ELSEVIER

THEO CHEM

Journal of Molecular Structure (Theochem) 395-396 (1997) 245-270

The use of the MM3* and ESFF force fields in conformational analysis of carbohydrate molecules in solution: The methyl cu-lactoside case.

Juan Luis Asensio, Manuel Martin-Pastor, Jestis Jimhez-Barbero*

Gmpo de Carbohidratos, Departamento de Quimica Orgdnica Biolrigica, Institute de Quimica Orgdnica, (C.S.I.C)., Juan de la Ciema 3,

28006 Madrid, Spain

Received 4 October 1996; accepted 24 October 1996

Abstract

The solution conformation of methyl a-lactoside has been studied through NMR spectroscopy and molecular mechanics calculations using the MM3* and ESFF force fields. The steady-state and transient NOES have been interpreted in terms of an ensemble average distribution of conformers, making use of the complete relaxation matrix approach. The molecular mechanics calculations have been performed at two dielectric constants (E = I and 80 Debyes) for both force fields in an exhaustive way, and in the case of MM3* have been complemented with calculations using the continuum solvent model GB/ SA. Relaxed energy maps and adiabatic surfaces have been generated for the different dielectric constants. The probability distribution of conformers has been estimated from these steric energy maps. MM3* molecular dynamics simulations in vacua

and with GB/SA have also been performed. The comparison between predicted and experimental results indicate that the p-( l- > 4) glycosidic linkage shows some fluctuations among three low-energy regions, although it spends ca. 90% of its time in the region close to the global minimum. The results indicate that MM3*, when used with high dielectric constants or with the GB/ SA solvent model, satisfactorily reproduces the conformational properties of methyl cY-lactoside in water solution. The use of ESFF only provides a qualitative agreement between experimental and theoretical results. 0 1997 Elsevier Science B.V.

Keywords: Molecular mechanics; Molecular dynamics; Methyl a-lactoside; Conformational analysis in solution; Nuclear

Magnetic Resonance Spectroscopy

1. Introduction

There is a growing interest in the study of the three- dimensional structure of oligosaccharides and glyco- conjugates [I]. The understanding of how these molecules are recognised by the binding sites of lec- tins, antibodies and enzymes is currently a topic of major concern [2]. To play a role in their biological functions, the three-dimensional structure of the

* Corresponding author

carbohydrate is of primary importance [3]. The extent and nature of the motion around the glycosidic lin- kages of oligosaccharides [4] remains an open ques- tion (51, and even detailed analysis of experimental and theoretical results have concluded either single rigid conformations [6] or conformational averaging [7] for different-or the same-carbohydrate struc-

tures. One of the most usual methods of establishing the solution conformation of biomolecules is the com- bination of NMR spectroscopy [8] and molecular mechanics and dynamics simulations [9], techniques

0166-1280/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved

PII SO 166- 1280(96)04956- I

246 J.L. Asensio et nl.Nournal of Molecular Structure (Theochern) 395-396 (1997) 245-270

which are also currently applied in the carbohydrate field [lo]. Nevertheless, there are still important pro- blems with the force field calculations which mainly arise from the lack of general valid parameter sets, which are usually insufficiently proven, and this is

particularly true for oligo- and polysaccharide moi- eties. In this context, different force fields have been used in the conformational analysis of these biomole- cules [6,1 l-191. Following our studies on the appli- cation and testing of different force fields for conformational analysis of carbohydrates in solution [20-221, we now report on the study of the three- dimensional structure of methyl fi-galactopyranosyl- (l- > 4)-cu-glucopyranoside (methyl cY-lactoside, 1) using MM3 * [23] and ESFF 1241 molecular mechanics and dynamics calculations and NMR spectroscopy. The regular MM3 force field [23] has been exten-

sively used by French et al. [13,25-271 to compare theoretical and experimental X-ray results for differ- ent mono- and oligo-saccharides with more than satis-

factory results. In addition, the results provided by MM3(92) have been recently compared to those pro- vided by other force fields [28]. Although the MM3* (which uses charge-charge instead of dipole-dipole interactions) has been recently employed in the con- formational study of a number of carbohydrates [29,30], to the best of our knowledge there is no reported systematic approach to the use of this force field in the oligosaccharide field such as that presented herein. In addition, the possibility of employing the GB/SA solvation model [31] in conjunction with MM3* makes its use particularly attractive for solu- tion studies [32]. Besides, we also present the results obtained when using the recently developed extensi- ble systematic force field (ESFF) force held [24].

2. Methods

2.1. NMR experiments

The methodology and NMR spectra used to study methyl u-lactoside have been already described by us [20]. Thus, only the relevant data will be described. The NMR spectra included 2D rotating frame NOE (ROESY, CAMELSPIN) experiments with mixing times of 100, 250, 400 and 700 ms. In addition, 2D- NOESY experiments with mixing times of 200, 300,

500, 900 and 1500 ms, and steady state NOES at 300 and 500 MHz, with a saturation delay of 10 s were also performed. For all the NOE experiments, the intensity of the partially overlapping H-3’ + H-4 sig- nal was compared to that of H-5’. Since H-3’ has a fixed distance to H-l ‘, and, in addition. it is possible to have an exact measurement of H-l ‘-H-5’ NOE, the independent contribution of H-3’ was calculated according to a relaxation matrix approach [33] (see below), and then H- 1 ‘-H-4 was estimated from direct subtraction. Since the relevant protons for the NOE calculations (H-3, H-4, H-6a, H-6b, H-3’, H-5’) are not affected by strong coupling effects, no effort was made to account for these effects. HSMQC-ROESY

experiments were also collected with spin locking times of 200, 300, 500 and 650 ms.

2.2. Conformational calculations: Molecular

mechanics

Glycosidic torsion angles are defined as Cp H- 1 ‘-C- l ‘-O- 1 ‘-C-4, and \k C- 1 ‘-O- 1 ‘-C-4-H-4. Relaxed (a,*) potential energy maps were calculated for 1 by using MM3* as integrated in MACROMODEL 4.5 [34]. This program differs from the regular MM3 force field [23] in the treatment of the electro- static terms, since it employs charge-charge instead of dipole-dipole interactions. The potential energy maps were also calculated by ESFF [24] as integrated in DISCOVER 94.0 [35]. Only the gg and gt orienta-

tions of the lateral chain were used for the glucose moiety [36], while tg and gt conformations were used for the galactose residue [37], since they have been shown to be rather more stable than the altema- tive tg or gg conformers, respectively. Thus, four combinations were taken into account, namely, gggt, gtgt, ggtg and gttg. The first two characters corre- spond to the glucose unit, and the other two to the galactose one. The starting position for the secondary hydroxyl groups was set as r (anti-clockwise) or c (clockwise). Also, the four more stable combinations were considered, rr, rc, cr or cc (clockwise) [ 131. The first character corresponds to the glucose ring while the second one belongs to the galactose one. There- fore, sixteen initial configurations were taken into account, and sixteen different relaxed energy maps were built for each dielectric constant and force field (E = 1 and E = 80 Debyes for MM3* and ESFF,

J.L. Asensio et al./Joumal of Molecular Structure (Themhem) 395-396 (1997) 245-270 247

and, in addition, the GB/SA solvent mode13’ for MM3*). In total, 6400 conformers were calculated for each E. The previous step involved the generation

of the corresponding rigid residue maps by using a grid step of 18”. Then, every a, q point of this map was optimised using 200 steepest descent steps, fol- lowed by 2500 conjugate gradient iterations. Follow- ing this protocol, the maximum rms derivative was smaller than 0.05 kcal mall’ A-‘. Despite the restric-

tion set around the glycosidic linkages (4000 kcal rad-‘), deviations smaller than 0.3” in WP values were observed in high-energy regions. From these relaxed energy maps, adiabatic surfaces were built by choosing the lowest energy structure for a given @,\k point. Therefore, in these surfaces, which repre- sent a two-dimensional projection of a multidimen- sional hyperspace, points coexist which come from different geometries in terms of the secondary hydro-

xyl and hydroxymethyls.

This relationship can be used in a simple way to trans-

form energy maps into probability maps. In our case, the sum of the numerator extends over sixteen points, which correspond to the sixteen relaxed maps. The sum on the denominator considers 6400 points, i.e. sixteen maps consisting of 400 points each.

2.5. NOE calculations

The first step in the NOE calculations was to estimate the interproton average distances, using a < r-6 > kl average.

2.3. Molecular dynamics

The estimated probability distributions from the 16 relaxed energy maps were used to calculate the aver- age distances according to the following equations:

< r+ >k[ = CP(W * r -%(w)

In addition, the average distances were also estimated

from the MD simulations, considering the interproton distance for every saved frame: < rmh >kl = C(l/n) * r-%(w), where n represents the total number of frames.

Geometries describing local MM3* minima were extensively minimised using conjugate gradients and then taken as starting structures for Molecular Dynamics simulations with MM3* as integrated in MACROMODEL 4.5 [34]. The simulations were repeated with E = 80 D and with the GB/SA solvent model described by Still et al. [31]. The simulations were performed at 300 K and a time step of 1 fs, using the SHAKE option [38]. The equilibration time was 100 ps, while the total simulation time was 3.6 ns. The temperature was controlled during the equilibration and simulation periods by coupling to a temperature bath, using an exponential decay con- stant of 0.1 ps [39]. Trajectory frames were saved every 0.5 or 1 ps.

2.4. Probability calculations

From the sixteen relaxed energy maps, calculated for each dielectric constant, the probability distribu-

tion was calculated for each WP point. Assuming that the entropy difference among the different conformers is negligible, the probability P of a given WP point is

[40] :

The steady-state 1D NOE and the transient 2D NOESY and 2D ROESY spectra were calculated according to the complete relaxation matrix method assuming isotropic motion, external relaxation of 0.1 s, and a correlation time 7, of 0.06 ns. All the spectra were simulated from the average relaxation rates (from < rm6 > u) calculated from both the relaxed relative energies (E = 1 and 80 D, also GB/ SA for MM3 *), and the MD trajectories (E = 80 D and GB/SA) at 300 K. In the case of the steady-state NOES, the calculations were performed by solving the simultaneous set of linear equations proposed by Noggle and Schrimer [41], while the NOESY and ROESY spectra were simulated using the protocol outlined by Cagas and Bush [42]. All the NOE calcu- lations were performed using software written in- house. The software is available from the authors and uses as input the coordinate files provided by MACROMODEL [34] or INSIGHT11 [43].

3. Results

3.1. MM3 * calculations

f’+p = CiW - E,/fW/&&+xp( - Ei+q/RT) Fig. 1 shows the structure of methyl cY-lactoside (1)

248 J.L. Asensio et al.Nournal oj’M&cular Structure (Theochem) 395-396 (1997) 245-270

OH 6’

OMe

OH

6

OH

Fig. I. View of methyl a-lactoside (1) showing the atomic numbering

along with the atomic numbering. The analysis of the sixteen relaxed energy maps calculated by MM3*, using E = 1 (not shown, see supplementary material) indicated that the shapes of the surfaces are strongly dependent on the initial configuration of the second- ary hydroxyls, and, although less pronounced, also on the hydroxymethyl groups. This fact illustrates the importance of considering both factors when the molecular mechanics calculations are performed at

low dielectric constant values. After energy minimi- sation, it can be observed that, in all cases, there is a broad low-energy region, which, depending on the orientation of the hydroxyl groups, can be described by different conformers with rather small energy bar- riers among them, and two separated smaller islands.

The adiabatic surface built from the sixteen relaxed maps is shown in Fig. 2. The isoenergy contours are drawn every kcal mol-‘. The total minimisation of the

Table 1

Relative steric energy differences (kcal mol-‘) and single point and ensemble average populations for the low-energy conformers of 1

Conformer (a/*)

la 2a 3a 4a lb 2b 3b 4b

l80/ - I8 54/- I6 %I- 179 3l/- 46 I6812 50/6 42/- 179 - 32/- 33d

AE,e= I

Pop. (%)

Ensemble average

pop. (a) AE,E = 80

Pop. (%)

Ensemble average

pop. (8)

AE, GBISA

Pop. (%)

Ensemble average

pop. (R)

3.6 0.0

<I 86

I 99’

4.3 0.0

<I 78

I 96’

4.5 0.0

<I 79

<I 99’

3.7 I.1 0.0 2.5 2.1 2.2 <I I4 93 2 3 2

<I 99‘ 85 9’ 6 9’

2.2 0.8 I .2 0.0 0.3 0.7

2 20 7 49 29 I5

3

3.9

<I

<I

96’ 3 77’ 20 77’

2.0

21

99‘

* MM3’:

b ESFF;

’ Minimum 2 + Minimum 4;

“Wq l9/-45,fors=80D.

J.L. Asensio et ai./Journai qf Moleculrrr Structwe (Throchem) 395-396 (1997) 245-270 249

(4 -180 -120 -60 0 60 120 180

i80-

80

-i80 -120 40 0 60 120 180

-16011-160 -180 -120 -60 0 60 12'3 160

60 60 60 60

0 0 3 O 0 3

-60 -60 80 .60

-120 -120 -120 -1M

-160 -180 -160 -180 -120 -60 0 60 120 180

-180 -160 -120 -60 0 120 180

cb 0

-t80 -180 -120 -60 0 60 120

60

0 3 O

60 -60

-120 -120

-180 -160 160 -160 -129

-180 40 0 120 180

Fig. 2. (A) Adiabatic maps calculated from the relaxed steric energy plots obtained by using MM3’ (left) E = 1 and its co~espondi~~ population

distribution (right) for methyl wlactoside. The level contours for the energy map are given every kcai mole’. The level contours for the

probability map are given at IO%%, I% and 0.1%. Q, is indicated horizontally, and q along the vertical axis. (B) Partial adiabatic maps obtained

for the different orientations of the hydroxymethyl groups of methyl rw-lactoside using MM3’ with F = 1. From left to right: gg,q, RR@, gfxl and

REP o~entatjons. Q, is indicated ho~zontally, and rk along the vertical axis.

250 J.L. Asensio et d/Journal of Molecular Structure (Theochem) 395-396 (1997) 245-270

4

Fig. 3. Stereoscopic views of the low-energy conformers of 1 using the MM3* and ESFF force fields. Approximate W\k angles are as follows:

From top to bottom: minimum 1 (180/ - 18), minimum 2 (54/ - 16), minimum 3 (36/ - 179), minimum 4 (31/ - 46).

geometries included in the different valleys of this map affords four main minima, below a steric energy level of 4 kcal mol-‘. Table 1 shows the values of the steric energies and of the estimated populations of these conformers of 1 obtained by using the MM3* force field. The single-point populations are calcu- lated from the energy values for the corresponding local minima, and not for the conformers having the exact WP value. Anyway, the torsion angle values for the different minima did not differ more than 10” from the figures of Table 1, independent of the dielectric constant used. The partial adiabatic maps obtained for the different orientations of the hydroxymethyl groups are shown in Fig. 2b. It can be observed that the cal- culated maps for a gg orientation of the hydroxy- methyl glucose residue are somehow more extended along the 9 axis than those for a gt conformation. In contrast, the orientation of the galactose lateral chain does not influence the general shape of the maps.

Fig. 3 shows views of the main four low-energy conformers of 1. The previously reported experimen- tal and theoretical structures for different /3( l- > 4) equatorial linked disaccharides [44,45] are included in the central low-energy region close to minima 2/4. This region is fairly extended and is defined by + values between - 60 and 80” and a set of + values ranging between - 60 and 60”, that is, ca. 14% of the total area. The other two regions around conformers 1 and 3 are narrower, and account for an additional 6% of the bidimensional energy surface. The energy bar- riers between minima 1 and 2 are of ca. 10 kcal mol-‘, while that between 2 and 3 is about 8 kcal mol-‘. The barrier between minima 2 and 4 is barely detectable. It is noteworthy to mention that the possibility of the existence of islands 1 and 3 in solution cannot be discarded since their existence has been reported, for some p-(1- > 4)-linked oligosaccharides, in the case of interglycosidic synthetic disaccharide

Table 2

J.L. A.serwio et ul./Journal of Molecular Structure (Theochem) 395-396 (1997) 245-270 251

Calculated probability distributions for the different configurations of 1 from relaxed steric energy maps using the MM3* and ESFF force fields

Configuration Pop. (%)

a b c d e

gggtcc 0.0 1.3

RRg’cr 63.5 23.4

gggrrc 0.0 0.5

RRRt” 0.9 9.2

ww 0.0 0.1

‘%vx?c’r 5.6 0.9

‘%wRr~ 0.0 0. I ‘sisrr 0.0 0.4

gtgtl’c 0.1 2.4

‘yt‘ytc’r 26.5 41.8

gtgtrc 0.0 0.9

grgtrr 0.5 16.0

gttgc’c 0.0 0.3

gttgu 2.6 1.8

gttgm 0.0 0.1

gttgrr 0.0 0.7

cc 0 4

CT 98 68

i-c 0 2

IY- 2 26

“MM3’,o=l,

hMM3*,e=80;

‘ MM3*, GBISA;

* ESFF, F = 80;

‘ESFF,o= I.

acetals [46] and for an oligosaccharide bound to a lectin [47].

Minimum 1, located in the right part of the map, shows two interresidue hydrogen bonds, namely HO- 2’-O-3 and HO-6’-0-6, with short H...O distances. Minimum 2, located in the central part of the surface, shows only one hydrogen bond, HO-3-O-5’, the one observed in crystalline lactose [44]. Minimum 4 does not show any interresidue hydrogen bond. According to the probability distribution, more than 95% of the population is concentrated around minimum 2.

Minima l-3 are in agreement with the operativity of the exo-anomeric effect [48]. The role of the posi- tion of the secondary hydroxyl groups in the distribu- tion was also calculated and demonstrated that most of the conformers of the adiabatic map have orientation cr (98%). Table 2 shows the probability distribution

for the sixteen initial configurations. It can be observed that the gggtcr and gtgtcr configurations dominate the probability distribution. Regarding the

0.4 6.2 0.2

66.8 6.5 1.1

0.0 6.4 0.1

2.2 6.9 3.0

0.2 4.5 0.6

20.5 4.6 I .7

0.0 4.x 0.1

0.0 4.8 3.1

0.1 7.6 0.1

1.3 7.9 0.1

0.0 8.0 I .o

0. I 8.5 23.1

0.0 5.6 0.2

2.1 5.5 0.1

0.0 6.0 I .9

0.0 5.9 63.4

I 24 1

97 25 3

0 25 3

2 26 93

orientation of the hydroxymethyl groups, MM3* at this low dielectric constant predicts a 70:30 ratio between the gg:gt rotamers for the glucose unit and a 9 I:9 equilibrium between the gt:rg conformers of the galactose hydroxymethyl moiety.

Sixteen relaxed energy maps were also calculated using E = 80 D. In this case, the sensitivity of the surface to the initial configuration is smaller than for E = 1. The corresponding adiabatic map is shown in Fig. 4a. The relative energies calculated for the local minima are also shown in Table 1. At this dielectric

constant, the energy barrier between minima 1 and 2 is now smaller, ca. 8 kcal mall’, while that between 2 and 3 decreases to about 5 kcal mol-‘. The region around conformer 2 still dominates the probability distribution, but the population is extended towards minimum 4 (minima 2 + 4, ca. 96%). Minimum 3 is populated to a ca. 3% extent. The areas which are populated now account for 12% of the complete potential energy surface. The probability in other

252 J.L. Asensio et a~./~ou~n~ll of Molecular Sttwture ~~heo~.he~lj 395-396 (1997) 245-270

(4 -160 -120 -55 0 60

180 120 180

160

120 120

60 60

O 3

0

-65 -60

-tm -120

-180 -160 -1.90 -120 -65 0 60 tm 165

-180 -120 -65 0 60 120 185

3

im 120

65 64

0 0

SO 40

420 -1m

-180 -185

-1643 -120 40 0 60 120 160

-180 -120 .60 0 60 tm 180 185

im

60

-180 -180 -120 40 0 60 120 185

3

-180 t80,

-tm 60 0 60 120 180 , , -180

120- - 120 i

60

0

80

420

30 -x%0 40 40 6 150. li0"

0 +

Fig. 3. (A) Adiabatic maps calculated from the relaxed sreric energy plots obtained by using MM3* (left) e = 80 and its corresponding population distribution (right) for methyl cy-lactoside. The level contours for the energy map are given every kcal mol“. The level contours

for the probability map are given at 1 O%, 1% and 0.1%. @J is indicated horizontally, and ‘4’ along the vertical axis. (B) Partial adiabatic maps

obtained for the different orientations of the hydroxymethyl groups of methyl or-lactoside using MM3’ with E = 80. From left to right: gggr,

ggrg, gfgr and gftg orientations. Q, is indicated horizontally, and * along the vertical axis.

J.L. Asensio et d/Journal of Molecular Structure (Theochem) 395-396 (1997) 245-270

(4

253

-180 160

-120 -64 0 80 120 160 160 0

160

120 120 120 120

60 60 60 60

0 0 3 O 3

0

-60 so 60 60

-120 -120 -120 -120

-160 -160 -160 -180 -120 -60

-lea 0 60 120 160 -160 -1M 40 0 60 120 160

4 0

60 60 60 66

3 O 0

3 O 0

40 40 40 40

-120 -120 -120 -126

-1.30 -160 -180 -1.30 -1110 -120 -80 0 60 120 160 0

+ +

Fig. 5. (A) Adiabatic maps calculated from the relaxed steric energy plots obtained by using MM3* (left), with the GB/SA solvent model and its corresponding population distribution (right) for methyl cx-lactoside. The level contours for the energy map are given every kcal mol-‘. The

level contours for the probability map are given at 10%. 1% and 0.1%. @J is indicated horizontally, and Q along the vertical axis. (B) Partial

adiabatic maps obtained for the different orientations of the hydroxymethyl groups of methyl or-lactoside using MM3’ with the GBlSA solvent

model. From left to right: gggt, ggtg, gtgt and gttg orientations. + is indicated horizontally, and f along the vertical axis.

254 J.L. Asensio et d/Journal of Molecular Structure (Theochem) 395-396 (1997) 245-270

(B) a b

120

60

-160 -1x) -60 0 60 120 16C ISO,.,.,.,.,.,. <SO

-120

-160 -160 -120 -60 0 60 120 1

6

120

60

0

.60

.12

j'"

I -0 rmo moo 3mo rmo

tit7-b8 (~4

Fig. 6. Trajectory plots of one MD simulation (MM3 *, E = 80, time 4 ns) for 1, starting from minimum 2. (A) Trajectory of the simulation in 3/q

space. (B) From left to right and top to bottom: (a) History of @. (b) History of \k. (c) History of hydroxymethyl w for the Glc moiety. (d) History

of hydroxymethyl w for the Gal moiety.

regions of the map is negligible. The associated prob- orientations of the lateral chains are shown in Fig. 4b. abilities for the different orientations of the hydroxyl The most abundant conformers have now cr (68%) and hydroxymethyl groups are collected in Table 2 and rr (26%) configurations for the hydroxyl groups, and the partial adiabatic maps for the four calculated while the ratio of rotamers for the glucose and

J.L. Asensio et al./Journal of Molecular Structure (Theochem) 395-396 (1997) 245-270 255

galactose hydroxymethyl groups is gg:gt, 36:64, and gt:tg, 955, respectively. In this case, the shape of the

four maps shown in Fig. 4b is fairly similar. The sixteen relaxed energy maps calculated using the

GBLSA continuum solvent model [31] for water showed again an important sensitivity to the initial configura- tion of the pendant groups, in some sense similar to that observed for E = 1. The corresponding adiabatic map is shown in Fig. 5a. The relative energies calculated for the local minima are also shown in Table 1. Using this solvent model, the energy barrier between minima 1 and 2 is now smaller, ca. 7 kcal mol-‘, while that between 2 and 3 is also about 7 kcal mall’. Conformers 2 and 4 completely dominate the probability distribu- tion (99%). The probability of the other minima is negligible. It is interesting that the central low-energy region is fairly more extended in @ than the corre- sponding regions calculated in vacua, towards values (a < 0) separated from the exo-anomeric orientation [6,48]. In fact, minimum 4 shows values of (cP/\k, - 29/ - 31). The probabilities for the different configurations of the pendant groups also are shown in Table 2. The cr

configuration is again the most populated (97%), while the rotameric distribution is now gg:gt, 90:10, for glu-

cose and gt:tg, 77:23, for galactose. Dowd et al. have calculated [25] the relaxed energy

maps for other p-( l- > 4)-linked disaccharides, 01- and @-cellobiose, using the regular MM3 force field, with E = 4. Although lactose and cellobiose differ in the configuration at C-4 of the non-reducing moiety, it can be deduced that the global shape of their maps are indeed similar to those calculated by us with the MM3* force field and E = 80 D. In particular, the

reverse-clockwise (rr) and the mixed clockwise- reverse-clockwise (CT) orientations occur more fre- quently in both cases. With regard to the orientation of the hydroxymethyl group of the reducing moiety, in our case also for E = 80 D, the gt rotamer is favoured as also found by Dowd et al. for E = 4 D [25]. Never- theless, the best agreement with the experimental results is found by us with MM3* and E = 1 D (vide infra). With regard to the position of the minima, our conformer 2, cP/q 54/ - 16 (E = 1) or 54/l (E = 80) closely resembles one of their conformers @/‘Jr 50/ -11. The same occurs for our conformer 1 a/*,

180/-18 similar to - 179/l, conformer 3 Q/q, 361 -179 close to 381172, and conformer 4 with @/‘I’, 31/ - 46, close to - 36/- 46.

3.2. Molecular dynamics

As a further step, the conformational stability of the

low-energy minima was studied by using molecular dynamics simulations. Conformation 2 was used as input geometry for independent 4 ns simulations at 300 K, using E = 80 D and the GB/SA solvent model. The trajectories are displayed in Figs. 6 and 7. In both cases, no chair-to-chair or chair-to-boat interconversions were observed. It can be observed that, for the simulation using E = 80 D, the trajectory remained (4 ns) in the corresponding low-energy region, with barely no fluctuations in the sampled +/ \k angles. No transitions were detected for the hydro- xymethyl groups, which remained in the initial gtgt

configuration (Fig. 6b). These observations may indi- cate that much longer simulation times should be necessary to simulate the experimentally observed transitions, at least for the hydroxymethyl groups. In fact, it has been recently reported that extremely long simulation times (ca. 100 ns) could be necessary to adequately sample the accessible regions for a disac- charide molecule [49]. On the other hand, when the simulation (3.6 ns) was performed using the GB/SA solvent model, an interconversion from minimum 2 to

3 could be observed after 3.5 ns (Fig. 7). In addition, even within the central region of the @+I’ map, a wider range of 9 and \k values were sampled during the simulation. The hydroxymethyl group of the glucose moiety experiments transitions between the gg and gt

orientations (Fig. 7b), while that of galactose moves between the gt and tg conformations, as observed experimentally (vide infra). Thus, at least from the molecular dynamics point of view, it seems that a more realistic view of the actual behaviour of methyl a-lactoside in water solution is accomplished when using the GB/SA solvent model than when a bulk

dielectric constant is employed.

3.3. ESFF force jield

The observation of the sixteen relaxed energy plots calculated by ESFF [24], using E = 1 (not shown, see supplementary material) indicated that the form of the low-energy regions are extremely dependent on the

initial configuration of the secondary hydroxyls and of the hydroxymethyl groups. The adiabatic surface built from the sixteen relaxed maps is shown in Fig. 8.

256 J.L. Asensio et al.Noumal of Molecular Structure (Theochem) 395-396 (1997) 245-270

a b

C

time (ps) time (pa)

Fig. 7. Trajectory plots of one MD simulation (MM3*, GB/SA solvent model, time 3.6 ns) for 1, starting from minimum 2. (A) Trajectory of the

simulation in W\Ir space. (B) From left to right and top to bottom: (a) History of a. (b) History of ‘I’. (c) History of hydroxymethyl w for the Glc

moiety. (d) History of hydroxymethyl w for the Gal moiety.

2.57

-180 -120 4.0 0 60 120 180 180

120

60 60

3 O

0

SO -80

-120 120

-180 -180 -180 -120 .60 0 60 120 180

0

-180 -120 40 0 80 120 180 180

120

60 60

0

60 -60

-120 -120

-180 -180 -180 -120 -80 0 60 120 180

-184 180

-1x) 60 0 60 120 180 180

120 120

80 60

O 3 0

-80 b0

-120 -120

-180 -180 -180 -120 -80 0 So 120 180

0

-180 -120 60 0 60 120 180

+

-180 -120 80 0 180

60 120 180 .*.*

120

60 60

3 o 0

60 -60

-120 -120

-180 -180 -180 -120 -80 0 60 120 180

0

fm3 -180

180 -120 -60 0 60 120 180

180

120 120

80 60

O 3 0

SO 60

-120 -120

-180 -180 -180 -120 40 0 80 120 180

0

Fig. 8. (A) Adiabatic map calculated from the relaxed steric energy plots obtained by using ESFF (left) e = 1 and its corresponding population

dist~bution (right) for methyl tu-lactoside. The level contours for the energy map are given every kcal mol-‘. The level contours for the

probability map are given at IO%, 1 oi and 0. I%. % is indicated horizontally, and ‘8 along the vertical axis. (B) Partial adiabatic maps obtained

for the different orientations of the hydroxymethyl groups of methyl a-lactoside using ESFF with E = I. From left to right: ,qggr. ggrg, grgr and grrg orientations. @ is indicated horizontally, and \k along the vertical axis.

258 J.L. A.sensio et ul.Nournul of Molecular Structure (Theochrm) 395-396 (1997) 245-270

3

-180 -120 40 0 60 120 180 180 160

120 120

60 60

0 0

-60 40

-120 -120

-160 -160 -160 -120 SO 0 60 120 180

3

-160 -120 40 0 60 Im 160 160- ., ., ., ., ., . 164

lrn- -tm

60- - 60

O-

_i@y

0

-60 40

-120 -120

-1.30 -120 40 0 60 120 160

-160 -120 40 0 60 120 180 la4 180

120 120

60 60

O 3

0

60 60

-120 -120

-160 -160 -160 -120 40 0 60 120 164

3

-160 -120 40 0 60 120 IBO 160 la0

IM 1x1

60 60

0 0

40 40

-120 -120

-160 -la0 -160 -120 -60 0 60 120 180

3

3

-160 -120 80 0 60 lm 160 160 180

120 IM

60 60

0 0

60 40

-120 -120

-I80 -180 480 -120 40 0 60 Im (80

-160 -120 -60 0 60 im ia0 180 180

lm im

60 60

0 0

40 40

-120 -120

-160 -la0 -180 -120 60 0 60 120 180

Fig. 9. (A) Adiabatic maps calculated from the relaxed steric energy plots obtained by using ESFF (left) E = 80 and its corresponding population

distribution (right) for methyl or-lactoside. The level contours for the energy map are given every kcal mol-‘. The level contours for the

probability map are given at lo%, 1% and 0.1%. Cp is indicated horizontally, and q along the vertical axis. (B) Partial adiabatic maps obtamed

for the different orientations of the hydroxymethyl groups of methyl wlactoside using ESFF with E = 80. From left to right: gggt, ggtg, gtgt and gttg orientations. + is indicated horizontally, and ‘4’ along the vertical axis.

J.L. Asensio et al.Noumal of Molecular Structure (Theochem) 395-396 (1997) 245-270 259

There are several local minima, four of them are below a steric energy level of 5 kcal mol-‘. Table 1 also shows the values of the steric energies and of the estimated populations of these four different confor- mers of 1 (Fig. 3) obtained by using the ESFF force field with E = 1. For this force field and dielectric constant, the central region appears clearly split into two well separated areas, one for positive @ (mini- mum 2) and a second one for negative @ values (mini- mum 4), thus separated from the exo-anomeric position. This region (minimum 4b, see Table 1) does not show any interresidue hydrogen bonds. The other two regions around conformers 1 and 3 are also detected. The energy barriers between minima 1 and 2 is of ca. 5 kcal mol-‘, while those between 2 and 3, and 2 and 4, are about 2 kcal mol-‘. According to the probability distribution, 85% of the population is con- centrated around minimum 1, with the HO-2’-0-3 and HO-6’-0-6 hydrogen bonds with H...O distances smaller than 1.9 A. In addition, 9% is located around minima 2/4 in the central low-energy region and 6% of the population is around minimum 3. As stated

below, there is null concordance with the experimen- tal results both in water [20,45] and in the solid state [44] (for 1 and different analogues). The role of the position of the secondary hydroxyl groups in the dis- tribution was also calculated and demonstrated that the probability distribution for the cr orientation of

the hydroxyls is rather different from those for the cc, rc and rr dispositions, since for cr, 90% of the population can be located around minimum 2 in the central region of the map. In the cr orientation, there is no possibility of formation of the HO-2’-0-3 hydrogen bond in minimum 1, since the atoms do not have the appropriate orientation, and, in addition, both hydrogen atoms come into close contact, destabilizing this conformer. This result illustrates the importance of using the widest possible range of initial structures in order to avoid erroneous conclusions. Table 2 shows the probability distribution for the sixteen initial configurations. It can be observed that the rr

configuration appears populated to more than 92% extent. The corresponding minimum, conformer 1, can not explain the experimental NOE data (vide infra). The Glc gt conformer dominates the probabil- ity distribution, while the conformational equilibrium for the Gal lateral chain is described by a 29:71 dis- tribution between the gt and rg rotamers.

Sixteen relaxed energy maps were also calculated using E = 80 D. In this case, as observed for the MM3 * calculations, the sensitivity of the surface to the initial configuration is much smaller than for E = 1. The corresponding adiabatic map is shown in Fig. 9. The relative energies calculated for the local minima are also shown in Table 1. The complexity of the potential energy surface is substantially reduced at E = 80. At

Table 3

Experimental and calculated steady-state NOES (H-l’. Sat. time = IO s) for 1 at 37°C in D20 solution at 500 MHz

Proton pair Intensity (70)

a b c d e f g

H-l ‘/H-2’ 6.5 6.4 6.6 6.5 6.6 6.1 6.6

H-l ‘/H-3’ 9.1 8.4 7.6 8.0 8.2 1.1 1.2

H-l ‘/H-4’ - 2.3 - 1.8 - I.7 - 1.8 - 1.7 - I.8 - 1.9

H-l ‘m-5’ 10.0 10.9 II.2 10.8 10.9 11.0 IO.9

H-l ‘/H-3 3.5 0.2 1.5 0.0 - 0.2 I.1 10.0 H-l ‘M-4 12.1 17.0 15.2 17.8 17.8 12.9 IO.7

H- 1 ‘/H-5 _ - 0.1 0.0 - 0.1 - 0.3 - 0.1 I .o

H- 1 ‘M-6a I .o 0.5 0.8 I .3 - 0.4 0.3 0.9

H-l ‘&I-6b 1.0 0.2 1.9 - 0.2 I .o 2.0 0.8

a exp.;

h MM3*-I;

’ MM3’-80;

d MM3 *-GBISA;

’ MM3 *;-80(MD);

f MM3*-GB/SA(MD);

g ESFF-80.

260 J.L. Asensio et al.Nournnl of Molecular Structure (Theochem) 395-396 (1997) 245-270

Table 4

Experimental and expected normalized NOESY intensities (%;) for 1 in D20

Proton Pair Mixing time (ms)

200

H-I’/H-2’” I .5

H-1’/H-2’h 0.4

H-l ‘/H-2” 0.4

H-l ‘/H-2”’ 0.2

H-1 ‘/H-3”’ I .2

H-I’/H-3’h 0.9

H-l ‘/H-3” I .o

H-l ‘/H-3’d 0.9

H-l ‘/H-5’ ’ I.8

H-I ‘m-5’ h I.6

H-l ‘/H-5” 1.6

H-l ‘/H-5’d I .6

H-It/H-3” 0.3

H-I ‘/H-3 h 0.0

H-I ‘/H-3c 0.0

H-I ‘/H-3d 0.8

H_ ] ‘m-4” I .9

H-l’/H-4h 2.2

H-l ‘W-4’ 1.7

H-l ‘/H-4d 1.2

H-I ‘IH-6” 0.5

H-I’/H-6h 0.8

H-l ‘/H-6’ 0.3

H-l ‘/H-6d 0.2

a exp.; h MM3 *, MM, E = 80;

’ MM3 *, MD, GB/SA;

* ESFF, E = 80.

300 500 900 1500

0.7 I .3 I .4 2.7

0.5 0.9 1 .s 2.3

0.5 0.9 I .5 2.3

0.4 1 .o I .9 3.7

I.3 2.9 5.1 7.5

I .3 2.2 4.0 6.9

I .4 2.3 4.3 7.6

1.3 2.2 4.0 6.9

2.0 3.7 5.9 8.7

2.3 4.0 7.1 Il.6

2.3 4.0 7.1 II.6

2.4 4.1 7.3 12.0

0.2 0.8 0.9 I .4

0.0 0.0 0.1 0.2

0.0 0.0 0.0 0.1

I.3 2.2 4.3 7.8

2.0 3.9 6.6 9.6

3.0 4.8 8.1 12.7

2.4 4.0 7.2 11.6

1.8 3.1 5.8 IO.1

0.7 0.9 I.6 2.8

I .2 I .6 2.7 4.9

0.7 I.3 2.2 3.9

0.5 0.8 1.2 1.3

this dielectric constant, the energy barrier between minima 1 and 2 is now smaller, ca. 2.5 kcal mol-‘, while that between 2 and 3 is also slightly higher than 2 kcal molF’. There is only a small barrier between minima 2 and 4 (1 kcal mol-‘). In addition, the region around conformers 214 dominates now the probability distribution (ca. 77%). Minima 1 and 3 are also importantly populated to 3 and 20% extent, respectively. The areas which are populated now account for 17% of the complete potential energy surface. The associated probabilities for the different orientations of the hydroxyl and hydroxymethyl groups are collected in Table 2. There is no pre- dominant orientation of the hydroxyl groups, while for the lateral chains, the predicted distributions are 83:17, gg:gr, for Glc, and 39:61, gt:tg, for Gal (Table 7).

3.4. Comparison between expected and experimental

NMR results

The validity of this conformational analysis has been tested with relaxation measurements, i.e., nuclear Overhauser enhancements [33,41]. These experiments for methyl cu-lactoside have already been described by us [20]; thus, only the relevant facts will be presented here. The experimental nuclear Overhauser enhancements, obtained via steady-state

measurements, and through NOESY and ROESY experiments are collected in Tables 3-5. The experi- mental NOE values were compared with the expected parameters, obtained as described in Section 2, for the corresponding probability distributions. Table 6 pre- sents the interproton average < r-6 > -u6 distances, obtained as indicated in Section 2, in comparison to

J.L. Asensio er al.Nournal of Molecular Structure (Theochem) 395-396 (1997) 245-270 261

Table 5

Experimental and expected normalized ROESY intensities (%I for 1 in DzO

Proton pair

H-I’IH-2’”

H-I’/H-2’h

H-l ‘IH-2”

H-I’/H-2’d

H-l ‘/H-3’”

H-I ‘/H-3’ h

H-I ‘&I-3”

H-I’IH-3’”

H_, ‘m-5’”

H-I’/H-5’h

H-I ‘/H-5”

H-I’/H-Sfd

H-If/H-3”

H-l’/H-3h

H- 1 ‘/H-3’

H-I’/H-3”

H- 1 ‘/H-4”

H-I’/H-4h

H-l’/H-4’

H- I ‘/H-4d

H-I ‘/H-6”

H-l ‘/H-6h

H-l’/H-6’

H-l ‘/H-6d

Mixing time (ms)

100

0.8

0.2

0.2

0.2

0.7

0.4

0.5

0.4

0.8

0.9

0.9

0.9

0.2

0.0

0.0

0.4

I .o

1.3

1.1

0.7

0.5

I.0

0.7

0.7

250 400 700

0.7 0.7 I.1

0.4 0.8 1.5

0.4 0.8 1.5

0.4 0.9 1.6

1.9 2.5 4.0

I .2 2.0 3.5

I .3 2.1 3.8

I .2 2.0 3.5

2.2 3.4 5.0

2.3 3.6 6.2

2.3 3.6 4.2

2.3 3.7 6.5

0.5 0.7 1.1

0.0 0.1 0.2

0.0 0.0 0.1

I .2 2.0 3.7

2.2 3.5 6.0

2.8 4.4 7.8

2.3 3.7 6.5

1.7 2.8 5.1

0.8 1.1 1.5

I .4 2.1 3.5

0.9 I .2 2.0

0.9 1.2 2.0

a exp.; b MM3’, MM, E = 80;

’ MM3*, MD, GB/SA;

d ESFF, E = 80.

the values of the interproton distances estimated from

the different experiments. In addition, Tables 3-5 present the expected NOES for the theoretical distributions using MM3* and ESFF at the different dielectric constants, using a full relaxation matrix approach.

3.5. MM3 results

Although the model is rather simple, both in the energy calculation (the solvent is approximated as a bulk dielectric constant or as a continuum) and in the NOE derivation (rigid body, isotropic motion), it can be observed that all the distributions either from the relaxed energy maps or from the molecular dynamics simulations show important agreements between expected and observed results. From the molecular mechanics maps, the best agreement for the NOES is

found when the calculations were performed with E = 80 D, particularly for the interresidue H- 1 ‘/H-3, H- 1 ‘/ H-4 and H-l’/H-6 NOES (Tables 3-6). In addition, a more than satisfactory agreement is found for the MD simulation performed using the GB/SA solvent model, although that calculated with E = 80 D also shows qualitative matchings between expected and observed NOES.

With regard to the conformation of methyl a-lacto- side in solution based on the interresidue NOES, those on H-3 and H-4 should be representative of popula-

tions around minima 3 and 2(4), respectively. The experimental H-I’-H-3 NOE is higher than that cal- culated from the relaxed energy maps and E = 80 D (Tables 3-6). This could mean that the population around minimum 3 is higher than that predicted by means of MM3* (E = 80, 3%). On the other hand, the theoretical NOE between H- 1’ and H-4 is higher than

262 J.L. Asensio et al./Journal of Molecular Structure (Theochem) 395-396 (1997) 245270

Table 6

Experimental (from NOE experiments) and calculated average distances (A) for 1

Proton pair Dist. (A)

a b c d e f h

H-I ‘12’

H-1’/3’

H-l ‘14’

H-l ‘15’ H-l’/3

H-l’/4

H- 1 ‘I5

H- 1’/6a

H- 1 ‘l6b

H-2’14

3.01 3.12 3.11 3.12 3.12 3.11 3.06 3.07

2.50 2.61 2.65 2.63 2.62 2.62 2.59 2.65

> 3.5 4.06 4.10 4.09 4.07 4.08 4.04 4.06

2.45 2.40 2.39 2.41 2.41 2.38 2.54 2.37

3.16 4.10 3.58 4.16 4.35 3.71 3.63 2.66

2.42 2.28 2.32 2.28 2.21 2.39 3.23 2.50

> 3.5 4.21 3.86 4.17 4.34 4.03 4.27 3.24

3.15 3.24 2.90 3.00 3.55 3.05 4.06 2.99

3.15 3.58 2.79 3.83 3.18 2.78 3.61 3.06

z 3.5 4.18 4.41 4.3 1 4.52 4.42 2.34 3.84

“ exp., 500 MHz;

‘MM3*-I;

’ MM3 ‘-80;

’ MM3 *&B/SAP:

’ MM3 ‘-80 (MD);

’ MM3 ‘-GB/SA(MD);

R ESFF-1;

h ESFF-80.

the experimental (Tables 3-6). That could mean that the actual population in the central region of the map is smaller than 96%. The NOES between H-l ’ and both H-6s are also satisfactorily explained. With regard to minimum 1, the key H-2’-H-4 NOE was not observed

in any of the steady-state or transient experiments per- formed. However, it is necessary to consider certain conformational population around this conformer to explain the H- 1 ‘-H-2’ NOES. Otherwise, the relaxation rate p for H-2’ is rather small and the calculated NOES involving this proton would be much higher than the experimental ones. Therefore, the experimental NOES can be explained by a population distribution around conformers 1, 2(4) and 3 of ca. 1, 91 and 8%, respec- tively. A more precise quantitation of the population of these states is precluded by the uncertainty in the time scale of motion around the linkages [5]. Nevertheless, it can be concluded according to our results, that the

MM3* force field, when used in these conditions (bulk dielectric constant, F = 80 D or with the GBI SA solvent model), does correctly reproduce the con- formational properties of 1.

3.6. MM3: Hydroxymethyl conformation

The observed couplings for the galactose

hydroxymethyl group agree with combinations of the gt and tg rotamers, being gt family populated [37] in extensions > 65%. On the other hand the couplings for the glucose lateral chain is in agreement with a ca. 60:40 distribution between the gg and gt

conformers [36,50]. The couplings for the Glc moiety are well explained by the calculation from the relaxed energy maps using E = 1. On the other hand, the use of E = 80 predicts the reverse order of populations with a preponderance of the gt family. A similar result has been presented by Dowd et al. [25] using the regular MM3 force field, with E = 4, for cr-cellobiose. Finally, the use of the GB/SA solvent model overestimates the presence of gg rotamers. With respect to the galactose lateral chain, the best agreement between theoretical and observed results are produced when the GB/SA solvent model is used. The use of a bulk dielectric constant (either 1 or 80 D) heavily overestimates the contribution of the gt rotamer. The MD simulation

with E = 80 D did not show any transitions of the hydroxymethyl lateral chains; thus, only the simula- tion carried out with the GB/SA model may be com- pared with the observed results. In this case, the gt conformer of Glc appears 65% of the time; thus, the population of this rotamer appears ca. 25% higher than experimentally observed. On the other hand,

J.L. Asensio et al./Journal of Molecular Structure (Theochem) 395-396 (1997) 245-270

Table I Calculated probability distributions from relaxed steric energy maps for the different hydroxymethyl conformations of 1

Configuration Pop. (%)

EXP MM3*;-1 MM3*-80 MM3*;-GBISA ESFF-I ESFF-80

263

&a 60 70 36 90 IO 83 glcgt 40 30 64 10 90 17 Rakr 70 91 95 77 29 39 &tK 30 9 5 23 71 61

the distribution for the Gal chain is fairly well pre-

dicted (Table 7).

3.7. ESFF results

The use of ESFF with E = 1 produces null concor-

dance between theoretical and experimental NOE results, since most of the population of conformers (85%) is predicted to be around minimum 1. As explained above, for this case, the H-2’/H-4 NOE should be large, and both H-l ‘/H-3 and H-l ‘/H-4 NOES should be almost non existent. However, the

opposite case is observed (Tables 3-6). With regard to the results of ESFF at high dielectric constants (E = SO), it can be observed that the predicted H- 1 ‘/H-3 NOE is similar to that predicted for H-l’/H-4. Thus, the population around minimum 3 is overestimated. As mentioned above, the predicted distribution by ESFF with E = 80 is 3:77:20 for conformers 1:2(4):3, while the experimental is close to 1:91:8.

Therefore, only qualitative agreement is obtained between the expected and experimental results.

3.8. ESFF: Hydroxymethyl conformation

For the Glc lateral chain, the use of either E = 1 or E = 80 overestimates the population of the gg family (99% or 83% predicted, versus 60% experimental).

For the galactose moiety, the reverse order of popula- tions between the gt and tg rotamers is calculated by the ESFF force field, independently of dielectric con- stant, (70:30 experimental, versus 25:75 (e = l), or 39:61 (F = 80), theoretical); see Table 7.

3.9. Concluding remarks

We have compared the results provided by different force fields for a single disaccharide molecule, methyl

a-lactoside, 1. This comparison has demonstrated that MM3*, when used with high dielectric constants or with the GB/SA solvent model, adequately reproduces

the NOE data obtained for 1. On the other hand, ESFF (E = 80 D) only provides a qualitative agreement between the theoretical and experimental results. When comparing these observations to those pre- viously obtained by us [20,21] using AMBER modi- fied for carbohydrates [19] or employing other force fields as CVFF [51] or CFF91 [52], one may conclude that MM3* may be recommended to deal with con- formational studies of oligosaccharides in solution, as

also stated for MM3 and solid-state studies [13,25- 27]. We have also reported [21] that the predictions of CVFF are in good agreement with the observed data, while those obtained by CFF91 and by AMBER/ Homans [ 191 have only qualitative concordance [20] with the experimental NOE results, as also deduced herein for ESFF. With regard to the lateral chains conformations, there is not a complete concordance between the experimental and theoretical data. Only partial agreement is found for the Glc chain when using MM3* (e = 1) and for the Gal one using MM3* with the GB/SA solvent model. In this sense,

it should be mentioned that the use of the AMBER! Homans force field (E = 80) [20] provides the best adjustment to the experimental facts. The use of CVFF (E = 80) also makes a satisfactory matching

between observed and predicted data [21]. Finally, and with respect to the conformation of

methyl ol-lactoside, our results indicate that the extent of flexibility around the &l- > 4) linkage of 1 in water solution is rather limited, although regions other than that around the global minimum are slightly present, and about 10% of the complete cP/\k potential energy surface is appreciably populated in solution. Therefore, besides the associated entropic loss, the molecular recognition of conformers of

264 J.L. Asensio et al.Nournal of Molecular Structure (Theochm) 395-396 (1997) 245-270

e * * +

Fig. 10. Relaxed steric energy plots obtained by using MM3* (E = I) for 1. The columns show variations in the orientations of the secondary

hydroxyl groups: from left to right cc, CT, rc, rr. The rows present the different orientations of the hydroxymethyl groups: from top to bottom:

gggt, g&q, gtgt, gttg. The level contours are given every kcal mol-‘. @ is indicated horizontally, and 9’ along the vertical axis.

J.L. Asensio et al.Nournal of Molecular Structure (Theochem) 395-396 (1997) 245-270

Fig. 1 I. Relaxed steric energy plots obtained by using MM3’ (F = 80) for 1. The columns show variations in the orientations of the secondary

hydroxyl groups: from left to right cc, CT, KC, rr. The rows present the different orientations of the hydroxymethyl groups: from top to bottom:

gggt, ggtg, gtgf, gttg. The level contours are given every kcal mol-‘. @ is indicated horizontally, and \Ir along the vertical axis.

* * * *

Fig. 12. Retaxed steric energy plots obtained by using MM3‘ (GRISA solvent model) for 1. The columns show variations in the orientations of

the secondary hydroxyl groups: from left to right cc, CT, rc, P-T. The rows present the different orientations of the hydroxymethyl groups: from top

to bottom: gggt, ggtg, gtgt, gtrg. The level contours are given every kcal mol.‘. rP is indicated horizontally, and ‘# along the vertical axis.

J.L. Asensio et al./Journal of Molecular Structure (Theochem) 395-396 (1997) 245-270

* *

gttw gttw

t *

Fig. 13. Relaxed steric energy plots obtained by using ESFF (.Y = 1) for 1. The columns show variations in the orientations of the secondary

hydroxyl groups: from left to right cc, cr. rc, i-t-. The rows present the different orientations of the hydroxymethyl groups: from top to bottom:

gggr, ggtg, grgt, gtfg. The level contours are given every kcal mol- ’ @ is indicated horizontally, and ‘4’ along the vertical axis.

268 J.L. Asensio et d/Journal of Molecular Structure (Theochem) 395-396 (1997) 245-270

*

t

gttgrc

t t t

Fig. 14. Relaxed steric energy plots obtained by using ESFF (E = 80) for 1. The columns show variations in the orientations of the secondary

hydroxyl groups: from left to right cc, CT, K, W. The rows present the different orientations of the hydroxymethyl groups: from top to bottom:

gggt, ggtg, gtgf, gttg. The level contours are given every kcal mol-‘. % is indicated horizontally, and \Ir along the vertical axis.

J.L. Asensio et al./Journal of Molecular Structure (Theochem) 395-396 (1997) 245-270 269

other regions should be accompanied by the formation of hydrogen bonds or by the establishment of stabilis- ing van der Waals contacts to override the energy barrier between the low-energy area and the different islands [53].

Acknowledgements

Financial support by DGICYT (Grant PB93-0127) is gratefully acknowledged. JLA and MMP thank Boehringer Ingelheim Espafia for a fellowship. We thank Prof. Martin-Lomas for his interest and support throughout this work.

Appendix ASUPPLEMENTARY MATERIAL

Figs. lo-14 show the relaxed steric energy plots obtained by MM3* (E = 1, 80, and the GB/SA solvent model) and ESFF (E = 1, 80) for the different sixteen initial configurations of the pendant groups of 1.

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