Introduction to NMR Spectroscopy of Carbohydrates - ACS Publications

Chapter 1

Introduction to NMR Spectroscopy of Carbohydrates

Downloaded by MT ALLISON UNIV on May 5, 2013 | http://pubs.acs.org Publication Date: March 9, 2006 | doi: 10.1021/bk-2006-0930.ch001

Johannes F. G. Vliegenthart Bijvoet Center, Department of Bioorganic Chemistry, Utrecht University, Padualaan 8,3584 C H Utrecht, The Netherlands

In this chapter an introductory overview is presented of advances in N M R spectroscopy of carbohydrates. The main emphasis is on the application of H - N M R spectroscopy for identification and structural studies of glycans. 1

Introduction The application of N M R spectroscopy to carbohydrates has a relatively long history, but its suitability for structural analysis has increased enormously in recent years (/). The N M R spectroscopy of biomolecules in general has undergone an almost complete revolution in the past 25 years. Spectacular developments in the instrumentation, pulse sequences, spectral interpretation, isotope labeling of compounds and molecular modeling techniques have led to new possibilities to determine the primary structure and the three-dimensional structure of biomolecules in solution (2). The high resolutions that can be obtained with the most advanced spectrometers allow the unraveling of details of the structure and

© 2006 American Chemical Society

In NMR Spectroscopy and Computer Modeling of Carbohydrates; Vliegenthar, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

1

2 render possible the study of the molecular dynamics in solution. Even for large molecules significant information can be derived. The most impressive progress has been made for proteins and nucleic acids. For these compounds the main chain, the side-chains of the constituting residues and the homo- and heterotypic interactions can be established with a high degree of accuracy. The advances in isotope labeling through cloning techniques and organic chemistry have stimulated the further development of the direct and indirect spectral detection of various nuclei. For carbohydrates and glycoconjugates H and C have proved to be extremely valuable to determine primary structures. In fact the characterization of (partial) structures of glycoprotein-derived N-glycans has greatly facilitated the unraveling of biosynthetic routes and studying the functional roles of these glycans in complex biological systems. Another important aspect concerns the confirmation of the identity of glycan structures that are supposed to be identical to known compounds. Owing to the inherent flexibility of carbohydrate chains, the characterization of the three-dimensional structure in solution is rarely feasible into the same detail as for proteins and nucleic acids. Nevertheless, interesting results have been obtained. For the study of the interaction of carbohydrates with complementary compounds N M R spectroscopy can be a valuable tool (7). The labeling of carbohydrates and glycan chains with isotopes is a bit more cumbersome for carbohydrates than for other bio-macromolecules. Glycanlabeling is eagerly waiting for further innovations. In addition to H and C spectra also those of the nuclei N , 0 , F , P (whether or not fully isotopically enriched) and of several metal ions in carbohydrates have been recorded. Obviously, resolution and sensitivity are different for the various nuclei and thereby decisive for the type of information that can be extracted from the spectra. This introductory chapter will mainly be focused on H - N M R spectra carbohydrates and glycoconjugates.


l

1 3

!

1 5

1 7

19

1 3

3 1

l

*H NMR spectra of glycans and the reporter group concept 2

Usually, the spectra of unprotected glycans are recorded in H 0 after full exchange of the exchangeable protons (3.4). Spectra recorded at N M R machines operating at 500 M H z or at higher frequencies contain sufficient details to be used as identity card (5). For the (partial) assignment of the resonances in novel compounds additional N M R experiments are needed. For the characterization of compounds described in literature, mostly comparison of the spectral data to reference data is sufficient. Two groups of signals can be distinguished. First, 2



3 the so-called bulk signal containing mainly the non-anomeric protons, present in a rather narrow spectral range between 3.2 and 3.9 ppm. Secondly, the structural-reporter-group signals that are found outside the bulk region (6-8). The chemical shift patterns of the structural reporter groups comprising chemical shifts and couplings are translated into structural information, based on a comparison to patterns in a library of relevant reference compounds. The comparison of N M R data for many closely related glycans resulted in empirical rules to correlate chemical shift values with carbohydrate structures. Successful application of this approach requires accurate calibration of the experimental conditions like sample temperature, solvent and pH (3.4). The structural reporter group signals can be subdivided into the following categories: Anomeric protons: shifted downfield, due to their relative unshielding by the ring oxygen atom. Protons that can be discerned outside of the bulk region, as a result of glycosylation shifts, or under influence of substituents such as sulfate, phosphate and acyl groups. Deoxysugar protons. Alkyl and acyl substituents like methyl and acetyl, glycolyl, pyruvate, respectively. The N M R database 'sugabase' to identify glycan structures has been founded on such assignments (8Ί0). It should be emphasized that definitive conclusions on the identity of novel compounds invariably require validation by experimental data from independent approaches. Since the reporter group signals are relatively insensitive for alterations in the structural elements remote from the corresponding locus, the structural-reporter group concept has proved its usefulness for the identification of numerous compounds. In particular, the concept is invaluable for the analysis of glycoconjugate-glycans that form an ensemble of closely related compounds.

2D-spectra To assign resonances in the region of the bulk signal and of coinciding structure reporter group signals, 2-D homonuclear correlation type of spectra, such as various C O S Y or T O C S Y experiments are needed. In this way spin systems corresponding to monosaccharide constituents can be traced. In general the interpretation of such spectra can start from an anomeric signal or from any other well-resolved signal. For compound I (see Fig. 1), a heptasaccharide methyl β-glycoside, corresponding to a low molecular mass glycan of a - . hemocyanin of the snail Helix Pomatia (77), the T O C S Y spectrum is depicted in Fig.2 (72). D


4

Mama 1-6

Fucal-6 \

\ Manp 1 -4GlcNAcp 1 -4GlcNAcp 1 -OMe

/ Manal-3 Xyipi-2 Figure 1. Structure of compound 1. The monosaccharide constituents are in the text and spectra abbreviated as: 4 = Manal-6, 4 = Manal-3, 3 = Μαηβ1-4, 2 = GlcNAcpi-4, 1 = GlcNAcfil-, X = Xylfil-2, F = Fucal-6, OMe = O-Methyl.


9

(p.p.m.) XH1 Β

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is

XH4 »

«

A

H

8

o

0

91

3H4 3H6R 3H3 (,WB& · oo l i ilo< - Ο 3H5 , e

ο ' 1H6S ^ > = : : : = ^ XH5eq

I

H

W P l H s W (fil « O h , , (ft * - - « XH4 X H 3 | XH5ax XH2

1

-

2H1

Τ 3H1 3MZ 4Ή1>

»0··[!ββ&"@ 2H6S/|\2H5 2H2/H3/H4 FH2° FH3 4Ή2 0 4H2

4

.*

H3

ίί~ β 4H3

—I— 3.8

l

l

Figure 2 H- H

' II' *

igrvmn ' ;·» «· «·

< L J 4

X

on »»·

*

™*0t-— 3H2 0 - -

XH3 XH2 «Ι» Φ — X H S a x

*•

Μβ^ίΜ

I 1 Ί'

XH5eq « »

Ί

4Ή4/Η5 · ««· 4H4

—I— 3.4

(p.p.m.)

TOCSY spectrum at 500 MHz, 281.5 K, mixing time 100ms.


5


Homomiclear C O S Y and T O C S Y spectra do not provide monosaccharide sequence information, due to the absence of coupling over the glycosidic linkage. Often N O E S Y or R O E S Y spectra are used for this purpose. In many cases the most intense N O E S Y peak identifies the linkage, but not always. In Fig. 3 the R O E S Y spectrum of compound 1 is presented (72).

„FH5§ • »3H2 0 it

ο

f—«000 FH3 FH4 m

§8 · ·

4H5 3H3

1

H

- 2 1H3/H5 ^ Ο Μ θ

1H1 S : : : : : : : : § ^ = ^ = = ^ îf 2H1 00 3H1 FH1^ \

4H1 j -

«0^«lOf

2H2/H3/H4

0| :::: f

*H3 | X

1H4lirÎH6R -

H

ifle

XH5ax 2

Cfi^"3)-β-Ι>Ό»1ρΚ 1 ->3)-a-l>Ga|p-(l - > 3 ) - a - L - R h « K l - > 2 ) - a - L - R h v - ( I ->2)-a-D-Galp-( 1 ->

Figure 5. Structure of the repeating unit of the exopolysaccharide of Streptococcus thermophilus S3.

C6/F6

HSQC


OAc—Ô

2.80

2.60

2.40

—

Introduction to NMR Spectroscopy of Carbohydrates - ACS Publications

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