NMR relaxation studies of oligosaccharides in solution: Reorientational dynamics and internal motion

journal of MOLECULAR

LIQUIDS ELSEVIER

Journal of MolecularLiquids78 (1998)255-261

NMR RELAXATION SOLUTION: INTERNAL

STUDIES

OF OLIGOSACCHARIDES

REORIENTATIONAL MOTION

DYNAMICS

IN

AND

Jozef Kowalewski a, Lena M~iler a'* and CrSran Widmalm b aPhysical Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, SWEDEN. bOrganic Chemistry, Arrhenius Laboratory, Stockholm University, S-I06 91 Stockholm, S W E D E N . Received 15 April 1998;Accepted 20 June 1998 S o m e recent studies of nuclear spin relaxation in small oligosaccharides are presented. The emphasis is on the carbon-13 work, but some data on proton cross-relaxation are also reported. © 1998 ElsevierScienceB.V. All rightsreserved.

1. I N T R O D U C T I O N Carbon-13 NMR relaxation studies are an important source of information on molecular dynamics in solution [1,2]. This is illustrated by some examples t a k e n from recent work on small oligosaccharides [3-5]. The layout of this communication is as follows. The theory of nuclear spin relaxation, with the emphasis on the case of carbon-13 is presented briefly, followed by a short discussion of the experimental techniques. In the results a n d discussion section, the relaxation studies of a trisaccharide (melezitose) [3], a pentasaccharide [4] and a disaccharide [5] are reviewed. 2. THEORY

The relaxation of proton-carrying carbon-13 spins is usually r a t h e r simple to interpret. In terms of physical interactions, the relaxation processes are caused by the dipole-dipole interaction with the directly bonded proton spins. If experiments are performed with simultaneous proton irradiation, the carbon-13 spin-lattice relaxation (the relaxation of the m a g n e t i s a t i o n component along the magnetic field) is a simple exponential process. The time constant of the process is called spin-lattice relaxation time and is denoted T r The relaxation of the magnetisatibn components p e r p e n d i c u l a r to the magnetic field (spin-spin relaxation) is also an exponential process, at least under certain conditions [6], with the tinge constant called spin-spin relaxation time and denoted T 2. A characteristic signature of the dipoledipole relaxation is that the irradiation of the proton spins increases the

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Pll SO 167-7322(98)

256 carbon signal intensity by a factor 1+77, nuclear Overhauser enhancement (NOE) factor. The strength of the carbon-proton dipolar interaction is characterised by the dipolar coupling constant d, the magnitude of which can be assumed known for a directly bonded case.

(l-to ~(rcYn h

(1)

where the symbols have their usual meaning. The relaxation rates depend on spectral densities, J(o)) , which are Fourier-Laplace transforms of the dipolar time correlation functions (TCF), G(t). Both quantities, G(t) and J(w), carry information on the reorientation of the C H bond axis. For small molecules and low viscosity solutions, the reorientation is rapid relative to the nuclear Larmor frequencies. One says that the extreme narrowing conditions, co~2~¢2<<1, apply. The symbol cok refers to the relevant combinations of Larmor frequencies and To is the rotational correlation time. For a rigid body undergoing rotational diffusion in small steps, % is a time constant for the exponential decay of the dipolar TCF. In the extreme narrowing regime, the relaxation rates T~ "I and 7'2"~ are equal and independent of the magnetic field.

- c4-

j

o

(2)

The symbol x J f is an effective correlation time, the term "effective" meaning that the details of the motional information are lost. For larger molecules and]or higher viscosity, the molecular reorientation is slower, ~2vc2>-1, and the 13C relaxation is dependent of B 0 ( coI = 71Bo ) : T~-1 = R~ = 1 ( D C C ) 2 [j(O)H _ COC)+ 3j(O~c) + 6 j ( w H + Wc )]

(3)

1

T2-~ = Ph = 8 ( D C C ) 2 [ 4 J ( O ) + J(O~H -- eriC) +

+3J(o) c) + 6J(o)n) + 6J(o) H + mc)]

rl

(Yn'~

6J(wH +COc)--J(WH-WC)

(4)

(5)

In this regime, the information content is clearly higher and testing of models for the spectral density functions becomes possible. Carbon-13 and nitrogen-15 relaxation outside of the extreme narrowing region is commonly interpreted using the model proposed by Lipari and Szabo (the "model-free

257

approach") [7]. The model is based on the following assumptions: 1) the global reorientation is isotropic. 2) there exist anisotropic, rapid local motions, uncorrelated with the global motion. 3) the local motions partly average out the DD interaction, the r e m a i n d e r is modulated by the global motion. The TCF of this model decays rapidly to a plateau; a slower exponential decay follows. This resembles in fact the correlation functions proposed, in another context, in early eighties by Levy et al. for proteins in solution [8]. The spectral densities of the Lipari-Szabo model are given by the following functional form:

2( S2~M +(1-S2)T / J(co) = 5~1 +--~-~VM2

1+ 0~2Z2 )

(6)

Here, S is the generalised order parameter, TM is the global reorientational correlation time and z-1 = ZM-1 + re-1 with ze denoting the local correlation time.

3. EXPERIMENTAL METHODS In an early 13C relaxation study of sucrose by McCain & Markley [9], they observed a slight B0-dependence of T 1 and NOE in aqueous solution below room temperature. Some time later, we noted that the mixed solvent, 7:3 molar ratio D20/DMSO-d6, provided a much stronger field dependence t h r o u g h the higher viscosity and thus slower molecular tumbling [10]. I n addition, the mixture is a cryosolvent and allows low t e m p e r a t u r e s to be reached. In this way, it becomes possible to study even small molecules u n d e r dynamic conditions outside of the extreme narrowing region and to m a k e use of the higher information content available through the use of equations (3) - (5). In all work from our laboratory discussed here, we used this mixed solvent. The details of the multiple-field NMR m e a s u r e m e n t s can be found in the original papers [3-5]. The general strategy of the 13C relaxation m e a s u r e m e n t s has been described in some detail elsewhere [6]. Briefly, the strategy is as follows: a) T 1 : for sensitivity reasons, one-dimensional (1D) polarisation transfer (PT) methods were often used. Two-dimensional (2D) techniques were only applied, if required to resolve signals, b) T2 : the C P M G method (with the suppression of the DD-CSA interference),slightly s u p e r i o r to Tip was applied. The considerations mentioned above concerning PT, 1D and 2D apply also for T 2. c) NOE: conventional gated decoupling was used, if required with proton-detection. For 1H cross-relaxation m e a s u r e m e n t s [5], we used 1D NOESY with a half-Gaussian soft pulse [11]. For fitting the multiple-field relaxation data to the Lipari-Szabo model, the following strategy was applied. We fitted relaxation p a r a m e t e r s averaged over ring carbons in each sugar residue, r a t h e r t h a n those for the individual carbon atoms. The data at each t e m p e r a t u r e were treated separately, r a t h e r t h a n assuming the Arrhenius relation for correlation times. We employed three p a r a m e t e r fits (S 2, ~M and ~e), if the order p a r a m e t e r was not too large (S2 < 0.8). Otherwise, two p a r a m e t e r fits (S2 and ~M) were used. In all cases, we used a fLxed DCC, based on rcH=109.8 pro.

258 4. R E S U L T S A N D D I S C U S S I O N

The first example is the trisaccharide melezitose, Figure 1 [3]. The averaged results for the ring carbons in the glucose unit g2, obtained at 303K are shown in Figure 2. For this molecule, .we found the dynamics of the three s u g a r units veiT similar, b o t h at 303K and at 323tC This is nicely d e m o n s t r a t e d by the fact that the diagrams for the other two sugar units, corresponding to Figure 2 for the g2, are very similar to the g2 data and to each other [31.

OHO~

~ , ,H ~OO 'H OH

H

9W

a

~7 ~-'+,

~C" ~

"...

~ ' 2 ..-.. L

H

HO

, 4.

6

~ '$ ~ I0~

12 B , Tesla

14

16

HO Figure 1. The structure of melezitose. The two glucose residues substituting positions 2 and 3 of the fructofuranose residue are labelled g2 and gS.

Figure 2. Plots of the calculated and experimental relaxation parameters for the g2 ring in melezitose at 303K as a function of magnetic field. Reproduced with permission from Magn. Reson. Chem., 33 (1995) 541 (copyright John Wiley & Sons ~imitedj.

At 303 K, we obtained Se = 0.84, x.~ = 0.67 ns. S ~ was also found t e m p e r a t u r e dependent, S ~ -~ 0.79 at 323t6 As opposed to 'bhe rings, the dynamics of exocyclic CH2OH groups was found to be r a t h e r diversified. I n particular, the C-1 hydroxymethyl group of fructose was found to be considerably less mobile t h a n the other exocyxlic groups, which perhaps can be explained by an intramolecular hydrogen bond. The second example is a pentasaccharide, Figure 3, from reference [4]. The dynamics of the rings is in this case rather different. The GN Gunit (S~ = 0.75 at 318K), linked to the central mannopyranoside residue (S2 = 0.91) through the hydroxymethyl group, is found significantly more mobile t h a n the GN 2 moiety (S2 = 0.83). The outer residues display even less motional restrictions. We note further that the global correlation times for the three inner rings are not quite identical. This may indicate a certain anisotropy in the global motion, not taken into consideration by the simple Lipari-Szabo model.

259

G2

H --

GN6

"OHA"

0 ~ ' ~

G2

GN2

_(,OH O

OH ~ O

~HR

AcHN~ o ~ O H H o ~ ~ , ~

X ~ O~

Hd)~)H

'HO'-'~::I~

~

HO

OH

OR

M Figure 3. A branched pentasaccharide, with two identical disaccharide units connected at positions 2 and 6 of the central mannopyranoside residue.

The finalexample is a disaccharide,methyl 3-O-a-D-mannopyranosyl-J]D-glucopyranoside,Figure 4, originallyinvestigatedin reference [5]. OH

HO~

HO H

o.1, .... o.1 ~ o.o,, 0.12

H2

H(~ ,..

---i'.-,---,--.,-'',-" \

0.06

Me

o.o2 0

R1

~3

Figure 4. The structureof methyl 3-Oa-D-mannopyranosyl-[~-D-glucopyranoside

6

0

10

12

14

16

eom

Figure 5. Plotsof calculatedand experimental cross-relaxation rates at 323K as a functionof the magnetic field.Reproduced with permission from J.Phys. Chem., 100 (1996)17103.(Copyright1996 American Chemical Society). Circles: intra-residue, squares: interresidue.

For this compound, we have reported [5] a combined carbon-13 and proton relaxation study. From carbon-13 T t and NOE data at two magnetic fields (4,7 T and 9.4 T), we estimated the S~ and Tu values at several different temperatures. Proton cross-relaxation rates (a,.) for intraresidue (Hr-H2.) and interresidue (Hr-Hs) proton pairs were also measured, at the same temperatures. The cross-relaxation rates are given by:

260 The spectral densities J*(co) in eq. (6) refer to the motion of the axis joining protons i and j and may in principle differ from the spectral densities obtained from the carbon measurements. We found, however, that the intraresidue cross-relaxation rates OHrS2,could very well be reproduced with J*(o~) based on ~Mand S 2from lSC, after adjusting the proton-proton distance. The adjusted Hr-H z distance was practically constant upon temperature variation, close to 262 pro. This distance is in good agreement with molecular mechanics (MM) modelling. The calculated and measured field dependence of the cross-relaxation rate at 323K are displayed in Figure 5. We can thus conclude that the two types of measurements are consistent with each other, when interpreted using the Lipari - Szabo model. The interresidue cross-relaxation rate am'm is an even more interesting case, as it can lead to conclusions regarding the flexibility of the glycosidic linkage. The trans-glycosidic cross-relaxation rate in sucrose in aqueous solution has been measured by Poppe and van Halbeek [12]. Their conclusion was that the field- and temperature-dependence of that cross-relaxation rate was inconsistent with the rigid glycosidic linkage. In our measurements for the disaccharide of Figure 4, the field- and temperature-dependence of O'H1.H3 and Ore.H2. are very similar (Figure 5). The single-parameter fit of the Hr-H 3 distance in eq. (6), with the spectral densities from carbon-13 measurements, yields again a distance that is only slightly temperature-dependent (245 - 248 pro) and in reasonable agreement the MM-modelling. Our conclusion is thus that the interresidue cross-relaxation rates provide in the present case no evidence of any other internal motions than those contributing to the carbon relaxation. Moreover, we find the generalised order parameter from the carbon measurement, S 2 -- 0.8, to be consistent with the accessible conformational space with a certain range of ¢ and ~, provided that the motion within the accessible space is much faster than the overall reorientation. 5. CONCLUSIONS

Multiple field, 13C relaxation studies allow the characterisation of reorientational dynamics in oligosaccharides. The interpretation based on the Lipari-Szabo model can clearly differentiate between different motional situations for different sugar rings and for different exocyclic hydroxymethyl groups. Combined carbon-13 and proton relaxation studies have a potential of clarifying the flexibility/rigidity of glycosydic linkages. ACKNOWI,EI~EMENTS This work has been supported by the Swedish Natural Science Research Council and by the Tempus program. The generous grants of spectrometer time at the Swedish NMR Center and the skilful assistance by Dr Toshi Nishida and Ms Charlotta Damberg are gratefully acknowledged. REFERENCES 1. J. Kowalewski, Ann. Rep. NMR Spectr., 22 (1990) 307; ibid., 23 (1991) 289. 2. G.C. Levy and D.J. Kerwood, Encyclopedia of Nuclear Magnetic Resonance, eds. D.M. Grant and R.K. Harris, Wiley, 1996, p. 1147.

261 3. L. M~iler, J. Lang, G. Widmalm and J. Kowalewski, Magn.Reson. Chem., 33 (1995) 541. 4. L. M~iler, G. Widmalm and J. Kowalewski, J. Biomol. NMR, 7 (1996) 1. 5. L. M~ler, G. Widmalm and J. Kowalewski, J. Phys. Chem., 100 (1996) 17103. 6. J. Kowalewski and L. M~iler, Methods for Structure Elucidation by HighResolution NMR, eds. Gy. Batta, K.E. KSv6r and C. Szantay, Elsevier, 1997, ch. 16. 7. G.Lipari and A. Szabo, J. Am. Chem. Soc., 104 (1982) 4546. 8. R.M. Levy, M. Karplus and J.A. McCammon, J. Am. Chem. Soc., 103 (1981) 994. 9. D.C. McCain and J.L. Markley, J. Am. Chem. Soc., 108 (1986) 4259. 10.H. Kovacs, S. Bagley and J. Kowalewski, J. Magn. Reson., 85 (1989) 530. ll.H. Kessler, U. Anders, G. Gemmecker and S. Steuernagel, J. Magn. Reson., 85 (1989) 1. 12.L. Poppe and H. van Halbeek, J. Am. Chem. Soc., 114 (1992) 1092.