Four‐bond deuterium isotope effects on the chemical shifts of amide nitrogens in proteins

An approach towards precision NMR measurements of four‐bond deuterium isotope effects on the chemical shifts of backbone amide nitrogen nuclei in proteins is described. Three types of four‐bond ¹⁵ N deuterium isotope effects are distinguished depending on the site of proton‐to‐deuterium substitution: ⁴ΔN(N_i‐1D), ⁴ΔN(N_i+1D) and ⁴ΔN(C_β,i‐1D). All the three types of isotope shifts are quantified in the (partially) deuterated protein ubiquitin. The ⁴ΔN(N_i+1D) and ⁴ΔN(C_β,i‐1D) effects are by far the largest in magnitude and vary between 16 and 75 ppb and ?18 and 46 ppb, respectively. A semi‐quantitative correlation between experimental ⁴ΔN(N_i+1D) and ⁴ΔN(C_β,i‐1D) values and the distances between nitrogen nuclei and the sites of ¹H‐to‐D substitution is noted. The largest isotope shifts in both cases correspond to the shortest inter‐nuclear distances. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of the enrichment of isotopically labelled molecules by mass spectrometry

A general method for the determination of the enrichment of isotopically labelled molecules by mass spectrometry (MS) is described. In contrast to other published procedures, the method described here takes into account and corrects for measurement errors such as the contribution at M ? 1 due to loss of hydrogen or lack of spectral resolution and provides an uncertainty value for the determined enrichment. The general procedure requires the following steps: (1) evaluation of linearity in the mass spectrometer by injecting the natural abundance compound at different concentration levels, (2) determination of the purity of the mass cluster using the natural abundance analogue, (3) calculation of the theoretical isotope composition of the labelled compound using different tentative isotope enrichments, (4) calculation of ‘convoluted’ isotope distributions for the labelled compound taking into account the purity of the mass cluster determined with the natural abundance analogue and (5) comparison of the isotope distributions measured for the labelled compound with those calculated for different isotope enrichments using linear regression. The method was applied to a series of commercially available ¹³C‐ and ²H‐labelled compounds and to a suite of singly ¹³C‐labelled β₂‐agonist prepared in‐house both by gas chromatography (GC)–MS, GC–tandem MS (MS/MS) and liquid chromatography–MS/MS with satisfactory results. It was observed that the main uncertainty source for the isotope enrichment was the uncertainty in the purity of the measured cluster as determined with the natural abundance compound. Copyright © 2014 John Wiley & Sons, Ltd.     

1H‐, 13C‐, and 15N‐NMR chemical shifts for selected glucosides and ribosides of aromatic cytokinins

The ¹H and ¹³C NMR resonances of 16 purine glucosides were assigned by a combination of one‐ and two‐dimensional NMR experiments, including gs‐COSY, gs‐HSQC, and gs‐HMBC, in order to characterize the effect of substituent and the position of glucose unit on the NMR chemical shifts. In addition, ¹⁵N NMR chemical shifts for selected derivatives were investigated by using ¹H? ¹⁵N chemical shift correlation techniques. To map the influence of sugar moiety on the directly bonded nitrogen atom, selected N⁹‐glucosides and their ribose analogs were compared. Characteristic long‐range ¹H? ¹⁵N coupling constants, measured by using ¹H? ¹⁵N gradient‐selected single‐quantum multiple bond correlation (GSQMBC), are also reported and discussed. All compounds investigated here belong to cytokinins, an important group of plant hormones. Copyright © 2010 John Wiley & Sons, Ltd.     

Disentangling Complex Mixtures of Compounds with Near‐Identical 1H and 13C NMR Spectra using Pure Shift NMR Spectroscopy

The thorough analysis of highly complex NMR spectra using pure shift NMR experiments is described. The enhanced spectral resolution obtained from modern 2D HOBS experiments incorporating spectral aliasing in the ¹³C indirect dimension enables the distinction of similar compounds exhibiting near‐identical ¹H and ¹³C NMR spectra. It is shown that a complete set of extremely small Δδ(¹H) and Δδ(¹³C) values, even below the natural line width (1 and 5 ppb, respectively), can be simultaneously determined and assigned.     

Deuterium isotope effects on 13C chemical shifts of negatively charged NH…N systems

Deuterium isotope effects on ¹³C chemical shifts are investigated in anions of 1,8‐bis(4‐toluenesulphonamido)naphthalenes together with N,N‐(naphthalene‐1,8‐diyl)bis(2,2,2‐trifluoracetamide) all with bis(1,8‐dimethylamino)napthaleneH⁺ as counter ion. These compounds represent both “static” and equilibrium cases. NMR assignments of the former have been revised. The NH proton is deuteriated. The isotope effects on ¹³C chemical shifts are rather unusual in these strongly hydrogen bonded systems between a NH and a negatively charged nitrogen atom. The formal four‐bond effects are found to be negative indicating transmission via the hydrogen bond. In addition, unusual long range effects are seen. Structures, ¹H and ¹³C NMR chemical shifts and changes in nuclear shieldings upon deuteriation are calculated using density functional theory methods. Copyright © 2013 John Wiley & Sons, Ltd.     

Deuterium Quantification through Deuterium‐Induced Remote 1H and 13C NMR Shifts

Partial labeling by deuterium may be quantified through simple integrations of those ¹H (200 or 400 MHz ) and ¹³C (100.6 MHz ) NMR resonances that are split into pairs by chemical shifts ⁿΔ=δ(deuterated)?δ(nondeuterated) as induced by deuterium across n>2 chemical bonds. The relative intensities of the two components of a pair are shown to be influenced to practically equal degrees by relaxation effects, so that a deuterium fraction may be determined from ¹H and ¹³C integral pairs at more remote molecular positions under the routine conditions of fast accumulative spectral acquisition.     

NMR analysis of mono‐ and difructosyllactosucrose synthesized by 1F‐fructosyltransferase purified from roots of asparagus (Asparagus officinalis L.)

Two novel oligosaccharides, mono‐ and difructosyllactosucrose {[O‐β‐D ‐fructofuranosyl‐(2 → 1)]_n‐β‐D ‐fructofuranosyl‐O‐[β‐D ‐galactopyranosyl‐(1 → 4)]‐α‐D ‐glucopyranoside, n = 1 and 2} were synthesized using 1^F‐fructosyltransferase purified form roots of asparagus (Asparagus officinalis L.). Their ¹H and ¹³C NMR spectra were assigned using several NMR techniques. The spectral analysis was started from two anomeric methines of aldose units, galactose and glucose, since they showed separate characteristic signals in their ¹H and ¹³C NMR spectra. After assignments of all the ¹H and ¹³C signals of two units of aldose, they were discriminated as galactose and glucose using proton–proton coupling constants. The HMBC spectrum revealed the galactose residue attached to C‐4 of glucose, fructose residue attached to the C‐1 of glucose, and further fructosyl fructose linkage extended from the glucosyl fructose residues. Copyright © 2002 John Wiley & Sons, Ltd.     

Aromatic 19F–13C TROSY—[19F, 13C]‐Pyrimidine Labeling for NMR Spectroscopy of RNA

We present the access to [5‐¹⁹F, 5‐¹³C]‐uridine and ‐cytidine phosphoramidites for the production of site‐specifically modified RNAs up to 65 nucleotides (nts). The amidites were used to introduce [5‐¹⁹F, 5‐¹³C]‐pyrimidine labels into five RNAs—the 30 nt human immunodeficiency virus trans activation response (HIV TAR) 2 RNA, the 61 nt human hepatitis B virus ? (hHBV ?) RNA, the 49 nt SAM VI riboswitch aptamer domain from B. angulatum, the 29 nt apical stem loop of the pre‐microRNA (miRNA) 21 and the 59 nt full length pre‐miRNA 21. The main stimulus to introduce the aromatic ¹⁹F–¹³C‐spin topology into RNA comes from a work of Boeszoermenyi et al., in which the dipole‐dipole interaction and the chemical shift anisotropy relaxation mechanisms cancel each other leading to advantageous TROSY properties shown for aromatic protein sidechains. This aromatic ¹³C–¹⁹F labeling scheme is now transferred to RNA. We provide a protocol for the resonance assignment by solid phase synthesis based on diluted [5‐¹⁹F, 5‐¹³C]/[5‐¹⁹F] pyrimidine labeling. For the 61 nt hHBV ? we find a beneficial ¹⁹F–¹³C TROSY enhancement, which should be even more pronounced in larger RNAs and will facilitate the NMR studies of larger RNAs. The [¹⁹F, ¹³C]‐labeling of the SAM VI aptamer domain and the pre‐miRNA 21 further opens the possibility to use the biorthogonal stable isotope reporter nuclei in in vivo NMR to observe ligand binding and microRNA processing in a biological relevant setting.     

Knight Shift in 13C NMR Resonances Confirms the Coordination of N‐Heterocyclic Carbene Ligands to Water‐Soluble Palladium Nanoparticles

The coordination of N‐heterocyclic carbene (NHC) ligands to the surface of 3.7 nm palladium nanoparticles (PdNPs) can be unambiguously established by observation of Knight shift (KS) in the ¹³C resonance of the carbenic carbon. In order to validate this coordination, PdNPs with sizes ranging from 1.3 to 4.8 nm were prepared by thermal decomposition or reduction with CO of a dimethyl NHC Pd^II complex. NMR studies after ¹³CO adsorption established that the KS shifts the ¹³C resonances of the chemisorbed molecules several hundreds of ppm to high frequencies only when the particle exceeds a critical size of around 2 nm. Finally, the resonance of a carbenic carbon is reported to be Knight‐shifted to 600 ppm for ¹³C‐labelled NHCs bound to PdNPs of 3.7 nm. The observation of these very broad KS resonances was facilitated by using Car–Purcell–Meiboom–Gill (CPMG) echo train acquisition NMR experiments.     

Linear Free‐Energy Relationships Applied to the 13C NMR Chemical Shifts in 4‐Substituted N‐[1‐(Pyridine‐3‐ and ‐4‐yl)ethylidene]anilines

Two series of 4‐substituted N‐[1‐(pyridine‐3‐ and ‐4‐yl)ethylidene]anilines have been synthesized using different methods of conventional and microwave‐assisted synthesis, and linear free‐energy relationships have been applied to the ¹³C NMR chemical shifts of the carbon atoms of interest. The substituent‐induced chemical shifts have been analyzed using single substituent parameter and dual substituent parameter methods. The presented correlations describe satisfactorily the field and resonance substituent effects having similar contributions for C1 and the azomethine carbon, with exception of the carbon atom in para position to the substituent X. In both series, negative ρ values have been found for C1′ atom (reverse substituent effect). Quantum chemical calculations of the optimized geometries at MP2/6‐31G++(d,p) level, together with ¹³C NMR chemical shifts, give a better insight into the influence of the molecular conformation on the transmission of electronic substituent effects. The comparison of correlation results for different series of imines with phenyl, 4‐nitrophenyl, 2‐pyridyl, 3‐pyridyl, 4‐pyridyl group attached at the azomethine carbon with the results for 4‐substituted N‐[1‐(pyridine‐3‐ and ‐4‐yl)ethylidene]anilines for the same substituent set (X) indicates that a combination of the influences of electronic effects of the substituent X and the π₁‐unit can be described as a sensitive balance of different resonance structures.     

Solid‐state 11B and 13C NMR,IR, and X‐ray crystallographic characterization of selected arylboronic acids and their catechol cyclic esters

Nine arylboronic acids, seven arylboronic catechol cyclic esters, and two trimeric arylboronic anhydrides (boroxines) are investigated using ¹¹B solid‐state NMR spectroscopy at three different magnetic field strengths (9.4, 11.7, and 21.1 T). Through the analysis of spectra of static and magic‐angle spinning samples, the ¹¹B electric field gradient and chemical shift tensors are determined. The effects of relaxation anisotropy and nutation field strength on the ¹¹B NMR line shapes are investigated. Infrared spectroscopy was also used to help identify peaks in the NMR spectra as being due to the anhydride form in some of the arylboronic acid samples. Seven new X‐ray crystallographic structures are reported. Calculations of the ¹¹B NMR parameters are performed using cluster model and periodic gauge‐including projector‐augmented wave (GIPAW) density functional theory (DFT) approaches, and the results are compared with the experimental values. Carbon‐13 solid‐state NMR experiments and spectral simulations are applied to determine the chemical shifts of the ipso carbons of the samples. One bond indirect ¹³C‐¹¹B spin‐spin (J) coupling constants are also measured experimentally and compared with calculated values. The ¹¹B/¹⁰B isotope effect on the ¹³C chemical shift of the ipso carbons of arylboronic acids and their catechol esters, as well as residual dipolar coupling, is discussed. Overall, this combined X‐ray, NMR, IR, and computational study provides valuable new insights into the relationship between NMR parameters and the structure of boronic acids and esters. Copyright © 2012 John Wiley & Sons, Ltd.     

NMR,MP2, and DFT study of thiophenoxyketenimines (o‐thio‐Schiff bases): Determination of the preferred form

Five new thiophenoxyketinimines have been synthesized. ¹H and ¹³C NMR spectra as well as deuterium isotope effects on ¹³C chemical shifts are determined, and spectra are assigned. DFT and MP2 calculations of both structures, chemical shifts, and isotope effects on chemical shifts are done. The combined analysis reveals that the compounds are primarily on a zwitterionic form with an NH⁺ and a S⁻ group and with a little of the neutral form mixed in. Very strong intramolecular hydrogen bonding is found and very high NH chemical shifts are observed. The theoretical calculations show that calculations at the MP2 level are best to obtain correct “C═S” chemical shifts.     

Combined 1H, 13C and 11B NMR and mass spectral assignments of boronate complexes of D‐(+)‐glucose,D‐(+)‐mannose,methyl‐α‐D‐glucopyranoside,methyl‐β‐D‐galactopyranoside and methyl‐α‐D‐mannopyranoside

Complex formation between N‐butylboronic acid and D ‐(+)‐glucose, D ‐(+)‐mannose, methyl‐α‐D ‐glucopyranoside, methyl‐β‐D ‐galactopyranoside and methyl α‐D ‐mannopyranoside under neutral conditions was investigated by ¹H, ¹³C and ¹¹B NMR spectroscopy and gas chromatography–mass spectrometry (GC–MS) D ‐(+)‐Glucose and D ‐(+)‐mannose formed complexes where the boronates are attached to the 1,2:4,6‐ and 2,3:5,6‐positions of the furanose forms, respectively. On the other hand, the boronic acid binds to the 4,6‐positions of the two methyl derivatives of glucose and galactose. Methyl α‐D ‐mannopyranoside binds two boronates at the 2,3:4,6‐positions. ¹¹B NMR was used to show the ring size of the complexed sugars and the boronate. GC–MS confirmed the assignments. Copyright © 2003 John Wiley & Sons, Ltd.     

Efficient Hyperpolarization of U‐13C‐Glucose Using Narrow‐Line UV‐Generated Labile Free Radicals

Free radicals generated by UV‐light irradiation of a frozen solution containing a fraction of pyruvic acid (PA) have demonstrated their dissolution dynamic nuclear polarization (dDNP) potential, providing up to 30 % [1‐¹³C]PA liquid‐state polarization. Moreover, their labile nature has proven to pave a way to nuclear polarization storage and transport. Herein, differently from the case of PA, the issue of providing dDNP UV‐radical precursors (trimethylpyruvic acid and its methyl‐deuterated form) not involved in any metabolic pathway was investigated. The ¹³C dDNP performance was evaluated for hyperpolarization of [U‐¹³C₆,1,2,3,4,5,6,6‐d₇]‐d ‐glucose. The generated UV‐radicals proved to be versatile and highly efficient polarizing agents, providing, after dissolution and transfer (10 s), a ¹³C liquid‐state polarization of up to 32 %.     

1H and 13C NMR and X‐ray diffraction data for a diosgenyl N,O‐protected glucopyranoside

The assignments of ¹H and ¹³C NMR chemical shifts together with x‐ray diffraction data for synthesized diosgenyl 3,4,6‐tri‐O‐acetyl‐2‐deoxy‐2‐tetrachlorophthalimido‐β‐D ‐glucopyranoside are described. The structure of this glycoside was established by using homo‐ and heteronuclear two‐dimensional NMR techniques. X‐ray diffraction data for this compound are also reported. Copyright © 2002 John Wiley & Sons, Ltd.     

A Simple and Efficient Procedure for Synthesis of Optically Active 1,2,4‐Triazolo‐[3,4‐b]‐1,3,4‐Thiadiazole Derivatives Containing l‐Amino Acid Moieties

Some new and optically active 1,2,4‐triazolo thiadiazoles bearing N‐phthaloyl‐l ‐amino acids were synthesized by reaction of 4‐amino‐5‐(3‐ or 4‐)pyridyl‐3‐mercapto‐(4H)‐1,2,4‐triazoles with N‐phthaloyl‐l ‐amino acids in the presence of phosphorus oxychloride. All the newly synthesized compounds were confirmed by IR, ¹H NMR, ¹³C NMR and elemental analysis.     

Synthesis,NMR spectroscopic characterization and structure of a divinyldisilazane‐(triphenylphosphine)platinum(0) complex: observation of isotope‐induced chemical shifts 1Δ12/13C(195Pt)

Tetramethyldivinyldisilazane‐(triphenylphosphine)platinum(0) was prepared, characterized in solid state by X‐ray crystallography and in solution by multinuclear magnetic resonance spectroscopy (¹H, ¹³C, ¹⁵N, ²⁹Si, ³¹P and ¹⁹⁵Pt NMR). Numerous signs of spin–spin coupling constants were determined by two‐dimensional heteronuclear shift correlations (HETCOR) and two‐dimensional ¹H/¹H COSY experiments. Isotope‐induced chemical shifts ¹Δ^12/13C(¹⁹⁵Pt) were measured from ¹⁹⁵Pt NMR spectra of the title compound as well as of other Pt(0), Pt(II) and Pt(IV) compounds for comparison. In contrast to other heavy nuclei such as ¹⁹⁹Hg or ²⁰⁷Pb, the “normal” shifts of the heavy isotopomers to low frequencies are found, covering a range of >500 ppb. Copyright © 2013 John Wiley & Sons, Ltd.     

Substituent effects on 13C?13C coupling constants and chemical shifts in 13C labelled polycyclic aromatic hydroxy compounds

The effects of an hydroxy substituent on ¹³C? ¹³C coupling constants and ¹³C chemical shifts have been measured in 1-hydroxynaphthalene-2-¹³C and 1-hydroxypyrene-1-¹³C. The changes observed in the ¹³C? ¹³C couplings show the effect of a substituent attached directly to the labelled carbon or to a carbon ortho to this. In both situations the effect is a decrease in the numerical magnitude of most of the long range ¹³C? ¹³C coupling constants.     

Conformational and long-range low field steric deuterium isotope effects on carbon chemical shifts

Comparison of the room and low temperature (203 K) ¹³C NMR spectra of 3-cis-bicyclo[4.4.0]decanone and its 2,2,4,4-tetradeuterio isotopomer provides information about the deuterium isotope effect on the conformational equilibrium in this compound. Four-bond low field deuterium isotope effects on some carbon chemical shifts are observed. These effects can be used for unambiguous assignment of the ¹³C lines to carbon atoms in alicyclic compounds.