X-ray structure of the key synthetic intermediate of a cancer-related sialyl-Tn antigen analogue

Article abstract of DOI:10.1007/s10870-010-9864-x

We report herein the synthesis and the X-ray crystal structure of 2-azido-2-deoxy-3,4-O-isopropylidene-α-d-galactopyranosyl-N- oxyphthalimide 3 which is the key-precursor of a cancer-related STn antigen analogue 5. Compound 3 crystallizes from a mixture of dichloromethane/pentane as colorless orthorhombic needles in P2 ₁ 2 ₁ 2 space group with the following cell parameters: a = 14.039(2) A; b = 16.574(3) A; c = 8.038(1) A. The hexapyranose ring of 3 adopts a twisted boat according to the Cremer and Pople puckering parameters (Cremer and Pople, J Am Chem Soc 97:1354, 1975) whereas the structure determination of the corresponding deprotected intermediate 4 (orthorombic colorless prisms, P2 ₁ 2 ₁ 2 ₁ space group, a = 4.927(3) A; b = 13.423(2) A; c = 22.744(3) A) has revealed a quasi perfect chair.

Full text of DOI:10.1007/s10870-010-9864-x

J Chem Crystallogr (2011) 41:204–208
DOI 10.1007/s10870-010-9864-x
ORIGINAL PAPER
X-Ray Structure of the Key Synthetic Intermediate
of a Cancer-Related Sialyl-Tn Antigen Analogue
•
Olivier Renaudet Christian Philouze
•
•
Corinne Bailly Andre Durif Pascal Dumy
•
´
Received: 20 April 2010 / Accepted: 5 August 2010 / Published online: 3 September 2010
Ó Springer Science+Business Media, LLC 2010
Abstract We report herein the synthesis and the X-ray
crystal structure of 2-azido-2-deoxy-3,4-O-isopropylidene-
a-D-galactopyranosyl-N-oxyphthalimide 3 which is the
key-precursor of a cancer-related STn antigen analogue 5.
Compound 3 crystallizes from a mixture of dichlorometh-
ane/pentane as colorless orthorhombic needles in P21212
space group with the following cell parameters: a =
blocks for the construction of therapeutic anti-cancer vac-
cines [4, 5]. Among other approaches, we have previously
reported in our laboratory that synthetic multiepitopic
vaccine candidates combining, on a suitable carrier mole-
cule, a cluster of TACAs and immunostimulant peptide
[6, 7] or lipopeptide epitopes [8–10] can promote a strong
immune response and protection against tumor cells. In the
course of our researches in this field, we were interested
recently on the sialyl-Tn (STn) antigen. This mucin-type
TACA is composed of a sialylated a-GalNAc residue
attached to the hydroxyl side-chain of serine or threonine
of glycoproteins expressed in many adenocarcinomas cell
surface [11].
˚
˚
˚
14.039(2) A; b = 16.574(3) A; c = 8.038(1) A. The hexa-
pyranose ring of 3 adopts a twisted boat according to the
Cremer and Pople puckering parameters (Cremer and Pople,
J Am Chem Soc 97:1354, 1975) whereas the structure
determination of the corresponding deprotected intermedi-
ate 4 (orthorombic colorless prisms, P212121 space group,
˚
a = 4.927(3) A; b = 13.423(2) A; c = 22.744(3) A) has
˚
˚
In order to ensure the controlled molecular assembly of
our vaccine prototype, we focused on the synthesis of the
aminooxy STn analogue 5 (Scheme 1) as cancer-related
carbohydrate antigen to be introduced in a multivalent
display into the aldehyde-containing cyclopeptide carrier
or conjugated with other molecules by chemoselective
oxime ligation [12–26]. This compound 5 was indeed used
successfully for the synthesis of mucin mimics following a
similar chemoselective procedure [27]. For this purpose,
we developed a synthetic route of 5 requiring the prepa-
ration the key synthetic intermediate 3 (Scheme 1). Herein,
we report the synthesis of this building block 3 which was
unexplored to date and compare its X-ray analysis with the
corresponding unprotected derivative 4.
revealed a quasi perfect chair.
Keywords Carbohydrate Á Cancer antigen Á Pyranose ring
Introduction
The glyco-patterning of the extracellular membrane is the
major difference between malignant and normal cells.
Tumors are indeed characterized by altered or over-
expressed carbohydrate markers, namely Tumor Associ-
ated Carbohydrate Antigens (TACAs), which are displayed
as repeated and multimeric units by proteins and lipids in
the cell membrane [1–3]. Recent reports have clearly
shown that these TACAs represent essential building
The envisioned chemoselective procedure requires the
incorporation of an aminooxy function to the anomer
position of the carbohydrate moiety. This can be easily
achieved from the fluoride derivative 1 which was gly-
cosylated with N-hydroxyphthalimide using boron tri-
fluoride etherate (Scheme 1) following a previously
reported protocol [28, 29]. We thus obtained the fully
protected glycosylated derivative as an anomer mixture
O. Renaudet (&) Á C. Philouze Á C. Bailly Á A. Durif Á P. Dumy
´
Departement de Chimie Moleculaire, UMR-CNRS 5250
and ICMG FR 2607, Universite Joseph Fourier, BP 53,
´
´
38041 Grenoble Cedex 9, France
e-mail: olivier.renaudet@ujf-grenoble.fr
123

J Chem Crystallogr (2011) 41:204–208
205
N
-PhthOH
HO
OH
BF3.Et2O
CO2H
O
OTBDPS
O
Et3N
CH2Cl2
OR
O
HO
AcHN
O
O
O
O
O
HO
O
N3
F
N3
OH
O
O
N
1
5
AcOH, Bu4NF
THF
HO
2: R = TBDPS
3: R = H
AcHN
O
O
NH2
CF3CO2H
CH2Cl2
OH
HO
HO
O
O
N3
O
N
4
O
Scheme 1 Synthesis of compounds 3 and 4
from which the desired alpha anomer 2 was recovered by
silica gel chromatography. The subsequently desilylation
of 2 afforded the regioselectively protected compound 3.
The latter derivative was next crystallized from a mixture
of dichloromethane and pentane. In order to evaluate the
influence of isopropylidene protecting group on the sugar
ring conformation, compound 3 was further deprotected by
acidolysis to afford 4 after crystallisation from methanol.
73.6, 73.1, 70.4, 62.6, 59.2, 27.7, 25.9. The electron spray
ionization mass spectrometry (ES-MS) was recorded on a
VG Platform II (Micromass). ES-MS analysis (positive
mode): calcd. 390.3, found 390.9 (M ? H?). The com-
pound 4 was prepared from 3 by acidolysis with a solution
of trifluoroacetic acid in dichloromethane and was crys-
1
tallized in methanol. H NMR (300 MHz, (CD3)2SO) d
(ppm): 7.88 (s, 4H), 5.51–5.48 (m, 2H), 4.94 (d, 1H,
J = 4.2 Hz), 4.59 (bt, 1H, J = 5.4 Hz), 4.42 (bt, 1H,
J = 6.4 Hz), 4.01–3.79 (m, 2H), 3.81 (dd, 1H, J = 3.7,
10.7 Hz), 3.58–3.50 (m, 1H), 3.45–3.37 (m, 1H); 13C NMR
(75 MHz, (CD3)2SO) d (ppm): 162.9, 134.9, 128.4, 123.3,
103.4, 72.9, 67.8, 67.4, 59.3, 59.0. ES-MS analysis (posi-
tive mode): calcd. 350.3, found 351.0 (M ? H?).
Experimental Section
Synthesis of 3 and 4
The glycosylation reaction between the D-galactosyl fluo-
ride 1 (450 mg; 0.92 mmol) and N-hydroxyphthalimide
(150 mg; 1 equiv.) was performed in dichloromethane in
the presence of triethylamine (136 lL; 1 equiv.) and
BF3 Á Et2O (470 lL; 4 equiv.) as promoter [28]. The alpha
anomer 2 (235 mg; 40% yield) was recovered after clas-
sical work-up and silica gel purification (eluant: hexane/
ethyl acetate 4:1). The tert-butyldiphenylsilyl (TBDPS)
protecting group of 2 (875 mg; 1.39 mmol) was finally
removed by treatment with acetic acid (150 lL; 2 equiv.)
and tetrabutyl ammonium fluoride (2.78 mL; 2 equiv.) to
afford 3 which was crystallized as single crystals in a
mixture of CH2Cl2 and pentane by slow evaporation of the
solvent at room temperature (325 mg; 60% yield). NMR
spectra were recorded using Bruker AC300 spectrometers.
1H NMR (300 MHz, CDCl3) d (ppm): 7.89–7.78 (m, 4H),
5.55 (d, 1H, J = 4.2 Hz), 5.01–4.97 (m, 1H), 4.55 (dd, 1H,
J = 5.8, 7.3 Hz), 4.42 (dd, 1H, J = 2.4, 5.8 Hz), 4.00–3.87
(m, 3H), 1.54 (s, 3H), 1.41 (s, 3H); 13C NMR (75 MHz,
CDCl3) d (ppm): 163.3, 134.8, 128.7, 123.8, 110.7, 103.2,
Crystal Mounting and Data Collection
We selected a needle-shape crystal of 0.36 9 0.15 9
0.12 mm and a prismatic crystal of 0.34 9 0.30 9
0.29 mm for 3 and 4, respectively. Both crystals were stuck
on a glass fiber using epoxy resin and mounted at
room temperature (293 K) on a Bruker-AXS-Enraf-Nonius
CAD-4 automated 4-circle diffractometer, working at
the monochromated (graphite) Cu Ka radiation k =
˚
1.54178 A. We used the CAD-4 software [30] to achieve
the data reduction, the cell determination and refinement.
For both crystals, the cell determinations were performed
using 25 reflections (with 20.27° \ H \ 23.80° for 3 and
20.02° \ H \ 36.68° for 4) and the data collections were
performed up to H = 75.00° (with 2229 reflections for 3
and 1848 for 4). Since no heavy atom was part of the
chemical formula, we did not collect Friedel pairs. The
data were corrected for decays and for the Lorentz and
123

206
J Chem Crystallogr (2011) 41:204–208
Table 1 Crystal data and structure refinement
Compound
Name
3
4
2-azido-2-deoxy-3,4-O-isopropylidene-a-
D-galactopyranosyl-N-oxyphthalimide
2-azido-2-deoxy-a-D-galactopyranosyl-
N-oxyphthalimide
CCDC deposit no.
Color/shape
700851
700852
Colorless/needle
Colorless/prism
C14H14N4O7
350.29
Chemical formula
Formula weight
C17H18N4O7
390.35
Temperature (K)
Crystal system
293
293
Orthorhombic
P21212
Orthorhombic
P212121
Space group
˚
˚
a = 14.039(2) A; b = 16.574(3) A;
˚
a = 4.927(3) A; b = 13.423(2) A;
˚
Unit-cell dimensions (25 reflections)
˚
c = 8.038(1) A
˚
c = 22.744(3) A
3
Unit-cell volume (A )
˚
1870.2(4)
1504.2(7)
Z
4
4
Density (calculated) (g/cm3)
1.386
1.547
Absorption coefficient (mm-1
Diffractometer/scan
)
0.935
1.090
Bruker-Enraf-Nonius CAD4/omega
3.15–74.89
Bruker-Enraf-Nonius CAD4/omega-2theta
3.29–74.85
h range for data collection (°)
Reflections measured
2229
1848
Independent/observed reflections
Data/restraints/parameters
Goodness-of-fit on F
2229/1644 [I [ 2r(I)]
1644/0/253
1848/1741 [I [ 2r(I)]
1741/0/227
1.971
1.929
Final R indices [I [ 2r(I)]
R indices (all data)
R1 = 0.0615, wR2 = 0.0804
R1 = 0.0768, wR2 = 0.0936
R1 = 0.0310, wR2 = 0.0460
R1 = 0.0318, wR2 = 0.0462
polarization effects. The crystals data are available in
Table 1.
presence of one alcohol functions in 3, whereas two addi-
tional alcohols are released from the acidolysis of the
protecting group in 4. Consequently, the latter structure
contains three intermolecular hydrogen bonds (Table 2;
Fig. 1), contrarily to the protected molecule 3 which shows
only one intramolecular hydrogen bond. As a result, the
secondary structure is modified.
Crystal Structures Determinations and Refinement
The structure was solved using direct methods from SIR-92
[31], C, N, O atoms were refined anisotropically by the full
matrix least-squares method implemented by TeXsan [32].
H atoms were calculated on idealised positions but H atoms
from the hydroxyl groups were located from the Fourier
map. Drawings of the molecular structures were performed
with the use of ORTEP [33].
Both compounds display numerous chemical groups
around the hexapyranose ring, such as the N-hydroxyph-
thalimido group at C-1, an azido group at C-2 and the
alcohol function from the sugar at C-6, which remain
unchanged after deprotection. The main structural differ-
ence between 3 and 4 is due the isopropylidene removal at
C-3 and C-4 to restore the hydroxyl group of the carbo-
hydrate in 4. While the expected distances and angles are
observed [34], a significant modification in the hexapyra-
nose ring conformation is, however, induced. Indeed, the
compound 4 stands as a classical chair conformation
(Fig. 2) by contrast with the compound 3 which shows a
clear distortion between a regular and a twist boat con-
formation (Fig. 3), presumably due to the constraint of the
fused five-membered ring acetal at C-3 and C-4.
Results and Discussion
While the chemical deprotection of the isopropylidene
group left most of the molecule unchanged, several varia-
tions were observed in the crystallographic parameters of
compounds 3 and 4. By contrast with the crystallographic
system which is orthorhombic in both molecules, the space
group changes from P21212 to P212121 for 3 and 4,
respectively, and the unit-cell dimensions are quite differ-
ent (Table 1). These variations can be attributed to the
This can be revealed by the calculation of the Cremer
˚
and Pople parameters [35] which are Q = 0.703(5) A,
U2 = 219.3(4)°, H = 87.1(4)° for compound 3 and Q =
123

J Chem Crystallogr (2011) 41:204–208
207
Table 2 Hydrogen bonds for compounds 3 and 4
Com-
pound
A
H
B
B-adc A_B
A–H H_B A–H_B
3
4
4
4
O(6) H(1) O(14)
1 2.938(5) 1.13 1.86 156.9
O(3) H(1) O(6) 75604 2.650(2) 0.93 1.73 173.1
O(6) H(3) O(3) 64604 2.780(2) 1.01 1.83 155.1
O(4) H(2) O(14) 75604 3.027(2) 1.01 2.09 152.8
Fig. 3 Ortep drawing for compound 3
axial orientation of the azido group in compound 3 whereas
an equatorial orientation would have been observed as in
compound 4.
As regard to the stereochemistry, the absence of heavy
atom in 3 and 4 precludes the direct determination of their
absolute configuration. However, we assume that the rela-
tive configuration of C-2 to C-6 in compounds 3 and 4 is
similar to the starting material, i.e. D-galactose, since both
compounds were obtained as enantio pure compounds
crystallising in acentric groups. Indeed, no inversion of
chirality was induced during the chemical route from this
natural carbohydrate. The main uncertainty could concern
the stereoselectivity of the glycosylation reaction between 1
and N-hydroxyphthalimide. Due to the absence of neigh-
bouring group participation in 1, i.e. the azido group at C-2
is considered as ‘‘non-participating’’ group in a glycosyla-
tion reaction, we expected the formation of the alpha ano-
mer as major stereoisomer by virtue of anomeric effect.
This was unambiguously showed by NMR experiments
since the coupling constant values (J) measured between the
protons H-1 and H-2 in 3 are in perfect agreement with the
alpha configuration (J = 4.2 Hz). A similar coupling con-
stant value was observed for 4, suggesting that the isopro-
pylidene deprotection had no influence on the anomer
configuration.
Fig. 1 Ortep view for compound 4 of the cell showing the four
molecules generated by the symmetry operations: (i) x, y, z; (ii)
1/2 - x, -y, 1/2 ? z; (iii) 1/2 ? x, 1/2 - y, -z; (iv) -x, 1/2 ? y,
1/2 - z and the hydrogen bonds between them (dotted line)
Conclusion
2-azido-2-deoxy-3,4-O-isopropylidene-a-D-galactopyrano-
syl-N-oxyphthalimide 3 is a new key building block
towards the synthesis of the cancer-related aminooxy STn
antigen 5. This compound was prepared from D-galactosyl
fluoride derivative 1 by a glycosylation reaction using
N-hydroxyphthalimide. The expected alpha anomer was
isolated by silica gel chromatography then compound
3 was obtained after desilylation and crystallisation.
Fig. 2 Ortep drawing for compound 4
˚
0.549(2) A, U2 = 266.1(3)°, H = 0.2(2)°. Together with
this conformational cycle distortion, the intramolecular
hydrogen bond between O-6 and O-14 might explain the
123

208
J Chem Crystallogr (2011) 41:204–208
8. Bettahi I, Dasgupta G, Renaudet O, Chentoufi AA, Zhang X,
Carpenter D, Yoon S, Dumy P, BenMohamed L (2009) Cancer
Immunol Immunother 58:187
9. Renaudet O, BenMohamed L, Dasgupta G, Bettahi I, Dumy P
(2008) ChemMedChem 3:737
Isopropylidene removal of 3 by acidolysis afforded com-
pound 4 in a quantitative yield. NMR, MS and X-ray
structures analyses are in perfect agreement with structures 3
and 4. Interestingly, different hexapyranose ring confor-
mations were observed, depending on the presence of pro-
tecting group between alcohols on C-3 and C-4. The fused
five-membered isopropylidene ring in compound 3 indeed
constrains the sugar cycle, leading to a distortion between a
regular and a twist boat, whereas unprotected compound 4
reveals a quasi-perfect chair. This observation was con-
firmed by the calculation of the Cremer and Pople puckering
parameters.
10. Renaudet O, Dasgupta G, Bettahi I, Shi A, Nesburn AB, Dumy P,
BenMohamed L (2010) PLoS One 5:e11216
11. Ragupathi G (1998) Cancer Immunol Immunother 46:82
12. Renaudet O (2008) Mini-Rev Org Chem 5:274
13. Boturyn D, Defrancq E, Dolphin GT, Garcia J, Labbe P,
´
Renaudet O, Dumy P (2008) J Pept Sci 14:224
14. Renaudet O, Boturyn D, Dumy P (2009) Bioorg Med Chem Lett
19:3880
15. Dendane N, Hoang A, Renaudet O, Vinet F, Dumy P, Defrancq E
(2008) Lab Chip 8:2161
´
´
16. Dulery V, Renaudet O, Wilczewski M, Vander Heyden A, Labbe P,
Dumy P (2008) J Comb Chem 10:368
17. Wilczewski M, Van der Heyden A, Renaudet O, Dumy P, Coche-
Supplementary Material
´
´
Guerente L, Labbe P (2008) Org Biomol Chem 6:1114
18. Renaudet O, Dumy P (2008) Eur J Org Chem 32:5383
19. Renaudet O, Dumy P (2006) Org Biomol Chem 4:2628
20. Garanger E, Boturyn D, Renaudet O, Defrancq E, Dumy P (2006)
J Org Chem 71:2402
Crystallographic data (CIF) for 2-azido-2-deoxy-3,4-O-iso-
propylidene-a-D-galactopyranosyl-N-oxyphthalimide and for
2-azido-2-deoxy-a-D-galactopyranosyl-N-oxyphthalimide
have been deposited with the Cambridge Crystallographic
Data Center as supplementary publication numbers CCDC
700851 and CCDC 700852, respectively. This material is
available free of charge via www.ccdc.can.ac.uk/conts/
retrieving.html (or from the Cambridge Crystallographic
Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK. Fax:
(?44) 1223-336033. E-mail: deposit@ccdc.cam.ac.uk).
21. Dubois M-P, Gondran C, Renaudet O, Dumy P, Driguez H, Fort S,
Cosnier S (2005) Chem Commun 34:4318
22. Renaudet O, Dumy P (2005) Bioorg Med Chem Lett 15:3619
23. Edupuganti OP, Renaudet O, Defrancq E, Dumy P (2004) Bioorg
Med Chem Lett 14:2839
24. Renaudet O, Dumy P (2004) Tetrahedron Lett 45:65
25. Renaudet O, Reymond J-L (2003) Org Lett 5:4693
26. Renaudet O, Dumy P (2002) Tetrahedron 58:2127
27. Marcaurelle LA, Shin Y, Goon S, Bertozzi CR (2001) Org Lett
3:3691
28. Renaudet O, Dumy P (2001) Tetrahedron Lett 42:7575
´
Acknowledgments This work was supported by the Centre
´
National pour la Recherche Scientifique (CNRS), Universite Joseph
Fourier (UJF) and the COST Action D-34.
29. Dulery V, Renaudet O, Philouze C, Dumy P (2007) Carbohydr
Res 342:894
30. Enraf-Nonius CAD-4 Software (1988) Bruker-AXS-Enraf-No-
nius, Delft, The Netherlands
31. Altomare A, Cascarano G, Giacovazzo C, Guagliardi A (1993) J
Appl Cryst 26:343
32. Molecular Structure Corporation TeXsan (1992–1997) Single
crystal structure analysis software, version 1.7. MSC, The
Woodlands, USA
33. Johnson CK (1976) ORTEPII. Report ORNL-5138. Oak Ridge
National Laboratory, Oak Ridge
34. Allen FH, Kennard O, Watson DG, Brammer L, Orpen AG,
Taylor R (1987) J Chem Soc Perkin Trans 2:S1–S2
35. Cremer D, Pople JA (1975) J Am Chem Soc 97:1354
References
1. Feizi T, Childs RA (1985) Trends Biochem Sci 10:24
2. Hakomori S (1984) Am Rev Immunol 2:103
3. Springer GF (1997) J Mol Biol 75:594
4. Danishefsky SJ, Allen JR (2000) Angew Chem Int Ed 112:882
5. Buskas T, Thompson P, Boons G-J (2009) Chem Commun
36:5335
´
6. Grigalevicius S, Chierici S, Renaudet O, Lo-Man R, Deriaud E,
Leclerc C, Dumy P (2005) Bioconjugate Chem 5:1149
ˇ
7. Renaudet O, Krenek K, Bossu I, Dumy P, Kadek A, Adamek D,
Vanek O, Kavan D, Gazak R, Sulc M, Bezouska K, Kren V
´
´
ˇ
ˇ
ˇ´
ˇ
ˇ
(2010) J Am Chem Soc 132:6800
123