15N NMR studies of amino acids and their reaction products with formaldehyde

Natural abundance ¹⁵N NMR spectroscopy has been used to investigate the effect of pH on the ¹⁵N chemical shifts of lysine and of ε-hydroxymethyllysine. A computer calcualtion which fits the chemical shifts of both α-and ε-nitrogen atoms versus pH has been used to predict the pK_a values. ¹⁵N chemical shifts and some ¹J(¹⁵NH) values of some other amino acids and of their reaction products with formaldehyde are also reported.     

o-Nitrobenzoyl group as a new amino protecting group for peptide synthesis and the synthesis of some anthranilyl peptides

N (o-nitrobenzoyl)amino acids can be coupled with other amino acids using DCC and the resulting product on hydrogenation gives peptides, containing the anthranilyl group as —NH₂ end group. N (anthranilyl)amino acids or peptides can also be obtained by reaction of isatoic anhydride on amino acids or peptides. The anthranilyl end group is easily cleaved by metal (Cu⁺²) catalysed hydrolysis to give α-amino acid peptides and the insoluble copper(II) anthranilate.     

Gas-Phase Conformational Map of the Amino Acid Isovaline

Four conformers of the non-proteinogenic α-amino acid isovaline, vaporized by laser ablation, are characterized by Fourier-transform microwave techniques in a supersonic expansion. The comparison between the experimental rotational and ¹⁴N nuclear quadrupole coupling constants and the ab initio calculated ones provides conclusive evidence for the identification of the conformers. The most stable species is stabilized by an N−H⋅⋅⋅O =C intramolecular hydrogen bond and a cis-COOH interaction, whereas the higher-energy conformers exhibit an N⋅⋅⋅H−O intramolecular hydrogen bond and trans-COOH, as in other aliphatic amino acids. The spectroscopic data herein reported can be used for the astrophysical purpose in a possible detection of isovaline in space.     

Through‐space 19F–15N couplings for the assignment of stereochemistry in flubenzimine

Through‐space ¹⁹F–¹⁵N couplings revealed the configuration of flubenzimine, with the CF₃ group on N4 pointing towards the lone pair of N5. The ¹⁹F–¹⁵N coupling constants were measured at natural abundance using a spin‐state selective indirect‐detection pulse sequence. As ¹⁵N‐labelled proteins are routinely synthesized for NMR studies, through‐space ¹⁹F–¹⁵N couplings have the potential to probe the stereochemistry of these proteins by ¹⁹F labelling of some amino acids or can reveal the site of docking of fluorine‐containing drugs. Copyright © 2016 John Wiley & Sons, Ltd.     

Protonation equilibria studies of the standard α-amino acids in NaNO3 solutions in water and in mixtures of water and dioxane

The protonation equilibria for 20 standard α-amino acids in solutions have been studied pH-potentiometrically. The dissociation constants (pK_a) of 20 amino acids and the thermodynamic functions (ΔG^∘, ΔH^∘, ΔS^∘, and δ) for the successive and overall protonation processes of amino acids have been derived at different temperatures in water and in three different mixtures of water and dioxane (mole fractions of dioxane were 0.083, 0.174, and 0.33). Titrations were also carried out in water ionic strengths of (0.15, 0.20, and 0.25) mol · dm⁻³ NaNO₃, and the resulting dissociation constants are reported. A detailed thermodynamic analysis of the effects of organic solvent (dioxane), temperature and ionic strength influencing the protonation processes of amino acids is presented and discussed to determine the factors which control these processes.     

Heteroditopic receptor based on crown ether and cyclen units for the recognition of zwitterionic amino acids

The molecular recognition of five targeted amino acids differing in the nature of the side (R)- group and in the size of the aliphatic chain, glycine (Hgly), phenylalanine (Hphe), glutamic acid (Hglu^?), 4-aminobutyric acid (Hgaba), and 6-aminohexanoic acid (Heahx), has been studied with a new heteroditopic receptor based in two distinct macrocycles, a cyclen and a crown ether moiety. The bismacrocycle L was synthesized via the bis-aminal route allowing to obtain the designed compound in gram scale and in good yield. Protonation constants of L and its binding constants with amino acids were determined by potentiometry in H₂O/MeOH (1:1 v/v) solutions at 298.2 K and I=0.10 mol dm^?3 in NMe₄NO₃. Stronger binding ability of the H_nLⁿ⁺ receptor for α-amino acids, Hgly and Hphen, than for the other studied substrates were found. Structural data derived from NMR studies showed that the binding of α-amino acids result from the cooperative participation of hydrogen bonds between the carboxylate group of amino acids and the polyammonium sites of cyclen, and the ion-dipole interactions between the ammonium group of the amino acids and the oxygen atoms of the crown ether.     

Oligonucleotides and nucleotidopeptides. XLII. Efficacy of the intramolecular influence of the carboxy groups of amino acids in nucleotidyl-(P → N)-amino acids (peptides) as a function of their positions

It has been established that remote carboxy groups in nucleotidyl-(5′ → N)-β-alanine and -alanylalanine do not affect the mechanism of the cleavage of the phosphoramide center. The situation is different in the case of nucleotidyl-(5′ → N^?)-lysine. It has been shown that in the 3′-phenylalanine derivative of dTMP, the influence of the α-carboxy group of the amino acid is only half as great as in the 5′-analog. The α-carboxy groups of the amino acids in oligonucleotidyl-(Pⁱⁿ → N)-amino acids also weaken the mechanism of the cleavage of the phosphoramide centers.     

Compound-Specific Isotope Analysis of Amino Acid Labeling with Stable Isotope Nitrogen (15N) in Higher Plants

Individual free amino acid δ¹⁵N values in plant tissue reflect the metabolic pathways involved in their biosynthesis and catabolism and could thus aid understanding of environmental stress and anthropogenic effects on plant metabolism. In this study, compound-specific nitrogen isotope analysis of amino acid by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) was carried out to determine individual free amino acid δ¹⁵N values. High correlations were observed between the δ¹⁵N values obtained by GC-C-IRMS and elemental analyzer-isotope ratio mass spectrometry (EA-IRMS) determinations, and the mean precision measured was better than 1 ‰. Cation-exchange chromatography was employed to purify the sample, and the difference between prior to and following passage through the resin was within 1 ‰. The amino acid δ¹⁵N values of plant leave samples following incubation in ¹⁵N-nitrate at different time points were determined. A typical foliar free amino acid ¹⁵N-enrichment pattern was found, and glutamine was the most rapidly labeled amino acid; other amino acids derived from the GS-GOGAT cycle were also enriched. The pyruvate family amino acids were labeled less quickly followed by the aromatic amino acids. This study highlighted that amino acid metabolism pathways had a major effect on the δ¹⁵N values. With the known amino acid metabolism pathways and δ¹⁵N values determined by the presented method, the influence of various external factors on the metabolic cycling of amino acid can be understood well.

     

The Shape of the Simplest Non-proteinogenic Amino Acid α-Aminoisobutyric Acid (Aib)

The simplest non-proteinogenic amino acid α-aminoisobutyric acid (Aib), an analogue of glycine and alanine, has been vaporized by laser ablation and probed by high-resolution Fourier transform microwave spectroscopic techniques. Comparison of the experimental rotational and ¹⁴N nuclear quadrupole constants with that predicted ab initio has allowed the identification of three conformers of Aib exhibiting three types of hydrogen-bond interactions I (NH⋅⋅⋅O=C, cis-COOH), II (OH⋅⋅⋅N, trans-COOH), and III (N−H⋅⋅⋅O−H, cis-COOH) within the amino acid backbone. The observation of conformer III, not detected previously for related proteinogenic amino acids with a nonpolar side chain in a supersonic expansion, indicates that the presence of the methyl groups should restrict the conformational relaxation from conformer Aib-III to Aib-I. For conformer Aib-II, the rotational spectra of the ¹³C isotopomers reveal a tunneling motion arising from the two equivalent methyl groups in the molecule. The observation of a single spectrum at the midpoint between those predicted for the two ¹³C of the methyl groups has been explained by considering a double-minimum potential function with a low-energy interconversion barrier for a large amplitude internal motion. This singular fact has been corroborated by the anomalous centrifugal distortion effects determined in conformer Aib-II.     

15N NMR spectroscopy acids: 7—solvent effects on α-and ω-amino

The natural abundance ¹⁵N NMR spectra of several α-and ω-amino acids were measured in various protic solvents. Increasing acidity of the solvents results in an upfield shift in the case of the α-amino acids, while ω-amino acids are almost insensitive to solvent effects.     

Chemo-enzymatic synthesis of isotopically labelled L-Valine,L-Isoleucine and allo-isoleucine

Syntheses of (3R)-[4,4,4-D₃]-L-valine, [¹⁵N]-L-isoleucine and [¹⁵N]-allo-isoleucine from homochiral 2-alkylated carboxylic acids are described. The approach involves a one-carbon homologation of the carboxylic acid to give the corrresponding β-substituted α-keto ester which is converted directly to the α-amino acid in a one-pot procedure involving two enzyme catalysed reactions (Candida cylindracea lipase to hydroluse the ester and leucine dehydrogenase to catalyse the reductive amination of the ketone). This strategy may be simply adapted for the selective labelling of each site of the L-amino acid.     

Evaluation of the influence of arsenic species on the nitrogen metabolism of a model angiosperm: nasturtium,Tropaeolum majus

How the various organic and inorganic arsenic species affect the nitrogen metabolism of a model plant, Tropaeolum majus, was studied in order to evaluate the toxicological impact of the various chemical forms of arsenic. For this purpose, the effects on the (a) entire nitrogen pool, (b) protein fraction, and (c) non‐protein fraction were distinguished. The arsenic‐dependent effects on the nitrogen cycle were assessed by using ¹⁵N‐labelled KNO₃ as a nutritive substance and optical emission spectroscopy to analyse how ¹⁵N is incorporated into the nitrogen cycle. In addition to the ¹⁵N‐tracer experiments, the uptake and metabolization of the arsenic compounds were examined. The work shows that biochemical indicator systems like ¹⁵N‐tracer studies are able to characterize the degree of the influence of metabolic processes by arsenic species. For example, the incorporated ¹⁵N concentration decreased linearly and independently of the ¹⁵N fraction with increasing dimethylarsinate (DMA) concentrations. This behaviour indicates that DMA has prevented the uptake of ¹⁵N and hence the formation of amino acids and proteins. Arsenite‐treated plants exhibited an elevated concentration of non‐protein ¹⁵N, which could be an indication either for a stimulated uptake of nitrate or for an interrupted amino acid/protein synthesis. Copyright © 2005 John Wiley & Sons, Ltd.     

Conformational study of phosphoserine in aqueous solution. I—13C n.m.r. results

¹³C chemical shifts and coupling constants of phosphoserine in aqueous solutions were studied as a function of pH values. Carboxyl and α-amino titration shifts agree with those observed in amino acids. The analysis of the coupling constants indicates that for rotation about the (C)? C? O? (P) axis the trans conformer predominates for all pH values. The fractional population of the gauche conformer reaches a maximum at pH=8.     

Long‐lived states in an intrinsically disordered protein domain

Long‐lived states (LLS) are relaxation‐favored spin population distributions of J‐coupled magnetic nuclei. LLS were measured, along with classical ¹H and ¹⁵N relaxation rate constants, in amino acids of the N‐terminal Unique domain of the c‐Src kinase, which is disordered in vitro under physiological conditions. The relaxation rates of LLS can probe motions and interactions in biomolecules. LLS of the aliphatic protons of glycines, with lifetimes approximately four times longer than their spin–lattice relaxation times, are reported for the first time in an intrinsically disordered protein domain. LLS relaxation experiments were integrated with 2D spectroscopy methods, further adapting them for studies on proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

The nuclear magnetic resonance spectra of N-nitroso-N-alkyl amino acids

Structurally related acyclic and cyclic N-nitroso-N-alkyl amino acids were prepared and their ¹H, ¹³C and ¹⁵N NMR spectra were investigated. The changes in the ¹³C NMR spectra of the N-nitroso α-amino acids after dissolving in solvents suggest that they posses the Z-configuration in the crystalline state; in solution some of them isomerize to mixtures of the Z- and E-isomers whose composition appear to depend on steric factors. The ¹³C chemical shifts were assigned on the basis of anisotropic effects of the nitrosamino group and configurational stability. The ¹³C chemical shifts were correlated with those of the nitrosamines of the same carbon skeletons and the effects of changing a methyl group to the carboxylic acid are deshielding on the α-carbons and shielding on the β-carbons.     

17O-NMR. of Enriched Acetic Acid,Glycine, Glutamic Acid and Aspartic Acid in Aqueous Solution. I. Chemical Shift Studies

The ¹⁷O-NMR. chemical shifts of the enriched amino acids glycine, aspartic acid and glutamic acid were measured in aqueous solution as a function of pH. High magnetic fields are necessary to resolve the α, β- and α, γ-carboxyl resonances of aspartic acid and glutamic acid, respectively. The chemical shifts of acetic acid were measured for comparative reasons. Ionization constants and titration shifts were obtained by nonlinear least-squares fits to one-proton titration curves. The average excitation energy approximation is discussed in terms of the observed changes in ¹⁷O-shielding on deprotonation. No intramolecular association between the α-amino group and the α-carboxyl group in the zwitterionic form is required to explain the high-frequency shift of the carboxylate ion. Also no indication of an intramolecular association between the α-amino group and the side-chain carboxyl groups of aspartic acid or glutamic acid was found.     

The use of15N-labelled amino acids in vivo and in vitro for transmination studies: Tryptophan transaminase in maize plants

Besides conventional methods, transminase activity can also be followed by an isotopically labelled substrate, i.e. by an amino acid with a¹⁵N labelled amino group. The application of the labelled substrate enables their study in vitro as well as in vivo. This method was applied for following the tryptophan transaminase activity in germinating maize plants. The enzym preparations used for in vitro analysis were purified on a Sephadex G 100 column. For in vivo experiments the¹⁵ N substrate was introduced by vacuum infiltration into the plants leaves. The amino acids originated by transamination are chromatographically separated and after purifying by paper electrophoresis, the atom excess¹⁵N is determined. In maize tissue originated glutamic acid (from infiltrated α-ketoglutarate substrate), which in the course of further reactions was transformed into aspartic acid and asparagine. The results found so far by studying the low activity of tryptophan-transaminase in vivo entitle us to improve our method of transaminase activity determination by using¹⁵N labelled-substrate.     

Structural assignments in isomeric pyrazoles based on 3J(15NH) coupling constants

Vicinal ¹⁵N–H coupling constants are used to characterize the isomeric pyrazoles obtained from the condensation of phenylhydrazine, ¹⁵N-enriched at the amino nitrogen atom, with various 1,3-diketones.     

Dediazoniations of arenediazonium ions part 25 Influence of Substituents on the Exchange of the Diazonio Group for External Molecular Nitrogen and on the N(α), N(β)-Rearrangement in the Diazonio Group

The N(α), N(β)-rearrangement of the two N-atoms which can be observed in solutions of [β-¹⁵N]-labelled p-substituted benzenediazonium ions follows dual substituent parameter treatments. The reaction yields a negative field and a positive resonance reaction constant (p_F =?3.35, p_R = 2.47). The magnitude of these constants is, within experimental error, the same as the respective reaction constants for solvolytic dediazoniation. The exchange of the diazonio group of ¹⁵N-labelled p-substituted benzenediazonium ion yields, however, field and resonance reaction constants which are close to zero. This result is attributed to cancellation of the reaction constants for the forward and reverse steps in the complex mechanism of the exchange reaction.     

Arenesulfonylation of dl-serine, l-proline, l-threonine, and dl-methionine in systems 1,4-dioxane—water and propan-2-ol—water

Kinetics of interaction of dl-serine, l-proline, l-threonine, and dl-methionine with 3-nitrobenzenesulfonyl chloride in water (40%)—1,4-dioxane and water (40%)—isopropanol mixed solvents was studied spectrophotometrically at 298 K. The main reactive form of α-amino acids is shown to be anionic. Under conditions of arenesulfonylation, the basicity of α-amino acids is crucial in determining the reaction rate. Arenesulfonylation rate constants are 20—50 times lower than the constants of N-acylation of the same α-amino acids with benzoyl chloride and 10⁴–10⁵ times higher than the rate constants of their reactions with benzoic acid 4-nitrophenyl ester.