High Resolution Proton NMR Spectra of Blood-Group Active

4 High Resolution Proton NMR Spectra of Blood-GroupActive Glycosphingolipids in DMSO-d Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on May 29, 2018 | https://pubs.acs.org Publication Date: July 31, 1980 | doi: 10.1021/bk-1980-0128.ch004

6

J. DABROWSKI—Max-Planck-Institut für Medizinische Forschung, Heidelberg, Jahnstrasse 29 H. EGGE—Institut für Physiologische Chemie, Universitat Bonn, Nussallee 11 P. HANFLAND and S. KUHN—Institut für Experimentell Hamatologie und Bluttransfusionswesen, Universitat Bonn, Annaberger Weg

It has been shown recently that the application of high resolution proton magnetic resonance spectroscopy is a useful technique for the structural elucidation of glycosphingolipids. Martin-Lomas and Chapman (1) and the group of Karlsson (2) investigated acetylated and methylated and reduced derivatives of glycosphingolipids. This group succeeded in analyzing non- derivatized cerebrosides (3) and higher glycosphingolipids by high resolution NMR spectroscopy. In order to avoid solubility problems, like formation of micelles and aggregates in aqueous solution, these products have been successfully measured in DMSO-d. In simpler glycosphingolipids such as glucosylceramide, galactosylceramide and lactosylceramide the signals of all protons that are linked to carbons bearing negatively charged substituents could be assigned. In this paper the results obtained with some of the more complex glycosphingolipids are presented. In the oligosaccharide moiety of these glycosphingolipids, well analyzed by conventional methods, the linkages of the sugar components glucose, galactose, N-acetyl-glucosamine and N-acetylgalactosamine exhibit a number of variations with respect to sequence, anomeric configuration and site of attachment. The observed changes of the H and H resonances of the sugar rings, resulting from the interaction of the neighboring sugar units, can be condensed to a number of rules. These rules greatly facilitated the complete structural elucidation of a hitherto unknown ceramidedecasaccharide isolated from rabbit erythrocyte membranes (4). 6

1

2

Methods The spectra were obtained at 338° K on the Bruker HX-360 spectrometer equipped with a Bruker 2000 computer with 32 K memory capacity. The operating frequency was 360 MHz and the spec0-8412-0556-6/80/ 47-128-055$5.00/ 0 © 1980 American Chemical Society Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

56

CELL SURFACE GLYCOLIPIDS

t r a widths amounted to 3.3 KHz. The deuterium-exchanged samples were d i s s o l v e d i n DMS0-d5 c o n t a i n i n g 2% D2O. The sample c o n c e n t r a t i o n amounted t o 0.2%. The f r e e i n d u c t i o n decays were m u t i p l i e d by a r e s o l u t i o n enhancement f u n c t i o n ( L o r e n t z i a n - t o Gaussian t r a n s f o r m a t i o n ) . The chemical s h i f t s of the H p r o tons of a l l sugar u n i t s were determined by s p i n - d e c o u p l i n g d i f ference spectroscopy (SDDS). 2

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on May 29, 2018 | https://pubs.acs.org Publication Date: July 31, 1980 | doi: 10.1021/bk-1980-0128.ch004

R e s u l t s and D i s c u s s i o n From the data presented i n Tables 1 and 2 , two sets o f r u l e s can be deduced concerning the i n f l u e n c e o f s u b s t i t u t i o n on the chemical s h i f t s o f the H and H protons of the sugar r i n g s . Considering one sugar i n an o l i g o s a c c h a r i d e c h a i n , the s h i f t s of H and H* of t h i s sugar w i l l be i n f l u e n c e d a) by the sugar u n i t s attached to C3, C4 or C6 and b) by the aglycon or sugar moiety to which i t i s g l y c o s i d i c a l l y l i n k e d i t self. 1

2

1

a)

The H s i g n a l of a 3-1 inked sugar ( c o u p l i n g constant 1 . 2 8 Hz) i s s h i f t e d about 0.05 - 0.07 ppm t o lower f i e l d a f t e r s u b s t i t u t i o n with another sugar u n i t . Further extension of the o l i g o s a c c h a r i d e chain to the nonreducing end has no apparent e f f e c t on the p o s i t i o n of t h i s s i g n a l . The only exception to t h i s r u l e i s observed i n the spectrum of the Forssman hapten (V) ( F i g . 5, Table 1 ) . This downf i e l d s h i f t , though r e g u l a r l y observed i n a l l compounds so f a r a n a l y z e d , i s n e i t h e r s p e c i f i c w i t h regard to the type nor the s i t e o f attachment of the s u b s t i t u t i n g sugar, l i k e p o s i t i o n 3 or 4 i n the s e r i e s presented here. 1

J

The H s i g n a l of an a-1inked galactose ( c o u p l i n g constant ^1.2 ^ Hz) i s s h i f t e d downfield by only 0.02 ppm a f t e r s u b s t i t u t i o n by another sugar. A g a i n , f u r t h e r extension o f the sugar c h a i n towards the non-reducing end has no e f f e c t on the p o s i t i o n of the s i g n a l , e x a c t l y as i n the case of the 3-1 inked sugars. 1

b)

In c o n t r a s t to the r e l a t i o n s j u s t d e s c r i b e d , the chemical s h i f t s of the anomeric protons are i n f l u e n c e d i n a r a t h e r s p e c i f i c way by the type of sugar or aglycon to which the sugar under o b s e r v a t i o n i s g l y c o s i d i c a l l y l i n k e d . The SH* values a l s o d i f f e r depending on the point of attachment t o another sugar u n i t . Thus, the H* s i g n a l of a non-terminal galactose l i n k e d 3 - g l y c o s i d i c a l l y t o p o s i t i o n 4 of glucose appears at 4.27 ppm whereas t h a t o f a galactose 3 - g l y c o s i d i c a l l y l i n k e d to p o s i t i o n 4 of N-acetylglucosamine appears a t 4.30 ppm. Thus, two d i s t i n c t l y resolved H s i g n a l s can be observed f o r the two 3 1-4 l i n k e d galactose residues i n compound (VI) (Table 1, F i g . 6 ) . In the same way, terminal 1

Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


4.

Table 1:

and

I

6

II

6

III

6

IV

J

i ,

2

Glc :

4.23 (7.3)

4.17 (7.7)

4.81 (4.0)

4.28 (7.7)

4.17 (8.1)

4.54 (8.1)

4.83 (3.6)

4.28 (7.7)

4.17 (8.1)

4.74 (3.6)

4.59 (8.5)

4.83 (3.6)

4.29 (7.7)

4.21 (7.7)

a

1,2'

)

1

p-3Gal 1 -

4.85 (3.8)

6 (J

)

3

1 *4GlcNac 1 — * 3 G a l

4.30 (7.3)

1

3

*3Gal 1

4.84 (3.6)

4.27 (8.0)

4.70 (8.4)

a Gal

VII

1 —*4

3 —•ICer

)

6

6 (J

1 —MGal

4.10 (7.7)

Gal

VI

57

Spectra

1 _^3Gal

1 — • 3GalNAc

6 (

NMR

Coupling Constants o f G l y c o s p h i n g o l i p i d s i n DMS0-d5.

GalNAc

V

Proton

DABROWSKI ET A L .

*lCer

4.17 (7.8)

•4GlcNAc 31

4.30 (7.4)

4.42 (8.1)

\

6

1.2

3 3 3 3 Gal l - * . 4 G l c N A c 1 ->3Gal l - * 4 G l c 1-^Cer

3 3

o

Gal 6 (Jl,2)

3 1 —MGlc

4.84 (3.6)

1 —*3Gal

1 4.30 (7.3)

/ *4GlcNAc

3l 4.67 (8.5)

4.30 (7.3)

4.67 (8.5)

4.27


4.17 (7.7)


58

C E L L SURFACE GLYCOLIPIDS

Table 2:

Chemical S h i f t s of H

of G l y c o s p h i n g o l i p i d s in DMSO-ds

2

Gal 1 — ^ - * 4 G a l I II III

3.66

Gal 1 —m3 IV

VII

3.33 3.34

3.59

Gal 1 —»» 4 GlcNAc 1 —»> 3Gal 3.42

a Gal 1 —*>3 Gal 3.59

3.44

3 1 —•4

3.42

1 -L-4

Glc

1-^1

Cer

2.98 3.06 3.05

1 —**4 Glc

3.42

1 —*

1 Cer

3.04

GlcNAc 1 3.45

x

6 3 3 3 Gal l-#4GlcNAc l->3Gal 1 - V l G l c 3 *3.45

Gal 1 —+3 3.59

Gal 3.42

1 — 4

GlcNAc

3.44

3.46

3.05

1

3.44


3 1-^lCer


4.

DABROWSKI ET A L .

Figure 1.

360-MHz

Figure 2.

Proton

NMR

59

Spectra

proton NMR spectrum of glucosylceramide in For other conditions, see Methods.

DMSO-d .

360-MHz proton NMR spectrum of lactosylceramide in DMSO-d

6


6


Figure 4.

360-MHz proton NMR spectrum of globotetraosylceramide in DMSO-d


6

DABROWSKI ET A L .

Proton

NMR

Spectra

61


4.

Figure 6.

360-MHz proton NMR spectrum of IV in DMSO-d

3

Gal-fl-neolactotetraosylcetamide

6



62


Figure 7.

360-MHz

proton NMR spectrum of a ceramidedecasaccharide from rabbit erythrocyte membranes


4.

DABROWSKI ET A L .

Proton

NMR

63

Spectra

galactose residues l i n k e d a - g l y c o s i d i c a l l y to p o s i t i o n 3 or 4 of another galactose u n i t can be d i s t i n g u i s h e d by the signals at 4.85 and 4.81 ppm, r e s p e c t i v e l y (Table 1, F i g s . 3 and 6 ) . Hence, s p e c i f i c features of B-blood-group a c t i v e glycosphingol i p i d s can i n p r i n c i p l e a l s o be deduced from the NMR s p e c t r a . The chemical s h i f t s of the H protons are s t r o n g l y i n f l u enced by the s u b s t i t u t i o n i n p o s i t i o n 3 o r 4. Thus, the s i t e s of attachment deduced by H* s h i f t s , as o u t l i n e d above, can be confirmed independently by the H v a l u e s . These values - a l l determined by the SDDS method - are shown i n Table 2. The sequences G a l - 3 - l - 4 - g a l - 3 - ( F i g . 3) and G a l - 3 - l - 3 - G a l - 3 - ( F i g . 6) can be c l e a r l y d i s t i n g u i s h e d by a l a r g e downfield s h i f t of 6H from 3.34 t o 3.42 ppm i n the a-1-3 s u b s t i t u t e d g a l a c t o s e . The same s h i f t can be observed i n compound VI with the sequence G l c N A c - 3 - l - 3 - G a l - ( F i g s . 3 and 6, Table 2). The r u l e s e x e m p l i f i e d above g r e a t l y f a c i l i t a t e d the s t r u c t u r a l e l u c i d a t i o n o f a ceramidedecasaccharide i s o l a t e d from r a b b i t e r y t h r o c y t e s ( F i g . 7, Tables 1 and 2 ) . The resonances at 4.17 ppm (1 proton) and 4.27 ppm (1 proton) i n the spectrum o f (VII) ( F i g . 7) c l e a r l y correspond t o the H* s i g n a l s a t t r i b u t e d to the sequence G a l - 3 - l - 4 - G l c - 3 - l - l - c e r a m i d e . The s i g n a l at 4.84 ppm, w i t h the i n t e n s i t y of two p r o t o n s , apparently belongs to two terminal galactoses l i n k e d a-1-3 to a g a l a c t o s e , as i n compound ( V I ) . The doublet at 4.30 ppm (3 protons) corresponds t o three H protons of galactose residues l i n k e d 3-1-4 t o N-acetylglucosamine. The i n d i c a t e d branching point i s supported by the r e s u l t s of SDDS. A f t e r i r r a d i a t i o n at 4.30 ppm two d i s t i n c t l y d i f f e r e n t spectra were obtained f o r the r e s p e c t i v e H protons a t 3.42 ppm and 3.45 ppm. W h i l s t overlapping resonances precluded an assignment o f these s i g n a l s on the b a s i s o f i n t e g r a l s a c l e a r d e c i s i o n can be reached by comparison w i t h the H resonances of compound ( V I ) . Since i n both 3-1-4 l i n k e d galactose residues the H s i g n a l appears at 3.42 ppm, the s i g nal a t 3.45 o b v i o u s l y has t o be a t t r i b u t e d to the doubly s u b s t i tuted g a l a c t o s e . Two of the glucosamine residues e x h i b i t a common H* s i g n a l at 4.67 ppm (2 p r o t o n s ) . T h i s shows t h a t they form part of the sequence G l c N A c - 3 - 1 - 3 G a l . On the other hand the s i g n a l at 4.42 ppm (1 proton) belongs t o the glucosamine l i n k e d 3-1-6 to the galactose at the branching p o i n t . Analogous u p f i e l d s h i f t s f o r H* resonances have been reported f o r 1-6 l i n k e d mannose {$) and glucose (6) d e r i v a t i v e s . The proposed s t r u c t u r e i s i n f u l l agreement w i t h the r e s u l t s obtained by mass spectrometry, immunodiffusion and a n a l y s i s of p a r t i a l l y methylated a l d i t o l a c e t a t e s (4). 2


2

2

1

2

2

2


64



References 1. Martin-Lomas M.; Chapman D. Chem. Phys. Lipids 10, 152 (1973). 2. Falk, K. E.; Karlsson, K. A.; Samuelsson, B. E. Arch. Biochem. Biophys. 192, 164 (1979); ibid. 191, 177 (1979); ibid. 192, 191 (1979). 3. Dabrowski, J.; Egge, H.; Hanfland P. Chem. Phys. Lipids, submitted. 4. Hanfland, P.; Egge, H.; Dabrowski, J.; Roelke, D., in Glycoconjugates, R. Schauer, P. Boer, E. Buddeke, M. F. Kramer, J. F. G. Vliegenthart, A. Wiegandt eds., J. Thieme Verlag Stuttgart 520 (1979). 5. Dorland, L.; Haverkamp, J.; Vliegenthart, J. F. G.; Strecker, G.; Michalski, J. -C.; Fournet, B.; Spik, G.; Montreuil, J. Eur. J. Biochem. 87, 323 (1978). 6. De Bruyn, A.; van Beeumen, J.; Anteunis A.; Verhegge, G. Bull. Soc. Chim. Belg. 84, 799 (1975); A. De Bruyn (1979) Dissertation, Rijksuniversiteit Gent, Belgium. RECEIVED

December 10, 1979.


High Resolution Proton NMR Spectra of Blood-Group Active

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