Nuclear magnetic resonance spectroscopy of carbohydrates

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Carbohydrate NMR spectroscopy is the application of nuclear magnetic resonance (NMR) spectroscopy to structural and conformational analysis of carbohydrates. This method allows the scientists to elucidate structure of monosaccharides, oligosaccharides, polysaccharides, glycoconjugates and other carbohydrate derivatives from synthetic and natural sources. Among structural properties that could be determined by NMR are primary structure (including stereochemistry), saccharide conformation, stoichiometry of substituents, and ratio of individual saccharides in a mixture. Modern high field NMR instruments used for carbohydrate samples, typically 500 MHz or higher, are able to run a suite of 1D, 2D, and 3D experiments to determine a structure of carbohydrate compounds.

Contents

Carbohydrate NMR observables

Chemical shift

Common chemical shift ranges for nuclei within carbohydrate residues are:

In the case of simple mono- and oligosaccharide molecules, all proton signals are typically separated from one another (usually at 500 MHz or better NMR instruments) and can be assigned using 1D NMR spectrum only. However, bigger molecules exhibit significant proton signal overlap, especially in the non-anomeric region (3-4 ppm). Carbon-13 NMR overcomes this disadvantage by larger range of chemical shifts and special techniques allowing to block carbon-proton spin coupling, thus making all carbon signals high and narrow singlets distinguishable from each other.

The typical ranges of specific carbohydrate carbon chemical shifts in the unsubstituted monosaccharides are:

Coupling constants

Direct carbon-proton coupling constants are used to study the anomeric configuration of a sugar. Vicinal proton-proton coupling constants are used to study stereo orientation of protons relatively to the other protons within a sugar ring, thus identifying a monosaccharide. Vicinal heteronuclear H-C-O-C coupling constants are used to study torsional angles along glycosidic bond between sugars or along exocyclic fragments, thus revealing a molecular conformation.

Sugar rings are relatively rigid molecular fragments, thus vicinal proton-proton couplings are characteristic:

Nuclear Overhauser effects (NOEs)

NOEs are sensitive to interatomic distances, allowing their usage as a conformational probe, or proof of a glycoside bond formation. It's a common practice to compare calculated to experimental proton-proton NOEs in oligosaccharides to confirm a theoretical conformational map. Calculation of NOEs implies an optimization of molecular geometry.

Other NMR observables

Relaxivities, nuclear relaxation rates, line shape and other parameters were reported useful in structural studies of carbohydrates. [1]

Elucidation of carbohydrate structure by NMR spectroscopy

Structural parameters of carbohydrates

The following is a list of structural features that can be elucidated by NMR:

NMR spectroscopy vs. other methods

Widely known methods of structural investigation, such as mass-spectrometry and X-ray analysis are only limitedly applicable to carbohydrates. [1] Such structural studies, such as sequence determination or identification of new monosaccharides, benefit the most from the NMR spectroscopy. Absolute configuration and polymerization degree are not always determinable using NMR only, so the process of structural elucidation may require additional methods. Although monomeric composition can be solved by NMR, chromatographic and mass-spectroscopic methods provide this information sometimes easier. The other structural features listed above can be determined solely by the NMR spectroscopic methods. The limitation of the NMR structural studies of carbohydrates is that structure elucidation can hardly be automatized and require a human expert to derive a structure from NMR spectra.

Application of various NMR techniques to carbohydrates

Complex glycans possess a multitude of overlapping signals, especially in a proton spectrum. Therefore, it is advantageous to utilize 2D experiments for the assignment of signals. The table and figures below list most widespread NMR techniques used in carbohydrate studies.

Heteronuclear NMR techniques in carbohydrate studies, and typical intra-residue (red) and inter-residue (blue) atoms that they link each to other. Heteronuclear correlations in saccharides.jpg
Heteronuclear NMR techniques in carbohydrate studies, and typical intra-residue (red) and inter-residue (blue) atoms that they link each to other.
Homonuclear NMR techniques in carbohydrate studies, and typical intra-residue (red) and inter-residue (blue) atoms that they link each to other. Homonuclear correlations in saccharides.jpg
Homonuclear NMR techniques in carbohydrate studies, and typical intra-residue (red) and inter-residue (blue) atoms that they link each to other.
NMR experimentDescriptionInformation obtained
1H 1D1D proton spectrummeasurement of couplings, general information, residue identification, basis for carbon spectrum assignment
13C BBProton-decoupled 1D carbon-13 spectrumdetailed information, residue identification, substitution positions
31P BB, 15N BBProton-decoupled 1D heteronuclei spectraadditional information
APT, 13C DEPTattached proton test, driven enhanced polarization transfer (edited 1D carbon-13 spectrum)assignment of CH2 groups
13C Gated, 31P GatedProton-coupled 1D carbon-13 and heteronuclei spectrameasurement of heteronuclear couplings, elucidation of anomeric configuration, conformational studies
1H,1H J-resolved2D NMR plot showing J-couplings in second dimensionaccurate J-couplings and chemical shift values for crowded spectral regions
1H DOSY2D NMR plot with proton spectra as a function of molecular diffusion coefficientmeasurement of diffusion coefficient, estimate of molecular size/weight, spectral separation of different molecules in a mixture
1H,1H COSYProton spin correlationproton spectrum assignment using vicinal couplings
COSY RCT, COSY RCT2Proton spin correlation with one- or two-step relayed coherence transferproton spectrum assignment where signals of neighboring vicinal protons overlap
DQF COSYDouble-quantum filtered proton spin correlationJ-coupling magnitudes & number of protons participating in the J-coupling
1H HD difSelective differential homodecouplingline shape analysis of the overlapped proton signals
TOCSY (HOHAHA)Total correlation of all protons within a spin systemdistinguishing of spin systems of residues
1D TOCSYTOCSY of a single signalextraction of a spin system of a certain residue
NOESY, ROESYHomonuclear Nuclear Overhauser effect correlation (through space)revealing of spatially proximal proton pairs, determination of a sequence of residues, determination of averaged conformation
1H NOE difSelective differential NOE measurementstudies of proton spatial contacts
1H,13C HSQCHeteronuclear single-quantum coherence, direct proton-carbon spin correlationcarbon spectrum assignment
1H,31P HSQCHeteronuclear single-quantum coherence, proton-phosphorus spin correlationlocalization of phosphoric acid residues in phosphoglycans
1H,13C HMBCHeteronuclear multiple-bond correlation, vicinal proton-carbon spin correlationdetermination of residue sequence, acetylation/amidation pattern, confirmation of substitution positions
1H,X 1D HMBCHMBC for a single signalassignment of proton around a certain carbon or heteroatom
1H,13C HSQC RelayImplicit carbon-carbon correlation via vicinal couplings of the attached protonsassignment of neighboring carbon atoms
1H,13C HSQC-TOCSYCorrelation of protons with all carbons within a spin system, and vice versaassignment of C5 using H6 and solving similar problems, separation of carbon spectrum into subspectra of residues
1H,X 1D NOEHeteronuclear NOE measurementheteronuclear spatial contacts, conformations

Research scheme

NMR spectroscopic research includes the following steps:

Approximate scheme of NMR (blue) and other (green) techniques applied to carbohydrate structure elucidation, and information obtained (in boxes) Glycan NMR investigation.jpg
Approximate scheme of NMR (blue) and other (green) techniques applied to carbohydrate structure elucidation, and information obtained (in boxes)

Carbohydrate NMR databases and tools

Multiple chemical shift databases and related services have been created to aid structural elucidation of and expert analysis of their NMR spectra. Of them, several informatics tools are dedicated solely to carbohydrates:

Simulation of the NMR observables

Comparative prediction of the 13C NMR spectrum of sucrose using various methods. Experimental spectrum is in the middle. Upper spectrum (black) was obtained by empirical routine. Lower spectra (red and green) were obtained by quantum-chemical calculations in PRIRODA and GAUSSIAN respectively. Included information: used theory level/basis set/solvent model, accuracy of prediction (linear correlation factor and root mean square deviation), calculation time on personal computer (blue). GlycoNMR simulation.jpg
Comparative prediction of the 13C NMR spectrum of sucrose using various methods. Experimental spectrum is in the middle. Upper spectrum (black) was obtained by empirical routine. Lower spectra (red and green) were obtained by quantum-chemical calculations in PRIRODA and GAUSSIAN respectively. Included information: used theory level/basis set/solvent model, accuracy of prediction (linear correlation factor and root mean square deviation), calculation time on personal computer (blue).

Several approaches to simulate NMR observables of carbohydrates has been reviewed. [1] They include:

Growing computational power allows usage of thorough quantum-mechanical calculations at high theory levels and large basis sets for refining the molecular geometry of carbohydrates and subsequent prediction of NMR observables using GIAO and other methods with or without solvent effect account. Among combinations of theory level and a basis set reported as sufficient for NMR predictions were B3LYP/6-311G++(2d,2p) and PBE/PBE (see review). It was shown for saccharides that carbohydrate-optimized empirical schemes provide significantly better accuracy (0.0-0.5 ppm per 13C resonance) than quantum chemical methods (above 2.0 ppm per resonance) reported as best for NMR simulations, and work thousands times faster. However, these methods can predict only chemical shifts and perform poor for non-carbohydrate parts of molecules. As a representative example, see figure on the right.

See also

Related Research Articles

<span class="mw-page-title-main">Glycome</span> Complete set of all sugars, free or bound, in an organism.

A glycome is the entire complement or complete set of all sugars, whether free or chemically bound in more complex molecules, of an organism. An alternative definition is the entirety of carbohydrates in a cell. The glycome may in fact be one of the most complex entities in nature. "Glycomics, analogous to genomics and proteomics, is the systematic study of all glycan structures of a given cell type or organism" and is a subset of glycobiology.

The nuclear Overhauser effect (NOE) is the transfer of nuclear spin polarization from one population of spin-active nuclei to another via cross-relaxation. A phenomenological definition of the NOE in nuclear magnetic resonance spectroscopy (NMR) is the change in the integrated intensity of one NMR resonance that occurs when another is saturated by irradiation with an RF field. The change in resonance intensity of a nucleus is a consequence of the nucleus being close in space to those directly affected by the RF perturbation.

In carbohydrate chemistry, a pair of anomers is a pair of near-identical stereoisomers or diastereomers that differ at only the anomeric carbon, the carbon atom that bears the aldehyde or ketone functional group in the sugar's open-chain form. However, in order for anomers to exist, the sugar must be in its cyclic form, since in open-chain form, the anomeric carbon atom is planar and thus achiral. More formally stated, then, an anomer is an epimer at the hemiacetal/hemiketal carbon atom in a cyclic saccharide. Anomerization is the process of conversion of one anomer to the other. As is typical for stereoisomeric compounds, different anomers have different physical properties, melting points and specific rotations.

In nuclear magnetic resonance (NMR) spectroscopy, the chemical shift is the resonant frequency of an atomic nucleus relative to a standard in a magnetic field. Often the position and number of chemical shifts are diagnostic of the structure of a molecule. Chemical shifts are also used to describe signals in other forms of spectroscopy such as photoemission spectroscopy.

<span class="mw-page-title-main">Nuclear magnetic resonance spectroscopy</span> Laboratory technique

Nuclear magnetic resonance spectroscopy, most commonly known as NMR spectroscopy or magnetic resonance spectroscopy (MRS), is a spectroscopic technique based on re-orientation of atomic nuclei with non-zero nuclear spins in an external magnetic field. This re-orientation occurs with absorption of electromagnetic radiation in the radio frequency region from roughly 4 to 900 MHz, which depends on the isotopic nature of the nucleus and increased proportionally to the strength of the external magnetic field. Notably, the resonance frequency of each NMR-active nucleus depends on its chemical environment. As a result, NMR spectra provide information about individual functional groups present in the sample, as well as about connections between nearby nuclei in the same molecule. As the NMR spectra are unique or highly characteristic to individual compounds and functional groups, NMR spectroscopy is one of the most important methods to identify molecular structures, particularly of organic compounds.

Carbon-13 (C13) nuclear magnetic resonance is the application of nuclear magnetic resonance (NMR) spectroscopy to carbon. It is analogous to proton NMR and allows the identification of carbon atoms in an organic molecule just as proton NMR identifies hydrogen atoms. 13C NMR detects only the 13
C
isotope. The main carbon isotope, 12
C
does not produce an NMR signal. Although ca. 1 mln. times less sensitive than 1H NMR spectroscopy, 13C NMR spectroscopy is widely used for characterizing organic and organometallic compounds, primarily because 1H-decoupled 13C-NMR spectra are more simple, have a greater sensitivity to differences in the chemical structure, and, thus, are better suited for identifying molecules in complex mixtures. At the same time, such spectra lack quantitative information about the atomic ratios of different types of carbon nuclei, because nuclear Overhauser effect used in 1H-decoupled 13C-NMR spectroscopy enhances the signals from carbon atoms with a larger number of hydrogen atoms attached to them more than from carbon atoms with a smaller number of H's, and because full relaxation of 13C nuclei is usually not attained, and the nuclei with shorter relaxation times produce more intense signals.

<span class="mw-page-title-main">Proton nuclear magnetic resonance</span> NMR via protons, hydrogen-1 nuclei

Proton nuclear magnetic resonance is the application of nuclear magnetic resonance in NMR spectroscopy with respect to hydrogen-1 nuclei within the molecules of a substance, in order to determine the structure of its molecules. In samples where natural hydrogen (H) is used, practically all the hydrogen consists of the isotope 1H.

Nuclear magnetic resonance spectroscopy of proteins is a field of structural biology in which NMR spectroscopy is used to obtain information about the structure and dynamics of proteins, and also nucleic acids, and their complexes. The field was pioneered by Richard R. Ernst and Kurt Wüthrich at the ETH, and by Ad Bax, Marius Clore, Angela Gronenborn at the NIH, and Gerhard Wagner at Harvard University, among others. Structure determination by NMR spectroscopy usually consists of several phases, each using a separate set of highly specialized techniques. The sample is prepared, measurements are made, interpretive approaches are applied, and a structure is calculated and validated.

<span class="mw-page-title-main">Anomeric effect</span> Tendency of some substituents on a cyclohexane ring to prefer axial orientation

In organic chemistry, the anomeric effect or Edward-Lemieux effect is a stereoelectronic effect that describes the tendency of heteroatomic substituents adjacent to a heteroatom within a cyclohexane ring to prefer the axial orientation instead of the less-hindered equatorial orientation that would be expected from steric considerations. This effect was originally observed in pyranose rings by J. T. Edward in 1955 when studying carbohydrate chemistry.

Two-dimensional nuclear magnetic resonance spectroscopy is a set of nuclear magnetic resonance spectroscopy (NMR) methods which give data plotted in a space defined by two frequency axes rather than one. Types of 2D NMR include correlation spectroscopy (COSY), J-spectroscopy, exchange spectroscopy (EXSY), and nuclear Overhauser effect spectroscopy (NOESY). Two-dimensional NMR spectra provide more information about a molecule than one-dimensional NMR spectra and are especially useful in determining the structure of a molecule, particularly for molecules that are too complicated to work with using one-dimensional NMR.

Nuclear magnetic resonance (NMR) in the geomagnetic field is conventionally referred to as Earth's field NMR (EFNMR). EFNMR is a special case of low field NMR.

In vivo magnetic resonance spectroscopy (MRS) is a specialized technique associated with magnetic resonance imaging (MRI).

<span class="mw-page-title-main">Fluorine-19 nuclear magnetic resonance spectroscopy</span> Analytical technique

Fluorine-19 nuclear magnetic resonance spectroscopy is an analytical technique used to detect and identify fluorine-containing compounds. 19F is an important nucleus for NMR spectroscopy because of its receptivity and large chemical shift dispersion, which is greater than that for proton nuclear magnetic resonance spectroscopy.

Carbohydrate conformation refers to the overall three-dimensional structure adopted by a carbohydrate (saccharide) molecule as a result of the through-bond and through-space physical forces it experiences arising from its molecular structure. The physical forces that dictate the three-dimensional shapes of all molecules—here, of all monosaccharide, oligosaccharide, and polysaccharide molecules—are sometimes summarily captured by such terms as "steric interactions" and "stereoelectronic effects".

Nuclear magnetic resonance decoupling is a special method used in nuclear magnetic resonance (NMR) spectroscopy where a sample to be analyzed is irradiated at a certain frequency or frequency range to eliminate or partially the effect of coupling between certain nuclei. NMR coupling refers to the effect of nuclei on each other in atoms within a couple of bonds distance of each other in molecules. This effect causes NMR signals in a spectrum to be split into multiple peaks. Decoupling fully or partially eliminates splitting of the signal between the nuclei irradiated and other nuclei such as the nuclei being analyzed in a certain spectrum. NMR spectroscopy and sometimes decoupling can help determine structures of chemical compounds.

<span class="mw-page-title-main">Nuclear magnetic resonance</span> Spectroscopic technique based on change of nuclear spin state

Nuclear magnetic resonance (NMR) is a physical phenomenon in which nuclei in a strong constant magnetic field are perturbed by a weak oscillating magnetic field and respond by producing an electromagnetic signal with a frequency characteristic of the magnetic field at the nucleus. This process occurs near resonance, when the oscillation frequency matches the intrinsic frequency of the nuclei, which depends on the strength of the static magnetic field, the chemical environment, and the magnetic properties of the isotope involved; in practical applications with static magnetic fields up to ca. 20 tesla, the frequency is similar to VHF and UHF television broadcasts (60–1000 MHz). NMR results from specific magnetic properties of certain atomic nuclei. High-resolution nuclear magnetic resonance spectroscopy is widely used to determine the structure of organic molecules in solution and study molecular physics and crystals as well as non-crystalline materials. NMR is also routinely used in advanced medical imaging techniques, such as in magnetic resonance imaging (MRI). The original application of NMR to condensed matter physics is nowadays mostly devoted to strongly correlated electron systems. It reveals large many-body couplings by fast broadband detection and should not be confused with solid state NMR, which aims at removing the effect of the same couplings by Magic Angle Spinning techniques.

Nucleic acid NMR is the use of nuclear magnetic resonance spectroscopy to obtain information about the structure and dynamics of nucleic acid molecules, such as DNA or RNA. It is useful for molecules of up to 100 nucleotides, and as of 2003, nearly half of all known RNA structures had been determined by NMR spectroscopy.

<span class="mw-page-title-main">Carbohydrate Structure Database</span>

Carbohydrate Structure Database (CSDB) is a free curated database and service platform in glycoinformatics, launched in 2005 by a group of Russian scientists from N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences. CSDB stores published structural, taxonomical, bibliographic and NMR-spectroscopic data on natural carbohydrates and carbohydrate-related molecules.

<span class="mw-page-title-main">Platinum-195 nuclear magnetic resonance</span>

Platinum-195 nuclear magnetic resonance spectroscopy is a spectroscopic technique which is used for the detection and characterisation of platinum compounds. The sensitivity of the technique and therefore its diagnostic utility have increased significantly starting from the 1970s, with 195Pt NMR nowadays considered the method of choice for structural elucidation of Pt species in solution.

Glycan nomenclature is the systematic naming of glycans, which are carbohydrate-based polymers made by all living organisms. In general glycans can be represented in (i) text formats, these include commonly used CarbBank, IUPAC name, and several other types; and (ii) symbol formats, these are consisting of Symbol Nomenclature For Glycans and Oxford Notations.

References

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Further reading