Nuclear magnetic resonance spectroscopy of carbohydrates

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.

Main article: Nuclear magnetic resonance

Carbohydrate NMR observables

Chemical shift
Main article: chemical shift

Common chemical shift ranges for nuclei within carbohydrate residues are:

  • Typical 1H NMR chemical shifts of carbohydrate ring protons are 3–6 ppm (4.5–5.5 ppm for anomeric protons).
  • Typical 13C NMR chemical shifts of carbohydrate ring carbons are 60–110 ppm

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:

  • Anomeric carbons: 90-100 ppm
  • Sugar ring carbons bearing a hydroxy function: 68-77
  • Open-form sugar carbons bearing a hydroxy function: 71-75
  • Sugar ring carbons bearing an amino function: 50-56
  • Exocyclic hydroxymethyl groups: 60-64
  • Exocyclic carboxy groups: 172-176
  • Desoxygenated sugar ring carbons: 31-40
  • A carbon at pyranose ring closure: 71-73 (α-anomers), 74-76 (β-anomers)
  • A carbon at furanose ring closure: 80-83 (α-anomers), 83-86 (β-anomers)

Coupling constants
Main article: coupling constant

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:

  • Equatorial to axial: 1–4 Hz
  • Equatorial to equatorial: 0–2 Hz
  • Axial to axial non-anomeric: 9–11 Hz
  • Axial to axial anomeric: 7–9 Hz
  • Axial to exocyclic hydroxymethyl: 5 Hz, 2 Hz
  • Geminal between hydroxymethyl protons: 12 Hz

Nuclear Overhauser effects (NOEs)
Main article: Nuclear Overhauser effect

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:

  • Chemical structure of each carbohydrate residue in a molecule, including
    • carbon skeleton size and sugar type (aldose/ketose)
    • cycle size (pyranose/furanose/linear)
    • stereo configuration of all carbons (monosaccharide identification)
    • stereo configuration of anomeric carbon (α/β)
    • absolute configuration (D/L)
    • location of amino-, carboxy-, deoxy- and other functions
  • Chemical structure of non-carbohydrate residues in molecule (amino acids, fatty acids, alcohols, organic aglycons etc.)
  • Substitution positions in residues
  • Sequence of residues
  • Stoichiometry of terminal residues and side chains
  • Location of phosphate and sulfate diester bonds
  • Polymerization degree and frame positioning (for polysaccharides)

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.
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:

  • Extraction of carbohydrate material (for natural glycans)
  • Chemical removal of moieties masking regularity (for polymers)
  • Separation and purification of carbohydrate material (for 2D NMR experiments, 10 mg or more is recommended)
  • Sample preparation (usually in D2O)
  • Acquisition of 1D spectra
  • Planning, acquisition and processing of other NMR experiments (usually requires from 5 to 20 hours)
  • Assignment and interpretation of spectra (see exemplary figure)
  • If a structural problem could not be solved: chemical modification/degradation and NMR analysis of products
  • Acquisition of spectra of the native (unmasked) compound and their interpretation based on modified structure
  • Presentation of results
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:

  • GlycoSCIENCES.de
    • over 2000 NMR spectra of mammalian glycans[2]
    • search of structure by NMR signals and vice versa
  • CSDB (carbohydrate structure database[3][4]) contains:
    • over 4000 NMR spectra of bacterial, plant and fungal glycans,
    • search of structure by NMR signals and vice versa
    • empirical spectra simulation routine optimized for carbohydrates,[5]
    • statistical chemical shift estimation based on HOSE algorithm optimized for carbohydrates,[6][7]
    • structure generation and NMR-based ranking tool.[8]
  • CASPER (computer assisted spectrum evaluation of regular polysaccharides).[9][10] contains:
    • chemical shift database,
    • empirical spectra simulation routine optimized for carbohydrates,
    • online interface.
    • structure matching tool. Both proton and carbon C and H chemical shifts can be used to access structural information.

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).

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

  • Universal statistical database approaches (ACDLabs, Modgraph, etc.)
  • Usage of neural networks to refine the predictions
  • Regression based methods
  • CHARGE
  • Carbohydrate-optimized empirical schemes (CSDB/BIOPSEL,CASPER).
  • Combined molecular mechanics/dynamics geometry calculation and quantum-mechanical simulation/iteration of NMR observables (PERCH NMR Software)
  • ONIOM approaches (optimization of different parts of molecule with different accuracy)
  • Ab initio calculations.

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

References
  1. Toukach F.V.; Ananikov V.P. (2013). "Recent advances in computational predictions of NMR parameters for structure elucidation of carbohydrates: methods and limitations". Chemical Society Reviews. 42 (21): 8376–8415. doi:10.1039/C3CS60073D. PMID 23887200.
  2. http://csdb.glycosciences.de
  3. http://csdb.glycoscience.ru
  4. Toukach Ph.V. (2011). "Bacterial Carbohydrate Structure Database 3: Principles and Realization". Journal of Chemical Information and Modeling. 51 (1): 159–170. doi:10.1021/ci100150d. PMID 21155523.
  5. http://csdb.glycoscience.ru/bacterial/index.html?help=nmr#empirical
  6. http://csdb.glycoscience.ru/bacterial/index.html?help=nmr#statistical
  7. Kapaev R.R.; Egorova K.S.; Toukach Ph.V. (2014). "Carbohydrate structure generalization scheme for database-driven simulation of experimental observables, such as NMR chemical shifts". Journal of Chemical Information and Modeling. 54 (9): 2594–2611. doi:10.1021/ci500267u. PMID 25020143.
  8. http://csdb.glycoscience.ru/bacterial/index.html?help=nmr#grass
  9. http://www.casper.organ.su.se/casper/
  10. P.-E. Jansson; R. Stenutz; G. Widmalm (2006). "Sequence determination of oligosaccharides and regular polysaccharides using NMR spectroscopy and a novel Web-based version of the computer program CASPER". Carbohydrate Research. 341 (8): 1003–1010. doi:10.1016/j.carres.2006.02.034. PMID 16564037.
  11. http://toukach.ru/nmrglyco.htm
  12. http://toukach.ru/glyco-db.htm

Further reading
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