Bulgarian Chemical Communications, Volume 41, Number 2 (pp. 122-126) 2009

Received July 4, 2008; Revised September 18, 2008

The development of new protective groups by means of stereospecific and selective reactions is very important for the practice especially in the synthesis of semi-synthetic antibiotics and biologically active peptides. The aim of the present investigation is to study the kinetics of enzyme hydrolysis of the Phenylacetyl (Phac) group in synthetic peptide substrates. Two model compounds with two and three Phac, respectively, were studied. The enzymatic hydrolysis by means of penicillin G acylase in pH range 6–9 was studied. The substrate concentration was between 0.4–40 mM. Maximum degree of substrate conversion 75.5% (pH 9), which is closed to the degree of conversion to pH 7.8 (74.5%), was obtained for 3 to 10 min. At pH 6.5 and 7 the degree of hydrolysis was 62.5 and 37.5%, respectively. The possibilities for the reaction of hydrolysis in the presence of salts of different complex agents CuSO₄.5H₂O, NiCl₂, Ni(NO₃)₂ and MnCl₂ were investigated, too. There was 100% degree of Phac group hydrolysis in the presence of 10% Ni(NO₃)₂ for 9 min and full shift of equilibrium to the direction of hydrolysis was monitored in the presence of 25% Ni(NO₃)₂.

Key words: enzyme kinetic; penicillin G acylase; peptide mimetics; phenylacetyl protecting group.

INTRODUCTION

Peptides and α-amino acids are widely used in the medical practice. The development of new protective groups by means of stereo specific and selective reactions is very important for the practice especially in the synthesis of semi-synthetic anti-biotics and biologically active peptides. Penicillin G acylase (PGA) is an enzyme that demonstrated high specificity on the phenyl acetyl residue. The enzyme’s hydrolase activity is of a great importance for the new substrates modifications [1–5].

The aim of the present investigation is to study the kinetics of enzyme hydrolysis of the amide function between Phenylacetyl (Phac) group and amino acid residue.

EXPERIMENTAL

PGA [E.C 3.5.1.11] from E. Coli was supplied by Fluka (Switzerland). The enzyme activity of 980 E/ml was determined according to [6]. In the reaction solution, the product was diluted to 98 E/ml. The solution with concentration 0.15 mg/ml was used. KOH, KCl, NaOH, HCl; NH₄OH, KH₂PO4; K₂HPO₄ and CH₃CN - gradient grade for HPLC were received from MERCK (Darmstadt, Germany); NiCl₂; Ni(NO₃)₂; CuSO₄.5H₂O; MnCl₂ were purchased from Reanal (Budapest , Hungary)

The IR spectra were recorded on a Perkin-Elmer Model 1600 Series FTIR instrument. The purity of the products and kinetics of the hydrolysis reactions of phenylacetyl group were checked by RP-HPLC on a Perkin-Elmer apparatus, C₁₈ column 4.6×250 mm ODS-A, S-5 micron 120 A. 50% 0.02 M KH₂PO₄:K₂HPO₄ pH = 7: 50% CH₃CN were used as eluents, rate 0.8 ml/min, λ = 220 nm and diode array detector.

The needed substrates Boc-Lys(Phac)-Val-OMe and Boc-Lys(Phac)-Lys(Phac)-Lys(Phac)-OH were obtained by conventional peptide synthesis in solution.

Boc-Lys(Phac)-Lys(Phac)-Lys(Phac)-OH was synthesized by drop-wise addition of Boc-Lys(Phac)-OH to C-terminal H-Lys(Phac)-OH by using of MA (mixed anhydrides) method:

1.00 mmol of Boc (or Z) amino acid (or peptide obtained from Boc-peptide by treatment with 10-fold excess of TFA) was dissolved in THF (10 ml) and after cooling to –10 ÷ –15°C and good stirring 1.00 mmol Et₃N and 0.98 mmol isobutyl chloro-formate were added. After 15 min 1.00 mmol of amino component without protecting groups dissolved in 1.1 mmol 2 N NaOH was added dropwise. The reaction mixture was stirred for 1 h at –10 ÷ –15°C and 24 h at room temperature. After that it was concentrated in vacuo and 2 N HCl was added to pH 2–3. The product was extracted with EtOAc (3×10 ml) and the organic layer was washed with 10% citric acid (3×10 ml) and H₂O to pH 7. The solvent was dried with Na₂SO₄ and removed in vacuo. The obtained products Boc-Lys(Phac)-Lys(Phac)-OH and Boc-Lys(Phac)-Lys(Phac)-Lys(Phac)-OH were oils.

Boc-Lys(Phac)-Val-OMe was obtained by the DCC/1-HOBt method:

1.00 mmol of HCl.H-Val-OMe was dissolved in DMF (10 ml) and after cooling to –5°C, neutralized to pH 7–7.5 with Et₃N. 1.2 mmoles of Boc-Lys(Phac)-OH; 1.20 mmol of DCC and 1.4 mmol of 1-HOBt were added. The reaction mixture was stirred for 24 h at room temperature. The obtained DC-urea was removed by filtration and then 30 ml of water were added. The product was extracted with EtOAc (3×10 ml) and the organic layer was washed with 5% NaHCO₃ (3×10 ml), H₂O (3×10 ml), 10% citric acid (3×10 ml) and H₂O to pH 7. The solvent was dried with Na₂SO₄ and removed in vacuo followed by recristalization in EtOAc/petro-leum ether.

The obtained product has the following IR spectral characteristics: [cm^–1] 3328 ν_N-H; 3067 ν_C-H (aromatic system); 2930 ν_C-H (CH₃); 2853 ν_C-H (CH₂); 1740 ν_C=O (ester); 1700 ν_C=O (uretan); 1680 ν_C=O (amide I); 1661 ν_C=O (amide II); 1574 δ_N-H (amide I); 1545 δ_N-H (amide II); 1390 and 1367 δ_C-H (isopropyl and tert-butyl groups); 1247 ν_C-O-C (uretan); 743 δ_C=C and 696 δ_C=C (monosubstituted aromatic system).

General procedure for the reaction of hydrolysis of Phac by means of PGA

20 ml of substrate with the needed concentration in 10% DMF/0.1 M KCl were alkalized till pH 9 with 4 M K₂HPO₄. The solution of PGA with needed concentration was added. The hydrolysis of substrate was monitored on HPLC at 0, 1, 2, 3, 5, 9, 15, 30 and 60 min after acidification. During the reaction pH of the reaction mixture was hold up by titration with solution with pH 11–12 of complex agent’s salt with needed concentration or NH₄OH.

RESULTS AND DISCUSSION

Two model reactions of hydrolysis of Phac were

Fig. 1. Reaction of hydrolysis of Phac group in dipeptide ester Boc-Lys(Phac)-Val-OMe with PGA.

Fig. 2. Reaction of hydrolysis of Phac group in tripeptide Boc-Lys(Phac)-Lys(Phac)-Lys(Phac)-OH with PGA.

D. L. Danalev and L. K. Yotova: Synthesis of model peptide substrates

It is well known that pH optimum of PGA according to its natural substrate benzylpenicillin sodium or potassium salt is 7.8–8.0 [7–9]. In our investigation the used substrates are two peptides which are not specific substrates for PGA, thus the reaction of hydrolysis was monitored at pH range 6 ÷ 9. Series of reactions were carried out with 0.4 mM substrate at different pH (Fig. 3).

Maximum degree of substrate conversion 75.5% (pH 9), which is closed to the degree of conversion at pH 7.8 (74.5%), was obtained for 3 and 10 min, respectively. At pH 6.5 and 7, the degree of hydro-lysis was 62.5 and 37.5%, respectively. By that reason, our next investigations on the reactions were carried out at the optimum value of pH 9 and at 37°C. Kinetic studies of the Phac group’s hydrolysis were done with different enzyme concentrations in the synthetic substrate Boc-Lys(Phac)-Lys(Phac)-Lys(Phac)-OH. The results are shown in Fig. 4. Maximum degree of substrate conversion 73.75% was obtained in the presence of 0.04 ml PGA for 8 min, which is closed to that obtained with 0.4 ml of PGA for 15 min (72.5%).

The obtained curves in the Figs. 3 and 4 clearly show that the reaction of Phac group hydrolysis is reversible. In order to attempt to shift the equi-librium of the reaction to the direction of full hydro-lysis of the protecting group in the model peptide substrates and to make this reaction non-reversible, the same reaction was carried out in the presence of metal ions, which are good complexing agents. Their addition to the system might lead to the formation of complex with released ε-amino function of Lys residues (Fig. 5). The obtained complex might precipitate the forming product and to shift the reaction to the right direction. The possibility of similar complex formation was dis-cussed previously by Schröder and Lubke [10].

Fig. 3. Kinetics of hydrolysis of 0.4 mM synthetic substrate with 0.4 ml PGA at different pH.

Fig. 4. Kinetics of hydrolysis of 5.0 mM synthetic substrate with different concentrations of PGA.

D. L. Danalev and L. K. Yotova: Synthesis of model peptide substrates

The kinetic results depending on the Ni(NO₃)₂ concentration, as a complex agent, are shown in Fig. 6. The figure clearly shows that the reaction equilibrium of hydrolysis of Phac group with stepwise addition of 10% Ni(NO₃)₂ was shifted to the direction of full hydrolysis of the protecting group for 9 min. In this case the substrate was 40 mM and the final concentration of Ni(NO₃)₂ – 1 M.

Fig. 5. Structure of the complex of ε-amino function of Lys residues with different metals.

Fig. 6. Kinetics of hydrolysis of different amounts of synthetic substrate Boc-Lys(Phac)-Lys(Phac)- Lys(Phac)-OH with 0.002 ml PGA at pH = 9.0 and 37°C by stepwise addition of Ni(NO₃)₂ at different concentrations.

Fig. 7. Kinetics of hydrolysis of 0.4 mM synthetic substrate Boc-Lys(Phac)-Val-OH with 0.4 ml PGA in the reaction mixture at pH = 9.0 and 37°C by partial addition of different complex agents’ salts.