Peptide enzymatic microsynthesis, using carboxypeptidase Y as the catalyst: application to stepwise synthesis of Leu-enkephalin

Biochimie 70 (1988) 791-802 © Soci6t6 de Chimie biologique/Elsevier, Paris

791

Research

article

Peptide enzymatic microsynthesis, using carboxypeptidase Y as the catalyst: application to stepwise synthesis of Leuenkephalin Florence HELLIO, Paul G U E G U E N and Jean-Louis MORGAT*

Centre d'Etudes Nucl#aires de Saday, D~partement de Biologic, Service de Biochimie, C.E.A., 91191 Gif-sur-Yvette C#dex, France (Received 8-9-198~ accepted after revision 18-1-1988)

Summary - - In order to develop the use of carboxypeptidase Y (CPD-Y, EC 3.4.12.1) as a catalyst for radioactive hormonal synthesis, the stepwise synthesis of a pentapeptide, Leu-enkephaiin, was studied on a microscale. Each peptide bond was formed by enzymatic catalysis, using microquantities of the precursors (amino acid or peptide esters as acyl-components and amino acid ester or amide as nucleophiles). The high condensation yields obtained suggests that CPD-Y can be a useful tool for preparation of radioactive hormonal peptides. enzymatic synthesis / microcatalysis /

carboxypeptidase Y

Introduction In recent years, the use of proteolytic enzymes tn pat~lyz~ Lv ~uL~a ~

tLha x e ~'~'"~'~*;'~".~..,..L...,,o f J~J ~.l .J L:Int l h=l t T b ,

gL C.V.IaI U~D

kI I Q. ~~

undergone intensive development in parallel with the increasing demand for hormonal peptides. Since Bergmann and Fraenkel-Conrat demonstrated, in 1937, that papain catalyzed peptide bonds formation [1], several papers devoted to successful protease-catalyzed syntheses have been published [2-6]. Enzymatic peptide synthesis presents certain advantages over chemical synthesis: 1) it requires limited or no side chain protection of the amino acids; 2) the reaction takes place under mild conditions with retention of stereospecificity; and 3) like most enzymatic reactions, it can be carried out in very small volumes. These remarks, in particular the last one, suggest that enzymatic peptide synthesis might be an attrac-

tive approach to the preparation of peptides labeled with radioactive amino acids. To investigate this potential application, we attempted to synthesize a pentapeptide, Leuenkephalin, whose sequence is T y r - G l y - G l y - P h e - L e u [7]. In 1979, Kullmann described the preparation of the peptide with all the peptide bonds established by protease-catalyzed reactions [8]. Widmer et al. used CPD-Y to catalyze the total synthesis of Met-enkephalin which differs from Leu-enkephalin by its C-terminal amino acid [9]. Whereas the published results concerned the enzymatic synthesis on a preparative scale, we tried to apply the stepwise synthesis principle to Leu-enkephalin synthesis using microquantities of amino acids. CPD-Y, a non-specific exoprotease, which not only hydrolyzes peptides but acts on peptide esters and peptide amides as well, was chosen as the catalyst [10-14]. At alkaline pl! values,

*Author to whom correspondence should be addressed. Abbreviations: CPD-¥-" carboxypeptidase Y; DFP: diisopropylfluorophosphate; DMF: dimethyiformamide; DMSO: dimethyl

suifoxide; EDTA: ethylenediaminetetraacetic acid; PMSF: phenylmethylsulfonyl fluoride; TEA: triethylamine; "lEAF: triethylamineformate.

792

F. Hellio et al.

CPD-Y exhibits some esterase activity but very low carboxypeptidase activity. This unique property of the enzyme can be exploited for peptide bonds synthesis according to Scheme 1 [15].

In the present study we focused our attention on the miniaturization of Leu-enkephalin stepwise synthesis. Initially, all the reactions involved were studied separately, in small

b

R~NH-CH-C-OH

+ EH

IRI Oa R~NH-CH-C-R 2 + EH <.__.~> R~NH-CH-C-E + R2H

~'--' R. O llll

e., O / a II

R~NH-CH-C-NH-CH-C-R 4 ÷ EH Scheme 1. Reaction schcmc for CPD-Y catalyzed peptide bond formation [15]. R0: blocking group, benzoyl (Bz); RI R3: side chain of an amino acid; R2: -OCH3, -OC2H5, -OC3H7; R4: -OH, -NH2, -OCH3; EH: CPD-Y.

The first step is the formation of an a c y l enzyme intermediate between the ester substrate and the enzyme. The second step is the main key to the whole process: the amine nucleophile has to compete successfully with water for the a c y l - e n z y m e bond, thus forming a peptide bond instead of hydrolyzing it to free carboxylic acid. An efficient nucleophilic reaction requires that the amino acid amino group not be ionized or in a zwitterion situation. Therefore, the amino acid carboxyl group has to be blocked. If this is done by an amide, further elongation of the peptide amide produced requires the removal of the amide group and its replacement by an ester group [16, 17]. On the other hand, if the amino acid carboxylic acid is blocked as an ester, the peptide produced is directly in an ester form and ready to react again with the enzyme, leading to consecutive additions of the same nucleophile. This is either a drawback [18] or an advantage when the sequence is made of the same residue in succession.

volumes, and the optimal conditions for peptide bond formation were defined. Then the different parameters were applied to perform the total synthesis of Leu-enkephalin.

Materials

and methods

CPD-Y from baker's yeast was purchased from Merck. Trypsin, pretreated by N-tosyl-L-phenylalanyl chloromethyl ketone, and a-chymotrypsin were obtained from Mann Research Laboratories. Amino acids and peptides were bought from Sigma or Bachem. All were of the L-configuration. The pH meter from Tacussel was equipped with an electrode, MI 410, from Microelectrodes Inc. Separation and purification of compounds were performed on a Gilson chromatographic system (high pressure liquid chromatography, HPLC). Two different columns were used: an analytical one, Bondapak Cla (Waters) ,and a semipreparative one, Nueleosil 10 p,C~s (Soci6t6 franqaise chromato-colonne). Acetonitrile, used for HPLC was bought from BDH. All other solvents were purchased from Merck or Prolabo.

Enzymatic microsynthesis of Leu-enkephalin Amino acid analyses were performed on an LKB 4400 autoanalyzer. Esterifications were performed in the presence of anhydrous ethanol and thionyl chloride. The reaction mixture was maintained 30 rain at 0oC, while stirred, and then overnight at room temperature. To determine the product yield, each reaction mixture was analyzed by HPLC (reverse~phase columns, solvent gradient: A = 50 mM TEAF, pH 3; B = 50 mM TEAF; pH 3, acetonitrile (20/80, v / v ) , flow rate 1 or 2 ml/min, depending upon the column used). Detection wavelength was 250 nm. Amino acid composition of isolated compounds was determined, after acid hydrolysis at 100oC for 24 h. The biological and immunological properties of the Leu-enkephalin obtained were controlled by Dr. E. Barres (Laboratoire de Physioiogie Nerveuse, CNRS, Gif-sur-Yvette) and Dr. J. M. Zajac (lnserm U266, CNRS UA498, UER des Sciences Pharmaceutiques et Biologiques, 4, av. de I'Observatoire, 75006 Paris), respectively.

Results In order to determine the optimal conditions for CPD-Y catalyzed Leu-enkephalin synthesis, each step was separately studied, in 50/zl volumes. The different reactions involved were coupling reactions, using esters as acyl components and amino acid ester or amino acid amide as amine components. In the latter case, since a peptide amide was obtained, we also studied enzymatic deamidation and chemical esterificaLI~'ZZ

I,~J

IJIt~L/UUI~,I~ 611 ~ , . q ~ t ~ l

ILIIglI.L g , U U I L I

IUIIf~LIUII

d~

the acyl component for the subsequent coupling step. The first reaction studied concerned the synthesis of B z - A r g - T y r - N H 2 , from B z - A r g -

793

O E t and T y r - N H 2 . B z - A r g - O E t was chosen as a solubilizing blocking group which could be easily removed by trypsin. We started by defining conditions for total hydrolysis of the B z - A ~ g - O E t alkyl group by CPD-Y (Table l). Under these conditions, 0.52 M tyrosinamide was added to the reaction mixture (Table I). As expected dipeptide B z - A r g T y r - N H 2 was synthesized, but 20% of the initial substrate B z - A r g - O E t were not transformed. It seemed that the formation of the a c y l - e n zyme intermediate between the alkyl ester and CPD-Y was partially affected by the presence of tyrosinamide. Since CPD-Y has a potential amidase activity, the nucleophile T y r - N H 2 may interact as a pseudo-substrate with the enzyme. This is suggested by the data in Table I, where the enzyme concentration was increased, resulting in the total transformation of B z - A r g - OEt. The product B z - A r g - T y r - N H 2 was then synthesized with a yield of 82%. After studying the formation of B z - A r g - T y r - N H 2 , we examined the conditions for its deamidation. For this purpose c~-chymotrypsin was chosen as the catalyst. Under the conditions presented in Table V, the deamidation yield of B z A r g - T y r - N H 2 reached 100%. The second reaction using CPD-Y as the catalyst, enabled us to obtain the tetrapeptide B z Arg-Tyr-Gly-Gly-OEt from B z - A r g - T y r - O E t and G l y - O E t . In contrast to previous condensations, the nucieophiie was in the ester form. Indeed, it was expected, using this amino acid derivative, that aiJ oligomer would be formed by incorporation of two G l y - O E t [18, 19], leading directly to a peptide ester. We started by

Table i. Synthesis of B z - A r g - T y r - N H 2. Concentrations of substrate, nucleophile, CPD-Y

Products formed

Yield (%)

100

B z - A r g - O E t (mM)

Tyr-NH2 (M)

CPD-Y (/zM)

40

-

35

Bz-Arg-OH

40

0.52

35

Bz-Arg-OH Bz-Arg-Tyr-NH2 Bz-Arg-OEt a

29 51 20

40

0.52

82

Bz-Arg-OH Bz-Arg-Tyr-NH2

18 82

Conditions: 0.1 M KCI, I mM EDTA, ethanol 10% (v), pH 9.6, total volume50 #!, 37°C, reaction quenched at 60 min with DFP. aCompound which was not transformed.

F. Hellio et al.

794

900

80

70

A

60

v

G) Ot 5O

\

cQ

o

n 4Q

-.-----" O B Z - R - Y - O H

0 ~ 0 _ _

u

a

.......+

o ~ Bz-R-Y-G-G-OEt

20 ,

. ~ J

[//~ ~ i 0

10

_-------. 20

~

/

~ 30

x

Bz-R-Y-G-OH

~ 40

$0

,.-,-Y-QSt 60

Time (minutes) Fig. 1, Time course of tetrapeptide B z - A r g - T y r - G l y - G l y - O E t synthesis. Conditions: 55 mM substrate, 0.5 M nueleophile, 4/zM CPD-Y, 0.1 M KCI, 1 mM EDTA, 10% ethanol (v), pH 9, total volume 50 ~1, 37oC. B z - R - Y - O H ( - O E t ) : B z - A r g Tyr-OH (-OEt); Bz-R-Y-G-OH (-OEt): Bz-Arg-Tyr-Gly-OH (-OEt); Bz-R-Y-G-G-OH (-OEt): BzArg-Tyr-Gly-Gly-OH (-OEt).

defining conditions for total transformation of B z - A r g - T y r - O E t into B z - A r g - T y r - O H , in the presence of CPD-Y. Under similar conditions, 0.5 M G l y - O E t was added to the reaction mixture, and the time course of its incorporation was followed (Fig. 1). In addition to the final product expected, B z - A r g - T y r - G l y - G l y - O E t , several other compounds appeared: tripeptide B z - A r g - T y r - G l y - O E t whose kinetics of formation are characteristic of an intermediate compound, and three hydrolysis products i.e., B z - A r g - T y r OH, B z - A r g - T y r - G l y - O H and B z - A r g T y r - G l y - G l y - O H . As far a~ the dipeptide

B z - A r g - T y r - O H is concerned, the fact that it accumulated from the beginning confirmed that CPD-Y has a higher affinity for B z - A r g - T y r OEt than for G l y - O E t [20]. Otherwise, it can be noted that, after 1 h of incubation, the s~abstrate B z - A r g - T y r - O E t was totally transformed, the tripeptide B z - A r g - T y r - G l y OEt was present in a negligible amount and the whole products of hydrolysis represented over 70% of the compounds. We have shown that this result was not due to an insufficient concentration of G l y - O E t . Indeed, the increase in G l y - O E t concentration over 0.5 M did not modify the percentage of the hydrolysis product.

g-~

f

795

e ~ ",,d-

tL d~ ¢'~1 0 0

.o v

O

~

~

,A

c-

'

o" .e,

.o

o

I

t¢1 '~'-

I

O v

O I

A

~w ~

m

~luld.i

El

O

.< I "O

<-

© I 0

N

[..,

,.

ud I

?

.o 8O

E

0 I

o

v~

O

<,

O

O I

O I

O0

O0

~ l l

II

"0

?

=

0 0 0 0 0 0

.o

_=

m i

i

T

O0 !1

0

O0 II

E

"O

~o M m

"O er -.1 O t'm

E O

0 0 0 0 0 0 I l l l l l I I I I I I

N

N

N

N

N

:= N~

< < < < < < I I I I I I ~

E C

="

•~, .~

I

l

l

l

l

I

I

I

I

I

I

"0 0 e~

0

I

t-

< < < < < < I N

I N

I I I I N N N N

"0 cO

796

F. Hellio et al.

The presence of non-esterified compounds resuited in part from the poor affinity of CPD-Y for G l y - O E t . The concentration of the main competitor, water, was reduced by adding ethanol, which also allowed the solubilization of the ester substrates. The influence of 5 other solvents was compared to that of ethanol. Experiments were performed in the presence of 16/zM instead of 4/xM CPD-Y, in order to shorten the reaction time from 1 h to 15 min. From the results obtained (Table II), the 5 solvents could be divided into 4 groups according to their activities: 1) D M F and DMSO which inhibited the catalytic properties of CPD-Y. In their presence, the enzyme no longer functioned which suggested partiai or total denaturation of the catalyst. 2) Butanediol-l,4 selectively modified CPD-Y in a way that disadvantaged aminolysis of the intermediate complex. Such a phenomenon may result from a modification of the enzyme affinity for the nucleophile, G l y - O E t . 3) In the presence of ethyleneglycol no significant increase in the B z - A r g , T y r - G l y G l y - O E t percentage was noticed. 4) When 40% glycerol was oresent in the reaction mixture, it notably favt.r..d the synthesis of B z - A r g -Tyr-Gly-Gly-OEt. However, it must be noted that the increase in the glycerol concentration did not improve the results. On the contrary the aminolysis reaction was affected (Table Ill). The most satisfactory glycerol concentration was 40% and, under these conditions, the product gJ/., /-lkl~-- lyl--~..Jly--K.Jly UI,~.I, ~lfll~ uutant~d with a yield of 50%. At this stage of the study, having bound the two G l y - O E t , it was conceivable to eliminate the B z - A r g group from B z - A r g - T y r - G l y G l y - O E t and further use T y r - G l y - G l y - O E t and T y r - G l y - G l y - P h e - O E t as ester sub-

strates. The absence of an N-blocking group in these peptides was not crucial. Indeed, to ensure a correct binding between CPD-Y and its substrate, the !atter has to meet the following requirement [16]: it must bind to at least 3 subsites of the enzyme S~, S'l, $2 [21]. Whereas T y r - O E t had to be blocked by the benzoylarginyl residue to meet this requirement, the tripeptide and the tetrapeptide were expected to correctly interact with CPD-Y. Thus the conditions for total hydrolysis of the B z - A r g group from Bz-Arg- Tyr-Gly-Gly-OEt, in the presence of trypsin, were defined (Table V). The third peptidic synthesis examined was the incorporation of P h e - N H 2 , starting from T y r - G l y - G l y - O E t . First, we studied conditions for total hydrolysis of the esterified tripeptide to T y r - G l y - G l y - O H , in the presence of CPD-Y. Then, 86 mM phenylalaninamide was incubated under the conditions found and the time course of its incorporation was followed (Fig. 2). It can be noted that free phenylalanine accumulated, which was not expected. It did not come from the free phenylalaninamide but was due to the exoprotease activity of CPD-Y towards the peptide T y r - G l y - G l y - P h e - O H , suggested by the decrease in its percentage. In parallel, T y r - G l y - G l y - O H was formed in the reaction mixture. Tetrapeptide T y r - G l y - G l y P h e - O H , itself, came from T y r - G l y - G ! y P h e - N H 2 , a component which disappeared if I_ . . . . . . . . llll~ WU~ s u f ~ l l c i e n tly I' O l l ~ . .t l.l .~ . . . .11. lt.~ . . .U.I..lial . . t I U I I t l:--~ Finally, if one compares the accumulation kinetics of T y r - G l y - G l y - O H and free phenylalanine, it appears that hydrolysis of T y r - G l y - G l y - O E t to the tripeptide and ethanol was very low. Synthesis conditions were excellent. It should be noted that, at pH 8, C P D - Y hydrolyz-

Table IV. Influence of Leu-NH2 concentration on CPD-Y-catalyzed Leu-enkephalin amide synthesis. Leu-NH2 concentration

(u)

0.10 0.12 0.25 0.46

Yield of products formed (%) Tyr-Gly-Gly-Phe-OH

Leu-enkephalin amide

75 47 35 10

25 53 65 75

Conditions: Substrate 25 mM, CPD-Y 16/.tM, 0.1 M KCI, 1 mM EDTA, 10% ethanol (v), pH 9.5, total volume 50 #l, 37oC. Reaction quenched at 60 min. After quenching the reaction with DFP, 10-30/zl of DMF were added to solubilize Leu-enkephalin amide.

Enzymatic microsynthesisof Leu-enkephalin

797

÷

lOq

90

80

+ F-NH2

~

i

60ir.G.G.

m

50

O Y-G-G-F-OH

/

° Q.

o

="

- e~

"r" Z I

40 •

2 E°

a

0

A J 30

Y-G-G- F-NH| 60

90

120

Time (minutes) Fig. 2. Time course of T y r - G l y - G l y - P h e - N H 2 synthesis. Conditioos: 32 mM substrate. 86 mM nucleophile, 226 ~M CPD-Y, 0.1 M KCI, I mM EDTA, 10% ethanol (v), pH 8, total volume 50 p,l, 37oC. F - O H (-NH~): P h e - O H (-NI-t2); Y - G - G - O H ( - O E t ) : T y r - G l y - G l y - O H ( - O E t ) ; Y - G - G - F - O H (-NH2): T y r - G l y - G l y - P h e - O H (-NH2).

ed the amide group of T y r - G l y - G l y - P h e NH2. Thus, after 1 h of incubation, when no more T y r - G l y - G l y - O E t substrate was left, T y r - G l y - G l y - P h e - O H represented 67% of the mixture compounds and the synthesis yield reached 73% (this value does not take into account the molar percentage of P h e - O H ) . The last elongation step, in our strategy, was the condensation of T y r - G l y - G l y - P h e - O E t

and Leu-NtI2. To start, the esterase activity of CFD-Y towards the peptide ester was studied. Under conditions of total hydrolysis of the ester to T y r - G l y - G l y - P h e - O H , the nucleophile 0.12 MLeu-NH2 was added to the reaction mixture. The time course of its incorporation is presented in Fig. 3. This figure shows that within 1 h, substrate T y r - G i y - G l y - P h e - O E t was totally trans-

798

F. Hellio et al.

'°t

"

Y-G-G-F-OH

i

•

60

a;c ~ 5o Q.

_~ 4o 0

Z

301-1

.~

O Y-G-G-F-L-NH2

20

10

0

~p/Y-G~G-

30

F-OEt

60

90

120

Time (minutes) Fig. 3. Time coarse of Leu-enkephalin amide synthesis. Conditions: 25 mM substrate, 0. ~2 M nueleophile, 16 tzM CPD-Y, 0.1 M KCI. i mM EDTA. 11)%ethanol (v), pH 9.5, to~al volume 50/~I, 37°C. Y - G - G - F - O H (-OEt): T y r - G l y - G l y - P h e - OH (-OEt); Y - G - G - F - L - N H - : T y r - G l ~ - G l y - P h e - L e u - N l - t2.

formed into Leu-enkephalin amide as well as into T y r - G l y - G l y - P h e - O H . After 1 h of incubation, the percentage of Leu-enkephalin decreased. This result was due to peptidyl amino acid amide hydrolase activity of CPD-Y towards the pentapeptide. It can also be noted that the hydrolysis preduct, T y r - G l y - G l y - P h e - O H , accumulated during the 5 first min of incubation and represented 43% of the mixture compounds after 1 h, indicating that Leu-NH2 concentration was insufficient. If this concentration was increased, the percentage of Leu-enkephalin amicie increased, while the percentage of the hydrolysis product decreased, as shown in Table IV.

The most favorable concentration of L e u - N H 2 was 0.46 M, since Leu-enkepha!in amide represented 75% of the compounds. Taking into account the presence of 10% T y r G l y - G l y - P h e - O H in the initial compound T y r - G l y - G l y - P h e - O E t , the synthesis yield reached 85%. After the incorporation of Leu-NH2, the study of T y r - G l y - G l y - P h e L e u - N H 2 deamidation wag necessary. We used CPD-Y as the catalyst, and successfully obtained Tyr-Gly- Gly-Phe-Leu-OH from Leuenkephalin amide (see conditions in Table V). After the optimization of each enzymatic reaction, the synthesis of L e u - e n k e p h a l i n was attempted, on a microsc'ale. Starting with

799

m

N o,..

0

Z~

0

0

z

I

tO

0

.-~

I

I

E e-

Z I

r~

0

i

I

I

I

I

I

I

I

I

I

N

I

I

t~

~.

I

._.

I

....

I

e~

F. Hellio et al.

800 Arg

_OEt

Gly

Gly

Tyr

Leu

Phe

H_ ~NH 2

Bz

Y

NH 2

Bz

CT

OH OEt

Bz

H_ _OEt

H_ DEt

OEt

Bz

OEt

H H

Y

H- _NH 2 _OH _OEt

H

H__NH 2

-NH 2 _OH

Scheme 2. Syr~thesis of Leu-enkephalin. Y: CPD-Y; T: trypsin; CT: chymotrypsin. *: chemical esterification.

9.8/zmol of B z - A r g - O E t , we successively incorporated the nucleophiles Tyr-NH2, G l y - O E t , Phe-NH2 and Leu-NH2 as outlined in Scheme 2. If the condensation product was in the amide form, it was deamidated prior to its chemical esterification with ethanol and thionylchloride. In the particular case of the incorporation of Phe-NH2, since the product formed, T y r - G l y - G l y - P h e - N H z , was deamidated by CPD-Y, the deamidation step was eliminated. After each reaction, the reactants were separated by HPLC to remove the hydrolysis 'by product' and the excess amine component. The reaction yields for esterifications were 100% and ester components were not purified. It can be seen from Table V that the synthesis of Leu-enkephalin is characterized by satisfactory reaction yields and from our synthetic pathway 50 nmol of Leu-enkephalin were obtained. The losses were due to the purification steps which reduce the total amount of Leu-

enkephalin. Amino acid analysis of the pentapeptide was consistent with the theoretical values and the enzymatically prepared molecule retained both full immunological and biological potencies. Conclusion

During the study presented here, we investigated the use of CPD-Y for peptide synthesis on a microscale. A pentapeptide, Leu-enkephalin, was chosen as object of the study, since several other attempts with much larger quantities have been published. Starting with 9.8/xmol of the precursor, B z - A r g - O E t , the stepwise synthesis of the molecule was performed and led to the recovery of 50 nmol of pure Leu-enkephalin. All condensation yields were over 50%, showing that enzymatic peptide synthesis is feasible on a microscale.

Enzymatic microsynthesis o f Leu-enkephalin

Differences were noticed according to the amino acids used as nucleophiles. Amino acid amides were incorporated with yields over 70%. However, the elongated peptide was in the amide form and two additional reactions (deamidation and esterification) had to be performed to obtain the peptide ester for the subsequent coupling step. In the particular case of P h e - N H 2 , after condensation of this nucleophile with T y r - G l y - G l y - O E t , the aminolysis product T y r - G l y - G l y - P h e - N H 2 was deamidated by CPD-Y. This result could be explained by the pH of the reaction (pH 8) which was close to the optimum pH for CPD-Y amidase activity. Other authors [9] have studied the condensation of B z - A r g - T y r - G l y - G l y - O E t and P h e - N H 2 but did not mention amidase activity of CPD-Y towards B z - A r g - T y r - G l y - G l y P h e - N H 2 . Whether this difference is due to the presence of the B z - A r g group or to enzyme preparations is not known. Apart from the distinct CPD-Y affinities for the different nucleophiles, the effect of water is predominant on the aminolysis of the a c y l - e n zyme intermediate, if the condensation reaction occurs in pure aqueous medium, where the water concentration reaches 55 M, hydrolysis reactions can be significant. Moreover, the lower the affinity of CPD-Y for the nucleophile, the more important these reactions are. To limit this phenomenon, it is possible to reduce the water concentration by adding organic miscible solvents to the reaction mixture. In this study, glycerol was used to enhance the condensation yield of B z - A r g - T y r - O E t and G l y - O E t . However, the solvent probably interferes with the enzyme conformation, an effect which can lead to positive or negative results as sho~:n in Table II. It would be interesting to define more precisely what is required at the enzyme level, for the retention of the catalytic properties. Then the difficulties might be overcome by using CPD-Y variants with an increased hydrophobicity after chemical modifications or by means of site-directed mutagenesis [19, 22-26]. From this study, it appears that high concentrations of nucleophiles are required to reach satisfactory synthesis yields. However, to keep the total amounts of reagents small, the reaction has to be performed in very reduced volumes. As a consequence, small amounts of elongated peptides are synthesized and difficulties as well as losses arise during purification. Nevertheless, enzymatic synthesis on a microscale can be an attractive method for the preparation of highly

801

labeled hormone peptides, since only minute amounts of these molecules are required for molecular physiology studies.

Acknowledgments We are indebted to Dr. P. Fromageot for his advice and for critically revising the manuscript. We would like to gratefully acknowledge E. Barres and J. M. Zajac for performing biological and immunological assays.

References 1 Bergmann M. & Fraenkel-Conrat H. (1937) J. Biol. Chem. 119,707-720 2 Bergmann M. & Fruton F. S. (1938) J. Biol. Chem. 124,321-329 3 Morihara K. & Oka T. (1977) Biochem. J. 163, 531-542 4 Jakubke H. D., Kuhl P. & Krnnecke A. (1985) Angew. Chem. Int. Ed. Engl. 24, 85-88 5 Wong C. H., Chen S. T. & Wang K. T. (1979) Biochem. Biophys. Acta 576, 247-249 6 Mitin Y. V., Zapevalova N. P. & Gorbunova E.Y. (1984) Int. J. Pept. Protein Res. 23, 528-534 7 Hugues J., Smith T.W., Kosterlitz H.W., . . . . . ~gm L. A., Morgan B. A. & Morris H. R. (1975) Nature 258, 577-579 8 Kuilmann W. (1980) J. Biol. Chem. 255, 8234-8235 9 Widmer F., Breddam K. & Johansen J. T. (1981) in: Proc. 16th Eur. Pept. Symp. (Brunfeldt K., ed.), Seripter, Copenhagen, pp. 46-55 10 Hayashi R., Moore S. & Stein W. H. (1973) J. Biol. Chem. 248, 2296-2302 11 Hayashi R., Bai Y. & HataT. (1975) J. Bhgchem. 77, 69- 79 12 Bai Y., Hayashi R. & HataT. (1975) J. Biochem. 77, 81-88 13 Bai Y., Hayashi R. & Hata T. (1975) J. Biochem. 78,617-626 14 Breddam K., Widmer F. & Johansen J. T. (1980) Carlsberg Res. Commun. 45,237-247 15 Widmer F. & Johansen J. T. (1979) Carlsberg Res. Commun. 44, 37-46 16 Breddam K. (1986) Carlsberg Res. Commun. 51, 83-125 17 Widmer F., Breddam K. & Johansen J. T. (1981) Carlsberg Res. Commun. 46, 96-106 18 Widmer F., Breddam K. & Johansen J. T. (1980) Carlsberg Res. Commun. 45,453-463 19 Widmer F., Breddam K. & Johansen J. T. (1983) Carlsberg Res. Commun. 48,231-237

802

F. Hellio et al.

20 Breddam K., Widmer F. & Johansen J. T. (1980) Carisberg Res. Commun. 45,361-367 21 Scheehter I. & Berger B. (1967) Biochem. Biophys. Res. Commun. 27, 157-162 22 Breddam K. (1983) Carlsberg Res. Commun. 48, 9-19 23 Breddam K. & Svendsen I. B. (1984) Carlsberg ICes. Commun. 49,639-645

24 Breddam K. (1984) Carlsberg Res. Commun. 49, 627- 638 25 Winther J, R., Kielland-Brandt M. C. & Breddam K. (1985) Carlsberg Res. Commun. 50, 273-284 26 Beth L. M., Nielsen J., Winther J. R., KieUandBrandt M. C. & Breddam K. (1986) Carlsberg Res. Commun. 51,459-465