Enzymatic peptide synthesis in organic media: a comparative study of water-miscible and water-immiscible solvent systems

Journal of Biotechnology, 15 (1990) 323-338 Elsevier

323

BIOTEC 00528

Enzymatic peptide synthesis in organic media: comparative study of water-miscible and water-immiscible solvent systems

a

Pere Claprs, P a t r i c k A d l e r c r e u t z a n d B o M a t t i a s s o n Department of BiotechnoloD,, Chemical Center, University of Lund, Lund, Sweden (Received 27 October 1989; accepted 13 January 1990)

Summary

Peptide synthesis was carried out in a variety of organic solvents with low contents of water. The enzyme was deposited on the support material, celite, from an aqueous buffer solution. After evaporation of the water the biocatalyst was suspended in the reaction mixtures. The chymotrypsin-catalyzed reaction between Z-Phe-OMe and Leu-NH 2 was used as a model reaction. Under the conditions used ([Z-Phe-OMe]0 < 40 mM, [Leu-NH2]0/([Z-Phe-OMe] 0 = 1.5) the reaction was first order with respect to Z-Phe-OMe. Tris buffer, p H 7.8, was the best buffer to use in the preparation of the biocatalyst. In water-miscible solvents the reaction rate increased with increasing water content, but the final yield of peptide decreased due to the competing hydrolysis of Z-Phe-OMe. Among the water-miscible solvents, acetonitrile was the most suitable, giving 91% yield with 4% (by vol.) water. In water-immiscible solvents the reaction rate and the product distribution were little affected by water additions in the range between 0% and 2% (vol. %) in excess of water saturation. The reaction rates correlated well with the log P values of the Correspondence to: Patrick Adlercreutz, Department of Biotechnology, Chemical Center, P.O. Box 124, S-221 00 Lund, Sweden, Tel. Int. + 46 46 104842, Fax Int. + 46 46 104713. Abbreviations: Z-Phe-OMe, N-benzyloxycarbonyl-L-phenylalanine methyl ester; Leu-NH2, L-leucine amide; P, partition coefficient in the octanol/water two-phase system; Pcph, partition coefficient in the octanol/water two-phase system for the continuous phase; Pi, interphase partition coefficient in the octanol/water two-phase system; Ps, partition coefficient in the octanol/water two-phase system for the substrate(s); p, partition constant between the by-product and product (retool 1-1); k, intrinsic first-order rate constant (g-1 h-1 of immobilized preparation); k', first-order rate constant (h-1). 0168-1656/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

324

solvent. The highest yield (93%) was obtained in ethyl acetate; in this solvent the reaction was also fast. Under most reaction conditions used the reaction product was stable; secondary hydrolysis of the peptide formed was normally negligible. The method presented is a combination of kinetically controlled peptide synthesis (giving high reaction rates) and thermodynamically controlled peptide synthesis (giving stable reaction products). Enzymatic peptide synthesis; Bioorganic synthesis; Organic media for enzymatic synthesis; a-Chymotrypsin

Introduction

The use of enzymatic methods for peptide synthesis is an expanding research field and these methods constitute an important complement to the methods using chemical reagents (Jakubke et al., 1985). Enzyme-catalyzed peptide bond formation can be performed by two approaches: kinetically-controlled synthesis or equilibrium-controlled synthesis. In kinetically-controlled synthesis an ester substrate (donor ester) is used. This substrate reacts with the enzyme, yielding an acyl enzyme intermediate. This complex can be deacylated either by water, yielding the hydrolysis product, or by a nucleophile (i.e. the amino group of an amino acid), forming a peptide bond (Scheme 1) (Jakubke, 1987). The reaction proceeds quickly and in aqueous media it must be stopped, because further incubation will result in secondary hydrolysis of the peptide formed. In the equilibrium-controlled synthesis (reversed hydrolysis), the yield can be increased by using manipulations based on the law of mass action. In both approaches, the characteristics of the reaction media are very important in order to obtain yields and substrate solubilities as well as high enzyme activity and stability. Organic solvents have been used for quite a long time in order to improve yields in enzyme-catalyzed peptide synthesis. Additions of water-miscible solvents increase the yields by decreasing the acidity of the ot-carboxy group of the carboxy compo-

RCO-NHR" + EH k3 k2

K 1

RCOOX + EH q

H2NR'

~ RCOOX.EH

, RCO-E + HOX k4 I

H20

RCOOH + EH Scheme 1

325 Leu-NH2 Z-Phe-OMe

Z-Phe-Leu-NH 2

ct-chymotrypsin _ H 20 MeOH Scheme2

Z-Phe-OH

nent, thereby shifting the equilibrium position towards peptide synthesis (Homandberg et al., 1978). The concentration of the solvent has usually been under 50% since deactivation of the enzyme is often observed when higher concentrations are used. Water-immiscible solvents can be used to extract the peptide formed from the enzyme-containing water phase, thereby shifting the reaction equilibrium towards peptide synthesis (Jakubke et al., 1985; Jakubke, 1987). The use of enzymes in organic media has attracted much interest in recent years (Laane et al., 1987a) and the methods for carrying out this kind of reactions have improved substantially. One method for biocatalyst preparation which often gives good results is to deposit the enzyme on a porous support material which is later used in organic media with low water contents. As enzymes are insoluble in almost all organic solvents they stay on the support, although they are not covalently linked to it. This kind of biocatalyst has recently been used successfully for peptide synthesis (Reslow et al., 1988). It was shown that peptide synthesis can be carried out in the water-miscible solvent acetonitrile with only a few percent of water present. The reaction rate decreased with increasing solvent concentration but the final yield increased. It is only at very high concentrations of water-miscible solvents that the positive effect of the decreased water activity (water concentration) on the equilibrium peptide yield becomes appreciable. There are a few other reports in the literature that peptide synthesis can be carried out in a favourable way using less than 10% water in water-miscible organic solvents (Isowa et al., 1981; Kisee et al., 1988; Noritomi et al., 1989). Here, the conditions for peptide synthesis in water-poor reaction mixtures with enzymes deposited on porous support materials were further investigated. Different reaction conditions were evaluated with respect to reaction rate, peptide yield and product distribution (peptide synthesis versus hydrolysis). Reaction systems containing water-miscible and water-immiscible solvents were compared. As a model reaction the chymotrypsin-catalyzed reaction between the donor ester Z-Phe-OMe and the nucleophile Leu-NH 2 was chosen (Scheme 2).

Materials and Methods

Chemicals a-Chymotrypsin (EC 3.4.21.1) from bovine pancreas, with a specific activity of 51 benzoyl-tyrosine ethyl ester units mg-1 of solid was obtained from Sigma Chemicals

326

(St Louis, MO, U.S.A.) and was used without further purification. Celite (30-80 mesh) was from BDH (Poole, U.K.). N-Benzyloxycarbonyl-L-phenylalaninemethyl ester and L-leucine amide were purchased from Bachem (Feinchemikalien AG, Switzerland). Acetonitrile HPLC grade was from Fisons (Loughborough, U.K.). All other chemicals used were of analytical grade.

Preparation of the immobilized enzyme The enzyme was immobilized by being dried onto the support. Chymotrypsin (31.8 mg m1-1) was dissolved in 50 mM buffer solution, pH 7.8 or 9. One ml of enzyme solution was mixed with 1 g of support material. After mixing, the preparation was dried under vacuum overnight.

Enzymatic peptide synthesis The preparation of immobilized enzyme (150 mg) was added to 2 ml of organic solvent containing a controlled amount of water (in the case of water-immiscible solvents) or buffer (in the case of water-miscible solvents) and the substrates. The initial concentrations of ester and nucleophile were 20 mM and 30 mM, respectively, unless otherwise stated. The reactions were carried out in 10-ml stoppered glass bottles placed on a reciprocal shaker (125 rpm) at 25°C. Fifty/~1 of samples were taken and mixed with 200 /~1 of acetic acid to stop any enzymatic reaction. Samples were evaporated and dissolved in eluent before HPLC analysis.

Measurement of log P values of the substrates The log P of the substrates Z-Phe-OMe and Leu-NH 2 were determined by shaking 25.1 mg (0.08 mmol) of Z-Phe-OMe and 15.6 mg (0.12 mmol) of Leu-NH 2 overnight at 25°C in a two-phase system containing 1-octanol (2 ml) and water (2 ml). The concentration of substrates in both phases was determined by HPLC in the conditions described in HPLC analysis. The calculated log P values for the substrate Z-Phe-OMe and nucleophile Leu-NH 2 were 3.2 and -0.21, respectively. Log P is a measure of hydrophobicity and in this case it means that Z-Phe-OMe is more hydrophobic than Leu-NH 2.

HPLC analysis The amounts of dipeptide, substrates, and by-product were analysed by HPLC (Shimadzu LC-6A) using a ODS-2 column (Tracer analitica). The donor ester, dipeptide and byproduct were eluted with acetonitrile/water/acetic acid (45 : 50:5, v/v) and detected using a UV detector at 254 nm. The yields were calculated from the peak areas of substrate, dipeptide and hydrolysis product. The nucleophile, Leu-NH2, was eluted with acetonitrile/water/trifluoroacetic acid (30:70:0.05, v/v) and UV detector at 215 nm.

Kinetic analysis In this study the kinetics were not studied in detail so it was not revealed which was the rate-limiting step, but it was found that the substrate consumption in the

327 model reaction with reasonable accuracy can be approximated as being a first order reaction with the rate constant k': -

d[RCOOX] = k ' [RCOOX] dt

(1)

where k' = k[E0], [E0] is the amount of enzyme. In this work the values of k(g -1 h -1) are given per g of immobilized preparation. In order to describe the product distribution the parameter p as defined by Jakubke (1987) was used. The partition constant p corresponds to the nucleophile concentration needed to obtain d[RCOOH] = d[RCO-NHR'], which means equal partition of the acyl enzyme into peptide and hydrolysis product. d[RCOOH] k4[H20 ] p d[RCO-NHR'] = k3[H2NR' ] - [H2NR']

(2)

To obtain p from the time-courses of the model reaction under different conditions, Eq. (2) and the relation d[RCO-NHR'] = d[H2NR' ] were used and the equation was integrated [H2NR']0 [RCOOH] -- p In [H2N R, ]0 - [RCO-NHR' ]

(3)

where [HENR']0 is the initial concentration of the nucleophile. The values of k and p were calculated for different reaction conditions from the experimental results using computer calculations. A computer program (Graph-Pad v.1.1, ISI Software) with non-linear and linear regression methods was used to estimate the parameters k (Eq. 1) and p (Eq. 3), respectively.

Results and Discussion

The chymotrypsin-catalyzed reaction between Z-Phe-OMe and Leu-NH 2 was carried out under a wide variety of conditions. The enzyme was adsorbed on celite, and the immobilized preparation was used as a suspension in an organic medium with different amounts of water present. A list of the solvents used is given in Table 1. Water-miscible soloents The reaction time-course of a typical series of experiments in a water-miscible solvent with different water content is shown in Fig. 1. In water-miscible solvents, like acetonitrile, the reaction rate increased with increasing water content of the medium. However, as noted before (Reslow et al., 1988) the yield of peptide decreased with increasing water content because of the competing hydrolysis of the ester substrate. The yield can also decrease due to secondary hydrolysis of the peptide formed but this reaction was of little importance under the conditions used in this study.

328 TABLE 1

Properties of the soloents used in this study Solvent

Log P

Dichloromethane Cyclohexanone Ethyl acetate 2-Butanone Tetrahydrofuran Acetone Acetonitrile DMF/Acetonitrile, DMF

1.25 0.81 0.66 0.29 0.46 - 0.24 - 0.34 -0.69 - 1.04

1:1 a

A m o u n t o f w a t e r to saturate solvent

S o l u b i l i t y in water

(~ (w/w))

(~ (w/w))

0.198 8.0 2.94 10.0 -

1.3 2.3 8.1 24.0 -

a A m i x t u r e of 50% ( v / v ) D M F / a c e t o n i t r i l e gives a l o g P = - 0 . 6 9 Pmi~ture = X1 log P1 + X 2 l o g P2, w h e r e X is t h e m o l e f r a c t i o n .

b y t h e s e m i e m p i r i c a l f o r m u l a log

Normally, enzymes in organic media obey the same kind of kinetics as in water solution and this has been shown to be the case for chymotrypsin (Reslow et al., 1987; Zaks and Klibanov, 1988). The values of the kinetic constants vary with the reaction conditions; for example they depend on the properties of the solvent. In this study the initial reaction rates were measured with different concentrations of the substrate Z-Phe-OMe. In the range studied (0-40 mM) the reaction rate was proportional to the substrate concentration indicating that the apparent g m value under these conditions was quite high (Fig. 2). These observations agree well with measurements of the time-course of the reactions. It was found that the disappearance of substrate correlated well with a first-order reaction (Fig. 3). In order to get a suitable measure of the reaction rates under different conditions the reactions were approximated to be first order reactions and the rate constants were calculated as described in Materials and Methods. In addition to the reaction rate, described by the first-order rate constant, the product distribution is also of great importance in peptide synthesis. To rationalize the results concerning product distribution, the p parameter was used as described in Materials and Methods. A low p value is desirable because it means that most of the substrate is converted to peptide and only a minor fraction is hydrolyzed. When preparing the biocatalysts, an aqueous buffer solution of the enzyme was mixed with the support and then water was removed at reduced pressure. The p H value of the aqueous solution is of great importance for the activity of the resulting biocatalyst in organic media (Zaks and Klibanov, 1985). The effects of different buffers on the chymotrypsin-catalyzed reaction between Z-Phe-OMe and Leu-NH 2 are shown in Fig. 4. It is clear that the choice of buffer greatly influences the performance of the biocatalyst with Tris giving the highest reaction rates (Fig. 4a) and the lowest p values (Fig. 4b). Tris buffers with p H values of 7.8 and 9.0 gave similar results. In both Tris and phosphate buffers the reaction rate increased with increasing water content in the range studied (0-20%), but for carbonate buffer a

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Fig. 1. Time-course of the reaction between Z-Phe-OMe and L e u - N H 2 catalyzed by a-chymotrypsin on celite. The reactions were carried out in acetonitrile with 4% ( v / v ) (molar fraction of water, x = 0.107) (a), 10% (x = 0.243) (b) and 20% (x = 0.421) (c) water content. D o n o r ester Z-Phe-OMe (rT), by-product Z-Phe-OH ( × ) and dipeptide Z-Phe-Leu-NH 2 (e) were analyzed by HPLC.

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[Z-Phe-OMe] (mM) Fig. 2. Initial reaction rate as a function of the substrate concentration for the reaction between Z-Phe-OMe (variable concentration) and Leu-NH 2 (60 mM) catalyzed by a-chymotrypsin on celite. The reactions were carried out in water-saturated ethyl acetate with 1% of extra water added. m a x i m u m was f o u n d at 10% water. T r i s buffer, p H 7.8, was c h o s e n for the rest o f the e x p e r i m e n t s p r e s e n t e d here b u t for m o s t e x p e r i m e n t s a l m o s t i d e n t i c a l results were o b t a i n e d with Tris buffer, p H 9.0. T h e p r o p e r t i e s of the solvent greatly influence the p e r f o r m a n c e of the enzyme. T h e m o d e l r e a c t i o n was carried o u t in various solvents w i t h v a r y i n g w a t e r contents. T h e e n z y m e was active in acetonitrile, t e t r a h y d r o f u r a n e a n d a c e t o n e b u t a l m o s t inactive in d i m e t h y l f o r m a m i d e . H o w e v e r , the e n z y m e was highly active in a 1 / 1

20

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Fig. 3. Time-course of substrate, Z-Phe-OMe, consumption in the enzymatic synthesis of Z-Phe-Leu-NH2. The reaction was carried out in acetonitrile with 4% (v/v) water content (O) and in water-saturated ethyl acetate with 1% (v/v) water added (11). Experimental (points) and theoretical values (solid lines) calculated from the first order model reaction (Eq. 1).

331 12 10" 8 T

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Water content (Vol. %) Fig. 4. Influence of buffers on the a-chymotrypsin-catalyzed coupling of Z-Phe-OMe and Leu-NH 2. The reactions were carried out in acetonitrile with the specified water contents and with the enzyme adsorbed on celite. The rate constant, k (a), and the product distributions, expressed as p (b), were measured using the following buffers (50 raM): phosphate, pH 7.8 (11), Tris, pH 7.8 (D), Tris, pH 9.0 (A) and carbonate, pH 9.0 (×). m i x t u r e of d i m e t h y l f o r m a m i d e a n d acetonitrile. T h e highest r e a c t i o n rates were o b t a i n e d in acetonitrile a n d a c e t o n e (Fig. 5a). T h e r a t e c o n s t a n t k increases s t r o n g l y with increasing w a t e r c o n t e n t in the w a t e r - m i s c i b l e solvents (Figs. 4a, 5a). T h i s is p r o b a b l y caused b y a n increase in the h y d r a t i o n o f the e n z y m e at h i g h e r w a t e r contents. T h e p values were c o n s i d e r a b l y higher in t e t r a h y d r o f u r a n e a n d a c e t o n e t h a n in acetonitfile a n d a c e t o n i t r i l e / d i m e t h y l f o r m a m i d e (Fig. 5b). I n a q u e o u s m e d i a the p a r t i t i o n c o n s t a n t p is n o r m a l l y c o n s t a n t since the c o n c e n t r a t i o n o f w a t e r c a n b e c o n s i d e r e d to b e c o n s t a n t a n d the w a t e r activity is close to 1.0. H o w e v e r , in the

332 14 :a 1210 T ,.=

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10 Water content (Vol. %)

20

b

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20

10

!

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Water content (% V/V) Fig. 5. Influence of the solvent on the a-chymotrypsin-catalyzed coupling of Z-Phe-OMe and Leu-NH 2. The enzyme was adsorbed on celite. With different amounts of water present the rate constant, k (a), and the product distributions, expressed as p (b), were measured using the following solvents: acetone (×), acetonitrile (ll), tetrahydrofuran (D) and dimethylformamide/acetonitrile, 1 : 1 (&). The buffer used was Tris-HC1, pH 7.8, for all solvents except dimethylformamide/acetonitrile, in which case Tris-HC1, pH 9, w a s used.

reaction m e d i a used in this investigation water c o n c e n t r a t i o n s or water activities m u s t be taken i n t o account. F r o m Eq. (2) it is seen that p = k 4 [ H 2 0 ] / k 3. T o be entirely correct the c o n c e n t r a t i o n of water i n the f o r m u l a should be replaced b y the water activity (Hailing, 1987) b u t i n this investigation water activities were n o t measured. I n the three pure solvents the p values increased linearly with i n c r e a s i n g water content, as expected, b u t with a c e t o n i t r i l e / d i m e t h y l f o r m a m i d e the p value varied only slightly i n the range studied.

333

Water-immiscible solvents When water-immiscible solvents are used instead of water-miscible ones, quite different systems are obtained. At low water contents water partitions between the solvent and the catalyst phase; when the solvent is water saturated, extra additions of water are mainly absorbed by the catalyst. This creates a minute aqueous phase, surrounding the enzyme, between the support material and the bulk solution. Polar reactants and products, like methanol, can be enriched in this polar phase, which can influence the performance of the enzyme. Furthermore, the formation of an aqueous phase can lead to mass transfer limitations, especially if high water contents are used in combination with substrates having a low water solubility. The knowledge about the microenvironment of enzymes when deposited on celite is too small to draw conclusions about enzyme distribution in the particle and mass transfer limitations. The model reaction was carried out in some different water-immiscible solvents with specified amounts of water present. The time-course of a typical reaction is shown in Fig. 6. Four different solvents were chosen for the model reaction. It is clear that the amount of extra water added influences the reaction much less than in the case with water-miscible solvents (Fig. 7a). Large differences in their rates were observed with 2-butanone giving the highest values irrespective of the water content. High rates were also obtained in ethyl acetate but in dichloromethane and cyclohexanone the reaction was considerably slower (Fig. 7a). The effects of solvents can be explained by considering the hydrophobicities of solvents and substrates. Laane et al. (1985) have introduced the use of log P values to characterize hydrophobicities in biocatalytic systems. Log P is defined as the partition coefficient of a substance in the o c t a n o l / w a t e r two-phase system (this P should not be confused with the p value concerning product distribution in peptide synthesis). According to Laane et al.

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Time (h) Fig. 6. Time-course of the reaction b e t w e e n Z - P h e - O M e a n d L e u - N H 2 in w a t e r - s a t u r a t e d ethyl a c e t a t e w i t h 1 ~ ( v / v ) of e x t r a w a t e r added. The e n z y m e was a d s o r b e d on celite a n d the d o n o r ester Z - P h e - O M e ([3), b y - p r o d u c t Z - P h e - O H ( × ) a n d d i p e p t i d e Z - P h e - L e u - N H 2 ( e ) were a n a l y z e d b y H P L C .

334 10

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8 T

6'

2i O#: 0.0

,

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1.0

2.0

Water content (Vol. %) 50

b

40 3O r. 20 10 0

,i. -

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'I' I

'~ 2

Water content (Vol. %) Fig. 7. Influence of solvent on the a-chymotrypsin-catalyzed coupling of Z-Phe-OMe and Leu-NH 2. The enzyme was adsorbed on celite, the solvents were saturated with water and specified additions of extra water were done. The rate constants, k (a) and the product distribution, expressed as p (b), were measured using the following solvents: 2-butanone (x), ethyl acetate (111), cyclohexanone (A) and dichloromethane ([2). (1987b) the reaction rate in biocatalytic systems containing an interface can be optimized using the following rules: 1. Ilog Pq,h -- log Psi should be maximal 2. Ilog Pi - log Psi should be minimal where log Pcph, log Ps and log Pi are the log P values for the continuous phase, the substrate and the interphase, respectively. L o g Pcph can be a p p r o x i m a t e d with the literature values for the pure solvents (Leo et al., 1971; H a n s c h and Leo, 1979) and the log P values for the substrates were determined experimentally (see Materials and Methods). I n the systems used it is difficult to estimate log P values of the

335

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Log P Fig. 8. The rate constant k as a function of the log P value of the solvent. The chymotrypsin-catalyzed reaction between Z-Phe-OMe and Leu-NH2 was carried out in 4 water-immiscible solvents (watersaturated without extra water additions). Data from Fig. 7a. interface so we concentrate on the first of the rules above. The rate of the model reaction depends primarily on the concentration of Z-Phe-OMe. Since the log P value of Z-Phe-OMe (3.2) is higher than for the solvents used it can be expected that the highest reaction rate should be obtained with the solvent having the lowest log P value. Although the number of solvents used is too small to draw any conclusions with certainty, there is a clear tendency that decreasing log P values result in increasing reaction rates (Fig. 8). Quite different p values (product distribution) were obtained in the different solvents. In ethyl acetate and dichloromethane low p values were reached, indicating a high selectivity for the peptide synthesis reaction. In contrast to the results with water miscible solvents, the p value did not increase with increasing water content (Fig. 7b). A slight decrease was observed instead with increasing water content. This can be expected since the water activity in the system should increase until the solvent is saturated and further water additions should have little influence on the activity and p value. The water activity could also strongly influence the activity of the enzyme and thereby the reaction rate; accordingly, the rate constants were little affected by water additions in excess.

Comparison of different solvent systems Among the water-miscible solvents acetonitrile was clearly the best one (Table 2). The varying yields of products from enzymatic reactions in the different organic solvents are not yet explained. This is due, among other things, to experimental problems in monitoring actual concentrations of substrate(s) and product(s), in the microenvironment of the enzyme. The yield in acetonitrile (91%) was considerably higher than the best yield obtained in the synthesis of N-acetyl-tyrosyl-leucine

336 TABLE 2

The highest peptide yields obtained in the different solvents The reaction between Z-Phe-OMe and Leu-NH 2 was carried out with chymotrypsin on celite for 72 h. Solvent

Maximal peptide yield (%)

Yield of hydrolysis product (%)

Water content (%(v/v))

Acetonitrile Tetrahydrofuran Acetone DMF DMF/Acetonitrile, 50 : 50 Ethyl acetate Dichloromethane 2-Butanone Cyclohexanone

91 65 70 9 76 93 90 76 38

9 34 30 2 20 7 10 24 59

4 4 4 20 4 0 2 0 1

amide with chymotrypsin in 95% ethanol (Kisee et al., 1988). This difference is probably due to differences in the preparation of the biocatalyst or the choice of solvent. High yields in similar peptide synthesis reactions have mainly been achieved in solvents like butanediol (Homandberg et al., 1978; Nilsson and Mosbach, 1984), which are known to stabilize enzymes. The best yield for reactions in water-immiscible solvents in the present study was obtained in ethyl acetate (Table 2). This agrees well with the high yields reported for similar reactions in water-ethyl acetate two-phase systems (Semenov et al., 1981; Khmelnitski et al., 1984; Nakanishi and Matsuno, 1986; Kuhl et al., 1987). Further advantages with ethyl acetate are that the reaction rate is high (Fig. 7a) and that the reaction is not sensitive to the water content in the range studied (water saturation plus 0-2% extra water). In conclusion, ethyl acetate was found to be the best solvent for chymotrypsincatalyzed coupling of Z-Phe-OMe and Leu-NH 2. Similar reactions with other substrates are currently being carried out. The synthetic method used in this study is a combination of kinetically controlled and thermodynamically controlled peptide synthesis. An ester is used as the carboxy component as in kinetically controlled synthesis. This choice of substrate gives high reaction rates which are needed, at least in water-miscible solvents because of the reduction in reaction rate observed when the water content is decreased to the levels used in this study. When kinetically controlled peptide synthesis is carried out in aqueous solution the reaction must be monitored and stopped at a suitable time or secondary hydrolysis of the peptide formed will decrease the peptide yield. However, in the systems used in this study the equilibrium position is much more favourable due to the low water concentration (or water activity; in water-miscible solvents) or the effective extraction of the reaction product (in water-immiscible solvents). Consequently secondary hydrolysis is suppressed and the stability of the peptides obtained is much better than in aqueous media. Even some of the

337 3

0

'~

I

I

I

0

20

40

60

80

Time (h) Fig. 9. Time-course of by-product formation. Production of Z-Pbe-OH in the reaction between Z-Phe-OMe and Leu-NH 2 catalyzed by a-chymotrypsin in acetonitrile as organic solvent with 4% of Tris-HC1 buffer, pH 7.8. Experiments were carried out in triplicate.

by-product formed in the hydrolysis of the ester substrate reacts with the nucleophile in a reverse hydrolytic reaction which further increases the peptide yield (Fig. 9).

Acknowledgements This project was supported by the Biotechnology Research Foundation (SBF). P. Clap6s wishes to express his gratitude to Consejo Superior de Investigaciones Cientificas (C.S.I.C.) (Spain) for its financial support. The authors wish to thank Scott Bloomer for linguistic advice.

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