights. The data for Z-GlyGlyOBut were not shown because this compound has no asymmetric carbon and it was unstable against heat ionization in EI-MS measurement. For the final product, Z-L-TyrGIy-GIy-L-Phe-L-LeuOEt, more physical and biochemical properties were measured. The results of amino acid analysis and assay of opioid activity after removal of protecting groups are presented in Table 3. The component values and opioid activity of the product were consistent with those of authentic Leu-enkephalin. IH-NMR data of the precursor of Leu-enkephalin was as follows: IH-NMR 6(CD3OD) ppm: 0.88 (3H, d, J = 6.1 Hz), 0.93 (3H, d, J = 6.4 Hz), 1.23 (3H, t, J = 7.0 Hz), ca. 1.6 (3H, m),

Physical data of the reaction intermediates and products Molecular Weight

El-MS m/z

Z-L-TyrGlyGlyOBu t Z-L-Phe-L-LeuOEt

485.5 440.5

412 (M+-Ot-Bu) 440 (M ÷)

Z-L-TyrGlyGly-L-Phe-L-LeuOEt Z-L-Tyr-L-LeuOEt Z-L-Tyr-L-Phe-L-LeuOEt Z-L-Tyr-L-Phe-L-Phe-L-LeuOEt

717.8 456.5 603.7 750.9

718 456 603 750

(M* + 1)a (M ÷) (M ÷) (M ÷)

[a]o;Temp (°C) +1.6°;24 (c = 2.25, MeOH) -19.9°;24 (c = 1.00, EtOH) -20.9°;25 (c = 1, EtOH) b -14.4°;21 (c = 0.66, MeOH) -17.6°;21 (c = 0.79, MeOH) -36.3°;21 (c = 0.76, MeOH) n.d.

a SIMS (MATRIX, m-nitrophenol) b H. Ogura and K. Takeda TM searched by the data base "PRF/SYNDB" of the Peptide Institute, Inc.

Enzyme Microb. Technol., 1990, vol. 12, April

279

Papers Table 3 Amino acid analysis and opioid activity of the synthesized product and authentic Leu-enkephalin Amino acid analysis Gly Exp. Sample Authentic Cal.

2.15 2.19 2

Leu

1 1 1

Tyr

0.95 0.98 1

Phe

1.13 0.96 1

Opioid activity 2° IC50 (riM) a

160 160

a Concentration of Leu-enkephalin that lowered specific binding of 8 nM [3Hlnaloxone with opiate-receptor sites in rat-brain tissue by 50%

ca. 2.8 (IH, dd, J = 13.7, 8.6 Hz), 2.94 (1H, dd, J = 14.0, 9.5 Hz), 3.05 (1H, dd, J = 13.7, 6.1 Hz), 3.18 (IH, dd, J = 14.0, 5.2 Hz), 3.7-3.9 (4H, m), 4.13 (2H, q, J = 7.0 Hz), 4.27 (1H, m), 4.42 (1H, dd, J = 9.5, 5.8 Hz), 4.66 (IH, dd, J = 9.5, 5.2 Hz), 4.99 (1H, d, J = 12.5 Hz), 5.06 (IH, d, J = 12.5 Hz), 6.69 (2H, d, J = 8.5 Hz), 7.03 (2H, d, J = 8.6 Hz), 7.2-7.3

(10H, m). Conclusions In the synthesis of Z-L-TyrGIyGly-L-Phe-L-LeuOEt from Z-L-TyrGiyGly and L-Phe-L-LeuOEt, the yield with the IME was higher than with free enzyme. The presence of the support increased the yield because LPhe-L-LeuOEt was specifically partitioned towards the inside of the support, which increases the rate of synthesis. Furthermore, the desired product was not hydrolysed appreciably because it precipitated in a buffer system or was extracted with an organic solvent in the biphasic system or saturated organic solvent. A new method to synthesize the precursor of Leuenkephalin enzymatically without the protection of a side chain of L-Tyr has been described. Kullmann jl synthesized Leu-enkephalin (and Met-enkephalin) enzymatically in 1979. However, in his method blocking of phenol residue of L-Tyr with benzylic acid is necessary. Wong e t a l . t2 synthesized Leu-enkephalin from Nps-L-TyrGlyGly and L-Phe-L-LeuOMe, and from Boc-L-Tyr(Bzl)GlyGly and L-Phe-L-LeuOTMB, both by papain-catalysed condensation reaction with the yields of 18% and 66%, respectively. By our method,

280

without the protection of a side chain of e-Tyr, Z-LTyrGlyGly-e-Phe-e-LeuOEt was produced with a yield of 68%.

Enzyme Microb. Technol., 1990, vol. 12, April

Acknowledgements We are greatly indebted to Daiwa Kasei K. K. for providing thermolysin, to Dr. K. Irie, Food Science and Technology, Kyoto University, for the El-MS measurements, to Dr. T. Ueno, associate professor, Pesticide Research Institute, Kyoto University, for the SIMS measurement, to Mr. R. Imamura, Faculty of Science, Kyoto University, for the =H-NMR measurement, and to Mr. F. Tani, Food Science and Technology, Kyoto University, for the analysis of opioid activity.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Bergmann, M. and Fraenkel-Conrat, H. J. Biol. Chem. 1937, 119, 707-720 Homandberg, G. A., Mattis, J. A. and Laskowski, M., Jr. Bioehemistry 1978, 17, 5220-5227 Klibanov, A. M., Samokhin, G. P., Martinek, K. and Berezin, I. V. Biotechnol. Bioeng. 1977, 19, 1351-1361 Nakanishi, K., Kamikubo, T. and Matsuno, R. Bio/Technol. 1985, 3, 459-464 Martinek, K., Semenov, A. N. and Berezin, I. V. Biochim. Biophys. Aeta 1981, 658, 76-89 Martinek, K. and Semenov, A. N. Biochim. Biophys. Acta 1981, 658, 90-101 Nakanishi, K. and Matsuno, R. Eur. J. Bioehem. 1986, 161, 533-540 Nakanishi, K., Kimura, Y. and Matsuno, R. Eur. J. Biochem. 1986, 161, 541-549 Nakanishi, K., Kimura, Y.and Matsuno, R. Bio/Technol. 1986, 4, 452-454 Hughes, J., Smith, T. W., Kosterlitz, H. W., Fothergill, L. A., Morgan, B. A. and Morris, H. R. Nature 1975, 258, 577-579 Kullmann, W. Biochem. Biophys. Res. Commun. 1979, 91, 693-698 Wong, C.-H., Chen, S.-T. and Wang, K-T. Bioehim. Biophys. Acta 1979, 576, 247-249 Bergmann, M. and Zervas, L. Chem. Ber. 1932, 65, 1192-1201 Ben-lshai, D. and Berger, A. J. Org. Chem. 1952, 17, 1564-1570 Ben-lshai, D. J. Org. Chem. 1954, 19, 62-66 Barman, T. E. in Enzyme Handbook Springer-Verlag, Berlin, 1969, vol. 2, pp. 620-622,625 Morihara, K. and Oka, T. J. Biochem. 1981, 89, 385-395 Morihara, K. and Tsuzuki, H. Biochim. Biophys. Acta 1966, 118, 215-218 Ogura, H. and Takeda, K. J. Chem. Soe. Jpn. 1981, 836-844 Pert, C. B. and Snyder, S. H. Pro¢. Natl. Acad. Sei. USA 1973, 70, 2243-2247