Single-crystal and powder X-ray diffraction and solid-state 13C NMR of p-nitrophenyl glycopyranosides, the derivatives of d-galactose, d-glucose, and d-mannose

Article abstract of DOI:10.1016/j.carres.2009.05.026

The X-ray diffraction patterns, ¹³C CP MAS NMR spectra, and powder X-ray diffraction analyses were obtained for selected p-nitrophenyl glycosides: α- and β-d-galactopyranosides (1 and 2), α- and β-d-glucopyranosides (3 and 4), and α- and β-d-mannopyranosides (5 and 6). In X-ray diffraction analysis of 1 and 2, characteristic shortening and lengthening of selected bonds were observed in the molecules of 1 due to anomeric effect, and in the crystal lattice of 1 and 2, hydrogen bonds of complex network were detected. In the crystal asymmetric unit of 1 there were two independent molecules, whereas in 2 there was one molecule. For 1 and 3-6 the number of resonances in solid-state ¹³C NMR spectra exceeded the number of the carbon atoms in the molecules, while for 2 there were distinct singlet resonances in its solid-state NMR spectrum. Furthermore, the powder X-ray diffraction (PXRD) performed for 1-3 and 5 revealed that 1, 3, and 5 existed as single polymorphs proving that the doublets observed in appropriate solid-state NMR spectra were connected with two non-equivalent molecules in the crystal asymmetric unit. On the other hand 2 existed as a mixture of two polymorphs, one of them was almost in agreement with the calculated pattern obtained from XRD (the difference in volumes of the unit cells), and the subsequent unknown polymorph existed in small amounts and therefore it was not observed in solid-state NMR measurements.

Full text of DOI:10.1016/j.carres.2009.05.026

Carbohydrate Research 344 (2009) 1734–1744
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
13
Single-crystal and powder X-ray diffraction and solid-state C NMR
of p-nitrophenyl glycopyranosides, the derivatives of D-galactose, D-glucose,
and D-mannose
a
a,*
b
c
Tomasz Gubica , Andrzej Temeriusz , Katarzyna Paradowska , Andrzej Ostrowski ,
a
a
Paulina Klimentowska , Michał K. Cyra n´ ski
Faculty of Chemistry, University of Warsaw, Pasteura 1, PL-02093 Warsaw, Poland
Department of Physical Chemistry, Faculty of Pharmacy, Medical University of Warsaw, Banacha 1, PL-02097 Warsaw, Poland
Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, PL-00664 Warsaw, Poland
a
b
c
a r t i c l e i n f o
a b s t r a c t
The X-ray diffraction patterns, 13C CP MAS NMR spectra, and powder X-ray diffraction analyses were
Article history:
Received 3 March 2009
Received in revised form 20 May 2009
Accepted 25 May 2009
obtained for selected p-nitrophenyl glycosides:
a- and b-D-galactopyranosides (1 and 2), a- and b-D-
glucopyranosides (3 and 4), and - and b- -mannopyranosides (5 and 6). In X-ray diffraction analysis
a
D
of 1 and 2, characteristic shortening and lengthening of selected bonds were observed in the molecules
of 1 due to anomeric effect, and in the crystal lattice of 1 and 2, hydrogen bonds of complex network were
detected. In the crystal asymmetric unit of 1 there were two independent molecules, whereas in 2 there
Available online 2 June 2009
Keywords:
Crystal structure
13
was one molecule. For 1 and 3–6 the number of resonances in solid-state C NMR spectra exceeded the
number of the carbon atoms in the molecules, while for 2 there were distinct singlet resonances in its
solid-state NMR spectrum. Furthermore, the powder X-ray diffraction (PXRD) performed for 1–3 and 5
revealed that 1, 3, and 5 existed as single polymorphs proving that the doublets observed in appropriate
solid-state NMR spectra were connected with two non-equivalent molecules in the crystal asymmetric
unit. On the other hand 2 existed as a mixture of two polymorphs, one of them was almost in agreement
with the calculated pattern obtained from XRD (the difference in volumes of the unit cells), and the sub-
sequent unknown polymorph existed in small amounts and therefore it was not observed in solid-state
NMR measurements.
Single-crystal X-ray diffraction
Powder X-ray diffraction
Solid-state NMR
Nitrophenyl glycopyranosides
Ó 2009 Elsevier Ltd. All rights reserved.
1
. Introduction
Glycosides are a group of natural compounds widely spread in
sidases,21–26 since during enzymatic decomposition the free agly-
con, that is, p-nitrophenol, is released and therefore the changes
of absorbance in UV–vis spectrum can be observed.
1
both flora and fauna. Therefore, glycosidases, the enzymes that
catalyze the decomposition of corresponding glycosides are a sub-
ject of intense investigations regarding their kinetics and mecha-
The important applications of p-nitrophenyl glycosides listed
above prompted us to investigate their structure using not only
1
3
the X-ray diffraction, but also the solid-state C NMR techniques
(Table 1). These techniques are complementary. Single-crystal
X-ray diffraction (XRD) measurements can provide the information
about the number of molecules in the independent unit cell of the
appropriate monocrystals, while the powder X-ray diffraction
(PXRD) experiments deliver information about the number of crys-
talline forms in the samples, and can associate the appropriate
polymorph with the data obtained from XRD. Similarly to powder
2
–16
nism of action.
The first step in these investigations is the isolation of glycosi-
dases from natural materials, often using the affinity chromatogra-
phy. In this technique, specific ligands, covalently bound to the
supporting, stationary phase which can recognize specific natural
substances are needed. Such ligands which can be used for isolat-
ing glycosidases are the p-aminophenyl glycosides.17 The simplest
precursors of p-aminophenyl glycosides are their p-nitrophenyl
1
3
X-ray diffraction (PXRD), the solid-state C NMR techniques relate
to macroscopic samples, not only to selected monocrystals, and
therefore they can reveal the number of magnetically unequal mol-
ecules in these samples providing the additional information about
the structure of the selected p-nitrophenyl glycosides. As discussed
counterparts, which can be easily converted into the corresponding
p-aminophenyl derivatives.1
8–20
The p-nitrophenyl glycosides are
also used as markers in the investigations of the kinetics of glyco-
2
7
by Harris, solid-state NMR has been successfully applied to the
studies of organic molecules in a variety of chemical systems,
*
Corresponding author. Tel.: +48 22 822 2325; fax: +48 22 822 5996.
E-mail address: atemer@chem.uw.edu.pl (A. Temeriusz).
0
008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.05.026

T. Gubica et al. / Carbohydrate Research 344 (2009) 1734–1744
1735
Table 1
Comparison of the results obtained for p-nitrophenyl glycosides 1–6 using single-crystal and powder X-ray diffraction, as well as solid-state 13C NMR
Compound
Signals in 13C CP MAS
NMR
The number of molecules in the crystal asymmetric
unit (XRD)
PXRD
1
2
3
4
5
6
p-Nitrophenyl
a
-
D
-
-
-
Split
Two
One polymorph
galactopyranoside
p-Nitrophenyl b-
galactopyranoside
p-Nitrophenyl
glucopyranoside
p-Nitrophenyl b-
glucopyranoside
p-Nitrophenyl
mannopyranoside
p-Nitrophenyl b-
mannopyranoside
D
-
Singlets
Split
One
Two polymorphs (the unknown one in small
amounts)
One polymorph
a-
D
Two30,31
D
-
Split
—
—
a-
D
Split
Two (hemihydrate)32 one (ethanol solvate)33
—
One polymorph (according to32
—
)
D
-
Split
appearing especially as polymorphs and solvates. Polymorphs may
yield observably different NMR spectra, and, in suitable cases, valu-
able information can be obtained from chemical shifts of multi-
plets. In particular, the solid-state 1 C NMR data were helpful in
the structural analysis of methyl glycopyranosides28 and therefore
it seemed interesting to characterize the anomeric pairs of p-nitro-
phenyl glycosides using NMR technique in conjunction with the
X-ray diffraction analyses.
one. Another, much weaker hydrogen bond is formed between
the water molecule and the nitro group of 1A (the Oꢀ ꢀ ꢀO distance
is 3.068(2) Å). Several hydrogen bonds of comparable strength
are also observed, which are formed between the hydroxyl groups
at the pyranosidic ring of neighboring molecules, where they are
either hydrogen bond donors or acceptors. In 2, similarly to 1,
the strongest hydrogen bonds are also formed by the water mole-
cule. Two of them involve the hydroxyl groups at C-3 of two neigh-
boring molecules. One of the hydroxyl groups plays a role of
hydrogen bond donor, another one of the acceptor (the Oꢀ ꢀ ꢀO dis-
tances are 2.683 (2) Å and 2.735 (2) Å, respectively). The subse-
quent hydrogen bond involves the hydroxyl group at C-6, which
plays a role of hydrogen bond acceptor (the Oꢀ ꢀ ꢀO distance is
2.838 (2) Å). The hydrogen bonds formed by the hydroxyl groups
at the pyranosidic ring are of similar strength in 2 as in 1 (their
3
This paper is a continuation of our previous investigations fo-
cused on the structural properties of nitrophenyl glycosides. In
2
9
our previous paper, we have described the structure of six glyco-
sides, the derivatives of -glucose and -galactose, focusing on per-
D
D
O-acetyl derivatives of these glycosides. The current paper deals
with the selected p-nitrophenyl glycosides with free hydroxyl
groups, which, contrary to per-O-acetyl derivatives, can be used di-
rectly in the enzyme studies. We have chosen the derivatives of the
monosaccharides, such as
D-galactose, D-glucose, and D-mannose,
which are most popular and widespread in nature.
Table 2
Crystal data and structure refinement for p-nitrophenyl
and p-nitrophenyl b-D-galactopyranoside (2)
a
-D-galactopyranoside (1)
2
2
. Results and discussion
Compound
1
2
.1. Single-crystal X-ray diffraction
Molecular formula
Molecular weight
Crystal system
2C12
620.52
Monoclinic
H
15NO
8 2
ꢀH O
C
12
H
15NO
319.27
Monoclinic
8
2
ꢀH O
For X-ray investigations the crystals of p-nitrophenyl
a-D-galac-
Space group
P2
1
P2
1
topyranoside (1) and p-nitrophenyl b- -galactopyranoside (2) were
D
Z (molecules/cell)
Dcalcd Mg/m
4
2
obtained by slow evaporation from ethanol/water solutions. The
experimental data and structure refinement parameters are given
in Table 2. Molecular structures and atom numbering are shown
in Figures 1 and 2, whereas selected bond lengths, bond angles,
and torsion angles are given in Table 3. The absolute configuration
of 1 was assigned following the information obtained from Koch
Light Laboratories Ltd, whereas that of 2 by the reference to the
unchanging chiral centers of the precursor in the synthetic
procedure.
3
1.555
1.554
Unit cell dimensions
a (Å)
b (Å)
c (Å)
b (°)
Volume (Å )
F(0 0 0) (e)
7.295 (2)
11.280 (2)
16.110 (3)
90.88 (3)
1325 (5)
652
10.898 (2)
4.635 (1)
13.513 (3)
91.13 (3)
682.4 (2)
336
3
Wavelength (Å)
0.71073
0.134
0.71073
0.135
Absorption coefficient
ꢁ1
(
mm
)
Both 1 and 2 crystallize in P2
of the unit cell is formed by two molecules of sugar in 1 (1A and
B) and by one molecule in 2. Moreover, both 1 and 2 crystallize
1
space group. An independent part
Crystal size (mm
3
)
0.3 ꢂ 0.2 ꢂ 0.15
0.4 ꢂ 0.2 ꢂ 0.15
H
range for data
3.57–25.00
3.74–24.99
collection (°)
1
Limiting indices
ꢁ6 6 h 6 8
ꢁ12 6 h 6 12
ꢁ4 6 k 6 5
ꢁ16 6 l 6 16
5053
1827 [Rint = 0.0342]
1827/1/224
1.062
with one water molecule, which interacts with the sugar molecules
by medium–strong hydrogen bonds. The packing in the crystals is
determined by rather complex intermolecular networks of hydro-
gen bonds, involving all oxygen atoms of the hydroxyl groups,
water molecules, nitro group, as well as a large number of intermo-
lecular van der Waals interactions such as C–Hꢀ ꢀ ꢀO and Nꢀ ꢀ ꢀO. The
selected interactions in the crystal lattice of 1 and 2 are shown in
Figures 3 and 4, respectively. The strongest hydrogen bonds in 1
are formed between the water molecule and hydroxyl groups at
C-6 (1B) and C-2 (1A) in which the water molecule plays a role
of H-bond acceptor or donor, respectively. The Oꢀ ꢀ ꢀO distances
are 2.696 (2) Å in the former case and 2.776 (3) Å in the latter
ꢁ
3 6 k 6 13
19 6 l 6 19
ꢁ
Reflections collected
9757
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
4584 [Rint = 0.0259]
4584/1/433
0.993
Final R indices [I > 2
r
(I)]
R
1
= 0.0277;
wR = 0.0630
= 0.0320;
wR = 0.0651
R
1
= 0.0284;
wR = 0.0655
= 0.0310;
wR = 0.0668
2
2
R indices (all data)
R
1
R
1
2
2
Extinction coefficient
Largest differences peak
0.024(2)
0.130 and ꢁ0.155
0.019(4)
0.146 and ꢁ0.161
3
and hole
Dq (e/Å )

1
736
T. Gubica et al. / Carbohydrate Research 344 (2009) 1734–1744
Figure 1. Molecular structure and atomic numbering of p-nitrophenyl
a
-
D-galactopyranoside (1A and 1B); the thermal ellipsoids are drawn at 50% probability.
Oꢀ ꢀ ꢀO distances range between 2.735 (2) Å and 3.065 (2) Å) and in-
volve the following interactions: O-41–Hꢀ ꢀ ꢀO-31, O-21–Hꢀ ꢀ ꢀO-21
and O-61–Hꢀ ꢀ ꢀO-11. In both cases, 1 and 2, a considerable number
of weaker interactions, for example, C–Hꢀ ꢀ ꢀO are also observed; the
shortest ones being around 3.2 Å.
formation. Consequently, the strength of the hydrogen bonds is
comparable to that of 1 and 2. In 3 the Oꢀ ꢀ ꢀO distances range be-
3
0,31
30
tween 2.740
and 3.109 Å whereas in 5 they are between
3
2
33
2.737 (6) and 3.128 (4) Å.
The difference between 1 and 2 can be clearly observed in the
geometry of the anomeric carbon atom (C-1). The C-1–O-5 and
C-1–O-1 bond lengths and O-1–C-1–O-5 bond angles are clearly
distinct. For 1 they range between 1.394 (2) Å and 1.400 (2) Å,
1.421(2) Å and 1.426(2) Å, and 113.6° (2) and 112.5° (1), respec-
tively, while for 2 they are equal to 1.415 (2) Å, 1.404 (2) Å,
and 106.5° (2), respectively. Such characteristic shortening of
Similarly to 1 and 2, both p-nitrophenyl
a-D-glucopyranoside
0,31
32,33
(
3)3
and p-nitrophenyl
a-
D
-mannopyranoside (5)
crystallize
in P2
1
space group. Two molecules also form an independent part
3
0,31
32
33
of 3
one molecule in an independent part. Both 3
essentially the same fragments participating in the hydrogen bond
and hemihydrate of 5, whereas ethanol solvate of 5 has
30,31
32,33
and 5
have

T. Gubica et al. / Carbohydrate Research 344 (2009) 1734–1744
1737
Figure 2. Molecular structure and atomic numbering of p-nitrophenyl b-D-galactopyranoside (2); the thermal ellipsoids are drawn at 50% probability.
Table 3
(
H
0
5
5)°, respectively, for molecules A and B. For other related systems,
Selected bond lengths, bond angles, and major torsion angles for p-nitrophenyl
galactopyranoside (1) and p-nitrophenyl b-D-galactopyranoside (2)
a
-D-
30
31
, Q and u are equal to 7.4° or 3.6° (6.2° or 4.8°), 0.561 or
3
0
31
30
.542
(0.562 or 0.538),
303.85° or 55.10°
(309.19° or
3
1
Atoms
Compound
8.04°) for two independent molecules of 3 and are equal to
3
2
33
32
1
A
1B
2
5.2° (6) or 5.3° (6) (4.9° (5)), 0.561 (6) or 0.557 (6) (0.528
3
3
32
33
(
4)), 80° (6) or 265° (7) (76° (6)) for 5.
Bond lengths (Å)
C-1–O-5
C-1–O-1
C-5–O-5
O-1–C-7
Bond angles (°)
O-1–C-1–O-5
C-1–O-5–C-5
1.394 (2)
1.421 (2)
1.444 (2)
1.367 (2)
1.400 (2)
1.426 (2)
1.446 (2)
1.360 (2)
1.415 (2)
1.404 (2)
1.442 (2)
1.367 (2)
2.2. 13C CP MAS NMR spectroscopy
The 1H and 13C NMR spectra were recorded for 1–6 in DMSO-d
6
solutions. The 2D experiments were helpful to achieve a complete
assignment of the proton and carbon chemical shifts (Table 4). The
113.6 (2)
115.2 (1)
119.2 (1)
112.5 (1)
113.9 (1)
120.7 (1)
106.5 (2)
111.4 (1)
119.3 (2)
1
3
C-1–O-1–C-7
C CP MAS NMR spectra were recorded for solid compounds at
0 kHz with RAMP shape CP sequence. The resonances in the so-
Torsion angles (°)
O-1–C-1–O-5–C-5
O-5–C-1–O-1–C-7
C-2–C-1–O-1–C-7
C-1–O-1–C-7–C-8
C-1–O-1–C-7–C-12
C-11–C-10–N-1–O-11
C-9–C-10–N-1–O-12
O-1–C-7–C-12–C-11
1
63.3 (2)
83.0 (2)
57.3 (2)
69.3 (2)
–169.4 (2)
–4.0 (3)
175.6(2)
14.0 (3)
14.6 (3)
178.3 (2)
–177.1 (1)
–84.1 (2)
156.2 (2)
12.1 (3)
–168.2 (2)
4.2 (3)
lid-state spectra were assigned following the same sequence as in
the solution spectra. The spectra of both 1 and 2, as well as those
of 3 and the ethanol solvate of 4, are shown in Figures5 and 6, respec-
tively. The resonances of aromatic carbon atoms could be easily dis-
tinguished from those of C–H carbon atoms of sugars. The chemical
shifts for solid p-nitrophenyl glycopyranosides 1–6 are collected in
Table 4, together with the differences
Besides the intramolecular (steric) effects, intermolecular inter-
actions should also be taken into account while analyzing the NMR
–154.6 (2)
–18.2 (3)
161.9 (2)
179.5 (2)
178.6 (2)
–177.7 (2)
5.8 (3)
177.5 (2)
D
= dsolution ꢁ dsolid
.
C-1–O-5 bond and lengthening of C-1–O-1 bond in 1 (
a
anomer) in
6
spectra. In the solids, as well as in DMSO-d solutions, the sugar hy-
comparison to 2 (b anomer) are undoubtedly connected with the
droxyl groups are involved in O–Hꢀ ꢀ ꢀO hydrogen bonds. Therefore,
chemical shifts for C–OH carbon atoms are close in both phases;
larger differences amounting to 2–3.5 ppm observed for C-5 and
C-6, probably result from conformational differences (frozen rota-
3
0,31
anomeric effect. The comparison of geometries of 3
and
3
2,33
5,
leads to the conclusion that the largest structural changes
are observed for the fragments whose configurations differ from
each other. It obviously concerns the C-4 carbon atom in 1 and the
C-2 and C-4 fragments in 2.
2
tion of CH OH moiety). The packing in the crystal is determined by
the intermolecular network of hydrogen bonds and a number of
van der Waals interactions. Water or ethanol molecules act as
additional hydrogen bond donors (4, 5). In the spectrum of 4 one
Interestingly, neither in 1, nor in 2, the nitro group is coplanar
with the phenyl ring. The angles between the best planes estimated
for these fragments are 2.4°, 14.8°, and 5.1° for 1A, 1B, and 2, respec-
tively. This effect has been observed and quantified in acetylated
gluco- and galactopyranosides29 and supportsthe view that theni-
tro group does not effectively conjugate with the benzene ring and
even weak interactions in the solid-state may easily twist it. This ef-
fect is also observed in 3, where the twist angle is equal to 12.8° or
1
3
can observe the
C resonances of the ethanol at 59.4 and
15.2 ppm (Fig. 6b); complex pattern of hydrogen bond interactions
involving the disordered ethanol molecules is reflected in the mul-
tiplicity of sugar resonances.
34
In the solid-state spectra of 1 and 3–6 the number of resonances
exceeded the number of carbon atoms in the molecule, suggesting
that there are at least two different molecules in the independent
unit cell. The doublets of C-1 and C-7, as well as of C-10, can be eas-
ily recognized (Fig. 5a). The main differences between these mole-
cules are the relative orientations of the pyranosidic and aromatic
rings, as indicated by the XRD data for 1, 3, and 5 (there is no X-ray
structure for 6). However, the structural differences may also result
from two orientations of the aromatic ring, the resonances of C-1
3
0,31
1
6.3° (for two independent molecules)
and in 5, where the angle
3
3
32
may range from 2.0° to 8.2°.
4
The sugar moieties of 1 and 2 adopt C
1
conformation. The Cre-
mer–Pople puckering parameter35
while the other puckering parameters, Q and u, are equal to
.579 (2) and 310 (3)°, respectively. For 1, , Q, and u are equal
to 3.1 (2)°, 0.543 (2) and 143 (3)° and 2.0 (2)°, 0.556 (2) and 23
H for 2 is equal to 4.2 (2)°,
0
H

1
738
T. Gubica et al. / Carbohydrate Research 344 (2009) 1734–1744
Figure 3. Crystal packing of p-nitrophenyl a-D-galactopyranoside (1); hydrogen bonds are indicated as dashed lines.
and C-7 appeared as singlets, whereas the resonances of C-8 and
C-12 are split.
(Table 1). The experimental powder X-ray diffraction patterns of
1 (Fig. 7), 3 (Fig. 8) and 5 (Fig. 9) closely match those calculated
from a single-crystal X-ray diffraction refinement in 2h angles.
The observed small variation in the relative peak intensities is sub-
jected to the preferred orientation effects. This confirms the pres-
ence of the particular crystal forms in the samples. Therefore, the
splitting of resonances in C CP MAS NMR spectrum of 1, 3, and
5 seems to be caused by two non-equivalent molecules in the inde-
pendent part of the crystal unit, and not by two polymorphs exist-
ing in these samples (Table 1).
In the solid-state spectrum of 2, distinct singlet resonances are
observed, suggesting that 2 consists of only one kind of magneti-
cally equal molecules. Therefore it seems, at this stage of investiga-
tions, that 2 exists as one polymorphic form which probably
corresponds to the data obtained from single-crystal X-ray diffrac-
tion. However, in the next section, some new details will be dis-
cussed according to the structure of 2.
1
3
In solutions, the p-nitrophenyl group (aglycon) can rotate freely,
in solids the equilibration is usually inhibited revealing specific
environments with different chemical shifts. Separate signals of
both C-8 and C-12, as well as of both C-9 and C-11, (the carbon
atoms in the aromatic ring) were visible in the solid-state spectra.
The splitting (of 3.1–10.5 ppm) is clearly observable especially for
C-8 and C-12. One can expect that the chemical shifts of C-7, C-8
and C-12 reflect the conjugation of free electron pairs of glycosidic
oxygen atom with the aromatic system (as suggested by the length
of the C-7–O-1 bond which is shorter than the typical single one),
and that they are sensitive to the twist angle of the aromatic ring.
However, there is no simple relationship between the length of
C-7–O-1 bond and chemical shift of C-7. Larger dihedral angle be-
tween the aromatic ring and the plane of C-1, C-7, and O-1 (glyco-
sidic oxygen atom) results in a relatively close approach between
the atoms of the sugar residues and C-8 and C-12 of the aromatic
ring (and can be relieved by the enlargement of the valence
angles).
Powder X-ray diffraction pattern for 2 (Fig. 10) indicated the
presence of two polymorphs, monohydrate of p-nitrophenyl b-
D-
galactopyranoside (monoclinic system with space group P2 and
1
cell parameters: a = 10.898(2) Å, b = 4.6350(9) Å, c = 13.513(3) Å,
3
b = 91.13(3)°, and V = 682.44 Å ) and another unknown crystal
form present in a small amount. Due to such a small content of this
unknown polymorphic form it was impossible to determine the
parameters of its unit cell. The unit cell parameters of the mono-
clinic form were refined from its diffraction patterns using a full-
3
6
pattern profile fitting method using the program GSAS (Fig. 11).
The parameters of the refined unit cell are a = 10.924(1) Å,
3
b = 4.6689(5) Å, c = 13.705(1) Å, b = 91.76(1)°, and V = 698.70 Å .
The resulting volume of the unit cell is slightly larger. This may
be the result of the greater water content in monoclinic form of
p-nitrophenyl b-
. Experimental
3.1. p-Nitrophenyl a-D-galactopyranoside (1)
D-galactopyranoside in 2.
3
2
.3. Powder X-ray diffraction
The confirmation of the crystal structures and NMR data for 1–3
and 5 was obtained from the powder X-ray diffraction patterns
This compound was purchased from Koch Light Laboratories
Ltd.

T. Gubica et al. / Carbohydrate Research 344 (2009) 1734–1744
1739
Figure 4. Crystal packing of p-nitrophenyl b-D-galactopyranoside (2); hydrogen bonds are indicated as dashed lines.
Table 4
The 13C NMR chemical shifts (d, ppm) for p-nitrophenyl glycopyranosides 1–6 in: DMSO-d
given in parentheses)
6
solutions (dsolution); solid phase (dsolid) and the differences
D
= dsolution ꢁ dsolid >1 ppm
(
Atom
Compound
1
2
3
4
5
6
d
solid
d
solution
d
solid
d
solution
d
solid
d
solution
d
solid
d
solution
d
solid
d
solution
d
solid
d
solution
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-11
C-12
98.7/96.7
—/ꢁ1.4)
68.3
98.05
100.4
70.3
101.29
70.95
98.8/97.7
(ꢁ1.2/—)
69.9 (1.3)
97.65
100.1/98.7
99.69
96.2 (2.4)
70.1
98.58
97.7
97.23
(
67.64
69.25
68.35
72.86
60.15
71.19
72.83
69.59
74.26
60.49
73.2/70.9
72.95
77.08
69.34
76.29
60.38
69.59
70.42
66.41
75.43
60.82
70.4/72.0
(—/ꢁ2.0)
69.98
(
ꢁ/2.1)
79.1/76.2
ꢁ2.0/—)
67.2/65.8
2.1/3.5)
74.5/73.2
1.8/3.1)
62.5/61.8
ꢁ2.1/ꢁ1.4)
160.7 (1.6)
72.4 (ꢁ3.2)
70.7 (ꢁ2.4)
75.0 (ꢁ2.1)
63.9 (ꢁ3.8)
165.1/162.6
74.8
73.97
72.1
71.5 (ꢁ1.1)
74.4 (ꢁ1.3)
73.09
(
70.3
68.91
68.1 (1.5)
75.1
66.0/64.9
(—/1.5)
73.3 (2.1)
65.5/63.7
(—/2.8)
77.9
66.50
(
77.5
76.56
77.73
(
61.3
61.16
59.1 (1.4)
161.2
60.3
60.9/59.9
162.8
60.79
(
162.43
116.85
125.61
141.41
125.61
116.85
161.4
113.9
124.8
140.2
127.0
113.9
163.32
117.35
126.55
142.40
126.55
117.35
162.16
116.81
125.63
141.47
125.63
116.81
162.28
116.42
125.60
141.53
125.60
116.42
160.1/158.8 (1.2/2.5)
120.1 (ꢁ3.3)
161.34
116.76
125.62
141.52
125.62
116.76
162.05
116.31
125.59
141.39
125.59
116.31
(
ꢁ2.7/—)
121.3/116.5
ꢁ4.5/—)
127.5/125.3
ꢁ1.9/—)
143.3/141.5
ꢁ1.9/—)
127.5/125.3
ꢁ1.9/—)
118.8/115.7
ꢁ2.0/1.2)
121.7/113.7
(ꢁ4.9/3.1)
125.0
117.2/113.6
(—/2.8)
126.5/123.7
(—/1.9)
119.1/117.6
(ꢁ2.8/ꢁ1.3)
122.8/121.8
(2.8/3.8)
140.4/139.1
(—/2.3)
125.3/124.0
(—/1.6)
114.5/113.9
(1.8/2.4)
(
124.5 (1.1)
(
141.5
125.0
142.2
143.1/141.5
(ꢁ1.6/—)
124.5
(
126.5/123.7
(—/1.9)
115.6/112.4
(—/4.0)
(
120.0/111.2
(ꢁ3.2/5.6)
115.1/113.5
(1.7/3.3)
(

1
740
T. Gubica et al. / Carbohydrate Research 344 (2009) 1734–1744
Figure 5. 13C CP MAS NMR spectra of (a): p-nitrophenyl
a-D-galactopyranoside (1) and (b): p-nitrophenyl b-D-galactopyranoside (2); rotation speed of 10 kHz.
3
.2. p-Nitrophenyl b-
D
-galactopyranoside (2)
3.6. Physical measurements
Deacetylation of p-nitrophenyl 2,3,4,6-tetra-O-acetyl-b-
D
-galac-
The 1H and 13C NMR spectra for DMSO-d
solutions were re-
6
2
9
1
13
topyranoside by Zemplén method gave 2 as white crystals; mp
corded on a Bruker DRX-400 spectrometer; the (2D) H– C corre-
lations were run using standard pulse programs from Bruker
2
D
0
37
23
D
1
ꢁ
80–182 °C, ½
a
2
ꢃ
ꢁ80.6 (c 0.5, H
2
O); lit. mp 180–182 °C, ½ ꢃ
a
1
3
79.5 (c 1, H
O).
library. Solid-state C cross-polarization (CP) magic angle spin-
ning (MAS) spectra were recorded on a Bruker DRX-400 spectrom-
eter at 100.61 MHz. Powder samples were spun at 10 kHz in a
3
.3. p-Nitrophenyl a-D-glucopyranoside (3)
4
2
mm ZrO rotor (a contact time of 2 ms, a repetition time of 6 s,
1
3
This compound was purchased from Sigma–Aldrich Chemie
GmbH.
and 600 scans). C chemical shifts were calibrated indirectly
through the glycine CO signal recorded at 176.0 ppm, relative to
TMS.
3
.4. p-Nitrophenyl b-
D
-glucopyranoside (4)
The X-ray diffraction measurements of 1 and 2 were performed
on a KUMA CCD
j-axis diffractometer with graphite-monochro-
This compound was obtained from p-nitrophenyl 2,3,4,6-tetra-
O-acetyl-b-
-glucopyranoside29 according to the procedure that
was the same as that used for obtaining 2. White crystals; mp
mated Mo K radiation (0.71073 Å) at 150 ± 2 K and 100 ± 2 K,
a
D
respectively. The crystals were positioned at 62.3 mm from the
KM4CCD camera. 664 frames were measured at 0.9° intervals with
a counting time of 20 s, and 664 frames were measured at 0.9°
intervals with a counting time of 40 s, respectively. The data were
corrected for Lorentz and polarization effects. The numerical
absorption correction was not applied. Data reduction and analysis
were carried out with the Kuma Diffraction (Wrocław, Poland) pro-
2
0
38
39
1
66–167 °C, ½
a
ꢃ
ꢁ102.3 (c 1, H
2
O) lit. mp 165–167 °C; lit.
D
2
D
5
½
a
ꢃ
ꢁ108.3 (H
2
O).
3
.5. p-Nitrophenyl
a-D-mannopyranoside (5) and p-Nitrophenyl
b-D
-mannopyranoside (6)
4
0
41
grams. The structures were solved by direct methods and re-
4
2
2
These compounds were purchased from Sigma–Aldrich Chemie
GmbH.
fined using SHELXL
reflections except for those with very negative F . The weighted
.
The refinement was based on F for all
2

T. Gubica et al. / Carbohydrate Research 344 (2009) 1734–1744
1741
Figure 6. 13C CP MAS NMR spectra of (a): p-nitrophenyl
a-
D-glucopyranoside (3) and (b): p-nitrophenyl b-D-glucopyranoside (4); rotation speed of 10 kHz.
2
R factor, wR, and all goodness-of-fit S values are based on F . The
non-hydrogen atoms were refined anisotropically, whereas the
H-atoms were placed in the calculated positions; their positions
and thermal parameters were refined isotropically. Scattering fac-
tors were taken from Tables 6.1.1.4 and 4.2.4.2.43 Crystal data to-
gether with the data collection and structure refinement details
are listed out in Table 2.
The powder X-ray diffraction patterns of 1–3 and 5 were re-
corded on a Seifert HZG-4 automated diffractometer using Cu
Ka1,2 radiation (1.5418 Å). The data were collected in the Brag-
Figure 7. PXRD pattern of 1 (a) and theoretically calculated pattern of p-nitrophenyl a-D-galactopyranoside (b).

1
742
T. Gubica et al. / Carbohydrate Research 344 (2009) 1734–1744
Figure 8. PXRD pattern of 3 (a) and theoretically calculated pattern of p-nitrophenyl a-D
-glucopyranoside (b).30
Brentano (h/2h) horizontal geometry (flat reflection mode) be-
1–3 and 5 (Table 1). According to the XRD measurements, 1
and 2 crystallize with one water molecule which interacts with
sugar molecules by hydrogen bonds. In 1 and 2 a complex hydro-
gen bond network is observed. There are two independent mole-
cules in the crystal unit cell in 1, whereas in 2 there is one
molecule. The most distinct structural differences between both
molecules of 1 (1A and 1B) are found in the torsion angles around
the hydroxyl groups at C-2, C-4, and C-6, while the most signifi-
cant differences between structures of 1 and 2 are connected with
the geometry at the anomeric carbon atom. Therefore, due to the
anomeric effect, characteristic lengthening and shortening of
appropriate bond lengths are observed between the two anomers.
In both 1 and 2, the nitro group is not coplanar with the benzene
ꢁ1
tween 4 and 40° (2h) in 0.04° steps, at 5 s step . The optic of the
HZG-4 diffractometer was a system of primary Soller slits between
the X-ray tube and the fixed aperture slit of 2.0 mm. One scattered-
radiation slit of 2 mm was placed after the sample, followed by the
detector slit of 0.2 mm. The X-ray tube operated at 40 kV and
4
0 mA. Powder XRD patterns were simulated from single-crystal
44
data using the program MERCURY
.
4
. Conclusions
The structural analyses of p-nitrophenyl glycopyranosides, the
derivatives of -galactose, -glucose, and -mannose, often used
D
D
D
ring. Moreover, the molecules of 1 and 2 adopt slightly disordered
in enzymology as markers, were performed using the following
experimental techniques: single-crystal X-ray diffraction (XRD)
4
C
1
conformations.
1
3
In solid-state NMR spectra, separate signals of C-8 and C-12
as well as of C-9 and C-11 are detected and this phenomenon
for 1 and 2, C NMR spectroscopy in both solid and solution
phases for 1–6, and the powder X-ray diffraction (PXRD) for
Figure 9. PXRD pattern of 5 (a) and theoretically calculated pattern of p-nitrophenyl a-D
-mannopyranoside (b).32

T. Gubica et al. / Carbohydrate Research 344 (2009) 1734–1744
1743
Figure 10. PXRD pattern of 2 (a) and theoretically calculated pattern of p-nitrophenyl b-D-galactopyranoside (b).
is connected with a frozen rotation of the p-nitrophenyl group,
the aglycon, in the solid phase in contrast to the solution phase
in which this group can rotate freely. In contrast, the resonances
of carbon atoms bearing hydroxyl groups are similar in both so-
lid and solution phases, because of the existence of hydrogen
bonds in both cases. Solid 4 exists as ethanol solvate, as sug-
The powder X-ray diffraction patterns reveal that 1, 3, and 5
consist of only one polymorphic phase which corresponds to
the appropriate XRD measurements, so the splitting of resonances
in the solid-state spectra of these compounds is caused not by
two polymorphs, but by two magnetically different molecules in
the separate polymorph. The PXRD pattern for 2 indicates the
existence of two polymorphic forms, one of them is unknown,
and the subsequent one is similar to the calculated pattern. The
slight difference between the XRD and PXRD of 2 is connected
with a bigger volume of the unit cell in the macroscopic sample
1
3
gested by the characteristic signals in the solid-state C NMR
spectrum. For and 3–6 two magnetically non-equivalent
1
molecules are detected, while separate signals have been noticed
for 2.
Figure 11. Experimental (ꢂ), calculated (red, solid line), and difference (green, lower line) PXRD profiles for the LeBail fitting of p-nitrophenyl b-
D-galactopyranoside (2).

1
744
T. Gubica et al. / Carbohydrate Research 344 (2009) 1734–1744
used in PXRD in contrast to the single-crystal sample employed in
XRD. Furthermore, this difference could be connected with the
greater water content in the polymorph of the macroscopic
sample.
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