Synthesis of a bis(sialic acid) 8,9-lactam

Article abstract of DOI:10.1021/jo950994p

Methyl [2-(trimethylsilyl)ethyl 5-acetamido-9-azido-4-O-benzoyl-3,5,9-trideoxy-D-glycero-α-D-galacto- 2-nonulopyranosid]onate was sialylated with methyl (ethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-2-thio-3-(phenylthio)-2,3,5-trideox y-D-erythro-α-L-gluco-2-nonulopyranosid)onate to give the corresponding bis(sialic acid) derivative in 23% yield. Removal of protecting groups, reduction of the azido group to an amino group, and removal of the auxiliary thiophenyl group gave the desired bis(sialic acid) 8,9-lactam. Comparison of the ¹H NMR spectra and energy-minimized (MM3) structures of the bis(sialic acid) lactam with those of the corresponding bis(sialic acid) lactone showed the conformations of the two compounds to be very similar (RMS = 0.027 A).

Full text of DOI:10.1021/jo950994p

J . Org. Chem. 1996, 61, 179-184
179
Syn th esis of a Bis(sia lic a cid ) 8,9-La cta m
Teddy Erce´govic and Go¨ran Magnusson*
Organic Chemistry 2, Chemical Center, The Lund Institute of Technology, University of Lund,
P.O. Box 124, S-221 00 Lund, Sweden
Received May 31, 1995 (Revised Manuscript Received September 28, 1995X)
Methyl [2-(trimethylsilyl)ethyl 5-acetamido-9-azido-4-O-benzoyl-3,5,9-trideoxy-D-glycero-R-D-galacto-
2-nonulopyranosid]onate was sialylated with methyl (ethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-2-
thio-3-(phenylthio)-2,3,5-trideoxy-D-erythro-R-L-gluco-2-nonulopyranosid)onate to give the corre-
sponding bis(sialic acid) derivative in 23% yield. Removal of protecting groups, reduction of the
azido group to an amino group, and removal of the auxiliary thiophenyl group gave the desired
bis(sialic acid) 8,9-lactam. Comparison of the 1H NMR spectra and energy-minimized (MM3)
structures of the bis(sialic acid) lactam with those of the corresponding bis(sialic acid) lactone showed
the conformations of the two compounds to be very similar (RMS ) 0.027 Å).
In tr od u ction
Glycosphingolipids that contain sialic acid moieties
(gangliosides) are present on the surface of mammalian
cells.1 Tumor cells often carry abnormal amounts of some
gangliosides, which has led to the concept of gangliosides
as tumor-associated antigens.2 It is well known that
gangliosides form δ-lactones under acidic conditions,3 and
furthermore, the high density of gangliosidic sialic acid
on many tumor cells may induce the formation of lactones
in vivo. The δ-lactones were suggested to be the true
immunogens in the preparation of antiganglioside anti-
bodies, but the hydrolytic lability of the lactones would
make them poor immunogens due to the difficulty of
maintaining
a high in vivo concentration during
immunization.2b,4
We have prepared ganglioside lactams5 (GM1-4) and
used them as immunogens in order to raise antibodies
that cross-react with the corresponding lactones on
murine melanoma cells. This work demonstrated that
G
M3-lactone is indeed present on the cell surface.6 The
F igu r e 1. Bis(sialic acid) lactone and the synthetic lactam
analogs 1 and 2.
lactams have proved to be hydrolytically stable and
structurally similar to their lactone counterparts.5
Ganglioside GD3, which carries two sialic acid moieties,
is abnormally frequent on human malignant melanoma
cells as compared to healthy cells.7 GD3 is capable of
forming several lactones, of which the 8,9-lactone is
formed most readily (Figure 1).8 We now describe the
synthesis of a bis(sialic acid) lactam (1), suitable for
further elaboration into GD3-lactam and other ganglioside
lactams containing a bis(sialic acid) moiety. The syn-
thesis is based on our novel sialyl donor9 12 and acceptor
6.
X Abstract published in Advance ACS Abstracts, December 15, 1995.
(1) (a) Hakomori, S. Ann. Rev. Biochem. 1981, 50, 733. (b) Karlsson,
K.-A. In Biological Membranes; Chapman, D., Ed.; Academic Press:
London, 1982; Vol. 4, pp 1-74. (c) Gigg, J .; Gigg, R. Top. Curr. Chem.
1990, 154, 77. (d) Sweeley, C. C. In Biochemistry of Lipids, Lipoproteins
and Membranes; Vance, D. E., Vance, J ., Eds.; Elsevier Science
Publishers B. V.: Oxford, 1991; pp 327-361.
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bayashi, Y.; Hanaoka, A.; Matsumoto, M.; Matsubara, T.; Tagawa, M.;
Wakabayashi, S.; Taniguchi, M. J . Biol. Chem. 1985, 260, 13328. (c)
Hakomori, S. Chem. Phys. Lipids 1986, 42, 209.
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190. (b) Gross, S. K.; Williams, M. A.; McCluer, R. H. J . Neurochem.
1980, 34, 1351. (c) Riboni, L.; Sonnino, S.; Acquotti, D.; Malesci, A.;
Ghidoni, R.; Egge, H.; Mingrino, S.; Tettamanti, G. J . Biol. Chem. 1986,
261, 8514. (d) Bassi, R.; Riboni, L.; Sonnino, S.; Tettamanti, G.
Carbohydr. Res. 1989, 193, 141. (e) Maggio, B.; Ariga, T.; Yu, R. K.
Biochemistry 1990, 29, 8729.
Resu lts a n d Discu ssion
Our synthetic strategy was based on stereoselective
8-O-sialylation of the acceptor 6, rather than introduction
of a nitrogen functionality in the 9-position of com-
mercially available bis(sialic acid) (for numbering, see
Figure 2). The latter forms a quite stable 8,9-lactone,10
(4) Nores, G. A.; Dohi, T.; Taniguchi, M.; Hakomori, S. J . Immunol.
1987, 139, 3171.
(7) (a) Livingston, P. J .; Ritter, G.; Calves, M. J . Cancer Immunol.
Immunother. 1989, 29, 179. (b) Pukel, C. S.; Lloyd, K. O.; Travassos,
L. R.; Dippold, W. G.; Oettgen, H. F.; Old, L. J . J . Exp. Med. 1982,
155, 1133. (c) Portoukalian, J .; Zwingelstein, G.; Abdul-Malak, N.; Dore´,
J . F. Biochem. Biophys. Res. Commun. 1978, 85, 916.
(8) Ando, S.; Yu, R. K.; Scarsdale, J . N.; Kusunoki, S.; Prestegard,
J . H. J . Biol. Chem. 1989, 264, 3478.
(9) (a) Erce´govic, T.; Magnusson, G. J . Chem. Soc., Chem. Commun.
1994, 831. (b) Erce´govic, T.; Magnusson, G. J . Org. Chem. 1995, 60,
3378.
(5) (a) Ray, A. K.; Nilsson, U.; Magnusson, G. J . Am. Chem. Soc.
1992, 114, 2256. (b) Wilstermann, M.; Kononov, L. O.; Nilsson, U.; Ray,
A. K.; Magnusson, G. J . Am. Chem. Soc. 1995, 117, 4742.
(6) (a) Ding, K.; Rose´n, A.; Ray, A. K.; Magnusson, G. Glycoconjugate.
J . 1992, 9, 303. (b) Magnusson, G.; Ding, K.; Nilsson, U.; Ray, A. K.;
Rose´n, A.; Sjo¨gren, H.-A. In Complex Carbohydrates in Drug Research;
Bock, K., Clausen, H., Eds.; Munksgaard: Copenhagen, 1994; pp 89-
100.
0022-3263/96/1961-0179$12.00/0 © 1996 American Chemical Society

180 J . Org. Chem., Vol. 61, No. 1, 1996
Erce´govic and Magnusson
Sch em e 2a
Sch em e 1a
a
Key: (a) p-MePhSO2Cl, CH2Cl2/pyridine, -80 °C, 48 h; (b)
NaN3, 18-crown-6, DMF, 60 °C, 20 h; (c) BzCl, Et3N, -30 °C, 18
h.
and it is by no means trivial to exploit this for regio-
selective introduction of nitrogen at C-9.
The sialic ester glycoside 311 was treated with 2 equiv
of p-toluenesulfonyl chloride at -80 °C, which gave the
9-O-tosylate 4 (91%) after chromatography (Scheme 1).
Regioselective 9-O-tosylations of sialic ester glycosides
have been conducted earlier.12 Treatment of the tosylate
4 with sodium azide in N,N-dimethylformamide in the
presence of 18-crown-6 ether as catalyst gave the 9-azido
compound 5 (83%).
a
Key: (a) p-MePhSO2Cl, pyridine, 0 f 20 °C, 22 h; (b) LiN3,
15-crown-5, THF, 45 °C, 24 h; (c) MeONa, MeOH, 20 °C, 2 h; (d)
BzCl, CH2Cl2/pyridine, -80 °C, 96 h.
which had disappeared when the reaction was complete.
It is thus expected that the transformation of 8 into 10
proceeds via the tosylate 9 (Scheme 2). 1H-NMR analysis
of the reaction mixture supported this assumption.
Tetrabutylammonium azide in acetonitrile gave rapidly
the desired product according to TLC analysis. However,
rearrangement into 10 occurred during workup of the
reaction mixture. De-O-acetylation of 10 gave 5 (89%),
identical with the product obtained by azide treatment
of 4 (Scheme 1). As a consequence of these studies, acyl
protection of HO-7 in sialic acids should be avoided and
sialylations in the 8-position be performed with acceptors
such as 6 that have both HO-7 and HO-8 unprotected,
as shown in Scheme 3.
The joining of two sialic acid units via an R2f8
glycosidic bond in high yield and Râ selectivity is difficult,
and consequently, many synthetic approaches have been
published.15 In a recent paper, we reported the synthesis
of the novel sialyl donor 12, which was found to be
superior to conventional donors. Sialylation yields were
higher, even with sterically hindered and unreactive
sialyl acceptors, and the Râ-selectivity was virtually
complete.9 Compound 12, which carries an auxiliary
phenylthio substituent16 in the 3-position, was synthe-
sized from N-acetylneuraminic acid in six steps and 47%
overall yield.9
The 9-azido diol 6 was sialylated by the donor 12 at
-40 °C in acetonitrile, using methyl sulfenyl bromide17
/silver trifluoromethane sulfonate as promotor, to give
the R2f8 bis(sialoside) 13 in 28% yield (Scheme 3).
When the concentration of the reactants was halved, the
yield of 13 was reduced to 23%. However, the main part
(63%) of acceptor 6 was thus recovered, and the yield,
based on consumed 6, was raised from 47% to 64% when
the more diluted reaction mixture was employed. Ac-
cording to TLC and NMR analysis, additional bis-
(sialosides) were formed, but these were extremely labile
and could not be isolated; their identity remains un-
At this point, the reactivity order was not settled for
the three secondary hydroxyl groups in 5. Treatment of
5 with benzoyl chloride in dichloromethane/pyridine at
-80 °C (Scheme 2) gave the desired monobenzoate 6
(55%) and the dibenzoate 11 (21%). In order to improve
the yield of 6, alternative benzoylation procedures were
investigated. Regioselective O-benzoylation has been
performed using benzoyl cyanide/triethylamine or 1-(ben-
zoyloxy)benzotriazole/triethylamine.13 Since benzoyl cya-
nide is reported to be unreactive in the absense of
triethylamine in aprotic solvents,13a we thought that
benzoyltriethylammonium cyanide was the reactive spe-
cies needed for regioselective benzoylation to occur.
Hence, we expected that the new combination benzoyl
chloride/triethylamine (Scheme 1) would constitute a
viable alternative. Treatment of 5 with the latter reagent
combination in dichloromethane at -30 °C gave the
desired sialyl acceptor 6 (95%) as the only product
formed. The order of reactivity of the four hydroxyl
groups in 3 was thus established to be HO-9 >> HO-4 >
HO-8 >> HO-7.
Prior to the successful preparation of 6 (Scheme 1), we
investigated the regioselective introduction of a 9-azido
group in the known diol 714 (Scheme 2). Treatment of 7
with a large excess of p-toluenesulfonyl chloride gave the
monotosylate 8 (92%). However, treatment of 8 with
lithium azide in tetrahydrofuran, using 15-crown-5 ether
as catalyst, caused the 7-O-acetyl group to migrate to the
8-position, and 10 (82%) was the only compound isolated.
Monitoring the reaction by TLC revealed the intermedi-
ate formation of a strongly UV-absorbing compound,
(10) (a) Abbas, S. Z.; Sugiyama, S.; Diakur, J .; Pon, R. A.; Roy, R.
J . Carbohydr. Chem. 1990, 9, 891. (b) Hasegawa, A.; Ishida, H.; Kiso,
M. J . Carbohydr. Chem. 1993, 12, 371.
(11) Hasegawa, A.; Ito, Y.; Ishida, H.; Kiso, M. J . Carbohydr. Chem.
1989, 8, 125.
(12) (a) Warner, T. G. Biochem. Biophys. Res. Commun. 1987, 148,
1323. (b) Isecke, R.; Brossmer, R. Tetrahedron 1994, 50, 7445.
(13) (a) Holy, A.; Sovcek, M.; Tetrahedron Lett. 1971, 185. (b) Abbas,
S. A.; Haines, A. H. Carbohydr. Res. 1975, 39, 358. (c) Kim, S.; Chang,
H.; Kim, W. J . J . Org. Chem. 1985, 50, 1751.
(14) Hasegawa, A.; Ito, Y.; Morita, M.; Ishida, H.; Kiso, M. J .
Carbohydr. Chem. 1989, 8, 135.
(15) (a) Okamoto, K.; Kondo, T.; Goto, T. Tetrahedron Lett. 1986,
43, 5229. (b) Kanie, O.; Kiso, M.; Hasegawa, A. J . Carbohydr. Chem.
1988, 7, 501. (c) Tsvektov, Y. E.; Schmidt, R. R. Tetrahedron Lett. 1994,
35, 8583.
(16) Ito, Y.; Ogawa, T. Tetrahedron 1990, 46, 89.
(17) Dasgupta, F.; Garegg, P. J . Carbohydr. Res. 1988, 177, C13.

Synthesis of a Bis(sialic acid) 8,9-Lactam
J . Org. Chem., Vol. 61, No. 1, 1996 181
Sch em e 3a
a
Key: (a) MeSBr, CF3SO3Ag, 3 Å MS, MeCN, -40 °C, 2 h; (b) MeONa, MeOH/toluene, 20 °C, 16 h; (c) Ph3P, THF/H2O, 40 °C, 20 h;
(d) Ra-Ni, EtOH, 20 °C, 3 h, then Ph3P, 1 h; (e) NaOH, MeOH/H2O, 20 °C, 24 h, Sephadex G10; (f) MeONa, MeOH, 20 °C, 24 h, then
HOAc, 20 °C, 22 h, freeze-drying.
known. All of the donor 12 was destroyed in the reaction.
Since 12 gave high yields (71-77%) with galactoside and
lactoside diol acceptors,9 the low yields obtained with
sialic acid diol acceptors indicate that the latter are
highly hindered. More stable, yet highly reactive, sialyl
donors are thus required in order to obtain high yields
in sialylations of sialic acid-derived acceptors in the
8-position. Such donors remain to be developed.
In our previous syntheses of ganglioside lactams, we
found that the conditions used for reduction of the azide
functionality caused the intermediate amino ester to
lactamize.5 However, reduction of the 9-azido group of
13, using hydrogen sulfide as reducing agent, gave almost
no lactamization. De-O-acylation of 13 (f 14, 80%),
followed by reduction of 14 with triphenylphosphine, gave
the desired 8,9-lactam 16 (presumably via the amine 15)
in 87% yield.
Reductive removal of the auxiliary phenylthio sub-
stituent of 16 was performed with Raney nickel in
ethanol. Initial attempts gave the desired lactam 1 in
<20% yield, due to absorption of 1 to the surface of the
Raney nickel particles. However, addition of triphenyl-
phosphine to the reaction mixture prior to filtration
caused desorption of 1 and an acceptable yield of 60%
was obtained. The yield might well be improved by
optimizing the type and amount of the desorption agent.
It deserves to be mentioned that the phenylthio group
could not be removed by traditional tin hydride re-
agents;16,18 either several products were formed or no
reaction occurred.
1 has provided useful information for our planned
synthesis of the tetrasaccharidic GD3-ganglioside lactam.
Furthermore, compound 1 is well suited as starting
material for the preparation of neoglycoprotein antigens,
similar in scope to GM3-lactam-BSA used for raising
monoclonal antibodies that crossreacted with GM3-lactone
on cell surfaces.6
The bis(sialic acid) lactone 179b was de-O-acylated, and
the crude product was freeze-dried from an acetic acid
solution to give lactone 18 (72%) for comparison with 1
concerning the solution conformations of the two com-
pounds. The lactone ring of 18 was partially opened by
methanol to give an equilibrium mixture (∼9:1) of 18 and
the corresponding bis(methyl ester), as indicated in
Figure 2C. FAB mass spectroscopy of the mixture gave
peaks at m/ z 697.3 (18) and m/ z 729.3 (bis(methyl
ester)), thus confirming the presence of the bis(methyl
ester).
Comparison of the 1H-NMR spectra (Figure 2) of 1 and
18 showed that coupling constants diagnostic of the sialic
acid- and lactam/lactone-ring conformations are very
similar (Figure 2 and Table 1). The conformational
similarity was corroborated by a molecular mechanics
calculation [MM3(92)]19 of 1 and 18 (Figure 2). Super-
imposition and RMS-fitting of the low energy conforma-
tions of 1 and 18 (using all ring atoms) showed them to
have very similar overall shapes (rms ) 0.027 Å), as
depicted in Figure 2.
(19) (a) Burkert, U.; Allinger, N. L. Molecular Mechanics; American
Chemical Society: Washington, D.C., 1982. (b) The MM3 force-field:
Allinger, N. L.; Yuh, Y. H.; Lii, J .-H. J . Am. Chem. Soc. 1989, 111,
8551. (c) Molecular construction and energy minimization were made
with the MacMimic/MM3 package: InStar Software, Ideon Research
Park, S-22370 Lund, Sweden.
Hydrolysis of the methyl ester function of 1 gave the
sodium salt 2 in 85% yield. The successful synthesis of
(18) Kondo, T.; Abe, H.; Goto, T. Chem. Lett. 1988, 1657.

182 J . Org. Chem., Vol. 61, No. 1, 1996
Erce´govic and Magnusson
F igu r e 2. (A) Stereoview of the superimposed low energy [MM3(92)]19 conformers of the methyl glycosides that correspond to 1
1
and 18. (B) 1H-NMR (500 MHz, CD3OD) spectrum of 1. (C) H-NMR (500 MHz, CD3OD) spectrum of 18 (contaminated by ∼10%
of the corresponding bis(methyl ester), obtained by reaction of 18 with the solvent).
Ta ble 1. Selected 1H NMR cou p lin g con sta n ts for 1 a n d
As with the mono(sialic acid)-containing lactams re-
ported earlier,5 bis(sialic acid)-containing lactams also
18
seem to be good substitutes for the natural sialic acid
lactones, thus making the lactams potentially useful as
hydrolytically stable immunogens for the preparation of,
e.g., anti-lactone antibodies.
coupling constant (Hz)
couplinga
1
18
H3a-H3e
H3a-H4
H3e-H4
H7-H8
H9A-H9B
H8-H9A
H8-H9B
H3′a-H3′e
H3′a-H4′
H3′e-H4′
12.6
11.9
4.6
12.7
12.3
4.6
8.6
7.7
Exp er im en ta l Section
13.1
11.0
5.9
11.8
11.6
5.2
Gen er a l. NMR spectra were recorded at 500 and 300 MHz.
Assignment of 1H NMR spectra was achieved using 2D-
methods (COSY, HETCOR). Optical rotations were measured
at +20 °C. Chemical shifts are expressed in ppm using
residual CHCl3, CHD2OD as reference. Reactions were moni-
tored by TLC using alumina plates coated with silica gel 60
F254 (Merck) and visualized using either UV light or by
charring with H3PO4 (aqueous 10% spray solution). Prepara-
tive chromatography was performed with Merck silica gel (35-
70 µm, 60 Å). CH2Cl2, toluene, and THF were distilled under
N2 over CaH2 and sodium benzophenone ketyl, respectively.
MeCN and NEt3 were stored over 3 Å molecular sieves and
filtered through a column of Al2O3 (activity I, Merck) im-
12.7
10.9
5.4
13.1
11.2
5.4
a
Assignments were made by COSY and HETCOR procedures.
The 1H NMR spectra of 1 and 18 require some
additional comments. In both compounds, the ring
hydrogen H-4′ is observed at an unusually large chemical
shift (∼4.4 ppm). This is probably due to close proximity
between H-4′ and the carbonyl oxygen of the lactam and
lactone rings. The H-4′-O-distance is ∼2.6 Å in the
energy-minimized structures obtained by the MM3-
calculations. Such oxygen-induced chemical shifts have
been observed in other saccharides.20
(20) (a) Tho¨gersen, H.; Lemieux, R. U.; Bock, K.; Meyer, B. Can. J .
Chem. 1982, 60, 44. (b) Bock, K.; Frejd, T.; Kihlberg, J .; Magnusson,
G. Carbohydr. Res. 1988, 176, 253. (c) Gro¨nberg, G.; Nilsson, U.; Bock,
K.; Magnusson, G. Carbohydr. Res. 1994, 257, 35.

Synthesis of a Bis(sialic acid) 8,9-Lactam
J . Org. Chem., Vol. 61, No. 1, 1996 183
mediately before use. Methanol was dried over 3 Å molecular
sieves >3 days before use. Compounds obtained as white
powders were precipitated with n-hexane from a chloroform/
diethyl ether solution.
3) δ 3.97 (ddd, 1 H, J ) 9.0, 2.6, 6.3 Hz), 3.82 (m, 1 H), 3.78 (s,
3 H), 3.66 (t, 1 H, J ) 10.1 Hz), 3.57 (dd, 1 H, J ) 12.7 Hz),
3.45 (m, 1 H), 3.40 (dd, 1 H, J ) 1.9 Hz), 3.37-3.27 (m, 3 H),
2.69 (dd, 1 H, J ) 13.1, 4.7 Hz), 1.99 (s, 3 H), 1.79 (t, 1 H, J )
12.5 Hz), 0.82 (m, 2 H), -0.05 (s, 9 H); 13C NMR (CDCl3) δ
173.6, 169.9, 98.6, 73.7, 70.6, 69.5, 68.3, 62.0, 53.6, 53.2, 53.0,
40.8, 29.7, 23.2, 18.0. HR FAB-MS calcd for C17H33N4O8Si (M
+ H) 449.2067, found 449.2069.
(b) To a stirred solution of 10 (233 mg, 0.437 mmol) in dry
methanol (3 mL) was added a solution of NaOMe in methanol
(0.050 mL, ∼2 M) under N2. After 2 h at rt, Amberlite IR-120
was added and the reaction mixture was filtered, washed with
methanol, concentrated, and chromatographed (toluene/EtOH
15:1 f 10:1, gradient) to give 5 (175 mg, 89%) as a white
powder.
Meth yl [2-(Tr im eth ylsilyl)eth yl 5-a ceta m id o-9-a zid o-
4-O-b en zoyl-3,5,9-t r id eoxy-D-glycer o-r-D-ga la cto-2-n on -
u lop yr a n osid ]on a te (6) a n d Meth yl [2-(Tr im eth ylsilyl)-
eth yl 5-acetam ido-9-azido-4,8-di-O-ben zoyl-3,5,9-tr ideoxy-
D-glycer o-r-D-ga la cto-2-n on u lop yr a n osid ]on a te (11). (a)
A stirred solution of 5 (132 mg, 0.295 mmol) in dry dichloro-
methane/pyridine 2:1 (1.5 mL) was cooled to -70 °C under Ar,
and benzoyl chloride (0.034 mL, 0.29 mmol) was added. The
mixture was stirred vigorously for 20 min and then kept at
-80 °C for 3 d, after which additional benzoyl chloride (0.007
mL, 60 µmol) was added. After 24 h at -80 °C, water (0.050
mL) was added, and the mixture was concentrated and
chromatographed (toluene/EtOH 80:1 f 50:1 f 10:1, gradient)
to give 11 (41 mg, 21%) and 6 (90 mg, 55%) as white powders
and recovered 7 (20 mg, 15%) as a syrup. Compound 6: [R]D
-31° (c 1.00, CHCl3); IR (neat) 2090, 1720 cm-1; 1H NMR (300
MHz, CDCl3/CD3OD ∼97:3) δ 7.99, 7.60, 7.45 (5 H), 5.12 (ddd,
1 H, J ) 12.2, 4.9, 10.5 Hz), 4.14-4.04 (m, 2 H), 3.90 (m, 1 H),
3.86 (s, 3 H), 3.63 (dd, 1 H, J ) 2.5, 12.8 Hz), 3.52-3.45 (m, 2
H), 3.41 (dd, 1 H, J ′ ) 6.3 Hz), 3.39 (m, 1 H), 2.82 (dd, 1 H, J
) 12.9 Hz), 2.10 (t, 1 H), 1.91 (s, 3 H), 0.86 (m, 2 H), -0.01 (s,
9 H); 13C NMR (CDCl3) δ 173.0, 169.5, 167.5, 133.9, 129.8,
128.7, 128.6, 98.4, 73.9, 70.5, 69.6, 69.4, 62.1, 53.6, 53.3, 51.7,
37.5, 23.0, 17.9; HR FAB-MS calcd for C24H37N4O9Si (M + H)
553.2329, found 533.2330.
Meth yl {2-(Tr im eth ylsilyl)eth yl 5-a ceta m id o-9-a m in o-
3,5,9-tr id eoxy-8-O-[5-a ceta m id o-3,5-d id eoxy-D-glycer o-r-
D-ga la cto-2-n on u lop yr a n osyl]-D-glycer o-r-D-ga la cto-2-
n on u lop yr a n osid }on a te 1′f9-La cta m (1). Compound 16
(8.7 mg, 0.0108 mmol) was stirred vigorously with Raney-Ni
(1 mL, 50% slurry in water) in ethanol (4 mL) at room
temperature for 3 h. Triphenylphosphine (0.35 g) was added,
and the stirring was continued for 1 h. The reaction mixture
was filtered through glass fiber paper (Whatman) on a Celite
pad (MeOH). The eluate was concentrated, and the residue
was chromatographed (CH2Cl2/MeOH/H2O 40:10:1) to give 1
(4.5 mg, 60%) as a white solid: [R]D -36° (c 0.34, CH3OH); IR
1
(neat) 1738, 1680, 1635 cm-1; H NMR (500 MHz, CD3OD) δ
4.42 (ddd, 1 H, J ) 10.9, 5.4, 10.0 Hz), 4.15 (ddd, 1 H, J ) 5.9,
8.6, 11.0 Hz), 3.92 (m, 1 H), 3.79 (s, 3 H), 3.73 (t, 1 H, J ) 10.3
Hz), 3.69 (dd, 1 H, J ) 13.1 Hz), 3.62 (m, 1 H), 3.56 (dd, 1 H,
J ) 1.5, 10.5 Hz), 3.53 (dd, 1 H), 3.44 (m, 1 H), 2.64 (dd, 1 H,
J ) 4.6, 12.6 Hz), 2.48 (dd, 1 H, J ) 12.7 Hz), 2.02, 2.01, (2 s,
6 H), 1.68 (dd, 1 H, J ) 11.9 Hz), 1.58 (dd, 1 H), 0.88 (m, 2 H),
0.01 (s, 9 H); 13C NMR (CD3OD) δ 175.8, 175.4, 171.1, 170.7,
100.7, 99.0, 74.5, 74.1, 73.5, 72.3, 72.0, 69.1, 69.0, 65.4, 62.9,
54.4, 53.8, 42.8, 42.0, 41.9, 23.0, 22.7, 19.2. HR FAB-MS calcd
for C28H50N3O15Si (M + H) 696.3011, found 696.3019.
Sod iu m {2-(Tr im eth ylsilyl)eth yl 5-a ceta m id o-9-a m in o-
3,5,9-tr id eoxy-8-O-[5-a ceta m id o-3,5-d id eoxy-D-glycer o-R-
D-ga la cto-2-n on u lop yr a n osyl]-D-glycer o-R-D-ga la cto-2-
n on u lop yr a n osid }on a te 1′f9-La cta m (2). To a stirred
solution of 1 (4.3 mg, 0.0062 mmol) in methanol (0.3 mL) were
added water (0.2 mL) and aqueous sodium hydroxide (0.050
mL, 0.2654 M, 0.0133 mmol). After 24 h at room temperature,
the mixture was chromatographed (Sephadex G10, H2O). The
eluate was freeze-dried to give 2 (3.7 mg, 85%) as a white
foam: [R]D +6.2° (c 0.32, H2O); 1H NMR (500 MHz, D2O) δ
4.33 (ddd, 1 H, J ) 5.4, 10.0, 11.0 Hz), 4.23 (ddd, 1 H, J ) 4.7,
6.4, 11.1 Hz), 4.07 (dd, 1 H, J ) 1.0, 10.6 Hz), 3.91 (t, 1 H, J
) 10.1 Hz), 3.86 (t, 1 H, J ) 10.2 Hz), 3.83 (m, 1 H), 3.83 (dd,
1 H, J ) 2.4, 11.8 Hz), 3.76 (dd, 1 H, J ) 1.2, 10.5 Hz), 3.70
(dd, 1 H, J ) 1.2, 6.6 Hz), 3.63 (m, 1 H), 3.54 (m, 1 H), 3.54
(dd, 1 H, J ) 1.1, 9.3 Hz), 2.66 (dd, 1 H, J ) 4.4, 12.2 Hz),
2.58 (dd, 1 H, J ) 13.2 Hz), 2.06, 2.05 (2 s, 6 H), 1.78 (dd, 1 H,
J ) 11.4 Hz), 1.59 (t, 1 H, J ) 12.2 Hz), 0.94 (m, 2 H), 0.02 (s,
9 H); HR FAB-MS calcd for C27H47N3O15SiNa (M + H):
704.2674, found 704.2675.
Compound 11: [R]D +8.2° (c 1.00, CHCl3); 1H NMR (300
MHz, CDCl3) δ 8.06-7.40 (m, 10 H), 6.21 (d, 1 H, J ) 10.1
Hz), 5.58 (ddd, 1 H, J ) 8.4, 2.9, 4.2 Hz), 5.18 (ddd, 1 H, J )
12.1, 4.7, 10.5 Hz), 4.13 (q, 1 H), 3.95 (m, 1 H), 3.87-3.80 (m,
3 H), 3.75 (dd, 1 H, J ) 10.5, 1.8 Hz), 3.37 (m, 1 H), 3.31 (s, 3
H), 2.70 (dd, 1 H, J ) 12.7 Hz), 2.10 (t, 1 H), 1.95 (s, 3 H), 0.91
(m, 2 H), 0.02 (s, 9 H); HR FAB-MS calcd for C31H41N4O10Si
(M + H) 657.2592, found 657.2583.
(b) To a stirred solution of 5 (200.4 mg, 0.447 mmol) in
dichloromethane (3 mL) was added dry triethylamine (0.090
mL, 0.65 mmol) at rt under Ar. After cooling to -50 °C and
addition of benzoyl chloride (0.055 mL, 0.48 mmol), the mixture
was left at -30 °C for 18 h. Methanol (0.010 mL) was added,
the mixture was concentrated, and the residue was chromato-
graphed (toluene/EtOH 80:1 f 40:1, gradient) to give 6 (234
mg, 95%) as a white powder.
Met h yl [2-(Tr im et h ylsilyl)et h yl 5-a cet a m id o-3,5-d i-
d eoxy-9-O-(p -t olu en esu lfon yl)-D-glycer o-r-D-ga la cto-2-
n on u lop yr a n osid ]on a te (4). A stirred solution of 311 (244.5
mg, 0.577 mmol) in dry dichloromethane/pyridine 2:1 (4.5 mL)
was cooled to -70 °C under an argon atmosphere, and p-TsCl
(226 mg, 1.18 mmol) was added. The reaction was left at -80
°C without stirring for 2 d, after which water (0.1 mL) was
added. Stirring for 10 min at -70 °C and 30 min at rt followed
by concentration to a brown syrup which upon chromatography
(dichloromethane/methanol 25:1) gave 4 (303 mg, 91%) as a
Meth yl [2-(Tr im eth ylsilyl)eth yl 5-a ceta m id o-4,7-d i-O-
a cetyl-3,5-d id eoxy-9-O-(p-tolu en esu lfon yl)-
D-glycer o-r-D-
1
white foam: [R]D -8.6° (c 0.93, CHCl3); H NMR (300 MHz,
CDCl3/CD3OD ∼97:3) δ 7.76 (d, 2 H, J ) 8.3 Hz), 7.32 (d, 2
H), 4.30 (dd, 1 H, J ) 2.2, 10.0 Hz), 4.12 (d, 1 H, J ) 5.7 Hz),
4.00 (ddd, 1 H, J ) 9.2 Hz), 3.79 (m, 1 H), 3.78 (s, 3 H), 3.66
(t, 1 H, J ) 10.1 Hz), 3.47 (m, 1 H), 3.42 (dd, 1 H, J ) 1.7 Hz),
3.36-3.26 (m, 2 H), 2.69 (dd, 1 H, J ) 13.0, 4.7 Hz), 2.41 (s, 3
H), 2.00 (s, 3 H), 1.79 (t, 1 H, J ) 11.4 Hz), 0.82 (m, 2 H), 0.00
(s, 9 H); HR FAB-MS calcd for C24H40NO11SSi (M + H+)
578.2091, found 578.2096.
ga la cto-2-n on u lop yr a n osid ]on a te (8). To an ice-cooled,
stirred solution of 714 (235.8 mg, 0.465 mmol) in dry pyridine
(5 mL) was added p-toluenesulfonyl chloride (0.30 g, 1.6 mmol)
under N2. After 5 h at 0 °C, the mixture was left at room
temperature for 17 h. Methanol was added, the mixture was
concentrated, and the residue was chromatographed (toluene/
EtOH 30:1) to give 8 (284 mg, 92%) as a white powder: [R]D
-3.3° (c 1.01, CHCl3); 1H NMR (300 MHz, CDCl3) δ 7.80, 7.33
(2 d, 4 H), 5.24 (d, 1 H, J ) 10.1 Hz), 4.98 (dd, 1 H, J ) 2.2,
8.7 Hz), 4.81 (ddd, 1 H, J ) 12.1, 4.8, 10.3 Hz), 4.17 (m, 1 H),
4.11 (q, 1 H, J ) 10.3 Hz), 4.08 (dd, 1 H, J ) 2.8, 10.8 Hz),
3.95 (dd, 1 H, J ′ ) 6.7 Hz), 3.85 (s, 3 H), 3.89-3.78 (m, 2 H),
3.40 (m, 1 H), 2.67 (dd, 1 H, J ) 13.0 Hz), 2.44 (s, 3 H), 2.08,
2.03 (2 s, 6 H), 1.99 (t, 1 H), 1.85 (s, 3 H), 0.89 (m, 2 H), 0.02
(s, 9 H); HR FAB-MS calcd for C28H44NO13SSi (M + H)
662.2302, found 662.2299.
Meth yl [2-(Tr im eth ylsilyl)eth yl 5-a ceta m id o-9-a zid o-
3,5,9-tr id eoxy-D-glycer o-r-D-ga la cto-2-n on u lop yr a n osid ]-
on a te (5). (a) A solution of 4 (256.0 mg, 0.443 mmol), 18-
crown-6 ether (44.4 mg, 0.168 mmol), and sodium azide (148
mg, 2.27 mmol) in dry N,N-dimethylformamide (1.5 mL) was
stirred vigorously at 60 °C for 20 h after which the reaction
mixture was filtered, coconcentrated with toluene, and chro-
matographed (CH2Cl2 f CH2Cl2/EtOH 12:1, gradient) to give
5 (165 mg, 83%) as a white powder: [R]D +1.1° (c 0.95, CHCl3);
A sample of 8 was treated with acetic anhydride/pyridine
1
IR (neat) 2090 cm-1; H NMR (300 MHz, CDCl3/CD3OD ∼97:
to give the fully acetylated derivative: 1H NMR (300 MHz,

184 J . Org. Chem., Vol. 61, No. 1, 1996
Erce´govic and Magnusson
CDCl3) δ 5.35-5.27 (m, 2 H), 4.35 (dd, 1 H, J ) 2.4, 11.5 Hz),
4.01 (dd, 1 H, J ) 7.1 Hz), 2.07, 2.06, 2.02 (3 s, 9 H), 1.88 (s,
3 H).
ized with acetic acid and concentrated, and the residue was
chromatographed (CH2Cl2/MeOH 25:1 f CH2Cl2/MeOH/H2O
60:10:1, gradient) to give 14 (38 mg, 80%) as a white solid:
[R]D -36° (c 1.0, CH3OH); 1H NMR (500 MHz, CD3OD) δ 7.67-
7.19 (m, 5 H), 4.63 (dt, 1 H, J ) 6.7 Hz), 4.12 (t, 1 H, J ) 10.1
Hz), 4.04 (dd, 1 H, J ) 10.6 Hz), 4.00 (dd, 1 H, J ) 1.4, 1.9
Hz), 3.86, 3.79 (2 s, 6 H), 3.77 (dd, 1 H, J ) 1.5, 7.3 Hz), 3.76
(dd, 1 H), 3.58 (dd, 1 H, J ) 2.8, 13.1 Hz), 3.47 (dd, 1 H, J )
9.2 Hz), 3.41 (m, 1 H), 3.33 (d, 1 H), 2.65 (dd, 1 H, J ) 4.6,
12.8 Hz), 2.02, 2.02 (2 s, 6 H), 1.66 (dd, 1 H, J ) 11.6 Hz),
Meth yl [2-(Tr im eth ylsilyl)eth yl 5-a ceta m id o-4,8-d i-O-
a cetyl-9-a zid o-3,5,9-tr id eoxy-D-glycer o-r-D-ga la cto-2-n on -
u lop yr a n osid ]on a te (10). To a stirred solution of 8 (20.0
mg, 0.0302 mmol) in dry tetrahydrofuran (0.6 mL) was added
lithium azide (22 mg, 0.45 mmol) and 15-crown-5 ether (0.010
mL). After 24 h at 45 °C, the mixture was filtered and
concentrated and the residue was chromatographed (toluene/
EtOH 50:1) to give 10 (13.2 mg, 82%) as a colorless syrup: [R]D
0.84 (m, 2 H), 0.01 (s, 9 H); HR FAB-MS calcd for C35H56N5O16
-
1
-26° (c 0.37, CHCl3); H NMR (300 MHz, CDCl3) δ 5.91 (d, 1
SSi (M + H) 862.3212, found 862.3224.
H, J ) 7.5 Hz), 5.28 (ddd, 1 H, J ) 9.1, 2.9, 3.8 Hz), 4.90 (ddd,
1 H, J ) 12.2, 4.6, 10.2 Hz), 4.86 (d, 1 H, J ) 4.7 Hz), 3.95 (m,
1 H), 3.89 (m, 1 H), 3.78 (s, 3 H), 3.77-3.59 (m, 4 H), 3.24 (dt,
1 H, J ) 7.3, 9.2 Hz), 2.59 (dd, 1 H, J ) 12.8 Hz), 2.16, 2.10,
2.01 (3 s, 9 H), 1.95 (t, 1 H), 0.86 (m, 2 H), 0.01 (s, 9 H); HR
FAB-MS calcd for C21H37N4O10Si (M + H) 533.2279, found
533.2264.
A sample of 10 was treated with acetic anhydride/pyridine
to give the fully acetylated derivative: 1H NMR (300 MHz,
CDCl3) δ 5.34-5.28 (m, 2 H), 3.66-3.62 (m, 2 H), 2.17, 2.16,
2.03 (3 s, 9 H), 1.88 (s, 3 H).
Meth yl {2-(Tr im eth ylsilyl)eth yl 5-a ceta m id o-9-a m in o-
3,5,9-t r id eoxy-8-O-[5-a cet a m id o-3,5-d id eoxy-3-(p h en yl-
th io)-D-er yth r o-r-L-glu co-2-n on u lop yr a n osyl]-D-glycer o-
r-D-ga la cto-2-n on u lop yr a n osid }on a te 1′f9-La cta m (16).
To a stirred solution of 14 (17.9 mg, 0.0208 mmol) in tetrahy-
drofuran/water 8:1 (0.23 mL) was added triphenylphosphine
(22.1 mg, 0.0843 mmol), and the mixture was kept at 40 °C
for 20 h. Chloroform/methanol (∼1:1) was added, the mixture
was concentrated, and the residue was chromatographed
(CH2Cl2/MeOH/H2O 50:10:1) to give 16 (14.5 mg, 87%) as a
white solid: [R]D -44° (c 1.0, CH3OH); 1H NMR (500 MHz,
CD3OD) δ 7.63-7.16 (m, 5 H,), 4.39 (t, 1 H, J ) 10.1 Hz), 4.22
(dt, 1 H, J ) 5.4, 9.9 Hz), 3.90 (t, 1 H, J ) 10.3 Hz), 3.90 (m,
1 H), 3.68 (s, 3 H), 3.58 (ddd, 1 H, J ) 11.8, 4.6, 9.9 Hz), 3.44
(m, 1 H), 3.00 (d, 1 H, J ) 10.4 Hz), 2.58 (dd, 1 H, J ) 12.7
Hz), 2.01, 2.00 (2 s, 6 H), 1.69 (dd, 1 H), 0.86 (m, 2 H), 0.01 (s,
9 H); HR FAB-MS calcd for C34H54N3O15SSi (M + H) 804.3045,
found 804.3024.
Met h yl {2-(Tr im et h ylsilyl)et h yl 5-a cet a m id o-3,5-d i-
deoxy-8-O-[5-acetam ido-3,5-dideoxy-D-glycer o-r-D-ga la cto-
2-n on u lop yr a n osyl]-D -glycer o-r-D -ga la ct o-2-n on u lo-
p yr a n osid }on a te 1′f9-La cton e (18). To a solution of 179b
(14.0 mg, 0.0145 mmol) in methanol (0.7 mL) was added
sodium methoxide in methanol (0.003 mL, ∼2 M, ∼0.006
mmol), and the mixture was left at room temperature for 24
h and then neutralized with acetic acid, concentrated, and
dried in vacuo for 3 h. The crude product was dissolved in
acetic acid (0.5 mL), and the mixture was left at room
temperature for 22 h. The mixture was freeze-dried and the
residue was chromatographed (SiO2, CH2Cl2/MeOH/H2O 50:
10:1 f 40:10:1, gradient) to give 18 as a syrup (7.2 mg, 72%):
[R]D -64° (c 0.14, CH3OH); 1H NMR (500 MHz, CD3OD) δ 4.88
(t, 1 H, J ) 11.7 Hz), 4.59 (dd, 1 H, J ) 5.2, 11.8 Hz), 4.36
(ddd, 1 H, J ) 5.4, 11.2, 10.3 Hz), 4.32 (ddd, 1 H, J ) 7.7, 11.6
Hz), 3.92 (m, 1 H), 3.79 (s, 3 H), 3.75 (t, 1 H, J ) 10.3 Hz),
3.73 (dd, 1 H, J ) 10.4, 1.2 Hz), 3.69 (dd, 1 H, J ) 8.0 Hz),
3.60 (m, 1 H), 3.53 (m, 1 H), 3.52 (dd, 1 H, J ) 1.7, 10.6 Hz),
3.49 (dd, 1 H, J ) 9.3 Hz), 2.64 (dd, 1 H, J ) 4.6, 12.7 Hz),
2.47 (dd, 1 H, J ) 5.4, 13.1 Hz), 2.02, 2.01 (2 s, 6 H), 1.69 (t,
1 H, J ) 12.3 Hz), 1.63 (dd, 1 H, J ) 11.2 Hz), 0.88 (m, 2 H),
0.02 (s, 9 H); HR FAB-MS calcd for C28H49N2O16Si (M + H)
697.2851, found 697.2865.
Meth yl {2-(Tr im eth ylsilyl)eth yl 5-a ceta m id o-9-a zid o-
4-O-b en zoyl-3,5,9-t r id eoxy-8-O-[m et h yl (5-a cet a m id o-
4,7,8,9-tetr a-O-acetyl-3-(ph en ylth io)-3,5-dideoxy-D-er yth r o-
r-L-glu co-2-n on u lopyr an osid)on ate]-D-glycer o-r-D-ga la cto-
2-n on u lop yr a n osid }on a te (13). A solution of 6 (84.7 mg,
0.153 mmol) and 12 (160.1 mg, 0.249 mmol) in acetonitrile (1.6
mL) was stirred for 40 min at rt with 3 Å molecular sieves
(0.2 g). Silver trifluoromethanesulfonate (72.8 mg, 0.283
mmol) in acetonitrile (0.3 mL) was added under Ar, and the
temperature was lowered to -40 °C, after which a solution of
methyl sulfenyl bromide17 in 1,2-dichloromethane (0.103 mL,
2.8 M, 0.28 mmol) was added dropwise over a period of 10 min.
After 2 h, diisopropylamine (0.1 mL) was added, the stirring
was continued for 5 min at -40 °C, and the mixture was
allowed to attain room temperature. The mixture was filtered
through a short column of silica gel (toluene/acetone 1:1), the
eluate was concentrated, and the residue was chromato-
graphed (toluene/acetone 8:1 f 6:1 f 4:1 f 3:1 f 2:1,
gradient) to give recovered 6 (53.6 mg, 63%) as a syrup and
13 (40.5 mg, 23%) as a white powder: [R]D +14.5° (c 1.00,
CHCl3); IR (neat) 2095, 1735, 1660 cm-1; 1H NMR (500 MHz,
CDCl3) δ 8.03-7.23 (m, 10 H), 6.05 (d, 1 H, J ) 8.8 Hz), 5.47
(dd, 1 H, J ) 10.6, 9.9 Hz), 5.38 (ddd, 1 H, J ) 2.8, 6.4, 8.9
Hz), 5.33 (d, 1 H, J ) 9.5 Hz), 5.27 (m, 1 H), 5.26 (dd, 1 H, J
) 1.6 Hz), 4.75 (dt, 1 H, J ) 2.4, 7.3 Hz), 4.37 (dd, 1 H, J )
12.3 Hz), 4.20 (q, 2 H), 4.15 (dd, 1 H, J ) 10.9 Hz), 4.12 (dd,
1 H), 3.99 (broad d, 1 H), 3.94 (dd, 1 H, J ) 13.3 Hz), 3.88 (m,
1 H), 3.84, 3.82 (2 s, 6 H), 3.78 (dd, 1 H, J ) 10.5, 1.4 Hz),
3.50 (d, 1 H), 3.48 (m, 1 H), 3.40 (dd, 1 H), 2.77 (dd, 1 H, J )
12.8, 4.9 Hz), 2.19, 2.10, 2.07, 2.03 (4 s, 12 H), 2.09 (1 H), 1.92,
1.89 (2 s, 6 H), 0.89 (m, 2 H), 0.02 (s, 9 H); 13C NMR (CDCl3)
δ 172.2, 171.1, 171.0, 170.6, 170.1, 169.7, 168.3, 168.2, 166.8,
135.4, 133.5, 132.0, 129.8, 129.3, 128.9, 128.5, 127.5, 100.7,
98.6, 75.4, 75.3, 73.0, 72.1, 69.5, 69.4, 68.5, 67.2, 62.7, 62.2,
58.1, 52.5, 52.2, 51.1, 50.0, 37.8, 23.2, 23.0, 20.9, 20.8, 20.7,
20.6, 18.1; HR FAB-MS calcd for C50H68N5O21SSi (M + H)
1134.3897, found 1134.3956.
Ack n ow led gm en t. This work was supported by the
Swedish Natural Science Research Council.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra
Meth yl {2-(Tr im eth ylsilyl)eth yl 5-a ceta m id o-9-a zid o-
3,5,9-tr id eoxy-8-O-[m eth yl (5-a ceta m id o-3,5-d id eoxy-3-
(p h en ylt h io)-D-er yth r o-r-L-glu co-2-n on u lop yr a n osid )-
on a t e]-D-glycer o-r-D-ga la cto-2-n on u lop yr a n osid }on a t e
(14). To a stirred solution of 13 (62.7 mg, 0.0553 mmol) in
toluene (0.8 mL) was added methanol (1.6 mL) and sodium
methoxide in methanol (0.020 mL, 0.04 mmol, ∼2 M) under
Ar at room temperature. After 16 h, the mixture was neutral-
1
and H NMR data with assigned signals for all title compounds
described in the Experimental Section (30 pages). This
material is contained in libraries on microfiche, immediately
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