Solid-state 17O NMR analysis of synthetically 17O-enriched D-glucosamine

Article abstract of DOI:10.1016/j.cplett.2020.137455

The hydroxyl groups of carbohydrates are critical determinants of their conformational dynamics and intermolecular interactions but are difficult to characterize by conventional ¹H nuclear magnetic resonance (NMR) approaches in solution. Here,

Full text of DOI:10.1016/j.cplett.2020.137455

ChemicalPhysicsLetters749(2020)137455
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Chemical Physics Letters
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Research paper
Solid-state 17O NMR analysis of synthetically 17O-enriched D-glucosamine
Kazuhiko Yamadaa,1, Yoshiki Yamaguchib,1, Yoshinori Uekusac,2, Kazumasa Aokid,3
,
⁎
Ichio Shimadad, Takumi Yamaguchie,f, Koichi Katoc,f,g,
a Interdisciplinary Science Unit, Multidisciplinary Sciences Cluster, Research and Education Faculty, Kochi University, Nankoku 783-8505, Japan
b Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan
c Institute for Molecular Science, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan
d Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
e School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi 923–1292, Japan
f Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467–8603, Japan
g Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444–8787, Japan
H I G H L I G H T S
D-glucosamine was chemically synthesized with 17O enrichment at position 6.
•
•
Its high-quality 17O NMR data were obtained by high-magnetic-field measurements.
•
This enabled estimation of chemical shift and electric field gradient tensors.
A R T I C L E I N F O
A B S T R A C T
Keywords:
The hydroxyl groups of carbohydrates are critical determinants of their conformational dynamics and inter-
molecular interactions but are difficult to characterize by conventional 1H nuclear magnetic resonance (NMR)
approaches in solution. Here, we report a solid-state 17O NMR analysis of synthetic glucosamine with 17O en-
richment at position 6. Based on magic-angle spinning and stationary spectral data obtained at varying magnetic
fields in conjunction with quantum chemical calculations, we successfully estimated 17O chemical shift and
electric field gradient tensors, providing benchmark for 17O NMR analyses of oligosaccharide structures.
Solid-state 17O NMR spectroscopy
D-Glucosamine
Hydroxyl group
Stable isotope enrichment
Chemical shift
Electric field gradient
Quantum chemical calculation
1. Introduction
interactions [10], and metabolism [11] of polysaccharides.
Carbohydrate structures are characterized by an abundance of hy-
Nuclear magnetic resonance (NMR) spectroscopy is indispensable
for carbohydrate researches as a useful tool not only to determine
covalent structures of oligosaccharides but also to characterize their
dynamic conformations and interactions at atomic level [1–3]. Using
solution NMR techniques, the information is provided through chemical
shifts (CSs), scalar couplings, nuclear Overhauser effects, dipolar cou-
plings, and paramagnetic effects, regarding CH and NH groups of oli-
gosaccharides [4]. In addition, solid-state NMR investigations of car-
bohydrates have been undergoing rapid development and providing
valuable information especially on structures [5–7], dynamics [8,9],
droxyl groups, which can be steric constraints and involved in hydra-
tion and specific hydrogen bonding, thereby serving as critical de-
terminants of their conformational dynamics and intermolecular
interactions. Despite such importance, the rapid exchange of carbohy-
drate hydroxyl hydrogens with solvent preclude their direct observation
by 1H NMR measurements in aqueous solution. Hence, information on
the carbohydrate hydroxyl groups have been obtained through indirect
approaches based on observation of deuterium-induced 13C isotope
shifts [12,13] and hydrogen-bond-mediated scalar couplings [14,15].
Solid-state 17O NMR spectroscopy offers an alternative, promising
⁎ Corresponding author.
E-mail address: kkatonmr@ims.ac.jp (K. Kato).
1 These authors contributed equally to this work.
2 Current affiliation: Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan.
3 Current affiliation: Medicinal Chemistry Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710,
Japan.
https://doi.org/10.1016/j.cplett.2020.137455
Received 8 March 2020; Received in revised form 3 April 2020; Accepted 6 April 2020
Availableonline08April2020
0009-2614/©2020ElsevierB.V.Allrightsreserved.

K. Yamada, et al.
ChemicalPhysicsLetters749(2020)137455
approach that enables direct observation of the hydroxyl oxygens of
carbohydrates, providing information on microenvironments sur-
rounding the hydroxyl groups through 17O CS and electric field gradient
(EFG) tensors. However, in general, 17O NMR observation is precluded
by low sensitivity due to low natural abundance (0.037%) and severe
line broadening due to quadrupole relaxation. D’Souza and Lowary
overcame the low-natural-abundance problem by 17O enrichment [16].
The quadrupole relaxation can be suppressed at higher magnetic fields
[17,18]. Indeed, Grandinetti and co-workers reported solid-state 17O
NMR data obtained at 19.5 T of position-specific 17O-enriched analogs
of glucose, galactose and Glc–Glc disaccharides, which were prepared
by nucleophilic substitution using 17O-labeled benzoic acid and its salt
[19].
solution was kept stirring for 4 h at 120 °C. The reaction was monitored
with silica gel TLC (ethyl acetate : water : isopropylamine = 18:1:1).
After cooling, the mixture was diluted with ethyl acetate (1.5 L). The
organic layer was washed with water (500 mL × 5) and brine
(500 mL × 1). The organic layer was dried over MgSO4, and evapo-
rated to remove solvents. The product [6-17O2]acetyl-1,3,4-triacetyl-N-
acetyl-α-D-glucosamine was obtained as a brown oil (4.54 g, 58%).
2.1.5. D-[6-17O]Glucosamine hydrochloride
[6-17O2]Acetyl-1,3,4-triacetyl-N-acetyl-α-D-glucosamine (4.54 g,
11.6 mmol) was dissolved in 2 M hydrochloric acid (116 mL) and
stirred for 4 h at 100 °C under argon atmosphere. The reaction was
monitored
with
silica
gel
TLC
(ethyl
acetate:water:-
Here, we extend the solid-state NMR approach to determine 17O-
derived parameters of D-glucosamine, which is a common metabolic
precursor of aminosugars including sialic acids and their derivatized
polysaccharides and glycoconjugates. Therefore, solid-state 17O NMR
characterization of D-glucosamine will serve as solid starting point of
structural studies of these glycoconjugates.
isopropylamine
=
18:1:1 and ethanol:tert-butyl alcohol:water:-
ammonia water = 8:2:1:1). After cooling, solvent was evaporated and
toluene (10 mL × 5) was added to the oil reside. After evaporation, a
crude brown solid was obtained. The crude solid was washed with
methanol giving colorless material (2.41 g, yield 96%). The purity
(> 95%) and anomeric configuration (α-anomer 95%) of the obtained
D-[6-17O]glucosamine hydrochloride were assessed based on solution
1H and solid-state 13C NMR spectra, respectively (Supplementary data).
2. Experimental section
2.2. Solid-state 17O NMR experiments
2.1. Sample preparations
All solid-state 17O NMR spectra of D-[6-17O]glucosamine hydro-
chloride were obtained on JEOL ECA 600, JEOL ECZR 800, and Bruker
Avance-900 spectrometers, operating at 81.356 MHz (14.1 T),
108.469 MHz (18.8 T), and 122.026 MHz (21.1 T), respectively. The
polycrystalline samples were packed into 2.5- mm, 3.2- mm, or 4.0- mm
rotors of zirconium oxide and/or silicon nitride. An external sample of
liquid water was used for pulse adjustments and chemical shift refer-
encing. For all the experiments, an Oldfield echo sequence [23] was
used, and high-power 1H decoupling was applied during the acquisition
periods. The recycle delay was typically 1 s, and the π/2 pulse length
was typically 1.0 μs –2.0 μs. Sample spinning frequencies, ωR, at 21.1 T
and 18.8 T were 20 kHz and 8 kHz, respectively, and the inter-pulse
delay times were rotor-synchronized. For the stationary 17O NMR ex-
periments, the inter-pulse delay times were set to 40 μs – 50 μs. All the
experimental NMR spectra were processed using TOP-SPIN (Bruker)
and/or Delta (JEOL Ltd.) software. Spectral simulations were per-
formed using the program written by the authors on MATLAB (The
MathWorks Inc.). To describe the relative orientation of 17O CS and EFG
tensors, expressed by the rotation parameters in terms of three Euler
angles α, β, and γ, the definition of the literature [24] was used.
2.1.1. 6-O-Tosyl-N-acetyl-D-glucosamine
Preparation of 6-O-tosyl-N-acetyl-D-glucosamine was performed
according to the previous report [20] with some modifications. To a
solution of N-acetyl-D-glucosamine (66.4 g, 0.30 mol) in pyridine
(860 mL), a solution of p-toluenesulfonyl chloride (68.6 g, 0.36 mol) in
pyridine (430 mL) was slowly dropped at 4 °C within 30 min, and the
mixed solution was stirred for 4 h at 4 °C under argon atmosphere. The
reaction was monitored on silica gel TLC (ethyl acetate : water:-
isopropylamine
= 18:1:1 and methanol:tert-butyl alcohol:water:-
ammonia water = 8:2:1:1). The reaction was quenched by adding
methanol (43 mL). The mixture was diluted with chloroform (2.1 L) and
the organic layer was washed with water (500 mL, 300 mL). The or-
ganic layer was dried over MgSO4, and concentrated in vacuo. To re-
move pyridine, toluene (40 mL × 3) was added and repeatedly eva-
porated. To the residue, CH2Cl2 (500 mL) was added to obtain crude
solid. The solid was further washed with CH2Cl2 (500 mL) to give white
solid (42.8 g, yield 38%).
2.1.2. 6-O-Tosyl-1,3,4-triacetyl-N-acetyl-α-D-glucosamine
To a solution of 6-O-tosyl-N-acetyl-D-glucosamine (37.5 g, 0.10 mol)
in pyridine (500 mL), acetic anhydride (70 mL, 0.74 mol) was slowly
dropped at 0 °C under argon atmosphere within 30 min. The mixture
was stirred for 5 h at 0 °C. The reaction was monitored on silica gel TLC
(ethyl acetate : water : isopropylamine = 18:1:1). The reaction was
quenched by adding methanol (24 mL). The solvent was removed by
evaporation. Then, toluene (30 mL × 3) was added and repeatedly
evaporated to remove pyridine. To the residue, methanol was added to
give crude solid. The solid was recrystallized from methanol (100 mL)
and a colorless crystal (27.8 g, yield 55%, m.p. 153 °C) was obtained.
2.3. Quantum chemical calculations
All quantum chemical calculations on 17O EFG and CS tensors were
performed with the Gaussin16 program package [25]. The crystal
structure of α-D-glucosamine hydrochloride, determined by X-ray dif-
fraction [26], was used for the input geometry, in which two neigh-
boring molecules were additionally included as the effect of inter-
molecular interactions. The Gauge-Induced Atomic Orbital (GIAO)
approach [27] was used for chemical shielding calculations. In NMR
experiments, the frequency of an NMR signal is observed relative to that
in a reference system. For 17O NMR, liquid water was used as the re-
ference. Because the quantum calculations give absolute chemical
shielding values, σii, it is convenient to convert them into chemical
shifts relative to water, δii, by using
2.1.3. Sodium [17O2]acetate
Sodium [17O2]acetate was synthesized using 1 g of [17O]H2O (17O:
89.9%, ICON) according to the reported procedure [21]. The reaction
was conducted under argon atmosphere. The yield of sodium [17O2]
acetate was 2.1 g (97%).
δii = 307.9 − σii [ppm],
[i]
2.1.4. [6-17O2]Acetyl-1,3,4-triacetyl-N-acetyl-α-D-glucosamine
Introduction of 17O at the 6 position of glucosamine unit was per-
formed following the previous procedure [22]. To a solution of 6-O-
tosyl-1,3,4-triacetyl-N-acetyl-α-D-glucosamine (10.02 g, 20.0 mmol) in
DMF (50 mL), 15-crown-5 (4.76 mL, 24.0 mmol) and sodium [17O2]
acetate (2.02 g, 24 mmol) were added under stirring at once. The
where the value of 307.9 ppm is the absolute chemical shielding con-
stant for the 17O nucleus in liquid H2O [28]. The quantum chemical
calculations yield EFG tensors in atomic units (a. u.), qii. In solid-state
NMR experiments, quadrupolar coupling interactions are expressed as
EFG tensors, whose principal components are defined as
2

K. Yamada, et al.
ChemicalPhysicsLetters749(2020)137455
Scheme 1. Synthesis of D-glucosamine hydrochloride with 17O enrichment at position 6.
|Vxx| < |Vyy| < |Vzz|.
[ii]
EFG tensors is effective for investigation of the local electronic struc-
tures of oligosaccharides. To further approach the CS and EFG tensors,
we also measured 17O MAS spectrum with ωR = 8 kHz (Fig. 1b). Using
this intermediate rotor regime, spinning sidebands appeared in the MAS
spectrum, which was well fitted by the theoretical 17O MAS spectrum
simulated by the second-order quadrupolar interaction with lower rotor
frequency [30]. This result indicated that the magnitude of the 17O CS
tensor for D-[6-17O]glucosamine was expected to be much smaller than
that of the EFG tensor.
To describe a traceless EFG tensor, two NMR parameters are used,
namely, the quadrupole coupling constant, CQ, and the asymmetry
parameter, ηQ. The following equations were employed for making a
direct comparison−b1 etween theoretical and experimental data:
CQ [Hz] = eVzzQh = −2.3496 Q [fm2]qzz [a. u. ]
[iii]
and
In principle, the analysis of stationary 17O NMR line shape is re-
quired to reveal 17O CS tensor components and the relative orientations
between CS and EFG tensors [31]. Therefore, the 17O stationary NMR
spectra of D-[6-17O]glucosamine were measured and analyzed at
14.1 T, 18.8 T, and 21.1 T (Fig. 2). The high-quality 17O NMR data were
obtained at the high magnetic field. All the obtained experimental 17O
NMR parameters for the oxygen at position 6 of glucosamine are
summarized in Table 1. It was also necessary to carry out the 17O NMR
experiments at higher magnetic fields, where the magnitude of EFG
tensors decreases while that of CS tensors increases in general. At a
lower magnetic field, e.g., at 11.7 T, it was nearly impossible to obtain
information on 17O CS tensors and the Euler angles, because that the
line shape was insensitive to the changes in their parameters.
For reference, the results of density functional theory (DFT) calcu-
lations for the 17O NMR parameters of α-D-[6-17O]glucosamine using
various basis sets are summarized in Table 2. While the calculated CQ
values were reasonably consistent in the experimental one, the ηQ value
was not reproduced by the DFT calculation. The calculated relative
orientations between the two NMR tensors tend to be in good agree-
ment with the experimental ones. Assuming that the orientations of the
17O EFG tensor components obtained from the present quantum che-
mical calculations were correct, the absolute orientations of the 17O CS
tensor components with respect to the molecular frame were de-
termined using the experimentally obtained relative orientations [31].
The 17O NMR tensor orientations thus determined for α-D-[6-17O]glu-
cosamine are depicted in Fig. 3. The largest 17O EFG tensor component,
ηQ = (Vxx − Vyy)/Vzz,
[iv]
where Q is the electric quadrupole moment of the 17O nucleus and the
factor of 2.3496 comes from unit conversion. In the present calcula-
tions, Q = −2.558 fm2 [29] was employed in all EFG calculations.
3. Results and discussion
We synthesized D-[6-17O]glucosamine efficiently using 17O-labeled
sodium acetate (Scheme 1). To realize high isotope enrichment, sodium
[
17O2]acetate was prepared through consecutive hydrolysis of triethyl
orthoacetate using [17O]H2O (17O: 89.9%) according to the reported
procedure [21]. The reaction of the obtained sodium [17O2]acetate with
a 6-O-tosylated glucosamine derivative allowed the selective introduc-
tion of 17O at 6 position of the glucosamine unit. A subsequent depro-
tection in hydrochloric acid gave D-[6-17O]glucosamine hydrochloride
in good yield.
In parallel, D-[6-18O]glucosamine was prepared under the same
procedure using [18O]H2O (18O: 95–98%, CIL) instead of [17O]H2O to
estimate the isotope enrichment ratio. By comparing signal intensities
(
13C-16O and 13C-18O) in the 13C NMR spectrum, the 18O enrichment
ratio in the synthesized D-[6-18O]glucosamine hydrochloride was de-
termined as 89%. Thus, the enrichment ratio of 17O in our D-[6-17O]
glucosamine was estimated to be 82%-84%.
The synthetic D-[6-17O]glucosamine was subjected to solid-state
NMR measurements and successfully provided 17O spectra with high
sensitivity. By the measurement at 21.1 T using magic-angle spinning
(MAS) with a fast rotor regime (ωR = 20 kH), a typical MAS line shape
arising from the central transition of second-order quadrupolar inter-
actions for half-integer quadrupolar nuclei was obtained (Fig. 1a). The
spectral analysis yielded the following parameters; δiso = −14(2) ppm,
CQ = 9.0(2) MHz, and ηQ = 0.91(5). Grandinetti and co-workers
previously reported a series of solid-state 17O MAS NMR data of car-
bohydrates [19], providing the 17O NMR parameters of methyl α-D-
[6-17O]glucopyranoside, relevant to the present compound, as follows:
V
ZZ, and the smallest component, VXX, lay in the molecular plane of C-
O-H, while the VZZ direction was approximately 20° off the C-O bond.
The least shielding component, δ11, and the most shielding component,
δ
33, slightly lay off the molecular plane, since the direction of δ22
component did not coincide with that of VYY, i.e., α = 16(10) and
γ = 12(30). The angle between δ33 and VZZ components was 108(8)°.
Although there remains room to improve quantum chemical calcula-
tions for magnitudes of 17O NMR tensors, the present experimental
data, in particular the 17O CS tensors, would be useful for the future
evaluation of ab initio calculations.
δ
iso = 9.5 ppm, CQ = 8.76 MHz, and ηQ = 1.00. The CQ and ηQ values
obtained for the oxygen at position 6 of glucosamine are similar to
those reported for methyl α-D-[6-17O]glucopyranoside, indicating their
comparable EFG tensors. In contrast, the isotropic chemical shifts of the
oxygen at position 6 were significantly different between glucose and
glucosamine, which suggested that a combined analysis of 17O CS and
4. Conclusions
We successfully interpreted the 17O spectral data, for estimating the
17O CS and EFG tensors, of D-glucosamine by the site-specific 17O
3

K. Yamada, et al.
ChemicalPhysicsLetters749(2020)137455
Fig. 2. Experimental (upper trace) and the best-fitted simulated (lower trace)
stationary solid-state 17O NMR spectra of D-[6-17O]glucosamine, measured at
(a) 14.1 T, (b) 18.8 T, and (c) 21.1 T. The chemical shift is relative to liquid
water.
Fig. 1. Experimental (upper trace) and the best-fitted simulated solid-state
(lower trace) 17O MAS NMR spectra of D-[6-17O]glucosamine, measured at (a)
21.1 T and ωR = 20 kHz and (b) 18.8 T and ωR = 8 kHz. The chemical shift is
relative to liquid water.
CRediT authorship contribution statement
enrichment in conjunction with the high-field NMR measurements and
the quantum chemical calculations. The analyses of 17O MAS and sta-
tionary NMR spectra provided the parameters associated with the
magnitudes and rotations of the CS and EFG tensors. Furthermore, the
orientations of the two 17O NMR tensors with respect to the molecular
frame were obtained by the combined analyses with NMR experiments
and DFT calculations. To the best of our knowledge, this is the first
experimental report on 17O CS tensors for carbohydrates, which pro-
vides a benchmark for future solid-state 17O NMR studies of oligo-
saccharides. This line of approach will be widely applicable to 17O NMR
analyses of oligosaccharides, offering sensitive probes for hydrogen
bonding and glycosidic linkage conformations.
Kazuhiko Yamada: Conceptualization, Investigation, Writing -
original draft. Yoshiki Yamaguchi: Conceptualization, Investigation,
Writing - original draft. Yoshinori Uekusa: Investigation. Kazumasa
Aoki: Supervision. Ichio Shimada: Supervision. Takumi Yamaguchi:
Investigation, Project administration, Writing - original draft. Koichi
Kato: Conceptualization, Supervision, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
4

K. Yamada, et al.
ChemicalPhysicsLetters749(2020)137455
Table 1
The experimentally obtained 17O CS tensors, EFG tensors, and Euler angles for D-[6-17O]glucosamine.
δ11 /ppm
δ22 /ppm
−2(7)
δ33 /ppm
−46(7)
δiso /ppm
−14(2)
CQ / MHz
ηQ
α / deg.
β / deg.
γ / deg.
6(5)
9.0(2)
0.91(5)
16(10)
108(8)
12(30)
Table 2
The calculated 17O CS tensors, EFG tensors, and Euler angles for the oxygen at position 6 of α-D-glucosamine. All the calculations were carried out by using B3LYP
exchange functional.
Basis set
δ
11/ppm
δ22/ppm
δ33/ppm
δ
iso/ppm
CQ/ MHz
ηQ
α/ deg.
β/ deg.
γ/ deg.
6–311++G**
cc-pVTZ
spAug-cc-pVTZ
D95**
−13.9
−17.9
−13.4
−20.6
−27.6
−28.4
−27.5
−37.9
−49.9
−54.9
−51.0
−60.3
−30.5
−33.7
−30.6
−39.8
10.3
10.0
9.97
10.5
0.76
0.76
0.75
0.62
0.4
3.3
0.7
4.4
107.1
109.5
105.5
114.2
3.8
5.8
4.7
−9.2
doi.org/10.1016/j.cplett.2020.137455.
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off the molecular plane, and δ11 component is approximately parallel to the C-O
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Acknowledgements
We are deeply grateful to the late Professor Kenji Koga for super-
vising the carbohydrate synthesis in this study. We sincerely thank Mr.
Yuichi Shimoikeda (JEOL Ltd.), Mr. Kenzo Deguchi (National Institute
for Materials Science) and Dr. Minoru Hatanaka (Bruker Japan) for the
help in the spectral measurements. The NMR measurements were per-
formed at JEOL Ltd., NIMS, and Yokohama City University (NMR
PLATFORM). The DFT calculations were performed at Research Center
for Advanced Computing Infrastructure, JAIST. This work was partly
supported by the MEXT/JSPS Grants in Aid for Scientific Research
(JP18K05175 and JP19H04569), Nanotechnology Platform Program
(Molecule and Material Synthesis) of MEXT and Joint Research by IMS.
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Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
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