Determination of the Conformation of Lewis Blood Group Oligosaccharides by Simulation of Two-Dimensional Nuclear Overhauser Data PERSEVERANDA CAGAS and C. ALLEN

BUSH

Depxtment of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21 228

SYNOPSIS

Through control of both the nmr probe temperature and of the solvent viscosity, phasesensitive two-dimensional 'H nuclear Overhauser data (NOESY) at 300 and 500 MHz are obtained with excellent signal-to-noise ratios for Lewis blood group penta- and hexasaccharides isolated from human milk. Relatively long mixing times are required to produce measurable NOE intensities in these oligosaccharides, which makes a full relaxation maxtrix analysis necessary. By measurements of selective TI for a few isolated 'H resonances, it was possible to generate a simulation of the complete NOESY spectrum at arbitrary mixing time for comparison with the experimental data. From an exhaustive search of the conformational space, it was found that only a small range of glycosidic dihedral angles for the nonreducing terminal Lewis blood group determinant fragments of the milk oligosaccharides LNF-2 and LND-1 produce simulated spectra agreeing within experimental error to the data. Conformational energy calculations reveal that each of these conformations is also one of minimum energy. It is concluded that the Lewis" and Lewisb oligosaccharides adopt relatively compact rigid structures in solution, as shown by the observation of cross peaks between protons in nonadjacent residues. Like the blood group A and H oligosaccharides, there exists only a small dependence of the conformation for Lewis" and Lewisb oligosaccharides on solvent. The apparent lack of dependence of conformation of these oligosaccharides on DMSO in D 2 0 suggests that modification of solvent viscosity with mixtures of DMSO : D,O may provide a useful general strategy of NOESY studies of oligosaccharides.

INTRODUCTIO N Complex oligosaccharides of the type found in glycoproteins, glycolipids, and bacterial polysaccharides have the capacity for storage of substantial information in their chemical structures. Unlike information storage in peptides and nucleic acids whose information is encoded in a linear sequence, complex carbohydrates feature different linkage isomers, anomeric configurations, and branching to encode information. Recent research into the biosynthetic pathway of asparagine N-linked glycopeptides reveals a complex mechanism with many glycosyl transferases and specific glycosidases that provide :C, 1990 John Wiley & Sons, Inc. CCC 0006-3525/90/11121123-16 $04.00 Biopolymers, Vol. 30, 1123-1138 (1990)

potential control points at which modulation of the structure could occur by a combination of genetic and environmental conditions. If, as these observations suggest, information relevant to biological function is stored in complex carbohydrate structures, one must consider the question of how the information is decoded and what is its biological function. One interesting theory that is supported by a growing body of experimental evidence is that the function of complex carbohydrates in animals is control of cell growth and differentiation, and that the information is decoded by lectins that may be either soluble or membrane bound.'.* Although precise biological details of this mechanism for control of cell growth and differentiation are unknown, enough is known to suggest just which kinds of complex oligosaccharides are involved. It is possible that N-linked glycopeptides play a role, 1123

1124

CAGAS AND BUSH

as well as glycosphingolipids such as gangliosides. Perhaps blood group oligosaccharides are the most likely candidates because of their close structural relationship t o the sialylated tumor antigens. Since the above-mentioned complex oligosaccharides are composed of the same constituent monosaccharides and since they share many common structural features, it is timely to study the conformation and dynamics of all these types of oligosaccharides along with considerations of their binding to lectins. Investigations of this type would provide information on what features of the structure and dynamics are relevant to their discrimination by the various lectins responsible for biological activity. While many studies of peptide conformation and dynamics by the methods of biophysical chemistry have appeared as a result of the importance of protein folding, the conformation of complex oligosaccharides has received less attention. Presently available data indicate that the general principles that determine the conformation of complex oligosaccharides differ substantially from the forces governing peptide folding?-5 It was originally proposed by Lemieux and Bock that oligosaccharides adopt single rigid conformations that could be deduced from a simple molecular modeling scheme called by ther.. workers the "hard sphere exoanomeric effect" (HSEA method) ,6 which incorporates the stereochemistry of the molecule in the bond lengths and bond angles along with a simple formulation of the nonbonded interactions and a torsional potential. Subsequently, other laboratories have studied the conformations of the blood group oligosaccharides originally treated by Lemieux and Bock as well as other types of oligosaccharide including the mannose-containing oligosaccharides that are found in the asparagine N-linked glycopeptides. More sophisticated molecular modeling methods have been applied to this problem along with experimental methods for testing the predictions7 The results of these tests of the proposal of Lemieux and Bock on various different oligosaccharides have been mixed, confirming their concept of rigid conformations in some cases but yielding conflicting results in others. Molecular modeling studies combined with nuclear Overhauser data from 'H-nmr spectroscopy applied to blood group substances, the structures originally studied by Lemieux and co-workers, has provided evidence supporting the proposal of rigid conformations for certain oligosaccharide structure^.^*^^^ But other workers using methodology that does not differ fundamentally from that used by our laboratory have found results that are not compatible with single rigid oligosaccharide conformation^.^-'^

The origin of these discrepancies remains obscure. It is possible that, in spite of the apparent rigor of the complete matrix analysis of the 'H nuclear Overhauser effect (NOE) data and of the qualitative reliability of the molecular modeling methods that there is an undiscovered flaw in the methodology used in the work cited above. The application of further experimental methods such as x-ray crystallography or additional nmr methods such as longrange 13C-lH coupling or T1 measurements may provide a test of the methodology. A second possibility is that the methodology is correct but that the extent of internal motion differs among the different classes of oligosaccharides that have been the target of these studies. If it is true that some types of oligosaccharides have single well-defined conformations while others are flexible, then it would be especially interesting to generalize the distinctions among these structural classes to detect any possible correlation of flexibility with biological function.' These considerations suggest the value of extension of rigorous studies to as many different types of oligosaccharides as possible. We have concentrated our attention on structures that are most likely to be rigid and in the present report we describe the application of NOE measurements with complete spin simulations and energy calculations on Lewis blood group oligosaccharides isolated from human milk including NOE data for Lewis" and Lewisb structures in two different solvents. Synthetic oligosaccharides containing these determinants were first studied by Lemieux et a1.6but no quantitative analysis of the NOE was given to critically evaluate the proposed conformation. Breg et al.13 have reported rotating frame nuclear Overhauser effect spectroscopy (ROESY) studies of these milk oligosaccharides but this experimental technique does not lend itself to quantitative ana1y~is.l~ Although Bechtel et al.15 have recently reported NOESY data on a Lewis" oligosaccharide, our interpretation differs from the conclusions reached by these workers. We also report the application to carbohydrates of recently developed methodology for two-dimensional (2D) NOESY measurement and simulation that will be especially useful in oligosaccharides. Since we believe the use of a complete spin analysis is important for the correct interpretation of NOE data on complex oligosaccharides, we have extended the matrix treatment of NOE previously used for steady-state NOE measurements to transient 2-6 NOESY data. The principal advantage of the 2D NOESY method over one-dimensional (1D) NOE difference spectroscopy lies in avoiding the difficulties of cross-saturation encountered with selective

CONFORMATION OF LEWIS BLOOD OLIGOSACCHARIDES

irradiation of close lying resonances in the latter method.

MATERIALS A N D METHODS Materials

Human milk was processed according to the method of Kobata and Ginsburg, 16~17 and the resulting mixture of oligosaccharides was fractionated on a BioGel P6 column using water as eluant. Lacto-N-difucohexaose 1 (LND-1) was subsequently isolated by reverse-phase high performance liquid chromatography ( H P L C ) of the gel fractions using a n Alltech CIScolumn. Lacto-N-fucopentaose 2 ( L N F - 2 ) was a gift of Dr. Ginsburg. Purity of the LNF-2 sample was established by HPLC to be a t least 90%. Structures of LND-1 and LNF-2 are shown in Figure 1. Samples ( 5 mg LND-1, 20 mg LNF-2) for nmr analysis were prepared by exchanging in D 2 0 followed by lyophilization for three cycles, after which the final solutions were made by dissolving the samples in 0.4 mL of either high-purity (99.96 atom 96 D ) D 2 0 (Merck, Sharp and Dohme, Co.) or 2:1 solution of DMSO-d, (99.96 atom % D, Merck, Sharp and Dohme) and D 2 0 . Spectra in D,O were referenced with respect to internal acetone a t 2.225 ppm, while those in DMSO : D 2 0 had tetramethylsilane ( T M S ) as a n internal reference. NMR Experiments

Proton spectra in D 2 0 and in DMSO : D 2 0 a t 24°C were assigned using double quantum filtered correlation spectroscopy (DQF-COSY) and 2D homonuclear Hartmann-Hahn (2D HOHAHA) spec-

Fuc (a-I-4)\ GlcNAc (B-l-3)-Gal

(0-1-4)-Glc

Gal (p-1-3)'

Lacto-N-fucopentaose 2 (LNF-2)

Fur ( a 1 +2)-Gal (0-1-3),, GlcNac (8-1-3)-Gal

(0-1-4)-Glc

Fuc (a-1-4)f

Lacto-N-difucohexaose 1 (LND-1)

Figure 1 . Structures of the milk oligosaccharides LND1 and LNF-2.

11 25

troscopy a t 300 MHz using an NT300 nmr spectrometer equipped with a 293C pulse programmer. Additional assignment data for LNF-2 in D2O were obtained by a proton-detected single bond 'H- I3C heteronuclear multiple quantum coherence (HMQC) experiment recorded on a Bruker AM 500 spectrometer ( National Magnetic Resonance Facility a t Madison) with the pulse sequence of Bax et a1." with WALTZ-16 l9 decoupling at the carbon frequency during acquisition and using time proportional phase incrementation" for phase-sensitive acquisition. The broad-band proton-decoupled I3C spectrum of LNF-2 in D 2 0 was obtained a t 75 MHz with MLEV-16 decoupling." DQF-COSY experiments were done in the phased mode using the method of States et a1." and the pulse sequence 90"tl-90"-900-tzwith appropriate phase cycling for coherence transfer through a double-quantum filter.23 The phased2' 2D HOHAHAZ4spectra were obtained in the low-power transmitter mode with a n EN1 411LA power amplifier and using the pulse sequence 90"-t,-SL,- (MLEV 17),-tz. A 5 kHz spin-locking field with 90-ms spin propagation time was used. The nuclear Overhauser experiments (2DNOESY) were done a t 5°C in D20, and a t 24°C in DMSO : D 2 0 ( 2 : l ) , with mixing times varying from 250 t o 350 ms. The experiments were acquired in the phase-sensitive mode" from GN-500 and NT300 spectrometers using the standard NOESY pulse sequence25 -90"-tl-90"-tm-90"-t,. A 2-s delay was used between acquisitions ( 16-32), with two dummy scans per acquisition. Typically, 220-256 points in tl and 1K points in t2 were obtained. The resulting free induction decays were apodized with 60-75" shifted sine-bell functions in t2and tl prior to Fourier transformation in each dimension. Zero filling was done in tl to yield a final matrix size of 1K X 1K points. All nmr data were processed, analyzed, and plotted with the FTNMR program (Hare Research, Woodinville, WA) run on a VAXstation 3200 (DEC) or a Personal Iris ( 4 D / 2 0 ) workstation (Silicon Graphics). I3C TI measurements a t 125.76 MHz for LNF-2 in D 2 0 a t 5°C were obtained by inversion recovery and analyzed with a three-parameter fit. Rotational correlation times were determined from T, data according to Kuhlmann et a1.26 Selective 'H spin lattice relaxation t,ime ( T I )experiments were made using a 22-ms 180" pulse from the decoupler to selectively invert an isolated peak, followed by a nonselective 90" pulse to measure return of the peak to equilibrium. The delays used were from 5 ps to 3s a t 25-50 ms intervals. An 8-s

1126

CAGAS AND BUSH

delay was used between acquisitions (16-64) and 8K points were acquired for each. Nonselective spin lattice relaxation times were obtained by the standard inversion recovery ~ e q u e n c e .In ~~ both . ~ ~selective and nonselective T I experiments the data were fitted to a single exponential to obtain T I values. T h e same conditions of temperature, solvent, and spectrometer frequency as the NOESY experiments were also observed.

Table I1 Experimental NOESY Cross-Peak Intensities for LNF-2 % NOE"

Cross Peak

A. LNF-2 in D20, 5"C, 350 ms mixing time, 500 MHz Fuc4 Hl/H2 Fuc4 Hl/GlcNAc H4 ca13H1/H3 Gal3 Hl/GlcNAc H3 Fuc4 H5/Gal" H2

7 6 6 4 4

(i2) (22) (f2) (t2) (9)

Quantitation of NOESY Peaks

T h e spectrum was baseline corrected with a fourthorder polynomial prior to quantitation. For measurement of cross- and diagonal-peak volumes, a square region that corresponds to the observed line width for each pertinent peak was chosen and all the points within the region summed. The ratio of the cross-peak volume to the diagonal peak volume of a single proton a t t, = 0 is the normalized crosspeak volume used in the NOESY simulations. Simulation of a NOESY spectrum a t a single mixing time is possible if selective T l ' s are known since the decay of the diagonal peak with mixing Table I Experimental NOESY Cross-Peak Intensities for LND- 1 Cross Peak

76 NOE"

A. LND-1 in D'O, 5"C, 250 ms mixing time, 500 MHz FUC' H1/H2b

FUC'H1/Ga13 H2 Fuc4 H1/H2 Fuc4 Hl/GlcNAc H4 ca13H1/H3 Gal3 Hl/ClcNAc H3 Fuc4 H5/Ga13 H2 FUC'H5/GlcNAc H2

4 (21) 4 (fl) 4 (fl) 3 (21) 3 (51) 3 (fl) 3 (fl) 3 (fl)

B. LND-1 in DMSO-d6 : D20, 24"C, 250 ms mixing time, 300 MHz ca13H1/H5 Fuc2 H1/Ga13 H2 Fuc4 Hl/H2 Fuc4 Hl/GlcNAc H4 GlcNAc H1/H5 Gal3 Hl/GlcNAc H3 Fuc4 H5/Ga13 H2 Fuc2 H5/GlcNAc H2

12 ( f 3 ) 11 ( 2 2 ) 9 (22) 7 (+2) 10 ( t 2 ) 10 (k2) 5 (f2) 7 (22)

* Cross-peak volume a t t, = 250 ms/diagonal peak volume of a single proton a t t, = 0 ms. A superscript a t the name of a sugar residue indicates to which position of the adjacent monosaccharide it is glycosidically linked, e.g., FUC'means Fuc is ( a - 1 + 2)-linked to Gal.

B. LNF-2 in DMSO-d6 : D20, 24"C, 250 ms mixing time, 500 MHz Fuc4 H1/H2 Fuc4 Hl/GlcNAc H4 GlcNAc H1/H5 Gal3 Hl/GlcNAc H3 Fuc4 H5/Ga13 H2

5 5 5 5 3

(51) (fl)

(f2) (i2) (k1)

a Cross-peak volume at t , = 350 ms/diagonal peak volume of' a single proton at t , = 0 ms. A superscript at the name of a sugar residue indicates to which position of the adjacent monosaccharide it is glycosidically linked, e.g., Fuc4 means Fuc is (a-1+ 4)-linked to GlcNAc.

time is the same as when that peak is selectively i n ~ e r t e d . ~ ~The . ~ ' . quantitation ~~ of NOESY cross peaks a t a single mixing time was therefore accomplished by first measuring the selective Ti's of isolated peaks. Then the volumes of crosspeaks and isolated diagonal peaks in the NOESY spectrum a t a particular mixing time were measured as described above. Finally, the volume of the isolated diagonal peak a t zero mixing time was calculated using the selective TI for that peak, which was then used to normalize the cross-peak volumes. The diagonal peak volume a t zero mixing time was determined using the relation

2)

v = voexp(

where V is the diagonal peak volume a t a particular mixing time, Vo is the diagonal peak volume a t t, = 0, TI, is the selective TI, and t, is the mixing time. For overlapping diagonal resonances, the volume a t zero mixing time was obtained from the average volume a t zero mixing time of the isolated diagonal peaks. The volumes of cross peaks on both sides of the diagonal were measured and averaged except when there was considerable tl noise a t one of the cross peaks. Each of the normalized NOE intensities listed in Tables I and I1 is reported with estimated

CONFORMATION OF LEWIS BLOOD OLIGOSACCHARIDES

experimental error, the origins of which will be discussed below. Simulation of NOESY Peak Intensities and Computer Modeling

The intensities in a NOESY spectrum for a particular mixing time t, can be calculated from the equation

where a ( t,) is the matrix of normalized NOE intensities, X , X -' is the eigenvector matrix (and its inverse) of the relaxation matrix R , and X is the matrix of eigenvalues of the relaxation matrix R .31-33 The relaxation rate matrix R contains the diagonal ( p , ) and cross-relaxation ( C T , , ~ )rates, which are dependent on the rotational correlation time ( T , ) , spectrometer frequency ( w ) , and the internuclear distance ( r , , ) ): 1

n

(4) where h is Planck's constant and y is the proton gyromagnetic ratio. The term p f represents contributions from nondipolar relaxation mechanisms. Neglecting the effect of internal motion, the spectral density function J , ( n u )can be expressed as

Our neglect of internal motion is a significant assumption in the treatment of these data and its validity will be considered below. The matrix of relaxation rates was built for a model, diagonalized using a standard procedure ( Quantum Chemistry Program Exchange, Program 62, Indiana University) and the resulting matrices of eigenvectors and eigenvalues directly used in Eq. ( 2 ) to yield the theoretical matrix of normalized peak intensities. The di-, tri-, and tetrasaccharide models used t o simulate NOE peaks in LND-1 and in LNF-2 were constructed from monosaccharides with coordinates from crystallographic data.34r35In the conformational search, each pyranoside ring was fixed in its crys-

1127

tallographic structure, and the glycosidic angle was also fixed a t 117". Conformation was determined as a function of the glycosidic angles \k and CP defined as the dihedral angles C1-O1-Cx-Cx-land Oring-C101-C,, respectively. The value of 7, was adjusted t o fit the intensity of a cross peak between protons within the same residue, e.g., Fuc H l / H 2 , Gal H l / H 3 . The actual NOE intensities as well as those relative to these intra-ring NOEs were used in the analysis, as will be explained below. Simulation of cross peaks was done in two steps. Initially, a disaccharide model for each linkage in the nonreducing ends of LNF-2 and LND-1, was searched in a 10" grid of \k and CP simulating the cross peak between protons directly across the glycosidic bond. All disaccharide conformations whose calculated interglycosidic NOEs were within experimental error of the corresponding observed values in LNF-2 and LND-1 (Tables I and 11) were then used in the second step of the simulation. For LND1, a tetrasaccharide model was used to search among the previously determined disaccharide conformations for the Fuc a-1,2-Gal, Fuc a-1,4-GlcNAc, and Gal P-1,3-GlcNAc linkages, by simulation of all NOE cross peaks listed in Table I. Similarly, a trisaccharide model was used to search among the conformations of the Fuc a-1,4-GlcNAc and Gal P-1,3GlcNAc linkages in LNF-2, simulating all NOE cross peaks given in Table 11. Together with the calculated 2D NOE intensities, nonselective 'H Tl's were also obtained and compared with experiment (Table 111). As shown in previous ~ t u d i e s , measurement ~.~ of TI is a useful supplement t o NOE measurements for determination of 3-dimensional structures of oligosaccharides. Conformational energy calculations in 10" intervals of \Er and CP were first made using the disaccharide models mentioned above, with the parameters of Momany et al.36 Disaccharide conformations within 10 kcal of the global minimum were then used in four- and six-dimensional calculations for the trisaccharide and tetrasaccharide models for LNF-2 and LND-1, respectively.

RESULTS Proton assignments for LNF-2 and LND-1 in DzO a t 25°C agree with those previously reported by Breg e t al.,13and the differences in chemical shifts a t 5°C are small. T h e chemical shifts in DMSO-d, : D 2 0 differ significantly from those in D20 as can be seen

1128

CAGAS AND BUSH

Table I11 Experimental Nonselective Spin-Lattice Relaxation Time (TI) Data for LND- 1 and LNF-2

diagonal peak volume of a single proton a t zero mixing time, are shown in Tables I and 11. Only those peaks t h a t were actually used in the simulations are shown. Experimental 'H nonselective T1values are listed in Table 111. Figures 2 and 3 show portions of the 500-MHz NOESY spectra of LND-1 in DzO a t a 250-ms mixing time and of LNF-2 in DzO a t a 350 ms mixing time, respectively. Cross sections in o1 a t selected chemical shifts in both spectra are given in Figure 4. The noise level in these spectra contributes an error of k15% in peak volumes. Figures 5 and 6 are maps of simulated NOES as a function of the conformation along a particular glycosidic linkage, determined by the glycosidic dihedral angles and \k. We consider the overall conformation of a n oligosaccharide as arising from a combination of the various conformations of the glycosidic linkages. Conversely, given a n overall conformation for a n oligosaccharide, we can examine the conformation of a specific glycosidic linkage, bearing in mind that this "local" arrangement may depend not only on the sugar units directly involved in t h a t particular glycosidic linkage but also on the conformation of the other glycosidic linkages in the molecule. This approach allows us to describe in two dimensions what is actually a multidimensional problem.

A. LND-1 in DzO, 5"C, 300 MHz

FUC'H I Fuc4 H1

760 (+16) 576 (217) 475 (212)

Fuc4 H5

B. LND-1 in DMSO-d, : DzO, 24"C, 300 MHz Fuc4 H1 Fuc2 H 1

916 (+14) 1060 (216)

C. LNF-2 in DzO, 5"C, 300 MHz 565 (28) 425 (29)

Fuc4 H1 Fuc4 H5

D. LNF-2 in DMSO-d, : D20, 24"C, 500 MHz Fuc4 H1 Fuc4 H5 GlcNAc H1

1734 (212) 1494 (210) 1737 (k13)

from Tables IVA and IVB. T h e normalized crosspeak intensities of the NOESY spectra, expressed in terms of the ratio of cross-peak volume to the Table IVA

'H-nmr Chemical Shifts" of LND-1 in DzO and DMSO-d, : DzO at 24°C

H1

Residue

H2

H3

H4

H5

H61, H62

3.736 3.830 3.854 4.141 3.733 3.645 3.64

4.351 4.866 3.57 3.72 3.520 3.945 3.58

1.278 1.267 nac na 3.91 3.87 3.91, 3.75

3.561 3.60 3.687 3.919 3.544 3.666 3.41

4.224 4.718 3.417 na 3.335 3.764 3.386

1.124 1.124 na na 3.72 na na

LND-1 in D20 FucZb Fuc4 ~

~

1

3

~

~

1

4

GlcNAc a-Glc P-Glc

5.151 5.023 4.663 4.417 4.612 5.219 4.663

3.763 3.812 3.613 3.571 3.851 3.578 3.282

3.710 3.929 3.806 3.719 4.134 3.830 3.642 LND-1 in DMSO-d, : DzO

Fuc2 Fuc4 ~

~

1

3

~

~

1

4

GlcNAc a-Glc P-Glc

4.954 4.832 4.538 4.265 4.497 5.006 4.427

3.560 3.596 3.485 3.427 3.715 3.319 3.068

3.56 3.714 3.600 3.510 3.951 3.640 3.414

a Chemical shifts in D 2 0 are with reference to internal acetone a t 2.225 ppm, while those in DMSO-d6 : D 2 0 use internal TMS. Accuracy is f0.005 ppm except for strongly coupled resonances (accuracy is kO.01 ppm). Assignments were obtained at 300 MHz. Superscript refers to which position of the next monosaccharide unit the residue is glycosidically linked. Chemical shift could not be assigned from DQF-COSY nor 2D-HOHAHA.

CONFORMATION OF LEWIS BLOOD OLIGOSACCHARIDES

Table IVB Residue

1129

'H-NMR Chemical Shifts" of LNF-2 in D 2 0 and DMSO-d6 :DzO at 24°C H1

H2

H3

H4

H5

H61, H62

3.797 3.880 4.149 3.759 3.664 3.653

4.875 3.574 3.710 3.546 3.92 3.61

1.178 3.73 3.73, 3.78 3.85, 3.94 3.86 3.94, 3.77

3.596 3.698 3.951 3.573 na 3.41

4.734 3.39 3.53 3.378 na 3.377

1.064 nad na 3.77 na na

LNF-2 in D 2 0 b Fuc4' ~

~

1

3

~

~

1

4

GlcNAc CY-G~C P-GIC

5.023 4.503 4.438 4.701 5.219 4.659

3.809 3.485 3.586 3.956 3.571 3.279

3.879 3.625 3.716 4.071 3.818 3.641 LNF-2 in DMSO-d6 : D 2 0

Fuc4 Gal3 Gal4 GlcNAc a-Glc P-Glc

4.847 4.361 4.285 4.630 5.021 4.439

3.617 3.335 3.458 3.787 3.336 3.083

3.699 3.377 3.533 3.909 3.648 3.417

(:hemica1 shifts in D 2 0 are with reference to internal acetone at 2.225 ppm, while those in DMSO-d, : D 2 0 use internal TMS. Accuracy is +0.005 ppm except for strongly coupled resonances (accuracy is f O . O 1 ppm). Assignments were obtained at 500 MHz. Assignments were confirmed by HMQC experiment (data not shown). Supersrript refers to which position of the next monosaccharide unit the residue is glycosidically linked. Chemical shift could not be assigned from DQF-COSY nor 2D-HOHAHA.

The results of NOE simulation a t 500-MHz and a 250-ms mixing time in D20, 5"C, for LND-1 are shown in Figure 5 ( a1-a3), while ( bl-b3) are obtained by simulation of NOEs for LND-1 a t 300 MHz in DMSO-d, : D 2 0 a t 250 ms mixing time, 24°C. The regions within the loops are those in which the calculated and observed ratio of the NOE between protons directly across the glycosidic bond to an intraring NOE agree within the error bounds specified in Table I. The calculated NOEs in this case were obtained using a disaccharide model for each linkage, as discussed in Materials and Methods. Specifically, the entire looped region in Figure 5 ( a 1 ) is that in which there is agreement between the calculated and observed ratio of the NOE between Gal3 H1/ GlcNAc H3 to that between Gal3 H1/ H3. In Figure 5 ( a') it is the region in which calculated and observed ratio of NOE between FUC'H1/Ga13 H2 to that between FUC'H1/ H2 agree. The looped region in Figure 5 ( a3) is that in which the ratio of NOE between Fuc4H1/ GlcNAc H4 to NOE between Fuc4 H1/ H2 agree. Results of the second stage in the simulation using a tetrasaccharide model to simulate all the NOEs listed in Table I are shown by the heavy dots within the loops. These are the regions in which there is agreement between observed and calculated NOE ratios, i.e., Gal3 H1/ GlcNAc H3 to Gal3 H l / H 3 , Fuc' H1-Gal3 H2 to FUC'H l / H 2 ,

Fuc4 H1/ GlcNAc H4 to FUC*H1/ H2, Fuc2 H5/ GlcNAcH2 toFuc2H1/H2, andFuc4H5/Ga13H2 to Fuc4 H1/ H2. The tetrasaccharide conformations along each glycosidic linkage have been plotted separately, but are actually dependent on the conformations of the other two linkages, as explained in the previous paragraph. The regions in which there is agreement within the error limits given in Table IIIA between observed and calculated nonselective 'H T , values of Fuc2 H1, Fuc4 H1, and Fuc4 H5 are shown by the points marked by x. Results from the simulation for LND-1 in DMSOd, : D 2 0 are similarly illustrated in Figure 5 ( bl-b3), except that for reasons to be explained below, the following NOE ratios were used instead of those mentioned above for D20 : Gal3 H1/ GlcNAc H3 to Gal3 H l / H 5 , Fuc2 H1/Ga13 H2 to Gal:' H l / H 5 , and Fuc2 H5/ GlcNAc H2 to GlcNAc H1/ H5. The other two NOE ratios are identical with the ones for D20.Points marked by x are conformations in which nonselective T Ivalues for Fuc2H1 and Fuc4 H1 agree with experimental values (Table IIIB.) Maps of simulated NOEs for LNF-2 in D 2 0 at 500 MHz, 350 ms mixing time, 5"C, and at 500 MHz in DMSO-d, : D20, 250 ms mixing time, 24°C as a function of the glycosidic dihedral angles @ and 9 are given in Figure 6 ( a l , a 2 ) and (b,, b 2 ) , respectively. In D20,the looped regions are those in which

1130

CAGAS AND BUSH

in which calculated and experimental T Ivalues for Fuc4 H1 and Fuc4 H5 agree (Table IIID ) . A summary of the conformations for LND-1 and LNF-2 in both solvents that were found to be consistent with the NOEs and 'H T 1is shown in Table V. As an independent method for determination of T ~ 13C , T 1were measured for LNF-2 at 5°C in D20, at 500 MHz. The sample of LND-1 was of inadequate quantity to obtain accurate 13CT I data. Consistent with earlier data,37T 1of the methine carbon atom resonances of LNF-2 are similar. The range of T Iwas observed to be 235 t 25 ms. Maps of the conformational energy for the Lewisb tetrasaccharide as a function of and J! for each linkage are shown in Figure 7 ( a-c ) , while energy maps for the Lewis" trisaccharide are given in Figure 8 (a, b ) . These plots were obtained in a similar manner as the NOE maps described earlier, wherein the overall ( tetrasaccharide or trisaccharide) conformation is broken down into conformations along each linkage by "collapsing" the other linkage ( s ).

I

5 2

4 8

4 4

1-

4 0

3 6

ppm

Figure 2. The 500-MHz NOESY spectrum of LND-1 in D20 at 5"C, 250 ms mixing time, with the 1D spectrum at the top. d @ P

there is agreement, within the error limits specified in Table 11, of observed and calculated ratios of ( a l ) NOE between Gal3 H1/ GlcNAc H3 to that between Gal3 H 1 / H 3 and ( a 2 ) NOE between Fuc4 H1/ GlcNAc H4 to that between Fuc4 H1/ H2. The points with heavy dots are those for which ratios of all observed (Table 11) and calculated (using trisaccharide model) NOEs agree. These are the two NOE ratios already mentioned, and the ratio of the remote NOE between Fuc4 H5/ Gal3 H2 to the intraring NOE between Fuc4 H l / H 2 . The points marked x are those for which calculated and observed nonselective 'H T 1values for Fuc4 H1 and FUC*H5 also agree, within the limits specified in Table IIIC. The results in DMSO-d, : D20 are similarly shown in (b,, b 2 ) ,with the exception that the intraring NOE between GlcNAc H1/ H5 was used instead of Gal3 H1/ H3 because of overlapping of the Gal3 H l / H 3 and H 1 / H 5 cross peaks in the NOESY spectrum. Again, x-marked points are those

gt!

IB

dl

B

f3

d D o .

cyu N I

5 2

4 8

4 4

4 0

m

3 6

PPm

Figure 3. The 500-MHz NOESY spectrum of LNF-2 in D 2 0 at 5"C, 350 ms mixing time. The 1D spectrum is also shown at the top.

CONFORMATION OF LEWIS BLOOD OLIGOSACCHARIDES

DISCUSSION

Cd’ HI

d

--n

=I

Fur‘ H5

fl

FUC-H5

11

a

1

5 2

-

-

7

4 8

4 4

113 1

4 0

3 6

PPm

Figure 4. Cross sections in w 1 of the NOESY spectra in Figures 2 and 3 at the chemical shifts of ( a ) FUC’H5 in LND-1 (Figure 2 ) ; ( b ) Fuc4 H5 in LND-1 (Figure 2 ) ; ( c ) Fuc4 H5 in LNF-2 (Figure 3 ) ; and ( d ) Gal3 H, in LNF-2 (Figure 3 ) .

T h e heavy dots represent conformations in which the conformational energy of the tetrasaccharide (or trisaccharide) is within 10 kcal of the global minimum. Table VI lists some distinct energy minima within 10 kcal for the Lewisb tetrasaccharide and the Lewis” trisaccharide.

As mentioned earlier, the chemical shifts of both compounds were found to be different in DzO and in DMSO : DzO solution. However, differences in chemical shifts between the D 2 0 and DMSO : DzO solution do not imply that the conformations of the oligosaccharides in these two solvents necessarily differ. On the contrary, we will argue below for the independence of the conformation of these two oligosaccharides on solvent. Earlier studies by Lemieux et a1.6 on the synthetic Lewis oligosaccharides have used an analysis of ‘H and 13C chemical shifts, i.e., interunit deshielding effects, and a semiquantitative interpretation of T I and 1D NOE data to support conformations previously predicted by HSEA calculations. In this work we determine the conformations of these oligosaccharides by a quantitative interpretation of 2D NOESY and T I data, independent of‘ energy calculations. The results of our quantitative comparisons of calculated and observed NOE and T Idata is in general agreement with these earlier results. In spite of the potential difficulties inherent in measuring NOE for molecules of intermediate size, our data show that through control of the temperature and the viscosity of the solvent, it is quite practical to obtain good NOESY data for oligosaccharides suitable for making detailed simulations. The expanded NOESY plot of LND-1 in DzO a t 250 ms mixing time and 5°C (Figure 2 ) shows several inter and intraring NOEs, including small NOEs between protons in nonadjacent sugar residues, i.e., the NOE between FUC’H 5 / GlcNAc H2 and that between FUC*H5/Ga13 H2. The same NOEs are also found in the 300-MHz NOESY spectrum of LND-1 in DMSO-d, : D 2 0 a t 250 ms mixing time, 24°C (data not shown). A similar type of NOE is found in the NOESY spectrum of LNF-2 in D 2 0 a t 350 ms mixing time, 5°C (Figure 3 ) , i.e., cross peak between Fuc4 H5/ Gal3 H2, in addition to the usual cross peaks between protons found in adjacent rings, and intraring NOEs. Typical cross sections along w1 (Figure 4)show good signals well above the noise level for these “long-range” cross peaks, which proved to be important in reducing the number of possible conformations in the conformational maps. Although it is more common practice in detailed comparisons of simulated with measured NOESY data to perform a series of experiments a t different mixing times, we have chosen to do simulations a t a single mixing time and use selective ‘H T Ivalues to normalize NOE cross peaks. In addition to the substantial decrease in experimental time, normal-

1132

300 350 250

CAGAS AND BUSH

r-i

--

200

a

150

-

100

-

50

-

w

..

.

.,

4-

-

4

ii

0

'

2

1 "

'

"

'

"

300 250

--

200

-

150

-

100

-

50

-

'

I

3 5 0 -

'

'

'

300

-

250

-

--f

200

-

a

150

-

100

-

50

-

..XX. x

-

ii

-

I

'

'

I

I

I

I

I

I

o

ZOO.

a

150.

-

.

'

'

I

l

1

Pii(l4)

F u c - ~ - ~ . ~ - C I C N A Elhkaga. rSm-l(DuSODZ0)

W a g s , LKO-l(DYSOD20)

.

250.

c! a

1

i

I

0

1

'..'p, .:x

i

0 1

1

..

1

100.

'

b3

Fuc-d-1.2-Gal

6

'

-

Pll(f2)

bz

300 250

'

T

X.

..

0

'

3 5 0 -

I

L

50

100

'

1

150

200

1

250

' 300

350

i!

150.

50.

loo 1 1

= I

1

200.

200.

1

0 1 0

150.

100.

50.

' 50

'

'

100

150

Pxi(g3)

I 200

Psi(f2)

I

250

L 300

1 1

350

c1

0 1 0

I

50

I

100

150

'

200

I

250

300

i

1 '

350

Psi(f4)

Figure 5. (al-a3) Maps of simulated NOEs (500 MHz, 250 ms mixing time, 5"C, D,O) as a function of conformation of each glycosidic linkage in the nonreducing end of LND1. Results using disaccharide models for each linkage are shown by the regions within the loops, within which calculated and observed values agree for (a, ) the ratio of NOE between Gal3 Hl/GlcNAc H3 to Gal3 H l / H 3 ; ( a z )ratio of NOE between FUC'H1/Ga13 H2 to that between FUC'H1/ H2; and ( aa) ratio of NOE between Fuc4 H1/ GlcNAc H4 to that between Fuc4 H1/ H2. Simulation of NOE ratios of all three interglycosidic NOEs and two remote NOEs (see Table IA) using a tetrasaccharide model yielded conformations for each linkage illustrated by the dots within the loops, while the overlapping X-marked points are those in which calculated and observed T I values agreed (see Table 111). Note that each dot shown above was obtained by plotting only that particular linkage in the tetrasaccharide without regard to the conformations of the other two linkages, i.e., each pair of angles for a particular linkage corresponds to several conformations for the other two linkages. ( blb3) Similar maps of simulated NOEs at 300 MHz, 250 ms mixing time in DMSO-d, : D,O at 24"C, except for the disaccharide models in (b,) the ratio of NOE between Gal3 H1/ GlcNAc H3 to that between Gal3 H l / H5, and in (b2)the ratio of NOE between FUC*H1/ Gal3 H2 to that between Gal3 H1/ H5 were used instead. As in (a1-a3), the dots represent tetrasaccharide conformations in which there is agreement between observed and calculated NOE ratios for all three interglycosidic and two remote NOEs listed in Table IB, and X-marked points are those in which there is agreement between observed and calculated T1 values as well.

izing by using selective T I allows one the flexiblity of measuring the diagonal intensity at any mixing time, particulary one in which there is less diagonal peak tailing. Theoretically, the diagonal peak intensities of all protons should be equal at zero mixing

time. Experimentally, the Vo's of different protons obtained using selective T I were found to differ by less than 10% from the average value. Associated with measuring volumes of peaks in an experimental spectrum are several sources of er-

CONFORMATION OF LEWIS BLOOD OLIGOSACCHARIDES

82

a1

I

I

350.

I

1

250.

I

350.

...... ..... . . . . .x xxx.... ....

300.

'

I

I

I

I

I

I

I

I

250.

' 2

200.

Y

150.

150.

100.

100.

50.

50.

0.

0.

0.

50.

100.

150.

200.

250.

300.

I

0.

350.

50.

I

1

100.

150.

I

200.

I

I

250.

300. 350.

Psi(f4)

Psi@)

bz

bl Linkage, LNF-Z(DMSO:D20)

Gal-bt-1.3-GlcNAc ~~

350.

I

I

I

I

I

I

c

r

... ...... ......x. :..: : - :%.. ... ...

300.

t

-x -xx x:

200.

h

s

Y . d

rl

a

150.

150.

zoo.

100.

50.

50.

loo. I

I

I

1

50.

100.

150.

200.

...

..

250.

Y

0. 0.

Linkage. LNF-Z(DMSO:DZO)

Fuc-al-1.4-GlcNAc

350.

*

250.

a

I

300.

-

200.

v

zw a

linkage, LNF-Z(D20)

Fuc-al-1.4-GlcNAc

linkage. LNF-2(D20)

Gal-bt-1.3-GlcNAc

zw

1133

I

I

250.

300.

1

350.

0.

I

I I

I

I

I

I

Psi(g3)

Figure 6. Maps of simulated NOEs as a function of conformation of each glycosidic linkage in the nonreducing end of LNF-2. ( a l , a 2 ) Results of simulation a t 500 MHz, 350 ms mixing time in D20a t 5"C, while ( bl , b l ) are for simulation at 500 MHz, 250 ms mixing time in DMSO-d, : D20 at 24°C. Conformations within the looped regions are those in which there is agreement between observed and calculated ratios of the interglycosidic NOE, i.e., from the anomeric proton to the aglycone proton, to an intraring NOE using disaccharide models for each linkage, while the dots are those in which there is agreement between observed and calculated ratios of the two interglycosidic and one remote NOEs (see Table 11) simulated using a trisaccharide model. Points marked by X are those in which observed and calculated TI values also agree. In the case of the trisaccharide, note that a conformation for one linkage actually corresponds to several conformations for the other linkage. See Results for detailed explanation.

1

I

I

1134

CAGAS AND BUSH

Table V Conformations of LND-1 (Lewisb)and LNF-2 (Lewis") Fragments Consistent with Observed NOE * and TI+ A. Lewisb Tetrasaccharide: Fuc(a-1 --t 2)-Gal(P-l + 3) [Fuc(a-1 + 4)1-GlcNAc-B-OMe

-70 -80

5 24

D20 DMSO-d6 : DZO

(+lo) (+-lo)

-100 (210) -110 (210)

(+lo) (+lo)

-80 -80

140 (210) 140 (*lo)

-70 (210) -80 (+lo)

140 (210) 130 (210)

@F"d

*FUC4

-70 (210) -70 (210)

140 (?lo) 140 (210)

B. Lewis Trisaccharide: Fuc(a-1 --t 4)[Gal(P-1 + 3)]-GlcNAc-@-OMe Temperature ("(2)

Solvent

5 24

DzO DMSO-dc : D20

aFuc2

@Gal

*Gal

-70 (210) -80 ( f 1 0 )

-100 (+-lo) -100 (+lo)

*F"&

~

* Refer to Tables I and I1 for observed NOES. t Refer to Table I11 for experimental T1values.

that were more than 50% overlapping, e.g., Gal3 H1/ H 3 and Gal3 H 1 / H 5 in LNF-2 in DMSO : DzO. In this case, another intraring NOE was used to calculate ratios, although the interglycosidic NOE remained the same (see Results). However, in the case of the Fuc4 Hl/GlcNAc H4 and Fuc4 H l / H 2 cross peaks in LNF-2 (both solvents) in which there was only a small region of overlap, the isolated peaks were integrated separately and to each was added one-half of the total integrated intensity of the overlapping portion. For LND-1 in DMSO-d, : D20, Fuc2 H2, H3, and H4 are strongly coupled (see Table

Tor. One is noise, which may not be uniform throughout the spectrum. For these oligosaccharides, the region below the intense diagonal peaks a t 3.44.0 ppm has greater tl noise compared to other sections of the spectrum. In the case where the peak in question was obviously distorted by tl noise, only the peak on the upper side of the diagonal was used. T o minimize errors from choosing the size of the region of integration, we obtained a n upper and lower limit for the peak volumes. Another potential source of error is overlap of cross peaks and strong coupling. No attempt was made to quantitate peaks

b

a Gal-bt-1.3-GlcNAc

P

Linkage. L e n 3 B tetrasaccharide

Fuc-al-1.2

Imkaga. Lewis

c-----77-q

350

I

.. .. ...

+

300

i

..

I

;

250

1

I

250

yp-7

350

I

... .. .... ...... ...... ...

r 1

Fuc-al-1.4

B tetraaaccharlde

... ...

50.

1

300

...

1

0 0

r

50

100

150

,

200

,

250

,

300

,

j

350

5

0

1

iI

;

I

I

I

.. ..

..

r I

I

r

100

I

1

I

t

100

h k a g e . Lens B tetrasaccharide

~ - v - - - - r - ~ - - - r - - ~

0 1

50

0

100

150

200

PSl(12)

P.I(E3)

250

300

350

0

" 50

100

1

150

200

Ps~(14)

Figure 7. Conformational energy (within 10 kcal/ mol of the global minimum) for the Lewisbtetrasaccharide, Fuc a- (1 + 2 ) -Gal (3- (1 + 3 ) [ Fuc a - ( 1 -+4 ) ] GlcNAc P-0-Me, as a function of the glycosidic angles @ and 9 for each of the following linkages: ( a ) Gal 6- ( 1+ 3 ) GlcNAc; ( b ) Fuc a- ( 1 2 )Gal; and ( c ) Fuc a- ( 1 + 4 ) GlcNAc. As in the NOE maps (Figures 5 and 6 ) , the angles for each linkage in the tetrasaccharide are plotted separately. --+

1

250

1

300

350

CONFORMATION OF LEWIS BLOOD OLIGOSACCHARIDES

1135

b

a Gal-bt-1.3-GlcNAc 350. I 1

4

linkage, Lewis A trisaccharide I

I

I

... ...

Fuc-al-1,4

350.

I

.

... ... ... ... ... ..

250.

.. ..

300.

... .... ... ...

.. .. ..

; ; 200.

c

w

linkage. Lewis A trisaccharide

Y

a a

.

150.

100.

50.

50.

n_ .

0.

0.

50.

100.

150.

200. 250. 300.

350.

0.

50.

100. 150.

200.

250.

300. 350

Psi(f4)

Psi(g3)

Figure 8. Conformational energy (within 10 kcal of the global minimum) for the Lewis" trisaccharide, Gal P- ( 1+ 3 ) [ Fuc a- ( 1+ 4) ] GlcNAc P-0-Me, as a function of the glycosidic angles @ and \k for each of the following linkages: ( a ) Gal 0-( 1 + 3 ) GlcNAc and ( b ) Fuc a- ( 1 + 4 ) GlcNAc. Angles for each linkage in the trisaccharide are plotted independently of each other (see Figures 5-7).

IVA ) ,which makes the measured volume of the FUC* H1/ H2 cross peak ambiguous due to distribution of enhancements among all three proton^.^' We therefore used another intraring NOE ( Gal3 H1/ H5 or GlcNAc H1/ H5) to calculate NOE ratios. The final Table VI

normalized peak intensities listed in Tables I and I1 reflect all these sources of error. In the NOE calculations a good estimate of T, is required by the strong dependence of the calculated NOE intensity on the value of 7,. By reproducing

Selected Distinct Energy Minima* for LND-1 (Lewis*) and LNF-2 (Lewis") Fragments Conformational Angles

Conformer

@Gal

*Gal

@Fuc2

*Fur2

@Furl

*F"C4

Relative Energy (kcal/mole)

A. Lewisb tetrasaccharide: Fuc(a-1 + 2)-Gal(P-1 + 3 ) [Fuc(a-l + 4)I-GlcNAc-P-OMe -50 - 70 -80 -50 -70 -50

-100 - 100 - 100 -100 -100 -100

-80 -90 -90 -90 -110 - 150

-80 150 140 140 160 90

-140 -150 -70 - 140 -80 -140

90 90 140 90 140 90

0.0 1.1 3.8 4.1 5.1 6.5

90 90

0.0

- 150

-70 -150

140 90

B. Lewis" trisaccharide: Fuc(a-1 + 4)[Gal(P-1 + 3)I-GlcNAc-fl-OMe -50 -90 -80 - 150

-100 -100 -100 -140

- 140

3.1 3.2 7.0

* Energies were calculated using the parameters of Momany et al.36Only the minima with relative energies 510 kcal/mol are listed.

1136

CAGAS AND BUSH

a n intraring NOE, a value for 7, was obtained. For LND-1 and LNF-2 in D 2 0 a t 5"C, 7, was estimated to be 1.1 t .1 and 1.0 ? .1 ns, respectively. The 7,'s obtained by simulation of intraring NOEs in DMSO4 : D 2 0 a t 24°C were 1.1 t .1 ns for LNF-2 and 2.0 k .1 ns for LND-1. From "C T1measurements (data not shown), 7, for LNF-2 in D 2 0 a t 5°C was estimated to be 0.50 ns, and calculated NOEs using this 7, were approximately 30% of those obtained using 7 , from intraring NOE. For both I3C T I and NOE, the derived value for r, was found t o be extremely sensitive to the value of T 1or NOE, due to the fact that these are close to their minimum values a t 500 MHz. It is possible that the lack of agreement between 7, values obtained from 13C T I and intraring NOEs comes from inapplicability of the isotropic motional model used in this analysis. However, in order to provide internal consistency we used r, obtained from intraring NOEs, and ratios of calculated inter- to intraring NOEs were compared with experimental ratios in the a n a l y ~ i s . ~ It - ~should - ~ ' also be noted that the range of 7, indicates slightly different values obtained from different rings in the oligosaccharides. Although these differences in 7, may be interpreted as arising from internal motion, inconsistent with our neglect of internal motion in Eq. ( 5 ) above, we wish t o point out that differences of 0.2 ns are extremely small. Although little is known about the time scale of internal motion in oligosaccharides, it is our interpretation that this much flexibility is so small as to be undetected in similar NOE studies of proteins or nucleic acid duplexes. Such a small amount of internal mobility can be detected only in oligosaccharides for which 7, is close to the reciprocal of the spectrometer frequency resulting in the great sensitivity of the NOE to 7,. T h e relatively long mixing times (250-350 m s ) used to observe NOEs in oligosaccharides require that all spins be considered in a complete relaxation matrix analysis.31s40 Use of the isolated spin-pair approximation is expected to result in large errors in calculated intensities a t long mixing times, where spin diffusion is ~ i g n i f i c a n t . ~ "41~ This ~ . ~ ~ is . even more important in the case of oligosaccharides since NOE intensities are low a s a result of wr, x 1. We also show here that useful information regarding three-dimensional structure is obtainable from a single NOESY spectrum, provided that multispin and motional effects are properly taken into account. Results of NOE and T1simulation for both LND1 and LNF-2 in both solvents indicate in each case the existence of a single small group of closely similar conformations that agree with the experimental

data. For LND-1 in D 2 0 ,the following values of the glycosidic angles were deduced: @.gal = -70 (+lo), '€'gal = -100(+10), @fuc2 = -SO(&lO), \kfuc2 = 140(?10), @fuc4 = -7O(-tlO), 'Pfuc4 = 1 4 0 ( t 1 0 ) . The conformation derived from NOE and 'H T1data agrees closely with minimum energy conformer 3 (Table VIA), shown in stereoview in Figure 9 ( a ) , and that predicted by HSEA calculations for a synthetic Lewis determinant.6It is also consistent with the qualitative interpretation of ROESY data13 for LND-1 in which there is back folding of the FUC' residue toward GlcNAc. As was observed in the ROESY data for LND-1, small NOEs were also seen in the NOESY spectra from the methyl group of GlcNAc to Fuc2 H 3 and Gal3 H1, indicating a trans orientation of N-H to GlcNAc H2. While it is not possible to obtain a quantitative interpretation of the NOE of methyl groups using our method due t o complications of their internal rotation, the proximity of the amide methyl group to these protons is apparent in our model of Figure 9 ( a ) . Rao et al.5 reported values of '€' and @ for the dihedral angles of Fuc2 and Gal3 in the milk pentasaccharide LNF1similar to those reported here for the same linkages in LND-1, implying that Fuc2 is tightly folded relative to GlcNAc in both LNF-1 and in LND-1. Breg e t al.I3 have used ROESY spectroscopy t o study these oligosaccharides and have encountered difficulty in quantitative interpretation of the data. No simple analogue of the matrix method for simulation of NOESY data is available for ROESY because of the effects of Hartmann-Hahn transfer. These effects are likely to make significant contributions t o the cross peaks in ROESY of oligosaccharides because the chemical shifts of the methine protons are not widely ~ e p a r a t e d .We ' ~ conclude that a better approach to nmr studies of the conformation of oligosaccharides for which w r , = 1 is to measure ordinary NOESY by adjusting temperature and solvent viscosity. T h e model of LNF-2 that is most closely consistent with NOE and 'H T l data in D 2 0 has the following glycosidic angles: @.gal = -70( * l o ) , \kgal = - l O O ( + l O ) , @fuc = -70(?10), P ' f, = 140(+10). This conformation is close to the energy minimum 3 in Table VIB, a stereo model of which is shown in Figure 9 ( b ) , which agrees with HSEA calculations for a synthetic Lewis" determinant.6 Our results show that the conformation of the Fuc a1,4(Gal @-1,3)-GlcNAc moiety is essentially the same in LNF-2 and LND-1, suggesting a very low degree of flexiblity in these molecules. We also found only small differences in the conformations in D 2 0 and in DMSO : D 2 0 (see Figures 5 and 6, Table V ) .

CONFORMATION OF LEWIS BLOOD OLIGOSACCHARIDES

1137

b

a

b Figure 9. Stereodiagrams of ( a ) the Lewisb tetrasaccharide for minimum energy conformation 3 (Table VIA) and ( b )the Lewis” trisaccharide for minimum energy conformation 3 (Table VIB). Both these conformations are consistent with observed NOES and T, values (Table V ) .

The same observation regarding solvent dependence was made for blood group A and H oligosaccharide^,^.^' which indicates the strong influence of nonbonded interactions on the conformation of these blood group o l i g o s a ~ c h a r i d e s . ~ * ~ ~ Bechtel et al.15 have noted that for a Lewis” trisaccharide a number of interproton distances deviated from those predicted by a rigid structure

model, assuming measurement a t initial rate, i.e., I A B = C r - 6 . Although the occurrence of motion cannot be totally discounted as was discussed above, it is likely that these “anomalous” interactions arise from spin diffusion effects due to the long mixing time (800 ms ) used by these authors. In simulations with mixing times as long as 800 ms, the calculated NOE show wide deviations from the initial rate ap-

1138

CAGAS AND BUSH

proximation. It is our interpretation that a full matrix analysis of NOE data for their oligosaccharide would lead to conclusions consistent with our results. Our conclusion that the nonreducing terminal fragments of these two Lewis blood group oligosaccharides adopt single rigid conformations differs from conclusions reached on mannose oligosaccharides by Carver and c o - w ~ r k e r s ~and * ' ~by Ha et a1.,I2 and by Tvaroska et al." on maltose. These workers conclude that their nmr nuclear Overhauser data and molecular modeling results are not consistent with a single conformation for the glycosidic linkages of these oligosaccharides. It is our interpretation that these discrepancies result not from any difference in the experimental and molecular modeling methods employed, but rather from real differences in the behavior of the different oligosaccharides studied. We acknowledge the facilities of the National Magnetic Resonance Facility at Madison, supported by NIH Grant RR02301, for making available the Bruker 500 MHz spect.rometer and for assistance in the measurements. This research was supported by NIH Grant GM-31449.

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Received J u n e 29, 1990 Accepted August 22, I990