Long‐range 19F–15N heteronuclear shift correlation: examination of J‐modulations associated with broad range accordion excitation

The first demonstrated example of ¹⁹F–¹⁵N long‐range heteronuclear shift correlation spectroscopy at natural abundance is reported. Because of the very large variation in the size of ²J(N,F) vs ³J(N,F) long‐range heteronuclear couplings, the utilization of one of the new accordion‐optimized long‐range heteronuclear shift correlations experiments is essential if all possible correlations are to be observed in a single experiment. A modified IMPEACH‐MBC pulse sequence was used in conjunction with an optimization range from 4 to 50 Hz to demonstrate the technique using a mixture of 2‐ and 3‐fluoropyridine, which had ²J(N,F) and ³J(N,F) long‐range couplings of ?52 and 3.6 Hz, respectively. Because of the size of the ²J(N,F) long‐range coupling constant, a J‐modulation of the long‐range correlation response is observed in the spectrum resulting in a ‘doublet’ in F₁ due to amplitude modulation. The size of the ‘doublet’ is shown to be a function of the parameter selection (t_1max,T_max,T_min and spectral width in F₁). This behavior is similar to F₁ ‘skew’ associated with long‐range correlation responses in ACCORD‐HMBC spectra which has been analyzed in detail previously. Copyright © 2002 John Wiley & Sons, Ltd.     

13C‐15N Connectivity networks via unsymmetrical indirect covariance processing of 1H‐13C HSQC and 1H‐15N IMPEACH spectra

Long‐range ¹H‐¹⁵N heteronuclear shift correlation methods at natural abundance to facilitate the elucidation of small molecule structures have assumed a role of growing importance over the past decade. Recently, there has also been a high level of interest in the exploration of indirect covariance NMR methods. From two coherence transfer experiments, A→B and A→C, it is possible to indirectly determine B?C. We have shown that unsymmetrical indirect covariance methods can be employed to indirectly determine several types of hyphenated 2D NMR data from higher sensitivity experiments. Examples include the calculation of hyphenated 2D NMR spectra such as 2D GHSQC‐COSY and GHSQC‐NOESY from the discrete component 2D NMR experiments. We now wish to report the further extension of unsymmetrical indirect covariance NMR methods for the combination of ¹H‐¹³C GHSQC and ¹H‐¹⁵N longrange (GHMBC, IMPEACH‐MBC, CIGAR‐HMBC, etc.) heteronuclear chemical shift correlation spectra to establish ¹³C‐¹⁵N correlation pathways.     

Simplifying LR‐HSQC spectra using a triple‐quantum filter: The LR‐HTQC experiment

Long‐range heteronuclear single quantum correlation (LR‐HSQC) experiments may be applied for detecting long‐range correlations but suffer from two disadvantages, common to all heteronuclear long‐range correlation experiments: (i) The information density in LR‐HSQC spectra may be too high to be used directly without “filtering out” shorter range correlations, and (ii) often, substantial differences in intensity among cross peaks exist, potentially hampering the visualization of weak, often crucial cross peaks. In this contribution, we propose a modified LR‐HSQC experiment, the LR‐HTQC experiment (Long‐Range Heteronuclear Triple Quantum Correlation) that partially solves the problems aforementioned. We show theoretically and experimentally that the LR‐HTQC experiment removes the intense cross peaks of CH spin pairs, substantially reduces the medium intensity of cross peaks originating from CHH' spin systems, whereas the typically weak intensity of cross peaks of CHH'H″ and C(H)_n, _{n > 3} spin systems is less affected. Consequently, the LR‐HTQC experiment affords simplified long‐range heteronuclear shift correlation spectra and scales down large intensity differences among different types of cross peaks, although a certain general reduction of signal intensities has to be accepted.     

Application of the randomly optimized RDSQC experiment for the elucidation of an unknown compound

The application of the randomly optimized RDSQC (Randomly optimized Direct correlation Single Quantum Coherence) experiment for the detection of direct correlations facilitated the characterization of an unknown compound. The expected structure consisted of purely aliphatic moieties. The actual, identified compound contained the desired structure plus an adenosine functionality with two protons whose direct proton‐carbon couplings were over 200 Hz. Application of a 130 Hz optimized direct heteronuclear GHSQC experiment afforded no correlations for the adenine responses. The RDSQC experiment allowed for the simultaneous optimization of multiple couplings in a range of 130 to 220 Hz producing a direct correlation spectrum with all the expected responses.     

Structural Characterization of a Phenoxazine Analog Formed as a By‐product of the Synthesis of a 3‐Amino‐N,N‐dimethylsalicylamide

A highly colored impurity formed at low levels during the preparation of 3‐amino N,N‐dimethyl salicylamide was isolated and identified as 2‐amino‐N,N,N′,N′‐tetramethyl‐3‐oxo‐3H‐phenoxazin‐4,6‐dicarboxamide using mass spectrometry and two‐dimensional NMR methods that included long‐range ¹H‐¹⁵N heteronuclear chemical shift correlation data to assemble the proton‐deficient “right‐half” of the 2‐aminophenoxazin‐3‐one nucleus. The structure was independently confirmed using computer‐assisted structure elucidation methods and by synthesis.     

Using LR-HSQMBC to observe long-range H–N correlations

The recently reported LR-HSQMBC experiment has been optimized for ¹H–¹⁵N long-range heteronuclear couplings. Several previously unreported four-bond correlations, consistent with the predicted by DFT calculations (0.2–0.3 Hz ⁴J_NH couplings), have been observed for strychnine using 2 Hz optimization of the LR-HSQMBC experiment. This experiment offers an advantage over accordion-optimized experiments such as IMPEACH and CIGAR for the observation of long-range ¹H–¹⁵N correlations in that the experiment is refocused and employs a CLIP pulse sequence element to bring the long-range correlations into phase, allowing broadband X-decoupling to be employed during acquisition.     

Posaconazole: Application of HSQC‐ADEQUATE from General Indirect Covariance Processing

Posaconazole is a structurally complex triazole antifungal agent that, by virtue of its structural complexity, provides a good test molecule for the evaluation of NMR structure elucidation methodologies. Although GHMBC and related long‐range ¹H–¹³C heteronuclear shift correlation techniques are extremely powerful, at the same time, when dealing with unknowns, they can be problematic in that there is no way to readily differentiate adjacent (² J_CH) correlations from longer range correlations, e.g., ³J_CH and ⁿJ_CH, n > 3. The 1,1‐ADEQUATE experiment, in contrast, provides unequivocal experimental access to adjacent carbon–carbon correlation information, albeit with a sensitivity penalty, as the experiment involves an adjacent ¹³C–¹³C out‐and‐back magnetization transfer. In part, the sensitivity penalty can be overcome by using unsymmetrical indirect covariance or general indirect covariance processing methods. The application of these methods through the coprocessing of multiplicity‐edited GHSQC and 1,1‐ADEQUATE data to generate an HSQC‐ADEQUATE correlation plot is demonstrated for posaconazole.     

Disentangling diffusion information of individual components in a mixture with a 3D COMPACT‐IDOSY NMR experiment

A new 3D diffusion‐ordered heteronuclear NMR experiment COMPACT‐IDOSY (cross‐polarization optimized multisite polarized accelerated time internally encoded diffusion ordered spectroscopy) has been designed and experimentally implemented on a mixture of flavonoids rutin and quercetin. The pulse sequence uses a cross‐polarization mixing period and diffusion encoding gradients internally incorporated into the coherence transfer interval of a long‐range heteronuclear correlation experiment. Substantial reduction in experimental time, good sensitivity and excellent resolution of signal overlap lead to the accurate determination of translational diffusion coefficients of individual components in the mixture. Copyright © 2012 John Wiley & Sons, Ltd.     

Measurement and Applications of Long‐Range Heteronuclear Scalar Couplings: Recent Experimental and Theoretical Developments

The use of long‐range heteronuclear couplings, in association with ¹H–¹H scalar couplings and NOE restraints, has acquired growing importance for the determination of the relative stereochemistry, and structural and conformational information of organic and biological molecules. However, the routine use of such couplings is hindered by the inherent difficulties in their measurement. Prior to the advancement in experimental techniques, both long‐range homo‐ and heteronuclear scalar couplings were not easily accessible, especially for very large molecules. The development of a large number of multidimensional NMR experimental methodologies has alleviated the complications associated with the measurement of couplings of smaller strengths. Subsequent application of these methods and the utilization of determined J‐couplings for structure calculations have revolutionized this area of research. Problems in organic, inorganic and biophysical chemistry have also been solved by utilizing the short‐ and long‐range heteronuclear couplings. In this minireview, we discuss the advantages and limitations of a number of experimental techniques reported in recent times for the measurement of long‐range heteronuclear couplings and a few selected applications of such couplings. This includes the study of medium‐ to larger‐sized molecules in a variety of applications, especially in the study of hydrogen bonding in biological systems. The utilization of these couplings in conjunction with theoretical calculations to arrive at conclusions on the hyperconjugation, configurational analysis and the effect of the electronegativity of the substituents is also discussed.     

Inversion of 1JCC correlations in 1,n‐ADEQUATE spectra

ADEQUATE experiments provide an alternative to the more commonly employed GHMBC experiment for the establishment of long‐range heteronuclear connectivities. The 1,1‐ADEQUATE experiment allows the unequivocal identification of both protonated and non‐protonated carbon resonances adjacent to a protonated carbon. The 1,n‐ADEQUATE experiment establishes correlations via an initial ¹J_CH heteronuclear transfer followed by an ⁿJ_CC out‐and‐back transfer, most typically, via three carbon–carbon bonds. Hence, the 1,n‐ADEQUATE experiment allows the equivalent of ⁴J_CH heteronuclear correlations to be probed when they are not observed in a GHMBC spectrum. Aside from the lower sensitivity of the 1,n‐ADEQUATE experiment relative to GHMBC experiments, the interpretation of the former is also complicated by the ‘leakage’ of ¹J_CC correlations into the spectrum that must be identified. A method for the inversion of ¹J_CC correlations to facilitate the interpretation of 1,n‐ADEQUATE spectra is presented that allows a single experiment to be performed to access ¹J_CC and ⁿJ_CC correlation information. Copyright © 2012 John Wiley & Sons, Ltd.     

Simple and Efficient Synthesis of Novel 3‐Substituted 2‐Thioxo‐2,3‐dihydro‐1H‐benzo[g]quinazolin‐4‐ones and Their Reactions with Alkyl Halides and α‐Glycopyranosyl Bromides

A series of 3‐substituted 2‐thioxo‐2,3‐dihydro‐1H‐benzo[g]quinazolin‐4‐ones 4a – e were synthesized from the reaction of 3‐aminonaphthalene‐2‐carboxylic acid 1 with isothiocyanate derivatives 2a – e . The alkylation of 4a – e with alkyl halides gave 3‐substituted 2‐alkylsulfanyl‐2,3‐dihydro‐1H‐benzo[g]quinazolin‐4‐ones 5a – o . S‐Glycosylation was carried out via the reaction of 4a – e with glycopyranosyl bromides 7a and 7b under anhydrous alkaline conditions. The structure of the compounds was established as S‐nucleoside and not N‐nucleoside. Conformational analysis has been studied by homonuclear and heteronuclear two‐dimensional NMR methods (2D DFQ‐COSY, heteronuclear multiple quantum coherence, and heteronuclear multiple bond correlation). The S site of alkylation and glycosylation was determined from the ¹H and ¹³C heteronuclear multiple quantum coherence experiments.     

A Ferrocenyl Kaleidoscope: Slow Interconversion of Six Diastereo‐atropisomers of 2,6‐Di‐tert‐butyl‐9,10‐diferrocenyltriptycene

The title triptycene, 6 , has been isolated as the product of 9,10‐cycloaddition of benzyne to 9,10‐diferrocenyl‐2,6‐di‐tert‐butylanthracene, 5 , whose X‐ray crystal structure is reported. Each ferrocenyl unit in 6 has access to the same three non‐equivalent molecular environments, and their rotations relative to the molecular paddlewheel give rise to six slowly interconverting atropisomers. Their dynamic behaviour in solution is a challenging NMR puzzle that can be successfully solved by taking advantage of the recently described very large diamagnetic anisotropy of the ferrocenyl moiety, together with the C₂ symmetry of particular atropisomers. Application of one‐ and two‐dimensional NMR techniques over a range of temperatures together, with a detailed analysis of the homo‐ and heteronuclear correlations in 6 , resulted in unequivocal mapping of the 99 ¹H and 162 ¹³C positions in the six interconverting systems. Variable‐temperature 2D‐EXSY measurements revealed that, while the stability of the atropisomers is almost identical, they are separated by energy barriers which the ferrocenyls must overcome in the course of their interconversions. The heights of two different rotational barriers have been identified and these experimental findings are in good agreement with DFT calculations.     

Complete 1H and 13C NMR chemical shift assignment of N1‐ and N3‐alkylnitrohistidines and of 1,4,6,7‐tetrahydroimidazo[4,5‐b]pyridines

¹H and ¹³C NMR data for 13 nitrohistidine derivatives are reported, providing a diagnostic method for the elucidation of the N¹‐( 2 ) and the N³‐substituted ( 3 ) regioisomers. Spectral assignments of constrained surrogates of His ( 4 ) and of the His–Gly dipeptide ( 5 and 6 ) are also described. The structure of the compounds was confirmed by NOESY and heteronuclear (¹³C, ¹H) short‐ and long‐range correlation experiments. Copyright © 2003 John Wiley & Sons, Ltd.     

NMR study on 9‐S and 9‐R oxirane derivatives of ascomycin

Structure elucidation of 9‐S and 9‐R oxirane derivatives of ascomycin, a 23‐membered immunomodulating macrolactam, was performed using NMR spectroscopy. The total ¹H and ¹³C signal assignments required the gradient‐selected versions of COSY (gs‐COSY), heteronuclear multiple quantum‐correlation spectroscopy (gs‐HSQC), heteronuclear multiple‐bond correlation spectroscopy (gs‐HMBC), and nuclear Overhauser methods. The data sets then were used to examine the dependence of ketone–hemiketal and cis–trans amide equilibria on the substitution pattern and the absolute configuration of the chiral oxirane. Copyright © 2002 John Wiley & Sons, Ltd.     

Frequency‐Switched Single‐Transition Cross‐Polarization: A Tool for Selective Experiments in Biomolecular NMR

Frequency‐switched single‐transition cross‐polarization (FS‐ST‐CP) provides a versatile tool for selective coherence transfer in heteronuclear NMR of biomolecules such as proteins and nucleic acids. This type of coherence transfer is spin‐state‐selective and can therefore benefit from the extension of the life‐times of selected coherences due to partial cancellation of interfering relaxation mechanisms. The limits of the selectivity of the transfer are discussed by theory and illustrated by experiment. The methods are particularly efficient to obtain quantitative structural and dynamic information for selected residues in medium‐sized nitrogen‐15 or carbon‐13 labeled macromolecules.     

The microstructure determination of ethylene‐styrene‐propylene terpolymers at triad level by high‐temperature two‐dimensional NMR spectra

To further extend temperature range of application and low temperature performance of the ethylene‐styrene copolymers, a series of poly(ethylene‐styrene‐propylene) samples with varying monomer compositions and relatively low glass‐transition temperatures (T_g = −28 – 22 °C) were synthesized by Me₂Si(Me₄Cp)(N‐t‐Bu)TiCl2/MMAO system. Since the ¹³C NMR spectra of the terpolymers were complex and some new resonances were present, 2D‐¹H/¹³C heteronuclear single quantum coherence and heteronuclear multiple bond correlation experiments were conducted. A complete ¹³C NMR characterization of these terpolymers was performed qualitatively and quantitatively, including chemical shifts, triad sequence distributions, and monomer average sequence lengths. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018 , 56, 340–350     

A long‐range 15N‐NMR study of the oxazolidinone antibiotic zyvox® and the major thermal degradation products

The structures of the major thermal degradation products of linezolid (Zyvox^®, PNU‐100766) are reported. Following hydrolytic decarboxylation, the remaining oxazolidinone‐derived skeleton of the antibiotic underwent a variety of acetyl migrations prior to deacetylation. Three of the degradation products are structural isomers. Structural characterization of the degradants was accomplished via the concerted analysis of 2D NMR, mass spectrometric, and infrared data. Direct and long‐range ¹H‐¹⁵N heteronuclear shift correlation experiments at natural abundance were performed on linezolid as well as each of the isolated products of the degradation cascade. ¹⁵N chemical shifts for linezolid and each of the degradants, as well as the observed long‐range ¹H‐¹⁵N coupling pathways are reported.     

NMR Characterization of Complex p‐Oligophenyl Scaffolds by Means of Aliasing Techniques to Obtain Resolution‐Enhanced Two‐Dimensional Spectra

The usefulness of computer‐assisted aliasing to secure maximal resolution of signal clusters in ¹H‐ and ¹³C‐NMR spectra (which is essential for structure determination by HMBC 2D NMR spectroscopy) in minimal acquisition time is exemplified by the complete characterization of the two complementary p‐octiphenyls 1 and 2 with complex substitution patterns. The need for digital resolution near 1 Hz/pt to dissect the extensive signal clusters in the NMR spectra of these refined oligomers excluded structure determination under routine conditions. High resolution was secured by exploiting the low signal density in the ¹³C dimension of HMBC spectra by using computer‐assisted aliasing to maximize signal density. Based on the observed shifts in DEPT and ¹H‐decoupled ¹³C‐NMR spectra of 1 and 2 , computer‐assisted aliasing allowed to reduce the number of required time increments by a factor of 20 to 30 compared to full‐width spectra with identical resolution. Without signal‐to‐noise constraints, this computer‐assisted aliasing reduced the acquisition time for high‐resolution NMR spectra needed for complete characterization of refined oligomers 1 and 2 by the same factor (e.g., from over a day to about an hour). With resolved signal clusters in fully aliased HSQC and HMBC spectra, unproblematic structure determination of 1 and 2 is demonstrated by unambiguous assignment of all C‐ and H‐atoms. These findings demonstrate that computer‐assisted aliasing of the underexploited ¹³C dimension makes extensive molecular complexity accessible by conventional multidimensional heteronuclear NMR experiments without extraordinary efforts.     

Spectroscopic separation of 13C NMR spectra of complex isomeric mixtures by the CSSF‐TOCSY‐INEPT experiment

Isomeric mixtures from synthetic or natural origins can pose fundamental challenges for their chromatographic separation and spectroscopic identification. A novel 1D selective NMR experiment, chemical shift selective filter (CSSF)‐TOCSY‐INEPT, is presented that allows the extraction of ¹³C NMR subspectra of discrete isomers in complex mixtures without physical separation. This is achieved via CSS excitation of proton signals in the ¹H NMR mixture spectrum, propagation of the selectivity by polarization transfer within coupled ¹H spins, and subsequent relaying of the magnetization from ¹H to ¹³C by direct INEPT transfer to generate ¹³C NMR subspectra. Simple consolidation of the subspectra yields ¹³C NMR spectra for individual isomers. Alternatively, CSSF‐INEPT with heteronuclear long‐range transfer can correlate the isolated networks of coupled spins and therefore facilitate the reconstruction of the ¹³C NMR spectra for isomers containing multiple spin systems. A proof‐of‐principle validation of the CSSF‐TOCSY‐INEPT experiment is demonstrated on three mixtures with different spectral and structural complexities. The results show that CSSF‐TOCSY‐INEPT is a versatile, powerful tool for deconvoluting isomeric mixtures within the NMR tube with unprecedented resolution and offers unique, unambiguous spectral information for structure elucidation. Copyright © 2014 John Wiley & Sons, Ltd.     

One‐ and two‐dimensional NMR characterization of N‐vinyl‐2‐pyrrolidone/methyl acrylate copolymers

N‐vinyl‐2‐pyrrolidone/methyl acrylate (V/M) copolymers were prepared by free‐radical bulk polymerization using benzoyl peroxide as an initiator. The copolymer composition of these copolymers was calculated from ¹H NMR spectra. The radical reactivity ratios for N‐vinyl‐2‐pyrrolidone (V) and methyl acrylate (M) were r_V = 0.09, r_M = 0.44. These reactivity ratios for the copolymerization of V and M were determined using the Kelen–Tudos and nonlinear least‐squares error‐in‐variable methods. The ¹³C{¹H} and ¹H NMR spectra of these copolymers overlapped and were complex. The complete spectral assignment of the ¹³C and ¹H NMR spectra were done with distortionless enhancement by polarization transfer and two dimensional ¹³C‐¹H heteronuclear single quantum correlation spectroscopic experiments. The two‐dimensional ¹H‐¹H homonuclear total correlation spectroscopic NMR spectrum showed the various bond interactions, thus inferring the possible structure of the copolymers. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 2225–2236, 2002