High-temperature Solid-state Catalytic Isotope Exchange Research Articles

Solid-State Catalytic Hydrogen/Deuterium Exchange in Mexidol

The reaction between spillover hydrogen (SH) and mexidol upon high-temperature solid state catalytic isotope exchange (HSCIE) is studied experimentally and via DFT B3LYP and MP2 calculations. The distribution of isotopes and the activation energy of the reaction are determined via NMR spectro-scopy and mass spectrometry for samples of the deuterium-labeled mexidol obtained via HSCIE at 50–120°C. The DFT calculated activation energy of hydrogen transfer on the charged alumina cluster HH+(‒Al–O–)3(OH)6 is found to agree with the experimental data. A mechanism is proposed for HSCIE between the adsorbed compound and charged Bronsted acid sites formed on the surface of the support upon exposure to hydrogen spillover.

Pharmacokinetics of HLDF-6-AA Peptide in the Organism of Experimental Animals

Pharmacokinetics of the promising antitumor peptide HLDF-6-AA (Ac-ThrGlyGluAsnHisArg-NH2) was studied using its uniformly tritiated derivative. Experiments were performed on male Wistar rats, Balb/c mice and Chinchilla rabbits. The tritium labeled peptide [3H]HLDF-6-AA with the molar radioactivity of 50 Ci/mmol was obtained by the reaction of high-temperature solid-state catalytic isotope exchange (HSCIE). Under intravenous bolus administration of HLDF-6-AA peptide to rats and rabbits, the characteristics of its pharmacokinetic profile in the blood were obtained and the values of the main pharmacokinetic parameters of HLDF-6-AA peptide, and its active metabolite HisArg-NH2 were calculated. It was shown that parameters of the retention time in the body and the rate of elimination for peptides HLDF-6-AA and HisArg-NH2 in rats and rabbits are close and in rats they are about 7 and 21 min, respectively. It was shown that repeated administration of the drug does not lead to a change in its regular cumulation and does not cause a change in its pharmacokinetics compared with a single administration. The linearity of the dependence of pharmacokinetic parameters on the amount of administered peptide in the range of 2–22 mg/kg was proved in experiments in rats. As a result of the study of the distribution of HLDF-6-AA peptide and its metabolite HisArg-NH2 between the blood and peripheral tissues of mice, it was shown that the maximum concentration of HLDF-6-AA peptide is observed in the kidney tissues and a somewhat smaller concentration in the omentum. It was found that 15–20 min after intraperitoneal administration of HLDF-6-AA to mice, the concentration of HisArg-NH2 peptide begins to exceed the concentration of HLDF-6-AA peptide, which is caused by its greater resistance to proteolytic hydrolysis. The highest concentration of HisArg-NH2 peptide is observed in the kidney and liver tissues.

Proteolytic Hydrolysis of the Antitumor Peptide HLDF-6-AA in Blood Plasma

HLDF-6 hexapeptide, which corresponds to the 41TGENHR46 fragment of human leukemia differentiation factor (HLDF), shows a wide range of neuroprotective, normalizing, anxiolytic, nootropic and antitumor activities. A promising drug with antitumor activity is being developed based on the N-acetyl, C‑amide form of HLDF-6 peptide, obtained by the solid phase method. Proteolytic hydrolysis of HLDF-6-AA peptide in blood plasma was studied using its derivatives labeled with hydrogen isotopes. The HLDF-6-AA peptide samples uniformly labeled with tritium and deuterium were obtained by the reaction of high-temperature solid-state catalytic isotope exchange at 170°С. [3H] HLDF-6-AA peptide was obtained with the molar radioactivity of 50 Ci/mmol and the average deuterium incorporation for [2H] HLDF-6-AA peptide was 2.90 atoms per peptide molecule. Proteolytic hydrolysis of HLDF-6-AA peptide in rat blood plasma was studied using radiochromatography. It was established that the main pathway of its proteolytic hydrolysis in rat blood plasma consists in cleavage of His-Arg-NH2 (HR-NH2) dipeptide from the C-end of the amide with the half-degradation period of 35 min.

The Qualitative Analysis of the Amide Derivative of HLDF-6 Peptide and Its Metabolites with the Use of Tritium- and Deuterium-Labeled Derivatives

The goal of the study was to elaborate the pharmacokinetics methods of the amide derivative of peptide HLDF-6 (TGENHR-NH2) and its range of nootropic and neuroprotective activity is wide. The hexapeptide 41TGENHR46 is a fragment of the HDLF differentiation factor. It forms the basis for the development of preventive and therapeutic preparations for treating cerebrovascular and neurodegenerative conditions. Pharmacokinetic and molecular mechanisms of the action of the HLDF-6 peptide were studied using tritium- and deuterium-labeled derivatives of this peptide, produced with the use of the high-temperature solid-state catalytic isotope exchange reaction (HSCIE). This reaction was employed to produce the tritium-labeled peptide [3H]TGENHR-NH2 with a molar radioactivity of 230 Ci/mmol and the deuterium-labeled peptide [2H]TGENHR-NH2 with an average deuterium incorporation equal to 10.5 atoms. It was shown by the NMR spectroscopy that the isotope label distribution over the labeled peptide's molecule was uniform, which allowed qualitative analysis ofboth the peptide itself and its fragments in the organism's tissues to be conducted. The newly developed pharmacokinetics method makes it possible to avoid almost completely losses of the peptides under study due to biodegradation during the analysis of tissues. These labeled peptides were used in mice, rats and rabbits to study the pharmacokinetics of the peptide and to calculate the values of its principal pharmacokinetic parameters. Characteristics of its pharmacokinetic profile in the blood were obtained, the hypothesis of pharmacokinetics linearity tested, its metabolism analyzed and its bioavailability value, 34%, calculated. It has been shown that the studied TGENHR-NH2 peptide shows high resistance to hydrolysis in the blood plasma, with dipeptidyl aminopeptidases making the largest contribution to its hydrolysis.

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 alpha-subunit was carried out for the HSCIE reaction with the hemoglobin complex and with the free alpha-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 alpha-subunits MFLSFPTTK (A(32-40)) and VDPVNFK (A(93-99)) for hydrogen replacement by tritium by almost an order of magnitude; hence, their availability to water (1.4 A) 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.

Interaction of the synthetic immunomodulatory dipeptide bestim with murine macrophages and thymocytes

The tritium-labeled dipeptide bestim (gamma-D-Glu-L-Trp) with a specific activity of 45 Ci/mmol was obtained by high-temperature solid-state catalytic isotope exchange. It was found that [3H]bestim binds with a high affinity to murine peritoneal macrophages (Kd 2.1 +/- 0.1 nM) and thymocytes (Kd 3.1 +/- 0.2 nM), as well as with plasma membranes isolated from these cells (Kd 18.6 +/- 0.2 and 16.7 +/- 0.3 nM, respectively). The specific binding of [3H]bestim to macrophages and thymocytes was inhibited by the unlabeled dipeptide thymogen (L-Glu-L-Trp) (Ki 0.9 +/- 0.1 and 1.1 +/- 0.1 nM, respectively). After treatment with trypsin, macrophages and thymocytes lost the ability to bind [3H]bestim. Bestim in the concentration range of 10(-10) to 10(-6) M reduced the adenylate cyclase activity in the membranes of murine macrophages and thymocytes.

Interaction of Synthetic Dipeptide Bestim with Mouse Thymocytes and Macrophages

Tritium-labeled dipeptide bestim (γ-D-Glu-L-Trp) with a specific activity of 45 Ci/mmol was obtained by the high-temperature solid-state catalytic isotope exchange (HSCIE) reaction. [3H]bestim was found to bind with high affinity to mouse peritoneal macrophages (Kd 2.1 ± 0.1 nM) and thymocytes (Kd 3.1 ± 0.2 nM) and also plasma membranes isolated from these cells (Kd 18.6 ± 0.2 and 16.7 ± 0.3 nM respectively). The specific bonding of [3H]bestim with macrophages and thymocytes was inhibited by unlabeled dipeptide thymogen (L-Glu-L-Trp) (Ki 0.9 ± 0.1 and 1.1 ± 0.1 nM respectively). Treatment of the macrophages and thymocytes with trypsin led to their loss of capacity to bind [3H]bestim. Bestim at concentrations range of 0.1–1000 nМ reduced the adenylate cyclase activity in macrophage and thymocyte membranes.

Isotopic effect of electron excitation in l-[ 3H]tryptophan

Isotopic effect of electron excitation in organic compounds was for the first time experimentally and theoretically analyzed. The nature of this phenomenon is due to zero-point energies (ZPE) for the ground and the excited states of the isotope substituted tryptophan. l-[ 3H]tryptophan ( l-[ 3H]Trp) with a tritium–hydrogen substitution ratio of 0.2–0.45 has been produced by high-temperature solid-state catalytic isotope exchange and was used in an experimental study. A comparison of the UV spectra of l-[ 3H]Trp and l-[ 1H]Trp showed a short-wavelength shift of the absorption maximum in the tritium-substituted compound that corresponds to an energy of 130 cal/mol for the electronic transition. To account for this effect, quantum chemical calculations of the geometric and electronic structures, frequencies of normal vibrations, and transition energies have been performed using of the restricted Hartree–Fock method (RHF), density-functional theory (RB3LYP), and configuration interaction (RCIS). The shifts of all allowed electronic transitions have been shown to shift towards the short-wavelength region and to be equal to 152 cal/mol for the first singlet–singlet transition (within the 280 nm region), to 242 cal/mol for the third transition (within the 220 nm region), and to 557 cal/mol for the singlet–triplet transition. All the shifts obtained by calculations are due to zero-point energies for the ground and the excited states of [ 3H]Trp and [ 1H]Trp.

New development in the tritium labelling of peptides and proteins using solid catalytic isotopic exchange with spillover-tritium.

The mechanism of the reaction of high temperature solid state catalytic isotope exchange (HSCIE) of hydrogen in peptides with spillover-tritium at 140-180 degrees C was analyzed. This reaction was used for preparing [(3)H]enkephalins such as [(3)H]DALG with specific activity of 138 Ci/mmol and [(3)H]LENK with specific activity of 120 Ci/mmol at 180 degrees C. The analogues of [(3)H]ACTG(4-10) with specific activity of 80 Ci/mmol, [(3)H]zervamicin IIB with specific activity of 70 Ci/mmol and [(3)H]conotoxin G1 with specific activity 35 Ci/mmol were produced. The obtained preparations completely retained their biological activity. [(3)H]Peptide analysis using (3)H NMR spectroscopy on a Varian UNITY-600 spectrometer at 640 MHz was carried out. The reaction ability of amino fragments in HSCIE was shown to depend both of their structures and on the availability and the mobility of the peptide chain. The reaction of HSCIE with the beta-galactosidase from Termoanaerobacter ethanolicus was studied. The selected HSCIE conditions allow to prepare [(3)H] beta-galactosidase with specific activity of 1440 Ci/mmol and completely retained its the enzymatic activity.

The solid-state catalytic isotope exchange of hydrogen in α-conotoxin G1 by the tritium spillover

Tritium-labeled α-conotoxin G1 with a molar radioactivity of 35 Ci/mmol and full biological activity (according to the binding to nicotinic acetylcholine receptor) was obtained by the high-temperature solid-state catalytic isotope exchange (HSCIE). The tritium distribution in the molecule of α-conotoxin G1 was revealed by3H NMR spectroscopy. Tritium was found in all amino acid residues except for the Asn4-Pro5-Ala6 fragment. The data on the comparative reactivity of C-H bonds, theab initio quantum-chemical calculation of the hydrogen exchange reaction, and the information on the spatial structures of α-conotoxin G1 in solution and in crystal state allowed us to establish that the reactivity of H atoms may be increased by their interaction with the electron donor O and N atoms at the transition state of the HSCIE reaction. A decrease in the rate of the HSCIE reaction could be caused by both a poor spatial accessibility of C-H bonds and a limited mobility of the peptide fragment containing these bonds.

The solid-state catalytic isotope exchange of hydrogen in dalargin

A [3H]Dalargin preparation with a molar radioactivity of 52 Ci/mmol was obtained by the high temperature solid-state catalytic isotope exchange (HSCIE) of tritium for hydrogen at 150°C. This tritium-labeled peptide was shown to completely retain its biological activity in the test of binding to opioid receptors from rat brain. The dissociation constant of the Dalargin-opioid receptor complex was found to be 4.3 nM. The dependences of the chemical yield and the molar radioactivity on the reaction time and temperature of HSCIE were determined. The activation energy of the HSCIE reaction for the peptide was calculated to be 32 kcal/mol. The amino acid analysis showed that tritium is distributed between all the amino acid residues of [3H]Dalargin at the HSCIE reaction, with the temperature growth significantly increasing the total tritium incorporation and, especially, enhancing the radioactivity incorporation into aromatic residues.

Glycolide deuteriation by hydrogen isotope exchange using the HSCIE method

Although poly(α-hydroxy acids)s derived from lactic and glycolic acids (PLAGA) are currently used for many surgical and pharmacological applications, their behaviour and their fate in vivo are still a source of concern according to some authors. As part of our attempts to label these polymers and monitor their fate by using radioactivity, investigation was made of the possibility to exchange the protons present in glycolide molecules by hydrogen isotopes using the High-temperature Solid-state Catalytic Isotope Exchange (HSCIE) method which was previously successful in the case of lactide. Accordingly H→H exchanges were carried out on glycolide in the presence of Pd/CaCO3 at various temperatures and 550 mbar pressure of deuterium. Substitution yields as high as 24% were obtained, depending primarily on temperature. PLAGA polymers with lactyl and glycolyl repeating units labeled with deuterium were synthesized and characterized. Copyright © 1999 John Wiley & Sons, Ltd.

Ab initio calculations of activation energy of the reaction of hydrogen exchange on strongly acidic centers

Ab initio calculations of fragments of the potential energy surfaces of hydrogen exchange reactions between H2, CH4, and alanine molecules and the H3O+ ion were performed by the restricted Hartree-Fock method, at the second-order Moller-Plesset level of perturbation theory, and by the method of coupled clusters using the 6–31G* and aug-cc-pVDZ basis sets. The one-center synchronous mechanism of hydrogen exchange reaction was studied and the activation energies and structures of transition states were determined. It was found that the geometric parameters of the H2 and CH4 molecules in the transition states are close to those of the H3 + and CH5 + ions. The higher the proton affinity of the reacting molecule in the reaction studied the lower the activiation energy of hydrogen exchange. The one-center mechanism studied can be used to describe the high-temperature solid-state catalytic isotope exchange (HSCIE) reaction. The results ofab initio calculations of synchronous hydrogen exchange between the H3O+ ion and hydrogen atoms in different positions of the alanine molecule are in good agreement with experimental data on the regioselectivity and stereoselectivity of the HSCIE reaction with spillover-tritium.

Experimental and ab Initio Studies on Solid-State Hydrogen Isotope Exchange with Spillover Tritium

This study utilizes ab initio calculations to investigate the reaction of high-temperature solid-state catalytic isotope exchange (HSCIE) between amino acids and spillover tritium. The Hartree−Fock approximation and second-order Moller−Plesset perturbation theory in conjunction with 6-31G* and aug-cc-pVDZ basis sets were used to calculate potential energy surfaces for the interactions between CH4, alanine, hydroxyproline, and the H3O+ ion. Ab initio calculations were used to estimate the activation energies and structures of the transition states of these reactions. The hydrogen exchange reaction occurs by a synchronous mechanism, with a transition state that is characterized by pentacoordinated carbon. The proposed one-center mechanism is in good agreement with observed retention of configuration of the asymmetric carbon atoms in the HSCIE reaction with spillover tritium in experiments. The regioselectivity and stereoselectivity of hydrogen isotope exchange in amino acids with spillover tritium can be pr...

Study of solid-state catalytic isotope exchange of hydrogen inl-hydroxyproline under the action of spillover tritium

Reaction of high-temperature solid-state catalytic isotope exchange (HSCIE) of hydrogen in L-hydroxyproline was studied byab initio quantum-chemical calculations. A one-center synchronous mechanism of isotope exchange between the amino acid and the H3O+ model acidic center was considered. The structures of transition states of the reaction and the activation energies were determined. Relative reactivity of the C−H bonds in the hydroxyproline molecule under conditions of HSCIE was studied. The results obtained are in agreement with experimental data on the stereoselectivity and regioselectivity of the HSCIE reaction,viz., the lower the calculated activation energy of isotope exchange, the larger the portion of hydrogen substituted by tritium in a given position of the amino acid molecule. The enhancement of the reactivity under conditions of solid-state isotope exchange can be associated with additional interaction between the exchanging H atoms and the electron-donor O and N atoms of the amino acid molecule in transition state.

Correlations between the quantum-chemical parameters of amino acids and regioselectivity of isotope exchange with the spillover hydrogen

The effective charges and the proton affinity of carbon atoms of α-amino acids were calculated by quantum-chemical methods. The relative reactivity of the C−H bonds of amino acids under conditions of high-temperature solid-state catalytic isotope exchange (HSCIE) was studied. Correlations between the electron structure of amino acids and the regioselectivity of the solid-state isotope exchange were established. The reactivity of the carbon atoms with high proton affinity increases under HSCIE conditions. An assumption was made that the interaction of a solid organic compound with the spillover hydrogen can be described as the electrophilic substitution at the saturated and aromatic carbon atoms.

The Preparation of Soft-Tritium-Labeled Proteins and Their Application for the Collagenolytic Activity Investigations

Tritium-labelled Type I collagen from rat skin with specific activity of 8 mCi/mg and mixture of the crab hepatopancrease collagenolytic enzymes with specific activity of 25 mCi/mg have been obtained by the method of high-temperature solid-state catalytic isotope exchange (HSCIE) without loss of native features. Tritium-labelled Type I collagen kept its ability to form native fibrills being resistant to trypsin hydrolysis. Tritium-labelled collagenase keeps about 70% of its proteolytic activity.

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.

The synthesis of tritium-labelled alanine by solid-state catalytic reactions

The solid-state catalytic hydrogenation (SCH) of unsaturated precursor compound and the high-temperature solid-state catalytic isotope exchange (HSCIE) showed that the total incorporation of isotope and its distribution between the α-and β-positions of the alanine molecule can be modified by changing the temperature of solid state reactions. Tritium incorporation at high-temperatures proceeds in a manner which retains the configuration of the asymmetric α-carbone atom. 3H NMR analysis of tritiated isotopomers was used for the investigation of solid-state reactions. The HSCIE reaction proceeds selectively at 140 °C and only part of the molecules is in the the tritium spillover reaction zone. At 220°C HSCIE proceeds evenly along the whole mass to give uniformly labelled alanine.

Tritium incorporation in α‐amino acids by isotope exchange using high‐temperature solid‐state catalysis

AbstractSolid‐state catalytic reactions provide a new effective method for the synthesis of tritium‐labelled biologically active compounds. We present the synthesis of tritium‐labelled amino acids through high‐temperature solid‐state catalytic isotope exchange (HSCIE). Under HSCIE, isotope exchange with gaseous tritium was shown to proceed at all hydrogen atoms in the molecules of solid organic compounds, which opens the possibility of producing biologically active compounds uniformly labelled with tritium at high molar activity. The configuration is retained upon the hydrogen atom substitution at asymmetrical carbon atoms under BSCIE conditions, allowing the synthesis of uniformly‐labelled amino acids in the optically active form.