Practical synthesis of sulfated analogs of lactosamine and sialylated lactosamine derivatives

Article abstract of DOI:10.1081/CAR-200030027

A series of β-D-Gal-(1 → 4)-β-D-GlcNAc-octyl, NeuAcα-(2 → 3)-β-D-Gal-(1 → 4)-β-D-GlcNAc-octyl, and their 6-O-sulfated and 6′-O-sulfated analogs (1-6) were synthesized in a concise manner starting from readily accessible monosaccharide intermediates. The syntheses involved formation of an orthogonally protected disaccharide and a trisaccharide from which all six compounds were derived. Copyright

Full text of DOI:10.1081/CAR-200030027

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Practical Synthesis of Sulfated Analogs of
Lactosamine and Sialylated Lactosamine
Derivatives
Anup Kumar Misra a , Geetanjali Agnihotri a , Soni Kamlesh
Madhusudan a & Pallavi Tiwari a
a Medicinal Chemistry Division , Central Drug Research Institute ,
Chattar Manzil Palace, Lucknow, 226 001, India
Published online: 17 Aug 2006.
To cite this article: Anup Kumar Misra , Geetanjali Agnihotri , Soni Kamlesh Madhusudan & Pallavi
Tiwari (2004) Practical Synthesis of Sulfated Analogs of Lactosamine and Sialylated Lactosamine
Derivatives, Journal of Carbohydrate Chemistry, 23:4, 191-199, DOI: 10.1081/CAR-200030027
To link to this article: http://dx.doi.org/10.1081/CAR-200030027
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JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 23, No. 4, pp. 191–199, 2004
Practical Synthesis of Sulfated Analogs of
Lactosamine and Sialylated Lactosamine
Derivatives#
*
Anup Kumar Misra, Geetanjali Agnihotri, Soni Kamlesh Madhusudan,
and Pallavi Tiwari
Medicinal Chemistry Division, Central Drug Research Institute, Lucknow, India
CONTENTS
ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
I. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
II. RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . 193
III. CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
#C.D.R.I. Communication no. 6456.
*
Correspondence: Anup Kumar Misra, Medicinal Chemistry Division, Central Drug Research
Institute, Chattar Manzil Palace, Lucknow 226 001, India; E-mail: akmisra69@rediffmail.com.
191
DOI: 10.1081/CAR-200030027
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright # 2004 by Marcel Dekker, Inc.

192
Misra et al.
ABSTRACT
A series of b-D-Gal-(1 ! 4)-b-D-GlcNAc-octyl, NeuAca-(2 ! 3)-b-D-Gal-(1 ! 4)-
b-D-GlcNAc-octyl, and their 6-O-sulfated and 60-O-sulfated analogs (1–6) were
synthesized in a concise manner starting from readily accessible monosaccharide inter-
mediates. The syntheses involved formation of an orthogonally protected disaccharide
and a trisaccharide from which all six compounds were derived.
Key Words: Sulfated lactosamine; Sialylated lactosamine; Sulfation.
INTRODUCTION
N-Acetyl lactosamine [Gal-b-(1 ! 4)-GlcNAc] and its sialylated (a2,3 or a2,6)
extention are quite common in cell surface glycans and glycolipids.[1] They are often
modified to express differentiation antigens and functional oligosaccharides, such as
Lewis X, sialyl Lewis X, which are found in human granulocytes and monocytes and
acts as ligands for E-, P-, and L-selectins.[2] Sulfate groups in carbohydrates play import-
ant roles in conferring highly specific functions like cell–cell interactions, signal transduc-
tion, immunogenic recognition, and embryonic development in glycoproteins, glycolipids,
and proteoglycans.[3] Keratan sulfate proteoglycan is a major component of the corneal
stroma, which is composed of N-acetylated lactosamine repeating unit with sulfate resi-
dues at 6-O position of GlcNAc or Gal or in both, plays an important role in maintaining
corneal transparency by organizing and providing proper hydration of the extracellular
matrix.[4] The biosynthesis of keratan sulfate takes place by involvement of four recently
cloned enzymes: b-N-acetylglucosaminyltransferase, b-galactosyltransferase, GlcNAc
6-O-sulfotransferase, and Gal 6-O-sulfotransferase.[5] All of them use N-acetyl lacto-
samine disaccharide as an acceptor to elongate the target glycosaminoglycans. Besides
these, N-acetylated lactosamine acts as acceptors to a number of glycosyltransferases in
the production of a number of glycoconjugates such as sialyl Lewis X, tumor related fuco-
sylated poly-N-acetyl lactosamines, blood group antigens, etc., for example, a-fucosyl-
transferases (FucT IV and VII) require sialylated N-acetyl lactosamine and its sulfated
analogs as acceptors to biosynthesize sialyl Lewis X and its sulfated analogs, ligands
for selectins.[6]
For a detailed mechanistic study of the above mentioned biosynthetic pathways to
make a variety of glyconjugates involving recently cloned several glycosyltransferases
and sulfotransferases enzymes, a large quantity of N-acetyl lactosamine and sialylated
N-acetyl lactosamine and their sulfated analogs are required, although the synthesis of a
reasonable quantities of complex oligosaccharides remains one of the most challenging
areas of chemistry. Therefore, a concise, efficient synthetic methodology for the synthesis
of N-acetyl lactosamine and sialylated N-acetyl lactosamine and their sulfated analogs
would extend the scope to get a large access of these compounds. A few reports have
been appeared in the literature regarding the synthesis of these classes of compounds
and most of them required lengthy multi-step sequences.[7] Chemo-enzymatic synthesis
shows great promise but requires access to a panel of glycosyltransferases and sulfotrans-
ferases.[8]

Practical Synthesis of Sulfated Analogs
193
We report herein a practical, high yielding chemical synthesis of the octyl glycosides
of N-acetyl lactosamine, sialylated N-acetyl lactosamine, and their 6-O-sulfated and 60-O-
sulfated analogs from the readily accessible protected monosaccharide precursors (8–16).
The key feature in this synthetic protocol is the use of common intermediate (10 and 17) to
get access to all target molecules (1–6) (Fig. 1).
RESULTS AND DISCUSSION
Glycosylation of octyl 2-acetamido-3-O-acetyl-2-deoxy-6-O-tert-butyldimethylsilyl-
b-D-glucopyranoside (8), prepared from N-acetyl-D-glucosamine in six steps and 2,3,4,6-
tetra-O-benzoyl-b-D-galactopyranosyl trichloroacetimidate (9)[9] using trimethylsilyl
trifluoromethanesulfonate (TMSOTf) in methylene chloride afforded the b-(1 ! 4)
linked disaccharide (10) in 78% yield which on treatment with sodium methoxide in
methanol furnished b-D-octyl lactosamine disaccharide (1) in 93% yield. De-silylation
of compound 10 using HF–pyridine[10] yielded disaccharide 11 in 78% that on
Figure 1. Synthesis of octyl glycosides of N-acetyl lactosamine, sialylated N-acetyl lactosamine,
and their 6-O-sulfated and 60-O-sulfated analogs from monosaccharide precursors (8–16).

194
Misra et al.
sulfation[11] (sulfur trioxide–pyridine complex; SO3 Pyr) followed by saponification
gave the 6-O-sulfate 3 in 74% yield. Treatment of compound 1 with benzaldehyde
dimethylacetal in presence of p-toluenesulfonic acid furnished octyl 4,6-O-benzylidene-
b-D-galactopyranosyl-(1 ! 4)-2-acetamido-2-deoxy-b-D-glucopyranoside (13) which
gave compound 14 after conventional acetylation using acetic anhydride–pyridine in
78% yield in two steps. Removal of the benzylidene acetal from compound 14 through
catalytic hydrogenolysis under neutral condition[11] followed by selective sulfation of
.
0
the 60-hydroxyl group (SO3 Pyr) and saponification afforded 6 -O-sulfate derivative 2
.
in 72% overall yield in three steps (Sch. 1).
Sialylation of the disaccharide triol acceptor 15, obtained from compound 13 by
selective silylation of 6-hydroxyl group (TBDMSCl/imidazole),[12] using the N-acetyl-
neuraminic acid donor[13] 16 and NIS/TfOH as promoter[14] afforded trisaccharide 17
1
in 56% yield. Characteristic proton and carbon signals in the H and 13C NMR spectra
[d 5.43 (s, PhCH), 5.05 (d, J ¼ 8.0 Hz, H-10), 4.60 (dd, J ¼ 7.8 Hz, H 2 1), 2.76 (dd,
J ¼ 12.0 Hz and 4.5 Hz, H-30e0), and 1.75 (t, J ¼ 12.0 Hz, H-3a00)] confirmed the structure
of 17. In order to improve the yield of the glycosylation, several other sialyl donors and
various promoters were examined but the yields were found to be similar. Trisaccharide
derivative 17 has been used as a common scaffold for the preparation of two sulfated
analogs 5 and 6. De-benzylidenation under catalytic hydrogenolysis followed by
removal of tert-butyldimethylsilyl group and acetyl groups together by using sodium
methoxide afforded sialylated lactosamine trisaccharide 4 in 82% yield. Use of acidic con-
dition to remove the benzylidene acetal resulted in some removal of acid sensitive sialic
acid residue.
Trisaccharide 17 was converted to 6-hydroxylated trisaccharide 19 in 77% overall
yield after a sequence of transformation, which involves removal of benzylidene acetal
Scheme 1. Reagents: (a) TBDMS–Cl, imidazole, DMF, rt, 7 hr, 72%; (b) TMSOTf, CH2Cl2,
˚
.
MS-4A, 2108C to rt, 3 hr, 78%; (c) HF–pyridine, THF, 0–58C, 4 hr, 78%; (d) SO3 Pyr complex,
pyridine, 6 hr, then Dowex 50W X8 (Naþ), 74%; (e) PhCH(OMe)2, p-TsOH, CH3CN, rt, 3 hr; (f)
Ac2O, pyridine, rt, 12 hr, 78% in two steps; (g) H2, Pd(OH)2–C (20%), MeOH, rt, 24 hr; (h)
þ
.
SO3 Pyr complex, pyridine, 6 hr, then Dowex 50W X8 (Na ), 72% in two steps; (i) 0.1 M
MeONa, MeOH, rt, 12 hr, 93%.

Practical Synthesis of Sulfated Analogs
195
under hydrogenolytic condition, conventional acetylation followed by removal of silyl
protection using HF–pyridine. Sulfation of 6-hydroxyl group of compound 19
followed by deacetylation furnished target 6-O-sulfated trisaccharide 5 in 78% yield.
In another approach, trisaccharide 17 was converted to 40,60-dihydroxylated trisacchar-
ide 18 by conventional acetylation followed by removal of benzylidene acetal using
10% Pd–C/H2 in 82% yield. Selective sulfation of 60-hydroxyl group of 18 using
.
SO3 Pyr followed by removal of silyl protection and acetyl group in one step
under saponification condition furnished 60-O-sulfated trisaccharide 6 in 74% yield
(Sch. 2).
Six target compounds (1–6) were prepared in 4 g scale following the above
1
mentioned reaction sequences. Selected H and 13C NMR and MS data for key com-
pounds are presented below.a Compounds 1–6 have been evaluated as acceptors
for several enzymes including various sulfotransferase and fucosyl transferase
acceptors.[2,3]
aPartial 1H NMR (300 MHz, D2O): the following common signals for the octyl aglycon were
observed in D2O solution: d 1.60–1.40 (m, 2H, OCH2CH2), 1.30–1.10 [m, 10 H, OCH2CH2(CH2)5-
CH3], 0.85 (t, 3H, octyl CH3). H-1 indicates the anomeric proton of the GlcNAc residue, H-10 the
anomeric proton of the Gal residue and onwards. 1: d 4.40 (d, J1,2 ¼ 8.1 Hz, 1H, H-1), 4.38
(d, J1 ,2 ¼ 7.0 Hz, 1H, H-10), 4.16 (m, 2H, H-2 and H-20), 1.96 (s, 3H, NHAc); 13C NMR: d
173.6, 105.2, 102.8, 81.0, 77.2, 76.6, 74.9, 74.4, 72.7, 70.8, 70.5, 62.6, 62.0, 56.8, 55.3, 39.1,
30.8, 30.6, 27.2, 23.8, 23.1, 14.5; TOFMS: calcd. for C22H41O11N (M þ Naþ) 518.5; found
0
0
518.5. 2: d 4.48 (d, J1,2 ¼ 8.4 Hz, 1H, H-1), 4.40 (d, J1 ,2 ¼ 7.5 Hz, 1H, H-10), 4.18 (dd, 2H,
H-6a0 ,b), 2.07 (s, 3H, NHAc); 13C NMR: d 175.0, 103.3, 101.6, 79.7, 75.2, 73.3, 72.9, 72.8, 72.6,
71.3, 71.1, 68.8, 67.7, 60.8, 55.7, 31.7, 29.1, 28.9, 25.6, 22.8, 22.6, 14.0; TOFMS: calcd. for
C22H40O14NSNa (M þ Naþ) 620.2; found 620.2. 3: 4.50 (d, J1,2 ¼ 7.5 Hz, 1H, H-1), 4.44
0
0
(d, J1 ,2 ¼ 8.1 Hz, 1H, H-10), 4.34 (bs, 2H, H-6a,b), 1.99 (s, 3H, NHAc); 13C NMR: d 175.5,
103.1, 101.7, 77.9, 75.8, 73.1, 73.0, 72.9, 72.8, 71.5, 71.2, 69.2, 66.9, 61.6, 55.7, 31.7, 29.1, 28.9,
25.6, 22.8, 22.6, 14.0; TOFMS: calcd. for C22H40O14NSNa (M þ Naþ) 620.2; found 620.2. 4: d
0
0
0
0
4.55 (d, J1,2 ¼ 7.8 Hz, 1H, H-1), 4.46 (d, J1 ,2 ¼ 7.2 Hz, 1H, H-1), 4.06 (dd, J ¼ 3.0 Hz and
9.9 Hz, 1H, H-3), 2.70 (dd, J ¼ 4.5 Hz and 12.3 Hz, 1H, H-3e00), 1.98 (s, 6H, 2NHAc), 1.75
(t, J ¼ 12.0 Hz, 1H, H-3a00); 13C NMR: d 175.6, 174.9, 174.5, 103.1, 101.6, 100.4, 78.8, 76.0, 75.7,
75.3, 73.4, 73.0, 72.8, 71.1, 69.9, 68.9, 68.6. 68.0, 63.1, 61.6, 60.6, 55.7, 52.2, 40.2, 31.7, 29.1
(2C), 28.9, 25.6, 22.8, 22.6 (2C), 14.0; TOFMS: calcd. for C33H57O19N2Na (M þ Naþ) 831.8;
found 831.8. 5: d 4.61 (d, J1,2 ¼ 7.8 Hz, 1H, H-1), 4.53 (d, J1 ,2 ¼ 7.8 Hz, 1H, H-10), 4.36 (q, 2H,
H-6a,b), 4.11 (dd, J ¼ 3.3 Hz and 9.1 Hz, 1H, H-2), 2.74 (dd, J ¼ 4.8 and 12.0 Hz, 1H, H-3e00),
2.01, 2.00 (2s, 6H, 2NHAc), 1.78 (t, J ¼ 12.0 Hz, 1H, H-3a00); 13C NMR: d 175.5, 175.0, 174.6,
102.6, 101.7, 100.3, 77.7, 75.9, 75.6, 73.4, 73.1, 72.9, 72.0, 71.2, 70.0, 69.0, 68.6, 68.0, 66.9,
63.0, 61.6, 55.8, 52.3, 40.1, 31.7, 29.1 (2C), 28.9, 25.6, 22.8, 22.6 (2C), 14.0; TOFMS: calcd. for
C33H56O22N2SNa2 (M þ Naþ) 933.8; found 933.8. 6: d 4.56 (d, J1,2 ¼ 7.8 Hz, 1H, H-1), 4.48
0
0
(d, J1 ,2 ¼ 7.8 Hz, 1H, H-10), 2.71 (dd, J ¼ 4.5 Hz and 12.0 Hz, 1H, H-3e00), 1.88 (s, 6H, 2NHAc),
1.76 (t, J ¼ 12 Hz, 1H, H-3a00); 13C NMR: d 175.5, 175.0, 174.4, 102.9, 101.6, 100.5, 79.7, 75.8,
75.2, 73.4, 73.2, 72.7, 72.3, 71.1, 69.8, 68.9, 68.7, 68.1, 68.0, 63.1, 60.8, 55.7, 52.2, 40.0, 31.7,
29.1 (2C), 28.9, 25.6, 22.8, 22.6 (2C), 14.0; TOFMS: calcd. for C33H56O22N2SNa2 (M þ Naþ)
933.8; found 933.8.
0
0

196
Misra et al.
Scheme 2. Reagents: (a) TBDMS–Cl, imidazole, DMF, rt, 5 hr, 72%; (b) NIS, TfOH, CH3CN–
˚
CH2Cl2 (5 : 1), MS-3A, 2208C, 18 hr, 56%; (c) H, Pd(OH)2–C (20%), MeOH, rt, 32 hr; (d)
0.1 M MeONa, MeOH, rt, 12 hr, then a few drops of water, rt, 12 hr, 82% in two steps; (e) Ac2O,
.
pyridine, rt, 12 hr, quantitative; (f) SO3 Pyr complex, pyridine, 08C–rt, 8 hr, then Dowex 50W
X8 (Naþ), 74%; (g) HF–pyridine, THF, 0–58C, 4 hr, 78%.
CONCLUSION
In conclusion, the 6- and 60-O-sulfated analogs of lactosamine and sialyllactosamine
were synthesized following a practical high yielding procedure utilizing a common
disaccharide scaffold. Most of the methodologies used in this synthetic scheme are very
convenient and high yielding thus providing this report a potential alternative to
the existing methods. This high yielding practical synthetic protocol for the synthesis
of these classes of molecules will certainly add value to the glycobiology.
ACKNOWLEDGMENTS
The authors thank the Director, Central Drug Research Institute (C.D.R.I.) for
his encouragement and support. R.S.I.C., C.D.R.I. is gratefully acknowledged for the

Practical Synthesis of Sulfated Analogs
197
instrumentation facilities. This work is partly supported by DST, New Delhi (Project no.
SR/FTP/CSA-10/2002). S.K.M. and P.T. thank C.S.I.R., New Delhi for providing
fellowships.
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Accepted March 30, 2004