introduction to nmr spectroscopy of carbohydrates

8/10/2019 Introduction to NMR Spectroscopy of Carbohydrates

1/19

Chapter 1

Introduction

toNMR

Spectroscopy

of Carbohydrates

Johannes

F.

G .

Vliegenthart

BijvoetCenter,Departmentof

Bioorganic

Chemistry,

Utrecht University,

Padualaan8 3584

C H

Utrecht,

The

Netherlands

In this chapter an introductory overview is presented of

advances in NMR spectroscopy of carbohydrates. The main

emphasis is on the application of

1

H - N M R

spectroscopy for

identification

and structural studies ofglycans.

Introduction

The

application

o f N M R

spectroscopy to carbohydrates has arelatively long

history,

but its suitability for structural analysis has increased enormously in

recent years (/).

TheN M Rspectroscopy ofbiomoleculesin 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

biomoleculesin solution (2). The high resolutionsthatcan be obtainedwith the

most advanced spectrometers allowthe unraveling of details of the structure and

2006

American Chemical

Society

1

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


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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

o f

the direct and indirect spectral detection of

various

nuclei.

For

carbohydrates and glycoconjugates

l

H and

1 3

C have proved to be

extremely valuable to determine primary structures. In fact the characterization

o f (partial) structures of glycoprotein-derived N-glycans has greatly facilitated

the unraveling of biosynthetic

routes

and studying the functional roles ofthese

glycans in complex

biological

systems. Another important aspect concerns the

confirmation

o f

the identity ofglycanstructures

that

are supposed to be identical

to known compounds.

Owing

to the inherent

flexibility

of carbohydrate chains, the characterization

o f 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.

G l y c a n -

labeling is eagerly waiting for further innovations. In addition to

H and

1 3

C

spectra also

those

of the nuclei

1 5

N ,

1 7

0,

1 9

F,

3 1

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

w i l l

mainly be focused on

l

H - N M R spectra

carbohydrates and glycoconjugates.

H

NMR

spectra

ofglycansand the reportergroupconcept

Usually,

the spectra of unprotected glycans are recorded in

2

H

2

0

after

full

exchange of the exchangeable protons

3.4).

Spectra recorded at

N M R

machines

operating at 500 MHz or at higher frequencies contain sufficient details to be

used as identity card (5). For the (partial) assignment of the resonances in novel

compounds additionalN 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,

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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

o fthe 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

l ike

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

glycosylationshifts, or under influence of substituents such as sulfate, phosphate

and

acyl

groups.

Deoxysugar

protons.

A l k y l

and

acyl

substituents

like

methyl and acetyl,

g lyco ly l ,

pyruvate,

respectively.

The NMR

database

'sugabase' to identify glycan structures has been

founded

on such assignments

(80).

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 -glycoside,

corresponding to a low molecular mass glycan of a

D

-.

hemocyanin of the

snail

Helix Pomatia

(77), the

T O C S Y

spectrum is depicted in

Fig.2(72).

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4

Mama

1-6

Fucal-6

\ \

Manp

1 -4GlcNA cp 1 -4GlcNA cp 1 -OM e

/

Manal-3

X y i p i - 2

Figure

1.Structureof

compound

1. The monosaccharideconstituents are in the

textand spectra abbreviated as: 4

9

=Manal-6, 4 =

Manal-3,

3 = 1 -4,

2 =

GlcNAcpi-4,

1 = GlcNAcfil-, X =

Xylfil-2,

F =

Fucal-6,

OMe =O-Methyl.

(p.p.m.)

1

'

I

2H1

3 H 1

4 1 >

XH 1 X H 5 e q X H 4 X H 3 X H 2

X H S a x

is

* on

I ^ ' II' *

H

A *

8

91

o

0

igrvmn

;

*0t-

e

3 H 4

3H6R 3 H 3 (,WB

3 H2 0 - - oo l i i lo< - 3 H 5 ,

< L J 4

' 1 H 6 S W P l H s W

^ > = : : : = ^

( f i l O h , , (f t * - -

X H 1

X H 5 e q X H 4 X H 3 | XH5a x

X H 2

- 0 [ & " @

2 H 6 S / | \ 2 H 5

2H2/H3/H4

3M Z

F H 2 F H 3

4 2

4

. *

H 3

4 4/ 5

0 ~

4H 2 4 H 3 4 H 4

I

3.8

I

3.4

(p.p.m.)

Figure

2

l

H-

l

H

TOCSY

spectrumat 500 M Hz, 281.5 K,

mixing

time100ms.

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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 areused forthis purpose. Inmany

cases themost intense

N O E S Y

peak identifiesthe linkage,but notalways.In

F i g .3 theR O E S Y

spectrum of

compound

1

is

presented (72).

2H1 00

3 H 1

F H 1 ^ \

4 1 ;

4 H 1 j -

? y

2 H 1

FH5 f 0 0 0

FH3 FH4

3 H 2 0 m 8

it 4H5 3H3

1 H

- 21 H 3 / H 5 ^

1 H1

S : : : : : : : : ^ = ^ = = ^ 0 ^ l O f 0 |

f

: : : :

i f l e

f 3H2 2 H 2 / H 3 / H 4 * H3 | X H 5 a x

H lirH6R

X H 2

- C f i ^ < f e 2 H 5

3 H 2

3H3

2 H 4 3 H 5

j

= ^ ^ \

C l H 6 S FH2]1H6R

4 2 3 H 6 S

( f i * '

4 H 2 3 H 3 . 3 H 4

I

4.2

3 -

4

(P .p m. )

Figure 3.ROESYspectrum of compoundIat 500M z, 281.5K,

mixing-time 155

ms.

* H -

1 3

C

Heteronuclear multiple

andsinglequantumcoherence

spectra,

H -

I 3

C H M Q C

and

H S Q C spectra provide important correlations.Owing

to

the usually large dispersion

of

the

1 3

C

signals,

these

spectra have

a

great value

fo r

the

interpretation. Often

in the

H M Q C spectra

the

one-bond correlations

are

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7

P-D-Gtf2 A c

0 4

1

4

6

- >3 )- - > 1 1 ->3)-a-l>Ga|p-(l- > 3 ) - a - L - R h K l- >2)-a-L-Rhv-( I

->2)-a-D-Galp-(

->

Figure 5.Structure of the repeatingunitoftheexopolysaccharideof

Streptococcus thermophilus S3.

HSQC

C6/F6 =

O A c

6

68 . 0

P P M

7 2 . 0

7 6 . 0

9 5 . 0

1 0 3 . 0

1 0 7 . 0

2 . 8 0

2.60 2.40 2.20 2.00 1.80 1.60 1.40 1.20 1.00

C2

-

A4

^ 5 ^ _

0 4

-

- - -D2

E3

C5

D S - c ^ ^ ^ ^ ; ; ; ; ; ; ; ; ; ^ : : ; : ; ; : ; - ; ; ^ . .

E 4

E S - ^ - I M

g ^ ^

' ^ B 5 / B 5

E2 - < > _

B' 3 5

B3

$ i B ) s S b . - . - ~ ~

C 3

B2 ^

~~ A3

D3

B'4

F2

. :. 3 . - - 4

5 .10 4.90 4.70 4.50 4.30 4.10 3.90 3.70 3.50 3.30

E1

D 1

C l


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8

carbon atom and the proton of the adjacent sugar residue

asw e ll

asbetweenthe

anomeric proton and the ring carbon atom

o f

the

adjacent

sugar residue.

In

particular

in

assigning

the structure of the

repeating units

in

polysaccharides, these spectra

are

very helpful. This

is

illustrated

for the

repeating unit of the exopolysaccharide ofLactobacillus delbrueckii

subspecies

bulgaricus ( F i g .7)

in the

H M B C

spectrum shown in

F i g .8 14).

A

P -D- G a l p - l - 4 ) - p -D-Gl p

1

I

6

-*4 )P -D-Glp- 1- > 4 ) - a-D- G l p - 1- 4 ) - p

-D

- G a l p - 1->

D E B

Figure 7. Structure of the repeatingunitof the exopolysaccharide of

Lactobacillus delbrueckii

subspecies

bulgaricus.

7 4 . 0 J

7 8 . 0

8 2 . 0 4

H M B C

CH-1.EC-6

EH-1.EC-5

EH-1.EC-3

CH-1.CC-3

> AH-1.AC-3

AH-1.AC-5

EH-1.BC-4

4SO

D H-1, E C 4

AH-1 .CC4

BH-1.DC-4

98.01

1 0 2 . 0 1

1 0 6 . 0

J

EH-1.EC-1

CH-1.CC-1

DH-1.DC-1- " * " 8tm

v

J

H-1. C-1

AH-1AC-1-p7--*fc

5 .2

5 ,0 4 .6 4 .8

4.4

P P M

4 . 2

Figure8. Partial 500-MHz

J

H-

]3

C undecoupledHBQC spectrumof the

polysaccharidefrom Lactobacillus

delbrueckii subspecies bulgaricus

at 353

K.

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However,

useful application

o f

these

spectra requires a preceding assignment of

the

l 3

C and

*H

resonances.

Field

strength

The development of

N M R

instruments, operating at higher

field

strengths,

has improved the resolution and sensitivity enormously. The advantages of

advanced instrumentation are particularly relevant for complex molecules

like

glycoproteins.

For polysaccharides built up of repeating units, spectral

improvements can be observed, but they are not spectacular. This can be

illustrated by comparison of 1-D (Fig. 10) and 2-D (Figs. 11-14) spectra

recorded at 500 and 900

M H z ,

respectively, for the exopolysaccharide

L. lactis

cremoris

having the repeating structure as shown in

F i g .

9 (75)

D B A

-*4- - 0- - 1 -*3)--31- 1

->4)-a-OGa^ 1

->

3

1

C

0

3

1

-3

Figure 9. Structure of the repeating

unit

of the exopolysaccharidefrom

Lactobacillus

lactis cremoris.

JID

i

I

jul

P M

5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4

Figure 10. I D*HNMR spectrumof the exopolysaccharide; uppertrace at 500

MHz,lower trace at 900 MHz.

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3-D NMRspectra

Owingto the small chemical shift dispersion in carbohydrate N M R spectra

these

molecules are

w e l l

suited for 3-DN M R .

In principle any two 2-Dmethodscan be combined into a 3-D experiment.

Non-selective homonuclear 3-D N O E S Y - T O C S Y was the first method applied

to an oligosaccharide (16, 17) Suitable 2-D alternatives have been developed

since then

(18, 19\

which are more efficient in thedatacollection.

The characterization of the spatial

structure

in solution ofglycans,free, in a

complexwith complementary molecules, or covalently bound to other molecules

l ike

proteins or

l ipids,

is relevant for gaining insight into the functioning o f

these

molecules and for their recognition by complementary molecules. In the last

case, it should be emphasized that by such interactions the conformational

equilibrium may undergo alterations in comparison to the free

state,

since

carbohydrates have an inherent flexibility and can

adapt

to the binding site.

Simi la r ly , in glycoconjugates the glycan conformation can be affected by

interaction with non-glycan part. Estimation of the overall solution structure

requires determination of the conformation of the constituting monosaccharides

and of all glycosidic linkages. An oligosaccharide chain manifests flexibility

throughout the whole molecule on a

short

time scale by fast vibrations at bonds

and angles and on a

longer

time scale bychanges of the dihedral angles. The

conformationally

relevant dihedral angles , and are indicated inF i g . 15.

Conformation

,0

'

.0

Figure

15. Conformational

angles ,

, and according to

1UPAC-IUB

).

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Information on the

rate

of tumbling and the internal flexibilities can be

deduced from the

longitudinal

relaxation time

T V

l

H-

l

H couplingconstantsare important for exploring the ring conformations

o f the constituents and for an estimation of the dihedral angle for the

glycosidic

linkages via an

exocyclic

hydroxymethyl group.

The skeleton protons of the constituting monosaccharide can be studied by

homonuclear

-

experiments. The smallest N O E s that can be measured

correspond to a distance between the protons of about 5 A. Several

intra-

residual

-* N O E s

can be observed, but only one or two wter-residual

N O E s

can be measured for consecutive monosaccharides. O n l y the latter are relevant

foran overall 3-D structure. N O E s between further remote constituents are

rare

in

non-rigid

chains.

Heteronuclear

H -

1 3

C N O E sare useful bygivingaccess to the calculation of

distances between carbon atoms and protons. The three-bond

H -

1 3

C

N O E s

are

suitable for the determination o fthe

glycosidic

linkage conformation.

Complications

in the analysis of

N O E s

arise from internal motions and

flexibility o f

the carbohydrate chain. When in comparison to the N O E

bui ld

up

rate,

fast internal motions occurwithina chain, they w i l laffect the N O E bui ldup

rates

and intensities. Since the observed

N M R parameters

are averages of the

different coexisting conformations, unrealistic results may be obtained by

translating the measured N O E sdirectly into distances.

Therefore, complementary approaches are necessary to identify the distinct

conformations and to determine their abundance,

e.g.

through a combination of

Molecular

Mechanics ( M M ) and

Molecular

Dynamics (MD) calculations. The

M M calculations are used to search for energy minima in vacuo in the

conformational space. The MD calculations are based on a derivative of the

potential energies to calculate the accelerations of the individual atoms using

Newton's law: F = m a.

Velocities

of the atoms are then calculated and after integration of a time

step

of

typically

1 - 2 fs, the molecular system

gets

new coordinates. This

process is repeated as long as the

M D

simulation lasts.

The obtained trajectories of a molecule through space in time make it

possible to extract time-dependent

parameters like

correlation time, diffusion

constants and free energies.

During

the MD simulation time-averaged NOE

constraints are applied. A time averaged interval of the MD trajectory is

evaluated

with

the N O E constraints, so the molecular system

gets

the opportunity

to

adapt

its conformation.

In

our laboratory, the M D simulations are carried out in water by

means

of

the

G R O M O S

program to probe the solution-structure (27).

Other approaches that y ie ld independent information on the conformations

involved

can be obtained from N MR residual dipolar couplings in aqueous,

dilute l iqu id

crystalline media (see chaptersby

restegrd

and

Widmalm

in this

volume)

and from uniform or specific

l3

C-labeling

of the constituents.

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The Manal-6 and Fucal-6 residues explore large

parts

of the

conformational space. The superimposed conformations of compound 1

are depicted inF i g . 16.

M a n - 4 '

M a n - 4

Figure

16. Superimposed conformations of compound

1

obtained

from

MD

simulation.

Pineapple

StemBromelain: Glycopeptideversus

Glycoprotein

The glycoprotein pineapple stem bromelain is a proteolytic enzyme carrying

asingle

N-glycosylation

site, only.

From

bromelain a glycopeptide was prepared

byexhaustive pronase digestion

(24)

(see F i g .17).

Mama

1-6

1

- 4 G lc N A c p

1

- 4 G lc N A c p

1-Asn-Glu-Ser-Ser(OH)

Fucal-3

/

Manal-3

X y i p i - 2

Figure 17. Structure of the glycopeptide derivedfrom pineapple

stem

bromelain.

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The conformational behavior of the glycopeptide was investigated by using

a

combination of

M D

simulations in water

with N O E S Y *H N M R

spectroscopy

(24, 25). Theoretical N O E S Y peak intensities were calculated on the basis of

models obtained by

M D

simulations through the C R O S S R E Lprogram (22). The

peptide moiety shows a large

flexibility

and extends in a larger area of the

conformational space than the glycan part. There are no strong interactions

between the glycan and peptide chain, although in the M D simulations of the

glycopeptide several hydrogen bonds were indicated. In particular, hydrogen

bonds were found for Asn and

G l c N A c

- 1 for 5.5%.

( -

Gl-07)

and

between Ser-1 and G l N A c- 1 (S1-0/ -G1-06) for 13.9% of the simulated time.

The presence of temporary hydrogen bonds illustrates the proximity of the

peptide and glycan parts, almost without inducing preferred conformations for

the peptide. In the glycan the

X y i p i - 2 M a n p l - 4 G l c N A c p i - 4 G l c N A c

part is

rather

r ig id ,

occurring in mainly one conformation. The

Manal-6Man

and the

Fucal-3

G l c N A c linkages are more

flexible

as can be concluded from the MD

simulation

and theSf values. The latter linkage occurs in two conformations at

average values of

=

-145, 100 and

, =

-140, -135. Transitions of the

glycosidic

linkages occur at a time scale of about 1 - 1 00 ns. In Fig. 18

superimposing the various conformations summarizes the results.

Figure 18.Snapshotsof

structures

of the glycopeptide takenateach20 ps of the

simulated trajectory. The conformations are superimposed on thecenterofmass

the

GlcNAc-1

residue. For the Manal-6Man linkage 6 gg and 7 gt

conformations are included.

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Interestingly, investigations

of the

intact bromelain glycoprotein showed

a

number of significant differences intheglycan conformation 26).

A l l

glycosidic

linkages showed

a

greatly reduced flexibility. This holds

in

particular

for the

M a n a l - 6 M a n

and

F u c c t l- 3 G l cN A c

linkages, probablydue to interactions with

the protein.

For the

F u c a l- 3 G lc N A c

linkage also

the

distribution between

the

populations

o f

the two main conformations isaffected.

C OOH

2x18.5

[11-53]

Man-3

GlcNAc 2

GlcNAc-1

0.77

0.82

0.84

0.02

0.04 0.04

/

2.0

[1.4-2.8]

Fuc

0.59

0.04

Man-4

5.4

0.4

9.5

J8.0-11]

2x87

[63

140]

Man-3

GlcNAc 2

GlcNAc-1

12.6

17.7

0.8

1.1

/ 25

[21-31]

Fuc

10.4

0.5

Figure 19. Effective rotation correlation timesofthemonosaccharideresidues

and theslowinternal rotation correlation timesof the glycosidic

linkagesin the

glycopeptide upper part)andthe glycoprotein

lower

part).

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Comparison of the effective rotation correlation times of the constituting

monosaccharides

and of the

slow-internal rotation correlation times

of the

glycosidiclinkages

demonstratethe

additivity of motions in

a

glycan chain going

from the centerofmassto theterminus/termini of the chain andtheeffectof a

large protein

versus

a

small oligopeptide.

The

rigidity

of the

N,N-

diacetylchitobiose unit

and the

site

of

interaction

(a or

site,

or

end-on

positioning) of the Asn-linked

G l c N A c

residue with

the

protein characterize

to a

large partthepresentation of the innerpartof the glycan. The additivity of the

motions of the individual

residues leads

to a relatively large flexibility at the

outer part(s)

of the

glycan

in a

glycoprotein. This

feature has

important

implications

for the

interaction with complementary molecules

and

recognition

phenomena asw e l l as on theaccessibilityforenzymes. The comparisonof the

conformations

of

free glycans

and

attached

to

small peptides with those

of

glycans in glycoproteins show clearly

that

the

location of the

center

of

mass

and

the interactions withtheprotein chainhaveagreat influence. By consequence,

conformational data obtained for

free

glycans or glycans attached to small

peptidesshould

not be

generalized

to

glycoproteins without carrying

out

further

experimental studies.

In summary: N M R has proven

to be an

invaluable tool in determing primary and

three-dimensional structuresof carbohydrates.

References

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introduction to nmr spectroscopy of carbohydrates

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