N-p-nitrobenzyloxycarbonyl galactosamine imidate as a glycosyl donor for the efficient synthesis of mucin core-2 analogue

Article abstract of DOI:10.1016/S0960-894X(00)00107-4

An efficient synthesis of the mucin core-2 analogue 1a was accomplished using N-p-nitrobenzyloxycarbonyl(PNZ)-protected trichloroacetimidate 4 as a novel glycosyl donor. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00107-4

Bioorganic & Medicinal Chemistry Letters 10 (2000) 793±795
N-p-Nitrobenzyloxycarbonyl Galactosamine Imidate as a Glycosyl
Donor for the Ecient Synthesis of Mucin Core-2 Analogue
Wensheng Liao, Conrad F. Piskorz, Robert D. Locke and Khushi L. Matta*
Molecular & Cellular Biophysics, Roswell Park Cancer Institute, Elm & Carlton Streets, Bu€alo, NY 14263, USA
Received 21 December 1999; accepted 9 February 2000
AbstractÐAn ecient synthesis of the mucin core-2 analogue 1a was accomplished using N-p-nitrobenzyloxycarbonyl(PNZ)-
protected trichloroacetimidate 4 as a novel glycosyl donor. # 2000 Elsevier Science Ltd. All rights reserved.
For the last several years we have been involved in the
study of glycosyltransferases, speci®cally a-l-fucosyl-
transferase, a(2,3)-sialyltransferase, and sulfotransferase
responsible for the synthesis of various mucin core 2-
linked carbohydrate chains. Our recent study indicates
that the core-2 branched structure Galb1,4GlcNAcb1,
6(Galb1,3)GalNAca is a preferred acceptor for 3-O-
sulfotransferase1a and a(2,3)-sialyltransferase1b in colon
tumor tissues. We have already described the associa-
tion of the novel a(1,2)-l-fucosylating activity with
the Lewis type a(1,3/4)-l-fucosyltransferase, which can
convert Galb1,3(Fuca1,4)GlcNAc (Lea) to Fuca1,2-
Galb1,3(Fuca1,4)GlcNAc (Leb).2 We are presently
investigating an unusual a(1,2)-l-fucosyltransferase
capable of converting Galb1,4(Fuca1,3)GlcNAc (Lex)
to Fuca1,2Galb1,4(Fuca1,3)GlcNAc (Ley) structure.
As we plan to use branched saccharides for this study,
molecules 1a and 1b become the target acceptors for this
enzyme (Fig. 1). Both acceptors can be used to identify
this unique enzyme in the presence of the commonly
occurring a(1,2)-l-fucosyltransferase. The present com-
munication describes the chemical synthesis of 1a.
without degradation of the resulting product and other
protecting groups. We have investigated various glyco-
syl donors and ®nally selected the N-p-nitrobenzyloxy-
carbonyl(PNZ)5-protected trichloroacetimidate 4, which
can be readily obtained from galactosamine hydro-
chloride 2 by ®ve successive high yield conversions.
Removal of the PNZ group is also convenient. Simple
treatment of N-PNZ-protected saccharides with sodium
hydrosulphite under neutral conditions provides the free
amines. The employment of PNZ avoids the vigorous
conditions required for removal of N-phthalimido
(NPhth) protection,6 which is widely used in the syn-
thesis of amino sugars.
Thus, treatment of 2 with one equivalent of NaOMe in
MeOH, followed by one equivalent of p-nitrobenzyl
chloroformate±Et3N (Scheme 1) gave the p-nitrobenzyl
carbamate 3, which was fully O-acetylated (Ac2O±pyr-
idine, DMAP). Selective removal of anomeric O-acetyl
group (hydrazine acetate, DMF) followed by treatment
with Cl3CCN±DBU a€orded the key donor 4, exclu-
sively as a-isomer.
Synthesis of 1a involves the construction of the Gal-
NAcb1,3GalNAc linkage. However, synthesis of this
linkage has not attracted much attention.3 Various
galactosamine donors have become available for the
synthesis of GalNAcb-linked compounds.4 The 1,2-
trans-glycosylation of amino sugars requires glycosyl
donors containing participating protective groups in the
C2-position. It is important to use a protecting group
that can be selectively removed under mild conditions
Compound 4 showed high reactivity when used as a
glycosyl donor in the presence of either triethylsilyl tri-
¯ate (Et3SiOTf) or boron tri¯uoride diethyl etherate
.
(BF3 Et2O) as a promotor (Scheme 2). Disaccharide 6
was obtained in high yield (89%) from the condensation
of 4 with 5 in CH2Cl2 employing the Et3SiOTf catalyst.
The newly formed b-linkage in 6 was con®rmed by
13C NMR data which included two signals for C-1B
and C-1A at d 101.01 (1JC,H=158.5Hz) and 99.43 (1JC,H
=
168.7 Hz), respectively. Glycosylation by 4 of the diol
11 (Et3SiOTf, 65 ꢀC) a€orded the b-(1!4) linked
disaccharide 12 in 78% yield. The glycosylation of 16
*Corresponding author. Tel.: +1-716-845-2397; fax: +1-716-845-
3458; e-mail: klmatta@yahoo.com
(BF3 Et2O, 60ꢀC) gave the b-(1!3) linked disaccharide
.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00107-4

794
W. Liao et al. / Bioorg. Med. Chem. Lett. 10 (2000) 793±795
Figure 1. This C-2 position is blocked for commonly occurring a(1,2)-l-fucosyltransferase.
Scheme 1. Reagents and conditions: (i) NaOMe±MeOH (1 equiv), rt; (ii) p-nitrobenzyl chloroformate±Et3N (1 equiv), 0 ꢀC; (iii) pyridine±Ac2O,
DMAP, 95%; (iv) hydrazine acetate, DMF, 0 ꢀC, 2 h, 87%; (v), CCl3CN (3 equiv)±DBU, CH2Cl2, 10 ꢀC, 90%.
Scheme 2. Reagents and conditions: (i) 4 (1.5 equiv), TESOTf (0.24 equiv), CH2Cl2, 4 A molecular sieves, rt, 15 min, 89%; (ii) Na2S2O4 (8 equiv),
MeCN:EtOH:H2O (v/v/v 1:1:1ꢀ), 10 min; (iii) Ac2O±MeOH; (iv) 75% acetic acid, 45 ꢀC, 2 h, 90%; (v) 80% acetic acid, 40 ꢀC, 2 h, 92%; (vi) 4 (1.2
equiv), TESOTf, CH2Cl2, 65 C, 1 h, 78%; (vii) pyridine±Ac2O, DMAP; (viii) 4 (1.2 equiv), BF3 Et2O (0.5 equiv), CH2Cl2, 60 ꢀC, 30 min, 82%.
.
17 in 82% yield. Removal of PNZ from compounds 6,
12 and 17 under mild condition by sodium hydro-
sulphite7 gave 7, 13 and 18 in 80, 77 and 78% yields,
respectively. Ensuing N-acetylation a€orded 8, 14 and
19 in quantitative yields. The above results indicate that
a combination of the N-PNZ moiety with anomeric tri-
chloroacetimidate activation8 in 4 a€ords an ecient
donor for galactosamine b-glycosylation.
acid (TfOH) condition10 did not proceed, possibly
owing to the poor solubility of 9 in CH2Cl2.
In order to increase solubility, the diol 10, which was
directly obtained by treatment of 6 with 80% acetic acid
(40 ꢀC, 2 h), was attempted as an acceptor and found to
be successful. Thus, glycosylation of 21 with 10 under
NIS±TMSOTf condition at 50 ꢀC in CH2Cl2 provided
the fully protected pentasaccharide 22 in 46% yield.
The 13C NMR DEPT135 spectra showed a downshift
of C-6 in the terminal GalNAca residue to d 69.1 ppm,
con®rming this position as the site of glycosylation. The
PNZ group in 22 was reductively cleaved with sodium
hydrosulphite. Removal of both the phthalimido and
acetate groups from 23 was accomplished by treat-
ment with hydrazine hydrate:ethanol (v/v, 1:4) at 90ꢀC,
With the methodology of e€ectively constructing Gal-
NAcb1,3GalNAc linkage in hand, we turned our atten-
tion to the synthesis of our target compound 1a
(Scheme 3). Initially we tried to use the diol 9, obtained
by hydrolysis of disaccharide 8 in 75% acetic acid at
45 ꢀC, as an acceptor. However, condensation with the
Lewisx donor 219 under N-iodosuccinimide (NIS)±tri¯ic

W. Liao et al. / Bioorg. Med. Chem. Lett. 10 (2000) 793±795
795
Scheme 3. Reagents and conditions: (i) 21 (1.5 equiv), NIS±TMSOTf, CH2Cl2, 4 A molecular sieves, 50 ꢀC, overnight, 46%; (ii) Na2S2O4 (8 equiv),
MeCN:EtOH:H2O, 10 min, 65%; (iii) hydrazine hydrate:MeOH (1:4 v/v), 90 ꢀC, 6 h, 90%; (iv) MeOH:Ac2O (1:1, v/v), 0 ꢀC, quantitative.
Crawley, S. C.; Palcic, M. M.; Hindsgaul, O. Carbohydr. Res.
1993, 243, 139.
followed by N-acetylation (Ac2O±MeOH) to furnish the
®nal product 1a. The structure of 1a was con®rmed by
1H, 13C NMR and FABMS spectroscopy.11
7. Typical procedure for removing PNZ group and N-acetyla-
tion: 6 (0.1 mmol) was dissolved in 4.5 mL CH3CN:EtOH:
H2O (v/v/v 1:1:1). Sodium hydrosulphite (140 mg) was added
and the stirring was continued for 10 min at room tempera-
ture. TLC (4:1 CH2Cl2:MeOH) indicated that the reaction was
complete. The solvent was concentrated and dried over
vacuum. Dry MeOH (2.5 mL) and Ac2O (2 mL) were added
successively. After 1 h, the solvent was removed and the resi-
due was puri®ed by column chloromatography (12:1 CH2Cl2:
MeOH) to a€ord compound 8 (55 mg, 80%).
Acknowledgements
We are grateful to the NIH (CA 35329) for ®nancial
support of this research. We thank Dr. E. V. Chan-
drasekaran for his helpful discussion in preparing this
manuscript.
8. Schmidt, R. R. Angew. Chem., Int. Ed. Engl. 1986, 25, 212.
9. Jain, R. K.; Matta, K. L. manuscript in preparation.
10. Konradsson, P.; Udodong, U. E.; Fraser-Reid, B. Tetra-
hedron Lett. 1990, 31, 4313.
References and Notes
11. Selected data for 4: 1H NMR (CDCl3, 400 MHz) d 8.78 (s,
1H, C=NH), 8.20 (d, 2H, J=9.2 Hz, arom H), 7.47 (d, 2H,
J=8.4 Hz, arom H), 6.45 (d, 1H, J=4.0 Hz, H-1), 5.50 (d, 1H,
J=2.4 Hz, H-4), 5.26 (dd, 1H, J=3.0, J=11.8 Hz, H-3), 5.19
(s, 2H, OCOCH2PhNO2), 4.94 (d, 1H, J=10.0 Hz, NHPNZ),
4.57 (m, 1H, H-2), 4.20±4.04 (m, 3H, H-5, H-6), 2.18, 2.01,
1. (a) Chandrasekaran, E. V.; Jain, R. K.; Vig, R.; Matta,
K. L. Glycobiology 1997, 7, 753; (b) Chandrasekaran, E. V.;
Matta, K. L. Manuscript in preparation.
2. Chandrasekaran, E. V.; Jain, R. K.; Rhodes, J. M.; Srnka,
C. A.; Larsen, R. D.; Matta, K. L. Biochemistry 1995, 34,
4748.
1
1.99 (3 s, 9H, 3 OAc); for 6: H NMR (CDCl3, 400 MHz) d
3. Barresi, F.; Hindsgaul, O. J. Carbohydr. Chem. 1995, 14 (8),
1043.
8.14 (d, 2H, J=7.2 Hz, arom H), 7.45 (d, 2H, J=8.8 Hz, arom
H), 7.44 (d, 2H, J=8.8 Hz, arom H), 6.85 (d, 2H, J=8.4 Hz,
arom H), 5.74 (d, 1H, J=8.8 Hz, H-10), 5.49 (s, 1H, MeO
PhCH), 5.35 (d, 1H, J=2.8 Hz, H-40), 4.85 (d, 1H, J=3.2 Hz,
H-1), 4.28 (d, 1H, J=2.8 Hz, H-4), 3.78, 3.41 (2 s, 6H, 2
OCH3), 2.17, 2.05, 1.94, 1.92 (4 s, 12H, 4 COCH3); 13C NMR
(CDCl3, 100.6 MHz) d 170.630, 170.46, 170.31, 170.24 (4 CO),
4. (a) Castro-Palomino, J. C.; Ritter, G.; Fortunato, S. R.;
Reinhardt, S.; Old, L. J.; Schmidt, R. R. Angew. Chem. Int.
Ed. Engl. 1997, 36, 1998; (b) Castro-Palomino, J. C.; Schmidt,
R. R. Tetrahedron Lett. 1995, 36, 6871; (c) Ellervik, U.; Mag-
nusson, G. Carbohydr. Res. 1996, 280, 251; (d) Jiao, H.;
Hindsgaul, O. Angew. Chem., Int. Ed. Engl. 1999, 38, 346; (e)
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Am. Chem. Soc. 1995, 177, 3302.
5. (a) Boullanger, P.; Jouineau, M.; Bouammali, B.; Lafont,
D.; Descotes, G. Carbohydr. Res. 1990, 202, 151; (b) Guibe-
Jampel, E.; Wakselman, M. Synth. Commun. 1982, 12, 219;
(c) For corresponding glucosamine derivative as glycosyl
donor, see: Qian, X.; Hindsgaul, O. J. Chem. Soc., Chem.
Commun. 1997, 1059.
0
0
101.01 (JC1 H1 =158.5 Hz, C-1 ), 99.43 (JC1H1=168.7 Hz, C-1),
69.34 (C-6), 61.74 (C-60), 55.48, 55.33 (2 OCH3), 23.31, 20.75,
20.70, 20.60 (4 COCH3); for 1: FABMS m/z 972.7 [M+Na]+;
1H NMR (D2O, 400 MHz) d 5.13 (d, 1H, J=3.6 Hz, H-10000),
4.81 (d, 1H, J=3.7 Hz, H-1), 4.58 (d, 1H, J=8.0 Hz, H-100),
4.53 (d, 1H, J=8.0 Hz, H-1000), 4.48 (d, 1H, J=7.6 Hz, H-10),
3.36 (s, 3H, OCH3), 2.06, 2.04, and 2.02 (each s, 9H,
3ÂNHAc), 1.20 (d, 3H, J=6.4 Hz, CMe); 13C NMR (D2O,
100.6 MHz) d 173.53, 173.10, 172.63 (3 CO), 101.68 (C-1000),
100.82 (C-10), 100.34 (C-100), 97.60 (C-10000), 97.12 (C-1), 54.69
(OMe), 21.29, 21.26, 21.04 (3ÂNHAc), 14.27 (C-60000).
6. (a) Lemieux, R. U.; Takeda, T.; Chung, B. Y. A.C.S. Symp.
Ser. 1976, 39, 90; (b) Spijker, N. M.; Westerduin, P.; van
Boeckel, C. A. A. Tetrahedron 1992, 48, 6297; (c) Kanie, O.;