15N Coupling Research Articles

ENDOR Spectroscopy Reveals the "Free" 5'-Deoxyadenosyl Radical in a Radical SAM Enzyme Active Site Actually is Chaperoned by Close Interaction with the Methionine-Bound [4Fe-4S]2+ Cluster.

1/2H and 13C hyperfine coupling constants to 5'-deoxyadenosyl (5'-dAdo•) radical trapped within the active site of the radical S-adenosyl-l-methionine (SAM) enzyme, pyruvate formate lyase-activating enzyme (PFL-AE), both in the absence of substrate and the presence of a reactive peptide-model of the PFL substrate, are completely characteristic of a classical organic free radical whose unpaired electron is localized in the 2pπ orbital of the sp2 C5'-carbon (J. Am. Chem. Soc. 2019, 141, 12139-12146). However, prior electron-nuclear double resonance (ENDOR) measurements had indicated that this 5'-dAdo• free radical is never truly "free": tight van der Waals contact with its target partners and active-site residues guide it in carrying out the exquisitely precise, regioselective reactions that are hallmarks of RS enzymes. Here, our understanding of how the active site chaperones 5'-dAdo• is extended through the finding that this apparently unexceptional organic free radical has an anomalous g-tensor and exhibits significant 57Fe, 13C, 15N, and 2H hyperfine couplings to the adjacent, isotopically labeled, methionine-bound [4Fe-4S]2+ cluster cogenerated with 5'-dAdo• during homolytic cleavage of cluster-bound SAM. The origin of the 57Fe couplings through nonbonded radical-cluster contact is illuminated by a formal exchange-coupling model and broken symmetry-density functional theory computations. Incorporation of ENDOR-derived distances from C5'(dAdo•) to labeled-methionine as structural constraints yields a model for active-site positioning of 5'-dAdo• with a short, nonbonded C5'-Fe distance (∼3 Å). This distance involves substantial motion of 5'-dAdo• toward the unique Fe of the [4Fe-4S]2+ cluster upon S-C(5') bond-cleavage, plausibly an initial step toward formation of the Fe-C5' bond of the organometallic complex, Ω, the central intermediate in catalysis by radical-SAM enzymes.

15N labeling and analysis of 13C-15N and 1H-15N couplings in studies of the structures and chemical transformations of nitrogen heterocycles.

This review provides a generalization of effective examples of 15N labeling followed by an analysis of 13C–15N (JCN) and 1H–15N (JHN) coupling constants in solution as a tool to study the structural aspects and pathways of chemical transformations (e.g., rearrangements and ring-chain tautomerisms) in monocyclic and fused nitrogen heterocycles. This approach allows us to significantly expand and supplement the scope of NMR techniques for heterocyclic compounds. Moreover, methods for the incorporation of 15N atoms into the cores of various N-heterocycles have been collected in this work.

Impact of two-bond 15N-15N scalar couplings on 15N transverse relaxation measurements for arginine side chains of proteins.

NMR relaxation of arginine (Arg) 15Nε nuclei is useful for studying side-chain dynamics of proteins. In this work, we studied the impact of two geminal 15N-15N scalar couplings on measurements of transverse relaxation rates (R 2 ) for Arg side-chain 15Nε nuclei. For 12 Arg side chains of the DNA-binding domain of the Antp protein, we measured the geminal 15N-15N couplings ( 2 J NN ) of the 15Nε nuclei and found that the magnitudes of the 2 J NN coupling constants were virtually uniform with an average of 1.2Hz. Our simulations, assuming ideal 180° rotations for all 15N nuclei, suggested that the two 2 J NN couplings of this magnitude could in principle cause significant modulation in signal intensities during the Carr-Purcell-Meiboom-Gill (CPMG) scheme for Arg 15Nε R 2 measurements. However, our experimental data show that the expected modulation via two 2 J NN couplings vanishes during the 15N CPMG scheme. This quenching of J modulation can be explained by the mechanism described in Dittmer and Bodenhausen (Chemphyschem 7:831-836, 2006). This effect allows for accurate measurements of R 2 relaxation rates for Arg side-chain 15Nε nuclei despite the presence of two 2 J NN couplings. Although the so-called recoupling conditions may cause overestimate of R 2 rates for very mobile Arg side chains, such conditions can readily be avoided through appropriate experimental settings.

Solid-State NMR Spectroscopy and Isotopic Labeling Target Abundant Dipeptide Sequences in Elastin’s Hydrophobic Domains

Isotopic labeling strategies are coupled with solid-state NMR spectroscopy to characterize elastin’s abundant glycines (Gly) and prolines (Pro) and the sequences that include these residues. Elastin is prepared with isotopic labels at Gly, Pro, and Val—three of its four most abundant amino acids. Solid-state 15N–1H nuclear magnetic resonance (NMR) spectra show four resolved glycine populations, whereas three features are observed for the prolines. Selection of the Val-Pro and Gly-Gly pairs found exclusively and abundantly in the hydrophobic domains confirms these assignments and also provides more information on the conformational ensembles of elastin. The 1HN and 15N temperature coefficients indicate that discrete secondary structures are present, even among significant populations with rapid conformational fluctuations typical of a random coil. 13C–15N couplings and the 13C chemical shift anisotropy (CSA) are also utilized to confirm assignments and structure, respectively. Implications for the evolving conformational ensemble model for elastin are also discussed.

Mixed Aggregates of the Dilithiated Koga Tetraamine: NMR Spectroscopic and Computational Studies

AbstractA combination of 1H, 6Li, 13C, and 15N NMR spectroscopies and density functional theory computations explores the formation of mixed aggregates by a dilithium salt of a C2‐symmetric chiral tetraamine (Koga's base). Lithium halides, acetylides, alkoxides, and monoalkylamides form isostructural trilithiated mixed aggregates with few exceptions. 6Li–13C and 6Li–15N couplings reveal heretofore undetected transannular contacts (laddering) with lithium acetylides and lithium monoalkylamides. Marked temperature‐dependent 15N chemical shifts seem to be associated with this laddering. Computational studies shed light on the general structures of the aggregates, their penchant for laddering, and the stereochemical consequences of aggregation.

Synthesis, spectroscopic and conformational analysis of 1,4-dihydroisonicotinic acid derivatives

Structural and conformational properties of 1,4-dihydroisonicotinic acid derivatives, characterized by ester, ketone or cyano functions at positions 3 and 5 in solid and liquid states have been investigated by X-ray analysis and nuclear magnetic resonance and supported by quantum chemical calculations. The dihydropyridine ring in each of the compounds exists in flattened boat-type conformation. The observed ring distortions around the C(4) and N(1) atoms are interrelated. The substituent at N(1) has great influence on nitrogen atom pyramidality. The 1H, 13C and 15N NMR chemical shifts and coupling constants are discussed in terms of their relationship to structural features such as character and position of the substituent in heterocycle, N-alkyl substitution and nitrogen lone pair delocalization within the conjugated system.

Long-Range 1H–15N J Couplings Providing a Method for Direct Studies of the Structure and Azide–Tetrazole Equilibrium in a Series of Azido-1,2,4-triazines and Azidopyrimidines

The selectively (15)N labeled azido-1,2,4-triazine 2*A and azidopyrimidine 4*A were synthesized by treating hydrazinoazines with (15)N-labeled nitrous acid. The synthesized compounds were studied by (1)H, (13)C, and (15)N NMR spectroscopy in DMSO, TFA, and DMSO/TFA solutions, where the azide-tetrazole equilibrium could lead to the formation of two tetrazoles (T, T') and one azide (A) isomer for each compound. The incorporation of the (15)N label led to the appearance of long-range (1)H-(15)N coupling constants (J(HN)), which can be measured easily by using amplitude-modulated 1D (1)H spin-echo experiments with selective inversion of the (15)N nuclei. The observed J(HN) patterns enable the unambiguous determination of the mode of fusion between the azole and azine rings in the two groups of tetrazole isomers (2*T', 4*T' and 2*T, 4*T), even for minor isoforms with a low concentration in solution. However, the azide isomers (2*A and 4*A) are characterized by the absence of detectable J(HN) coupling. The analysis of the J(HN) couplings in (15)N-labeled compounds provides a simple and efficient method for direct NMR studies of the azide-tetrazole equilibrium in solution.

Selective measurements of long-range homonuclear J-couplings in solid-state NMR

We demonstrate here that the principle of frequency-selective spin-echoes can be extended to the measurements of long-range homonuclear scalar J-couplings in the solid-state. Singly or doubly frequency-selective pulses were used to generate either a J-modulated experiment (S) or a reference experiment (S0). The combination of these two distinct experiments provides experimental data that, in favorable cases, are insensitive to incoherent relaxation effects, and which can be used to estimate long-range homonuclear J-couplings in multiple spin-systems. The concept is illustrated in the case of a uniformly 13C and 15N labeled sample of l-histidine, where the absolute value of homonuclear J-couplings between two spins separated by one, two or three covalent bonds are measured. Moreover, we show that a 2J(15N–C–15N) coupling as small as 0.9Hz can be precisely measured with the method presented here.

A solid-state NMR investigation of the colossal expansion material, Ag3Co(CN)6

Presented here is a solid-state NMR investigation of the so-called “colossal expansion” material, Ag3Co(CN)6, a compound that exhibits some of the largest positive and negative thermal expansion properties reported. This study explores the 13C, 15N, and 59Co NMR properties of this material at room temperature and at variable temperatures with the goal of probing the effects of this colossal expansion behaviour on these properties. We found that the flexible nature of the crystal framework leads to a distribution of electric field gradients, and that, oddly enough, no strong correlation is observed between the NMR parameters of Ag3Co(CN)6 and its colossal expansion nature. The 59Co isotropic chemical shift increased and the 59Co nuclear quadrupolar coupling constant decreased with increasing temperature, but neither of these relationships were extraordinary when compared to other octahedral Co(III) complexes. The link between the colossal expansion and the NMR properties of Ag3Co(CN)6 may be the distribution of lattice parameters and hence unusually broad features in the 59Co NMR spectra. The high order of symmetry at the cobalt site resulted in a small quadrupolar coupling constant less than 1 MHz in magnitude. We also observed a |1J(107/109Ag,15N)| value of 96 Hz, the largest 107/109Ag–15N coupling constant reported to date.

Interactions of Intermediate Semiquinone with Surrounding Protein Residues at the QH Site of Wild-Type and D75H Mutant Cytochrome bo3 from Escherichia coli

Selective (15)N isotope labeling of the cytochrome bo(3) ubiquinol oxidase from Escherichia coli with auxotrophs was used to characterize the hyperfine couplings with the side-chain nitrogens from residues R71, H98, and Q101 and peptide nitrogens from residues R71 and H98 around the semiquinone (SQ) at the high-affinity Q(H) site. The two-dimensional ESEEM (HYSCORE) data have directly identified N(ε) of R71 as an H-bond donor carrying the largest amount of unpaired spin density. In addition, weaker hyperfine couplings with the side-chain nitrogens from all residues around the SQ were determined. These hyperfine couplings reflect a distribution of the unpaired spin density over the protein in the SQ state of the Q(H) site and the strength of interaction with different residues. The approach was extended to the virtually inactive D75H mutant, where the intermediate SQ is also stabilized. We found that N(ε) of a histidine residue, presumably H75, carries most of the unpaired spin density instead of N(ε) of R71, as in wild-type bo(3). However, the detailed characterization of the weakly coupled (15)N atoms from selective labeling of R71 and Q101 in D75H was precluded by overlap of the (15)N lines with the much stronger ~1.6 MHz line from the quadrupole triplet of the strongly coupled (14)N(ε) atom of H75. Therefore, a reverse labeling approach, in which the enzyme was uniformly labeled except for selected amino acid types, was applied to probe the contribution of R71 and Q101 to the (15)N signals. Such labeling has shown only weak coupling with all nitrogens of R71 and Q101. We utilize density functional theory-based calculations to model the available information about (1)H, (15)N, and (13)C hyperfine couplings for the Q(H) site and to describe the protein-substrate interactions in both enzymes. In particular, we identify the factors responsible for the asymmetric distribution of the unpaired spin density and ponder the significance of this asymmetry to the quinone's electron transfer function.

Getting to know the nitrogen next door: HNMBC measurements of amino sugars

Long-range 1H– 15N correlations detected by the heteronuclear multiple-bond correlation (HMBC) experiment are explored for the characterization of amino sugars. The gradient-enhanced HMBC, IMPACT-HMBC, and a modified pulse sequence with the 1 J-filters removed, IMPACT-HNMBC, are compared for sensitivity and resolution. 15N chemical shifts and long-range proton correlations are reported using the IMPACT-HNMBC experiment for N-acetyl-glucosamine, N-acetyl-galactosamine, and for a series of glucosamine analogs with an N-sulfo substitution, unmodified amino group, and 6- O-sulfonation. As is common with sugars, for all the compounds examined both anomeric forms are present in solution. For each compound studied, the 15N chemical shifts of the α anomer are downfield of the β form. For the N-acetylated sugars, the β anomer has a unique long-range 15N correlation to the anomeric proton not observed for the α anomer. Though N-sulfonation results in a significant change in the 15N chemical shift of the glucosamine analogs, 6- O sulfo substitution has no significant effect on the local environment of the amino nitrogen. For N-acetylated sugars in D 2O solution, peaks in the 15N projection of the HMBC spectrum appear as triplets as a result of J-modulation due to 2H– 15N coupling.

Interpretation of Indirect Nuclear Spin−Spin Couplings in Isomers of Adenine: Novel Approach to Analyze Coupling Electron Deformation Density Using Localized Molecular Orbitals

Adenine, an essential building block of nucleic acids present in all living systems, can occur in several tautomeric forms. The phenomenon of tautomerism can be investigated by several experimental methods, including nuclear magnetic resonance. In this study, long-range (1)H-(13)C and (1)H-(15)N coupling constants for N-alkyl derivatives related to four tautomers of adenine are investigated in DMSO and DMF solutions. To investigate the structural dependence of the coupling constants and to understand how polarization propagates in the system, Fermi contact (FC) terms were calculated for the individual isomers and analyzed by using density functional theory (DFT), and the coupling pathways were visualized using real-space functions. The coupling electron deformation densities (CDD) of several (1)H-X (X = (13)C, (15)N) pairs are evaluated and compared. In order to analyze the CDD in more detail, a new approach to break down the CDD into contributions from Boys or Pipek-Mezey localized molecular orbitals (LMOs) has been developed. A similar approach has been applied to split the value of the FC contribution to the J coupling into the LMO contributions. On the basis of chemical concepts, the contributions of sigma-bonds, pi-electrons, and lone pairs of electrons are discussed. The lone pair of electrons at the nitrogen atom contributes significantly to the (1)H-C horizontal line(15)N coupling, whereas the (1)H-C=N-(13)C coupling is affected in a somewhat different way. Surprisingly, the contribution of the intervening C horizontal lineN bond to the FC term for (1)H-C=(15)N coupling originates exclusively in sigma-electrons, with a vanishingly small contribution calculated for the pi-electrons of this fragment. This behavior is rationalized by introducing the concept of "hard and soft J elements" derived from the polarizability of the individual components.

Does tautomeric equilibrium exist in 4-nitroso-5-pyrazolones?

The structure and relative stabilities of the tautomers and isomers of 4-nitroso-pyrazolones were investigated at HF, DFT and MPn ( n = 2, 4) quantum chemical levels. The effect of the medium on the relative stabilities of the different structures of the studied compounds was estimated using the polarizable continuum model. 1H, 13C and 15N NMR chemical shieldings and coupling constants were calculated using the GIAO approach at B3LYP/6-31+G(d,p) level and MP2/6-31+G(d,p) optimized geometries. The calculated relative energies of the tautomers and isomers of 3-methyl-4-nitroso-5-pyrazolone and 1-ethyl-3-methyl-4-nitroso-5-pyrazolone suggest that in solution the oxime tautomer is favoured. The second compound exists as a mixture of syn- and anti-oxime isomers. The coexistence of the oxime forms in solution corresponds to the available experimental NMR data.

The Largest 15N–15N Coupling Constant Across an NHN Hydrogen Bond

Tug of war: Treatment of the doubly 15N-labeled anion receptor 6-nitro-2,3-dipyrrol-2-ylquinoxaline with solid sodium hydride in CD2Cl2/[D6]DMSO leads to the monodeprotonated anion (see picture). The NMR data indicate the formation of a very strong intramolecular NHN hydrogen bond, with a scalar 15N–15N coupling constant of 16.5 Hz, the largest value observed to date. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2008/z704411_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

NMR spectra of silatranes and M ← N (M = C, Si, Ge, Sn, Pb) bond lengths in atranes: Substituent effects

The literature data on X substituent influence on the 1H, 29Si and 15N NMR chemical shifts ( δ) and coupling constants ( J) of Si-substituted silatranes ▪, as well as M–N bond lengths ( d) in atranes ▪ (M = C, Si, Ge, Sn, Pb) have been analyzed. It was established for the first time that the δ, J and d values depend not only on the inductive and resonance effects but also on the polarizability of X substituents. The polarizability contribution ranges from 8% to 25%.

A Karplus equation for 3JHCCN in amino sugar derivatives

The 1H– 15N coupling constants of a suite of organic-soluble amino sugar derivatives have been measured by one-dimensional and two-dimensional 1H/ 15N heteronuclear single quantum, multiple bond correlation (HSQMBC), and the values so obtained are compared with those measured by analysis of 1H spectra of 15N-labeled amino sugar derivatives. A number of bicyclic amino sugar models have been studied, including methyl 2- (and 3-)amino-4,6- O-benzylidene-2- (and 3-)deoxy-α- d-hexopyranosides in chair or skew conformations, and methyl 2,6-anhydro-3-deoxy-3-phthalimido-α- d-mannopyranoside in a locked, almost classical boat conformation. The magnitudes of the vicinal 1H– 15N coupling constants 3 J HCCN have been correlated with 1H/ 15N dihedral angles φ computed for the favored conformations by molecular dynamics with molecular mechanics energy minimization. Non-linear regression of the coupling constants on the dihedral angles has yielded a Karplus equation: 3 J HCCN = 3.1 cos 2 φ − 0.6 cos φ + 0.4. The coefficients of the terms in this equation have been compared with those reported for 15 other pairs of nuclei, and the coefficient of the important cos 2 φ term found to be numerically smallest for 3 J HCCN.

Unambiguous assignment of trifluoromethylpyrazole regioisomers by 19F–15N correlation spectroscopy

Analysis of the 19F chemical shifts of trifluoromethylpyrazole regioisomers has shown that while chemical shift is in general a reliable predictor of regiochemistry in this series, there is a narrow chemical shift range in which the two isomers overlap and the regiochemistry cannot be assigned with certainty. We have examined the usage of 19F--15N correlation spectroscopy as a method to provide a second unambiguous confirmation of regiochemistry of 3- and 5-trifluoromethylpyrazole regioisomers. In the case of 3-trifluoromethyl analogs, one expects a three-bond coupling to the pyridine type nitrogen (N-2). In the case of the 5-trifluoromethyl pyrazole the situation is exactly reversed, with the fluorines of the trifluoromethyl moiety being 3-bonds from the pyrrole type nitrogen (N-1). We have observed that 3-trifluromethyl analogs follow the expected pattern of readily observable 3-bond coupling. However, 19F--15N couplings in 5-trifluromethyl analogs do not follow the normal pattern of 3-bond coupling. Using this information we have been able to develop unambiguous methods to distinguish the isomers.

1H, 13C and 15N NMR spectral characterization of twenty‐seven 1,2‐diaryl‐(4E)‐arylidene‐2‐imidazolin‐5‐ones

1H, 13C and 15N NMR chemical shifts and couplings (n)J(H,C) in DMSO-d(6) at 30 degrees C have been determined for 1,2-diaryl-(4E)-arylidene-2-imidazolin-5-one derivatives 1-27. Their chemical shift assignments are based on PFG DQF 1H,1H COSY, PFG 1H,13C HMQC as well as PFG 1H,13C and 1H,15N HMBC experiments. For compounds 1-10 including aryl fluorine substituent(s) also the couplings (n)J(F,C) (n = 1 - 4) are reported.

Solution structures of chiral lithium amides with internal sulfide coordination: sulfide versus ether coordination in chiral lithium amides

A series of chiral lithium amides with chelating sulfide groups has been investigated by NMR spectroscopy. These chiral lithium amides have previously been found to mediate higher enantioselectivity in the asymmetric addition of alkyllithium reagents to aldehydes than the corresponding lithium amides containing ether groups. The chiral lithium amido sulfide chelates form homodimers in both diethyl ether and tetrahydrofuran. In diethyl ether both sulfur atoms coordinate to the same lithium while the other lithium in the dimer is solvent coordinated. Tetrahydrofuran favours equivalently solvated homodimers. Mixed dimeric complexes are formed with excess n-butyllithium in both diethyl ether and tetrahydrofuran. The structural information was obtained from observed 6Li, 15N couplings and cross peaks in the 6Li, 1H HOESY spectrum. DFT calculations at B3LYP/6-31G(d) level of theory indicate that the Li–S chelate is much less stable than the Li–O chelate. However the calculations indicate that the stability of the dimeric chelates are in agreement with the NMR results of the diethyl ether and tetrahydrofuran solutions.

Dynamic 15N NMR studies of iron phosphine complexes containing coordinated dinitrogen

AbstractThe 15N‐labelled iron dinitrogen complexes trans‐[FeH(N2)(PP)2]+[BPh4]− (PP = dppe, depe, dmpe) and cis‐[FeH(N2)(PP3)]+[BPh4]− were prepared in situ by exchange of unlabelled coordinated dinitrogen with 15N2. 15N NMR chemical shifts and coupling constants are reported. The 15N spectra exhibit separate signals for the metal‐bound and terminal nitrogen atoms of the coordinated N2. The 15N resonances display 15N, 15N coupling as well as 31P, 15N coupling and long‐range 15N, 1H coupling when there is a metal‐bound hydrido ligand. Exchange between free and coordinated dinitrogen was monitored by magnetization transfer between 15N‐labelled sites using an inversion–transfer–recovery experiment. Exchange between the metal‐bound and terminal nitrogen atoms of coordinated N2 was also monitored by magnetization transfer and this could proceed by N2 dissociation or by an intramolecular process. Copyright © 2003 John Wiley & Sons, Ltd.