Long-range Heteronuclear Coupling Constants Research Articles

How to measure long-range proton-carbon coupling constants from 1 H-selective HSQMBC experiments.

Heteronuclear long-range scalar coupling constants (n JCH ) are a valuable tool for solving problems in organic chemistry and are especially suited for stereochemical and configurational analyses of small molecules and natural products. This tutorial will focus on the step-by-step implementation of several 2D 1 H frequency selective HSQMBC experiments for the easy and accurate measurement of either the magnitude or both the magnitude and the sign of long-range n JCH couplings. The performance of these experiments will be showcased with several scenarios in a range of different experimental conditions.

ASAP-HSQC-TOCSY for fast spin system identification and extraction of long-range couplings.

Based on Ernst-angle-type excitation and Acceleration by Sharing Adjacent Polarization (ASAP), a fast HSQC-TOCSY experiment is introduced. In the approach, the DIPSI-2 isotropic mixing period of the ASAP-HSQC is simply shifted, which provides a TOCSY period without additional application of rf-energy. The ASAP-HSQC-TOCSY allows the acquisition of a conventional 2D in about 30 s. Alternatively, it allows the acquisition of highly carbon-resolved spectra (several Hz digital resolution) on the order of minutes. An ASAP-HSQC-TOCSY-IPAP variant, finally, allows the sign-sensitive extraction of heteronuclear long-range coupling constants from a pair of highly resolved spectra in less than an hour. Pulse sequences, several example spectra, and a discussion of results are given.

CSSF-CLIP-HSQMBC: measurement of heteronuclear coupling constants in severely crowded spectral regions.

A new pulse program development, a chemical shift selective filtration clean in-phase HSQMBC (CSSF-CLIP-HSQMBC), is presented for the user-friendly measurement of long-range heteronuclear coupling constants in severely crowded spectral regions. The introduction of the chemical shift selective filter makes the experiment extremely efficient at resolving overlapped multiplets and produces a clean selective CLIP-HSQMBC spectrum, in which the desired coupling constants can easily be measured as an extra proton–carbon splitting in f2. The pulse sequence is also provided as a real-time homonuclear decoupled version in which the heteronuclear coupling constant can be directly measured as the peak splitting in f2. The same principle is readily applicable to IPAP and AP versions of the same sequence as well as the optional TOCSY transfer, or in principle to any other selective heteronuclear experiment that relies on a clean 1H multiplet.

Improved Isotopic Profiling by Pure Shift Heteronuclear 2D J-Resolved NMR Spectroscopy.

Quantitative information on the carbon isotope content of metabolites is essential for flux analysis. Whereas this information is in principle present in proton NMR spectra through both direct and long-range heteronuclear coupling constants, spectral overlap and homonuclear coupling constants both hinder its extraction. We demonstrate here how pure shift 2D J-resolved NMR spectroscopy can simultaneously remove the homonuclear couplings and separate the chemical shift information from the heteronuclear coupling patterns. We demonstrate the power of this method on cell lysates from different bacterial cultures and investigate in detail the branched chain amino acid biosynthesis.

Measurement of long-range heteronuclear coupling constants using the peak intensity in classical 1D HMBC spectra.

In this contribution, we show that the magnitude of heteronuclear long-range coupling constants can be directly extracted from the classical 1D HMBC spectra, as all multiplet lines of a cross-peak always and exclusively vanish for the condition Δ=k/n JCH . To the best of our knowledge, this feature of the classical HMBC has not yet been noticed and exploited. This condition holds true, irrespective of the magnitude and numbers of additional active and passive homonuclear n JHH' couplings. Alternatively, the n JCH value may also be evaluated by fitting the peak's intensity in the individual spectra to its simple sin(πn JCH Δ)exp(-Δ/T2eff ) dependence. Compared to the previously proposed J-HMBC sequences that also use the variation of the cross-peak's intensity for extracting the coupling constants, the classical HMBC pulse sequence is significantly more sensitive.

Broadband homonuclear decoupled HSQMBC methods.

Long-range heteronuclear coupling constants convey invaluable information for stereochemical and conformational analysis of molecules from synthetic and natural origin. Here, we report a real-time Zangger-Sterk CPMG-HSQMBC method for the precise and direct measurement of multiple-bond heteronuclear couplings. It is demonstrated that the real-time acquisition strategy combined with multiple slice selective excitation can provide substantial improvement in sensitivity (or reduction of experimental time) as compared to other variants of broadband homonuclear decoupled HSQMBC methods published previously. Scope and limitations of the different strategies applied for decoupling are reviewed. Moreover, practical guidelines for the choice of the most appropriate method are also presented. Applications are given on a metal complex incorporating P-heterocycles and two diglycosyl-selenides for the extraction of n J(1 H, 31 P) and n J(1 H, 77 Se), respectively.

S3 HMBC hetero: Spin-State-Selective HMBC for accurate measurement of long-range heteronuclear coupling constants

A novel method, Spin-State-Selective (S3) HMBC hetero, for accurate measurement of heteronuclear coupling constants is introduced. The method extends the S3 HMBC technique for measurement of homonuclear coupling constants by appending a pulse sequence element that interchanges the polarization in 13C-1H methine pairs. This amounts to converting the spin-state selectivity from 1H spin states to 13C spin states in the spectra of long-range coupled 1H spins, allowing convenient measurement of heteronuclear coupling constants similar to other S3 or E.COSY-type methods. As usual in this type of techniques, the accuracy of coupling constant measurement is independent of the size of the coupling constant of interest. The merits of the new method are demonstrated by application to vinyl acetate, the alkaloid strychnine, and the carbohydrate methyl β-maltoside.

Determination of unresolved heteronuclear scalar coupling constants by J(up)-HSQMBC

Long-range heteronuclear scalar coupling constants provide important structural information, which is necessary for obtaining stereospecific assignment or dihedral angle information. The measurement of small proton–carbon splittings is particularly difficult due to the low natural abundance of carbon-13 and the presence of homonuclear couplings of similar size. Here we present a real-time J-upscaled HSQMBC, which allows the measurement of heteronuclear coupling constants even if they are hidden in the signal linewidth of a regular spectrum.

PSYCHE CPMG-HSQMBC: An NMR Spectroscopic Method for Precise and Simple Measurement of Long-Range Heteronuclear Coupling Constants.

Among the NMR spectroscopic parameters, long-range heteronuclear coupling constants convey invaluable information on torsion angles relevant to glycosidic linkages of carbohydrates. A broadband homonuclear decoupled PSYCHE CPMG-HSQMBC method for the precise and direct measurement of multiple-bond heteronuclear couplings is presented. The PSYCHE scheme built into the pulse sequence efficiently eliminates unwanted proton-proton splittings from the heteronuclear multiplets so that the desired heteronuclear couplings can be determined simply by measuring frequency differences between peak maxima of pure antiphase doublets. Moreover, PSYCHE CPMG-HSQMBC can provide significant improvement in sensitivity as compared to an earlier Zangger-Sterk-based method. Applications of the proposed pulse sequence are demonstrated for the extraction of (n)J((1)H,(77)Se) and (n)J((1)H,(13)C) values, respectively, in carbohydrates; further extensions can be envisioned in any J-based structural and conformational studies.

Implementing multiplicity editing in selective HSQMBC experiments

Even C/CH2 and odd CH/CH3 carbon-multiplicity information can be directly distinguished from the relative positive/negative phase of cross-peaks in a novel ME (Multiplicity-Edited)-selHSQMBC experiment. The method can be extended by a TOCSY propagation step, and it is fully compatible for the simultaneous and precise determination of long-range heteronuclear coupling constants. Broadband homonuclear decoupling techniques can also be incorporated to enhance sensitivity and signal resolution by effective collapse of J(HH) multiplets.

Precise measurement of long-range heteronuclear coupling constants by a novel broadband proton-proton-decoupled CPMG-HSQMBC method.

A broadband proton–proton-decoupled CPMG-HSQMBC method for the precise and direct measurement of long-range heteronuclear coupling constants is presented. The Zangger–Sterk-based homodecoupling scheme reported herein efficiently removes unwanted proton–proton splittings from the heteronuclear multiplets, so that the desired heteronuclear couplings can be determined simply by measuring frequency differences between singlet maxima in the resulting spectra. The proposed pseudo-1D/2D pulse sequences were tested on nucleotides, a metal complex incorporating P heterocycles, and diglycosyl (di)selenides, as well as on other carbohydrate derivatives, for the extraction of nJ(1H,31P), nJ(1H,77Se), and nJ(1H,13C) values, respectively.

Suppressing one‐bond homonuclear 13C,13C scalar couplings in the J‐HMBC NMR experiment: application to 13C site‐specifically labeled oligosaccharides

Site-specific (13)C isotope labeling is a useful approach that allows for the measurement of homonuclear (13)C,(13)C coupling constants. For three site-specifically labeled oligosaccharides, it is demonstrated that using the J-HMBC experiment for measuring heteronuclear long-range coupling constants is problematical for the carbons adjacent to the spin label. By incorporating either a selective inversion pulse or a constant-time element in the pulse sequence, the interference from one-bond (13)C,(13)C scalar couplings is suppressed, allowing the coupling constants of interest to be measured without complications. Experimental spectra are compared with spectra of a nonlabeled compound as well as with simulated spectra. The work extends the use of the J-HMBC experiments to site-specifically labeled molecules, thereby increasing the number of coupling constants that can be obtained from a single preparation of a molecule.

BASHD‐J‐resolved‐HMBC, an efficient method for measuring proton–proton and heteronuclear long‐range coupling constants

Natural products often possess various spin systems consisting of a methine group directly bonded to a methyl group (e.g. -CHa-CHb(CH3)-CHc-). The methine proton Hb splits into a broadened multiplet by coupling with several vicinal protons, rendering analysis difficult of (n)JC-H with respect to Hb in the J-resolved HMBC-1. In purpose of the reliable and easy measurements of (n)JC-H and (n)JH-H in the aforesaid spin system, we have developed a new technique, named BASHD-J-resolved-HMBC. This method incorporates band selective homo decoupled pulse and J-scaling pulse into HMBC. In this method, high resolution cross peaks can be observed along the F1 axis by J-scaling pulse, and band selective homo decoupled pulse simplified multiplet signals. Determinations of (n)JC-H and (n)JH-H of multiplet signals can easily be performed using the proposed pulse sequence.

Low‐power composite CPMG HSQMBC experiment for accurate measurement of long‐range heteronuclear coupling constants

A modified version of CPMG-HSQMBC pulse scheme is presented for the measurement of long-range heteronuclear coupling constants. The method implements adiabatic inversion and refocusing pulses on the heteronucleus. Low-power composite 180° XY-16 CPMG pulse train is applied on both proton and X nuclei during the evolution of long-range couplings to eliminate phase distortions due to co-evolution of homonuclear proton-proton couplings. The pulse sequence yields pure absorption antiphase multiplets allowing precise and direct measurement of the (n)J(XH) coupling constants regardless from the size of the proton-proton couplings. The applicability of the method is demonstrated using strychnine as a model compound. The selective 1D version of the method is also presented.

IPAP–HSQMBC: Measurement of long-range heteronuclear coupling constants from spin-state selective multiplets

A new NMR approach is proposed for the measurement of long-range heteronuclear coupling constants ( n J XH , n > 1) in natural abundance molecules. Two complementary in-phase (IP) and anti-phase (AP) data are separately recorded from a modified HSQMBC experiment and then added/subtracted to provide spin-state-selective α/ β–HSQMBC spectra. The magnitude of n J XH can be directly determined by simple analysis of the relative displacement between α- and β-cross-peaks. The robustness of this IPAP–HSQMBC experiment is evaluated experimentally and by simulation using a variety of different conditions. Important aspects such as signal intensity dependence and presence of unwanted cross-talk effects are discussed and examples on the measurement of small proton–carbon ( n J CH ) and proton–nitrogen ( n J NH ) coupling constants are provided.

Synthesis and conformational analysis of carbasugar bioisosteres of α- l-iduronic acid and its methyl glycoside

The synthesis of two novel carbasugar analogues of α- l-iduronic acid is described in which the ring-oxygen is replaced by a methylene group. In analogy with the conformational equilibrium described for α- l-Ido pA, the conformation of the carbasugars was investigated by 1H and 13C NMR spectroscopy. Hadamard transform NMR experiments were utilised for rapid acquisition of 1H, 13C-HSQC spectra and efficient measurements of heteronuclear long-range coupling constants. Analysis of 1H NMR chemical shifts and J H,H coupling constants extracted by a total-lineshape fitting procedure in conjunction with J H,C coupling constants obtained by three different 2D NMR experiments, viz., 1H, 13C-HSQC-HECADE, J-HMBC and IPAP-HSQC-TOCSY-HT, as well as effective proton–proton distances from 1D 1H, 1H T-ROE and NOE experiments showed that the conformational equilibrium 4 C 1⇌ 2 S 5a⇌ 1 C 4 is shifted towards 4 C 1 as the predominant or exclusive conformation. These carbasugar bioisosteres of α- l-iduronic acid do not as monomers show the inherent flexibility that is anticipated to be necessary for biological activity.

High resolution‐HMBC (HR‐HMBC), a new method for measuring heteronuclear long‐range coupling constants

A useful pulse sequence for measuring long-range C--H coupling constants (J(C-H)) named high resolution-HMBC (HR-HMBC) has been developed. In this pulse sequence, the J-scaling pulse [(nt(1))/2-180 degrees (H/C) - (nt(1))/2] is incorporated after the spin evolution period, and then followed by an (1)H 180 degrees pulse to reverse the magnetization of J(C-H) couplings. As a result, splittings of the cross peaks due to the long-range J(C-H) are realigned with separations of nJ(C-H) along the F(1) dimension, and thus even the small long-range J(C--H) values can easily be determined. The efficiency of measuring the long-range J(C--H) using the proposed pulse sequences has been demonstrated in application to the complicated natural product, portmicin.

H(C)P and H(P)C triple‐resonance experiments at natural abundance employing long‐range couplings

Modified two-dimensional (2D) triple-resonance H(C)P and H(P)C experiments based on INEPT/HMQC and double-INEPT schemes are applied to the study of organophosphorus compounds at natural abundances. The implementation of effective (1)H--(13)C gradient selection, additional purging pulsed field gradients, spinlock pulses, and improved phase cycling is demonstrated to allow weak correlation signals based on long-range couplings to be readily observed. Through the combination of two heteronuclear long-range coupling constants, (n)J(CH) and (n)J(PC) in H(C)P experiments or (n)J(PH) and (n)J(PC) in H(P)C experiments, protons can be correlated to a second heteronucleus through 4-7 chemical bonds. These experiments thus overcome the inherit limitations of classical (1)H-X HMBC experiments, which require a nonzero value of the heteronuclear coupling constant (n)J(XH). Ultra-broadband inversion composite pulses are successfully employed in the H(P)C INEPT/HMQC and H(P)C double-INEPT pulse sequences to increase the utility of the experiments and the quality of obtained spectra. This work extends and completes a set of 2D phase-sensitive triple-resonance experiments applicable at natural abundances, and also offers insight into the methodology of triple-resonance experiments and the application of pulsed field gradients. A one-dimensional triple-resonance experiment employing carbon detection is suggested for accurate determination of small (n)J(PC).

Molecular and solid-state structure of methyl [2-(acridin-9-ylimino)-3-( tert-butylamino)-4-oxothiazolidin-5-ylidene]acetate

The molecular and solid-state structures of methyl [2-(acridin-9-ylimino)-3-( tert-butylamino)-4-oxothiazolidin-5-ylidene]acetate ( 4) were determined by X-ray diffraction, 1H NMR, 13C NMR, CI mass spectroscopy and B3LYP calculations using the basis sets 6-31G(d, p), 6-311+G(d, p) and 6-311++G(2d, 2p). In particular, calculations of long-range heteronuclear coupling constants were found to be extraordinarily indicative for the E/ Z configuration of a distal segment. Compound 4 (C 23H 22N 4O 3S, M r = 434.52) crystallized in the P 1 ¯ space group wherein: a = 11.973(4), b = 12.398(3), c = 15.289(4) Å and α = 85.80(2)°, β = 73.70(3)°, γ = 87.93(2)°, V = 2172.2(11) Å 3, Z = 4 and calculated density = 1.329 g cm −3. The formation of dimers, chains and the crystal packing between the molecules by means of intermolecular hydrogen bonds and π–π interactions between neighboring acridine moieties are discussed. NMR spectral analysis and quantum chemical calculations were found to be in good agreement with the results of the X-ray study. The observation of 4 indicates the dominate role that the N-2 nitrogen of the isothiosemicarbazide intermediate plays in its formation via the reaction of 4-(acridin-9-yl)-1-( tert-butyl)thiosemicarbazide with dimethyl acetylenedicarboxylate (DMAD). This dominance is a result of the steric influences of the substituents on the competing nitrogen atoms.

A Simple Method for Measuring Long-Range 1H−13C Coupling Constants in Organic Molecules

This study presents a simple method for measuring long-range heteronuclear coupling constants between protons and proton-bearing carbons. The approach involves recording two conventional 1D-TOCSY experiments in which the offset of the selective proton pulse is set on the low- and high-frequency 13C satellites of an isolated proton signal, Hi. Long-range heteronuclear coupling values between the 13Ci bonded to Hi and the protons Hj,k...n that belong to the same spin system were easily determined from the relative displacement of the relayed Hj,k...n signals in the satellite-selective TOCSY spectra. The sense of the displacement indicated the sign of the coupling constants.