Syntheses of the 4-nitrophenyl glycosides of hyalobiuronic acid and chondrosine.

Article abstract of DOI:10.1016/S0008-6215(02)00114-3

4-Nitrophenyl [sodium beta-D-glucopyranosyluronate]-(1-->3)-2-acetamido-2-deoxy-beta-D-gluc opyranoside (1) and 4-nitrophenyl [sodium beta-D-glucopyranosyluronate]-(1-->3)-2-acetamido-2-deoxy-beta-D-galactopyran oside (2) were prepared from the zwitterions hyalobiuronic acid [beta-D-glucopyranuronic acid-(1-->3)-2-amino-2-deoxy-D-glucopyranose] and chondrosine [beta-D-glucopyranuronic acid-(1-->3)-2-amino-2-deoxy-D-galactopyranose], respectively. Compounds 1 and 2 were not hydrolysed by bovine testicular hyaluronidase.

Full text of DOI:10.1016/S0008-6215(02)00114-3

Carbohydrate Research 337 (2002) 1229–1233
www.elsevier.com/locate/carres
Structure of the complex of
heptakis(2,6-di-O-methyl)-b-cyclodextrin with
(2,4-dichlorophenoxy)acetic acidꢀ
Frantzeska Tsorteki, Dimitris Mentzafos*
Physics Laboratory, Agricultural Uni6ersity of Athens, Iera Odos 75, GR-11855 Athens, Greece
Received 23 January 2002; received in revised form 18 April 2002; accepted 3 May 2002
Abstract
The structure of the complex formed by heptakis(2,6-di-O-methyl)-b-cyclodextrin and (2,4-dichlorophenoxy)acetic acid was
studied by X-ray diffraction. The dichlorophenyl moiety of the guest molecule was found outside the host hydrophobic cavity in
the primary methoxy groups region whereas the oxyacetic acid chain penetrates the cavity from the primary face. The host
molecules stacks along the a crystal axis forming a column. In the space between three successive hosts of the column, a guest
molecule is accommodated. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Inclusion compound; Crystal structure; Heptakis(2,6-di-O-methyl)-b-cyclodextrin; (2,4-Dichlorophenoxy)acetic acid; Screw column
1. Introduction
formationally similar to native b-CD as there still exist
the hydrogen bonds between the O-2 methoxy and O-3
hydroxy groups of adjacent glucose units, keeping the
macrocycle rigid. However, steric hindrance due to
methylation of the O-2 hydroxy groups does not allow
for a dimerization, as in the majority of the crystalline
b-CD complexes. The methylation extends the depth of
(2,4-Dichlorophenoxy)acetic acid (2,4-D, 2), a syn-
thetic compound causing many responses common to
natural auxins, is classified as a plant growth regulator
and not as a hormone, since it is not produced by the
plants.2 It is the first selective herbicide discovered and,
as it is chemically very stable, it has been widely used
commercially. Although it exhibits auxin activity at low
concentrations, as all the phenoxyacetic acids, it be-
comes phytotoxic at relatively higher concentrations
and thus it was used as a defoliant in the war in
,
the hydrophobic torus, estimated to be 10–11 A com-
,
pared with 7.8 A for the native b-CD, enabling it to
form complexes with larger substrates.7 In spite of this
ability of the host, the guest molecules are not found
inside the DMbCD macrocycle cavity in four reported
structures. In three complexes, those of p-iodophenol/
DMbCD, p-nitrophenol/DMbCD8 and 2-naphthoic
acid/DMbCD,9 the guest was found outside the cavity
in the region of the primary methoxy groups, while in
carmofur (1-hexylcarbamoyl-5-fluorouracil)/DMbCD10
it was located in the area of the secondary rim of CD.
In three others, those of 1,7-dioxaspiro[5.5]undecane/
DMbCD,11 adamantanol/DMbCD7,12 and acetic acid/
DMbCD13 complexes, the guest was located inside the
cavity. In a continuing effort to study inclusion com-
pounds of plant growth regulators in cyclodextrins, we
prepared the complex of 2,4-D/DMbCD, the crystal
structure of which is now presented.
Vietnam.3
Heptakis(2,6-di-O-methyl)cyclomaltohep-
taose (DMbCD, 1), a chemically modified cyclomalto-
heptaose (b-cyclodextrin, b-CD) derivative arising by
methylation of the 2 and 6 hydroxyl groups, exhibits an
increased solubility in water and organic solvents,4
compared to the native b-CD, enabling to use it as a
carrier for poorly soluble molecules in hydrophobic
solvents or in water.5,6 The DMbCD molecule is con-
ꢀ Part III in the series: Inclusion compounds of plant
growth regulators in cyclodextrins. For Part II, see: Ref. 1.
* Corresponding author
E-mail address: mentz@aua.gr (D. Mentzafos).
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00114-3

1230
F. Tsorteki, D. Mentzafos / Carbohydrate Research 337 (2002) 1229–1233
2. Experimental
tions with 2qmax B100°. The scan mode was q–2q.
Three standard reflections monitored every 97 reflec-
tions showed an overall decay of 14.2% of the intensity.
Lorenz, polarization and decay corrections were ap-
plied to the intensity data.
Crystallization.—2,4-D (obtained from Fluka) and
DMbCD (purchased from Cyclolab) was dissolved in
water (concentrations 0.02 M) at a 1:1 host:guest mole
ratio. The resulting mixture was heated at 50 °C and
left at this temperature for a period of three days, at the
end of which colorless crystals of the title complex
suitable for X-ray data collection had formed.
X-ray data collection.—Final lattice parameters, de-
termined by 32 reflections, are given in Table 1 along
with other information on data collection and structure
refinement. Data collection was done on a crystal,
sealed in a Linderman glass capillary, on a Syntex P21
four-circle diffractometer upgraded by Crystal Logic,
attached to a Rigaku rotating anode generator, using a
graphite monochromatized Cu Ka radiation. Prelimi-
nary data collection has indicated the crystal to be
orthorhombic. As a consequence, one quarter of the
sphere was collected giving 8941 (8245 unique) reflec-
Structure solution and refinement.—The structure was
solved by molecular replacement using the skeleton
atom coordinates of the DMbCD molecule of the p-ni-
trophenol/DMbCD complex.8 Sequential difference
electron density maps (Dz) revealed the positions of the
remaining non-hydrogen atoms of the host, all the
atoms of the guest and the oxygen atom of one water
molecule. The refinement, based on F2, proceeded by
using the SHELXL-97 program.14 Hydrogen atoms
linked to carbon atoms of the host and guest molecules
were used at calculated positions with CꢀH distance
,
,
,
0.98 A for the tertiary, 0.97 A for the secondary, 0.96 A
,
for the primary and 0.93 A for the benzyl H-atoms,
while their thermal parameters have been set to 1.2×
Uiso of the isotropic thermal parameter of the corre-
sponding C atom. All the non-hydrogen atoms of the
host molecule, the chlorine atoms of the guest and the
oxygen atom of the water molecule were refined an-
isotropically. Extinction correction was applied, and 19
reflection intensities exhibiting poor agreement were
given zero weight during the final refinement cycles.
The refinement converged at R=0.0753 and 0.1479 for
observed (Io\2.0|(Io)) and all reflections (Table 1),
respectively. An atomic numbering of the host and
guest molecules is given in Scheme 1. A top and side
views of the complex are given in Fig. 1. C-mn and
O-mn denoting the mth atom within the nth glucosidic
residue of the host.
Table 1
Crystal data and structure refinement for 2,4-D/DMbCD
Empirical formula
C64H91O35·C8H6O3Cl2·
(H2O)0.35
Formula weight
Temperature (K)
1557.38
293(2)
,
Wavelength, u Cu Ka (A)
1.54180
Crystal system, space group Orthorhombic, P212121
,
Unit cell dimensions (A)
a=14.886(7)
b=18.980(4)
c=28.515(6)
8057(4)
−3
,
Volume (A
Z
)
4
Dcalcd (Mg/m−3
)
1.284
Absorption coefficient
(mm−1
F(000)
Crystal size (mm)
q Range for data collection
(°)
1.486
3. Results and discussion
)
3316
0.3×0.4×0.5
2.80–50.01
Molecular geometry and conformation of DMiCD.—
Two methyl groups, the primary C-91 and the sec-
ondary C-72, and one methoxy group, the O-65ꢀC-95,
are disordered over two sites, the occupancies of their
major sites being 0.69, 0.66 and 0.54, respectively. The
DMbCD atoms exhibit an increased thermal move-
ment, about the same as the atoms of the guest
molecule. This is not unusual, as it is observed also in
the complex of DMbCD with 1,7-dioxaspiro-
[5.5]undecane11 and the anhydrous DMbCD.15 The
thermal parameters of C-93 are particularly high but
attempts to find more than one atomic sites failed. Four
methoxy groups have a gauche–gauche orientation
pointing outwards of the cavity, see the torsion angles
(Table 2). The O-63ꢀC-63 and O-64ꢀC-64 and both
sites of the disordered O-65ꢀC-65 groups exhibit a
gauche–trans orientation pointing towards the
DMbCD cavity. Thus the opening is limited at the
primary side of the CD cavity. The methyl groups of
Limiting indices (°)
−145h50
−185k55
−285l528
8941/8245 [R(int)=0.0414]
Reflections collected/unique
Completeness to q
Refinement method
50.01, 99.7%
Full-matrix least-squares on
F2
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I\2|(I)]
R indices (all data)
Absolute structure parameter 0.97 (6)
Extinction coefficient
8245/0/888
1.057
R1=0.0753, wR2=0.1900
R1=0.1479, wR2=0.2414
0.00076 (11)
0.242 and −0.191
Largest diff. peak and hole
−3
,
(e A
)

F. Tsorteki, D. Mentzafos / Carbohydrate Research 337 (2002) 1229–1233
1231
Scheme 1. An atomic numbering of the host and guest molecules.
is not quite well understood.8,9 An explanation is given
by Harata pretending that, because of the many methyl
groups of the hydrophobic cavity of the DMbCD
molecule, unlike that of the native b-CD, a guest
molecule can favorably be accommodated in the inter-
stitial site when its shape and size are well suited to the
space.18 Besides, the guest molecules of all these com-
plexes have a common feature: they consist of a planar
ring with hydrophilic moieties bonded to it. Note that
the adamantol molecule, having a ball shape, is quite
included inside the DMbCD cavity.7,11 The characteri-
zation of these molecules, not inserting inside the
DMbCD cavity, as ‘guests’ is valid only if we accept
that a guest must not be necessarily fully trapped inside
the cavity to be designated as such.
the secondary side are oriented away from the torus, an
arrangement favorable for the intramolecular O-
3nꢀH···O-2(n+1) hydrogen bonds12 varying between
,
2.754 and 2.972 A and giving the rigid shape to the
truncated cone of the host. The distances of the glyco-
sidic O-4n atoms from their mean plane range between
,
−0.173(6) and 0.134(5) A, their values being signifi-
cantly greater than the observed in b-CD.1,16 The O-
,
4n···O-4(n+1) distances vary between 4.36 and 4.44 A,
the O-4(n−1)···O-4n···O-4(n+1) angles range from
126.3 to 132.2°, while the distances of the approximate
center K of the O-4n heptagon vary between 4.91 and
,
5.19 A and the O-4n···K···O-4(n+1) angles range from
50.2 to 52.5°. These values indicate that although the
O-4n atoms mean plane of the DMbCD molecule is
more puckered than in the native b-CD, its shape is
nearly round. The tilt angles of glycopyranose residues,
defined as the angles between the O-4n mean plane and
the individual mean planes formed by the O-4(n−1),
C-1n, C-4n and O-4n atoms, range between 1.2 (9) and
23.5 (7)°, being all positive as in the p-iodophenol and
p-nitrophenol/DMbCD isomorphous complexes8 and
in two DMbCD-hydrates.17 Therefore, the glucose units
are tilted towards the approximate sevenfold molecular
axis.
A hydrogen bond is formed between the O-1 atom of
the carboxyl group of the guest and the water molecule
,
(2.78 A), found inside the cavity and having the low
occupancy factor of 0.35, but between the host and
guest atoms only van der Waals contacts exist. In the
p-iodophenol and the p-nitrophenol/DMbCD com-
plexes,8 H-bonds are observed between hosts and guests
of the same asymmetric unit while in the 2-naphthoic
acid/DMbCD complex, where the carboxyl group is
pointing away from the host molecule, such a H-bond
is formed between the guest and an adjacent host
molecule.9 The molecular geometry of 2,4-D is nearly
the expected, the values of the bond lengths and angles
not differing significantly from the generally accepted.
The dichlorophenyl group, the chlorine atoms and the
The guest molecules.—The main feature of the struc-
ture of the 2,4-D/DMbCD complex is that the
dichlorophenyl group of the 2,4-D molecule is located
outside the hydrophobic cavity at the primary side of
the host and the hydrophilic moiety, the oxyacetic
group, penetrates into the cavity from the primary face
(Fig. 1). This arrangement may be attributed to the
difficulty of the aromatic ring, substituted at 2 and 4
positions by chlorine atoms, to fit into the rigid cavity
and, therefore, staying in the space between three host
molecules. This arrangement, common to all three crys-
tal structures that are isomorphous to the title complex,
,
ether oxygen atom lie on a plane within 0.038 (17) A;
the carboxyl group is nearly perpendicular to this plane
forming an angle of 75 (1)° with it.
The molecular packing.—The complexes are stacked
along the a crystal axis and the O-4n best planes of the
host molecule forms an angle of 25.4° with the bc plane.
The O-4n best planes of two adjacent DMbCD

1232
F. Tsorteki, D. Mentzafos / Carbohydrate Research 337 (2002) 1229–1233
molecules of the same stack form an angle of 50.8°, two
primary methoxy groups of one DMbCD molecule
entering into the cavity of the other (Fig. 2). As a result
the DMbCD cavity is closed from the secondary side,
the host and guest molecules forming a compact
column. The molecular packing consists of such
columns linked by the b and c screw axis through van
der Waals contacts, and it is characterized as ‘cage
type’,8,9 but it may be designated also as a ‘screw
column’, as the host molecules of these compact
columns are linked by a screw axis.
This molecular packing is found also in the carmo-
fur/DMbCD complex10 and the anhydrous DMbCD
structure.15 In the former complex, the fluorouracil
moiety of the guest molecule, disordered over two sites,
is located outside the cavity, in the region of the
secondary side of the host. The long alkyl chain of the
hexyl group of the major site penetrates into the host
cavity, ending in the primary methoxy groups region,
while this of the minor site was found completely
outside the cavity. The orientation of the guest
molecule is quite different from the observed one in the
title complex, but at any rate the fluorouracil moiety
does not enter in the cavity. The molecular packing of
the complexes of DMbCD with guests having an aro-
matic or planar moiety is a ‘screw column’. To our
knowledge, only three DMbCD structures have been
found till now not having a ‘screw column’ molecular
packing: the adamantanol/DMbCD complex where the
hosts are stacked along a column,7,12 the 1,7-dioxas-
piro[5.5]undecane/DMbCD complex with hosts form-
ing layers11 and the acetic acid/DMbCD complex being
of herringbone-type.13 In all those structures, the guest
molecules are entrapped in the cavity and an aromatic
moiety is in any case not involved. The water forms
,
weak hydrogen bonds with the O-64 (2.94 A) and
,
O-65A (3.03 A) atoms of the host. In the ‘screw
column’ structures8–10 the water molecules are also
Fig. 1. A front- and side-view of the complex. C-mn and
O-mn denote the mth atom within the nth glycoside residue.
located inside the cavity.
Table 2
Selected torsion angles (°) for 2,4-D/DMbCD complex
Torsion
angles
Site
n=1
n=2
n=3
n=4
n=5
n=6
n=7
C-3nꢀC-2nꢀO A
-2nꢀC-7n
C-1nꢀC-2nꢀO A
-2nꢀC-7n
C-4nꢀC-5nꢀC A
-6nꢀO-6n
O-5nꢀC-5nꢀC A
-6nꢀO-6n
C-5nꢀC-6nꢀO A
-6nꢀC-9n
−97.8 (11)
142.2 (10)
48.7 (13)
−97.1 (14) −111.0 (19)
−136.7 (19)
−92.0 (14)
−97.1 (13)
−99.2 (13)
140.3 (11)
49.4 (12)
−96.9 (12)
142.3 (10)
51.7 (11)
B
142.1 (13)
102.5 (19)
125.3 (19)
143.9 (12)
140.3 (12)
B
46.2 (14) −160.9 (13) −170.8 (16)
170.3 (16)
−141.7 (18)
50.1 (19)
98.1 (19)
84 (3)
B
−68.4 (11)
−74.9 (12)
76.6 (16)
173 (2)
77.5 (18)
−72.8 (11)
−66.9 (10)
B
88.9 (18)
126 (2)
−172.5 (11)
−104.1 (19)
−171.8 (11) −175.7 (10)
B
176 (4)

F. Tsorteki, D. Mentzafos / Carbohydrate Research 337 (2002) 1229–1233
1233
Fig. 2. A stereo diagram of the channel of the complex. The directions of the axis are indicated.
4. Uekama K.; Irie T. In Cyclodextrins and their Industrial
Uses; Duchene D., Ed.; Editions de Sante´: Paris, 1987; pp
395–439.
5. Szejtli J. J. Incl. Phenom. 1983, 1, 135–150.
6. Szejtli J. J. Incl. Phenom. 1992, 14, 25–36.
7. Stezowski J. J.; Czugler M.; Eckle E. In Ad6. Incl. Sci.;
Szejtli J., Ed.; Reider Editions: Dordrecht, 1981; pp
151–161.
8. Harata K. Bull. Chem. Soc. Jpn. 1988, 61, 1939–1944.
9. (a) Harata K. Chem. Commun. 1999, 191–192;
(b) Harata K. J. Chem. Soc., Chem. Commun. 1993,
546–547.
4. Supplementary material
Full crystallographic details, excluding structure fac-
tors, have been deposited with the Cambridge Crystal-
lographic Data Center, deposition No CCDC 178967.
These data may be obtained, on request, from the
CCDC, 12 Union Road, Cambridge CB2 1EZ, UK.
Tel.: +44-1223-336408; fax: +44-1223-336033; e-mail:
deposit@ccdc.cam.ac.uk.
10. Harata K.; Hirayama F.; Uekama K.; Tsoucaris G.
Chem. Lett. 1988, 1585–1588.
11. Rysanek N.; Le Bas G.; Villain F.; Tsoucaris G. Acta
Crystallogr. 1992, C48, 1466–1471.
12. Czugler M.; Eckle E; Stezowski J. J. J. Chem. Soc.,
Chem. Commun. 1981, 1291–1292.
13. Selkti M.; Navaza A.; Villain F.; Charpin P.; De Rango
C. J. Incl. Phenom. 1977, 27, 1–12.
Acknowledgements
The authors are indebted to Dr. I. M. Mavridis for
several pertinent comments.
14. Sheldrick, G. M. SHELXL-97, Program for the refine-
ment of Crystal Structures, University of Go¨ttingen, Ger-
many, 1993
15. Steiner T.; Saenger W. Carbohydr. Res. 1995, 275, 73–82.
16. Kokkinou A.; Makedonopoulou S.; Mentzafos D. Car-
bohydr. Res. 2000, 328, 135–140.
17. Stezowski J.; Parker W.; Hilgenkamp S.; Gdaniec M. J.
Am. Chem. Soc. 2001, 123, 3919–3926.
18. Harata K. Chem. Re6. 1998, 98, 1803–1827.
References
1. Kokkinou A.; Yannakopoulou K.; Mavridis I. M.;
Mentzafos D. Carbohydr. Res. 2001, 332, 85–94.
2. Salisbury F. B.; Ross C. W. Plant Physiology;
Wadsworth: Belmont, CA, 1992; pp 361–372.
3. Devlin R.; Witham F. Plant Physiology; Willard Grant
Press: Boston, 1983; pp 354–356.