Irrr. J . Peprid< Profebi Res. 39, 1992, 207-710
Effect of electrophilic and nucleophilic substituents on the protonation microequilibria of tyrosine derivatives PAL SIPOS*. GABOR PEINTLER** and GEZA TOTH***
* Departriierit of Inorganic and Analytical Chemistry, and * * General and Physical Chemistry, A . Jbzsef Univer.ritj,. * * * Isotope Laboratory, Biological Research Center, Hungarian Acadeny qf Sciences, Szeged, Hungai?. Received 27 August, accepted for publication 12 October 1991 The microscopic protonation constants of 10 tyrosine-like, unusual amino acids used in the syntheses of opioid peptides have been determined by using a combined pH-metric-spectrophotometric method, at 0.10 niol dni (NaC1) ionic strength and 25.0". The role of the different electrophilic and nucleophilic substituents on the individual basicity of the aliphatic aniine and phenolic hydroxylate basic centers is discussed in detail. The interactivity parameters between these two groups correlate fairly well with the structure of the skeleton and the distance between the two basic centers. but they were found to be substituent-independent. This finding made it possible to extend the calculations to compounds having non-overlapping protonation equilibria. ~
Key words; microequilibria; protonation; tyrosine derivates
The aminoterminal residue of opioid peptides is tyrosine. Any modification of Tyr decreases the biological activity of these peptides (1). Recently some new tyrosine analogues were synthesized (2, 3) and used for synthesis of new opioid peptide analogues (4). In our present study we investigated the effect of substituents on the tyrosine skeleton on the basicity of the constituent basic centers. Such studies have been carried out by combining the usual pH-metric and group specific methods, like UV-vis spectrophotometry (5-8), optical rotary dispersion (9), and N M R (10) techniques for several tyrosine-like amino-acids, making it possible to reveal their protonation-depronation microequilibria. The data prescribed in this publication could be used to determine correlations between the protonation microconstants of the tyrosine-like compounds and the biological activities of these opioid peptide analogues. These correlations will be published elsewhere (4). EXPERIMENTAL PROCEDURES Muterids The compounds presented in Table 1 were prepared in our laboratory [ 111, IV, ( 2 ) ; V (1 1); VI (3); VII (12)] or were Aldrich products (I, 11, VIII, IX). The purity of the sample was checked by TLC, N M R , and standard mi-
croanalytical methods. The exact concentrations of their solutions were measured and calculated by the Gran method (13). All other chemicals were of analytical grade. Methods The macroscopic proton dissociation constants were determined by pH-metric titration of samples (10 cm3) in the p H range 3-1 1. The ligand concentration was 2.5 x mol d m - 3 and the ionic strength was adjusted to 0.10 mol d m - with NaCl. These measurements were made on a home-made, computer-operated automatic device (14) with Radiometer G202B glass electrode and Radelkis OP-082OP double junction Ag/AgCl reference electrode (separating solution: 0.10 mol d m NaCI). Since the ligands tend to undergo oxidation, all measurements were performed in nitrogen atmosphere. The electrode system was calibrated by using the modified Nernst equation, as previously described (1 5 ) . In all cases the temperature \\ as 25.0 5 0.1 O . Absorption spectra in the UV region were recorded on a Varian 634 spectrophotometer. For the pH-spectrophotometric titration 2-5 x 10- .Iin01 dm ligand concentration and 1 cm pathlength were used. The concentration protonation constants b = [ H,A]/ [H]'[A] were calculated with the aid of the PSEQUAD computer program (16). ~
207
P. Sipos eta1 T.\BLF 1 The it11 esrrpred r:iiiipoiiiid
Effect of electrophilic and nucleophilic substituents on the protonation microequilibria of tyrosine derivatives PAL SIPOS*. GABOR PEINTLER** and GEZA TOTH***
* Departriierit of Inorganic and Analytical Chemistry, and * * General and Physical Chemistry, A . Jbzsef Univer.ritj,. * * * Isotope Laboratory, Biological Research Center, Hungarian Acadeny qf Sciences, Szeged, Hungai?. Received 27 August, accepted for publication 12 October 1991 The microscopic protonation constants of 10 tyrosine-like, unusual amino acids used in the syntheses of opioid peptides have been determined by using a combined pH-metric-spectrophotometric method, at 0.10 niol dni (NaC1) ionic strength and 25.0". The role of the different electrophilic and nucleophilic substituents on the individual basicity of the aliphatic aniine and phenolic hydroxylate basic centers is discussed in detail. The interactivity parameters between these two groups correlate fairly well with the structure of the skeleton and the distance between the two basic centers. but they were found to be substituent-independent. This finding made it possible to extend the calculations to compounds having non-overlapping protonation equilibria. ~
Key words; microequilibria; protonation; tyrosine derivates
The aminoterminal residue of opioid peptides is tyrosine. Any modification of Tyr decreases the biological activity of these peptides (1). Recently some new tyrosine analogues were synthesized (2, 3) and used for synthesis of new opioid peptide analogues (4). In our present study we investigated the effect of substituents on the tyrosine skeleton on the basicity of the constituent basic centers. Such studies have been carried out by combining the usual pH-metric and group specific methods, like UV-vis spectrophotometry (5-8), optical rotary dispersion (9), and N M R (10) techniques for several tyrosine-like amino-acids, making it possible to reveal their protonation-depronation microequilibria. The data prescribed in this publication could be used to determine correlations between the protonation microconstants of the tyrosine-like compounds and the biological activities of these opioid peptide analogues. These correlations will be published elsewhere (4). EXPERIMENTAL PROCEDURES Muterids The compounds presented in Table 1 were prepared in our laboratory [ 111, IV, ( 2 ) ; V (1 1); VI (3); VII (12)] or were Aldrich products (I, 11, VIII, IX). The purity of the sample was checked by TLC, N M R , and standard mi-
croanalytical methods. The exact concentrations of their solutions were measured and calculated by the Gran method (13). All other chemicals were of analytical grade. Methods The macroscopic proton dissociation constants were determined by pH-metric titration of samples (10 cm3) in the p H range 3-1 1. The ligand concentration was 2.5 x mol d m - 3 and the ionic strength was adjusted to 0.10 mol d m - with NaCl. These measurements were made on a home-made, computer-operated automatic device (14) with Radiometer G202B glass electrode and Radelkis OP-082OP double junction Ag/AgCl reference electrode (separating solution: 0.10 mol d m NaCI). Since the ligands tend to undergo oxidation, all measurements were performed in nitrogen atmosphere. The electrode system was calibrated by using the modified Nernst equation, as previously described (1 5 ) . In all cases the temperature \\ as 25.0 5 0.1 O . Absorption spectra in the UV region were recorded on a Varian 634 spectrophotometer. For the pH-spectrophotometric titration 2-5 x 10- .Iin01 dm ligand concentration and 1 cm pathlength were used. The concentration protonation constants b = [ H,A]/ [H]'[A] were calculated with the aid of the PSEQUAD computer program (16). ~
207
P. Sipos eta1 T.\BLF 1 The it11 esrrpred r:iiiipoiiiid
