ESI‐MS/MS analysis of protonated N‐methyl amino acids and their immonium ions

Methylation is one of the important posttranslational modifications of biological systems. At the metabolite level, the methylation process is expected to convert bioactive compounds such as amino acids, fatty acids, lipids, sugars, and other organic acids into their methylated forms. A few of the methylated amino acids are identified and have been proved as potential biomarkers for several metabolic disorders by using mass spectrometry–based metabolomics workstation. As it is possible to encounter all the N‐methyl forms of the proteinogenic amino acids in plant/biological systems, it is essential to have analytical data of all N‐methyl amino acids for their detection and identification. In earlier studies, we have reported the ESI‐MS/MS data of all methylated proteinogenic amino acids, except that of mono‐N‐methyl amino acids. In this study, the N‐methyl amino acids of all the amino acids ( 1 ‐ 21 ; including one isomeric pair) were synthesized and characterized by ESI‐MS/MS, LC/MS/MS, and HRMS. These data could be useful for detection and identification of N‐methyl amino acids in biological systems for future metabolomics studies. The MS/MS spectra of [M + H]⁺ ions of most N‐methyl amino acids showed respective immonium ions by the loss of (H₂O, CO). The other most common product ions detected were [MH‐(NH₂CH₃]⁺, [MH‐(RH)]⁺ (where R = side chain group) ions, and the selective structure indicative product ions due to side chain and N‐methyl group. The isomeric/isobaric N‐methyl amino acids could easily be differentiated by their distinct MS/MS spectra. Further, the MS/MS of immonium ions inferred side chain structure and methyl group on α‐nitrogen of the N‐methyl amino acids.     

Electron impact fragmentation mechanisms of 2,4,6-trinitrotoluene derived from metastable transitions and isotopic labeling

Precursors of the major ions in the electron impact spectrum of 2,4,6-trinitrotoluene have been determined by observations of metastable peaks and isotope shifts. In 2,4,6-trinitrotoluene with adjacent CH₃ and NO₂ groups, the facile transfer of a methyl hydrogen to oxygen followed by loss of an OH group gives rise to the m/z 210 ion which plays a key role in the six important fragmentation sequences that have been identified. A similarity of the reactions in the mass spectrometer to the decomposition pathways induced by other stimuli has been observed.     

Substituent chemical shifts of the organotin moiety in compounds of the type RSnMenCl3 − n (n = 0 to 3; R = n-alkyl)

The ¹³C and ¹¹⁹Sn NMR spectra of 33 organotin compounds of the type RSnMe_nCl_{3 ? n} and related types are discussed. The substituent effects of the groups SnMe₃, SnMe₂Cl, SnMeCl₂ and SnCl₃ (and of some related groups) on the carbon chemical shifts in the alkyl group R have been determined; the SnMe₃ group causes a small upfield shift of the carbon attached to it, while the other groups cause downfield shifts. The shifts show a monotonic change on replacing methyl groups in Me₃Sn by chlorine atoms. The effects on carbons further removed from the tin atom are discussed. Variation in R causes little change in ⁿJ(Sn? C) or δ(¹¹⁹Sn).     

13C NMR study on the methoxy carbon chemical shifts in chloro-substituted anisoles and guaiacols

The ¹³C NMR chemical shifts of methoxy carbons in chlorinated anisoles and guaiacols have been measured for acetone-d₆ solutions. Multiple linear regression analysis, and also ‘simple sum rule’ calculations, have been used to estimate the effects of the chlorine atoms (the position and degree of substitution) on the chemical shifts. The most important effects have shown to be due to the chlorine atoms adjacent to the methoxy and hydroxy substituents. For chlorinated guaiacols, the greatest effect is due to the chlorine atom adjacent to the methoxy group. For chlorinated anisoles, the substituents adjacent to the methoxy group (2,6-disubstitution) cause large effects. For both groups of compounds, the chemical shifts are also greatly influenced by the number of chlorine substituents. Using the three most important independent variables, the average differences between the observed and calculated chemical shifts are ca 0.2 ppm for anisoles and 0.1 ppm for guaiacols. For chloroguaiacols, the corresponding difference was only 0.1 ppm when calculations were performed using single substituent effects.     

2,6‐Dimethoxy‐7,9‐dimethylpurinium iodide hemihydrate

In the title compound, C₉H₁₃N₄O₂⁺·I⁻·0.5H₂O, the non‐H atoms of the ionic components lie on a mirror plane in Cmca, with the O atom of the partial water molecule lying on a twofold rotation axis. Whereas one of the methoxy methyl groups is directed away from the adjacent N‐methyl group, the other methoxy methyl group is directed towards its adjacent N‐methyl group. The conformation of the methoxy methyl groups provides an explanation for the outcomes of intramolecular thermal rearrangements of 2,6‐dialkoxy‐7,9‐dimethylpurinium salts.     

Carbon-13 NMR spectra of saturated heterocycles: XI—Tetrahydropyrans (oxanes)

The ¹³C NMR spectra of 62 oxanes (tetrahydropyrans) with and without methyl substituents at various ring positions, some of them bearing in addition (or instead) ethyl, vinyl, ethynyl, carbomethoxy and methylol substituents at C-2, have been recorded, and the 294 resulting chemical shifts have been correlated by multiple linear regression analysis. Axial and equatorial α-, β-, γ-, δ-, gem- and vic-parameters for shifts caused by methyl groups at all ring positions, and similar parameters for Et,—CH?CH₂,—C?CH, CO₂Me and CH₂OH groups at C-2, are reported. Standard deviations of the parameters are, in most cases, within 0.3 ppm and the agreement of calculated and experimental shifts is excellent. This is probably the largest parameter set of this type extant. ¹³C NMR spectra of a number of additional substituted tetrahydropyrans, and of 3,6-dihydro-2H-pyrans and 3,4-dihydro-2H-pyrans, are tabulated and discussed.     

NMR Studies of Host–guest Complexes Between Monocarboxylic Acids and Amide-based Cyclophanes in Chloroform

Formation of host–guest complexes with acetic acid and benzoic acid was studied by NMR for amide-based octaazacyclophanes having pendant methyl ester arms; the cyclophanes were tetramethyl 2,9,18,25-tetraoxo-1,4,7,10,17,20,23,26-octaaza[10.10]paracyclophane-4,7,20,23-tetraacetate, its meta-isomer and analogues. Amide NH proton and CH₂ proton adjacent to amide C = O in every cyclophane host showed down-field NMR shifts in the presence of the guest acids in CHCl₃-d, suggesting the formation of 1:1 complexes in which the carboxyl group of an acid molecule formed two hydrogen bonds with the amide NH and C = O moieties of a host molecule. Since the complex formation competed with the dimerization of the guest acids, the monomer–dimer equilibrium was restudied by NMR and the equilibrium constant was determined to be 330 M^? 1 for acetic acid and 518 M^? 1 for benzoic acid. By using these values, the formation constants of the host–guest complexes were determined to be 8–51 M^? 1. The close contact between the host and guest molecules via hydrogen bonding was consistently confirmed by NMR shifts due to the ring current of aromatic group.     

Long range 13C shift effects (δ and ϵ) in methylcyclohexanes

¹³C n.m.r. spectra of a number of methyl substituted cyclohexanes, some of them conformationally homogeneous, have been recorded in CDCl₃ and used to determine shift effects engendered by the introduction of methyl groups on carbon atoms remote from the site of substitution. Sizeable changes in shifts are found, including a substantial effect of an equatorial methyl group on an axial methyl group δ to it (+0.6₇ ppm, ‘δ_ea’). The effects reported are of consequence in investigations of conformational problems by ¹³C n.m.r. techniques.     

Prototropic processes in benzaurins. <Superscript>19</Superscript>F and <Superscript>1</Superscript>H NMR spectra of fluoro- and methylsubstituted 4-hydroxyphenyl-diphenylcarbinols,related fuchsones and benzaurins

Tautomerism of benzaurins and hydration are studied. ¹H and ¹⁹F chemical shifts have been determined for a number of substituted 4-hydroxyphenyl-diphenyl carbinols containing fluorine in a 3-, 3*- or 4*-position, and for similar compounds containing additional methyl groups in a position of 3, 3** or 4**. The same data have been obtained for the fuchsones prepared by dehydration of the above carbinols. On this basis chemical shifts of fluorine in different positions have been evaluated as a monitor of the transformation of 4-hydroxyphenyl group to the semiquinone moiety. The ¹⁹F NMR can be used to monitor the transformation of 4**-fluorobenzaurin and the related 3,3*-disubstituted and 3,3*,5,5*-tetramethylsubstituted compounds to the corresponding carbinols due to the addition of a water molecule and to study the tautomerism of the two latter benzaurins as well as that of 3,3*,4**trifluorobenzaurin. Furthermore, fluorine and methyl group chemical shifts are sensitive to syn-anti-isomerism in substituted fuchsones.     

1H NMR spectra. Part 29§: proton chemical shifts and couplings in esters—the conformational analysis of methyl γ‐butyrolactones

The ¹H NMR spectra of 35 cyclic and acyclic esters are analysed to give the ¹H chemical shifts and couplings. The substituent chemical shifts of the ester group were analysed using three‐bond (γ) effects for near protons and the electric field, magnetic anisotropy and steric effect of the ester group for more distant protons. The electric field is calculated from the partial atomic charges on the O?C = O atoms, and the asymmetric magnetic anisotropy of the carbonyl group acts at the midpoint of the C = O bond. The values of the anisotropies Δχ_parl and Δχ_perp were for the aliphatic esters 10.35 and ?18.84 and for the conjugated esters 7.33 and ?15.75 (×10^?6 ų/molecule). The oxygen steric coefficients found were 104.4 (aliphatic C = O), 45.5 (aromatic C = O) and 16.0 (C–O) (×10^?6 Å⁶/molecule). After parameterisation, the overall RMS error for the data set of 280 entries was 0.079 ppm. The strongly coupled ¹H NMR spectra of the 2‐methyl, 3‐methyl and 4‐methyl γ‐butyrolactones were analysed and the methyl conformational equilibrium obtained from the observed couplings. The observed versus calculated density functional theory (DFT) ΔG(ax‐eq) was 1.0 (1.01), 0.34 (0.54) and 0.65 (0.71) kcal/mol res. The shielding effect of a methyl cis to a proton in the five‐membered lactone rings is ?0.40 ±0.05 ppm and deshielding trans effect 0.12 ±0.05 ppm, which is common to both five and six membered rings. The cis/trans isomerism in the vinyl esters methyl acrylate, crotonate and methacrylate and methyl furoate was examined using the ¹H chemical shifts. The calculated shifts of both the cis and trans isomers were in good agreement with the observed shifts. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of the preferred tautomeric form of 4-nitrohistidine

Spectrophotometric pKa determinations of methyl derivatives of N^α-acetyl-4-nitrohistidine methyl ester 1 have been used to determine the position of the tautomeric equilibrium of 1 . The N₁-H tautomer is the predominant form with an equilibrium constant K_T of 48. The conclusion is supported qualitatively by the study of ¹H-nmr and ¹³C-nmr chemical shifts of the imidazole ring atoms and their changes from neutral to acidic media.     

Nitrogen-15 magnetic resonance spectroscopy XVI—natural-abundance spectra of aromatic amines

The ¹⁵N chemical shifts of aniline, the toluidines, xylidines, and several halogen and oxygen substituted anilines have been measured at the natural abundance level of ¹⁵N. Substituent parameters obtained by multiple regression analysis show that the methyl group induces comparable upfield shifts at the ortho and para positions (2·37 and 2·55 ppm/methyl, respectively) and a small (0·77 ppm/methyl) upfield shift at the meta position. The chemical shifts correlate reasonably well with ¹⁹F shifts of similarly substituted fluorobenzenes, with C-1 of the anilines themselves and with Hammett sigma values. While the shifts of C-methyl substituted anilines do not correlate with the methyl resonances of corresponding polymethylbenzenes, those of the halo- and alkoxyanilines show a reasonable parallelism with corresponding ¹³C-methyl shifts. The results are interpreted in terms of possible modes of transmission of electron density in an alternating and additive manner through the sigma framework.     

13C and 15N spin–lattice relaxation and chemical shift studies of pentane-2,4-diamine

¹³C and ¹⁵N NMR chemical shift and spin–lattice relaxation data have been measured for both meso- and racemic-pentane-2,4-diamine. At high pH (12), relaxation is consistent with hindered rotation of the NH₂ group due, in part, to the formation of intramolecular hydrogen bonds. At low pH (2), relaxation is consistent with relatively unhindered rotation of the NH₃⁺ group. Rotational jump rates and barriers are reported, determined from the NT₁ ratios between ¹⁵N and ¹³C nuclei. In all cases, the ratios for the racemic diastereomer are higher than those of the meso compounds; this is interpreted in terms of conformationally more stable intramolecular hydrogen bond formation in the meso compound. Chemical shifts for the diastereomeric amines show that ¹⁵N shifts move downfield on protonation along with methyl and methylene carbons, while the methine carbon resonances move upfield.     

Conformational equilibrium in 8-methyl-cis-2-thiahydrindane and 8-methyl-cis-2-oxahydrindane by 13C NMR spectroscopy

The preferred conformation of 8-methyl-cis-thiahydrindane has been both estimated by ¹³C NMR chemical shifts and determined by low temperature ¹³C NMR spectroscopy to be the conformer with the methyl group equatorial with respect to the cyclohexane ring. This result is in disagreement with the interpretation of the temperature dependence of the CD spectra of (+) and (?) 8-methyl-cis-2-thiahydrindane, whereby the conformation with the methyl group axial with respect to the cyclohexane ring was claimed to be the preferred conformation. The preferred conformation of the related oxygen heterocycle, 8-methyl-cis-2-oxahydrindane, has been estimated by ¹³C NMR chemical shifts to be the conformer with the methyl group axial with respect to the cyclohexane ring. Possible reasons for these observations are discussed.     

Carbon-13 spin–lattice relaxation in strongly associated molecules: Carboxylic acids

¹³C spin–lattice relaxation times determined for the protonated carbons of carboxylic acids and methyl esters give indications of solution dimerization with the free acids. Since isopthalic and fumaric acids have two carboxyl functions they are able to polymerize in solution. Unlike the case for molecular aggregation due to weak hydrogen bonding in solution (e.g. alcohols, phenols), the ¹³C T₁ values of mono carboxylic acids are not significantly affected by dilution to c. 10^?2 M. Variable temperature T₁ measurements of both the mono and dibasic acids gave activation energies for molecular reorientation of the order of 2 kcal mol^?1, considerably lower than E_a for hydrogen bonded alcohols and comparable with E_a for the unassociated methyl esters of propionic and benzoic acids.     

Substituent chemical shift correlations. Hammett σp+ values and shifts for exocyclic protons in para-substituted benzene derivatives

The methyl ¹H NMR shifts for series of para-substituted N,N-dimethylanilines as their conjugate acids in trifluoroacetic acid, and series of para-substituted N,N,N-trimethylphenyl-ammonium iodides in acetonitrile and in deuterium oxide, and the methylene shifts for series of para-substituted N,N-diethylanilines as their conjugate acids in deuteriosulfuric acid, are shown to be linearly related to the Hammett σ_p+ parameter. It is proposed that this dependence reflects a response of the chemical shift of the proton of the probe moiety to the electron density at the point of attachment of the probe to the aromatic ring and that this response is determined by the electric field effect of the charge at the point of attachment. Literature data are cited to indicate that Hammett σ_p+–¹H NMR shift relationships may be general for probe moieties lacking a through-resonance mechanism for interaction between the probe and the aromatic ring.     

Conformational study of methyl esters of some aliphatic erythro- and threo-dichlorocarboxylic acids

The average conformations of methyl esters of some aliphatic erythro- and threo-dichlorocarboxylic acids in dilute carbon tetrachloride solutions have been determined from the vicinal proton–proton coupling constants and ¹H NMR shifts. The ¹³C shift differences between the erythro and threo forms are compared and discussed with regard to the differences in the average conformations.     

Saturated amine oxides: part 10. hydroacridines: part 32. linear 17O/13C and 13C/13C chemical shift correlations between saturated azaheterocyclic N‐methylamine N‐oxides,and the methiodides of their parent amines

Two kinds of good linear correlations were found between the chemical shifts of saturated six‐membered azaheterocyclic N‐methylamine N‐oxides and the chemical shifts of the methiodides of their parent amines. One of the correlations occurs between the ¹⁷O chemical shift of the N⁺―O^– oxygen in the N‐oxides and the ¹³C chemical shift of the N⁺―CH₃ methyl group analogously situated in the appropriate methiodide (r = 0.9778). This correlation enables unambiguous configuration assignment of the N⁺―O^– bond, even if the experimentally observed ¹⁷O chemical shift of only one N‐epimer is available, provided the ¹³C chemical shifts of both N⁺―CH₃ groups in the methiodide are known and assigned; furthermore, it can be used also for the estimation of ¹⁷O chemical shifts of the N⁺―O^– oxygens in N‐epimeric pairs of N‐oxides, for which observed ¹⁷O data hardly become available. The second correlation is observed between the ¹³C chemical shift of the N⁺―CH₃ methyl group in the N‐oxides and the ¹³C chemical shift of the N⁺―CH₃ methyl group analogously situated in the appropriate methiodide (r = 0.9785). It can be used for safe configuration assignment of the N⁺―CH₃ group and, indirectly, also of the N⁺―O^– bond in an amine N‐oxide, even if no ¹⁷O NMR data, and the ¹³C chemical shift of only one N‐epimer is available. Copyright © 2015 John Wiley & Sons, Ltd.     

Carbon-13 NMR. The analysis of the relaxation times T1 of benzofuran and of a series of its methyl derivatives. Correlations between molecular motions and structural properties

Carbon-13 relaxation times (T₁) and nuclear Overhauser enhancements (η) have been measured for benzofuran and a series of its methyl derivatives. The contributions of dipolar (T₁ ^DD) and spin rotation (T₁^SR) mechanisms have both been determined. The temperature dependence of T₁ has been studied. The relationships between molecular motions and structural properties have been emphasized. The overall motional anisotropy of the benzofuran molecule is increased by substitution in positions 2 and 5. The internal rotation of a methyl group may change depending on its position in the molecule and on the influence of other methyl groups in its close neighbourhood.     

Fluoroazoles. III. Synthesis and 1H and 1–9F nmr spectra of 3-, 4-, and 5-fluoro-1-methylpyrazole

The three monofluoro derivatives of N-methylpyrazole have been synthesized. 3-Fluoro-1-methylpyrazole and 4-fIuoro-1-methylpyrazole were prepared from the appropriate amines by diazotization and photochemical irradiation of the diazonium salts in tetrafluoroboric acid. 5-Fluoro-1-methylpyrazole was obtained from 1-acetyl-3-fluoropyrazole and methyl fluorosulfonate, and also by direct methylation of 3(5) fluoropyrazole with dimethyl sulfate. The ^1–9F chemical shifts of these N-methylated fluoroazoles cover a great range (ca. 50 ppm) and show a good correlation with the chemical shifts of H₃, H₄, and H₅ protons of 1-methylpyrazole. An unexpected long-range coupling ⁵J (F-CH₃) is observed in 3-fluoro-1-methylpyrazole.