Stereoselective synthesis of Neu5Acalpha(2-->5)Neu5Gc: the building block of oligo/poly(-->5-O(g)(lycolyl)Neu5Gcalpha2-->) chains in sea urchin egg cell surface glycoprotein.

Article abstract of DOI:10.1021/jo025988p

The synthesis of a sialic acid dimer derivative, Neu5Acalpha(2-->5)Neu5Gc, is described. The synthetic strategy is based on the use of allyl alcohol to achieve an exclusive alpha-sialylation product. The allyloxy group is also a latent glycolic acid that provides the subsequent coupling with neuraminate with minimal protection-deprotection manipulations.

Full text of DOI:10.1021/jo025988p

Ster eoselective Syn th esis of
Neu 5Acr(2f5)Neu 5Gc: Th e Bu ild in g Block
of Oligo/P oly(f5-OglycolylNeu 5Gcr2f)
Ch a in s in Sea Ur ch in Egg Cell Su r fa ce
Glycop r otein
Gang-Ting Fan,† Chen-Chang Lee,†
,‡
Chun-Cheng Lin,*, and J im-Min Fang‡
†,§
Academia Sinica, Institute of Chemistry, Nankang,
Taipei 115, Taiwan, Department of Chemistry, National
Taiwan University, Taipei 106, Taiwan, and Department of
Chemistry, National Changhua University of Education,
Changhua 500, Taiwan
cclin@chem.sinica.edu.tw
Received May 29, 2002
Abstr a ct: The synthesis of a sialic acid dimer derivative,
Neu5AcR(2f5)Neu5Gc, is described. The synthetic strategy
is based on the use of allyl alcohol to achieve an exclusive
R-sialylation product. The allyloxy group is also a latent
glycolic acid that provides the subsequent coupling with
neuraminate with minimal protection-deprotection ma-
nipulations.
FIGURE 1. Synthetic plan of oligo/poly(f5-OglycolylNeu5GcR2f)
chains.
The species-specific interactions between sperm and
the cell surface molecules of an egg play a central role in
the fertilization of many organisms. In the case of sea
Successful synthesis of the novel (f5-Oglycolyl-Neu5-
1
GcR2f) polysialic acids in reasonable quantities would
n
certainly facilitate their relevant biological studies and
urchins, the binding of motile sperm with egg jelly coat
will trigger an acrosomal reaction for fertilization.2,3 The
applications. In addition to the variants of (1f5)oligo-
8
sialic acid, there is so far only one report on the synthesis
recent studies reveal that the egg jelly coat contains
polysialylated glycoproteins, in which the polysialic acid
of a dimeric sialic acid derivative, Neu5AcR(2f5)-
9
Neu5Gc. The reported method requires many tedious
chains, (f5-OglycolylNeu5GcR2f)
to more than 40), incorporate the repeated units of
N-glycolylneuraminic acid (Neu5Gc) with the novel 2,5-
n
(where n ranges from
protection-deprotection steps to prepare the thiosialic
acid donor and glycolic acid acceptor. Moreover, the
coupling reaction between the thiosialic acid derivative
and benzyl glycolate affords a mixture of R- and â-ano-
mers (3:1). Though the R-anomer can be isolated by
column chromatography, it would be desirable to devise
an efficient and stereoselective synthesis of glycolylsialic
acid in the exclusive R-form. We describe herein an
expedient method for the synthesis of Neu5AcR(2f5)-
Neu5Gc derivative (2) as an approach to oligo/poly(f5-
4
glycoside linkages (Figure 1).4 For the short oligo (f5-
,5
Neu5GcR-2f) chain, the nonreducing termini is capped
3
3
by 9-O-sulfated N-glycolyneuraminic acids. This sulfated
oligosialic acid is a component of a GalNAc-containing
O-linked glycoprotein. Unlike R2-8- or R2-9-linked
6
polysialic acid chains on bacterial or mammalian cells,
n
the (f5-OglycolylNeu5GcR2f) chains are resistant to exo-
and endosialidases.7
O
n
glycolyl-Neu5GcR2f) .
*
To whom correspondence should be addressed. Fax: +886-2-
Our synthetic strategy (Figure 1) is to use 2-allylsialic
2
7835007.
acid derivative 6 as the pivotal compound leading to the
sugar donor 3 and acceptor 4. Allyl alcohol is chosen for
†
Academia Sinica.
National Taiwan University.
National Changhua University of Education.
‡
§
10
sialylation to ensure a high R-selectivity, and transfor-
(1) Mengerink, K. J .; Vacquier, V. D. Glycobiology 2001, 11, 37R-
4
3R.
(2) Vacquier, V. D.; Moy, G. W. Proc. Natl. Acad. Sci. U.S.A. 1997,
(7) Kitazume, S.; Kitajima, K.; Inoue, S.; Troy, F. A., II; Lennarz,
W. J .; Inoue, Y. Biochem. Biophys. Res. Commun. 1994, 205, 893-
898.
7
4, 2456-2460.
(3) Kitazume-Kawaguchi, S.; Inoue, S.; Inoue, Y.; Lennarz, W. J .
Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 3650-3655.
(8) (a) Szabo, L.; Smith, B. L.; McReynolds, K. D.; Parrill, A. L.;
Morris, E. R.; Gervay, J . J . Org. Chem. 1998, 63, 1074-1078. (b)
Ramamoorthy, P. S.; Gervay, J . J . Org. Chem. 1997, 62, 7801-7805.
(c) Gervay, J .; Flaherty, T. M. Nguyen, C. Tetrahedron Lett. 1997, 38,
1493-1496.
(
4) (a) Lennarz, W. J . In Sialobiology and Other Novel Forms of
Glycosylation; Inoue, Y., Lee, Y. C., Troy, F. A., II, Eds.; Gakushin
Publishing Co.: Osaka, 1999; pp 1-5. (b) Kitazume, S.; Kitajima, K.;
Inoue, S.; Haslam, S. M.; Morris, H. R.; Dell, A.; Lennarz, W. J .; Inoue,
Y. J . Biol. Chem. 1996, 271, 6694-6701.
(9) Ren, C.-T.; Chen, C.-S.; Wu, S.-H. J . Org. Chem. 2002, 67, 1376-
(
5) Kitazume, S.; Kitajima, K.; Inoue, S.; Troy, F. A., II; Cho, J .-W.;
Lennarz, W. J .; Inoue, Y. J . Biol. Chem. 1994, 269, 22712-22718.
6) Troy, F. A., II. In Biology of the Sialic Acids; Rosenberg, A., Ed.;
Plenum Press: New York, 1995; pp 95-144.
1379.
(10) (a) van der Vleugel, D. J . M.; van Heeswijk, W. A. R.; Vliegent-
Hart, J . F. G. Carbohydr. Res. 1982, 102, 121-130. (b) Roy, R.;
Laferri e´ re, C. A. Can. J . Chem. 1990, 68, 2045-2054.
(
1
0.1021/jo025988p CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/18/2002
J . Org. Chem. 2002, 67, 7565-7568
7565

SCHEME 1a
SCHEME 2a
a
Reagents and conditions: (i) PyBOP, Et3N, DMF, 0 °C, 2 h,
7
0%; (ii) Ac2O, pyridine, rt, 16 h, 80%; (iii) NaIO4, cat. RuCl3‚xH2O,
CCl4/CH3CN/H2O, rt, 20 min, 67%.
a
Reagents and conditions: (i) MeOH, cat. TFA, rt, 48 h, 94%
yield; (ii) Ac2O, pyridine, rt, 48 h, 98%; (iii) AcCl, HCl(g), 0 °C, 36
h; (iv) allyl alcohol, silver salicylate, rt, 2 h, 88% overall yield for
two steps; (v) NaIO4, cat. RuCl3‚xH2O, CCl4/CH3CN/H2O, rt, 2 h,
8
3%; (vi) Me4NOH, BuOH, 105 °C, 24 h, 97%; (vii) Me3SiCl, MeOH,
rt, 20 h, 62%.
mation of the allyloxy group into glycolic acid moiety can
1
1
be achieved by an oxidative cleavage of the double bond.
According to the known procedure,12 sialic acid 5 was
subjected to esterification and peracetylation. The fully
protected sialic acid was then treated in AcCl solution
saturated with HCl gas to give the chloro derivative,13
which was replaced by allyl alcohol in the presence of
silver salicylate to give an exclusive R-anomer of com-
1
0
4
pound 6 (Scheme 1). On treatment with NaIO and
ruthenium trichloride hydrate,11 an oxidative cleavage
of the olefinic double bond in 6 occurred to give acid 3 in
8
4
3% yield. On the other hand, hydrolysis of 6 with Bu -
NOH in refluxing butanol afforded the fully deprotected
compound 7 (97%).1 By using chlorotrimethylsilane as
0b
1
4
both catalyst and dehydrating agent, the selective
esterification of 7 was realized to give 4 in 62% yield.
Attempts to prepare ester 4 by direct N,O-deacetylations
of 6 with methanesulfonic acid in MeOH15 resulted in a
complicated mixture.
With sialyl acid 3 and sialylamine 4 in hand, we
proceeded to study their solution-phase coupling reaction.
After meticulous investigations (such as activation of 3
as N-succinimidyl ester or by using EDC with/without
HOBt and triethylamine), we found that benzotriazol-1-
yloxytripyrrolidinophosphonium hexafluorophosphate (Py-
BOP) was the coupling reagent of choice. The sialic acid
dimer 8 was thus obtained in 70% yield from the coupling
F IGURE 2. Attempted enzymatic synthesis of Neu5AcR-
(2f5)Neu5Gc. Reagents and conditions: (i) N-hydroxysuccin-
imide, EDC, CH
2
Cl
2
, rt, 24 h, 92%; (ii) mannosamine hydro-
Cl , rt, 20 h, 76%; (iii) pyruvic acid, sialic
chloric salt, Et N, CH
3
2
2
acid aldolase, phosphate buffer (pH ) 7), 37 °C, 36 h.
aldolase in formation of sialic acid derivatives,16 we also
considered the possibility of using the enzymatic method
to synthesize Neu5Gc dimers such as compound 11 in
Figure 2. Acid 3 was activated as the N-succinimidyl
ester (92%) and subjected to the coupling reaction with
mannosamine to give 10 (76%), Neu5AcR(2f2)Man,
under basic conditions. The corresponding acid 12 was
obtained in 96% yield by hydrolysis of 10 using a catalytic
amount of NaOMe in MeOH. Unfortunately, many at-
tempts failed to effect the coupling reactions of 10 (or
3
reaction of 3 and 4 using PyBOP and Et N in DMF
solution (Scheme 2). The hydroxyl groups of 8 were
protected as acetates, giving 9, and the subsequent
oxidative cleavage of the olefinic double bond11 afforded
the target molecule 2 in 67% yield. The prolonged
oxidation should be avoided as it caused further cleavage
of the glycosidic bond.
1
2) with pyruvic acid by the catalysis of N-acetyl-
As various C-2-modified mannosamine derivatives
have been shown to be the substrates of sialic acid
neuraminic acid aldolase (EC 4.1.3.3, from microorgan-
ism). This outcome was presumably because the sialate-
mannosamine conjugates 10 and 12 had bulky substit-
uents at C-2 that could not fit into the active site of
aldolase.1
(11) Carlsen, P. H. J .; Katsuki, T.; Martin, V. S.; Sharpless, K. B.
J . Org. Chem. 1981, 46, 3936-3938. For a recent method, see: Travis,
B. R.; Narayan, R. S.; Borhan, B. J . Am. Chem. Soc. 2002, 124, 3824-
6, 17
3
825.
In conclusion, we have demonstrated in Schemes 1 and
(12) (a) Marra, A.; Sina y¨ , P. Carbohydr. Res. 1989, 190, 317-322.
2
a straightforward method for the synthesis of the sialic
(b) Marra, A.; Sina y¨ , P. Carbohydr. Res. 1989, 187, 35-42.
(
13) Chappell, M. D.; Halcomb, R. L. Tetrahedron 1997, 53, 11109-
1
1120.
(16) Lin, C.-C.; Lin, C.-H.; Wong, C.-H. Tetrahedron Lett. 1997, 38,
(
(
14) Brook, M. A.; Chan, T. H.Synthesis 1983, 201-203.
15) Sugata, T.; Kan, Y.; Nagaregawa, Y.; Miyamoto, T.; Higuchi,
2649-2652.
(17) Fitz, W.; Schwark, J .-R.; Wong, C.-H. J . Org. Chem. 1995, 60,
3663-3670.
R. J . Carbohydr. Chem. 1997, 16, 917-925.
7
566 J . Org. Chem., Vol. 67, No. 21, 2002

acid dimer derivative 2, Neu5AcR(2f5)Neu5Gc. The
R-anomer of 2-allyl sialate 6 serves as the common
precursor of the sugar donor (acid 3) and acceptor (amine
J ) 1.2, 2.8, 5.6, 12.8 Hz), 4.28 (1 H, dddd, J ) 1.2, 2.8, 5.6,
2.8 Hz), 5.12 (1 H, dddd, J ) 1.2, 1.2, 3.2, 10.8 Hz), 5.24 (1 H,
dddd, J ) 1.2, 1.2, 3.2, 17.2 Hz), 5.86 (1 H, dddd, J ) 5.6, 5.6,
1
1
6
1
0.8, 17.2 Hz); 13C NMR (100 MHz, DMSO-d
3.60, 64.21, 68.19, 68.30, 68.45, 71.49, 74.94, 98.35, 116.55,
6
) δ 52.70, 53.05,
4
). The allyloxy group is used as a latent glycolic acid
upon oxidative cleavage of the olefinic double bond. This
method thus provides a route to oligo(f5-Oglycolyl-Neu5-
+
8
34.52, 169.38; HRMS (FAB) calcd for C13H24NO (M + H )
322.1502, found 322.1503.
GcR2f)
n
by iterative coupling of 2 with 2-allyl neurami-
Met h yl [2-Allyl-5-glycolyla m id o-3,5-d id eoxy-5-Oglycolyl-
nate and oxidative cleavage of the olefinic double bond.
We are currently engaged in this endeavor.
[m eth yl (5-a ceta m id o-4,7,8,9-tetr a -O-a cetyl-3,5-d id eoxy-R-
D-glycer o-D-ga la cto-2-n on u lop yr a n osyl)on a te]-R-D-glycer o-
D-galacto-2-n on u lopyr an osid]on ate (8). Under an atmosphere
of argon, benzotriazole-1-yloxytripyrrolidinophosphonium hexa-
fluorophosphate (PyBOP, 374 mg, 0.72 mmol) was added in one
portion to a solution of acid 3 (200 mg, 0.36 mmol) in anhydrous
DMF (0.8 mL) at 0 °C. The resulting solution was stirred at 0
Exp er im en ta l Section
Gen er a l Con sid er a tion s. Chemicals used were reagent
grade and were used as supplied except where noted. Ethers
were distilled from sodium benzophenone ketyl; (chlorinated)
°
C for 10 min, and a solution of amine 4 (152 mg, 0.47 mmol)
and triethylamine (75 µL, 0.54 mmol) in DMF (1.2 mL) was
added slowly. The reaction was stirred at 0 °C for 2 h and then
partitioned between EtOAc and water. The organic layer was
2
hydrocarbons, and amines from CaH . Unless otherwise stated,
all reactions requiring anhydrous conditions were performed
under an atmosphere of argon. Analytical thin-layer chroma-
tography was performed using silica gel 60 F254 glass plates
washed with brine, dried over anhydrous MgSO
and purified by silica gel column chromatography (CHCl
4
, concentrated,
/MeOH,
9:1-9:1, v/v) to give compound 8 as a colorless oil (217 mg,
3
(
Merck); compound spots were visualized by UV light (254 nm)
and/or by staining with a yellow solution containing Ce(NH
NO (0.5 g) and (NH Mo O (24.0 g) in 6% H SO (500
mL) or a red solution containing p-anisaldehyde (3.7 mL), acetic
acid (15 mL), and concentrated H SO (50 mL) in ethanol (1350
mL). Flash column chromatography was performed on silica gel
2
7
Cl
)
9
)
3
(
)
2
-
23
4
0%): TLC (MeOH/CHCl
2
, 1:4) R
); H NMR (400 MHz, CDCl ) δ 1.88 (3 H, s), 1.92 (1 H, dd, J
11.6, 13.2 Hz), 2.01 (3 H, s), 2.02 (3 H, s), 2.04 (1 H, dd, J )
) 0.63; [R]
2
+1.3 (c 0.3, CH -
3
f
D
(
3
)
6
4
)
6
7
O
24‚4H
2
2
4
1
3
2
4
.6, 13.6 Hz), 2.09 (3 H, s, OAc), 2.12 (3 H, s), 2.61 (1 H, dd, J
5.2, 13.6 Hz), 2.83 (1 H, dd, J ) 4.8, 13.6 Hz), 3.45 (1 H, br),
.47 (1 H, dd, J ) 1.6, 10.8 Hz), 3.49-3.53 (1 H, m), 3.69-3.78
3 H, m), 3.80 (3 H, s), 3.81 (3 H, s), 3.84-3.96 (3 H, m, H-5,
H-8), 3.92 (1 H, dddd, J ) 1.2, 1.2, 5.6, 12.8 Hz), 4.00 (1 H, dd,
J ) 5.6, 12.8 Hz), 4.03 (1 H, dd, J ) 1.6, 10.8 Hz), 4.11-4.18 (1
H, m), 4.22-4.30 (2 H, m), 4.28 (1 H, dddd, J ) 1.2, 1.2, 5.6,
1
13
6
0 (40-63 µm, Merck). Chemical shifts of H and C NMR
spectra are reported relative to CDCl [δ 7.24, δ (central line
3
H
C
of t) 77.0]. Correlation spectroscopy (COSY) was often applied
to the NMR peak assignments. Low- and high-resolution mass
spectra were recorded under fast atom bombardment (FAB)
conditions. Optical rotations were measured on a digital pola-
1
2.8 Hz), 4.36 (1 H, dd, J ) 5.6, 12.8 Hz), 4.53 (1 H, d, J ) 4.4
Hz), 4.98 (1 H, ddd, J ) 5.2, 9.6, 9.6 Hz), 5.14 (1 H, dddd, J )
.2, 1.2, 3.2, 10.4 Hz), 5.23 (1 H, dddd, J ) 1.2, 1.2, 3.2, 17.2
Hz), 5.24-5.30 (2 H, m), 5.33 (1 H, d, J ) 10.0 Hz), 5.83 (1 H,
D
rimeter with a cuvette of 10 cm length. [R] values are given in
-1
2
-1
1
0
deg cm
g .
1
Compounds 6 and 7 were prepared from sialic acid according
to the published procedures.
Meth yl (2-Ca r boxylm eth yl-5-a ceta m id o-4,7,8,9-tetr a -O-
a cetyl-3,5-d id eoxy-R-D-glycer o-D-ga la cto-2-n on u lop yr a n o-
sid )on a te (3). To a biphasic solution of compound 6 (0.50 g, 0.94
1
0,12
13
dddd, J ) 5.6, 5.6, 10.4, 17.2 Hz), 6.53 (1 H, d, J ) 8.0 Hz);
C
NMR (100 MHz, CDCl /CD OD) δ 19.70, 19.70, 19.79, 20.15,
3
3
21.58, 36.17, 48.76, 51.88, 52.27, 52.40, 61.74, 62.57, 63.09, 64.66,
66.32, 66.49, 67.58, 68.50, 69.01, 70.55, 71.63, 71.71, 72.83, 98.01,
98.09, 116.22, 133.14, 167.13, 169.25, 169.73, 170.25 (2×),
mmol) and NaIO
mL)/H O (3 mL) was added ruthenium(III) chloride hydrate (0.01
g, 0.05 mmol). The reaction mixture was stirred vigorously at
room temperature for 2 h and then diluted with CH Cl (10 mL).
The aqueous layer was separated and washed with CH Cl (20
mL × 3). The combined organic phase was dried over anhydrous
MgSO , filtered over a short pad of Celite, concentrated, and
purified by silica gel column chromatography (CHCl /MeOH,
4 4 3
(0.82 g, 3.85 mmol) in CCl (2 mL)/CH CN (2
2
171.00, 171.50, 171.64; HRMS (FAB) calcd for C35
+ H ) 853.3090, found 853.3102.
H
53
N
2
O
22 (M
+
2
2
Met h yl [2-Allyl-5-glycolyla m id o-4,7,8,9-t et r a -O-a cet yl-
3,5-d id eoxy-5-Oglycolyl-[m eth yl (5-a ceta m id o-4,7,8,9-tetr a -O-
a cet yl-3,5-d id eoxy-R-D-glycer o-D-ga la ct o-2-n on u lop yr a n -
osyl)on a t e ]-R-D-glyce r o-D-ga la ct o-2-n on u lop yr a n osid ]-
on a te (9). To a solution of compound 8 (217 mg, 0.25 mmol) in
pyridine (2 mL) was added neat acetic anhydride (3 mL)
dropwise of over a period of 1 min at 0 °C. The mixture was
stirred at room temperature for 16 h, concentrated under
reduced pressure, and then partitioned between EtOAc and
water. The organic layer was washed with brine, dried over
4
2
2
4
3
9
:1-2:1, v/v) to give product 3 as a white foam (0.43 g, 83%):
23
TLC (MeOH/CHCl
3
(1:4)) R
f
) 0.21; [R]
) δ 1.66 (3 H, s), 1.69 (1 H, dd, J
12.4, 12.4 Hz), 1.92 (3 H, s), 1.97 (3 H, s), 2.00 (3 H, s), 2.07
3 H, s), 2.57 (1 H, dd, J ) 4.8, 12.4 Hz), 3.67-3.73 (1 H, m),
.73 (3 H, s), 3.80-3.95 (3 H, m), 4.01 (1 H, dd, J ) 6.0, 12.4
D
-6.2 (c 1.2, MeOH);
1
H NMR (400 MHz, DMSO-d
6
)
(
3
anhydrous MgSO , concentrated, and purified by silica gel
Hz), 4.17 (1 H, dd, J ) 3.2, 12.4 Hz), 4.72 (1 H, ddd, J ) 4.8, 9.6,
column chromatography (CHCl /MeOH, 29:1-9:1, v/v) to give
3
1
6
2.4 Hz), 5.13 (1 H, dd, J ) 1.6, 8.4 Hz), 5.24 (1 H, ddd, J ) 3.2,
product 9 as a pale yellow oil (208 mg, 80%): TLC (MeOH/CHCl ,
3
.0, 8.4 Hz), 7.69 (1 H, d, J ) 9.2 Hz); 13C NMR (100 MHz,
23
1
1:9) R ) 0.68; [R]
f
D
-8.5 (c 0.5, CH Cl ); H NMR (400 MHz,
CDCl ) δ 1.84 (3 H, s), 1.93 (1 H, dd, J ) 12.4, 12.4 Hz), 1.98 (1
2
2
CDCl
3
) δ 20.66, 20.66, 20.73, 21.05, 22.94, 37.24, 49.16, 53.23,
3
6
1
2.27, 62.78, 67.03, 68.52, 69.04, 72.18, 98.24, 168.01, 170.03,
70.80, 170.93, 174.05, 171.25, 174.76; HRMS (FAB) calcd for
H, dd, J ) 12.8, 12.8 Hz), 1.98 (3 H, s), 2.00 (9 H, s), 2.07 (3 H,
s), 2.08 (3 H, s), 2.09 (3 H, s), 2.11 (3 H, s), 2.61 (1 H, dd, J )
4.8, 12.8 Hz), 2.62 (1 H, dd, J ) 4.8, 12.4 Hz), 3.75 (3 H, s),
3.73-3.77 (1 H, m), 3.81 (3 H, s), 3.80-3.85 (1 H, m), 4.02-4.13
(7 H, m), 4.21-4.26 (2 H, m), 4.27 (1 H, dd, J ) 2.8, 9.6 Hz),
4.82-4.89 (2 H, m), 5.12 (1 H, ddd, J ) 1.6, 1.6, 2.8, 10.8 Hz),
5.19 (1 H, dd, J ) 1.6, 8.0 Hz), 5.24 (1 H, dddd, J ) 1.6, 1.6, 3.2,
17.2 Hz), 5.28 (2 H, br), 5.36-5.40 (2 H, m), 5.81 (1 H, dddd, J
+
C
22
H
32NO15 (M + H ) 550.1772, found 550.1779.
Meth yl (2-Allyl-5-am in o-3,5-dideoxy-R-D-glycer o-D-galacto-
-n on u lop yr a n osid )on a te (4). To a solution of compound 7
0.30 g, 1.0 mmol) in dry methanol (5 mL) under an atmosphere
2
(
of argon was added chlorotrimethylsilane (0.28 mL, 2.2 mmol).
The mixture was stirred at room temperature for 20 h. The
volatiles were removed under reduced pressure, and the residue
1
3
) 5.6, 5.6, 10.8, 17.2 Hz), 6.12 (1 H, d, J ) 10.0 Hz); C NMR
(100 MHz, CDCl ) δ 20.66, 20.68, 20.71, 20.75, 20.77, 20.80,
was subjected to flash chromatography (CHCl
3
/MeOH, 29:1-9:
3
1
, v/v) to give product 4 as a pale yellow foam (194 mg, 62%):
20.94, 21.05, 23.07, 37.43, 38.06, 48.73, 49.02, 52.61, 53.16, 62.21,
62.55, 63.72, 65.85, 67.14, 67.45, 68.42, 68.55, 68.59, 68.80, 72.55,
72.87, 98.34, 98.45, 117.23, 113.45, 167.58, 168.35, 168.51,
2
3
TLC (MeOH/CHCl
3
/H
2
O (5:5:1)) R
f
) 0.57; [R]
D
-12.5 (c 2.4,
1
MeOH); H NMR (400 MHz, CD
3
OD) δ 1.75 (1 H, dd, J ) 12.4,
1
1
3
2.4 Hz), 2.70 (dd, 1 H, J ) 4.8, 12.4 Hz), 2.96 (1 H, dd, J )
0.0, 10.0 Hz), 3.55 (1 H, ddd, J ) 4.8, 10.0, 12.4 Hz), 3.71-
.79 (3 H, m), 3.84 (3 H, s), 3.86-3.93 (2 H, m), 3.97 (1 H, dddd,
169.85, 169.94, 169.99, 170.17, 170.27, 170.48, 170.53, 170.58,
+
170.83; HRMS (FAB) calcd for C43
found 1021.3526.
H
61
N
2
O
26 (M + H ) 1021.3513,
J . Org. Chem, Vol. 67, No. 21, 2002 7567

Meth yl [2-Ca r boxym eth yl-5-glycolyla m id o-4,7,8,9-tetr a -
O-acetyl-3,5-dideoxy-5-Oglycolyl-[m eth yl (5-acetam ido-4,7,8,9-
t et r a -O-a cet yl-3,5-d id eoxy-R-D-glycer o-D-ga la ct o-2-n on -
u lop yr a n osyl)on a te]-R-D-glycer o-D-ga la cto-2-n on u lop yr a n -
osid ]on a te (2). By a procedure similar to that for compound 3,
gel filtration (LH20, CHCl
colorless foam (125.4 mg, 76%): H NMR (400 MHz, CDCl
3
/MeOH 1:1) to give the product as
1
3
/
MeOH) for R-anomer δ 1.35 (1 H, t, J ) 7.2 Hz), 1.87 (3 H, s),
1.98 (1 H, t, J ) 12.6 Hz), 2.03 (3 H, s), 2.04 (3 H, s), 2.15 (3 H,
s), 2.16 (3 H, s), 2.73 (1 H, dd, J ) 4.8, 12.6 Hz), 3.17 (1 H, dd,
J ) 7.2, 7.2 Hz), 3.19 (1 H, dd, J ) 7.2, 7.2 Hz), 3.54 (1 H, t, J
) 9.2 Hz), 3.80-3.85 (2 H, m), 3.86 (3 H, s), 3.98-4.18 (5 H, m),
4.23-4.42 (4 H, m), 4.83-4.95 (1 H, m), 5.13 (1 H, d, J ) 1.6
Hz), 5.32 (1 H, dd, J ) 2.0, 8.8 Hz), 5.36-5.45 (1 H, m); for
â-anomer δ 1.35 (1 H, t, J ) 7.2 Hz), 1.87 (3 H, s), 1.98 (1 H, t,
J ) 12.6 Hz), 2.03 (3 H, s), 2.04 (3 H, s), 2.15 (3 H, s), 2.16 (3 H,
s), 2.73 (1 H, dd, J ) 4.8, 12.6 Hz), 3.28-3.36 (1 H, m), 3.46 (1
H, t, J ) 9.2 Hz), 3.70-3.75 (1 H, m), 3.80-3.85 (2 H, m), 3.86
(3 H, s), 3.98-4.18 (5 H, m), 4.23-4.42 (5 H, m), 4.83-4.95 (1
a mixture of compound 9 (30 mg, 0.03 mmol) with NaIO
mg, 0.12 mmol) and RuCl ‚xH O (0.3 mg, 1.5 µmol) in CCl
mL)/CH CN (0.1 mL)/H O (0.15 mL) was stirred vigorously at
room temperature for 20 min. The reaction mixture was
extracted with CH Cl twice. The combined organic phase was
dried over MgSO and subjected to silica gel column chroma-
tography (CHCl
a white foam (20.4 mg, 67%): TLC (MeOH/CHCl
4
(25.8
3
2
4
(0.1
3
2
2
2
4
3
/MeOH, 19:1-9:1, v/v) to give the product 2 as
3
(1:9)) R
f
)
2
3
1
0
1
2
.45; [R]
D
-10.0 (c 1.0, CH
2
Cl
2
); H NMR (400 MHz, CDCl
3
) δ
.89 (3 H, s), 1.99-2.08 (2 H, m), 2.03 (3 H, s), 2.04 (3 H, s),
H, m), 5.32 (1 H, dd, J ) 2.0, 8.8 Hz), 5.36-5.45 (1 H, m, H-8);
+
.05 (3 H, s), 2.05 (3 H, s), 2.13 (3 H, s), 2.13 (3 H, s), 2.14 (3 H,
LRMS (ESI) calcd for C28
H
43
N
2
O
9
(M + H ) 711.25, found 711.07.
s), 2.14 (3 H, s), 2.67 (1 H, dd, J ) 4.8, 12.8 Hz), 2.74 (1 H, dd,
J ) 4.8, 12.8 Hz), 3.81-3.84 (1 H, m), 3.83 (3 H, s), 3.87 (3 H,
s), 4.04 (1 H, dd, J ) 6.4, 12.4 Hz), 4.05-4.18 (7 H, m), 4.25 (1
H, dd, J ) 2.8, 12.4 Hz), 4.28 (1 H, dd, J ) 2.0, 8.4 Hz), 4.37 (1
H, d, J ) 18 Hz), 4.91 (1 H, ddd, J ) 4.8, 9.6, 12.0 Hz), 4.98 (1
H, ddd, J ) 4.8, 9.6, 12.0 Hz), 5.22 (1 H, dd, J ) 2.0, 8.8 Hz),
2-Deoxy-2-[m eth yl (5-acetam ido-3,5-dideoxy-R-D-glycer o-
D-ga la cto-2-n on u lop yr a n osyl)on a te]m a n n op yr a n ose (12).
To a solution of compound 10 (42 mg, 0.06 mmol) in anhydrous
MeOH (1 mL) under Ar at room temperature was added sodium
methoxide (9 mg, 0.18 mmol). The reaction was kept at room
2
temperature for 48 h and purified by gel filtration (P-2, H O) to
5
.25 (1 H, d, J ) 9.6, 9.6 Hz), 5.33-5.34 (2 H, m), 5.36 (1 H,
give the product as white foam (30.5 mg, 96%): H NMR (400
1
1
3
ddd, J ) 2.8, 6.4, 8.8 Hz), 6.17 (1 H, d, J ) 9.6 Hz); C NMR
MHz, MeOH) δ 1.90 (1 H, dd, J ) 4.8, 12.0 Hz), 1.96 (1 H, dd,
J ) 9.6, 12.4 Hz), 2.14 (3 H, s), 2.86 (1 H, dd, J ) 4.8, 12.8 Hz),
2.92 (1 H, dd, J ) 4.8, 12.8 Hz), 3.50-4.30 (30 H, m), 4.80-5.10
(
100 MHz, CDCl
3
) δ 20.82, 20.86 (2×), 20.93, 20.96, 21.14, 21.24,
2
6
9
1
(
1
3.29, 37.56, 37.80, 45.38, 48.85, 49.35, 53.22, 53.40, 62.35, 62.72,
3.90, 67.28, 67.32, 68.18, 68.41, 68.64, 68.90, 70.11, 72.84, 73.04,
5.56, 95.57, 167.76, 167.95, 168.78, 170.05, 170.09, 170.12,
70.28, 170.43, 170.62, 170.67, 170.18, 171.00, 199.80; HRMS
MALDI) calcd for C42
+
(2 H, m); LRMS (ESI) calcd for C19
551.17, found 551.59.
H
32
N NaO (M + Na )
2
15
+
H
59
N
2
O
28 (M + Na ) 1061.3073, found
Ack n ow led gm en t. We thank the Academia Sinica,
National Science Council in Taiwan (NSC 90-2323-B-
061.3072.
-Deoxy-2-[m et h yl (5-a cet a m id o-4,7,8,9-t et r a -O-a cet yl-
,5-dideoxy-R-D-glycer o-D-galacto-2-n on u lopyr an osyl)on ate]-
2
0
01-005 and NSC 90-2113-M-001-056), and National
3
m a n n op yr a n ose (10). To a suspended mixture of D-mannose
amine hydrochlorate (50 mg, 0.23 mmol) and triethylamine (64
µL, 0.46 mmol) in anhydrous DMF (0.7 mL) was added the
N-hydroxysuccinimide-linked compound 3 (150 mg, 0.23 mmol)
under Ar at 0 °C. The reaction was stirred at 0 °C to room
temperature for 20 h and isolated by silica gel chromatography
Taiwan University for their financial support.
Su p p or tin g In for m a tion Ava ila ble: 1H and 13C spectra
of compounds 2, 3, 4, 8, and 9. This material is available free
of charge via the Internet at http://pubs.acs.org.
(CHCl
3
/MeOH, 49:1-4:1). The product was further purified by
J O025988P
7
568 J . Org. Chem., Vol. 67, No. 21, 2002