V. S. Kozik

HIGH TEMPERATURE SOLID STATE CATALYTIC ISOTOPE EXCHANGE WITH DEUTERIUM AND TRITIUM

A method for synthesizing tritium- or deuterium-labeled amino acids, peptides and biogenic amines through high temperature solid state catalytic isotope exchange (HSCIE) is proposed. The dependence of the degree of isotope exchange in HSCIE on the structure of the compound, the reactivity of hydrogen at different carbon atoms and the conditions of the process has been examined. If HSCIE is performed in the temperature range of 373 to 413K, the selectivity of isotopic label incorporation comes to 70% or higher. When the tritium label is introduced into peptides, they retain the configuration of asymmetric atoms, even upon the substitution of tritium for hydrogen at the α-carbon atoms of the amino acid residues. HSCIE at 453–513K leads to an even distribution of the isotopic label over the organic compound molecule. The results of3H NMR spectroscopy highlighting the distribution of the tritium label in the organic compound molecules are presented. The configuration of asymmetric atoms in amino acids is preserved to a high extent upon 80–90% substitution of isotopes for hydrogen atoms.

Solid State Isotope Exchange with Spillover Hydrogen in Organic Compounds

The term spillover, as used in heterogeneous catalysis, refers to the transport of active particles which are either absorbed or formed in one phase and transported to another phase where, under these reaction conditions, such particles are not absorbed or formed. An example is that of hydrogen, which after dissociative adsorption on platinum particles can migrate onto a nonorganic support such as aluminum oxide, barium sulfate, or others. Such active hydrogen atoms were given the name spillover hydrogen (SH).1 The first direct evidence of spillover was obtained during the reduction of wolfram trioxide to tungsten bronze at room temperature:2 the reaction proceeds in a mechanical mixture of 0.5% Pt/ Al2O3 + WO3. It was assumed that the hydrogen dissociated on the platinum and migrated through the alumina onto WO3 in the form of atoms or H+ ions. The solid state hydrogenation of asymmetric crystals of 2-isopropyl-5-methylphenol (also known as thymol) occurs with the participation of SH and leads to the formation of a series of asymmetric menthols and menthones.3 Despite the fact that processes involving SH have been known for over 40 years, the nature of this phenomenon has not been fully investigated or clarified. According to various hypotheses, hydrogen can migrate in the form of a solvated proton,4 as a proton-electron pair,5 or as atomic hydrogen.6 The main difficulty in explaining the SH phenomenon is that the SH concentration is too small to detect using direct instrumental analysis such as modern spectroscopic methods. Because the importance of SH in heterogeneous catalysis cannot be underestimated, the debate regarding the nature of the activated hydrogen particles and their diffusion is still ongoing.1

Evenly tritium labeled peptides in study of peptide in vivo and in vitro biodegradation

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50–150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a μg scale of biological samples both in vitro and in vivo.

Solid phase isotope exchange of deuterium and tritium for hydrogen in human recombinant insulin

Reaction of a high-temperature solid-phase catalytic isotope exchange in peptides and proteins under the action of the catalytically activated spillover hydrogen was studied. The reaction of human recombinant insulin with deuterium and tritium at 120–140°C resulted in an incorporation of 2–6 isotope hydrogen atoms per one insulin molecule. The distribution of the isotopic label by amino acid residues of the tritium-labeled insulin was determined by the oxidation of the protein S-S-bonds by performic acid, separation of polypeptide chains, their subsequent acidic hydrolysis, amino acid analysis, and liquid scintillation counts of tritium in the amino acids. The isotopic label was shown to be incorporated in all the amino acid residues of the protein, but the higher inclusion was observed for the FVNQHLCGSHLVE peptide fragment (B1–13) of the insulin B-chain, and the His5 and His10 residues of this fragment contained approximately 45% of the whole isotopic label of the protein. Reduction of the S-S-bonds by 2-mercaptoethanol, enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius, and HPLC fractionation of the obtained peptides were also used for the analysis of the distribution of the isotopic label in the peptide fragments of the labeled insulin. Peptide fragments which were formed after the hydrolysis of the Glu-Xaa bond of the B-chain were identified by mass spectrometry. The mass spectrometric analysis of the isotopomeric composition of the deuterium-labeled insulin demonstrated that all the protein molecules participated equally in the reaction of the solid-phase hydrogen isotope exchange. The tritium-labeled insulin preserved the complete physiological activity.

Study of the solid-state hydrogen isotope exchange ofl-alanine

The solid-state reaction of isotope exchange ofl-alanine (l-Ala) with spillover-hydrogen activated on a Rh(Pd)-supported catalyst was studied. The reactivity of the carbon atoms and the activation energies of isotope exchange of the hydrogen at the C(2) and C(3) atoms of thel-Ala molecule were determined using tritium NMR. Theab initio calculations of the activation energy of a model reaction between the alanine molecule and a hydroxonium cation were carried out. The mechanism and plausible structures of the transition states of this reaction were proposed.

A comparative analysis of the distribution of glyprolines after their administration by different ways

The distribution of the glyprolines, Pro-Gly-Pro and Thr-Lys-Pro-Arg-Pro-Gly-Pro (Selanc), was analyzed and compared in tissues of rat organs after different ways of their administration using the peptides uniformly labeled with tritium. Comparative data on changes of concentrations of the peptides in the rat organs after their intraperitoneal, intranasal, intragastric, and intravenous administration are given. The intranasal administration of both peptides was shown to be optimal for delivery of glyprolines molecules in the CNS. A high affinity of the studied glyprolines for gastric tissues was found for all the ways of their administration. We suggest that high efficacy of action of glyprolines on homeostasis of the gastric mucosa was partially provided by accumulation of these peptides (to high concentrations) in gastric tissues.

Solid phase reaction of hemoglobin with spillover hydrogen

The reaction of high-temperature solid-state catalytic isotope exchange (HSCIE) between bovine hemoglobin and spillover hydrogen (SH) was studied. It was shown that, in the field of subunit contact, there is a significant decrease in ability for hydrogen exchange by SH. A comparison of the distribution of the isotope label in the hemoglobin α-subunit was carried out for the HSCIE reaction with the hemoglobin complex and with the free α-subunit. To this end, enzymatic hydrolysis of protein under the action of trypsin was carried out. The separation of tritium-labeled tryptic peptides was achieved by HPLC. Changes in availability of polypeptide chain fragments caused by complex formation were calculated using a molecular model. The formation of the protein complex was shown to lead to a decrease in the ability of fragments of α-subunits MFLSFPTTK (A32−40) and VDPVNFK (A93−99) for hydrogen replacement by tritium by almost an order of magnitude; hence, their availability to water (1.4 Å) twice decreased on the average. The decrease in ability to an exchange of hydrogen by spillover tritium on the formation of hemoglobin complex was shown to be connected with a reduction in availability of polypeptide chain fragments participating in spatial interactions of subunits with each other. Thus, the HSCIE reaction can be used not only for the preparative obtaining of tritium-labeled compounds, but also for determining the contact area in the formation of protein complexes.

Stereoselective effects in the solid-phase hydrogenation of unsaturatedl-hydroxyproline derivatives

The solid-phase catalytic hydrogenation of (R-4tert-butoxy-Δ1-pyrroline-2-carboxylic) acid under the action of hydrogen spillover was studied. The reaction proceeds stereoselectively with the predominant formation of thel-amino acid. The configuration of the asymmetric center formed is determined by that of the asymmetric C(4) atom. The major portion of the isotope label is incorporated into the allylic C(3) and C(5) positions, and the β-H atoms are more mobile. Using quantum-chemical calculations, the geometric structure of thel-hydroxyproline molecule was calculated, and the spin-spin coupling constants for this tritium-labeled amino acid were determined.