Hydration of Sugars in the gas phase: Regioselectivity and conformational choice in N-acetyl glucosamine and glucose

Article abstract of DOI:10.1002/chem.200901830

The influence of an acetamido group in directing the preferred choice of hydration sites in glucosamine and a consequent extension of the working rules governing regioselective hydration and conformational choice, have been revealed through comparisons between the conformations and structures of "free" and multiply hydrated phenyl N-acetyl-β-D-glucosamine (sβpGlcNAc) and phenyl β-D-glucopyranoside (βpGlc), isolated in the gas phase at low temperatures. The structures have been assigned through infrared ion depletion spectroscopy conducted in a supersonic jet expansion, coupled with computational methods. The acetamido motif provides a hydration focus that overwhelms the directing role of the hydroxymethyl group; in multiply hydrated βpGlcNAc the water molecules are all located around the acetamido motif, on the "axial" faces of the pyranose ring rather than around its edge, despite the equatorial disposition of all the hydrophilic groups in the ring. The striking and unprecedented role of the C-2 acetamido group in controlling hydration structures may, in part, explain the differing and widespread roles of GlcNAc, and perhaps GalNAc, in nature.

Full text of DOI:10.1002/chem.200901830

FULL PAPER
DOI: 10.1002/chem.200901830
Hydration of Sugars in the Gas Phase: Regioselectivity and Conformational
Choice in N-Acetyl Glucosamine and Glucose
Emilio J. Cocinero,[a] E. Cristina Stanca-Kaposta,[a, c] Mark Dethlefsen,[a] Bo Liu,[a]
David P. Gamblin,[b] Benjamin G. Davis,*[b] and John P. Simons*[a]
Abstract: The influence of an acetami-
do group in directing the preferred
choice of hydration sites in glucosa-
mine and a consequent extension of
the working rules governing regioselec-
tive hydration and conformational
choice, have been revealed through
comparisons between the conforma-
tions and structures of “free” and mul-
tiply hydrated phenyl N-acetyl-b-d-glu-
cosamine (bpGlcNAc) and phenyl b-d-
glucopyranoside (bpGlc), isolated in
the gas phase at low temperatures. The
structures have been assigned through
infrared ion depletion spectroscopy
conducted in a supersonic jet expan-
sion, coupled with computational meth-
ods. The acetamido motif provides a
hydration focus that overwhelms the
directing role of the hydroxymethyl
group; in multiply hydrated bpGlcNAc
the water molecules are all located
around the acetamido motif, on the
“axial” faces of the pyranose ring
rather than around its edge, despite the
equatorial disposition of all the hydro-
philic groups in the ring. The striking
and unprecedented role of the C-2
acetamido group in controlling hydra-
tion structures may, in part, explain the
differing and widespread roles of
GlcNAc, and perhaps GalNAc, in
nature.
Keywords:
hydrogen
carbohydrates
bonding
·
·
IR spectroscopy · solvation
Introduction
found in the exo-skeletons of many insects and crustaceans;
it also provides the core linkage (to asparagine residues) in
all N-linked glycoproteins. GlcNAc residues are key build-
ing blocks in many proteoglycans, which are involved in a
host of biological functions, for example, heparan sulphate,
a hetero-glycosaminoglycan which incorporates an alternat-
ing co-polymer rich in glucosamine and uronic acid resi-
dues.[1,2]
N-Acetyl glucosamine (GlcNAc) and glucose (Glc) are the
primary building blocks of many biopolymers. The disac-
charide unit, cellobiose (Glc-b1,4-d-Glc), for example, pro-
vides the basic, repeat structural unit of cellulose which is a
key component of plant cell walls. Similarly, chitobiose
(GlcNAc-b1,4-d-GlcNAc) provides the repeat unit in chitin,
Despite their important role in many living organisms,
there have been very few spectroscopic[3] or structural inves-
tigations of 2-deoxy amino sugars, such as GlcNAc or
GalNAc,[4,5] although the conformational structures and
anomeric preferences of the corresponding sugars, Glc and
Gal, and many other hexapyranose carbohydrates, have
been the focus of a large number of molecular dynamics
simulations and NMR measurements (see Kraꢀtler et al.[6]
for a very thorough review of the recent literature). There is
now a general acceptance that their (dynamical) conforma-
tional preferences in aqueous solutions are strongly influ-
enced by hydrogen-bonded interactions, both externally,
with the solvent and internally, within the carbohydrate.[4,7,8]
Until recently, however,[9–11] there were no benchmark ex-
perimental investigations of their intrinsic conformational
structures (or those of oligosaccharides containing them) in
the absence of a bulk solvent, or of the influence of explicit
[a] Dr. E. J. Cocinero, Dr. E. C. Stanca-Kaposta, M. Dethlefsen,
Dr. B. Liu, Prof. J. P. Simons
Chemistry Department, University of Oxford
Physical and Theoretical Chemistry Laboratory
South Parks Road, Oxford OX1 3QZ (UK)
Fax : (+44)1865-275410
E-mail: John.Simons@chem.ox.ac.uk
[b] Dr. D. P. Gamblin, Prof. B. G. Davis
Chemistry Department, University of Oxford
Chemistry Research Laboratory, 12 Mansfield Road
Oxford OX1 3TA (UK)
Fax : (+44)1865-285002
E-mail: Ben.Davis@chem.ox.ac.uk
[c] Dr. E. C. Stanca-Kaposta
Institut fꢀr Experimentalphysik, Freie Universitꢁt Berlin
Arnimallee 14, 14195 Berlin (Germany)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200901830.
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and specific hydration on their conformational preferences
in the gas phase. Those that had been available were almost
all computational and many were dependent on the quality
of the assumed force field.[6,12–14]
In the last few years it has become possible to derive this
information experimentally through vibrational spectrosco-
py conducted under molecular beam conditions, combined
with molecular mechanics, density functional and ab initio
theoretical calculations.[9–11] Investigations of this kind have
led to a set of predictive “working rules”, governing binding
site selectivity, conformational choice and the influence of
spatially localized hydrophilicity (vs hydrophobicity) of indi-
vidual mono- and oligosaccharides.[9] Such information may
be at least as important as that obtained in aqueous solution
since it provides a reference point (a ground state) against
which the effect of the introduction of environment, includ-
ing biologically relevant environments, may be gauged.
Much of the biological activity of oligosaccharides and gly-
cans, for example, in cell adhesion and signaling, is mediated
through membrane bound protein–carbohydrate interac-
tions.[15–17] This can involve direct hydroxyl hydrogen-bond-
ing and purely dispersive “CH–p” binding,[18–20] both of
which may, or may not displace the water molecules sur-
rounding the carbohydrate or the protein[21] or binding may
alternatively be mediated by bound water molecules.[22,23] In
all of these scenarios it is likely that the carbohydrate con-
formations associated with complexed or explicitly, and
sometimes only partially, hydrated structures in the gas
phase might provide a more precise model than dynamical
structures in aqueous solution.
Scheme 1. Schematic representation of phenyl N-acetyl-b-d-glucosamine
and the notation indicating possible conformations of the exo-cyclic hy-
droxymethyl group.
selectivity (>98% b) by virtue of participation of the C-2
imide. Use of pentaacetyl-d-glucosamine bearing an acet-
amide at C-2 resulted in reduced yield in part due to com-
peting oxazoline formation. Removal of the phthalimide
and installation of the acetamide at C-2 followed by Zꢃm-
plen deprotection proceeded cleanly to give bpGlcNAc in
54% overall yield from 1.
The present work reports the first experimental investiga-
tions of the intrinsic gas-phase conformation and structure
of an amino sugar, phenyl N-acetyl-b-d-glucosamine1
(bpGlcNAc) shown in Scheme 1, and its singly and multiply
hydrated complexes, isolated at low temperatures in a mo-
lecular beam. The influence of the C-2 acetamido group in
directing the preferred choice of hydration sites, and a con-
sequent extension of the working rules governing selective
hydration and conformational choice, are revealed through
comparisons with multiply hydrated phenyl b-d-glucopyra-
noside (bpGlc) and recent investigations[9,10] of other hydrat-
ed monosaccharides.
Scheme 2. Synthesis of bpGlcNAc.
UV and IR spectroscopy of bpGlcNAc: The resonant two-
photon ionization (R2PI) spectrum of bpGlcNAc recorded
under molecular beam conditions is shown in Figure 1 to-
gether with two of its UV “hole-burn” spectra, recorded
with the probe laser tuned either to the band origin at
36894 cmꢀ1, labeled “1”, or the vibronic band at
36960 cmꢀ1, labeled “2”. The hole-burn spectra are rather
noisy but while the first clearly displays each of the leading
four bands in the vibronic progression, the next band, 2, is
lost in the noise; the same result was obtained when the UV
probe was tuned onto the other vibronic bands lying be-
tween peaks 1 and 2. The situation is reversed in the second
hole-burn spectrum where band 2 is noticeably stronger but
the first four bands can only just be discerned. These spectra
suggest the population of more than one conformer in the
cold molecular beam, associated with inter-leaved vibronic
spectra.
Results
Synthesis: b-Phenyl N-acetylglucosamine (bpGlcNAc), bear-
ing an appropriate C-1 phenyl chromophore, was synthe-
sized using the known phthalimide protected peracetyl
donor 1 (see Scheme 2). Lewis acid-mediated glycosylation
of phenol proved superior to the use of either GlcNAc-de-
rived halo sugar donors or the use of pentaacetyl-d-glucosa-
mine and proceeded in good yield and with excellent stereo-
1
The phenyl group provides the UV chromophore required for the 2-
photon ionisation process; DFT and ab initio calculations have con-
firmed that its presence has a negligible influence on the conformation-
al landscape of the carbohydrate.[10]
The infrared ion depletion (IRID) spectra shown in the
top half of Figure 2 reinforce this interpretation. The two
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Hydration of Sugars in the Gas Phase
FULL PAPER
experimental spectra, labeled 1
and 2, which were recorded
with the UV probe laser cen-
tred on the corresponding vi-
bronic bands, although having
several features in common are
not the same. A careful analysis
of the two IRID spectra and
comparisons with the predic-
tions of the DFT and ab initio
calculations shown in the lower
half of Figure 2, lead to their
association with one or more of
the three lowest energy con-
formers of the glucosamine.
The global minimum confor-
mation, ccG+gꢀ, is predicted
to lie at an energy 2.0 kJmolꢀ1
below that of the ccGꢀg+ con-
former and 3.2 kJmolꢀ1 below
the ccTg+ conformer.2 Their
free energies calculated at
298 K follow the same rank
Figure 2. Top: IR ion dip spectra of bpGlcNAc recorded with the UV laser centred on the vibronic bands la-
beled 1 and 2 in Figure 1. Bottom: computed vibrational spectra and structures of the four lowest energy con-
formers. Their relative energies in kJmolꢀ1, at 0 K, and free energies, at 298 K, are shown in brackets.
order. In all three of these conformers, a strong hydrogen
ꢀ
bond to the acetamido group, OH3!O=C NH with an
ꢀ
OH3 O distance ꢁ1.75 ꢄ, intensifies and broadens the OH
vibrational band s3 and displaces it by some 300 cmꢀ1 to-
wards lower wavenumber, at ꢁ3300 cmꢀ1. It also “rotates”
the acetamido group towards the pseudo plane of the pyra-
nosyl ring and the dihedral angle, H2-C2-N-H is ꢁ768 in all
three conformers. The fourth lowest-lying conformer, with a
relative energy of 5.6 kJmolꢀ1 also has a ccG+gꢀ confor-
ꢀ
mation but since there is no OH3!O=C NH hydrogen
bond the acetamido group can adopt an out-of-plane “per-
pendicular-NH-up” orientation and the H2-C2-N-H angle
moves to ꢁꢀ168. The computed shift of the NH vibration
to higher wavenumber also reflects the loss of the weak
NH!O1 hydrogen bonding present in the lower energy
conformers.
The IRID spectrum 1 is in reasonable accord with the vi-
brational spectrum predicted for the global minimum con-
former but the assignment of spectrum 2 is more subtle. The
appearance of a new band at ꢁ3595 cmꢀ1 and the broaden-
ing of its nearest neighbour, centred around 3610 cmꢀ1, indi-
cate contributions from more than one conformer. Further-
more, while the band at 3450 cmꢀ1, associated with the
“free” NH mode, is unchanged there are small but measura-
ble displacements in the sharp band located at ~3640 cmꢀ1
(to lower wavenumber) and the broad band associated with
s3 (to higher wavenumber). Comparisons with the UV hole-
burn and the computed vibrational spectra favour its assign-
ment principally to a combination of the ccGꢀg+ and
ccTg+ conformers, together with a minor component asso-
ciated with the global minimum conformer, ccG+gꢀ. The
Figure 1. R2PI spectrum of bpGlcNAc (bottom trace) and UV/UV hole-
burn spectra recorded with the probe laser centred on the vibronic peaks
1 and 2 (upper traces).
The notation is similar to that employed in previous publications.[9,10]
“cc” indicates the counter-clockwise orientation of the OH groups on
the pyranosyl ring and G+gꢀ, Gꢀg+, Tg+ indicate the orientation of
the exocyclic hydroxy (g+, gꢀ) methyl (G+, Gꢀ, T) group—see
Scheme 1. Alternative orientations of the acetamido group are desig-
nated as “parallel” (dihedral angle, H2-C2-N-H ~908), “perpendicular-
NH up” (H2-C2-N-Hꢁ08), or “perpendicular-NH down” (H2-C2-N-H
ꢁ1808).
2
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J. P. Simons, B. G. Davis et al.
“new” band appearing at ꢁ3595 cmꢀ1 can then be assigned
to the displaced mode s6, in the ccTg+ conformer, shifted
by ꢁ50 cmꢀ1 to lower wavenumber through the weak
lar, are not identical which indicates contributions to the
R2PI spectrum from more than one structure. Comparisons
with the computed spectra are in good qualitative agree-
ment with an assignment in which each of the two IRID
spectra includes a contribution, in comparable but slightly
different proportions, from the two lowest lying structures.
Each one retains the ccG+gꢀ conformation of the mono-
saccharide; in the global minimum, the pre-existing OH3!
O=C bond remains intact with the water molecule bound in
an addition complex, to the acetamido group through the
ꢀ
OH6!OH4 interaction, OH6 O4 ꢁ2.07 ꢄ.
Phenyl b-d-glucopyranoside, bpGlc, displays a similar
conformational landscape, presenting the same rank order
for its three lowest energy conformers and the same counter
clockwise “cc” orientation of the hydroxyl groups, OH4,
OH3 (and OH2),[10] promoted by the weak interaction,
OH2!O1, OH2-O1ꢁ2.2 ꢄ. In bpGlcNAc, where OH2 is
replaced by an acetamido group, the orientation is imposed
by the much stronger OH3!O=C hydrogen bond. This is
also reflected in the position of the band s4, which lies at
~3630 cmꢀ1 in the three lowest energy conformers of
bpGlc[10] but is shifted to ~3610 cmꢀ1 in the corresponding
conformers of bGlcNAc, reflecting the enhanced co-opera-
ꢀ
hydrogen bond sequence OH3!O=C NH!OHW. The
acetamido group re-orients into the pseudo plane of the pyr-
anosyl ring and the dihedral angle, H2-C2-N-H is ꢁ1208. In
the other structure, which lies very close in energy, the
water molecule inserts into the OH3!O=C bond and it is
ꢀ
bound through the sequence OH3!OHW!O=C NH; the
ꢀ
tivity in the OH4!OH3!O=C NH chain.
acetamido group now rotates out of the plane into a “per-
pendicular-NH down” orientation with an angle H2-C2-N-H
ꢁ1728. Assignment of both structures to a singly hydrated
complex is confirmed by the appearance of only one sharp
feature at ꢁ3720 cmꢀ1, which is associated in the addition
complex, with the antisymmetric OHW vibration, sW(a), and
in the insertion complex with the “free” OHW vibration, sWf.
The hydrated complexes, bpGlcNAc·(H2O)n=1–3: When the
AHCTUNGTRENNUNG
Ar gas stream was seeded with water vapour, the time of
flight mass spectrum of photo-ionised bpGlcNAc displayed
a series of peaks corresponding to singly, and multiply hy-
drated complexes. Their R2PI spectra shown in Figure 3 are
all broadened, though there is a hint of residual structure,
and each one is shifted by ꢁ200 cmꢀ1 to lower wavenumber
compared to the bare carbohydrate.
Figure 3. R2PI spectra of singly, doubly and triply hydrated complexes of
+
Figure 4. IR ion dip spectra recorded in the bpGlcNAc·ACTHNUTRGNEUNG
(H2O)+ ion chan-
bpGlcNAc recorded in the corresponding bpGlcNAc·
N
nel with the UV probe laser tuned to 36,781 cmꢀ1 (top) and to
36,809 cmꢀ1 (bottom), and computed vibrational spectra, structures and
channels; the corresponding spectrum of “bare” bpGlcNAc is also shown
for comparison.
relative energies (kJmolꢀ1) of the two lowest energy bpGlcNAc·
ACHTUNGTRENNUNG(H2O)
complexes.
The mono-hydrate, bpGlcNAc·
ed in the bpGlcNAc·
ACHTUNGTRENNUNG
G
The di-hydrate, bpGlcNAc·ACHTGUNETRN(NUG H2O)2: Figure 5 presents the
IRID spectrum recorded in the bpGlcNAc·ACTHNGUTERNNU(G H2O)2 in chan-
nel, with the UV probe laser tuned near to the peak of the
R2PI absorption (like the spectra shown in Figure 4, detun-
ing the probe toward lower wavenumber, again led to a
slight change in the spectra profile). Comparisons with the
probe laser tuned to different positions near (36809 cmꢀ1),
and slightly below (36781 cmꢀ1) the peak of the R2PI spec-
trum, are shown in Figure 4, together with the computed vi-
+
brational spectra of the two lowest energy bpGlcNAc·
structures. The two experimental IRID spectra, while simi-
ACHTUNGRTEN(NUNG H2O)
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Hydration of Sugars in the Gas Phase
FULL PAPER
computed vibrational spectra, also shown in Figure 5, indi-
cate its association predominantly, with the global minimum
energy structure together with a minor component associat-
ed with its closest neighbour, which has the same free
energy at 298 K and a relative energy ꢁ1 kJmolꢀ1 (at 0 K).
Each one retains the preferred ccG+gꢀ conformation of
the bare monosaccharide and accommodates a bound water
dimer (and not two separate water molecules) inserted be-
tween the acetamido and the OH3 groups, breaking the
The tri-hydrate, bpGlcNAc·ACHTNGUTERN(NUG H2O)3: The IRID spectra re-
corded at the two UV probe frequencies in the trihydrated
ion channel are shown in Figure 6, together with the com-
puted vibrational spectra of its two lowest-lying structures
which have almost identical relative energies but lie
ꢂ3.6 kJmolꢀ1 below their nearest neighbours. As with the
other hydrated complexes, the two experimental IRID spec-
tra differ slightly; note particularly, the change in the rela-
tive intensity of the band peaking at ꢁ3440 cmꢀ1, associated
with (a) strongly hydrogen bonded OH group(s). Compari-
sons with the two computed spectra reveal a composite
spectrum reflecting comparable populations of each of the
two low energy structures but there is no correspondence
with the vibrational spectra associated with the higher
energy structures (shown in the Supporting Information).
In the global minimum structure one water molecule is in-
serted between OH3 and the acetamido C=O group located
above the plane of the pyranosyl ring, while the other two
are bound as a dimer inserted between OH3 and the acet-
amido NH group located below the plane, to create the
ꢀ
OH3!O=C NH bond to create extended co-operative
ꢀ
loops, OH4!OH3!OHW1!OHW2!O=C NH. The water
dimer lies above the pyranosyl ring in the global minimum
structure, where the acetamido plane adopts a “perpendicu-
lar-NH down” configuration with a dihedral angle H2-C2-N-
Hꢁ1628 (Figure 5b). In the neighbouring structure the
plane rotates through ꢁ1808 into a “perpendicular-NH up”
orientation with a dihedral angle ꢁ18, and the bound water
dimer lies below the pyranosyl ring (see Figure 5c for com-
parison). In both cases, the location of the bound water mol-
ecules is dictated by the location of the carbonyl group. The
corresponding “perpendicular-NH up” conformer of bare
bpGlcNAc (H2-C2-N-H ꢁ168) lies well above the global
minimum with a calculated relative energy of 5.6 kJmolꢀ1,
see Figure 2, and it is not populated in the molecular beam.
ꢀ
cyclic hydrogen-bonded sequence: OH3!OHW1!O=C
NH!OHW2!OHW3!OH3 (Figure 6b). The alternative
structure depicted in Figure 6c accommodates a branched
water trimer located below the pyranosyl plane between
OH4 and OH6. It incorporates
a doubly hydrogen bonded cen-
tral water molecule connected
to the C=O group, to create the
sequence:
Like the dihydrate, the hy-
droxymethyl group continues to
retain its G+gꢀ conformation
and the acetamido plane is ori-
ented roughly perpendicular to
the plane of the pyranosyl ring,
either “NH-down” in the global
minimum, or “NH-up” in the
alternative structure, with H2-
C2-N-H dihedral angles of 178
or 118.
bpGlc·ACHTGNURTNEUN(G H2O)n=2: The original
investigations of phenyl b-d-
glucopyranoside (bpGlc) were
limited to its singly hydrated
complex.[10] Two structures were
detected: in the more strongly
populated one, associated with
the global minimum structure,
the water molecule was bound
+
Figure 5. a) IR ion dip spectrum recorded in the bpGlcNAc·
(H2O)2 ion channel with the UV probe laser
tuned to 36,809 cmꢀ1 and the computed vibrational spectra, structures, relative energies and free energies
(kJmolꢀ1) of the four lowest energy bpGlcNAc·
(H2O)2 complexes. (b,c) “side-on” views of the two, lowest
energy populated structures, “NH-down” (b) and “NH-up” (c).
AHCTUNGTRENNUNG
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J. P. Simons, B. G. Davis et al.
to create the extended se-
quence, OH2!OH3!OH4!
OHW1!OH6!OHW2!O5, in
conformity with the general
“propensity rules” identified
earlier for singly and multiply
hydrated monosaccharides. The
alternative structures, each of
which would accommodate a
bound dimer rather than two
separate water molecules, are
energetically less favoured and
are unpopulated.
Although it was possible to
detect R2PI signals in the
+
bpGlc·
G
ion channel
they were too weak unfortu-
nately, to enable an IRID spec-
trum to be recorded at an ade-
quate signal/noise level.
Discussion
Although the intrinsic confor-
mational landscapes of bpGlc
and its N-acetamido derivative,
bpGlcNAc, are quite similar,
the enhanced hydrogen bonding
to the acetamido group, OH3!
+
Figure 6. a) IR ion dip spectra recorded in the bpGlcNAc·
1) to 36,781 cmꢀ1 and 2) to 36,809 cmꢀ1 and the computed vibrational spectra, structures, relative energies and
free energies (kJmolꢀ1) of the two lowest energy bpGlcNAc·
(H2O)3 complexes. b,c) “side-on” views of the
ACHTUGNTRNEN(UGN H2O)3 ion channel with the UV probe laser tuned,
ꢀ
O=C NH, dominates and an-
chors the orientation of the pyr-
anosyl OH groups, locking
AHCTUNGTRENNUNG
two, lowest energy populated structures, “NH-down” (b) and “NH-up” (c).
them into a counter-clockwise
orientation around the ring.
More importantly, it also pro-
to the hydroxymethyl group, inserted between OH4 and
OH6. This promoted a conformational switch from ccG+
gꢀ to cGꢀg+, to create an extended, counter-clockwise
(OH-O)n chain. In the less favoured, alternative structure
the closest agreement with experiment indicated retention
of the ccG+gꢀ conformation with the bound water mole-
cule attached to the hydroxymethyl group, but this time in-
serted on the opposite side, between OH6 and the ring
oxygen, O5.[10]
vides a focus for the location of bound water molecules. In
hexoses such as glucose, galactose and mannose, hydration
regioselectivity is controlled primarily by the flexibility of
the exocyclic hydroxymethyl group, and secondarily by the
orientation (equatorial or axial) of the pyranosyl hydroxyl
groups.[9] Water molecules bound to phenyl a/b-d-glucosides
(a/bpGlc) and -mannosides (a/bpMan) inserted preferential-
ly into the attractive “pocket” between OH4 and OH6,
which either pre-exists (in apMan, cGꢀg+) or is newly cre-
ated through re-orientation of the hydroxymethyl group and
a consequent conformational switch (to cGꢀg+ in bpMan
and a/bpGlc).[9,24] When this site is filled the second water
The IRID spectrum of the dihydrate, bpGlc·ACTHNUTRGNE(UNG H2O)2, is
shown in Figure 7a, together with the computed vibrational
spectra of its lowest energy structures. The spectrum associ-
ated with the global minimum is in good correspondence
with observation, but the others are not able to reproduce
the experimental spectrum; since the relative energy (and
free energy) of the global minimum structure lies well below
that of its nearest neighbour, its assignment to the global
minimum structure is clear. In order to best accommodate
the bound water molecules, now located on each side of the
(re-oriented) hydroxymethyl group, the monosaccharide
conformation is again reshaped, from ccG+gꢀ to cGꢀg+,
molecule, in bpGlc·ACTHNUTRGENN(UG H2O)2 or bpMan·AHCTUNGTRNE(NUNG H2O)2 for example,
occupies the vacant neighbouring site between OH6 and the
ring oxygen, O5. When OH4 is axial rather than equatorial,
as in a- or b-galactosides, enhanced hydrogen bonded inter-
actions between OH4 and its nearest neighbour, either OH3
or OH6, prevent insertion of a water molecule into the (4,6)
site and instead, the neighbouring (6, O5) site becomes the
first port of call for an (in)bound water molecule.[9,24]
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Hydration of Sugars in the Gas Phase
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When neither an acetamido
nor a hydroxymethyl group is
present, for example, in xylose
or fucose, regioselectivity can
still be provided by the pre-ex-
isting counter-clockwise (OH-
O)n chain. Bound water mole-
cules located further round the
pyranose ring select peripheral
sites associated with the weak-
est link in the chain,[9] between
OH3 and OH2 in the a-anom-
ers and OH2 and O1 in the b-
anomers. Thus, in general the
degree of control exerted by
the groups around the pyrano-
syl ring lies in the order acet-
amido
>
hydroxymethyl
>
pyranosyl (OH-O)n chain. The
striking and unprecedented role
of the C-2 acetamido group in
controlling the hydrated struc-
tures determined here may, in
part, explain the differing and
widespread roles of GlcNAc
and perhaps GalNAc, in nature.
+
Figure 7. a) IR ion dip spectra of hydrated bpGlc recorded in the bpGlc·ACTHNUTRGNE(UNG H2O)2 ion channel and the comput-
ed vibrational spectra, structures, relative energies and free energies (kJmolꢀ1) of the four lowest energy
bpGlc·ACHTUNGTRENNUNG(H2O)2 complexes. b) Side-on view of the lowest energy, populated structure.
Experimental Section
Spectroscopy: A detailed description
of the molecular beam experimental
strategy has been published previously.[9] The carbohydrate samples were
mixed with graphite powder, vaporized through laser ablation at 1064 nm
(pulse energy ꢁ4 mJ) in front of the nozzle (0.8 mm orifice) of a pulsed
valve (Jordan Valve), and seeded into a free jet argon expansion (ꢁ3–
5 bar Ar). Hydrated complexes were formed by seeding the argon with
water vapor (>0.25%) allowing the investigation of their size-selected
structures. The free jet expansion passed through a 2 mm skimmer to
form a collimated molecular beam, crossed by one, or two tuneable laser
beams in the extraction region of a linear time-of-flight mass spectrome-
ter (Jordan). Mass-selected resonant two photon ionization (R2PI) spec-
tra were recorded using the frequency-doubled output of a pulsed Nd/
YAG-pumped dye laser (Continuum Surelite III/Sirah PS-G, 1.5 mJ per
pulse UV). Conformer-specific spectra were obtained through UV hole-
burning (UV-HB) and IR ion dip experiments, employing UV/UV and
IR/UV double resonance spectroscopy. The UV-HB experiments em-
ployed the frequency-doubled output of an excimer-pumped dye laser
(Lambda-Physik EMG 201/Lambda-Physik FL3002, 0.5 mJ UV). IR ex-
periments employed radiation in the range 3000–3800 cmꢀ1, generated by
difference frequency mixing of the fundamental of a Nd/YAG laser with
the output of a dye laser in a LiNbO3 crystal (Continuum Powerlite 8010/
ND6000/IRP module) or by a tunable OPO/OPA laser system (LaserVi-
sion); all laser pulses were ꢁ10 ns duration. The delay between the
pump and the probe laser pulses was ꢁ150 ns in both the IR ion dip and
UV-HB experiments.
Conclusion
In the 2-deoxyamino sugar, bpGlcNAc, bound water mole-
cules are marshalled into place by the acetamido group
which, through the orientation of the C=O and NH, now
provides the dominant control. Indeed, so great is its pro-
pensity to act as a hydration focus that in bpGlcNAc·ACTHNUTRGNE(UNG H2O)3
all three water molecules “loop” the [NHAc-2,OH-3] motif,
leaving the remaining motifs “dry”. It can act both as a hy-
drogen bond donor and an acceptor and like the hydroxy-
methyl group in bpGlc or bpMan, it can fine-tune the pre-
ferred choice of binding site by rotating into alternative con-
figurations: “in-plane” (H2-C2-N-H ꢁ908), “perpendicular-
NH up” (H2-C2-N-H ꢁ08) or “perpendicular-NH down”
(H2-C2-N-H ꢁ1808). These properties generate hydrate
structures where the bound water molecules are located
either above, or below, or above and below the pyranosyl
ring, see Figures 5 and 6. Similarly, in doubly hydrated
bpGlc, the bound water molecules are also located above
the ring rather than around its edge, directed by the “per-
pendicular” Gꢀg+ orientation of the hydroxymethyl group.
Thus, despite the equatorial orientation of all hydrophilic
groups in both bpGlc and bpGlcNAc, it is in fact, the
“axial” a and b faces of the carbohydrates that are the even-
tual sites of hydration.
Computation and assignment: Conformational and structural assignments
of the experimental IRID spectra were performed through comparison
with calculations using a combination of molecular mechanics, ab initio
and density functional theory (DFT) methods. Initial structures were gen-
erated by an extensive molecular mechanics conformational search using
the Monte Carlo multiple minimization procedure as implemented in the
Chem. Eur. J. 2009, 15, 13427 – 13434
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
13433

J. P. Simons, B. G. Davis et al.
MacroModel software (MacroModel v.8.5, Schrçdinger, LLC21).[25] Rele-
vant structures, filtered on the basis of their relative energies, information
gained from previous studies on carbohydrates and their experimental vi-
brational signatures, were selected for geometry optimizations using the
HF/6-31+G* method, as implemented in the Gaussian 03 program pack-
age.[26] The most stable conformers were re-optimized at the B3LYP/6-
31+G* level for the unhydrated sugars and 6-311+G* for the hydrated
ones. Their relative energies were subsequently calculated more accurate-
ly, at the MP2/6-311++G** level of theory, to take proper account of
dispersion interactions. Zero-point and free energy corrections were per-
formed using the harmonic frequency calculations performed on the
B3LYP structures; for comparison with the experiments, the frequencies
predicted for the O-H and N-H modes were scaled by factors of 0.9734
and 0.96, respectively.
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Acknowledgements
Financial support provided by the Engineering and Physical Sciences Re-
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Received: July 2, 2009
Revised: August 10, 2009
Published online: November 9, 2009
13434
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ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 13427 – 13434