Study of solid-phase catalytic isotopic exchange of hydrogen in alpha-conotoxin G1 under the effect of spillover-tritium

Tritium-labeled alpha-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 alpha-conotoxin G1 was revealed by 3H 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, the ab initio quantum-chemical calculation of the hydrogen exchange reaction, and the information on the spatial structures of alpha-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.     

Solid phase isotope exchange with spillover hydrogen in amino acids,peptides, and proteins

We summarize here information on the theoretical and experimental study of high-temperature (150–200°C) solid phase catalytic isotope exchange (HSCIE) carried out with amino acids, peptides, and proteins under the action of spillover hydrogen. Main specific features of the HSCIE reaction, its mechanism, and its use for studying spatial interactions in polypeptides are discussed. A virtually complete absence of racemization makes this reaction a valuable preparative method. The main regularities of the HSCIE reaction with the participation of spillover tritium have been revealed in the case of peptides and proteins, and the dependence of reactivity of peptide fragments on the spatial organization of their molecules has been studied. An important peculiarity of this reaction is that HSCIE proceeds at 150–200°C with a high degree of chirality retention in amino acids and peptides. This is provided by its reaction mechanism, which consists in a synchronous one-center substitution at the saturated carbon atom characterized by the formation of pentacoordinated carbon and a three-center bond between the carbon and the incoming and outgoing hydrogen atoms.Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 3–21.Original Russian Text Copyright © 2005 by Zolotarev, Dadayan, Borisov.     

The solid-state catalytic synthesis of tritium labeled amino acids,peptides and proteins

Summary New catalytic reaction between a solid bioorganic compound and activated spillover tritium (ST), based on High-temperature Solid-state Catalytic Isotopic Exchange (HSCIE) was examined. The HSCIE mechanism and determination of the reactivity of hydrogen atoms in amino acids, peptides and proteins was investigated. Quantum mechanical calculations of the reactivity of hydrogen atoms in amino acids in the HSCIE reaction were done. The carbon atom with a greater proton affinity undergoes a greater exchange of hydrogen for tritium in HSCIE. The electrofilic nature of spillover hydrogen in the reaction of HSCIE was revealed. The isotope exchange between ST and the hydrogen of the solid organic compound proceeds with a high degree of configuration retention at the carbon atoms. The HSCIE reaction enables to synthesize tritium labeled proteins with a specific activity of 20–30 mCi/mg and kept biological activity.Presented at the 3rd International Congress on Amino Acids, Peptides and Analogues. Vienna, August, 23–27, 1993     

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.     

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 (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 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.     

Trypsin revisited: crystallography AT (SUB) atomic resolution and quantum chemistry revealing details of catalysis

A series of crystal structures of trypsin, containing either an autoproteolytic cleaved peptide fragment or a covalently bound inhibitor, were determined at atomic and ultra-high resolution and subjected to ab initio quantum chemical calculations and multipole refinement. Quantum chemical calculations reproduced the observed active site crystal structure with severe deviations from standard stereochemistry and indicated the protonation state of the catalytic residues. Multipole refinement directly revealed the charge distribution in the active site and proved the validity of the ab initio calculations. The combined results confirmed the catalytic function of the active site residues and the two water molecules acting as the nucleophile and the proton donor. The crystal structures represent snapshots from the reaction pathway, close to a tetrahedral intermediate. The de-acylation of trypsin then occurs in true SN2 fashion.     

Solid phase catalytic hydrogen isotope exchange 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 degrees 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 dependencies 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.     

Solid phase isotope exchange with spillover hydrogen in amino acids, peptides, and proteins

We summarize here information on the theoretical and experimental study of high-temperature (150-200 degrees C) solid phase catalytic isotope exchange (HTSPCIE) carried out with amino acids, peptides, and proteins under the action of spillover hydrogen. Main specific features of the HTSPCIE reaction, its mechanism, and its use for studying spatial interactions in polypeptides are discussed. A virtually complete absence of racemization makes this reaction a valuable preparative method. The main regularities of the HTSPCIE reaction with the participation of spillover tritium have been revealed in the case of peptides and proteins, and the dependence of reactivity of peptide fragments on the spatial organization of their molecules has been studied. An important peculiarity of this reaction is that HTSPCIE proceeds at 150-200 degrees C with a high degree of chirality retention in amino acids and peptides. This is provided by its reaction mechanism, which consists in a synchronous one-center substitution at the saturated carbon atom characterized by the formation of pentacoordinated carbon and a three-center bond between the carbon and the incoming and outgoing hydrogen atoms.     

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.     

Hydrogen bonding of flavoprotein: II. Effect of hydrogen bonding at hetero atoms of reduced flavin on its reactivity

The effect of hydrogen bonding at hetero atoms of reduced flavin on its reactivity was studied by ab initio molecular orbital calculations. Among the atoms in the isoalloxazine nucleus of lumiflavin, C(4a) was found to be the most reactive with neutral electrophiles such as molecular oxygen, whereas no reactivity of N(5) can be expected, because of its negative charge. The reactivity of C(4a) is markedly enhanced by hydrogen bonding at N(1) and N(3) in a hydrophobic environment, while it is decreased when hydrogen bonding occurs at all the hetero atoms, as in the case of an aqueous solution of flavin.     

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 (B_1–13) 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.     

A computational study of structure-reactivity relationships in Na-adduct oligosaccharides in collision-induced dissociation reactions

Elucidating the fragmentation mechanisms in oligosaccharides using theoretical calculations is useful in analyzing the experimentally obtained mass spectra. Semi-empirical and ab initio quantum mechanics calculations were used to study the relationship between the structure and reactivity and the chemical properties of oligosaccharides. In these calculations, sodium-cationized oligosaccharides were investigated to determine Na+ ion affinity at several binding positions; in addition, the dependence of the glycosidic bond cleavage on the Na+ position was examined. The calculated structures reported in this study are directed at interpreting experimentally observed fragment ions, resulting from the cleavage of the glycosidic bonds. The calculated results for oligosaccharides containing between three and five monosaccharide units (27 oligosaccharides) were compared with experimental data generated by matrix-assisted laser-desorption/ionization (MALDI) using a quadrupole ion trap (QIT) with a time-of-flight (TOF) mass spectrometer (MS).     

Ab initio studies of the reaction of hydrogen transfer from DNA to the calicheamicinone diradical

BACKGROUND: The biological activity of enediyne chemotherapeutic (anti-cancer) agents is attributed to their ability to cleave duplex DNA. Part of the reaction of cleavage is the abstraction of hydrogens from the deoxyribose moiety of DNA by the biradical formed via a Bergman rearrangement. METHODS: The mechanism of the reaction of abstraction of two hydrogen atoms from two deoxyribophosphate molecules by the calicheamicinone biradical is studied with ab initio calculations at Hartree-Fock and post-Hartree-Fock level. The Titan program is used to perform the calculations. RESULTS: It is found that the reactions are exothermic and thus thermodynamically reasonable. CONCLUSIONS: The mechanism of DNA cleavage by the enediyne-containing drugs is likely to proceed by the abstraction of the hydrogens from deoxyribose by the biradical formed by the drug. Further studies should determine in which way the modification of the drug's structure would make this reaction even more exothermic and, thus, more likely to occur.     

Reaction field effects on proton transfer in the active site of actinidin

The feasibility of the inclusion of reaction field effects in accurate ab initio self-consistent field-molecular orbital calculations was studied in the case of proton transfer in the active site of actinidin. The effects of the polarizability of the environment were included, using the direct reaction field model, which treats the environment as a set of interacting polarizable atoms. Up to 1000 of these atoms could be treated but about 300 were sufficient. The full geometry of the active site and the environment was taken into account. The stabilization of the ion pair was calculated to be 3.5 kcal. but this value may be 10 kcal depending on the geometry used. The effect of the static field from the long alpha-helix present in the enzyme was also studied. Dispersion effects are shown to be unimportant. The orientational polarizability of side chains and water molecules was not included.     

The Hydrolysis Mechanism of Formamide Revisited: Comparison Between ab initio, Semiempirical and DFT Results

The hydrolysis of amides is a model reaction to study peptide hydrolysis. This process has been previously considered in the literature at the ab initio level. In this work, we revisit different reaction mechanisms (water-assisted, non-assisted, neutral and acid-catalyzed) with various theoretical methods : semiempirical, ab initio and Density Functional. The ab initio calculations are carried out at a computational level which is substantially higher than in previous studies. We describe the structure of the transition states and discuss the influence of the catalyst. We also compute the activation free energies for these processes at the Density Functional Theory level. Comparison of the methods allows to outline the main trends of these theoretical approaches which may be useful to design new computational strategies for investigating biological reaction mechanisms through the use of combined Quantum Mechanics/Molecular Mechanics methods.     

Torsion lability of the O=C-N-H fragment in single dipeptide molecules of L-amino acids

On the basis of ab initio MP2 and semi-empirical PM3 quantum-chemical calculations the bistability of the nonplanar O=C-N-H fragment in the structure of simple amides and dipeptides is discussed. The influence of the nature of amino acids on the structural polymorphism of the peptide group in dipeptides is shown.     

Monensin A acid complexes as a model of electrogenic transport of sodium cation

New Monensin A acid complexes with water molecule, sodium chloride and sodium perchlorate were obtained and studied by X-ray and (1)H, (13)C NMR and FT-IR methods as well as ab initio calculations. The crystal structure of the complexes indicates the complexation of the water molecule and Na(+) cation in the pseudo-cycle conformation of the Monensin acid molecule stabilised by intramolecular hydrogen bonds. Important for stabilisation of this structure is also the intermolecular hydrogen bonds with water molecule or the coordination bonds with Na(+) cation. It is demonstrated that the counterions forming intermolecular hydrogen bonds with OH groups influence the strength of the intramolecular hydrogen bonds, but they have no influence on the formation of pseudo-cyclic structure. Spectroscopic studies of the complexes in dichloromethane solution have shown that the pseudo-cyclic structure of the compounds is conserved. As follows from the ab initio calculations, the interactions between the Na(+) cation and the electronegative oxygen atoms of Monensin acid totally change the molecular electrostatic potential around the supramolecular Monensin acid-Na(+) cationic complex relative to that of the neutral Monensin acid molecule.     

Ab initio self-consistent field and potential-dependent partial equalization of orbital electronegativity calculations of hydration properties of N-acetyl-N'-methyl-alanineamide

Using a recently developed parallel computation algorithm, ab initio self-consistent field (SCF) calculations were carried out to estimate the relative hydration energies for 12 low-energy conformations of N-acetyl-N'-methyl-alanineamide. The requisite SCF calculations were carried out using 6-31G and 6-31G* basis sets, both in the absence and presence of a perturbing potential arising from a model solvent. The alpha R, alpha L, polyproline II (PII), and pi helical conformations were preferentially stabilized by the solvent potential, whereas conformations with intramolecular hydrogen-bonding C5 and C7 were preferred in the gas phase. Average vicinal nmr coupling constants (JNH-C alpha H), calculated using the total energies of the various solvated conformations, were consistent with observed coupling constants for this peptide in aqueous solution. Substantial alteration of the solute charge density occurred upon equilibration with the reaction field, as was exemplified in changes both in the molecular dipole moments and in atom-centered multipoles, when the molecule was transferred from a medium of low dielectric constant to one of high dielectric constant. In order to model these changes in charge density with an empirical scheme, we have implemented a novel monopolar representation of the solute charge density based on a potential-dependent form of partial equalization of orbital electronegativities (PDPEOE). In the atom-centered point charge PDPEOE representation, charge flows from one region of the solute to another in response to external fields. Hydration energies calculated using the PDPEOE representation are similar to those calculated by the SCF procedure. Also, the PDPEOE calculations yielded changes in molecular dipole moments upon solvation that agreed closely with the changes in the calculated ab initio SCF dipole moments.     

Gas phase hydrogen/deuterium exchange reactions of peptide ions in a quadrupole ion trap mass spectrometer

Hydrogen/deuterium exchange reactions of protonated and sodium cationized peptide molecules have been studied in the gas phase with a MALDI/quadrupole ion trap mass spectrometer. Unit-mass selected precursor ions were allowed to react with deuterated ammonia introduced into the trap cell by a pulsed valve. The reactant gas pressure, reaction time, and degree of the internal excitation of reactant ions were varied to explore the kinetics of the gas phase isotope exchange. Protonated peptide molecules exhibited a high degree of reactivity, some showing complete exchange of all labile hydrogen atoms. On the contrary, peptide molecules cationized with sodium exhibited only very limited reactivity, indicating a vast difference between the gas phase structures of the two ions. © 1997 Wiley-Liss Inc.