Deuterium Exchange Rates Research Articles

Nuclear Magnetic Resonance Study on the Microenvironments of Histidine Residues of Ribonuclease T<sub>1</sub> and Carboxymethylated Ribonuclease T<sub>1</sub><xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

The 270-MHz 1H NMR spectra and fluorescence of ribonuclease T1 and carboxymethylated ribonuclease T1 were measured in aqueous solution. Histidine C4 proton resonances were assigned to individual residues. From the pH dependences of the chemical shifts of histidine C2 and C4 protons, the pKa values of histidine residues were obtained by the non-linear least-squares method. The hydrogen leads to deuterium exchange rates of histidine C2 protons were determined as a measure of the accessibility of histidine residues to the solvent. Each histidine residue of ribonuclease T1 was found to interact with a carboxylate group of an aspartic or glutamic acid residue; in particular, His-40 was shown to interact with Glu-58. Upon carboxymethylation of Glu-58, His-92 and His-27 are more shielded from the solvent while His-40 remains exposed to the solvent. The 67.9-MHz 13C NMR spectra were measured for the 13C-enriched preparation of carboxymethylated ribonuclease T1. From the pH dependence of 13C chemical shift, the pKa value of the carboxymethylated Glu-58 was found to be unusually low, suggesting the formation of an ionic or hydrogen bond between this carboxymethyl group and a positively charged group, possibly of Arg-77.

Conformational analysis of somatostatin and selected analogs in oriented polyoxyethylene by infrared dichroism

AbstractInfrared dichroic studies and deuterium exchange measurements of somatostatin and some of its analogs incorporated in uniaxially oriented polyoxyethylene are described. Band positions and dichroic ratios in the N‐H stretching and amide I and II regions are similar to those of flexible and nonordered peptides like valinomycin and poly[Glu(ONa)]. This information, together with fast deuterium exchange rates, suggests that somatostatin exists in a flexible nonordered conformation in polyoxyethylene. One analog, di‐S3,14‐acetamidomethyl dihydrosomatostatin, was found to exist in both nonordered and β‐like conformations.

Kinetic studies with the use of proton-magnetic-resonance spectroscopy of the specific α-deuteration of amino acids by Escherichia coli aspartate aminotransferase

Escherichia coli aspartate aminotransferase was exposed to aspartate or phenylalanine without oxo acid in buffered 2H2O. The alpha-hydrogen of the amino acids underwent first-order exchange with respect to both substrate and enzyme. P.m.r. spectroscopy gave consistent reaction-rate constants. The deuterium-exchange rate was only moderately increased by addition of oxo acids and was of the same order as the transamination rate. No beta-deuteration was observed. The C(alpha)-H-bond-breaking step is discussed as a part of the entire transamination mechanism.

Isotopic hydrogen exchange in 1- and 2-aryl-5-methyltetrazoles

The rate constants for basic deuterium exchange of the methyl group (kD) in 2-phenyl-5-methyl-tetrazole (I) and 1-aryl-5-methyltetrazole (II) and its derivatives with a polar substituent (R) in the phenyl ring were measured. The increased CH acidity of II as compared with I [kD(II)/kD(I)∼20] is in agreement with the calculated and experimental values regarding the character of the electron-density distribution in the molecules. The effect of R on the rate of deuterium exchange of the methyl group correlates with the σ° constants (ρ=3.0, r = 0.997). The results of measurement of the kinetic isotope effect during deuterium (tritium) exchange in II (kD/kT∼1.8) are discussed in connection with the peculiarities of the stepwise reaction mechanism.

Isotopic hydrogen exchange in methyl derivatives of five-membered aromatic heterocycles

The kinetics of isotopic exchange of the hydrogen atoms of the methyl groups in an alcohol solution of potassium ethoxide were studied for an extensive series of methyl derivatives of azoles and di-, tri-, and tetrazoles. The electronic effect of one or several heteroatoms and substituents on the rate of deuterium exchange of five-membered heterocycles is satisfactorily conveyed by the correlation relationship previously established for a series of substituted toluenes and six-membered heteroaromatic compounds (ρ 25°=7.6). The limitations that exist in a number of cases are discussed in connection with the peculiarities of the electron-density distribution in five- and six-membered heterocycles.

Proton-nuclear-magnetic-resonance study of the conformation of neurotoxin II from Middle-Asian cobra (Naja naja oxiana) venom.

A proton nuclear magnetic resonance (NMR) study at 100 and 300 MHz of neurotoxin II from the venom of Middle-Asian cobra Naja naja oxiana has been performed in 2H2O and H2O solutions. By means of chemical modification and double resonance all the aromatic residue resonances have been assigned. From the NMR titration curves, pK values of histidine 4 and histidine 31 residues have been determined. For one of the two neighbouring tryptophan residues pH dependence (in the 2-8-pH range) of the chemical shifts of indole protons has been revealed. According to the different sensitivity of the linewidth of indole NH resonances to pH in H2O solution, the accessibility of each of the tryptophan residues has been estimated. Temperature dependence has been observed for the linewidth of the aromatic resonances of the tyrosine 24 residue. Deuterium exchange rates have been measured for amide protons as well as for C(2)H histidine resonances. The NMR data obtained have allowed the conclusions to be made that the two histidine residues and one of the tryptophan residues should be localized on the surface of the protein globule, that arginine residues should be present in the environment of histidine 4, that histidine 31 and the buried tryptophan are possibly localized in close spatial proximity and that the side chain of tyrosine 24 is buried within the protein globule.

ChemInform Abstract: PROTON MAGNETIC RESONANCE SPECTRA OF METAL AMMINE COMPLEXES. VI. THE TRANS‐EFFECT IN COBALT(III) AMINE COMPOUNDS AS STUDIED BY THE RATE OF HYDROGEN‐DEUTERIUM EXCHANGE

The rate of the isotopic deuterium exchange has been measured for several cobalt(III) amine complexes in D2O. The substituents CN−, NO2−, and possibly SO32− decelerate the exchange of the amine protons trans to these ligands, leading in some cases to a smaller rate for these protons than for the cis ones. On the other hand, the trans-proton labilizing ligands include F−, Cl−, Br,−, H2O, CO32−, OCONH2−, N3−, CH3NH2, CH3COO−, and the malonate, fumarate, and oxalate ions. The above observation clearly indicates that the HD exchange rate can be a measure of the trans-effect of these ligand molecules.

Nuclear magnetic resonance titration curves of histidine ring protons. A direct assignment of the resonances of the active site histidine residues of ribonuclease.

One of the four titrating histidine ring C-2 proton resonances of bovine pancreatic ribonuclease has been assigned to histidine residue 12. This was accomplished by a direct comparison of the rate of tritium incorporation into position C-2 of histidine 12 of S-peptide (residues 1 to 20) derived from ribonuclease S, with the rates of deuterium exchange of the four histidine C-2 proton resonances of ribonuclease S under the same experimental conditions. The same assignment was obtained by a comparison of the NMR titration curves of ribonuclease S, the noncovalent complex of S-peptide and S-protein (residues 21 to 124) with the results for the recombined complex in which position C-2 of histidine 12 was fully deuterated. The second active site histidine resonance was assigned to histidine residue 119 by consideration of the NMR titration results fro carboxymethylated histidines and 1-carboxymethylhistidine 119 ribonuclease. This assignment is a reversal of that originally reported, and has important implications for the interpretation of NMR titration data of ribonuclease.

The log kexovs. log kendo correlation as a mechanistic criterion; on the mechanisms of solvolysis of norbornyl derivatives and base-catalyzed isotope exchange of norbornanones

Rate data for the acetolysis of exo-norbornyl-sulfonates have been correlated with those for the corresponding endo isomers. It is shown that the slopes of the log kexovs. log kendo plots reflect the difference in delocalization between the transition states derived from the exo and endo isomers, respectively. The log kexovs. log kendo plot, which is comprised of the parent norbornyl sufonate and its derivatives substituted at the 5, 6, and 7 positions, has a slope of 1.11 ± 0.08, which establishes that σ bridging is absent in the transition state obtained from the exo isomer. A similar analysis of base-catalyzed hydrogen–deuterium exchange rates of norbornanones reveals that exo proton exchange is more sensitive to substituent effects than the corresponding endo process.

Proton Magnetic Resonance Spectra of Metal Ammine Complexes. VI. The trans-Effect in Cobalt(III) Amine Compounds as Studied by the Rate of Hydrogen-Deuterium Exchange

Abstract The rate of the isotopic deuterium exchange has been measured for several cobalt(III) amine complexes in D2O. The substituents CN−, NO2−, and possibly SO32− decelerate the exchange of the amine protons trans to these ligands, leading in some cases to a smaller rate for these protons than for the cis ones. On the other hand, the trans-proton labilizing ligands include F−, Cl−, Br,−, H2O, CO32−, OCONH2−, N3−, CH3NH2, CH3COO−, and the malonate, fumarate, and oxalate ions. The above observation clearly indicates that the HD exchange rate can be a measure of the trans-effect of these ligand molecules.

Reactions between azolium anions and electrophilic reagents. Part II. Direct thiation of 1,2-disubstituted pyrazolium anions with sulphur

Pyrazolium salts (1) afford Δ3-pyrazoline-5-thiones (2) in good yields when treated with sulphur and sodium hydride in dimethylformamide. Pyrazolium salts (1a) with two different N-substituents produce two isomeric Δ3-pyrazoline-5-thiones (2a and b) in a ratio reflected by the ratio between the deuterium exchange rates of H-3 and H-5 in the starting material (1a).

Stereochemistry of hydrogen–deuterium exchange α to the sulphinyl group in 2H-naphtho[1,8-bc]thiophen derivatives

The rate of deuterium exchange in MeONa—MeOD of α-protons having an equal stereochemical relation to a sulphinyl group was found to follow an opposite trend in 2H-naphtho[1,8-bc]thiophen derivatives (6)–(8) to that previously observed in 2-thiabicyclo[2.2.1]heptane derivatives (1) and (2), for which tetrahedral α-sulphinyl carbanions of different stability had been proposed as intermediates in the exchange process. The experimental results were rationalized by assiging to tetrahedral and planar α-sulphinyl carbanions a separate role in determining, according to their relative stability, the rate of proton abstraction or the stereochemistry of the exchange, or both.

Aryl Carbanions. A 13C Nuclear Magnetic Resonance Study of Deuterium Exchange in Acenaphthenes and Related Aromatic Hydrocarbons

The base-catalyzed hydrogen–deuterium exchange rates for the aromatic and benzylic centers in acenaphthene, 2,3-dihydrophenalene, 1,8-dimethylnaphthalene, 1,1,2,2,-tetramethylacenaphthene, and diphenylmethane have been determined using 13C n.m.r. The exchange rates serve as a probe of carbanion stability and reactivity, clearly indicating the importance of ring strain effects on carbanion stability.

The conformational characteristics in solution of the synthetic cyclic hexapeptide 5L-ala-D-ala.

AbstractAn attempt to elucidate the solution conformation(s) of the synthetic cyclic hexapeptide 5L‐ala·D‐ala is described. Nuclear magnetic resonance (nmr) spectra are recorded for the purpose of measuring the vicinal coupling constant between the amide and α‐protons in each residue and to observe the deuterium exchange rate and temperature dependence of the chemical shift of each amide proton. Low‐energy cyclic conformations, whose individual residues are in conformations consistent with the observed amide to α‐proton coupling constant, are searched for in an approximate theoretical treatment. The two lowest energy, all trans peptide bond conformations generated are distinguishable by the presence or absence of a single intramolecular hydrogen bond. The observed temperature independence of the chemical shift of one of the amide protons is consistent with the presence of a single intramolecular hydrogen bond, while the observation of similar deuterium exchange rates for each of the amide protons indicates their comparable availability to solvent. Consequently, it is concluded that 5L‐ala·D‐ala is in rapid equilibrium between conformations with and without a single internal hydrogen bond and possesses considerable conformational flexibility in solution.

CH-acidität—VII : Experimentelle und quantenchemische studien zur kinetischen CH-acidität der methyl-aza-acene

Hydrogen deuterium exchange rates of the α-hydrogen atoms of 14 methylazines measured in pyrrolidine-D are reported. The experimental CH-acidity order does not correlate with HMO, PPP or Variable β,γ PPP localization energies. The deficiency of β electron methods is presumably due to the field effect of nitrogen atoms and their lone pairs. Some simple models are proposed in order to treat this effect in terms of σ-π separation.

Fluctuation of the lysozyme structure

The kinetics of hydrogen-deuterium exchange in hen egg-white lysozyme (muramidase) has been followed in aqueous solutions of various pH values and in solutions with various concentrations of lithium chloride, by an infrared absorption measurement. It was found that, in every case, 34% of the total peptide hydrogen atoms exchange relatively slowly with a rate of a first-order reaction. This amount corresponds to 44 peptide groups per molecule, and this is equal to the number of peptide NH-groups which are found to be involved in hydrogen bonds with the carbonyls of other peptide groups in the lysozyme molecule in the crystalline state. Each rate constant determined is in good agreement with the value expected from two simple assumptions. (1) The scheme of the isotope exchange reaction is N ⇌ D → D ∗ (⇌ N ∗ ), where N is the native form of the molecule, D a denatured (unfolded) form, and ∗ indicates the deuterated products. (2) The N ⇌ D fluctuation rate is much higher than the rate of the isotope exchange reaction D → D ∗ . It has been shown that the N ⇌ D transition postulated here is the same as that which can be followed by circular dichroism measurement and by some other physical measurements. The effect of lithium chloride on the exchange reaction rate is solely attributable to the change in the N ⇌ D equilibrium caused by the salt, whereas the effect of pH (in the 5 to 8 range) is wholly ascribed to the catalytic action of the OH − anion on the D → D ∗ reaction rate. From the deuterium exchange rate observed, an effective value of the mole fraction of the D form is estimated to be 3 × 10 −6in the solution with no lithium chloride at 20 °C and of pH = 5 to 8.

Study of acid deuterium exchange in series of 3-hydroxypyridine derivatives

1. A study was made of the basic deuterium exchange of 3-hydroxypyridine and its alkyl derivatives. The constants for the rate of hydrogen exchange in the 2-, 6-, and 4-positions of the ring were determined, in which connection the 2-position of theβ-pyridinol ring proved to be the most reactive. 2. The introduction of methyl groups into the ring increases the rate of deuterium exchange in the 2- and 6-positions of theβ-pyridinol ring. 3. Deuterium exchange of the protons of the methyl groups of the substituents occurs under intense heating.

Complex Formation from Calcium Hydroxide and Carbohydrate in Alkaline Solutions

The abnormally high dissolution of CaO in aqueous solution of carbohydrate such as glucose or xylose is attributed to the complex formation by the following facts. Solubility of calcium hydroxide in glucose solutions is proportional to the concentration of glucose, as their molar ratio is one to one. The value of the molar ratio is further confirmed by chemical analysis of the solid sample, deposited by pouring ethanol in the glucose solution saturated with Ca(OH)2.NMR spectra changing with time show clearly that the rates of deuterium exchange of the skeletal CH at C-1 and C-2 positions of glucose or xylose in D20 solution containing Ca(0D)2 are rapid enough as compared with those in D20 solution containing NaOD. Thus, it can be concluded that the complex is made of calcium hydroxide coordinated the carbohydrate in eriediol type in alkaline solution.Such a structure of Ca-complex is also supportedjby experimental results that the optical activities of D-glucose or D-fructose solutions decrease linearly with the amount of added CaO and that the IR spectra of the solid complex show characteristic absorptions suitable to this structure.

CH-Acidität—IV: Die kinetische CH-acidität der isomeren monomethylazulene

Hydrogen deuterium exchange rates are reported for the isomeric methylazulenes and for some benzenoid arylmethanes with pyrrolidine-D. The relative rates follow the Brönsted catalysis law and are compared with the acidities of related arylammonium cations.

NUCLEAR MAGNETIC RESONANCE STUDIES OF CONFIGURATION AND LIGAND CONFORMATION IN PARAMAGNETIC OCTAHEDRAL COMPLEXES OF NICKEL(II) VI:Meso- andRac-2,3-Butane(dinitrilo)-N,N,N′,N′-tetraacetate Complexes

Abstract The effect of ligand stereochemistry upon the rate of intra-molecular interchanges, called acetate scrambling, are investigated by proton magnetic resonance. Ni(rac-BDTA)2- exhibits slow partial unwrapping followed by nitrogen inversion (k ≃ 1 × 10−5 sec−1 at 72°C). Ni(rac-BDTA)2- probably undergoes this process at a similar rate. Because of the great conformational preference of the methyl groups for the pseudo-equatorial position in the chelate ring, the contribution of Δ,Λ-conversion to the acetate scrambling in Ni(rac-BDTA)2- is negligible. Ni(meso-BTDA)2- undergoes Δ,Δ-conversion rapidly (k = 1.5 × 103 sec−1) at 72°C. A pseudo-axial methyl group selectively reduces the deuterium exchange rates of one out-of-plane acetate ring by a factor of 3 relative to those observed for the “unhindered” out-of-plane acetate ring. The deuterium exchange on a specific out-of-plane acetate ring is stereospecific with the pseudo-axial proton exchanging three times faster than the pseudo-equatorial proton.