Preferred conformation of C-lactose at the free and peanut lectin bound states

Article abstract of DOI:10.1021/ja982193k

A conformational analysis of methyl α-C-lactoside (5) is carried out (Table 1), which suggests the preferred solution conformation of 5 to be described as a mixture of conformers A (φ = 300°; ψ = 60°) and B (φ = 300°; ψ = 300°) to a first approximation. A

Full text of DOI:10.1021/ja982193k

J. Am. Chem. Soc. 1998, 120, 11297-11303
11297
Preferred Conformation of C-Lactose at the Free and Peanut Lectin
Bound States
R. Ravishankar,† A. Surolia,† M. Vijayan,*,† Sungtaek Lim,‡ and Yoshito Kishi*,‡
Contribution from the Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560 012, India,
and Department of Chemistry and Chemical Biology, HarVard UniVersity,
Cambridge, Massachusetts 02138
ReceiVed June 23, 1998. ReVised Manuscript ReceiVed September 25, 1998
Abstract: A conformational analysis of methyl R-C-lactoside (5) is carried out (Table 1), which suggests the
preferred solution conformation of 5 to be described as a mixture of conformers A (φ ) 300°; ψ ) 60°) and
B (φ ) 300°; ψ ) 300°) to a first approximation. Applying a modified Karplus equation for the vicinal
coupling constants observed for the H.a and H.1′ protons of 5, the dihedral angle (φ ) O.5′-C.1′-C.a-C.4)
was deduced to be approximately +280°. Nuclear Overhauser effect (NOE) experiments were then conducted
on 5-D2 and its parent methyl R-O-lactoside in a 7:3 mixture of pyridine-d5 and methanol-d4, qualitatively
demonstrating that the conformational characteristics of both methyl R-C- and R-O-lactosides at the free states
are represented as a mixture of two conformers A and B, but their relative population may be different. Through
X-ray analysis, it has been definitively established that the conformation (φ ) 297(7)° and ψ ) 120(2)°) of
C-lactose bound to peanut lectin is practically identical to the conformation (φ ) 291(6)° and ψ ) 118(9)°)
of its parent O-lactose bound to the same protein.
Previous work1 from one of these laboratories has experi-
mentally established that the C-glycosidic bond of a C-glyco-
side preferentially adopts the exo-anomeric conformation2 like
the O-glycosidic bond of its parent O-glycoside does. The
conformational preference of C-aglyconic bond can be pre-
dicted primarily on the basis of steric considerations. To a
first approximation, the C-monosaccharides, C-disaccharides,
and C-trisaccharides have been shown to have conformational
properties close to those of the corresponding parent O-gly-
cosides.
There has been disagreement over whether C- and O-lac-
tosides share the same conformational characteristics. Using
nuclear Overhauser effect (NOE) experiments coupled with
force-field calculations, Jime´nez-Barbero and co-workers3 com-
pared the conformation of C-lactose with that of methyl R-O-
lactoside. They concluded that (1) C-lactose preferentially
adopts the exo-anomeric conformation around the C-glycosidic
bond and (2) C-lactose exhibits different conformational proper-
ties around the C-aglyconic bond from those of methyl R-O-
lactoside. On the other hand, Berthault and co-workers4 used
a strong off-resonance rf irradiation as a spin-lock in the mixing
time of ROESY experiments and found that methyl â-C-lac-
toside shares the same conformational characteristics, including
those around the aglyconic bond, with its parent methyl â-O-
lactoside.
In this paper, we report the conformational properties of
methyl R-C-lactoside with CH2- and CD2-bridges in reference
to its parent methyl R-O-lactoside and present definitive
evidence that C-lactose bound to peanut lectin adopts a virtually
identical conformation to the one observed for O-lactose.
Conformational Analysis of Methyl R-C-Lactoside. As
mentioned, the C-glycosidic bond of all the C-monosaccharides,
C-disaccharides, and C-trisaccharides exhibits a clear preference
for the exo-anomeric conformation like the O-glycosidic bond
of their parent O-saccharides.1 This preference is more
pronounced for the axial C-glycosidic bond than for the
corresponding equatorial C-glycosidic bond.5
Accepting this conformational preference of the C-glycosidic
bond, the conformational analysis of methyl R-C-lactoside can
be done, to a first approximation, by focusing only on the
C-aglyconic bond. This implies that the same analysis can be
applied to C-lactose and also to methyl R-C-cellobioside and
C-cellobiose, because the C.1 and C.4′ substituents of these
saccharides are not directly involved in this analysis. To clearly
evaluate and present through-space steric interactions, a diamond-
lattice analysis was introduced.6 For example, in the analysis
of methyl R-C-lactoside (5), only the three ideal staggered
conformers around the C-aglyconic bond, A (φ ) 300°; ψ )
60°),7 B (φ ) 300°; ψ ) 300°), and C (φ ) 300°; ψ ) 180°),
* To whom correspondence should be addressed.
† Indian Institute of Science.
‡ Harvard University.
(1) Wei, A.; Boy, K. M.; Kishi, Y. J. Am. Chem. Soc. 1995, 117, 9432
and references therein.
(2) The term exo-anomeric conformation is used to describe the
conformation of O- or C-glycosidic bond with the glycosidic oxygen or
carbon being anti-periplanar to the C.1-C.2 bond.
(3) (a) Espinosa, J.-F.; Mart´ın-Pastor, M.; Asensio, J. L.; Dietrich, H.;
Mart´ın-Lomas, M.; Schmidt, R. R.; Jime´nez-Barbero, J. Tetrahedron Lett.
1995, 36, 6329. (b) Espinosa, J.-F.; Can˜ada, F. J.; Asensio, J. L.; Dietrich,
H.; Mart´ın-Lomas, M.; Schmidt, R. R.; Jime´nez-Barbero, J. Angew. Chem.,
Int. Ed. Engl. 1996, 35, 303. (c) Espinosa, J.-F.; Can˜ada, F. J.; Asensio, J.
L.; Mart´ın-Pastor, M.; Dietrich, H.; Mart´ın-Lomas, M.; Schmidt, R. R.;
Jime´nez-Barbero, J. J. Am. Chem. Soc. 1996, 118, 10862. (d) Espinosa,
J.-F.; Montero, E.; Vian, A.; Garc´ıa, J. L.; Dietrich, H.; Schmidt, R. R.;
Mart´ın-Lomas, M.; Imberty, A.; Can˜ada, F. J.; Jime´nez-Barbero, J. J. Am.
Chem. Soc. 1998, 120, 1309.
(4) Rubinstenn, G.; Sina¨y, P.; Berthault, P. J. Phys. Chem. A 1997, 101,
2536.
(5) (a) Goekjian, P. G.; Wu, T.-C.; Kishi, Y. J. Org. Chem. 1991, 56,
6412. (b) Wei, A.; Kishi, Y. J. Org. Chem. 1994, 59, 88.
(6) Babirad, S. A.; Wang, Y.; Goekjian, P. G.; Kishi, Y. J. Org. Chem.
1987, 52, 4825.
10.1021/ja982193k CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/27/1998

11298 J. Am. Chem. Soc., Vol. 120, No. 44, 1998
RaVishankar et al.
Figure 1. Three ideal staggered conformers of methyl R-C-lactoside. The C-glycosidic and C-aglyconic bonds are φ ) 300° and ψ ) 60° for A,
φ ) 300° and ψ ) 300° for B, and φ ) 300° and ψ ) 180° for C, respectively.
Table 1. Major Steric Interactions and Estimated Destabilization Energy for the Conformers A-C of Methyl R-C- and O-Lactosides
conformer A
1,3-diaxial-like interaction
conformer B
conformer C
Ca
Ob
Ca
Ob
Ca
Ob
C.4-C.3/C.1′-O.5′
C.3-O.3/C.a-C.1′
single-axial-like interaction
C.a-C.4/C.6-H
1.9c
1.9c
1.9c
1.9c
C.4-C.5/C.1′-O.5′
1.9c
1.9c
C.5-C.6/C.a-C.1′
3.7c
1.9c
0.9d,e
0.4d,e
C.4-C.3/C.1′-H
C.4-C.a/C.6-H
C.a-C.1′/C.5-H
C.3-O.3/C.a-H
0.9d,f
0.9h
0.9d
0.4d
5.0
0.9d,f
0.4h
0.9d
0
C.4-C.5/C.1′-H
C.3-O.3/C.a-H
C.1′-O.5′/C.4-H
C.1′-O.5′/C.6-H
2.4g
0.4d
0.4d
0.4d
7.3
2.4g
0
0.4d
0.4d
6.7
Totali
4.7
4.2
4.1
a Methyl R-C-lactoside. b Methyl R-O-lactoside. c 1,3-Diaxial destabilization energy reported for OMe/CH2R (1.9 kcal/mol) and Me/Me (3.7
kcal/mol) substituted cyclohexanes was adopted.10 d One-half of A-value reported for CH2R (1.8 kcal/mol) and OR (0.8 kcal/mol) substituted
cyclohexanes was used.10 e With an imaginative six-membered ring including the C.a-C.4-C.5, it is seen that the single-axial-like interaction of
C.5-C.6/C.a-H is already counted in the single-axial-like interaction of C.a-C.4/C.6-H. The destabilization energy is estimated to be 0.9 for
both in the C-series but to be 0.4 for the former and 0.0 for the latter in the O-series, and 0.4 is used for this estimation. f With an imaginative
six-membered ring including the O.5′-C.1′-C.a-C.4, it is seen that the single-axial-like interaction of C.a-C.1′/C.3-H is already counted in the
single-axial-like interaction of C.4-C.3/C.1′-H. g One-half of A-value (4.9 kcal/mol) reported for C(Me)3-cyclohexane was used.9 The single-
axial-like interaction of C.4-C.a/C.6-H is the same type of interaction as this, but involves the same protons as the C.4-C.5/C.1′. h With an
imaginative six-membered ring including the C.4-C.5-C.6, it is seen that the single-axial-like interaction of C.5-C.6/C.a-H is already counted
in the single-axial-like interaction of C.4-C.a/C.6-H. For estimation of the destabilization energy, see the footnote e. i A total destabilization
energy (kcal/mol) estimated.
are considered (Figure 1). By focusing on 1,3-diaxial-like and
single-axial-like interactions, the steric destabilization of these
conformers can then be analyzed (Table 1).8 Clearly, the
conformer C suffers from several steric destabilizations. Among
the single-axial-like interactions, the interaction between the C.1′
hydrogen and one of the C.6 hydrogens (or C.6 oxygen) is
assumed to be most significant; the C.1′ and C.6 hydrogens
need to occupy the same point in a diamond lattice, and this
steric situation can be compared with that of tert-butylcyclo-
hexane.9 The conformer B suffers from steric congestion due
to one 1,3-diaxial-like and four single-axial-like interactions,
whereas the conformer A suffers steric congestion due to two
1,3-diaxial-like and one single-axial-like interactions. Overall,
none of the three ideal staggered conformers is free of
unfavorable steric interactions.
Using (1) 1,3-diaxial interaction energy reported for Me/Me-
1
and OMe/CH2R-substituted cyclohexanes,10 (2)
/
of the
2
1
A-value reported for tert-butylcyclohexane,9 and (3) /2 of the
A-values reported for CH2R- and OR-cyclohexanes,10 the relative
stability of A, B, and C is estimated to be 4.7, 5.0, and 7.3
kcal/mol, respectively (Table 1). However, as a hydrogen bond
between the C.3-OH and the O.5′ may provide some stabiliza-
tion, the total destabilization of A may be less than 4.7 kcal/
mol. Overall, this analysis qualitatively suggests that the
preferred conformation of methyl R-C-lactoside (5) is predomi-
nantly a mixture of conformers A and B.11 It should be noted
that this conformational analysis provides only a rough gross
picture for the conformational characteristics of these substrates,
and the actual low-energy conformers are likely to deviate from
the conformers A and B.
The relative stability among these three ideal staggered
conformers is not predictable in a straightforward manner.
The conformational analysis of methyl R-O-lactoside and
O-lactose, and methyl R-O-cellobioside and O-cellobiose, can
be done in a similar manner (Table 1). Thus, the relative
stability of the three conformers corresponding to A, B, and C
is estimated to be 4.2, 4.1, and 6.7 kcal/mol, respectively. Once
again, a hydrogen bond between the C.3-OH and the O.5′ might
make the total destabilization of A less than 4.2 kcal/mol. In
addition, the C.3-O.3/O.a-C.1′ 1,3-diaxial-like steric desta-
bilization of A might not be as pronounced as it would be in a
general case because of the antiparallel orientation of the polar
carbon-oxygen bonds involved.
Overall, this analysis qualitatively suggests that the preferred
conformation of methyl R-C- and R-O-lactosides, as well as
(10) Corey, E. J.; Feiner, N. F. J. Org. Chem. 1980, 45, 765.
(11) Extensive variable-temperature 1H NMR experiments were per-
formed on C-monoglycosides to demonstrate that these substrates exist as
mixture of conformers.5a
(7) All dihedral angles are defined according to the IUPAC Joint
Commission on Biochemical Nomenclature (JCBN), symbols for specifying
the conformation of polysaccharide chains, 1981 recommendation. Repro-
duced in Eur. J. Biochem. 1983, 131, 5; φ ) O.5′-C.1′-O.4-C.4 and ψ
) C.1′-O.4-C.4-C.3.
(8) The single-axial-like interaction of C.2′-O.2′/C.a-H is not included
in Table 1 because it exists in all the three staggered conformers A-C.
(9) With an imaginative six-membered ring including O.5′-C.1′-C.a-
C.4 on the conformer C, it is seen that the C.5 and C.6 carbons correspond
to the quaternary and primary carbons of the tert-butyl group of tert-
butylcyclohexane. Thus, the single-axial-like interaction between the C.1′-H
and the C.4-C.5-C.6-C.6-H (or OH) is assumed to be roughly
1
represented by
/ of the A-value (4.9 kcal/mol) of tert-butylcyclohexane
2
reported by Manoharan, M.; Eliel, E. L. Tetrahedron Lett. 1984, 25, 3267.
The single-axial-like interaction of C.4-C.a-C.1′-C.1′-H/C.6-H is the
same type of interaction as this. However, since this interaction involves
the same protons as the single-axial-like interaction of C.4-C.5/C.1′-H,
the steric destabilization is counted only once.

Preferred Conformation of C-Lactose
J. Am. Chem. Soc., Vol. 120, No. 44, 1998 11299
Scheme 1. Synthesis of Methyl R-C-Lactoside and
C-Lactose with the CH2- and CD2-Bridgesa
C-glycosidic bond of 5 preferentially adopts the exo-anomeric
conformation. This conformation can be refined further; using
a modified Karplus equation14 and coupling constants of 8.2
and 0 Hz yields a dihedral angle (φ ) O.5′-C.1′-C.a-C.4) of
approximately +280°. On the other hand, the vicinal coupling
constants observed for the C.4 and C.a protons of 5 (Chart 1)
experimentally demonstrate that the C-aglyconic bond does not
preferentially adopt any one of the three ideal staggered con-
formers A-C. This observation implies that the steric repul-
sion existing in the conformers A-C is avoided by rotating
primarily the C-aglyconic bond away from the ideal staggered
position rather than the C-glycosidic bond. This phenomenon,
i.e., the steric destabilization present in the ideal staggered
conformers that primarily results in the deformation of the
C-aglyconic bond over the C-glycosidic bond, is uniformly
observed for all the C-saccharides studied thus far and impor-
tantly, the same phenomenon is known for the parent O-
saccharides as well.15 It is worthwhile to note that the vicinal
coupling constants, including the bridging methylene protons,
are remarkably close to those observed for methyl R-C-
cellobioside,16 as expected from the conformational analysis
given in the previous section.
NOE experiments are expected to provide more defined
information on these characteristics. Namely, an NOE between
the C.1′ and C.4 protons is expected for the conformer A,
between the C.1′ and C.3 protons for the conformer B, and
between the C.1′ and C.6 protons for the conformer C.
However, considering the previous observations made on methyl
R-C-maltoside and its analogues,16 we opted to use the bis-
deuterated substrate 5-D2. NMR experiments were first at-
tempted in D2O, but many resonances were severely overlapped,
and a definitive conclusion on the absence or presence of
NOEs between the C.1′ proton and the C.3, C.4, and C.6 pro-
tons could not be drawn. Therefore, we searched for a suit-
able solvent system in which all the C.1′, C.3, C.4, and C.6
protons of both methyl R-C- and R-O-lactosides were sep-
arated, and a 7:3 mixture of pyridine-d5 and methanol-d4 met
this need.17 As seen in the spectrum of methyl R-C-lactoside
shown in Figure 2,18 a pronounced cross-peak between the
C.1′ and C.4 protons, as well as between the C.1′ and C.3
protons, was observed. The resonances of C.6 protons were
well separated in this solvent system, and the NOE between
the C.1′ and each of the C.6 protons could be examined; there
a Reagents and yields: (a) (1) NaClO2, NaH2PO4, (Me)2CdCHMe,
t-BuOH; (2) LiAlD4, ether, 63% overall yield. (b) (1) Ph3P, Im, I2,
88%; (2) n-BuLi, -90 °C, followed by addition of galactolactone, 81%;
(3) MeSH, CH2Cl2, BF3‚OEt2, dropwise, 71% (17% of SM recovered).
(c) Ph3SnH, AIBN, 96%. (d) H2/Pd(OH)2 on C, MeOH/EtOAc, 85%.
(e) (1) allyl alcohol, Sc(OTf)3, 100 oC; DMSO, KOBu-t, 95 °C; acetone,
1N HCl, 80%; (2) H2/Pd(OH)2 on C, MeOH/EtOAc, 87%.
Chart 1. Vicinal Coupling Constants Observed for Methyl
R-C-Lactoside in a Mixture of Pyridine-d5 and Methanol-d4
that of C- and O-lactoses, is described primarily as a mixture
of conformers A and B.
(14) Haasnoot, C. A. G.; de Leeuw, F. A. A. M.; Altona, C. Tetrahedron
1980, 36, 2783.
(15) Lemieux, R. U.; Koto, S. Tetrahedron 1974, 30, 1933.
(16) Wang, Y.; Goekjian, P. G.; Ryckman, D. M.; Miller, W. H.; Babirad,
S. A.; Kishi, Y. J. Org. Chem. 1992, 57, 482.
(17) The conformational preference of C-glycosidic and C-aglyconic
bonds in D2O is well compared to those in a mixture of pyridine-d5 and
methanol-d4; cf. the chart shown below with the one in Chart 1.
Substrates for NMR Studies. Methyl R-C-lactoside (5) and
C-lactose (6)12 were synthesized as shown in Scheme 1. LiAlD4
or LiAlH4 reduction of the carboxylic acid derived from the
aldehyde 1,13 followed by the succeeding steps, furnished methyl
R-C-lactoside (5) and C-lactose (6) with the CD2- or CH2-bridge.
NMR Analysis. The NMR studies were performed on
methyl R-C-lactoside (5) in D2O and a mixture of pyridine-d5
and methanol-d4, respectively, at approximately 20 °C.
The vicinal coupling constants (J1,2 ) 3.6 Hz, J2,3 ) 9.5, J3,4
) 10.5, J4,5 ) 10.6, J1′,2′ ) 9.3, J2′,3′ ) 9.2, J3′,4′ ) 3.4, J4′,5′
)
0) observed for the ring protons clearly show that both
tetrahydropyran rings of 5 adopt a chair conformation, as do
the corresponding rings of parent methyl R-O-lactoside and
O-lactose.
The vicinal coupling constants observed for the C.a and C.1′
protons of 5-H2 (Chart 1) experimentally demonstrate that the
This chart shows vicinal coupling constants observed for 5 in D2O and for
4 in benzene-d6. It was noted in many cases that the conformational
preference of C-glycosidic and C-aglyconic bonds was not affected
significantly with the protecting groups on the hydroxy groups;1 for example,
the vicinal coupling constants observed for 4-H2 are an additional example
for this general observation.
(18) Chemical shifts of methyl R-C-lactoside were assigned by a COSY
experiment: 1H NMR (pyridine-d5/CD3OD, 7/3) δ 2.37 (H.4), 3.84 (H.1′),
3.9 (H.5′), 3.96 (H.2), 3.99 (H.3′), 4.11 (H.6), 4.17 (H.6′), 4.20 (H.5), 4.21
(H.2′), 4.29 (H.3), 4.34 (H.6), 4.37 (H.6′), 4.40 (H.4′), 5.11 (H.1).
(12) (a) Preuss, R.; Jung, K.-H.; Schmidt, R. R. Liebigs Ann. Chem. 1992,
377. (b) Dietrich, H.; Schmidt, R. R. Liebigs Ann. Chem. 1994, 975.
(13) Daly, S. M.; Armstrong, R. W. Tetrahedron Lett. 1989, 30, 5713.

11300 J. Am. Chem. Soc., Vol. 120, No. 44, 1998
RaVishankar et al.
was no cross-peak detected between the C.1′ and one of the
two C.6 protons, but there was a faint, barely detectable cross-
peak between the C.1′ and the other C.6 proton. Overall, this
result points out that the conformational characteristics of
methyl R-C-lactoside should be described primarily as a mix-
ture of conformers A and B. It is worth noting that the
conformational characteristics of both glycosidic and agly-
conic bonds of C-glycoside 7 have been shown to correspond
nicely to those of 5.19
The conformational characteristics of methyl R-O-lactoside
and O-lactose,20 as well as those of methyl R-O-cellobioside
and O-cellobiose,21 have been studied using NMR spectroscopy,
primarily NOE, coupled with force-field calculations, and it is
generally agreed that these glycosides exist as a mixture of
conformers corresponding primarily to A and B. However, to
compare the conformational characteristics of methyl R-C-
lactoside with those of its parent methyl R-O-lactoside in the
same environment, an NOE experiment of methyl R-O-lactoside
was done in a 7:3 mixture of pyridine-d5 and methanol-d4
(Figure 322). All of the C.1′, C.3, C.4, and C.6 protons of methyl
R-O-lactoside were separated, and a pronounced cross-peak was
clearly detected between the C.1′ and C.4 protons, as well as
between the C.1′ and C.3 protons, but not between the C.1′ and
C.6 protons.
The relative population of conformers A and B could be
estimated from the magnitude of NOE observed. However,
correlation of the magnitudes of NOE with internuclei distance
is not straightforward.23 Nevertheless, it is worthwhile to note
that the cross-peak observed between C.1′ and C.3 protons was
more intense than the cross-peak observed between C.1′ and
C.4 protons for methyl R-C-lactoside, whereas the cross-peak
observed between the C.1′ and C.3 protons was less intense
than the cross-peak observed between C.1′ and C.4 protons for
its parent methyl R-O-lactoside. These results may give a
qualitative, rather than a quantitative, indication that the con-
formational characteristics of both methyl R-C- and R-O-lac-
tosides could be described as a mixture of conformers A and
B, but their relative population may be different.
Protein-Bound Conformation of C-Lactose. Many crystal
structures have been reported for the bound form of O-lactose
and plant or animal lectins.24 In these X-ray crystal structures,
O-lactose was shown to bind through the galactose moiety and
leave the glucose moiety outside the binding pocket.25 A large
number of hydrogen bonds to the protein and to the bound water
molecules, as well as hydrophobic interactions with the side
Figure 2. NOESY spectrum of methyl R-C-lactoside in a 7:3 mixture
of pyridine-d5 and methanol-d4 at approximately 20 °C with (A) 2.2-
5.2/3.4-4.5 ppm region and (B) 3.6-4.5/3.6-4.5 ppm region.
(19) As the case of 5, the vicinal spin-coupling constants were used for
the conformational study for the glycosidic bond of 7-H2, whereas the NOE
experiment was conducted on the corresponding bis-deuterated substrate
7-D2
chains of some amino acids, hold the galactose inside the
binding pocket. Interestingly, even though the glucose residue
resides outside the binding pocket, the conformational preference
around the aglyconic bond appears to be shared by all of the
protein-bound O-lactoses studied.
Jime´nez-Barbero and co-workers used TR-NOESY and TR-
ROESY NMR experiments to study the bound-state conforma-
(24) (a) Banerjee, R.; Das, K.; Ravishankar, R.; Suguna, K.; Surolia,
A.; Vijayan M. J. Mol. Biol. 1996, 259, 281. (b) Liao, D. I.; Kapadia, G.;
Ahmed, H.; Vasta, G. R.; Herzberg, O. Proc. Natl. Acad. Sci. U.S.A. 1994,
91, 1428. (c) Lobsanov, Y. D.; Gitti, M. A.; Leffler, H.; Barondes, S. H.;
Rini, J. M. J. Biol. Chem. 1993, 268, 27034. (d) Sixma, T. K.; Pronk, S.
E.; Kalk, K. H.; van Zanten, B. A. M.; Berghuis, A. M.; Hol, W. G. J.
Nature 1992, 355, 561. (e) Shaanan, B.; Lis, H.; Sharon, N. Science 1991,
254, 862. (f) Montfort, W.; Villafranca, J. E.; Monzingo, A. F.; Ernst, S.
R.; Katzin, B.; Rutenber, E.; Xuong, N. H.; Hamlin, R.; Robertus, J. D. J.
Biol. Chem. 1987, 262, 5398.
in a mixture of pyridine-d5 and methanol-d4 for the conformational study
of the aglyconic bond. The synthesis of these substrates and the NOESY
spectrum are included in Supporting Information.
(20) For example, see: Ferna´ndez, P.; Jime´nez-Barbero, J. Carbohydr.
Res. 1993, 248, 15.
(21) For example, see: Peters, T.; Meyer, B.; Stuike-Prill, R.; Somorjai,
R.; Brisson, I.-R. Carbohydr. Res. 1993, 243, 49.
(22) Chemical shifts of methyl R-O-lactoside were assigned by a COSY
experiment: 1H NMR (pyridine-d5/CD3OD, 7/3) δ 4.02 (H.2), 4.06 (H.5),
4.07 (H.3′), 4.08 (H.5′), 4.15 (H.4), 4.29 (H.6′), 4.35 (H.6), 4.38 (H.6′),
4.4 (H.2′), 4.41 (H.3), 4.43 (H.6), 4.43 (H.4′), 5.0 (H.1′), 5.04 (H.1).
(23) For example, see the discussion given in ref 4.
(25) Interestingly, S-lac lectins, the lactose-binding animal lectins, are
reported to interact with more than one monosaccharide residue through
hydrogen bonding, unlike the C-type lectin family.24b

Preferred Conformation of C-Lactose
J. Am. Chem. Soc., Vol. 120, No. 44, 1998 11301
Figure 4. Stereoview of the Fo - Fc electron density corresponding
to the C-lactose molecule. The map is contoured at 2.5 σ level.
cule have almost identical structures. The peanut lectin tetramer
in the two complexes has the same structure, with an rms
deviation of 0.28 Å in CR positions.
The electron density corresponding to the sugar molecules
is well defined (Figure 4), as indeed was the case with the
crystals of the O-lactose complex.26 A representation of the
location of the C-lactose molecule in a subunit is illustrated in
Figure 5. A close-up of the sugar binding site with the C-lactose
molecule is shown in Figure 6. For comparison, the location
of the O-lactose molecule is also shown in this figure. The
mode of binding in the two complexes is the same with an rms
deviation as low as 0.61 among all of the atoms in the two
sugar molecules. The peanut lectin/C-lactose interactions are
given in Table 2. They are identical to those in the O-lactose
case.
In summary, the first X-ray crystal structure of C-lactose
bound to peanut lectin has definitely established the protein-
bound conformation (φ ) 297(7)°, ψ ) 120(2)°)27 of C-lactose.
The C-glycosidic bond adopts the almost perfect exo-anomeric
conformation, whereas the C-aglyconic bond exists in the
midpoint between the conformers A and C. Most importantly,
the conformation of C-lactose bound to peanut lectin is virtually
identical to the conformation of its parent O-lactose (φ ) 291-
(6)°, ψ ) 118(9)°) bound to the same protein. Interestingly,
the conformation of C-lactose at the protein-bound state is
remarkably similar to one of the two major conformations at
the free state described by Berthault and co-workers.4,28,29
Strictly speaking, the present study is only applied for the case
of C-lactose bound to peanut lectin. The conclusion gained in
the present studies, i.e., that the conformation of C- and
O-lactoses bound to peanut lectin are virtually identical, is per-
fectly consistent with the conclusions derived from our previous
work on the H-type II blood group1, and it is tempting to claim
that this is a general phenomenon unless an unusual steric or
electronic factor(s) exists within molecules.
Figure 3. NOESY spectrum of methyl R-O-lactoside in a 7:3 mixture
of pyridine-d5 and methanol-d4 at approximately 20 °C with (A) 3.4-
5.2/3.9-5.4 ppm region and (B) 3.3-4.8/4.8-5.2 ppm region.
tion of C- and O-lactose with a galactose-binding protein ricin-
B3c and, more recently, with an Escherichia coli â-galactosidase3d
and concluded that the conformation of the C-glycoside is
different from its corresponding O-glycoside in bound states.
In light of the conformational characteristics of methyl R-C-
and R-O-lactosides described in the previous section, coupled
with the information available through several X-ray structures,
Jime´nez-Barbero’s conclusion is rather surprising.
Obviously, the definitive answer should be obtained through
an X-ray analysis of a protein-bound C-lactose. For this pur-
pose, we chose peanut lectin because a high-resolution X-ray
structure was already available for O-lactose bound to peanut
lectin in one of these laboratories.24a
Experimental Section
Experiments for the Synthesis Shown in Scheme 1. 1H and 13C
NMR spectra were recorded at 500 and 125 MHz, respectively, and
chemical shifts are reported in parts per million. The residual solvent
(26) The C.1 OH group of C-lactose in the subunits 1, 2, and 4 was
found to adopt the R configuration, whereas that in the subunit 3 was found
to adopt the â configuration. Interestingly, the same phenomenon was
observed for the parent O-lactose case.24a
(27) The φ and ψ values given here are averaged over the four subunits
of the protein.
(28) With the fixed angle φ, Berthault and co-workers4 searched for the
best agreement between experimental and back-calculated NOE distances,
resulting in two refined structures with φ ) 295°/ψ ) 130° and φ ) 295°/ψ
) 300°.
Crystals of the peanut lectin/C-lactose complex were grown
under the conditions similar to those used for the corresponding
O-lactose complex.24a The structure of the crystals of peanut
lectin/C-lactose complex was solved and refined using 2.7 Å
resolution X-ray intensity data. The four subunits in the mole-
(29) The conformation of O-lactose bound to peanut lectin is well
compared with the crystal structure (φ ) 266°; ψ ) 95°) of O-lactose
monohydrate: Beevers, C. A.; Hans, N. H. Acta Crystallogr. 1971, B27,
1323-1325.

11302 J. Am. Chem. Soc., Vol. 120, No. 44, 1998
RaVishankar et al.
Figure 5. Stereoview of the peanut lectin monomer. The C-lactose molecule is in ball-and-stick representation. The figure was generated using
MOLSCRIPT.38
Flash chromatography separations were performed using E. Merck
Kieselgel 60 (230-400 mesh) silica gel.
Reagents and solvents are commercial grade and were used as
supplied, with the following exceptions: benzene, ether, and THF were
distilled from sodium benzophenone ketyl. Methylene chloride was
distilled from phosphorus pentoxide. Toluene was distilled from
sodium.
All reactions sensitive to air or moisture were conducted under argon
or nitrogen. Reaction vessels for moisture-sensitive reactions were
flame-dried or oven-dried and allowed to cool under inert atmosphere.
2-D2. To a solution of aldehyde 113 (0.55 g, 1.15 mmol) and
Figure 6. Stereoview of the superposition of the peanut lectin/C-lactose
interactions. The crosses represent water molecules and the broken lines,
hydrogen bonds. The O-lactose molecule (light lines) is also shown
for comparison.
2-methyl-2-butene (5 mL) in t-BuOH (15 mL) was added a solution
of NaClO2 (1.1 g) and NaH2PO4 (1.0 g) in H2O (8 mL). After 2 h at
21 °C, the mixture was concentrated and extracted with ether. The
extracts were dried (MgSO4), filtered, and concentrated to afford the
crude acid.
Table 2. Protein-Sugar Interactions
A solution of the acid (0.57 g, 1.15 mmol) in ether (30 mL) was
treated at 0 °C with 0.19 g (4.5 mmol) of LiAlD4. After 2 h at 40 °C,
the mixture was quenched with 10% NaOH solution, dried (MgSO4),
filtered (Celite), and concentrated. The residue was purified by
chromatography on SiO2 (hexanes/EtOAc, 2:1) to give 0.35 g (63%)
of the alcohol 2-D2 as an oil: 1H NMR (CDCl3) δ 7.37-7.20 (15H,
m), 5.01 (1H, d, J ) 11.2), 4.8-4.42 (6H, m), 3.89 (1H, t, J ) 9.5),
3.75-3.68 (1H, m), 3.7-3.57 (3H, m), 3.37 (3H, s), 1.84 (1H, t, J )
10.7). Note: the corresponding 2-H2 is known in the literature.12,13
3-D2. To a solution of alcohol 2-D2 (0.26 g, 0.54 mmol), triphen-
ylphosphine (0.42 g, 1.62 mmol), and imidazole (0.11 g, 1.62 mmol)
in benzene (10 mL) was added iodine (0.41 g, 1.62 mmol) at 21 °C.
After 1 h at 80 °C, the mixture was cooled and sequentially treated
with saturated NaHCO3 solution, iodine, and saturated Na2S2O3 solution.
The organic layer was separated, dried (MgSO4), and concentrated.
Chromatography on SiO2 (hexanes/EtOAc, 8:1) gave 0.28 g (88%) of
the iodide as an oil.
subunit subunit subunit subunit
protein atom
sugar atom
1a
2a
3a
4a
A. Hydrogen Bonds
Asp83 OD1
Gly104 N
Asn127 ND2 galactose O3
Asp83 OD2
Ser211 OG
Ser211 OG
Asp80OD2
Ser211 OG
Gly213 N
galactose O3
galactose O3
2.85
3.19
2.68
2.73
2.83
3.00
3.14
3.45
2.62
2.60
2.64
2.97
2.67
2.85
2.75
3.42
3.26
3.16
3.03
2.47
2.84
2.79
2.65
2.89
3.08
3.49
2.77
2.78
3.06
2.81
2.69
2.76
3.16
3.30
3.06
2.62
galactose O4
galactose O4
galactose O5
galactose O6
glucose O3
glucose O3
B. Water bridges
galactose O2 connected to N Gly 104 and
OE2 Glu 129 through water bridges
C. Residues within 4 Å from the Sugar
A solution of the iodide (0.28 g, 0.47 mmol) in THF (10 mL) was
deoxygenated three times by freeze-pump-thaw cycle and cooled to
-90 °C, followed by the addition of n-BuLi (0.28 mL, 0.61 mmol, 2.2
M in hexanes). The solution was stirred for 45 min at -90 °C before
a solution of the galactolactone (0.39 g, 0.71 mmol) in THF (2 mL)
was added. The mixture was allowed to warm to -50 °C over 1 h,
quenched with aqueous NH4Cl, and extracted with ether. The extracts
were dried (MgSO4), concentrated, and chromatographed on SiO2
(hexanes/EtOAc, 7:1 to 3:1) to afford 0.38 g (81%) of the hemiketal
as an oil.
Asp80, Ala82, Asp83, Gly103, Gly104, Tyr125,
Asn127, Ser211, Leu212, and Gly214
a Distance in Å.
peak was used as an internal reference. Fast atom bombardment (FAB)
mass spectra were obtained using 3-nitrobenzyl alcohol or glycerol as
the matrix. Sodium iodide was added when indicated. Fourier
transform infrared spectra were obtained as films on sodium chloride
plates.
Analytical thin-layer chromatography (TLC) was performed with
E. Merck precoated TLC plates, silica gel 60F-254, layer thickness
0.25 mm. Preparative TLC (PTLC) separations were carried out on
E. Merck precoated plates, silica gel 60F-254, layer thickness 0.50 mm.
A solution of the hemiketal (0.38 g, 0.38 mmol) in CH2Cl2 (15 mL)
was cooled to -78 °C and methanethiol (10 mL) was condensed into
the reaction mixture. A solution of BF3‚OEt2 (9 mL, 0.27 mmol, 0.03

Preferred Conformation of C-Lactose
J. Am. Chem. Soc., Vol. 120, No. 44, 1998 11303
M in CH2Cl2) was added at -30 °C via a syringe pump over a period
of 2 h. The reaction mixture was quenched with NaHCO3 before excess
methanethiol was removed in vacuo. The residue was extracted with
CH2Cl2, and the extracts were dried (MgSO4) and concentrated.
Chromatography on SiO2 (hexanes/EtOAc, 7:1 to 3:1) provided 0.28 g
(71%) of methyl thioketal 3-D2 and 0.064 g (17%) of the recovered
hemiketal as oils.
Table 3. Crystallographic Data and Refinement Statistics
data collection statistics
resolution (Å)
2.7
no. of observations
no. of unique reflections
data completeness (%)
R-mergea (%)
124179
33099
93.81
12.9
refinement statistics
resolution (Å)
no. of protein atoms
no. of sugar atoms
no. of solvent atoms
no. of other ions
R-cryst (F > 0σF) (%)
R-free (F > 0σF) (%)
root-mean-square deviation from
ideal geometry
4-D2. A stirred solution of thioketal 3-D2 (0.28 g, 0.27 mmol), AIBN
(4.4 mg, 0.03 mmol), and triphenyltin hydride (0.24 g, 0.68 mmol) in
toluene (15 mL) was immersed in a preheated oil bath (110 °C). After
1 h at 110 °C, the reaction mixture was concentrated and chromato-
graphed on SiO2 (hexanes/EtOAc, 20:1 to 3:1) to give 0.26 g (96%) of
the disaccharide 4-D2 as an oil: 1H NMR (C6D6) δ 7.48-7.05 (35H,
m), 5.18-4.27 (14H, m), 4.78 (1H, d, J ) 3.3), 4.15 (1H, dd, J )
10.9, 2.7), 4.06 (1H, dd, J ) 10.2, 9.6), 3.92-3.88 (2H, m), 3.81-
3.66 (4H, m), 3.67 (1H, dd, J ) 9.1, 3.4), 3.56-3.53 (2H, m), 3.41
(1H, dd, J ) 9.2, 2.8), 3.23 (3H, s), 2.50 (1H, t, J ) 11.0); 13C NMR
(CDCl3) δ 139.06, 138.63, 138.48, 138.38, 138.31, 137.97, 128.40,
128.23, 128.08, 127.92, 127.88, 127.81, 127.74, 127.58, 127.55, 127.47,
127.40, 127.19, 96.16, 84.94, 81.91, 79.24, 77.27, 76.57, 75.27, 74.84,
74.60, 73.92, 73.46, 73.07, 72.81, 72.09, 70.57, 70.05, 68.66, 54.81,
40.1; HRMS (FAB, NaI) calculated for C63H66D2O10Na ([M + Na]+)
1009.4834, found 1009.4836.
10-2.7
6976
92
330
4 Mn2+, 4 Ca2+
18.8
22.7
bond length
bond angle
0.008
1.619
a R-merge ) ∑|I - I |/∑ I .
) 129.12, b ) 126.75, c ) 76.90). The data were processed using the
MAR-XDS31 suite of programs. The data collection statistics are in
Table 3.
5-D2. A solution of benzyl ether 4-D2 (20 mg, 20 mmol) in
MeOH/EtOAc (1:1, 5 mL) was treated with Pd(OH)2 on C (20 mg)
under a hydrogen atmosphere for 2 h at 21 °C. The mixture was filtered
(Celite) and concentrated. The residue was chromatographed on SiO2
(CH3CN/H2O, 5:1) to give 5.8 mg (85%) of the polyol 5-D2: 1H NMR
(pyridine-d5/CD3OD, 95/5) δ 5.13 (1H, d, J ) 3.6), 4.45 (1H, d, J )
3.4), 4.43 (1H, dd, J ) 11.4, 4.7), 4.37 (1H, d, J ) 11.9), 4.36 (1H, t,
J ) 10.0), 4.31 (1H, t, J ) 9.2), 4.25 (1H, dd, J ) 10.6, 5.2), 4.22
(1H, m), 4.14 (1H, dd, J ) 11.9, 5.3), 4.03 (1H, dd, J ) 9.2, 3.4), 3.99
(1H, dd, J ) 9.5, 3.6), 3.93 (1H, t, J ) 5.6), 3.91 (1H, d, J ) 9.3),
2.40 (1H, t, J ) 10.5); 13C NMR (CD3OD) δ 101.33, 80.53, 79.41,
76.15, 74.84, 74.01, 72.46, 72.29, 71.05, 63.75, 63.21, 55.36, 49.43,
41.57; HRMS (FAB) calculated for C14H25D2O10 ([M + H]+) 357.1727,
found 357.1729.
Structure Refinement. The coordinates of the peanut lectin tetramer
in the O-lactose complex24a were used as the starting model. Refine-
ment was carried out with data in the range 10-2.7 Å using the program
XPLOR.32 Noncrystallographic restraints were used in all but the final
cycles of refinement. A difference map calculated after a few cycles
of refinement showed clear density for the C-lactose molecules in all
of the four subunits. Several rounds of fitting and refinement were
done during which extensive use was made of omit-type maps.33,34 All
maps were inspected using FRODO.35 After the fitting of the sugar in
the electron density maps, water molecules were added in steps using
2Fo - Fc and Fo - Fc maps. The model converged to a final R-value
of 18.8% and R-free of 22.7%. The quality of the model was monitored
using PROCHECK.36 More than 90.0% of the residues are in the most
favorable region of the Ramachandran map37 with no residue in the
disallowed region. The relevant geometric parameters are in Table 3.
The coordinates and the structure factors have been deposited in the
Brookhaven Protein Data Bank.
6-H2. A solution of 4-H2 (0.56 g, 0.57 mmol) and Sc(OTf)3 (42
mg, 0.09 mmol) in allyl alcohol (20 mL) was heated to 100 °C for 6
h. The mixture was cooled, treated with NaHCO3, and concentrated.
The residue was diluted with ether and brine. The organic layer was
separated, dried (MgSO4), and concentrated to give 0.65 g of the crude
allyl lactoside. To a solution of the above allyl acetal in DMSO (4
mL) was added t-BuOK (0.6 g, 5.3 mmol). After 1 h at 90 °C, the
mixture was extracted with ether, and the extracts were washed with
water. Drying (MgSO4) and concentration afforded 0.53 g of the crude
enol ether. A solution of the enol ether in acetone/1N HCl (9:1, 20
mL) was heated to 55 °C for 1 h. The mixture was treated with
NaHCO3 before the acetone was evaporated off. The residue was
extracted with ether, and the extracts were dried (MgSO4), concentrated,
and chromatographed on SiO2 (hexanes/EtOAc, 4:1 to 2:1) to provide
442 mg (80% from 4-H2) of the benzyl protected lactose as an oil.
Acknowledgment. The diffraction data were collected at the
National Area Detector supported by the Department of Science
and Technology (DST) and the Department of Biotechnology
(DBT). Facilities at the Super Computer Education and Research
Centre and the DBT supported Interactive Graphics Based
Molecular Modeling Facility were used for computations. Finan-
cial support from DST (M.V.) and from the National Institutes
of Health (NS-12108; Y.K.) is gratefully acknowledged.
Supporting Information Available: Synthesis of 7, diagram
of vicinal coupling constants of 7-H2, NOESY spectrum of 7-D2,
and Ramachandran map of the peanut lectin bound C-lactose
(5 pages, print/PDF). See any current masthead page for
ordering information and Web access instructions.
A solution of the benzyl ether (0.30 g, 0.31 mmol) in MeOH/EtOAc
(1:1, 20 mL) was treated with Pd(OH)2 on C (50 mg) under a hydrogen
atmosphere for 12 h at 21 °C. The mixture was filtered (Celite),
concentrated, and chromatographed on SiO2 (CH3CN/H2O, 5:1) to
afford 92 mg (87%) of C-lactose 6-H2.12
JA982193K
Experiments for the X-ray Analysis
(30) Majumdar, T.; Surolia, A. Prepr. Biochem. 1978, 8, 119-131.
(31) Kabsch, W. J. Appl. Crystallogr. 1993, 26, 795-800.
(32) Brunger, A. T. XPLOR Version 3.1 Manual, 1992, Yale University.
(33) Vijayan, M. In Computing in Crystallography; Diamond, R.,
Ramaseshan, S., Venkatesan, K., Eds.; Indian Academy of Sciences:
Bangalore, 1980, 19.01-19.26.
Crystallization. The protein was prepared by affinity chromatog-
raphy on cross-linked arabinogalactan.30 The crystals of the peanut
lectin/C-lactose complex were grown from a hanging drop of 5 mg/
mL protein in 0.05 M sodium phosphate buffer, pH 7.0, containing
0.2 M sodium chloride, 0.02% sodium azide, 10 mM C-lactose, and
12% (w/v) PEG 8000, equilibrated against 40% (w/v) PEG 8000 in
the same buffer.
(34) Bhat, T. N.; Cohen, G. H. J. Appl. Crystallogr. 1984, 17, 244-
248.
(35) Jones, T. A. J. Appl. Crystallogr. 1978, 11, 268-272.
(36) Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J.
M. J. Appl. Crystallogr. 1993, 26, 283-291.
Data Collection. X-ray intensity data were collected and processed
to 2.7 Å on a MAR image plate detector system mounted on a Rigaku
RU200 X-ray generator from a single crystal (space group P21212, a
(37) Ramachandran, G. N.; Sasisekharan, V. AdV. Protein Chem. 1968,
23, 283-438.
(38) Kraulis, P. J. Appl. Crystallogr. 1991, 24, 946-950.