Solvent interactions and conformational choice in a core N-glycan segment: Gas phase conformation of the central, branching trimannose unit and its singly hydrated complex

Article abstract of DOI:10.1021/ja801892h

The intrinsic conformational preferences and structures of the branched trimannoside, α-phenyl 3,6-di-O-(α-D-mannopyranosyl)-α-D- mannopyranoside (which contains the same carbohydrates found in a key subunit of the core pentasaccharide in N-glycans) and i

Full text of DOI:10.1021/ja801892h

Published on Web 07/17/2008
Solvent Interactions and Conformational Choice in a Core
N-Glycan Segment: Gas Phase Conformation of the Central,
Branching Trimannose Unit and its Singly Hydrated Complex
E. Cristina Stanca-Kaposta,† David P. Gamblin,‡ Emilio J. Cocinero,† Jann Frey,†
Romano T. Kroemer,# Antony J. Fairbanks,‡ Benjamin G. Davis,*,‡ and
John P. Simons*,†
Department of Chemistry, UniVersity of Oxford, Physical and Theoretical Chemistry Laboratory,
South Parks Road, OX1 3QZ Oxford, United Kingdom, Sanofi-AVentis, CRVA, 13 quai Jules
Guesde, BP14, 94403 Vitry-sur-Seine, France, and the Department of Chemistry, UniVersity of
Oxford, Chemistry Research Laboratory, 12 Mansfield Road, OX1 3TA Oxford, United Kingdom
Received March 13, 2008; E-mail: john.simons@chem.ox.ac.uk; Ben.Davis@chem.ox.ac.uk
Abstract: The intrinsic conformational preferences and structures of the branched trimannoside, R-phenyl
3,6-di-O-(R-D-mannopyranosyl)-R-D-mannopyranoside (which contains the same carbohydrates found in a
key subunit of the core pentasaccharide in N-glycans) and its singly hydrated complex, have been
investigated in the gas phase isolated at low temperature in a molecular beam expansion. Conformational
assignments of their infrared ion dip spectra, based on comparisons between experiment and ONIOM
(B3LYP/6-31+G(d):HF/6-31G(d)) and single-point MP2 calculations have identified their preferred structures
and relative energies. The unhydrated trimannoside populates a unique structure supported by two strong,
central hydrogen bonds linking the central mannose unit (CM), and its two branches (3M and 6M) closely
together, through a cooperative hydrogen-bonding network: OH4(CM)fOH6(3M)fOH6(6M). A closely
bound structure is also retained in the singly hydrated oligosaccharide, with the water molecule bridging
across the 3M and 6M branches to provide additional bonding. This structure contrasts sharply with the
more open, entropically favored trimannoside structure determined in aqueous solution at 298 K. In principle
this structure can be accessed from the isolated trimannoside structure by a simple conformational change,
a twist about the R(1,3) glycosidic linkage, increasing the dihedral angle ψ[C1(3M)-O3(3M)-C3(CM)-
C2(CM)] from ∼74° to ∼146° to enable accommodation of a water molecule at the centrally bound site
occupied by the hydroxymethyl group on the 3M ring and mediation of the water-linked hydrogen-bonded
network: OH4(CM) fOHWfOH6(6M). The creation of a “water pocket” motif localized at the bisecting
axis of the trimannoside is strikingly similar to the structure of more complex N-glycans in water, suggesting
perhaps a general role for the “bisecting” OH4 group in the central (CM) mannose unit.
molecules.4 In such a scenario, questions arise as to the
mechanism through which “bare” glycans acquire each solvent
Introduction
Recent spectroscopic studies of carbohydrates in the absence
of solvent have highlighted clearly delineated conformational
landscapes which display well-defined minima,1 but comparisons
between the intrinsic, gas-phase structures of carbohydrates and
their structures in aqueous solution have led to the conclusion
that the inherent conformational preferences of oligosaccharides
and consequently their secondary structures are modified by
solvent interactions.2,3 Molecular dynamics investigations of
naturally occurring N-glycans in aqueous solution have identified
enduring structural motifs supported by explicitly bound water
molecule. Is their acquisition specific and selective, and is there
a midpoint of partial solvation that is a tipping point in the
balance between inherent and solvated structural preferences?
To what extent do the glycan structures that are selected by
nature contain consistent pockets or “holes” for solvent?
Our earlier studies of hydration in small, unbranched glycans
revealed a preference for water binding at weakly hydrogen-
bonded OH sites which were “unpicked” by the solvent to create
a stronger, cooperatively hydrogen-bonded network, often
leading to conformational relaxation and a globally strengthened
structure.5,6 A key branched motif found in the core of most
hydrated N-linked glycans could be strengthened by acceptance
† University of Oxford, Physical and Theoretical Chemistry Laboratory.
# Sanofi-Aventis, CRVA.
‡ University of Oxford, Chemistry Research Laboratory.
(1) Simons, J. P.; C¸ arc¸abal, P.; Davis, B. G.; Gamblin, D. P.; Hu¨nig, I.;
Jockusch, R. A.; Kroemer, R. T.; Marzluff, E. M.; Snoek, L. C. Int.
ReV. Phys. Chem. 2005, 24, 489–532.
(4) (a) Woods, R. J.; Pathiaseril, A.; Wormald, M. R.; Edge, C. J.; Dwek,
R. A. Eur. J. Biochem. 1998, 258, 372–386. (b) Wormald, M. R.;
Petrescu, A. J.; Pao, Y-L.; Glithero, A.; Elliott, T.; Dwek, R. A. Chem.
ReV. 2002, 102, 371–386.
(2) Kirschner, K. N.; Woods, R. J. Proc. Natl. Acad. Sci. U.S.A. 2001,
98, 10541–10545.
(5) C¸ arc¸abal, P.; Jockusch, R. A.; Hu¨nig, I.; Snoek, L. C.; Kroemer, R. T.;
Davis, B. G.; Gamblin, D. P.; Compagnon, I.; Oomens, J.; Simons,
J. P. J. Am. Chem. Soc. 2005, 127, 11414–11425.
(3) Almond, A.; Sheehan, J. K. Glycobiology 2003, 13, 255–264.
9
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A R T I C L E S
Stanca-Kaposta et al.
(IR) vibrational spectroscopy, using the double resonance IR
ion dip (IRID) detection strategy.
Methods
Vaporization. Transfer of the trimannoside into the gas phase
followed essentially the same procedures described in an earlier
investigation of its R(1,3) and R(1,6) dimannoside components.8
Powdered samples were ground with graphite powder (graphite/
trisaccharide mass ratio ≈ 1:10), deposited as a thin uniform surface
layer on a graphite substrate, and placed in the vacuum chamber
close to the exit of a pulsed, cylindrical nozzle expansion valve
(0.8 mm diameter). Molecules were desorbed from the surface using
the fundamental of a pulsed and focused Nd:YAG laser (2-4 mJ/
pulse) and entrained and cooled in an expanding argon jet (∼4 bar
backing pressure) before passing into the detection chamber through
a 2 mm diameter skimmer. Hydrated complexes were formed by
seeding the carrier gas with water vapor prior to the expansion
(∼0.25% H2O in Ar).
Figure 1. Branch point trisaccharide of structure 1 and its R-phenyl
pyranoside derivative 2. The labels CM, 3M, and 6M identify the different
mannosyl residues as discussed in the text.
of a water molecule into its branch “fork”. The recurrence of
this site in many fully solvated glycan structures would suggest
the existence of a “water pocket” in N-glycans that conserves
a bisecting water molecule as a beneficial structural element,
akin to the conserved water molecules found in lectin binding
sites.7
In the past few years, it has become possible to determine
experimentally the intrinsic conformational structures of car-
bohydrates isolated at low temperatures in the gas phase using
a combination of vibrational spectroscopy, density functional
theoretical (DFT), and ab initio calculation, and to identify the
preferred structures and conformations of their hydrated com-
plexes under the same conditions. These have revealed directly,
their unperturbed conformational preferences and, perhaps more
importantly, a role for water in modulating their structures, at
least for the limited series of monosaccharides studied to date.5,6
They have also revealed some correlation between the preferred
waterbindingsitesandthoseassociatedwithprotein-carbohydrate
molecular recognition and led to a proposed set of “predictive
rules” for both.5
The successful investigation of the intrinsic gas-phase con-
formations of two disaccharides, the R(1,3) and R(1,6) diman-
nosides8 which correspond to the base sugars of the first two
branches of the N-glycan, Man9(GlcNAc)2, has now led to the
conformational analysis of the complete branch point in the
representative trisaccharide, 3,6-di-O-(R-D-mannopyranosyl)-R-
D-mannopyranose, 1 (which incorporates the R(1,3) and R(1,6)
dimannoside units, see Figure 1), and also of its singly hydrated
complex isolated at low temperatures in the gas phase. Valuably,
its preferred conformation in aqueous solution at 298 K is
already available through residual dipolar coupling NMR
measurements by Prestegard and his co-workers9 and Almond
and Duus.10 The conformational and structural assignments in
the gas phase described here are based upon a combination of
DFT, ab initio, and ONIOM11 computation coupled with and
also led by conformer-specific and mass-selective near-infrared
Spectroscopy. Conformer-specific IR spectra were recorded
through a combination of mass-selected resonant two photon
ionization (R2PI), double resonance IR-UV hole burning (HB)
and IR ion dip (IRID) spectroscopy, following the procedures
described earlier.8 The individual IRID spectra provided the
distinctive patterns of OH stretching modes through which the
structural assignments could be made.
Computational Strategies. Conformational and structural as-
signments were performed through, and also led by comparisons
between the experimental vibrational spectra and those determined
through quantum chemical calculations on a wide range of
conformers following procedures described earlier.5,8 These were
identified using a combination of force field conformational searches
and quantum chemical calculations as implemented in the SYBYL
(Tripos),12 Macromodel,13 and Gaussian 0314 program packages,
respectively.
Results, Discussion and Structural Assignments
The target phenyl mannoside 2, designed as a representative
minimal motif, contained two key features: the branch point of
the N-glycan core and a “tag” chromophore needed for its
detection and structural assignment via R2PI and IRID spec-
troscopy and provided by the phenyl group. The use of
participating O-2 acetyl protection in the intermediate mono-
mannosyl donor, 3 (Scheme 1), allowed excellently stereose-
lective (exclusively R) installation of the branch units 3M and
6M. Regioselective mannosylation of 7 at O-3 and O-6 was
achieved through the use of regioselective pivaloylation at O-6
and O-3, protection of O-2 and O-4 as benzyl ethers, and
subsequent removal of the pivaloyl esters. This protecting group
controlled access to O-6 and O-3 proved to be a superior strategy
to that based either on regioselective O-4 or O-2 protection via
stannylene acetals or on direct regioselective O-3, O-6 bis-
mannosylation methods.
Thus, the “branch point” trimannosyl unit, specifically phenyl
3,6-di-O-(R-D-mannopyranosyl)-R-D-mannopyranoside (2), was
prepared by glycosylation of mannosyl diol acceptor 7 using
two equivalents of mannosyl trichloroacetimidate donor 3
leading exclusively to the formation of O-6 and O-3 R-glycosyl
linkages. The diol acceptor itself 7 was prepared by the reaction
of benzyl mannoside 4 with two equivalents of pivaloyl chloride
in pyridine at 0 °C which gave compound 6 in 56% yield
(6) C¸ arc¸abal, P.; Patsias, Th.; Hu¨nig, I.; Liu, B.; Kaposta, E. C.; Snoek,
L. C.; Gamblin, D. P.; Davis, B. G.; Simons, J. P. Phys. Chem. Chem.
Phys. 2006, 8, 129–136.
(7) Toone, E. J. Curr. Opin. Sruct. Biol. 1994, 4, 719–728.
(8) C¸ arc¸abal, P.; Hu¨nig., I.; Gamblin, D. P.; Liu, B.; Jockusch, R. A.;
Kroemer, R. T.; Snoek, L. C.; Fairbanks, A. J.; Davis, B. G.; Simons,
J. P. J. Am. Chem. Soc. 2006, 128, 1976–1981.
(9) (a) Sayers, E. W.; Prestegard, J. H. Biophys. J. 2000, 79, 3313–3329.
(b) Tian, F.; Al-Hashimi, H. M.; Craighead, J. L.; Prestegard, J. H.
J. Am. Chem. Soc. 2001, 123, 485–492.
(12) SYBYL 7.3; Tripos International: St. Louis, Missouri.
(13) Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.; Lipton,
M.; Caufield, C.; Chang, G.; Hendrikson, T.; Still, W. C. J. Comput.
Chem. 1990, 11, 440–467.
(14) Frisch, M. J.; et al. Gaussian 03, Revision B.03; Gaussian, Inc.:
Pittsburgh, PA, 2003.
(10) Almond, A.; Duus, J.Ø. J. Biomol. NMR 2001, 20, 351–363.
(11) (a) Maseras, F.; Morokuma, K. J. Comput. Chem. 1995, 16, 1170–
1179. (b) Dappich, S.; Komaromi, I.; Byun, S.; Morokuma, K. J.;
Frisch, M. J. J. Mol. Struct. 1999, 451, 1–21.
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Core Glycan Conformations
A R T I C L E S
Scheme 1a
a (i) (CH3)3CCOCl, py, 0 °C, 56%; (ii) BnOC(NH)CCl3, TMSOTf, DCM/cyclohexane, 70%; (iii) NaOMe, MeOH, reflux, 78%; (iv) BF3 ·OEt2, 4 Å MS,
DCM, 75%; (v) H2, Pd(OH)2, EtOH then Ac2O, py, 98% (2 steps); (vi) PhOH, BF3 ·OEt2, DCM, 74%; (vii) NaOMe, MeOH, 91%.
through regioselective esterification of OH-3 and OH-6 over
OH-2 and OH-4. Subsequent benzylation on OH-2 and OH-4
could only be accomplished under mild Lewis acidic conditions
with benzyl trichloroacetimidate to give 7 in 70% yield; base-
assisted methods failed. Removal of pivaloyl protection using
stoichiometric amounts of sodium methoxide in refluxing
methanol gave diol acceptor 7. The glycosylation reaction
between the trichloracetimidate donor 3 and the diol acceptor
7 afforded the trisaccharide in 75% yield. Benzyl ether hydro-
genolysis through treatment of 7 with Pearlman’s catalyst
(Pd(OH)2) and hydrogen, followed by acetylation allowed
protecting group exchange and gave peracetylated trimannoside
9. The anomeric phenyl tag was introduced at this late stage on
Figure 2. Description of the layers for the ONIOM (B3LYP/6-31+G(d):
treatment of 9 with phenol and boron trifluoride diethyl etherate
to furnish the phenyl R-mannoside 10, in 74% yield. Global
deacetylation gave the target tagged trisaccharide 2.
HF/6-31G(d)) calculations: the gray-colored atoms (phenyl tag and the
central mannose (CM) sugar ring excluding the OH groups) were treated
at the HF/6-31G(d) level, whereas the other atoms were treated at the
B3LYP/6-31+G(d) level. The labeling identifies the mannose rings
designated on the upper structure as CM, 3M, and 6M and discussed in
the text.
Due to the large number of atoms in the phenyl trimannoside
2 and the high flexibility of the glycosidic linkages, the number
of possible conformers is very high, and a full search of the
conformational landscape would be correspondingly computa-
tionally expensive. A more efficient method allowed the
experiment to guide the conformational search. Thus, several
random conformational searches were carried out using different
initial structures, and filtering of the relevant structures was
performed based on the information obtained from the experi-
mental infrared spectrum, which indicated the presence of
cooperative hydrogen-bonded networks. The conformers result-
ing from the initial force field search were therefore scanned
for such networks, including inter-ring hydrogen-bonding
interactions, and the selected structures were then submitted for
ONIOM calculations at the B3LYP/6-31+G(d):HF/6-31G(d)
level (see Figure 2). Frequencies and relative energies for the
most stable conformers were then calculated using the same
ONIOM approach, but their relative zero-point corrected ener-
gies and free energies (at 298 K assuming harmonic frequencies)
were evaluated through single-point calculations at the MP2/
6-31+G(d) level. For comparison with the experiment, the O-H
stretch frequencies were scaled by the factor 0.9734.
mannose rings. The lowest-energy conformers obtained from
the conformational search as well as representatives of the
different prototype structures described above were submitted
for higher level ONIOM (B3LYP/6-31+G(d):HF/6-31G(d))
geometry optimizations, frequency calculations, and MP2/6-
31+G(d) single-point energy calculations, corrected for zero
point and free energy, to identify their different IR signatures
associated with alternative positions of the water molecule.
The experimental R2PI spectra of the phenyl trimannoside
(2, TriMan) and its singly hydrated complex (TriMan ·W1), both
recorded in their mass selected parent ion channels, are shown
in Figure 3. Apart from a small displacement, ∼20 cm-1 to
lower wavenumber in the hydrate they present very similar
profiles, displaying a few sharp bands superimposed upon a
broad underlying continuum and closely resembling the corre-
sponding spectral profiles of the “phenyl tagged” R(1,3) and
R(1,6) dimannosides.8 Their associated vibrational (IRID)
spectra, recorded in the O-H stretch region are shown in Figures
4 and 5. These both remained essentially the same when the
UV probe was tuned across their R2PI spectra, indicating their
predominant association in each case with a single structure
only, and highlighting thereby a strong conformational prefer-
ence in both the “bare” and the hydrated branching motifs.
Furthermore, as for the dimannosides,8 both the resolved features
and the underlying continua in the two R2PI spectra must be
A similar procedure was applied to the water complex, where
the experimental vibrational spectrum again guided the initial
structures submitted for quantum chemical calculations. Several
varieties of “prototype” structure were examined, incorporating
a centrally sited water molecule, or a water molecule bound to
OH groups located on the periphery or bridging across the
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A R T I C L E S
Stanca-Kaposta et al.
bands lying at higher wavenumbers. Although their associated
structures do not correspond to the lowest calculated energy,
one of them lies close to the minimum free energy (calculated
at 298 K). Given the flexibility of the trimannoside at the pre-
expansion temperature and the theoretical approximations, the
computed relative energies will be of less significance than the
free energies,15and in any case the “true observable” is the
vibrational spectrum.
The two conformers providing the best fit to experiment in
TriMan present very similar structures and are held together
by two central, co-operatively linked hydrogen bonds. They
connect OH4 on the central mannosyl (CM) ring to the
hydroxymethyl group on the R(1,3) mannosyl “wing”, labeled
3M, which is itself connected to the other hydroxymethyl group
on the R(1,6) mannosyl “wing”, labeled 6M. Comparison
between the experimental and computed spectra identifies the
two strongly displaced bands as the symmetric (∼3400 cm-1
)
and antisymmetic (∼3440 cm-1) stretching vibrational modes
associated with the central chain, OH4(CM)fOH6(3M)f
OH6(6M). Thus, in the absence of water and at low temperature,
the trisaccharide adopts a tightly bound “closed” conformation
held together by strong intramolecular hydrogen bonds that link
the component mannose units. The R(1,3) arm also preserves
the conformational structure and bonding displayed in the R(1,3)
dimannoside substructure 3M-CM and in the corresponding
conformer of the substructure 3M monosaccharide, see Figure
6.5,8
Figure 3. R2PI spectra of (a) TriMan 2 and (b) TriMan ·W1.
associated with the same carrier, which favors assignment of
the unresolved background to the excitation of congested hot-
band transitions.
Several alternative types of structural possibilities were
envisaged for the singly hydrated trimannoside. The water
molecule might bridge across the 3M and 6M branches (parts
b and d of Figure 5) or across OH groups located on the
periphery of the trisaccharide (Figure 5c), conserving the
“closed” centrally hydrogen-bonded structure. Alternatively, the
trimannoside framework might open up, for example by rotation
of the 3M wing about the R(1,3) glycosidic linkage (Figure 5e)
The experimental IRID spectrum of TriMan is compared in
Figure 4 with the ONIOM-computed vibrational spectra associ-
ated with several of its computed lowest energy and free energy
conformational structures. Two of the computed spectra (parts
d and e of Figure 4) are in remarkably good agreement with
the experimental spectrum which presents two intense, strongly
displaced and broadened bands at low wavenumbers, located
at ∼3400 cm-1 and ∼3440 cm-1, together with a cluster of
Figure 4. IRID spectrum of TriMan (a) and the predicted vibrational spectra (b, c, d, e, f) computed for its five lowest-energy conformers using the ONIOM
(B3LYP/6-31+G(d):HF/6-31G(d)) approach; their structures are shown on both sides of the figure. The zero-point corrected relative energies and free
energies at 298 K (in kJ mol-1) calculated at the MP2/6-31+G(d) level are shown in brackets. The blue dots identify the strong hydrogen bonds in the
structure and their corresponding features in the spectrum.
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Core Glycan Conformations
A R T I C L E S
Figure 5. IRID spectrum of singly hydrated TriMan (a) and the vibrational spectra computed for alternative hydrated structures shown on both sides of the
figure: (b) bridged I, (c) peripheral, (d) bridged II, and (e) central. Their zero-point corrected relative energies and free energies at 298 K (in kJ mol-1
)
calculated at the MP2/6-31+G(d) level are shown in brackets. The blue dots and arrows indicate the two, three, or four strong hydrogen bonds in each class
of singly hydrated TriMan structures and the corresponding features in the predicted and experimental spectra.
in Figure 5a displays four strongly displaced bands it must be
associated either with a “peripheral” hydrate structure, for
example insertion of the water molecule between OH4 and OH3
on the 6M ring (Figure 5c), or with the structure shown in Figure
5b where the water molecule links the 3M and 6M rings. By
good fortune, this structure (5b) also has the lowest calculated
relative energy (at 0 K and free energy at 298 K, see Supporting
Information for comparisons with other low-energy structures),
and its computed IR spectrum is in good accord with that from
experiment. It provides much the best agreement with the cluster
of bands recorded at higher wavenumber, associated with the
weakly H-bonded OH vibrational modes while the quartet at
lower wavenumbers, associated with the more strongly H-
bonded modes, presents a very similar pattern to the recorded
spectrum. Indeed a slight increase in the “anharmonicity scaling”
would bring the bands into very close accord, and it represents
the most probable assignment.
Figure 6. Schematic comparison between the determined conformational
structures of (a) the R-mannoside monomer5 3M (b) the dimannoside, Man
R(1,3) Man 3M-CM8 and (c) the full trimannoside (2, TriMan) determined
here. Despite variations in the contact partner, key features are maintained
in the 3M branch even as the complexity of the system increases.
allowing the water molecule into its interior to replace the
central, hydrogen-bonded hydroxymethyl (3M) group and create
the water-mediated chain, OH4(CM)fOHWfOH6(6M). In two
of these alternatives (parts b and c of Figures 5) the central
intramolecular hydrogen bonds are complemented by two
additional intermolecular H-bonds associated with insertion of
the water molecule between two of the peripheral OH groups;
this would lead to the appearance of four strongly displaced
bands in the IR spectrum. The structure shown in Figure 5d
would be signaled by the appearance of three strongly displaced
bands, and in the centrally hydrated structure (Figure 5e) the
two strong hydrogen bonds would be replaced by two new
hydrogen bonds in which case the IR spectrum would continue
to present two strongly displaced bands only.
The experimental IRID spectrum shown in Figure 5a actually
presents two pairs of closely spaced, blended bands at low
wavenumber, one presenting an asymmetric contour displaying
a peak centered at ∼3330 cm-1 and a subsidiary shoulder at
∼3365 cm-1 and the other, two clearly defined peaks at ∼3425
cm-1 and 3450 cm-1 in addition to the cluster at higher
wavenumbers. Since the experimental IRID spectrum shown
Branched N-Linked Glycan Conformations in Aqueous
Solution: Comparisons and Speculations. The “closed” confor-
mational structures of the bare trimannoside and its singly
hydrated complex displayed at low temperature in the gas phase
contrast with the “open” structures determined in aqueous
solution at 298 K, using NMR techniques coupled with MD
calculations.9,10 In the gas phase at low temperatures the
preferred conformations of carbohydrates and their hydrated
complexes are predominantly supported by cooperative intra-
and intermolecular hydrogen bonding4 as found for example in
the present study: however, in aqueous solution at ambient
temperatures in the absence of explicit solvation this may no
longer be the case.1,2 Nonetheless, it is notable that one of the
set of computed singly hydrated structures, the centrally hydrated
structure (Figure 5e) shown again in Figure 7a, adopts an
(entropically favored) open conformation in remarkably close
correspondence with that determined in aqueous solution. The
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A R T I C L E S
Stanca-Kaposta et al.
Figure 7. Comparison between (a) the computed open conformational structure of the monohydrate, TriMan ·W1, (b) a snapshot of the Man9(GlcNAc)2
structure, including the preferred water binding sites together with their occupancy (in %) based on molecular dynamics calculations, and (c) the computed
16
rmsd conformation of the core pentasaccharide segment, Man3(GlcNAc)2 based upon NMR measurements and MD simulations in aqueous solution at
∼298 K4 (black rods for the trisaccharide, light gray rods for the phenyl tag and gray thin wire frame) for the core pentasaccharide.
Table 1. Glycosidic Dihedral Angles of Phenyl Trimannoside in the
Isolated and Singly Hydrated “Open” Form in the Gas Phase at
explicit hydration site incorporates a “keystone” water molecule
Low Temperature and of Methyl Trimannoside in Aqueous
mannose N-glycan, Man9(GlcNAc)2 where the most persistent
H-bonded to OH4(CM) and two of the extended oligomannoside
Solution at ∼298 K.
arms (Figure 7b);4 and the average structure of its core
φ, ψ (1,3)a
φ, ψ, ω (1,6)b
pentasaccharide segment, Man3(GlcNAc)2. This is highlighted
in Figure 7c which compares the “frozen” singly hydrated
trimannoside structure (Figure 7a) with the computed rmsd
structures of the core pentasaccharide unit stripped of its
extended mannoside arms (based upon NMR measurements and
MD simulations of the N-glycan in an aqueous environment at
∼298 K).4 The similarity between the open structure and the
relative orientations of the 3M and 6M mannosyl “wings” in
the trimannoside and in the corresponding unit in the core
pentasaccharide is remarkable and provides some support for
the conformation of the key branched (trimannoside) segment
being driven by the creation of a water “pocket” in the branch
“fork”. Indeed in the absence of the extended mannoside
armssin the core pentasaccharide or the trimannosidesit would
not be difficult for the central water molecule linked to
OH4(CM) to slip into the water pocket. The repeated recurrence
of this motif in many fully solvated glycan structures suggests
a more general interplay between favored glycan structures and
hydration, with water pockets that conserve bisecting water
molecules acting as beneficial structural elements.
isolated
84, 74
81, 146
60(80), -180(-100)
71, 178, 60
69, 189, 60
64, 180(60), 60(180)
“open” hydratee
aqueous9
φΗ, ψΗ (1,3) c
φΗ, ψΗ, ω′ (1,6)d
isolated
-34, -170
-35, -28
-60, 0 ( 30
-46, 182, -64
-47, 189, -64
-60, 180, -60(60)
ω′(-60):(60) ) gg:gt ≈ 1
“open” hydratee
aqueous10
a φ[O5(i)-C1(i)-O3(i - 1)-C3(i - 1)]; ψ[C1(i)-O3(i)-C3(i -
1)-C2(i - 1)]. b φ[O5(i)-C1(i)-O6(i - 1)-C6(i - 1)]; ψ[C1(i)-O6(i)-
C6(i - 1)-C5(i - 1)]; ω[O6(i)-C6(i)-C5(i)-C4(i)]. c φH[H1(i)-C1(i)-
O1(i - 1)-C3(i - 1)]; ψH[C1(i)-O1(i)-C3(i - 1)-H3(i - 1)].
d φH[H1(i)-C1(i)-O1(i - 1)-C6(i - 1)]; ψH[C1(i)-O1(i)-C6(i -
1)-H6(i
-
1)]; ω′[O6(i)-C6(i)-C5(i)-O5(i)]. e “Open” hydrated
complex structure shown in Figure 7.
structure is generated by rotation of the 3M wing in the bare
molecule (shown in Figure 6c) about the dihedral angle ψ
[C1(3M)-O3(3M)-C3(CM)-C2(CM)] associated with the
R(1,3) glycosidic linkage, and insertion of the water molecule
into its interior. The average dynamical values of the inter-ring
glycosidic dihedral angles determined in aqueous solution at
298 K (unfortunately using different definitions for the dihedral
angles φ, ψ (1,3) and φ, ψ, ω (1,6)) are listed in Table 1; they
are remarkably close to the “frozen” dihedral angles in the open
singly hydrated complex.
The angular orientation about the R(1,6) linkage, which
retains a gg configuration in the gas phase both in the isolated
trisaccharide and its generated hydrated complex, is also very
similar to the corresponding average gg configuration in aqueous
solution.
Acknowledgment. We thank Drs. Pierre C¸ arc¸abal, Bo Liu, Eoin
Scanlan and Lavina Snoek for their contributions to the development
and completion of this research. We are also grateful for the support
provided by EPSRC, the Leverhulme Trust (Grant No. F/08788G),
the Spanish Ministry of Education and Science (E.J.C.), the STFC
Laser Loan Pool, the Oxford Supercomputing Centre and the
Physical and Theoretical Chemistry Laboratory.
Supporting Information Available: Detailed description of
the synthesis and characterization of 2, 3, 5, 6, 7, 8 and 10;
Cartesian coordinates, total energies and larger-scale displays
of the vibrational spectra and assignments of the conformations
shown in Figures 4 and 5 and additional low-lying structures
for the monohydrate; a table with all the structures for which
the vibrational spectra are shown, displayed on a larger scale;
and the complete ref 14. This material is available free of charge
via the Internet at http://pubs.acs.org.
Even more striking comparisons can be made between the
calculated structures of the open singly hydrated trimannoside
unit (Figure 7a); the “water-bridged” structure of the high
(15) Shubert, V. A.; Baquero, E. E.; Clarkson, J. R.; James, W. H., III;
Turk, J. A.; Hare, A. A.; Worrel, K.; Lipton, M. A.; Schofield, D. P.;
Jordan, K. D.; Zwier, T. S. J. Chem. Phys. 2007, 127, 234315.
(16) Kindly provided by M. R.Wormald.
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10696 J. AM. CHEM. SOC. VOL. 130, NO. 32, 2008