'Naked' and hydrated conformers of the conserved core pentasaccharide of N-linked glycoproteins and its building blocks

Article abstract of DOI:10.1021/ja4056678

N-glycosylation of eukaryotic proteins is widespread and vital to survival. The pentasaccharide unit -Man₃GlcNAc₂- lies at the protein-junction core of all oligosaccharides attached to asparagine side chains during this process. Although its absolute conservation implies an indispensable role, associated perhaps with its structure, its unbiased conformation and the potential modulating role of solvation are unknown; both have now been explored through a combination of synthesis, laser spectroscopy, and computation. The proximal -GlcNAc-GlcNAc- unit acts as a rigid rod, while the central, and unusual, -Man-β-1,4-GlcNAc- linkage is more flexible and is modulated by the distal Man-α-1,3- and Man-α-1,6- branching units. Solvation stiffens the 'rod' but leaves the distal residues flexible, through a β-Man pivot, ensuring anchored projection from the protein shell while allowing flexible interaction of the distal portion of N-glycosylation with bulk water and biomolecular assemblies.

Full text of DOI:10.1021/ja4056678

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‘Naked’ and Hydrated Conformers of the Conserved Core
Pentasaccharide of N‑linked Glycoproteins and Its Building Blocks
Conor S. Barry,†,# Emilio J. Cocinero,‡,⊥,# Pierre C
̧arca̧
bal,§,# David P. Gamblin,†,#
E. Cristina Stanca-Kaposta,‡,∇ Sarah M. Remmert,‡ María C. Fernandez-Alonso,∥ Svemir Rudic,†,‡
́
́
John P. Simons,*,‡ and Benjamin G. Davis*,†
†Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, United
Kingdom
‡Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1
3QZ United Kingdom
§Institut des Sciences Molec
∥Centro de Investigaciones Biolog
́
ulaire d’Orsay-CNRS, Universite
́ ̂
Paris Sud, Batiment 210, 91405 Orsay Cedex, France
́
icas, Calle Ramiro de Maeztu, 9, 28040, Madrid, Spain
S
* Supporting Information
ABSTRACT: N-glycosylation of eukaryotic proteins is wide-
spread and vital to survival. The pentasaccharide unit
−Man3GlcNAc2− lies at the protein-junction core of all
oligosaccharides attached to asparagine side chains during this
process. Although its absolute conservation implies an indis-
pensable role, associated perhaps with its structure, its unbiased
conformation and the potential modulating role of solvation are
unknown; both have now been explored through a combination of
synthesis, laser spectroscopy, and computation. The proximal
−GlcNAc-GlcNAc− unit acts as a rigid rod, while the central, and unusual, −Man-β-1,4-GlcNAc− linkage is more flexible and is
modulated by the distal Man-α-1,3− and Man-α-1,6− branching units. Solvation stiffens the ‘rod’ but leaves the distal residues
flexible, through a β-Man pivot, ensuring anchored projection from the protein shell while allowing flexible interaction of the
distal portion of N-glycosylation with bulk water and biomolecular assemblies.
have exposed the influence of the proximal chitobiose
(−GlcNAc-GlcNAc−) stem on the local peptide conformation
and suggested an explanation of nature’s choice of N-acetyl-D-
glucosamine (GlcNAc) over the more plentiful D-glucose. Does
the chosen chitobiose stem play other structural roles? Does
the notably rare β-mannoside (Man) central linkage between
this stem and the trimannoside (Man3) unit impart any unique
structural or functional benefits and do these units (and other
building blocks) function independently or in concert? Similar
NMR and molecular dynamic investigations of the naturally
occurring Man9GlcNAc2 N-glycan in aqueous solution have
suggested enduring structural features in the core pentasac-
charide, incorporating locally bound water molecules.12,13 Does
the core pentasaccharide present any favored water pockets and
are its shape and stiffness influenced by solvent-mediated
interactions?
INTRODUCTION
■
N-glycosylation of proteins is a highly conserved process in all
eukaryotes whereby complex oligosaccharides (glycans) are
cotranslationally appended to the side-chain asparagine γ-
nitrogen of the consensus motif NXS/T (Figure 1).1,2 N-
Glycans display a rich structural diversity3 and are vital for both
the correct folding of nascent glycoproteins, where interactions
with the chaperone lectins calnexin and calreticulin are key,4
and also for the correct functioning of the mature
glycoproteins, where glycans play crucial roles in such processes
as protein targeting, intercellular signaling, and host−pathogen
recognition.5,6 Common to these diverse roles and the many
structural possibilities for N-glycans is the conserved presence
of an invariant core pentasaccharide (Man3GlcNAc2−) (Figure
1a). This presence in all mammalian N-glycoproteins
(regardless of protein location or function) and the highly
conserved nature of N-glycosylation among eukaryotes suggests
an indispensable (and perhaps general) role for the core
pentasaccharide in facilitating the proper functioning of N-
glycans.4,7,8 Despite its ubiquity, however, the underlying
physical reasons for its conservation are not yet fully
understood.9
One approach to these various questions is to explore the
structural mechanics of the individual building blocks, in
isolation and then linked together, using a combination of
synthetic oligosaccharide assembly, mass- and conformer-
selected infrared laser spectroscopy under molecular beam
NMR measurements of model glycopeptides in aqueous
Received: June 6, 2013
Published: October 15, 2013
solution, coupled with molecular dynamics investigations,10,11
© 2013 American Chemical Society
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Figure 1. The pivotal role of the core pentasaccharide in N-glycoproteins. (a) The conserved core pentasaccharide motif, (Man3GlcNAc2 shown in
gray box), is attached to asparagine residues of glycoproteins (N, S, and T denote asparagine, serine, and threonine, X denotes any amino acid but
not proline). Proteins are cotranslationally modified with a tetradecasaccharide which is tailored by glycosyl-hydrolase and transferase enzymes to
create diverse glycans with varying antennae but all based upon the conserved core pentasaccharide (gray box). (b) The structures and symbol
representations of the core pentasaccharide 4 and the building blocks 1−3 from key regions of 4 used in this study. The naming convention A−E is
used in this manuscript to identify the individual glycosyl residues. The site of the benign chromophore used in this study mimics the location of the
protein scaffold or truncated glycan (shown by a red star).
conditions, molecular mechanics, and quantum chemical
calculations. This allows determination of their preferred
(inherent) conformational structures when isolated in the gas
phase to provide basic structural benchmarks; then inter-
rogation of the locations and structural consequences of their
microhydration; and finally, their response to a bulk, aqueous
environment.
METHODS
■
Experimental Section. Full details of all synthetic procedures and
characterization are provided in the Supporting Information (SI). The
carbohydrates were vaporized by laser desorption into an expanding
supersonic jet of argon before passing through a 2 mm skimmer to
create a collimated molecular beam which then intersected tunable UV
and IR laser beams in the extraction region of a linear time-of-flight
mass spectrometer (Jordan). Conformer-specific, mass-selected spectra
were recorded in the UV and IR using UV−UV and IR−UV ion dip
(IRID) double resonance spectroscopy.14
Early stages of this approach have previously been applied to
the NXS sequon14 and the trimannoside unit.15 We now report
the culmination of this strategy, applied here to dissect the
unbiased structure of the entire core pentasaccharide and then
to understand the effects of hydration upon it. Target
oligosaccharide building blocks (1−4, Figure 1b) were designed
in a form that provided a chromophore required for detection
through mass-selected ultraviolet (UV) photoionization.16
Syntheses of the chitobiose (GlcNAc-β-1,4-GlcNAc) stem 1,
the connecting disaccharide (Man-β-1,4-GlcNAc) 2, the
extended trisaccharide (Man-β-1,4-GlcNAc-β-1,4-GlcNAc) 3,
and finally the complete core pentasaccharide (Man3GlcNAc2)
4, all as their phenyl (Ph) glycosides, were achieved on the
gram-scales required for their gas-phase interrogation. Their
conformational preferences have been determined in the gas
phase using infrared ion depletion (IRID) laser spectroscopy16
coupled with molecular mechanics, density functional and ab
initio theoretical calculations. Those of their discretely hydrated
(and ‘blocked’) complexes were determined through theoretical
calculation. Comparisons with their preferred conformations in
aqueous solution, reported for chitobiose17 and the trimanno-
side (Man3)18−20 and computed here for the core pentasac-
charide through molecular mechanics calculations, reveal a
unique insight into the inner workings of the core
pentasacccharide and the molecular scaffolding provided by it
and its components.
Computational. The calculations began with completely unre-
stricted and exhaustive surveys of the conformational landscapes of
each of the carbohydrate ‘building blocks’ and their singly hydrated
complexes, using a molecular mechanics method (MMFFs force
field).21 Structures with relative energies <15 kJ mol−1, together with
additional representative structures that might have a significant
population in the cooled adiabatic expansion (see SI), were
reoptimized through density functional theory calculations (B3LYP/
6-311+G*) using the Gaussian 09 program package22 (and two
supercomputers, i2BASQUE and SGIker, employing up to 96
processors per calculation) to provide a more accurate energy ranking
of the lowest-energy structures and their associated harmonic
vibrational spectra. Zero-point corrected relative energies were
computed through subsequent single point ab initio calculations
(MP2/6-311++G**), and final optimizations were based upon
comparisons with the experimental spectra themselves, to provide
feedback and guide the ‘fine-tuning’ of the predicted structures.
The core pentasaccharide conformational structure was also
explored exhaustively, using the molecular mechanics OPLS200523
and GLYCAM06/AMBER24 force fields. (OPLS2005 possessed the
fewest low-quality stretch, bend, and torsional parameters when
benchmarked for the carbohydrate segments against all other native
force fields found in the Macromodel software; GLYCAM06/AMBER
(not included in Macromodel) is referenced specifically, for
carbohydrate structures). All stereogenic centers were preserved in
the computational search, and the ring-opening method of Still25 was
used to explore additional ring conformations (full details of all
calculations are given as SI).
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Figure 2. Schematic of synthetic strategy toward the core pentasaccharide and its building blocks. Synthesis of the core pentasaccharide 4 and its
building blocks 1−3 shown using symbol representations. The chromophore used in this study, phenyl, is shown by a red star. For full synthetic
details and structures see Figure S1 for the steps describing the conversion of 5 to 1−3 and intermediate 9; Figure S2 for conversion of 18, 19, and 9
onward to 4. All glycosylations were accomplished with >98% stereoselectivity for α- (αG) or β- (βG) glycosidic linkages through the use of
participatory C-2 ester substituents (Ac or Lev).
levulinate ester may be selectively deprotected. Subsequent
stereospecific inversion of the configuration at C-2 (I in Figure
2) would then allow conversion of this unnatural residue from
the β-D-gluco to the natural β-D-manno configuration. We
chose to accomplish this critical inversion through SN2
displacement at a late stage of our syntheses, envisaging that,
despite the strategic risk, this might afford more rigid
intermediates that, by virtue of their reduced conformational
flexibility, would be less prone to unwanted decomposition
(e.g., elimination) pathways.
Synthesis of the Proximal (GlcNAc2 1), Central (Man-
GlcNAc 2), and Extended Stem (Man-GlcNAc-GlcNAc 3)
Building Blocks. A suitably chromophoric ‘stem’ unit of the
core pentasaccharide was prepared as the phenyl β-chitobioside
1 (Figures 2 and S1) from parent monosaccharide sugar D-
glucosamine 5. A common thioglycoside divergent precursor
641 was prepared with participatory phthalimide N-2 protection
in six steps from D-glucosamine. 6 was divergently elaborated to
both donor 742 and acceptor 843 (through OH-4-regioselective
reductive benzylidene ring opening); use of a trichloroacetimi-
date donor group in 7 allowed its activation by TMSOTf
without disruption (or aglycon exchange) of the S-ethyl (−SEt)
group in acceptor 8 (Figures 2 and S1) to assemble 9. The
phenyl chromophore was then introduced using phenol
through activation of the −SEt group in 9 with N-
iodosuccinimide (NIS) and TMSOTf; global deprotection of
resulting phenyl β-glycoside 10, afforded the free, chromo-
phore-tagged stem (GlcNAc)2 disaccharide unit 1.
RESULTS AND DISCUSSION
■
Design of the Glycan Targets. The target core
pentasaccharide 4 and its building blocks 1−3 (Figure 1b)
were synthesized as phenyl β-glycosides, to provide a UV
chromophore,16 installed at the structurally benign reducing
terminus. The target sugars were all required in gram-scale
quantities to provide sufficient material for spectroscopic
investigation in the gas phase. While such scales have been
accomplished for certain smaller mono- and disaccharides,
large-scale synthesis is rare on larger glycans, such as the core
pentasaccharide, and required the development of efficient
synthetic routes. This dictated a global approach to all targets
that would allow flexible reuse of key building blocks and
intermediates in more than one target (Figure 2).
An additional key synthetic challenge was the formation of
the central β-D-mannoside linkage in 2−4, notoriously difficult
in carbohydrate chemistry.26−29 Several elegant methods have
been reported for the direct stereoselective synthesis of β-D-
mannosides,30−38 however selectivities can sometimes be low,
giving unwanted α-mannoside side products32 that are difficult
to remove through purification. Notably, <90% β-selectivity is
the best reported to date for direct β-mannosylation of the OH-
4 position of a GlcNAc precursor that would correspond to the
synthesis of the core pentasaccharide.34,39,40 Since this would
place severe limitations on the effective construction of grams
of the chromophore-equipped targets, an alternative strategy,
with potential for greater selectivity for the β-mannoside
linkage, was chosen. This took advantage of neighboring group
participation of a levulinate ester stereodirecting group (Lev) at
the C-2 position of an unnatural glucoside residue to allow the
formation of a corresponding β-glucoside linkage. The
The central disaccharide Man-β-1,4-GlcNAc unit 2 contains
the unusual and challenging β-D-mannoside linkage. An
acceptor 12 already containing the phenyl chromophore was
prepared from 6 (using phenol and NIS/TMSOTf-mediated
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Figure 3. The IR ion depletion (IRID) spectra, computed vibrational spectra and structures of 1, 2, and 3. σ1,2, ... and σNH indicate the OH and NH
vibrational mode assignments; relative energies (at 0 K) and free energies (at 298 K), kJ mol−1, are shown in brackets. Structural assignments are
based first on the correspondence between the experimental (IRID) and computed vibrational spectra and second on their relative energies, favoring
the most stable conformer(s). (a) The computed vibrational spectrum of the trans conformation of 1 predicted as the global minimum,
corresponded best with the experimental IRID spectrum associated with the major conformer (1-trans) while the lowest-lying cis conformation
corresponded to the IRID signature of the minor conformer (1-cis). (b) The IRID and computed vibrational spectra associated with the three
lowest-energy conformations of ManC-β1,4-GlcNAcB 2; the two lowest (which differ only in the orientation of the hydroxymethyl group on the
mannopyranoside ring) both display 2-cis conformations. The commonly observed B-ring acetamido → OH-3 interaction in the two units 1 and 2
lends further support to the ‘building block’ approach adopted here. (c) The calculated spectrum associated with the lowest energy, cis−cis
conformer of 3 is in good qualitative agreement with the IRID spectrum of the trisaccharide 3, ManC-β1,4-GlcNAcB-β1,4-GlcNAcA.
activation followed by regioselective reductive benzylidene ring
opening to reveal OH-4). 12 was glycosylated using the thio-D-
glucoside donor 11 containing a C-2 Lev stereodirecting group
(Figures 2 and S1); activation (NIS/TMSOTf) gave
disaccharide 13 with excellent (>98%) β-D-stereoselectivity.
The crucial C-2 epimerization (vide supra) from β-D-gluco- to
β-D-manno-configuration (13 → 14, Figure S1) was accom-
plished in 80% yield over three steps (selective Lev cleavage,
triflate formation, and ultrasound-assisted SN2 displacement
with acetate).44−47 The necessity for ultrasound treatment to
promote triflate displacement was consistent with the expected
stability afforded by the conformational restriction of the
glucoside 13 by its 4,6-O-benzylidene acetal; this stability also
ensured minimal side reaction through competing elimination.
Global deprotection of 14 yielded chromophore-tagged 2 in
70% yield over four steps.
Utilizing the intended divergency of common synthetic
intermediates (Figure 2), a strategically similar synthetic route
gave 3; this reused intermediates used for 1 and 2. Thus, Lev-
bearing donor 11 (used for 2) was again used for the ultimate
installation of the challenging β-D-mannoside. 11 was combined
with (GlcNAc)2-disaccharide 10 (used for 1) in which the
phenyl chromophore was already present; 10 was converted to
a suitable acceptor 15 through regioselective 4,6-O-benzylidene
opening to reveal OH-4B (Figure S1). 11 and 15 gave
trisaccharide 16 containing the desired β(1,4)-linkage (>98%
stereoselectivity). Creation of the desired β-D-mannoside
linkage (16 → 17) successfully employed the same inversion-
epimerization strategy used for 2; global deprotection gave 3 in
a very good yield of 55% over 7 steps.
Synthesis of the Chromophore-Tagged Core Penta-
saccharide 4. Consistent with our strategy, the most
challenging target, the core pentasaccharide 4, was created
(Figures 2 and S2) using previously generated intermediate
(GlcNAc)2 S-ethyl disaccharide 9. This (GlcNAc)2 disaccharide
was elaborated to a suitable acceptor 20, again through
regioselective protecting group manipulation to reveal OH-4′
and coupled in a convergent manner with disaccharide donor
22. 22 itself was assembled from D-manno trichloroacetimidate
1848 and the thioglucoside diol 19.49 Although 19 contains two
potential sites for reaction (OH-2 and OH-3), TMSOTf-
activated glycosylation proceeded with both excellent regio-
(OH-3 over OH-2) and stereo- (>98% α-manno) selectivities
to give 2150 in 76% yield. Advantageously, this left free the OH-
2; direct installation of the stereodirecting Lev group onto OH-
2 gave 22.
Unusually, our intended disaccharide 20 + disaccharide 22 [2
+ 2] glycosylation utilized a donor and an acceptor both
containing the same −SEt anomeric substituent; ordinarily this
would give rise to unwanted coincident activation during
glycosylation. However, by using an iterative glycosylation
strategy,51 thioglycoside 22 was successfully preactivated first
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(with triflic anhydride and diphenylsulfoxide in the presence of
stabilizing base DTBMP, allowing its successful use as a donor).
Subsequent addition of 20 then led to its reaction only as an
acceptor nucleophile (without −SEt activation) and gave
tetrasaccharide 23, in good yield and with excellent stereo-
selectivity for the critical central β-linkage (>98%). This
therefore directly provided 23 bearing a subsequently useful
−SEt group, thereby avoiding functional group manipulations
at this reducing terminus.
Before installation of the final α(1,6)-D-mannosyl unit of the
core pentasaccharide, we performed the critical inversion of the
central unit from β-D-gluco- to β-D-manno-configuration (23 →
24) (Figures 2 and S2) as for blocks 2 and 3 (chemoselective
Lev deprotection, triflate formation, and ultrasound-mediated
SN2 displacement with acetate), again in excellent yields. Next,
benzylidene removal revealed OH-4 and OH-6 in 25.
Regioselective OH-6 glycosylation with mannosyl donor 18
(used for 22) gave pentasaccharide 26; here the use of a
compatible trichloroacetimidate donor again led to retention of
the useful −SEt group at the reducing terminus. Acetylation
and subsequent NIS/TMSOTf-mediated activation of the −SEt
with phenol installed the chromophore into protected, phenyl
β-pentasaccharide 27. Three high-yielding deprotection reac-
tions gave the final chromophore-tagged, core pentasaccharide
4 in gram-scale quantities.
hydrogen-bonded OH groups. Although poorly resolved, its
contour is in qualitative correspondence with the IR spectrum
associated with its minimum-energy structure. This presents cis-
oriented ManC-GlcNAcB and GlcNAcB-GlcNAcA segments,
linked together by two very strong hydrogen bonds that
connect the terminal ManC and the central GlcNAcB units, and
a third that links GlcNAcB with GlcNAcA. The next
conformation lies 12.2 kJ mol−1 higher in energy; it only
presents a single inter-residue hydrogen bond, and its calculated
vibrational spectrum does not reproduce the experimental band
at low wavenumber (Figure S11).
Hydrated and ‘Blocked’ Conformations of 1−3. The
structural information obtained from IRID spectroscopy was
developed further through computation, to explore the effect of
explicit hydration and ‘blocking’ (to cap hydroxyls that would
be absent in extended oligosaccharides). The lowest-energy
computed structures of the monohydrate 1·H2O and also 2·
H2O (Figures 4a,b, S6, and S8) now both present trans
The Gas-Phase IRID Spectra and Structures of
Building Blocks 1−3. The R2PI spectrum of 1, the chitobiose
(GlcNAcB-β1,4-GlcNAcA) stem that links the core pentasac-
charide to protein, presented two overlapping components
(shown later in Figure 5 and in Figure S3). Their associated
IRID spectra were in best accord with its two lowest-energy
conformers, termed 1-trans and 1-cis (Figure 3a), in which the
two coplanar pyranose rings are anti- or syn-periplanar,
respectively (see also Figure S5).
The glucopyranoside rings in the 1-trans conformer are
supported by two inter-ring hydrogen bonds, OH-3A → OH-
6B(O-5B), while a third, stronger OH-3B → OC(NH)B bond
pulls the B-ring acetamido group into the plane of the pyranose
ring. The A-ring acetamido group remains ‘free’ with its amide
plane presenting a near perpendicular orientation. The 1-cis
conformer (Figure 3a), which has a similar relative energy at 0
K but a higher free energy at 298 K, is supported by a strong
OH-6B → OH-6A hydrogen bond; a further inter-ring
interaction, NHB → OH-3A contributes to a cooperative
chain, OH-4B → OH-3B → OC(NH)B → OH-3A → O
C(NH)A, to provide additional stabilization. Since both
acetamido groups are now able to interact with their
neighboring OH-3A/OH-3B groups, both are rotated toward
the plane of their respective pyranose rings.
Figure 4. Hydrated and ‘blocked’ structures of the core pentasacchar-
ide building blocks. The computed lowest-energy structures of (a,b),
the monohydrates 1·H2O and 2·H2O and (c,d) the “blocked” subunits
2-B and 2-B·H2O, in which the 6-OHC group is modified to the
methyl ether, and (e) the blocked subunit 3-B. Note the effect of
blocking; in 2-B·H2O it removes the water bridge present in 2·H2O
(and 1·H2O), and in 2-B and 3-B it switches the conformation about
the ManC-β1,4C-GlcNAcB linkage from cis to trans. Gray dotted circles
indicate ‘blocked’ sites, explored here through the use of a methyl
ether capping group. Red dotted circles indicate a water binding site.
The disaccharide unit −ManC-β1,4-GlcNAcB− 2 straddles
the unusual β-mannoside linkage. Its IRID spectrum is in good
accord with a superposition of the vibrational spectra associated
with its two lowest-energy conformers (2-cis), but is quite
unlike that of the lowest-lying 2-trans conformer (9.9 kJ mol−1
higher, Figure 3b). The 2-cis conformation is supported by two
inter-ring hydrogen bonds, OH-2C → O-3B and OH-6C → O-
6B, and the B-ring acetamido group is again hydrogen bonded
to its nearest neighbor, OH-3B → OC(NH)B.
conformations, supported by the bound water molecule which
bridges across the two pyranose rings. In 1·H2O this creates an
extended cooperative chain, OH-4B→OH-3B → OC(NH)B
→ H2O → OH-6A → O-1A, which greatly strengthens the inter-
ring binding by linking NHB to O-6A and, as a consequence,
also strengthens the supporting inter-ring bond, OH-3A → OH-
6B(O-5B) (r[OH3A···O6B] 2.16 → 2.07 Å). Its relative energy
now lies 4.5 kJ mol−1 below that of the lowest-lying cis hydrate,
cf. 0.6 kJ mol−1 in the ‘bare’ unsolvated disaccharide 1 (Figure
S6). This contrasts markedly with hydrated cellobiose (Glc-
β1,4-Glc) where a cis conformation is retained.52 Explicit
hydration therefore locks the trans conformation of 1,
The IRID spectrum of the −ManC-β1,4-GlcNAcB-β1,4-
GlcNAcA− trisaccharide 3, (Figure 3c), which extends from
∼3200 to ∼3600 cm−1, displays three broad maxima below
3500 cm−1 and congested weaker features at higher wave-
numbers, indicating contributions from strong and weakly
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enhancing the rigidity of the ‘stem’. NMR studies of chitobiose
in an aqueous environment at 298 K also identify a very similar
average conformation in solution.17 The two acetamido groups
continue to adopt a trans relative disposition, consistent with
the maintenance of a rigid conformation about the inter-ring
glycosidic bond,17 reinforced perhaps by transiently bound,
bridging water molecules.53,54
The central glycosyl residue in the core pentasaccharide,
ManC, is itself glycosylated on hydroxyl groups OH-6 and OH-
3, thereby precluding donor hydrogen bonds from these
positions.15,55 The dramatic structural consequences of this are
revealed in the finding that the lowest-energy conformer of the
‘blocked’ Man (6-OMe)C-β1,4-GlcNAcB− disaccharide 2-B
(and also its hydrate, 2-B·H2O) adopts a trans orientation, in
contrast to the strongly preferred cis conformation of 2
(compare Figures 3b and 4c); the lowest-energy 2-B-cis
conformer now lies ∼5 kJ mol−1 above the global minimum.
Unlike 2-cis (Figure 3b), the trans conformer of 2-B is only
supported by a single, weak inter-ring hydrogen bond, OH-3B
→ O-5C. The trans conformation is also retained in the hydrate
(Figure 4d) where the water molecule localizes on the GlcNAcB
ring, inserted between the acetamido group and O-3 rather
than forming an inter-ring bridge, again in contrast to hydrated
chitobiose.52 The reduced inter-ring bonding increases the
flexibility about the ManC-GlcNAcB glycosidic linkage, to
provide a fluxional ‘pivot’.
Similarly, in 3-B the blocked ManC OH-6 group lacks the
hydrogen bond found in 3 that linked the ManC and GlcNAcB
units (Figure 4e). The ManC-GlcNAcB segment again switches
from cis to trans to create a more flexible local structure about
the β-ManC ‘pivot’, very similar to that of 2-B, and the
trisaccharide adopts a more extended structure, with r[O-4C···
O-1A] increasing from 11.2 to 14.5 Å.
Gas-Phase Spectra and Structures of the Core
Pentasaccharide 4. The R2PI spectrum of the core
pentasaccharide 4 (Figure 5a) is centered at the same
wavenumber as the trans conformer of 1 and 3 (also trans
about GlcNAcB-GlcNAcA), providing circumstantial evidence
for a possible preference for a trans GlcNAcB-GlcNAcA
conformation in 4 also. Since the pentasaccharide contains 14
hydroxy and 2 acetamido groups, structures that optimize
global OH···O and NH···O hydrogen bonding are to be
expected in the gas phase: not surprisingly, its IRID spectrum
(Figure 5b) presents a broad red-shifted quasi-continuum,
ranging from ∼3100 to ∼3700 cm−1. This suggests a highly
congested set of overlapping bands associated with a large
number of both strong, and weak, hydrogen bonded OH and
acetamido groups.
Figure 5. Spectra of the core pentasaccharide. (a) Resonant two-
photon ionization (R2PI) spectra of the chitobiose stem, 1, the
trisaccharide linker, 3, and the complete core pentasaccharide, 4. (b)
The IRID spectrum of 4.
interactions with the proximal GlcNAc2 unit, which presented a
cis conformation about the −GlcNAcB-GlcNAcA− linkage
(although trans conformations, generally associated with more
extended structures, were also present at higher relative
energies). The addition of water molecules in the gas phase,
Figure 6a(ii), disrupted the intramolecular hydrogen-bonding
pattern. The lowest-energy conformers of the triply hydrated
complex, Figure 6a(ii), presented more extended structures,
now with a trans configuration about the −GlcNAcB-GlcNAcA−
linkage (Figure 4), supported by water bridging across the
inter-ring bond and extended cooperative hydrogen-bonded
networks (which were conserved in both low and high energy
structures).
In bulk water, the intramolecular hydrogen bonds were
largely ‘washed out’; the (near identical) lowest-energy
structures predicted by OPLS2005 and GLYCAM06/
AMBER, Figure 6a(iii), again presented a trans GlcNAcB-
GlcNAcA− stem, but now part of a fully extended structure in
which the distal Man3 unit was completely unfurled. Strikingly,
apart from the difference in the dihedral angle ω(H1-C1-O6-
C6) between ManC and ManE, the preferred structure of the
solvated core pentasaccharide was very similar to that of the
core unit in the high-mannose glycan, Man9GlcNAc2,
determined through coupled NMR measurements and
molecular dynamics simulation using the GLYCAM93/
AMBER force field, Figure 6a(iv,v).12,13,18−20 There were also
remarkable similarities between the conformations of the distal
Man3− head unit in bulk water, found here, and the isolated
(hydrated) Man3 unit, determined in solution18−20 and
predicted through investigations in the gas phase,15 Figure
6a(iv), and also between the conformation (and flexibility) of
Molecular mechanics simulations, undertaken partly in
response to the limitations of the experiment (see also
Supporting Information Methods and Results and Figures
S13 and S14), also predicted a preference for a large number of
hydrogen bonds (defined by r[OH···O] < 2.5 Å, θ[OH···O] >
120°) in the isolated pentasaccharide, see Figure 6a(i), and also
its triply hydrated complex, Figure 6a(ii), with 6−8 being the
optimal number for the isolated molecule (using the
OPLS2005 force field, Figure S13). The simulations in bulk
water, Figure 6a(iii), predicted disruption of the intramolecular
networks, leaving only 2−4 hydrogen bonds in the low-energy
ensemble (Figure S13).
As a consequence, the unsolvated pentasaccharide 4,
preferentially adopted compact structures with the distal
Man3 head unit folded back, supported by hydrogen-bonded
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Figure 6. The spectra and structures of the core pentasaccharide 4. (a) The lowest-energy structures of the core pentasaccharide calculated on the
OPLS2005 and GLYCAM06/AMBER potential energy surfaces: (i) the isolated molecule; (ii) the triply hydrated complex (the water molecules
were initially located at binding sites based upon the preferences of singly hydrated 1, 2, and the trimannosyl ManE(ManD)ManC− head unit15); and
(iii) in bulk water, (hydrogen bonds shown in red). (iv) An overlay of the “open” conformer of the trimannosyl ManE(ManD)ManC− head unit and
the core pentasaccharide in (v); (v) the preferred aqueous structure of the high mannose glycan, Man9GlcNAc2, determined through NMR
measurements and molecular dynamics simulations.12,13 Red dots represent transiently bound water molecules. (b) Distributions of the longest
intramolecular distances (for conformers with energies <30 kJ mol−1) in the core pentasaccharide, predicted by molecular mechanics (OPLS2005)
simulations: isolated, unsolvated (red), explicitly hydrated (green), and in bulk water (black).
the ManC-GlcNAcB segments in the ‘blocked’ units 2-B and 3-B
and in the core pentasaccharide 4.
hydrated, with conserved structural motifs taking shape, in
particular the trans conformation of the proximal chitobiose
unit, to provide an extended, well-defined structure in aqueous
solution.
The analysis of intramolecular distances (using the
OPLS2005 simulations), shown in Figures 6b and S14,
provided a way of estimating molecular size56 and hence the
favorability of extended versus compact structures. The longest
end-to-end distribution for the unsolvated, isolated core
pentasaccharide reflected flexibility, but the most favored
distance (∼16.7 Å) indicated a compact structure correspond-
ing to a geometric cross-section (σ ∼ 230 Å2) similar to the gas
kinetic cross-section (σ ∼ 260 Å2) reported for the doubly
sodiated core pentasaccharide ion,3 which is also likely to be
compact. Addition of three water molecules resulted in a broad
distribution with peaks at ∼21.5 and 23.5 Å, indicating a more
extended but still flexible structure. In bulk water, however, the
structures were exclusively extended and relatively inflexible,
with a narrow spread of end-to-end distances peaked at ∼23.4
Å. The experiments and simulations have revealed a folded
‘naked’ core pentasaccharide that uncurled as it became
DISCUSSION AND CONCLUSIONS
■
A recent bioinformatics analysis57 of thousands of glycoproteins
listed in the PDB58 found that many N-glycans showed
significantly similar substructures close to the protein,
suggesting their use as fragments in glycan modeling (and
supporting the approach employed in the present, exper-
imentally based study). The N-glycans also displayed a rigid
protein-proximal GlcNAcB-GlcNAcA stem.
The acetamido groups of the stem appear to play a key role;
in the gas phase the preferred trans conformation of 1 is
strengthened by microhydration through an inter-ring bridging
water molecule linking the GlcNAcB acetamide to O-6A. In bulk
water, molecular dynamic simulations predict the absence of a
direct inter-ring bond between these groups, which suggests the
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Figure 7. An extrapolated structural model for the N-glycan core in an aqueous environment. In the absence of water (a), the pentasaccharide would
adopt a compact conformation, but the interactions between the building blocks of the core pentasaccharide and water (b) lead, in solution (c), to a
rigid chitobiose stem that is anchored at one end to the peptide10,11,59 and at the other, through a flexible ‘pivot’, to a β-mannosyl structure that
projects the information-rich distal head of the glycan for interaction with other partners. The red dots indicate favored water-bridging sites identified
here or in previous investigations.15
trans chitobiose stem is held in a rigid extended conformation
through ‘filling’ of a conserved water pocket at the local
bridging site. The other GlcNAcA acetamide is predicted to be
unbonded, leaving it free to act as a hydrogen-bond donor/
acceptor and ‘anchor’ the glycan to the peptide backbone of the
glycoprotein.10,11,59 Hexoses (such as the more abundant D-
glucose, Glc) that lack this acetamide could not form such
strong hydrogen bonds (if they were to play the roles of
GlcNAcA and GlcNAcB) and would not match the rigidity of
the GlcNAc2 stem nor the potential for anchoring with the
protein backbone.10,11,59
found on N-glycoproteins60 while still being able to stimulate
proper folding of the underlying N-glycoprotein. The selection
of chitobiose, with its two acetamido groups rather than
cellobiose for the stem, appears to be critical, facilitating
hydrogen-bonded interactions with neighboring amino acid
residues10,11 and suggesting a mechanism by which the core
pentasaccharide influences the structural stability of the
adjacent peptide chain and its folding kinetics.7 The central
ManE(ManD)ManC− unit, which unfurls upon hydration,
presents the information-rich outer extremities of N-glycans
to the environment (for solubility, recognition, or transport).
The next structural feature, provided by the central β-
mannoside ManC-GlcNAcB, also contains a glycosidic linkage
formed through OH6C that precludes formation of the OH6C
→ O6B inter-ring hydrogen bond found in the isolated unit 2.
Instead of the compact cis conformation adopted by 2,
supported by two inter-ring hydrogen bonds, the ‘deletion’ of
one of them has the key effect of greatly increasing the
flexibility of the β-mannoside linkage, which adopts a trans
conformation that is unaffected by discrete hydration. Its
inherent flexibility allows the β-mannosyl residue to act
effectively as a pivot between the rigid chitobiose stem and
the outer trimannosyl head.
ASSOCIATED CONTENT
* Supporting Information
Synthetic methods; NMR data; spectroscopic and computa-
tional methods; computed relative energies, vibrational
frequencies, and structural data. This material is available free
of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
■
Corresponding Authors
John.Simons@chem.ox.ac.uk
Ben.Davis@chem.ox.ac.uk
Unsolvated structures of 4 are compacted by many
intramolecular hydrogen bonds (Figure 6a(i)). However,
discrete hydration sees the structure begin to unfurl with a
water molecule stabilizing the rigid chitobiose stem (vide
supra), while the trimannosyl head unit folds over to
encapsulate water in another pocket. In a fully aqueous
environment, a picture emerges (Figure 7) of an N-linked
glycan accommodating an extended core pentasaccharide
structural unit that incorporates a rigid proximal chitobiose
stem, anchored at one end to the adjoining protein10,11,59 and
at the other connected through a more flexible β-mannoside
pivot to the branched mannosyl D and E arms.
This combines both structural integrity and freedom of
movement to allow interaction with binding partners. The
extended display of the N-glycan would leave the distal branch-
tip sugars free to function as potential ligands for receptors and
also provide the potential for ‘levered’ interactions promoting a
conformational ‘response’ in the underlying protein fold. The
latter could explain the apparent ability of the calnexin/
calreticulin chaperone system to bind the tips of the sugars
Present Addresses
⊥
́
́
Departamento de Quimica Fisica, Facultad de Ciencia y
́
́
Tecnologia, Universidad del Pais Vasco, (UPV−EHU),
Apartado 644, E-48940, Bilbao, Spain.
∇Institut fur Experimentalphysik, Freie Universitat Berlin,
̈
̈
Arnimallee 14, 14195 Berlin, Germany.
Author Contributions
#These authors contributed equally
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to the following for their support: the
Leverhulme Trust for the award of an Emeritus Fellowship
(J.P.S.); the Royal Society and the Wolfson Foundation for a
Research Merit Award (B.G.D.); the EPSRC (B.G.D., J.P.S.);
the Spanish Ministry (MICINN) for a project CTQ2011-22923
́
and “Ramon y Cajal” Contract (E.J.C.); the STFC for the
provision of equipment from the Laser Loan Pool; the Oxford
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