Inr. J . Peptide Prorein Res. 40. 1992, 1 10- 1 13

Hydrolysis of peptide esters by different enzymes S I E G M U N D REISSMANN and G E O R G GREINER

Instirute qj' Bi0chet~7i.~tr~ aud Bioph,~~.~ics, Friedrich Schiller t',tirersitj.

cf Jeria, Jetia, German-v

Received 21 August 1991, accepted for publication 22 March 1992

The combined use in peptide synthesis of the Fnioc-group with methyl, benzyl or p-nitro benzyl esters is not practical because of the elimination of the Fmoc-group under basic conditions and by catalytic hydrogenation. Nevertheless the solution synthesis of peptides requires those combinations in some cases. For this purpose we have investigated enzymatic hydrolysis of some tri and tetrapeptide esters. The hydrolysis were carried out under pH-control. We measured deprotection of the carboxyl group by thermitase, porcine liver csterase, carboxypeptidase A and x-chymotrypsin. The main problems are to suppress proteolytic degradation of the peptide bond and to bring thc protected peptides into solution. To solve both problems we used diniethylforniamide and dimethylsulfoxide as cosolvents. The ratios between esterolytic and proteolytic activity were estimated under various cosolvent concentrations. Advantages of this method are to avoid side reactions of alkaline instable side chains (e.g. asparagine, glutamine), cleavage of base labile protecting groups and racemization by alkaline saponification. The enzymatic deprotection was followed by HPLC, HPTLC and titration. On a preparative scale this method gives good yields and sufficently pure products. Kej, ir.ords: enzymatic: cstcr h!drol! sis: Fmoc; pcptide s!ntliesis: thcrmitase

The use of enzymes for the cleavage of protecting groups opens new possibilities in the strategy and tactics of peptide chemistry and allows mild, sclcctive deblocking without side reactions. Enzymes have been used for the deblocking of amino (l), carboxy (2) and sulfur protecting groups (3). The combination of the Fmoc-group with methyl, benzyl or p-nitro benzyl ester groups is still impossible because of the elimination of the Fniocgroup under basic conditions and by catalytic hydrogenation. Nevertheless, the solution synthesis of peptides requires those combinations in some cases. For this purpose we have tried to carry out enzymatic saponification of some tri and tetrapeptide esters. Because of the high esterolytic activity first attempts were made to use thermitase for the hydrolysis of peptide esters (4) and for resolution of amino acids (5). For a comparison of different enzymes we estimated the esterolytic abilities of thermitase, porcine liver esterase, carboxypeptidase A and x-chymotrypsin to find the optimal conditions for preparative enzymatic hydrolysis of Fmoc-peptide esters. The main problems are first to suppress proteolytic degradation of the peptides and second to bring the protected peptides into solution. We report here one method to accomplish these goals. In addition to the described advantages the enzy110

niatic cleavage of esters prevents the racemization observed by the commonly used alkaline saponification (6). This advantage is of interest despite the relatively high racemization rates by the fragment condensation with some classical coupling methods. The reason why the optical purity of the fragments will become more important is the improvement of the coupling methods including enzymatic coupling for racemization free peptide synthesis. MATERIALS AND METHODS E~zqw?es For hydrolysis of the peptide esters we used: porcine liver esterase (PLE), EC 3.1.1.1 (130 Ujmg) from Boehringer Mannheim, z-chymotrypsin (CT) EC 3.4.21.1 (45 U,'nig) from SERVA, carboxy peptidase A (CPA) EC 3.4.17.1 (30 U/mg) from REANAL, thermitase (T) EC 3.4.21.14 (30 Ujmg) from the Institute of Biotechnology of Berlin. Pep tides Boc-Ala-Phe-OMe was synthesized by the DCCI/ HOBt-method from Boc-Ala-OH and Phe-OMe.HCL in a yield of 88 "/". It was purified as usual by extraction

Hydrolysis of peptide esters with 5 % KHS04 and 10% NaHC03, m.p. 84-86' (ethyl acetate/hexane); [ a I D= 19.5 (c 1, MeOH, 25"); TLC: Rf = 0.58 (chloroform/methanol 9: 1; silica gel, Merck). The synthesis of Boc-Val-Asn-Phe-OMe; Z-Val-Asn-Phe-OMe; Fmoc-Val-Asn-Phe-OMe; Fmoc-Val-Asn-Phe-Ser-OMe; Boc-Val-Asn-Phe-SerOMe; Boc-Asn-Phe-OMe; Boc-Phe-Ser-OMe; BocLeu-Val-Ile-OBzl(N02); Boc-Val-Ile-OBzl(N02) will be published elsewhere. Estimation of the MICHAELIS constant The KMvalue of Boc-Ala-Phe-OMe was determined by the rate of NaOH consumption during esterolysis by thermitase (T 5 5 " ; pH 8.0; 0.5 mM CaCh, 10% DMF, substrate concentration 20-0,5 mM. The Michaelis constant was calculated from the Lineweaver-Burk-plot.

HPLC For HPLC a system from Shimadzu was used: delivery system LC-6A autosampler SIL 9A, UVjVIS detector SPD 6AV and a Nucleosil C-18 reverse phase column ( 5 pm, 4 x 250 mm, Macherey-Nagel, FRG). The flow rate was 1 mL/min, isocratic conditions: acetonitrile/lO mM, ammonium acetate; 7/3. Estimation of esterolytic activity Enzymatic hydrolysis was carried out in an autotitrator TTT2, autoburette ABU 13 (0.2 mL) and titrigraph REA 300 from Radiometer/Copenhagen. The rate of NaOH consumption during hydrolysis of Boc-Ala-PheOMe (30 mM) as used to estimate the esterolytic activity of the enzymes. The following conditions were used: T(55"; pH 8.0; 0.5 mM CaC12); CT(37"; pH 8.0; 0.5 mM CaC12); CPA (37"; pH 7.5; 0.5 M NaCI), PLE (37"; pH 8.3; 1.5 M NaCI). To guarantee precise pH measurement, the electrodes were stored 24 h before use in the reaction medium if the addition of organic cosolvents was necessary. For preparative hydrolysis of peptide esters by thermitase we used the following conditions: 100 mL50% DMSOor40% DMF;0.5 mM CaC12; pH 7.5; 2 mmol substrate; 3 mg therniitase; 55 O . Estimation of the proteolytic activity Estimation of the proteolytic activity of T, a-CT and CPA was performed by a modified azocasein test (7). The pH of 0.2% azocasein in 0.1 M Tris buffer was readjusted to 8.0 after the addition of organic cosolvent. After an incubation time of 10 min, the reaction was stopped by the addition of 20% TFA. Proteolytic activity was estimated by the change in absorbance at 366 nm.

RESULTS AND DISCUSSION The low solubility of Fmoc peptide esters requires the addition of large amounts of organic cosolvents like DMF, DMSO etc. for the enzymatic hydrolysis of peptide esters. Therefore the influence of organic solvents

DMF concentrotion (percent) FIGURE 1 Dependence on the D M F concentration of the relative esterolytic activities of thermitase ( 0- - - 0 ) ,x-chymotrypsin (0- - -0). porcine liver esterase .( - -).and carboxypeptidase A (A - - -A);Substrate: Boc-Ala-Phe-OMe (30 mM).

on the enzymatic reaction had to be investigated. Fig. 1 shows the effect of addition of D M F on the relative esterolytic activity of T, a-CT, PLE and CPA. The esterolytic activities of these enzymes were estimated by the rate of the NaOH consumption during the hydrolysis of Boc-Ala-Phe-OMe. Because of its good solubility and its low KM constant (0.8 mM) this easily synthesized dipeptide methyl ester is a very useful substrate for investigation of enzymatic hydrolysis by thermitase. To get the relative enzyme activity the value

10

20

30

40

50

60

70

Organic solvent concentration (percent) FIGURE 2 Dependence of the total enzyme activity of thermitase on the concentration of various dipolar, aprotic solvents; Dioxane (A- - -A),

DMF(.---.),DMSO(O---O),55";pH8.0;0.5mMCaC1~; Substrate: Boc-Ala-Phe-OMe (30 mM).

111

S. Reissmann and G. Greiner

"

10

20

30

LO

50

60

70

OMSO concentration (percent) FIGURE 3 Dependencc of thc relative proteolytic ( 0 - - - 0 ) and cstcrol>tic (0 - - -0) activity of thcrrnitase on DMSO concentration: lo",, DMSO = 100", enzyme activity; 55' enzyme activity: proteolytic estcrolltic substrate: 0.2", azocasein Boc-Ala-Phe-OMe (30 niM) buffer: 0 . 1 Tris, ~ pH 8.0, pH-stat. pH 8.0. 0.5 mM CaCI-. 0.5 nlM CaCl2

in DMF was fixed to lOO",. According to Fig. 1 thermitase is much more stable in the presence of D M F than all other enzymes studied. A comparison of the influence of various aprotic, dipolar cosolvents, usually used in peptide chemistry. on the esterolytic activity of thermitase is given in Fig. 2. DMSO stabilizes thermitase much more than dioxane or D M F and is therefore recommended. Fig. 3 shows the dependence of the proteolytic and esterolytic activity of thermitase on the DMSO concentration. Proteolytic activity was checked with the azocasein test. The apparent increase of proteolytic activity of thermitase by the addition of DMSO up to 15% may be caused in a better enzyme substrate interaction resulting from unfolding of the azocaseine molecule. In the presence of 50"" DMSO, thermitase has only esterolytic activity. Probably the proteolytic

reaction mechanism of thermitase is more influence by organic cosolvents than the esterolytic one. The suppressed proteolytic and the high absolute esterolytic activity in the presence of 50% DMSO make thcrmitase suitable for enzymatic hydrolysis of peptide esters without damage to the peptide bond. Purity of the hydrolyzed peptide esters was checked by HPLC and HPTLC. No degradation of the peptide bond was observcd (Fig. 4). Table 1 shows various peptide esters which were hydrolysed by thermitase. Thermitase is a thermophilic, alkaline protease which was isolated from ThermoactinoniJres vulgaris (8) and has properties similar to those of subtilisin. The broad substrate specifity combined with high regio and stereo specifity recommend this enzyme for our purpose. Because of its high esterolytic activity attempts were first made to use thermitase for hydrolysis of peptide esters. In addition to the hydrolysis of methyl-, ethyl-, rert.-butyl- and benzyl esters by thermitase (4) we were also successful in the hydrolysis of p-nitro benzyl esters. All common amino acids at the C-terminal end, excluding D amino acids, proline and A'-unsubstituted amino acid esters, were accepted. Hydrophobic amino acids in P I and PZ position are favoured. In spite of the reaction temperature of 55" and 50% DMSO, hydrolysis of the Fmoc- and Z-derivatives had to be carried out in suspension. In this case, diffusion processes often limit the enzymatic hydrolysis and decrease the reaction rate. Effective stirring is required to guarantee a homogeneous reaction milieu. No inhibition by the substrate was observed at the concentration used (20 mM). The pH-stat technique circumvents the application of buffer systems, which can cause complications in preparative runs, and simplifies the purification procedure. At the end of the enzymatic hydrolysis we obtained a nearly clear solution, which was filtered and acidified to pH 2.0. The precipitated crude product kvas purified by extraction or crystallization in the usual manner. Thermitase was successfully used for the enzymatic hydrolysis of Fmoc-Val-Asn-Phe-OMe in gram scale kvithout side reactions and in good yield and purity as shoLvn in Fig. 4. Because of the weakly basic conditions

~

Compound Boc-Val-Asn-Phe-OMe Z-Val-Asn-Phe-OMe Fmoc-Val-Asn-Phe-OMe Fmoc-Val-Am-Phe-Scr-OMe Boc-Val-Asn-Phe-Scr-OMe

** 5 5 112

, 0.5 mM CaC12, pH-stat pH

Compound 45 105 I40 100

90

t [min]*

Boc- Ala-Phe-OMe Boc- Asn-Phe-OMc Boc-Phe-Ser-OMe Boc-Leu-Val-Ilc-OBzl(N0~) Boc-Val-Ilc-OBzl(N02)

8 0, 50", DMSO, tubstrate 10 mM, enzlme conc 30 pg mL:

90°, product formation

3 65 25 16

28

Hydrolysis of peptide esters advantages are the high stability against organic solvents, the high esterolytic activity, the low autolysis, the low side chain specifity and the high temperature optimum. The addition of 50% DMSO of40% DMF suppresses the proteolytic activity completely. This method avoids degradation of base sensitive side chains and racemization and allows the application of base labile protecting groups during the alkaline saponification of peptide esters. It gives good yields and sufficently pure products in preparative scale work.

REFERENCES

----id

hoc-Val-Asn-Phe

-U

FIGURE 4 Preparative hydrolysis of Fmoc-Val-Asn-Phe-OMe by thermitase. a) Time curve for enzymatic hydrolysis, 100mL 50% DMSO; 0.5 mM CaCI2:3 mg thermitase; 55"; pH-stat, p H 7.5; 2 mmol FmocVal-Asn-Phe-OMe (sample from the crude digest of the esler). b HPLC; Nucleosil 100, 5 pm; 4 x 250 mm; 1 mL/min; 220 nm; acetonitrile/lO mM amnionium acetate 7/3.

(pH 7.5) no cleavage of the Fmoc-group or the deamidation of the asparagine residue could be observed. With the help of this tripeptide we achieved the solution synthesis of the insect neurohormone Pyr-Val-AsnPhe-Ser-Pre-Asn-Trp-NH2 (manuscript in preparation).

CONCLUSION Thermitase is more useful for enzymatic hydrolysis of Fmoc peptide esters than the other enzymes tested. Its

1. Fuganti, C1. & Grasselli, P. (1986) Terruhedron Leu. 27, 31913194 2. Chen, S.-T., Lo, L.-Ch., Wu, S.-H. & Wang, K.T. (1990) h r . J . Peptide Protein Res. 35, 52-54 3. Hermann, P. & Greiner, G. (1986) WP DD 236 346, 4.6.1986 4. Hermann, P. & Salewski, L. (1983) in Peptides IY82 (Blaha, K. & Malon, P., cds.), pp. 399-402, Walter de Gruyter, Berlin 5. Lankiewicz, L., Kasprzykowski, F., Grzonka, Z . , Kettmann, U . & Hermann, P. (1989) Bioorg. Chert?. 17, 275-280 6. Kenncr, G.W. & Seely, J.H. (1972)J. A m . Chenz. Soc. 94. 32593260 7. Barrett, A.J. & Kirschke, H . (1981) Methods EnqwtoL 80, 535561 8. Kleine, R. & Rothe, U. (1977) Actu Bid. Med. Germ. 36, K27K33

Address: Dr. Georg Greiner Friedrich- Schiller-University Biological Faculty Institute of Biochemistry and Biophysics Philosophcnweg 12 0-6900 Jcna Germany

113