Synthesis of phosphorylated and sulfated glycosyl serines in the linkage region of the glycosaminoglycans

Article abstract of DOI:10.1021/jo001540h

We synthesized novel acidic glycans having acidic groups located in the linkage region of the glycosaminoglycans (GAGs). The targeted compounds, β-D-Xyl(2P)-Ser (1), β-D-Gal(±6S)-(1→4)-β-D-Xyl(2P)-Ser (3 and 2), β-D-Gal(±6S)-(1→3)-β-D-Gal- (1→4)-β-D-Xyl(2P)-Ser (5 and 4), and β-D-Gal-(1→3)-β-D-Gal(6S)-(1→4)-β-D-Xyl(2P)-Ser (6) contain phosphate and/or sulfate at the specified positions. Some of them (3, 5, and 6) are the first synthesized examples of natural-type glycoconjugates that simultaneously possess phosphate and sulfate as well as carboxylic acid.

Full text of DOI:10.1021/jo001540h

3074
J . Org. Chem. 2001, 66, 3074-3083
Syn th esis of P h osp h or yla ted a n d Su lfa ted Glycosyl Ser in es in th e
Lin k a ge Region of th e Glycosa m in oglyca n s
J un-ichi Tamura*,†,‡ and J unko Nishihara†
Department of Environmental Sciences, Faculty of Education & Regional Sciences, Tottori University,
Tottori, 680-8551 J apan, and CREST, J apan Science and Technology Corporation (J ST)
jtamura@fed.tottori-u.ac.jp
Received October 30, 2000
We synthesized novel acidic glycans having acidic groups located in the linkage region of the
glycosaminoglycans (GAGs). The targeted compounds, â-D-Xyl(2P)-Ser (1), â-D-Gal((6S)-(1f4)-â-
D-Xyl(2P)-Ser (3 and 2), â-D-Gal((6S)-(1f3)-â-D-Gal-(1f4)-â-D-Xyl(2P)-Ser (5 and 4), and â-D-Gal-
(1f3)-â-D-Gal(6S)-(1f4)-â-D-Xyl(2P)-Ser (6) contain phosphate and/or sulfate at the specified
positions. Some of them (3, 5, and 6) are the first synthesized examples of natural-type
glycoconjugates that simultaneously possess phosphate and sulfate as well as carboxylic acid.
In tr od u ction
(2) The characteristic phosphate was discovered at O-2
of Xyl in chondroitin 4-sulfate by Oegema et al. in 1984.2a
Fransson’s,2b Danishefsky’s,2c and Sugahara’s1d groups
also isolated the phosphate on Xyl from heparan sulfate
or chondroitin sulfate in 1985, 1988, and 1992, respec-
tively. In 1997, Fransson et al.3 reported that the content
of the phosphate dynamically changed along with the
glycan elongation of decorin. They observed that the
phosphate was gradually accumulated during elongation
up to the trisaccharide (Gal-Gal-Xyl); however, the
isolated tetrasaccharide (GlcA-Gal-Gal-Xyl) contained a
lower amount of phosphate at O-2 of Xly. This fact
suggests that the phosphate on Xyl is strongly related
to the glycan elongation, especially to GlcA transfer.
The use of these truncated oligosaccharides having
sulfate as well as phosphate will elucidate the biological
mechanisms of the glycan elongation. However, naturally
occurring phosphatases make it difficult to isolate the
oligosaccharides having labile phosphate. Furthermore,
oligosaccharides containing both phosphate and sulfate
at specific positions can hardly be obtained from nature
for biochemical use. These facts prompted us to synthe-
size the phosphorylated and sulfated oligosaccharides of
GAG. We now report a facile synthesis of phosphorylated
and/or sulfated mono-, di-, and triaosyl serines which
model GAG at the reducing terminus (1-6, Figure 2).
Some efforts have been made to synthesize the linkage
oligosaccharide of GAG containing either phosphate or
sulfate. In 1992, Goto and Ogawa4 synthesized the
monosulfated oligosaccharide: GlcAâ(1f3)-Gal(4S)â-
(1f3)-Galâ(1f4)-Xylâ-Ser. They also reported the syn-
thesis of a disulfated glycosyl serine: GlcAâ(1f3)-GalNAc-
(4S)â(1f4)-GlcAâ(1f3)-Gal(4S)â(1f3)-Galâ(1f4)-Xylâ-
Ser in the following year.5 In 1993, Nilsson et al.6
synthesized the di-, tri-, and tetrasaccharides possessing
phosphate at O-2 of Xyl as methyl glycosides: Galâ(1f4)-
Xyl(2P)â-OMe, Galâ(1f3)-Galâ(1f4)-Xyl(2P)â-OMe and
GlcAâ(1f3)-Galâ(1f3)-Galâ(1f4)-Xyl(2P)â-OMe. In 1994,
Glycosaminoglycans are composed of a common tetra-
saccharide and a repeating disaccharide region, as shown
in Figure 1. The former covalently binds to the hydroxyl
group of a serine residue in the so-called core protein.
The latter separates GAG into two categories, chondroitin
sulfate/dermatan sulfate and heparin/heparan sulfate,
based on the type of hexosamine residues. The OH and
NH groups on the repeating disaccharides are specifically
decorated with sulfates that are responsible for the
biological activities.
The common tetrasaccharide possesses sulfate1 at
O-4,6 of both Gals, as well as phosphate1d,2 at O-2 of Xyl.
The roles of these acidic groups as well as that of the
common tetrasaccharide are unknown. However, a few
recent efforts have suggested that their functions are
related to glycan elongation.
(1) GAG extends the saccharide chain from the reduc-
ing terminus with the help of the corresponding trans-
ferase and UDP-monosaccharide. GAG is sorted into the
heparin-type and chondroitin-type in the fifth saccharide
transfer: R-GlcNAc and â-GalNAc, respectively. It is
remarkable that we do not find sulfate at the common
tetrasaccharide of heparin and heparan sulfate.1 Thus,
the sulfate in the linkage region might orient the elonga-
tion of GAG toward the chondroitin-type.
* To whom all correspondence should be addressed. Tel & Fax:
+81-857-31-5108.
† Tottori University.
‡ CREST.
(1) (a) Sugahara, K.; Yamashina, I.; De Waard, P.; Van Halbeek,
H.; Vliegenthart, J . F. G. J . Biol. Chem. 1988, 263, 10168-10174. (b)
Sugahara, K.; Masuda, M.; Harada, T.; Yamashina, I.; De Waard, P.;
Vliegenthart, J . F. G. Eur. J . Biochem. 1991, 202, 805-811. (c)
Sugahara, K.; Mizuno, N.; Okumura, Y.; Kawasaki, T. Eur. J . Biochem.
1992, 204, 401-406. (d) Sugahara, K.; Ohi, Y.; Harada, T.; De Waard,
P.; Vliegenthart, J . F. G. J . Biol. Chem. 1992, 267, 6027-6035. (e) De
Waard, P.; Vliegenthart, J . F. G.; Harada, T.; Sugahara, K. J . Biol.
Chem. 1992, 267, 6036-6043. (f) Sugahara, K.; Ohkita, Y.; Shibata,
Y.; Yoshida, K.; Ikegami, A. J . Biol. Chem. 1995, 270, 7204-7212. (g)
Cheng, F.; Heinegård, D.; Fransson, L.-Å.; Bayliss, M.; Bielicki, J .;
Hopwood, J .; Yoshida, K. J . Biol. Chem. 1996, 271, 28572-28580. (h)
Kitagawa, H.; Oyama, M.; Masayama, K.; Yamaguchi, Y.; Sugahara,
K. Glycobiology 1997, 7, 1175-1180.
(3) Moses, J .; Oldberg, Å.; Cheng, F.; Fransson, L.-Å. Eur. J .
Biochem. 1997, 248, 521-526.
(2) (a) Oegema, T. R. J r.; Kraft, E. L.; J ourdian, G. W.; Van Valen,
T. R. J . Biol. Chem. 1984, 259, 1720-1726. (b) Fransson, L.-Å.;
Silverberg, I.; Carlstedt, I. J . Biol. Chem. 1985, 260, 14722-14726.
(c) Rosenfeldt, L.; Danishefsky, I. J . Biol. Chem. 1988, 263, 262-266.
(4) Goto, F.; Ogawa, T. Tetrahedron Lett. 1992, 33, 5099-5102.
(5) Goto, F.; Ogawa, T. Pure Appl. Chem. 1993, 4, 793-801.
(6) Nilsson, M.; Westman, J .; Svahn, C.-M. J . Carbohydr. Chem.
1993, 12, 23-37.
10.1021/jo001540h CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/07/2001

Phosphorylated and Sulfated Glycosyl Serines of GAG
J . Org. Chem., Vol. 66, No. 9, 2001 3075
F igu r e 1. Structure of glycosaminoglycans.
Sch em e 1a
F igu r e 2. Targeted glycosyl serines (1-6).
J acquinet’s group7 reported the syntheses of phosphory-
lated or sulfated tetraosyl serylglycins as acetamides:
GlcAâ(1f3)-Galâ(1f3)-Galâ(1f4)-Xyl(2P)â-(NHAc)Ser-
Gly and GlcAâ(1f3)-Gal(4S)â(1f3)-Galâ(1f4)-Xylâ-
(NHAc)SerGly. Very recently, we reported the synthesis
of the phosphorylated and/or sulfated diaosyl serines:
Gal((6S)â(1f4)-Xyl(2P)â-Ser.8 This was the first syn-
thesis of natural-type oligosaccharides containing both
sulfate and phosphate.
a
(a) HBr-AcOH, CH2Cl2; (b) TiCl4, CH2Cl2; (c) AgOTf, MS 4A,
DCE; (d) H2NNH2•AcOH, toluene-EtOH; (e) i, Bu2SnO, dioxane;
ii, ClCH2COCl, CH2Cl2; (f) AcCl, pyridine, CH2Cl2; (g) i,
(i-Pr)2NP(OBn)2, 1H-tetrazole, CH2Cl2; ii, m-CPBA; (h) H2NNH2•
AcOH, toluene-EtOH; (i) 1. Pd-C, AcOH, H2, MeOH; 2. Et3N-
MeOH-H2O.
superiority of the levulinoyl ester by exhibiting a better
neighboring group effect at O-2 of Xyl: a similar glycos-
ylation7 using a xylosyl chloride containing the chloro-
acetyl group at O-2 and the hydroxyl group of L-seryl-
glycine showed less stereoselectivity. All the levulinoyl
groups of 11 were then removed with hydrazine acetate10
to give the triol 12 (quantitative), which was purified on
a column of Sephadex LH-20. Nilsson’s group6 demon-
strated the regioselective monochloroacetylation at O-4
of methyl â-D-xylopyranoside via a stannylene acetal
intermediate. We applied this procedure to the glycopep-
tide 12 and obtained the desired chloroacetate at O-4 (13)
in 62% yield. We then regioselectively masked O-3 of 13
using a limited amount of acetyl chloride to afford 14
(78%). The remaining hydroxyl group at O-2 of 14 was
phosphorylated by the phosphoramidite method,7,11 using
commercially available (i-Pr)2NP(OBn)2 followed by oxi-
dation with mCPBA7,11 to yield 15 in 88%. We were able
to obtain the first targeted compound 1 through hydro-
genation in the presence of Pd-C and subsequent
saponification with Et3N in 90% yield (two steps). For
further glycan elongation, we chemoselectively removed
the chloroacetyl group of 15 with hydrazine acetate and
obtained the common acceptor 16 in 88% yield.
Resu lts a n d Discu ssion
In general, we have adopted a stepwise elongation from
the reducing terminus for the effective synthesis. Our
targeted compounds were also suitably designed for
enzymatic studies. As shown in Scheme 1, D-xylose was
levulinated with levulinic anhydride and DMAP in py-
ridine to give 7 in 86% yield. TMSOTf-mediated direct
condensation of the known N-benzyloxycarbonyl-serine
benzyl ester (10)9 and 7 afforded the desired 11 in only
29% yield. To increase the coupling yields, the Koenigs-
Knorr-type glycosylations were examined. We converted
7 to the corresponding bromide and chloride (8 and 9)
with HBr-AcOH and TiCl4 in 88 and 87% yields,
respectively. These halides were immediately used for the
following glycosylation with 10 in the presence of AgOTf
and 4A molecular sieves (MS 4A) in DCE at 0 °C.
Although the bromide 8 gave 11 only in low yield (∼35%,
data not shown), the chloride 9 stereoselectively afforded
11 in 73% yield. It seems that the bromide was too labile
to use in the reaction. This result also proved the
For the syntheses of diaosyl serines 2 and 3, we
prepared the galactosyl donor 18 that is suitably designed
(7) Rio, S.; Beau, J .-M.; J acquinet, J .-C. Carbohydr. Res. 1994, 255,
103-124.
(8) Tamura, J .; Nishihara, J . Bioorg. Med. Chem. Lett. 1999, 9,
1911-1914.
(10) Nakano, T.; Ito, Y.; Ogawa, T. Tetrahedron Lett. 1991, 32,
1569-1572.
(9) Ono, N.; Yamada, T.; Saito, T.; Tanaka, K.; Kaji, A. Bull. Chem.
Soc. J pn. 1978, 51, 2401-2404.
(11) Yu, K. L.; Fraser-Reid, B. Tetrahedron Lett. 1988, 29, 978-
982.

3076 J . Org. Chem., Vol. 66, No. 9, 2001
Tamura and Nishihara
Sch em e 3a
Sch em e 2a
a
(a) MBzCl, DMAP, pyridine; (b) NIS, TfOH, MS 4A, DCE-
Et2O; (c) TBAF, AcOH, THF; (d) SO3•Me3N, DMF; (e) 1. Pd-C,
AcOH, H2, MeOH; 2. Et3N-MeOH-H2O, 3. aqueous NaOH; (f) 1.
Pd-C, H2, MeOH; 2. NaOH, aqueous MeOH.
for sulfation at O-6, as shown in Scheme 2. Commercially
available methyl 1-thio-â-D-galactopyranoside was regi-
oselectively silylated at the primary position with tert-
butylchlorodiphenylsilane (TBDPSCl) to give 17 in 92%
yield. The remaining hydroxyl groups were 4-methylben-
zoylated in 86% yield. We now had the pivotal galactosyl
donor 18 in hand, and galactosylation was pursued. The
NIS-TfOH-mediated condensation12 with thiogalactoside
18 and the xylosyl serine 16 afforded the desired â-linked
diaosyl serine 19â in 52% yield, together with the
R-glycoside 19R (18%). The TBDPS group of 19 was
removed with tetrabutylammonium fluoride (TBAF) and
AcOH to yield 20 in 85% yield. Removal of the benzyl
groups of 20â by Pd-catalyzed hydrogenolysis followed
by saponification with Et3N and NaOH afforded the
nonsulfated diaosyl serine 2 in 41% yield (two steps). On
the other hand, we sulfated the primary hydroxyl group
of 20â with SO3‚NMe3 in DMF to give 21 (90%). The
deprotection procedures (hydrogenolysis and saponifica-
tion with NaOH) furnished the sulfated diaosyl serine 3
in 59% yield. To our knowledge, this is the first example
of the synthesis of a natural-type oligosaccharide con-
taining sulfate, phosphate, and a carboxylic acid group.8,13
In a similar way we synthesized triaosyl serines 4 and
5 that have no sulfate on the inner galactosyl moiety
(Scheme 3). The known methyl thiogalactoside (22)4 and
the acceptor 16 were subjected to the same coupling
conditions as described above. Different from the syn-
thesis of 19, we observed low stereoselectivity in this
coupling reaction; the R- and â-glycosides 23R and 23â
were obtained in 46 and 37% yields, respectively. The
low stereoselectivity is perhaps due to the rigid and
sterically hindered benzylidene acetal on the donor which
hinders the â-face. The allyl ether of 23â was then
removed with iridium complex chemistry, followed by
hydrolysis in high yield to give the O-3′ unmasked diaosyl
serine 24 (95%). The final glycosylation was carried out
a
(a) NIS, TfOH, MS AW300, DCE-Et2O; (b) 1. [Ir (COD)(PMe-
Ph2)2PF6], H2, THF; 2. I2, NaHCO3, THF-H2O; (c) TBAF, AcOH,
THF; (d) 1. Pd-C, H2, MeOH; 2. Et3N-MeOH-H2O; (e) 1.
SO3•Me3N, DMF; 2. Pd-C, H2, MeOH; 3. NaOMe, MeOH.
with 18 and 24. The usual NIS-TfOH-mediated coupling
procedure afforded inseparable triaosyl serine isomers.
Removal of the silyl ether protection on the anomeric
mixture allowed us to separate the stereoisomers: 26R
and 26â (9 and 31% in two steps, respectively). An
alternative coupling reaction using commercially avail-
able methyl 2,3,4,6-tetra-O-acetyl-1-thio-â-D-galactopy-
ranoside (25) instead of 18 showed a similar tendency;
27R and 27â were obtained in 11 and 39% yields,
respectively. It is remarkable that the glycosylation with
the corresponding imidate 3414 afforded no coupling
products, but gave a complicated reaction mixture. To
obtain nonsulfated 4, the benzylic protecting groups of
26â were removed by the usual hydrogenolysis, and that
product was exposed to a methanolic solution of NaOMe.
To our surprise, the acetyl group remained at O-2 on the
inner galactosyl moiety, even after longer reaction times.
However, saponification with Et3N could effectively
remove all the acylic protecting groups from the same
starting material to give 4 (49% in deprotection steps).
On the other hand, the primary hydroxyl group of 26â
was quantitatively sulfated as already described. The
sulfate was then exposed to deprotection procedures. In
contrast to the synthesis of 4, NaOMe successfully
afforded the sulfated and phosphorylated triaosyl serine
5 (53% in deprotection steps).
To synthesize the final targeted compound 6, we
employed the galactobiosyl donor 36. As shown in Scheme
4, the known 4-methoxyphenyl 3-O-allyl-â-D-galacto-
pyranoside4 was acetylated in a conventional manner in
order to purify it as the triacetate (28). Saponification of
28 with Et3N in aqueous MeOH was carried out over-
night, which left an acetate group at O-2. Subsequent
silylation with TBDPSCl afforded 29 in 85% yield to-
(12) Veeneman, G. H.; van Leeuwen, S. H.; van Boom, J . H.
Tetrahedron Lett. 1990, 9, 1331-1334.
(13) Vos et al. synthesized a mimetic of heparin repeating-pentasac-
charide having sulfate, phosphate, and carboxylic acid. Vos, J . N.;
Westerduin, P.; van Boeckel, C. A. A. Bioorg. Med. Chem. Lett. 1991,
3, 143-146.
(14) Amvam-Zollo, P. H.; Sinay¨, P. Carbohydr. Res. 1986, 150, 199-
212.

Phosphorylated and Sulfated Glycosyl Serines of GAG
J . Org. Chem., Vol. 66, No. 9, 2001 3077
Ta ble 1. 1H NMR Ch em ica l Sh ifts of 1-6 a t 400 MHz (a )
Sch em e 4a
XS
P
1
GXS
P
2
GXS
SP
3
GGXS
P
4
GGXS
GGXS
SP
6
S
P
5
Xyl1
Gal2
1-H
2-
3-
4.42
3.67
3.43
3.52
4.39
3.65
3.57
3.75
3.25
3.93
4.30
3.33
3.46
3.73
3.50
3.64
3.55
4.45
3.72
3.58
3.77
3.30
3.96
4.35
3.36
3.51
3.82
3.78
4.04
4.04
4.49
3.77
3.63
3.82
3.33
4.00
4.42
3.56
3.70
4.07
3.59
3.54-
3.72
4.49
3.47
3.54
3.80
b
4.45
3.72
3.59
3.78
3.30
3.97
4.39
3.52
3.64
4.09
3.56
3.62-
3.68
4.45
3.46
3.53
3.81
3.77
4.03
4.03
4.16
4.04
3.91
4.38
3.65
3.51
3.71
3.22
3.88
4.34
3.45
3.60
4.03
3.72
3.97
3.97
4.37
3.36
3.42
3.68
c
4-
5ax- 3.20
5eq-
1-H
2-
3-
4-
3.82
5-
6a-
6b-
1-H
2-
3-
4-
Gal3
5-
6a-
6b-
R
âa
âb
b
b
4.15
3.94
3.84
c
c
Ser
4.07
3.87
3.84
4.02
3.88
3.82
4.12
3.98
3.89
4.14
4.08
3.84
a
(a) 1. Et3N-MeOH-H2O; 2. TBDPSCl, imidazole, DMF; (b)
a-c Referenced by DHO in D2O as (a) δ 4.65, (b) 3.54-3.72, (c)
3.42-3.57.
MBzCl, DMAP, pyridine; (c) 1. [Ir (COD)(PMePh2)2PF6], H2, THF;
2. I2, NaHCO3, THF-H2O; (d) TMSOTf, MS AW300, CH2Cl2; (e)
1. CAN, CH3CN-H2O; 2. CCl3CN, DBU, CH2Cl2; (f) TBAF, AcOH,
THF; (g) 1. SO3•Me3N, DMF; 2. Pd-C, H2, MeOH; 3. Et3N-
MeOH-H2O; 4. NaOMe, MeOH; 5. aqueous NaOH.
of the phosphate and sulfate groups in the linkage region
in the biosynthesis of GAG.
gether with 2,4-diol 30 (10%) (two steps). The C-4
hydroxyl group of 29 was quantitatively 4-methylben-
zoylated to give 31, and the allyl group of 31 was also
quantitatively removed as described above to afford 32.
The donor (imidate) 34 and the acceptor 32 were coupled
in the presence of TMSOTf to yield galactobiosides 35R
and 35â as well as an acetylated acceptor (33) in 6, 43,
and 24% yields, respectively. The glycosylation with the
alternative thioglycoside 25 showed a similar tendency
(35R, 7%; 35â, 36%; 33, 8%). We have converted the
â-anomer 35â into the diaosyl imidate 36 via the removal
of the 4-methoxyphenyl group15 and trichloroacetimidoy-
lation (both in 93% yields). The imidate 36 and the
acceptor 16 were coupled by the TMSOTf method at -20
°C to give 37R and 37â in 23 and 18% yields, respectively.
The TBDPS group of 37â was chemoselectively removed
as described above to give 38 in 75% yield. Sulfation at
O-6 of the inner galactosyl moiety (93%) and the depro-
tection procedures [(1) Pd-C, H2, (2) Et3N-MeOH-H2O,
(3) NaOMe/MeOH, and then (4) aqueous NaOH] toward
final target 6 were successfully executed (65%, from 38).
All the targeted compounds (1-6) afforded satisfactory
1H NMR data which are summarized in Table 1.
Exp er im en ta l Section
Gen er a l Meth od s. Optical rotations were measured at 22
( 3 °C with a HORIBA polarimeter in solutions of the specified
solvents. 1H NMR assignments were supported by two-
dimensional HH COSY experiments with a J EOL LA 400 MHz
spectrometer. Signal assignments such as 13 stand for a proton
at C-1 of sugar residue 3. The FAB mass spectra were
measured with a triple-stage quadrupole mass spectrometer
(Finnigan MAT TSQ 700) equipped with the FAB ion source.
Silica gel chromatography, analytical TLC, and preparative
TLC (PTLC) were carried out on a column of silica gel 60 (E.
Merck) or glass plates coated with silica Gel F254 (E. Merck),
respectively. Gels for size-exclusion chromatography (Sephadex
LH-20, Biobeads S-X1) were purchased from Pharmacia and
BIO•RAD, respectively. Molecular sieves (MS) were purchased
from GL Science, Inc. and activated at 180 °C under vacuum
prior to use. Melting points were determined with a Yanaco
melting point apparatus and are uncorrected. All reactions in
organic solvents were performed under an argon atmosphere.
1,2,3,4-Tetr a -O-levu lin oyl-R-D-xylop yr a n ose (7). To a
solution of D-xylose (25.0 g, 167 mmol) in pyridine (500 mL)
were added with stirring at room temperature a 1.07 M
solution of levulinic anhydride in 1,2-dichloroethane (DCE)
(709 mL) and a catalytic amount of DMAP. The mixture was
stirred overnight and then ice was added. To the solution was
added CHCl3, and the organic phase was washed with aqueous
NaHCO3, brine, dried over anhydrous MgSO4, and concen-
trated. The residue was eluted from a column of silica gel (1:
2-1:5-1:10 toluene-EtOAc) to give syrupy 7 (77.54 g, 86%)
which was used for subsequent reactions without further
Con clu sion
The novel and acidic glycosyl serines of GAG (1-6)
containing phosphate and/or sulfate at the specified
positions were synthesized by employing sophisticated
protecting groups and modern glycosylation methods.
This paper is the first report of the syntheses of these
glycosyl serines. These glycosyl serines can be directly
utilized for enzymatic studies and will elucidate the roles
1
purification: Rf 0.60 (1:10 MeOH-EtOAc); H NMR (CDCl3)
δ 6.22 (d, 1H, J 1,2 ) 3.96 Hz, H-1), 5.49 (t, 1H, J 2,3 ) J 3,4
9.09 Hz, H-3), 5.11-4.98 (m, 1H, H-4), 5.00 (dd, 1H, H-2), 3.91
(dd, 1H, J 4,5eq) 5.94 Hz, J gem ) 11.21 Hz, H-5eq), 3.71 (t, 1H,
J 4,5ax ) 11.21 Hz, H-5ax), 2.86-2.40 (m, 16H, CH2), 2.21, 2.17,
2.18 (3s, 12H, 4MeCO). Anal. Calcd for C25H34O13: C, 55.35;
H, 6.32. Found: C, 55.29; H, 6.47.
)
1-Br om o-2,3,4-tetr a -O-levu lin oyl-R-D-xylop yr a n ose (8).
To a solution of 7 (2.05 g, 3.37 mmol) in DCE (21 mL) was
(15) Fukuyama, T.; Laird, A. A.; Hotchkiss, L. M. Tetrahedron Lett.
1985, 26, 6291-6292.

3078 J . Org. Chem., Vol. 66, No. 9, 2001
Tamura and Nishihara
12.20 Hz, PhCH2), 5.12 (s, 2H, PhCH2), 4.81 (dt, 1H, J 3,4
J 4,5ax ) 8.57 Hz, J 4,5eq ) 4.95 Hz, H-4), 4.63 (m, 1H, SerR),
4.25-4.20 (m, 2H, H-1, Serâa), 4.10 (d, 2H, J ) 3.3 Hz, ClCH2),
3.89 (dd, 1H, J gem ) 11.87 Hz, H-5eq), 3.76-3.66 (m, 1H,
Serâb), 3.69 (t, 1H, J 2,3 ) J 3,4 ) 8.58 Hz, H-3), 3.32 (dd, 1H,
J 1,2 ) 6.93 Hz, H-2), 3.09 (dd, 1H, H-5ax), 1.69 (m, 2H, OH).
Anal. Calcd for C25H28ClNO10: C, 55.82; H, 5.25; N, 2.60; Cl,
6.59. Found: C, 55.84; H, 5.38; N, 2.36; Cl, 6.88.
added 35% HBr in AcOH (16 mL) with stirring at 0 °C
overnight. The organic phase was diluted with DCE, was
washed with ice-cold brine, ice-cold aqueous NaHCO3, and ice-
cold brine again, dried over anhydrous MgSO4, and concen-
trated to give 8 (1.69 g, 88%) as a syrup: Rf 0.50 (1:4 toluene-
EtOAc). The residue was used for glycosylation without further
purification.
1-Ch lor o-2,3,4-tetr a -O-levu lin oyl-R-D-xylop yr a n ose (9).
To a solution of 7 (1.02 g, 1.88 mmol) in DCE (7 mL) was added
TiCl4 (0.41 mL, 3.73 mmol) with stirring at room temperature
overnight. To the solution was added CHCl3. The organic phase
was washed with brine, dried over anhydrous MgSO4, and
concentrated. The residue was eluted from a column of silica
gel (1:1-1:2 toluene-EtOAc) to give 9 (751.9 mg, 87%) as a
syrup: Rff 0.70 (1:10 MeOH-EtOAc). The chloride 9 was used
for glycosylation without further purification.
)
N-Ben zyloxycar bon yl-O-(3-O-aceth yl-4-O-ch lor oacetyl-
â-D-xylop yr a n osyl)-L-ser in e Ben zyl Ester (14). To a solu-
tion of 13 (1.70 g, 3.16 mmol) and pyridine (1.02 mL, 12.6
mmol) in CH2Cl2 (70 mL) was added acetyl chloride (251 µL,
3.54 mmol) with stirring at 0 °C. After 40 min, ice was added
and the organic phase was washed with M HCl, brine, aqueous
NaHCO3, and brine again, and dried over MgSO4. The crude
material obtained in the usual manner was purified on a
column of silica gel (7:1-6:1-3:1-1:3 toluene-EtOAc) to give
14 (1.42 g, 78%) as a syrup: Rf 0.73 (1:2 toluene-EtOAc); [R]D
) -19.8 (c 1.00, CHCl3); 1H NMR (CDCl3) δ 7.36 (bs, 10H, Ar
H), 5.78 (d, 1H, J ) 8.58 Hz, NH), 5.27-5.17 (m, 2H, PhCH2),
5.12 (s, 2H, PhCH2), 5.12 (t, 1H, J 2,3 ) J 3,4 ) 8.58 Hz, H-3),
4.89 (dt, 1H, J 3,4 ) J 4,5ax ) 8.58 Hz, J 4,5eq ) 4.95 Hz, H-4),
4.60 (m, 1H, SerR), 4.33 (d, 1H, J 1,2 ) 6.59 Hz, H-1), 4.21 (dd,
1H, J gem ) 10.88 Hz, J R,âa ) 3.96 Hz, Serâa), 4.03 (s, 2H,
ClCH2), 3.91 (dd, 1H, J gem ) 11.87 Hz, H-5eq), 3.83 (m, 1H,
Serâb), 3.77 (m, 1H, OH), 3.44 (m, 1H, H-2), 3.23 (dd, 1H,
H-5ax), 2.09 (s, 3H, Me). Anal. Calcd for C27H30ClNO11: C,
55.91; H, 5.21; N, 2.42; Cl, 6.11. Found: C, 55.91; H, 5.26; N,
2.46; Cl, 6.31.
N-Ben zyloxyca r bon yl-O-(3-O-a cetyl-4-O-ch lor oa cetyl-
2-O -d i b e n z y lo x y p h o s p h i n y l-â-D -x y lo p y r a n o s y l)-L -
ser in e Ben zyl Ester (15). To a solution of 14 (7.81 g, 13.47
mmol) and 1H-tetrazole (1.89 g, 26.9 mmol) in CH2Cl2 (300
mL) was added dibenzyl diisopropylphosphoramidite (6.79 mL,
20.2 mmol) in CH2Cl2 (50 mL) with stirring at room temper-
ature. After 50 min, the reaction mixture was ice-cooled and
m-CPBA (6.97 g, 40.4 mmol) was added. The reaction mixture
was diluted with CHCl3 after 30 min. The organic phase was
washed with aqueous Na2S2O3, aqueous NaHCO3, and brine
and dried over MgSO4. The crude material obtained in the
usual manner was eluted from a column of silica gel (8:1-7:
1-6:1-5:1-5:2 toluene-EtOAc) to give 15 (9.91 g, 88%) as a
syrup: Rf 0.40 (2:1 toluene-EtOAc); [R]D ) -14.4 (c 1.08,
CHCl3); 1H NMR (CDCl3) δ 7.33-7.22 (m, 20H, Ar H), 6.99 (d,
1H, J ) 8.78 Hz, NH), 5.27, 5.17 (ABq, 2H, J ) 12.20 Hz,
PhCH2), 5.19 (dd, 1H, J 2,3 ) 8.54 Hz, J 3,4 ) 8.78 Hz, H-3), 5.11,
4.93, 4.91 (3s, 2H × 3, 3PhCH2), 4.87 (m, 1H, H-4), 4.62 (d,
1H, J R,âa ) 9.03 Hz, SerR), 4.42 (m, 1H, Serâa), 4.41 (d, 1H,
J 1,2 ) 6.83 Hz, H-1), 4.11 (dt, 1H, J 2,P ) 8.54 Hz, H-2), 3.99 (s,
2H, ClCH2), 3.91 (dd, 1H, J 4,5eq ) 5.12 Hz, J gem ) 11.96 Hz,
H-5eq), 3.68 (dd, 1H, J R,âb ) 2.68 Hz, J gem ) 9.51 Hz, Serâb),
3.26 (dd, 1H, J 4,5ax ) 8.78 Hz, H-5ax), 1.89 (s, 3H, Me). Anal.
Calcd for C41H43ClNPO11: C, 58.61; H, 5.16; N, 1.67. Found:
C, 58.74; H, 5.25; N, 1.68.
N-Ben zyloxyca r bon yl-O-(2,3,4-tetr a -O-levu lin oyl-â-D-
xylop yr a n osyl)-L-ser in e Ben zyl Ester (11). Meth od A.
N-Benzyloxycarbonyl-L-serine benzyl ester9 (10, 357.6 mg,
1.086 mmol) and 7 (490.9 mg, 0.904 mmol) were dissolved in
CH2Cl2 (5 mL). The solution was cooled to -20 °C, and
TMSOTf (350 µL, 1.94 mmol) was added with stirring. After
6 h, saturated NaHCO3 and CHCl3 were added to the solution.
Insoluble materials were filtered off, and the filtrate was
extracted with CHCl3. The residue obtained by a usual workup
was eluted from columns of LH-20 (1:1 CHCl3-MeOH) and
silica gel (5:1-3:1-2:1-3:2-1:1-1:3-1:5-1:10 toluene-
EtOAc) to give 11 (199.6 mg, 29%) as a syrup. Meth od B. To
a light-shielded mixture of 4A MS (180 mg) and AgOTf (93.0
mg, 0.36 mmol) was added a solution of 10 (47.1 mg, 0.143
mmol) in DCE (2 mL) with stirring. One hour later, the
suspension was cooled to 0 °C and a solution of 9 (79.1 mg,
0.171 mmol) in DCE (2 mL) was added dropwise. The mixture
was stirred for 4 d at room temperature, then diluted with
CHCl3, aqueous NaHCO3 and brine, and filtered through
Celite. The filtered solution was worked up as usual. Purifica-
tion on a column of LH-20 as in method A afforded 11 (79.3
mg, 73%): Rf 0.35 (1:2 toluene-EtOAc); [R]D ) -26.9 (c 0.98,
1
CHCl3); H NMR (CDCl3) δ 7.35 (bs, 10H, Ar H), 6.27 (d, 1H,
J ) 8.58 Hz, NH), 5.22, 5.15 (ABq, 2H, J ) 8.58 Hz, PhCH2),
5.11 (s, 2H, PhCH2), 5.11 (m, 1H, H-3), 4.84-4.76 (m, 2H,
H-2,4), 4.52 (m, 1H, SerR), 4.44 (d, 1H, J 1,2 ) 6.60 Hz, H-1),
4.32 (dd, 1H, J gem ) 10.23 Hz, J R,âa <1.0 Hz, Serâa), 3.90 (dd,
1H, J 4,5eq ) 4.95 Hz, J gem ) 11.88 Hz, H-5eq), 3.76 (dd, 1H,
J R,âb )2.97 Hz, Serâb), 3.23 (dd, 1H, J 4,5ax ) 8.25 Hz, H-5ax),
2.8-2.4 (m, 12H, CH2), 2.17, 2.11, 2.06, (3s, 3H × 3, MeCO).
Anal. Calcd for C38H45NO15
: C, 60.39; H, 6.00; N, 1.85.
Found: C, 60.18; H, 6.06; N, 2.02.
N-Ben zyloxyca r b on yl-O-â-D-xylop yr a n osyl-L-ser in e
Ben zyl Ester (12). To a solution of 11 (492.0 mg, 0.651 mmol)
in toluene (12.5 mL) and EtOH (62.5 mL) was added hydrazine
acetate (957 mg, 9.75 mmol) with stirring. After 40 min,
volatiles were removed under diminished pressure and the
residue was eluted from a column of LH-20 (1:1 CHCl3-
MeOH) to give 12 (quantitative) as a syrup, which was used
for the next reaction without further purification: Rf 0.50 (1:
10 MeOH-EtOAc); 1H NMR (CD3OD) δ 7.39 (bs, 10H, Ar H),
5.28, 5.19 (ABq, 2H, J ) 12.54 Hz, PhCH2), 5.15 (s, 2H,
PhCH2), 4.56 (m, 1H, SerR), 4.41 (dd, 1H, J gem ) 10.0 Hz, J R,âa
) 3.63 Hz, Serâa), 4.22 (d, 1H, J 1,2 ) 7.59 Hz, H-1), 3.86 (dd,
1H, J 4,5eq ) 5.28 Hz, J gem ) 11.22 Hz, H-5eq), 3.78 (dd, 1H,
J R,âb ) 3.30 Hz, Serâb), 3.59-3.46 (m, 2H, H-3,4), 3.23-3.15
(m, 2H, H-2,5ax).
N-Ben zyloxyca r b on yl-O-(4-O-ch lor oa cet yl-â-D-xylo-
p yr a n osyl)-L-ser in e Ben zyl Ester (13). A mixture of 12
(849.4 mg, 1.841 mmol) and Bu2SnO (659.6 mg, 2.65 mmol)
in dioxane (42.5 mL) was boiled under reflux for 1.5 h and
then was evaporated to dryness. The residue was diluted with
42.5 mL of CH2Cl2, and a solution of chloroacetyl chloride (0.17
mL, 1.95 mmol) in CH2Cl2 (3.9 mL) was added with stirring.
After 80 min, the reaction mixture was worked up as usual
and eluted from a column of silica gel (2:1-1:1 toluene-EtOAc)
to give 13 (612.4 mg, 62%) as a syrup: Rf 0.45 (1:2 toluene-
EtOAc); [R]D ) -31.9 (c 0.695, CHCl3); 1H NMR (CDCl3) δ 7.35
(bs, 10H, Ar H), 6.25 (m, 1H, NH), 5.23, 5.15 (ABq, 2H, J )
N-Ben zyloxyca r bon yl-O-(3-O-a cetyl-2-O-d iben zyloxy-
ph osph in yl-â-D-xylopyr an osyl)-L-ser in e Ben zyl Ester (16).
To a solution of 15 (460.0 mg, 547.5 µmol) in EtOH (24 mL)
and toluene (6 mL) was added hydrazine acetate (151.2 mg,
1.64 mmol) with stirring. After 90 min, the volatiles were
evaporated and the residue was eluted from a column of LH-
20 (1:1 CHCl3-MeOH) to give 16 (366.5 mg, 88%) as a syrup:
Rf 0.58 (1:5 toluene-EtOAc); [R]D ) -21.3 (c 0.92, CHCl3); 1H
NMR (CDCl3) δ 7.34-7.23 (bs, 20H, Ar H), 6.98 (d, 1H, J )
9.03 Hz, NH), 5.23, 5.14 (ABq, 2H, J ) 12.44 Hz, PhCH2), 5.11
(s, 2H, PhCH2), 4.95-4.88 (m, 4H, 2PhCH2), 4.90 (t, 1H, J 2,3
) J 3,4 ) 8.53 Hz, H-3), 4.61 (m, 1H, SerR), 4.43 (dd, 1H, J gem
) 11.49 Hz, J R,âa )2.44 Hz, Serâa), 4.38 (d, 1H, J 1,2 ) 6.38
Hz, H-1), 4.11 (dt, 1H, J 2,P ) 6.38 Hz, H-2), 3.89 (dd, 1H, J 4,5eq
) 4.88 Hz, J gem ) 11.95 Hz, H-5eq), 3.71-3.64 (m, 2H, H-4,
Serâb), 3.22 (dd, 1H, J 4,5ax ) 9.27 Hz, H-5ax), 2.68 (m, 1H, J
) 5.61 Hz, OH), 1.94 (s, 3H, Me). Anal. Calcd for C39H42
-
NPO13: C, 61.33; H, 5.54; N, 1.83; P, 4.06. Found: C, 61.26;
H, 5.64; N, 1.77; P, 3.96.
O-(2-O-P h osp h on o-â-D-xylop yr a n osyl)-L-ser in e, Tr i-
sod iu m Sa lt (1). To a solution of 15 (16.5 mg, 19.6 µmol) in

Phosphorylated and Sulfated Glycosyl Serines of GAG
J . Org. Chem., Vol. 66, No. 9, 2001 3079
1
MeOH (2 mL) were added a catalytic amount of Pd-C and 3
drops of AcOH. The reaction mixture was stirred under an H2
atmosphere for 2 d and filtered on Celite, and the volatiles
were removed under diminished pressure. The residues were
diluted with MeOH (0.4 mL), H2O (0.2 mL), and Et3N (0.2 mL)
and the solution for 2 d. The volatiles were removed in a same
manner as above. The crude materials were eluted from a
column of LH-20 [1% (NH4)2CO3] and AG500W-X8 (Na+) to
give 1 (5.6 mg, 90%): Rf 0.10 (1:2:1 n-BuOH-AcOH-H2O);
[R]D ) -34.2 (c 0.79, H2O). For 1H NMR data, see Table 1.
FABMS m/z 361.75 (calcd for C8H14NPO10Na2 362.02, [M -
Na + 2H]+), 383.72 (calcd for C8H13NPO10Na3 384.01, [M +
H]+), 405.81 (calcd for C8H12NPO10Na4 405.99, [M + Na]+).
Meth yl 6-O-ter t-Bu tyld ip h en ylsilyl-1-th io-â-D-ga la cto-
p yr a n osid e (17). To a solution of methyl 1-thio-â-D-galacto-
pyranoside (303.4 mg, 1.44 mmol) and imidazole (223.5 mg,
3.28 mmol) in DMF (4.5 mL) was added tert-butylchlorodiphe-
nylsilane (0.4 mL, 1.5 mmol) with stirring. After 4 h, the
reaction mixture was diluted with EtOAc. The crude material
obtained in the usual manner was eluted from a column of
silica gel (3:1-1:1-1:2 toluene-EtOAc) to give 17 (592.3 mg,
92%) as a syrup: Rf 0.66 (1:4 MeOH-CHCl3); [R]D ) +0.88 (c
2.49, CHCl3); 1H NMR (CDCl3) δ 7.73-7.66 (m, 4H, Ar H),
7.47-7.38 (m, 6H, Ar H), 4.22-4.18 (m, 2H, ring H), 3.98-
3.90 (m, 2H, ring H), 3.76-3.71 (m, 1H, ring H), 3.62-3.56
(m, 2H, ring H), 2.89 (d, 1H, J ) 3.17 Hz, OH), 2.73 (d, 1H, J
) 6.59 Hz, OH), 2.49 (d, 1H, J ) 1.71 Hz, OH), 2.21 (s, 3H,
Me), 1.11 (s, 9H, t-Bu). Anal. Calcd for C23H32SiSO5‚0.1H2O:
C, 61.31; H, 7.22; S, 7.12. Found: C, 61.16; H, 7.27; S, 7.14.
Meth yl 6-O-ter t-Bu tyld ip h en ylsilyl-2,3,4-tr i-O-(4-m e-
th ylben zoyl)-1-th io-â-D-ga la ctop yr a n osid e (18). To a solu-
tion of 17 (592.3 mg, 1.32 mmol) in pyridine (6 mL) were added
4-methylbenzoyl chloride (1.57 mL, 11.9 mmol) and a catalytic
amount of DMAP, with stirring at 60-70 °C overnight. Then,
MeOH (2 mL) was added to the reaction mixture in a water
bath and stirring was continued for 2 h. The crude material
obtained from the usual workup was eluted from a column of
silica gel (n-hexane-20:1-15:1-7:1-2:1 n-hexanes-EtOAc)
to give 18 (910.9 mg, 86%) as a syrup: Rf 0.40 (3:1 n-hexanes-
EtOAc); [R]D ) +176.7 (c 1.45, CHCl3); 1H NMR (CDCl3) δ
7.90-7.82 (m, 4H, Ar H), 7.69-7.64 (m, 4H, Ar H), 7.49-7.36
(m, 5H, Ar H), 7.29-7.25 (m, 3H, Ar H), 7.18-7.03 (m, 6H, Ar
H), 6.05 (d, 1H, J 3,4 ) 3.41 Hz, H-4), 5.76 (dd, 1H, J 1,2 ) 9.76
Hz, J 2,3 ) 10.01 Hz, H-2), 5.63 (dd, 1H, H-3), 4.64 (d, 1H, H-1),
4.09 (brt, 1H, H-5), 3.83 (dd, 1H, J 5,6a ) 5.85 Hz, J gem ) 10.24
Hz, H-6a), 3.76 (dd, 1H, J 5,6b ) 8.05 Hz, H-6b), 2.47, 2.35, 2.30,
2.26 (4s, 3H × 4, SMe, PhMe), 0.99 (s, 9H, t-Bu). Anal. Calcd
for C47H50SiSO8‚0.1H2O: C, 70.12; H, 6.30; S, 3.98. Found: C,
69.91; H, 6.10; S, 3.98.
CHCl3); H NMR (CDCl3) δ 7.97-7.95 (m, 2H, Ar H), 7.82-
7.80 (m, 2H, Ar H), 7.67-7.65 (m, 2H, Ar H), 7.32-7.11 (m,
24H, Ar H), 7.03-7.01 (m, 2H, Ar H), 5.82 (dd, 1H, J 1,2 ) 2.44
Hz, J 2,3 ) 10.97 Hz, H-22), 5.79 (d, 1H, H-12), 5.59 (dd, 1H,
J 3,4 ) 3.36 Hz, H-32), 5.49 (d, 1H, H-42), 5.21, 5.15 (ABq, 2H,
J ) 12.44 Hz, PhCH2), 5.18 (bt, 1H, J ) 7.05 Hz, H-31), 5.09
(s, 2H, PhCH2), 4.93-4.82 (m, 4H, 2PhCH2), 4.62 (dd, 1H, J R,âa
<1 Hz, J R,âb ) 7.54 Hz, SerR), 4.47 (d, 1H, J gem ) 9.51 Hz,
Serâa), 4.34 (d, 1H, J 1,2 ) 7.56 Hz, H-11), 4.23 (brt, 1H, J )
7.07 Hz, H-52), 4.13 (dd, 1H, J 4,5eq ) 5.36 Hz, J gem ) 10.98 Hz,
H-5eq1), 4.00 (m, 1H, H-21), 3.78-3.67 (m, 3H, H-41,6a2,Serâb),
3.59 (m, 1H, H-6b2), 3.38 (m, 1H, H-5ax1), 2.52 (brt, 1H, J )
6.83 Hz, OH-62), 2.44, 2.34, 2.28 (3s, 3H × 3, 3PhMe), 1.42 (s,
3H, Me). Anal. Calcd for C69H70NPO21: C, 64.72; H, 5.52; N,
1.09; P, 2.42. Found: C, 64.77; H, 5.59; N, 1.27; P, 2.70.
20â: Rf 0.38 (1:1 toluene-EtOAc); [R]D ) +74.7 (c 0.91,
1
CHCl3); H NMR (CDCl3) δ 7.95-7.93 (m, 2H, Ar H), 7.85-
7.83 (m, 2H, Ar H), 7.69-7.67 (m, 2H, Ar H), 7.29-7.18 (m,
23H, Ar H), 7.05-6.95 (m, 3H, Ar H), 5.73 (d, 1H, J 3,4 ) 3.17
Hz, H-42), 5.70 (dd, 1H, J 1,2 ) 7.81 Hz, J 2,3 ) 10.25 Hz, H-22),
5.59 (dd, 1H, H-32), 5.17-5.09 (m, 4H, 2PhCH2), 5.14 (m, 1H,
H-31), 4.94-4.86 (m, 4H, 2PhCH2), 4.79 (d, 1H, H-12), 4.57 (d,
1H, J R,âb ) 8.78 Hz, SerR), 4.35 (d, 1H, J gem ) 9.51 Hz, Serâa),
4.26 (d, 1H, J 1,2 ) 6.83 Hz, H-11), 4.08 (m, 1H, H-21), 3.97 (t,
1H, J ) 6.59 Hz, H-52), 3.80-3.69 (m, 3H, H-41,5eq1,6a2), 3.61-
3.53 (m, 1H, H-6b2,Serâb), 3.11 (m, 1H, H-5ax1), 3.00 (brt, 1H,
J ) 6.72 Hz, OH-62), 2.42, 2.35, 2.28 (3s, 3H × 3, 3PhMe),
1.84 (s, 3H, Me). Anal. Calcd for C69H70NPO21: C, 64.72; H,
5.52; N, 1.09; P, 2.42. Found: C, 64.58; H, 5.53; N, 1.17; P,
2.58.
O-[O-â-D-Ga la ctop yr a n osyl-(1f4)-(2-O-p h osp h on o-â-
D-xylop yr a n osyl)]-L-ser in e, Tr isod iu m Sa lt (2). To a solu-
tion of 20â (18.4 mg, 14.4 µmol) in MeOH (3 mL) were added
a catalytic amount of Pd-C and 3 drops of AcOH. The reaction
mixture was stirred under an H2 atmosphere for 4.5 h and
filtered on Celite, and the volatiles were removed under
diminished pressure. The residue was diluted with MeOH (0.6
mL), H2O (0.3 mL), and Et3N (0.3 mL) and the solution for 2
h. To the reaction mixture was added 0.5 M NaOH (0.4 mL)
at 0 °C. After 18 h, the reaction was worked up by adding 50%
AcOH followed by evaporation. The crude materials were
eluted from columns of LH-20 (1% AcOH) and AG500W-X8
(Na+) to give 2 (6.4 mg, 82%): Rf 0.41 (1:1:1 MeOH-EtOAc-
H2O); [R]D ) -27.5 (c 0.08, H2O). For 1H NMR data see Table
1. FABMS: m/z 523.80 (calcd for C14H25NPO15Na2 524.08, [M
- Na + 2H]+), 545.79 (calcd for C14H24NPO15Na3 546.06, [M
+ H]+), 567.81 (calcd for C14H23NPO15Na4 568.04, [M + Na]+).
N -Be n zyloxyc a r b on yl-O-{O-[2,3,4-t r i-O-(4-m e t h yl-
b e n zoyl)-6-O-su lfo-â-D-ga la ct op yr a n osyl]-(1f4)-(3-O-
a cetyl-2-O-d iben zyloxyp h osp h in yl-â-D-xylop yr a n osyl)}-
L-ser in e Ben zyl Ester , Sod iu m Sa lt (21). To a solution of
20â (27.6 mg, 21.6 µmol) in DMF (1.5 mL) was added SO3‚
NMe3 (88.9 mg, 639 µmol). The reaction mixture was stirred
overnight at 50-60 °C and was subjected to columns of LH-
20 (1:1 CHCl3-MeOH) and AG50W-X8 (Na+) (8:1 MeOH-
H2O) to give 21 (26.7 mg, 90%), which was used for deprotec-
tion procedure without further purification: Rf 0.63 (1:4
MeOH-EtOAc).
N -Be n zyloxyc a r b on yl-O-{O-[2,3,4-t r i-O-(4-m e t h yl-
b e n zoyl)-R,â-D -ga la c t o p y r a n osy l]-(1f4)-(3-O -a c e t yl-
2-O -d ib e n zy lo x y p h o s p h in y l-â-D -x y lo p y r a n o s y l)}-L -
ser in e Ben zyl Ester (20R a n d 20â). To a solution of 18 (406.4
mg, 0.506 mmol) and 16 (313.4 mg, 0.410 mmol) in DCE (8
mL) was added 4A MS (500 mg). After stirring at room
temperature for 50 min, a solution of NIS (477 mg, 2.12 mmol)
and TfOH (27 µL, 0.31 mmol) in a mixture of DCE (5 mL) and
Et2O (5 mL) was added at -20 °C and the solution stirred for
30 min. Saturated NaHCO3 and CHCl3 were added to the
reaction mixture, and insoluble materials were filtered on
Celite. The organic phase was washed with aqueous Na2S2O3,
aqueous NaHCO3, and brine. The crude material obtained in
the usual manner was eluted from columns of S-X1 (toluene)
and then silica gel (20:1-10:1-7:1-5:1-3:1-2:1-1:1 n-hex-
anes-EtOAc) to give 19R (Rf 0.60 (3:1 toluene-EtOAc) (10.0
mg)), 19â (Rf 0.57 (3:1 toluene-EtOAc) (117.3 mg)), and a
mixture of them (311.5 mg). The yields of 19R and 19â were
estimated to be 18 and 52%, respectively, from the following
deprotection procedure. A part of the mixture of 19R and 19â
(37.7 mg) was diluted in THF (1 mL). To this were added AcOH
(27 µL) and 1 M TBAF (0.24 mL) with stirring for 4.5 h. The
reaction mixture was worked up as usual. PTLC (1:1 toluene-
EtOAc) purification of the crude materials gave 20R (8.9 mg)
and 20â (18.1 mg) (85%).
O-[O-(6-O-Su lfo-â-D-ga la ct op yr a n osyl)-(1f4)-(2-O-
ph osph on o-â-D-xylopyr an osyl)]-L-ser in e, Tetr asodiu m Salt
(3). To a solution of 21 (21.1 mg, 15.3 µmol) in MeOH (3 mL)
was added a catalytic amount of Pd-C. The reaction mixture
was stirred under an H2 atmosphere for 1 d and filtered on
Celite, and the volatiles were removed under diminished
pressure. The residue was diluted with 50% MeOH (2 mL),
and 6 drops of 0.5M NaOH was added at 0 °C. After 2 d, the
reaction was worked up by adding 50% AcOH and evaporation.
The crude materials were purified on columns of LH-20 (1%
AcOH) and AG500W-X8 (Na+) to give 3 (5.8 mg, 59%): Rf 0.55
(1:1:1 MeOH-EtOAc-H2O); [R]D ) -21.7 (c 0.71, H2O). For
1H NMR data see Table 1. FABMS: m/z 626.3 (calcd for C14H24
NPSO18Na3 626.01, [M - Na + 2H]+), 648.0 (calcd for C14H23
NPSO18Na4 648.00, [M + H]+), 669.9 (calcd for C14H22NPSO18
Na5 669.98, [M + Na]+).
-
-
-
20R: Rf 0.55 (1:1 toluene-EtOAc); [R]D ) +112.4 (c 0.89,
N-Ben zyloxyca r bon yl-O-{O-(2-O-a cetyl-3-O-a llyl-4,6-O-

3080 J . Org. Chem., Vol. 66, No. 9, 2001
Tamura and Nishihara
ben zylid en e-R,â-D-ga la ct op yr a n osyl)-(1f4)-(3-O-a cet yl-
2-O -d ib e n zy lo x y p h o s p h in y l-â-D -x y lo p y r a n o s y l)}-L -
ser in e Ben zyl Ester (23R a n d 23â). To a solution of the
known methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-1-thio-
â-D-galactopyranoside4 (22, 98.1 mg, 258 µmol) and 16 (98.5
mg, 129 µmol) in DCE (1.4 mL) was added MS AW300 (200
mg). After stirring at room temperature for 70 min, a solution
of NIS (174 mg, 774 µmol) and TfOH (14 µL, 155 µmol) in a
mixture of DCE (1.2 mL) and Et2O (1.2 mL) was added at -20
°C. After 30 min, the reaction mixture was worked up and
purified as described for the synthesis of 19 to give 23R (64.7
mg, 46%) and 23â (52.1 mg, 37%).
C54H58NPO19: C, 61.42; H, 5.52; N, 1.33; P, 2.93. Found: C,
61.34; H, 5.63; N, 1.29; P, 2.99.
N-Ben zyloxyca r bon yl-O-{O-[2,3,4,-tr i-O-(4-m eth ylben -
zoyl)-R,â-D-ga la ctop yr a n osyl]-(1f3)-O-(2-O-a cetyl-4,6-O-
ben zylid en e-â-D-ga la ct op yr a n osyl)-(1f4)-(3-O-a cet yl-2-
O-d ib en zyloxyp h osp h in yl-â-D-xylop yr a n osyl)}-L-ser in e
Ben zyl Ester (26R a n d 26â). To a solution of 18 (96.5 mg,
120 µmol) and 24 (97.5 mg, 92.3 µmol) in DCE (2 mL) was
added MS AW300 (120 mg). After stirring at room temperature
for 40 min, a solution of NIS (125 mg, 554 µmol) and TfOH (8
µL, 92 µmol) in a mixture of DCE (1 mL) and Et2O (1 mL)
was added at -20 °C and the solution was stirred for 30 min.
The reaction mixture was worked up and purified as described
for the synthesis of 19 to give an anomeric mixture of triaosyl
serines (113.6 mg). The mixture was diluted with THF (2 mL);
then AcOH (36 µL) and 1 M TBAF (0.31 mL) were added to
the solution. The reaction mixture was stirred for 10 h and
was worked up as described for the synthesis of 20. The crude
materials obtained were eluted from a column of silica gel (2:
1-3:2-1:2-1:5 toluene-EtOAc) and purified by PTLC (1:2 ×
2 toluene-EtOAc) to give 26R (12.3 mg, 9%) and 26â (45.7
mg, 31%).
23R: Rf 0.53 (1:1 toluene-EtOAc); [R]D ) +52.9 (c 1.32,
1
CHCl3); H NMR (CDCl3) δ 7.52-7.50 (m, 2H, Ar H), 7.37-
7.21 (m, 23H, Ar H), 5.94-5.85 (m, 1H, -CHdCH2), 5.55 (s,
1H, PhCH), 5.50-5.27 (m, 2H, dCH2), 5.23-5.14 (m, 2H,
PhCH2), 5.20 (m, 1H, H-31), 5.17 (m, 2H, H-12,22), 5.11 (s, 2H,
PhCH2), 4.94-4.88 (m, 4H, 2PhCH2), 4.62 (brd, 1H, J ) 8.78
Hz, SerR), 4.46 (dd, 1H, J gem ) 9.51 Hz, J R,âa ) 2.19 Hz, Serâa),
4.33 (d, 1H, J 1,2 ) 7.32 Hz, H-11), 4.30 (d, 1H, J 3,4 ) 3.18 Hz,
H-42), 4.24 (d, 1H, J gem ) 12.44 Hz, H-6a2), 4.13 (d, 2H, OCH2),
4.06 (m, 1H, H-6b2), 4.02 (m, 1H, H-21), 3.91 (dd, 1H, J 4,5eq
)
5.37 Hz, J gem ) 11.46 Hz, H-5eq1), 3.83 (dd, 1H, J 2,3 ) 10.73
Hz, H-32), 3.78 (m, 1H, H-41), 3.68 (dd, 1H, J R,âb ) 2.93 Hz,
Serâb), 3.60 (s, 1H, H-52), 3.28 (dd, 1H, J 4,5ax ) 10.25 Hz,
26R: Rf 0.61 (1:3 toluene-EtOAc); [R]D ) +126.0 (c 1.23,
1
CHCl3); H NMR (CDCl3) δ 7.98-7.96 (m, 2H, Ar H), 7.69-
7.65 (m, 4H, Ar H), 7.52-7.00 (m, 29H, Ar H), 6.89-6.87 (m,
2H, Ar H), 5.83-5.79 (m, 2H, H-33,43), 5.65 (d, 1H, J 1,2 ) 3.66
Hz, H-13), 5.56 (dd, 1H, J 2,3 ) 10.49 Hz, H-23), 5.30 (dd, 1H,
J 1,2 ) 8.05 Hz, J 2,3 ) 10.25 Hz, H-22), 5.20, 5.15 (ABq, 2H, J
) 12.44 Hz, PhCH2), 5.17 (s, 1H, PhCH), 5.12 (m, 1H, H-31),
4.94-4.84 (m, 4H, 2PhCH2), 4.60 (m, 1H, SerR), 4.48 (d, 1H,
H-12), 4.44 (dd, 1H, J gem ) 9.51 Hz, J R,âa ) 2.44 Hz, Serâa),
4.34 (d, 1H, J 1,2 ) 7.32 Hz, H-11), 4.28 (brt, 1H, J ) 6.96 Hz,
H-5ax1), 2.06, 1.88 (2s, 3H × 2, 2Me). Anal. Calcd for C57H62
-
NPO19: C, 62.46; H, 5.70; N, 1.28. Found: C, 62.25; H, 5.71;
N, 1.08.
23â: Rf 0.33 (1:1 toluene-EtOAc); [R]D ) -17.4 (c 1.38,
1
CHCl3); H NMR (CDCl3) δ 7.48-7.16 (m, 25H, Ar H), 5.89-
5.79 (m, 1H, -CHdCH2), 5.50 (s, 1H, PhCH), 5.27, 5.23 (m,
2H, dCH2), 5.22-5.10 (m, 4H, 2PhCH2), 5.16 (m, 1H, H-22),
5.14 (m, 1H, H-31), 4.95-4.85 (m, 4H, 2PhCH2), 4.61 (m, 1H,
H-53), 4.18 (d, 1H, J gem ) 10.98 Hz, H-6a2), 4.16 (d, 1H, J 3,4
)
SerR), 4.47 (d, 1H, J 1,2 ) 7.81 Hz, H-12), 4.45 (dd, 1H, J gem
)
3.41 Hz, H-42), 4.13 (m, 1H, H-21), 3.93 (d, 1H, H-6b2), 3.91
(dd, 1H, J 4,5eq ) 5.37 Hz, J gem ) 11.71 Hz, H-5eq1), 3.85 (dd,
1H, H-32), 3.80 (m, 1H, H-41), 3.72-3.67 (m, 2H, H-6a3,Serâb),
9.51 Hz, J R,âa ) 2.44 Hz, Serâa), 4.34 (d, 1H, J 1,2 ) 7.56 Hz,
H-11), 4.26 (dd, 1H, J 5,6a < 1.0 Hz, J gem ) 10.98 Hz, H-6a2),
4.23 (d, 1H, J 3,4 ) 3.17 Hz, H-42), 4.14 (m, 1H, H-21), 4.07-
4.02 (m, 3H, H-6b2, OCH2), 3.92 (dd, 1H, J 4,5eq ) 5.37 Hz, J gem
3.57 (m, 1H, H-6b3), 3.35 (s, 1H, H-52), 3.25 (dd, 1H, J 4,5ax
)
10.24 Hz, H-5ax1), 2.76 (m, 1H, OH-63), 2.43 (s, 3H, PhMe),
2.27 (s, 6H, 2PhMe), 2.21, 1.83 (2s, 3H × 2, 2Me). Anal. Calcd
for C84H86NPO27‚0.5H2O: C, 63.78; H, 5.56; N, 0.89; P, 1.96.
Found: C, 63.90; H, 5.52; N, 0.91; P, 1.90.
) 11.71 Hz, H-5eq1), 3.80 (ddd, 1H, J 3,4 ) 9.27 Hz, J 4,5ax
)
10.23 Hz, H-41), 3.69 (dd, 1H, J R,âb ) 3.41 Hz, Serâb), 3.55 (dd,
1H, J 2,3 ) 10.25 Hz, H-32), 3.38 (s, 1H, H-52), 3.25 (dd, 1H,
H-5ax1), 2.06, 1.91 (2s, 3H × 2, 2Me). Anal. Calcd for C57H62
-
26â: Rf 0.40 (1:3 toluene-EtOAc); [R]D ) +81.9 (c 1.27,
1
NPO19: C, 62.46; H, 5.70; N, 1.28; P, 2.83. Found: C, 62.50;
H, 5.74; N, 1.27; P, 2.77.
CHCl3); H NMR (CDCl3) δ 7.98-7.96 (m, 2H, Ar H), 7.80-
7.78 (m, 2H, Ar H), 7.67-7.65 (m, 2H, Ar H), 7.52-7.43 (m,
2H, Ar H), 7.32-7.13 (m, 27H, Ar H), 7.03-7.01 (m, 2H, Ar
H), 5.79 (dd, 1H, J 1,2 ) 7.81 Hz, J 2,3 ) 10.49 Hz, H-23), 5.72
(d, 1H, J 3,4 ) 3.17 Hz, H-43), 5.50 (dd, 1H, H-33), 5.41 (s, 1H,
PhCH), 5.20-5.05 (m, 2H, 2PhCH2), 5.16 (m, 1H, H-22), 5.09
(d, 1H, H-13), 5.07 (m, 1H, H-31), 4.93-4.83 (m, 4H, 2PhCH2),
4.59 (m, 1H, SerR), 4.42 (d, 2H, J ) 8.05 Hz, H-12,Serâa), 4.29
(d, 1H, J 1,2 ) 7.56 Hz, H-11), 4.29 (brs, 1H, H-42), 4.23 (d, 1H,
J gem ) 11.71 Hz, H-6a2), 4.11 (m, 1H, H-21), 3.99 (d, 1H, H-6b2),
3.96 (brt, 1H, J ) 6.70 Hz, H-53), 3.90 (dd, 1H, J 2,3 ) 10.49
N -Be n zyloxyca r b on yl-O-[O-(2-O-a ce t yl-4,6-O-b e n z-
ylid e n e -â-D-ga la ct op yr a n osyl)-(1f4)-(3-O-a ce t yl-2-O-
d ib e n zyloxyp h osp h in yl-â-D-xylop yr a n osyl)]-L-se r in e
Ben zyl Ester (24). A suspension of a catalytic amount of (1,5-
cyclooctadiene)bis(methyldiphenylphosphine)iridium(I) hexaflu-
orophosphate in THF (2.5 mL) was stirred under an H2
atmosphere which was then replaced by argon. This manipu-
lation was repeated a few times. Then, a solution of 23â (99.6
mg, 90.9 µmol) in THF (4 mL) was added to the above solution
of the iridium complex. After stirring for 2 h, H2O (1.5 mL),
NaHCO3 (305.5 mg, 3.64 mmol), and I2 (46.1 mg, 182 µmol)
were added to the reaction mixture. The solution was stirred
for 15 min and was diluted with CHCl3. The organic phase
was washed with aqueous NaHCO3, aqueous Na2S2O3, and
brine. The crude material obtained in the usual manner was
eluted from a column of silica gel (5:2-2:1-1:1-1:2-5:1
toluene-EtOAc) to give 24 (91.1 mg, 95%) as a syrup: Rf 0.30
Hz, J 3,4 ) 2.97 Hz, H-32), 3.84 (dd, 1H, J 4,5eq ) 5.37 Hz, J gem
)
11.46 Hz, H-5eq1), 3.79-3.71 (m, 2H, H-41,6a3), 3.66 (dd, 1H,
J R,âb ) 3.17 Hz, J gem ) 11.46 Hz, Serâb), 3.59 (m, 1H, H-6b3),
3.38 (s, 1H, H-52), 3.17 (dd, 1H, J 4,5ax ) 10.25 Hz, H-5ax1), 2.94
(m, 1H, OH-63), 2.43, 2.0.34, 2.27 (3s, 3H × 3, 3PhMe), 1.87,
1.64 (2s, 3H × 2, 2Me). Anal. Calcd for C84H86NPO27: C, 64.16;
H, 5.51; N, 0.89; P, 1.97. Found: C, 64.06; H, 5.58; N, 0.96; P,
1.96.
1
(1:5 toluene-EtOAc); [R]D ) +25.7 (c 0.79, CHCl3); H NMR
N -Be n zyloxyca r b on yl-O-[O-(2,3,4,6-t e t r a -O-a ce t yl-
R,â- D-ga la ctop yr a n osyl]-(1f3)-O-(2-O-a cetyl-4,6-O-ben z-
ylid en e-â-D-ga la ctop yr a n osyl)-(1f4)-(3-O-a cetyl-2-O-d i-
ben zyloxyp h osp h in yl-â-D-xylop yr a n osyl))-L-ser in e Ben z-
yl Ester (27R a n d 27â). To a solution of commercially
available methyl 2,3,4,6-tetra-O-acetyl-1-thio-D-galactopyra-
noside (25, 120.3 mg, 0.318 mmol) and 24 (172.8 mg, 0.164
mmol) in DCE (6 mL) was added MS AW300 (500 mg). After
stirring at room temperature for 35 min, a solution of NIS (221
mg, 0.983 mmol) and TfOH (17 µL, 0.194 mmol) in a mixture
of DCE (3 mL) and Et2O (3 mL) was added at -20 °C and the
solution was stirred for 20 min. The reaction mixture was
worked up as described for the synthesis of 19. The crude
materials obtained were eluted from a columns of S-X1
(CDCl3) δ 7.38-7.37 (m, 2H, Ar H), 7.31-7.30 (m, 3H, Ar H),
7.25-7.13 (m, 20H, Ar H), 7.02 (d, 1H, J ) 8.78 Hz, NH), 5.45
(s, 1H, PhCH), 5.13, 5.08 (ABq, 2H, J ) 12.44 Hz, PhCH2),
5.07 (dd, 1H, J 2,3 ) 9.51 Hz, J 3,4 ) 9.27 Hz, H-31), 5.03 (s, 2H,
PhCH2), 4.90-4.79 (m, 4H, 2PhCH2), 4.88 (m, 1H, H-22), 4.54
(m, 1H, SerR), 4.37 (dd, 1H, J gem ) 9.76 Hz, J R,âa ) 2.20 Hz,
Serâa), 4.35 (d, 1H, J 1,2 ) 7.80 Hz, H-12), 4.29 (d, 1H, J 1,2
)
7.31 Hz, H-11), 4.21 (d, 1H, J gem ) 12.44 Hz, H-6a2), 4.11 (d,
1H, J 3,4 ) 3.90 Hz, H-42), 4.09 (m, 1H, H-21), 3.99 (d, 1H,
H-6b2), 3.85 (dd, 1H, J 4,5eq ) 5.37 Hz, J gem ) 11.71 Hz, H-5eq1),
3.72 (ddd, 1H, J 4,5ax ) 9.75 Hz, H-41), 3.64 (dd, 1H, J R,âb ) 2.17
Hz, Serâb), 3.60 (m, 1H, H-32), 3.38 (s, 1H, H-52), 3.19 (dd,
1H, H-5ax1), 2.01, 1.87 (2s, 3H × 2, 2Me). Anal. Calcd for

Phosphorylated and Sulfated Glycosyl Serines of GAG
J . Org. Chem., Vol. 66, No. 9, 2001 3081
(toluene) and silica gel (5:1-3:1-5:2-2:1-1:1 toluene-EtOAc)
and purified by PTLC (1:2 × 2 toluene-EtOAc) to give 27R
(23.9 mg, 11%) and 27â (88.9 mg, 39%).
788.07, [M - Na + 2H]+), 810.1 (calcd for C20H33NPSO23Na4
810.05, [M + H]+), 832.1 (calcd for C20H32NPSO23Na5 832.03,
[M + Na]+).
4-Meth oxyp h en yl 2,4,6-tr i-O-a cetyl-3-O-a llyl-â-D-ga la c-
top yr a n osid e (28). Crude 4-methoxyphenyl 3-O-allyl-â-D-
galactopyranoside4 was acetylated with pyridine and Ac2O.
After the usual workup, the crude material obtained was
recrystallized from n-hexanes-EtOAc: mp 130-131 °C; [R]D
) +23.6 (c 1.52, CHCl3); 1H NMR (CDCl3) δ 6.97-6.93 (m, 2H,
Ar H), 6.83-6.79 (m, 2H, Ar H), 5.85-5.75 (m, 1H, CHdCH2),
5.46 (d, 1H, J 3,4 ) 3.66 Hz, H-4), 5.34 (dd, 1H, J 1,2 ) 8.05 Hz,
J 2,3 ) 10.01 Hz, H-2), 5.28-5.17 (m, 2H, dCH2), 4.86 (d, 1H,
H-1), 4.24-4.13 (m, 3H, H-6a, OCH2), 3.96-3.90 (m, 2H, H-5,-
6b), 3.77 (s, 3H, OMe), 3.59 (dd, 1H, H-3), 2.17, 2.11, 2.08 (3s,
3H × 3, 3MeCO). Anal. Calcd for C22H28O10: C, 58.39; H, 6.25.
Found: C, 58.38; H, 6.26.
4-Meth oxyp h en yl 2-O-Acetyl-3-O-a llyl-6-O-ter t-bu tyl-
d ip h en ylsilyl-â-D-ga la ct op yr a n osid e (29) a n d 4-Met h -
oxyp h en yl 3-O-Allyl-6-O-ter t-bu tyld ip h en ylsilyl-â-D-ga -
la ctop yr a n osid e (30). Triacetate 28 (590.6 mg, 1.31 mmol)
was diluted with MeOH (4 mL), H2O (2 mL) and Et3N (2 mL),
and the reaction mixture was stirred for 16 h at room
temperature. The volatiles were removed under diminished
pressure, and the residue was diluted with DMF (8 mL). To
this solution were added imidazole (216.6 mg, 3.18 mmol) and
tert-butylchlorodiphenylsilane (0.55 mL, 2.11 mmol). The
reaction mixture was stirred overnight. The crude material
obtained in the usual manner was eluted from a column of
silica gel (20:1-10:1-8:1-3:1-1:1 toluene-EtOAc) to give 29
(670.6 mg, 85%) as a crystalline solid together with 30 (77.0
mg, 10%) as a syrup.
29: mp 116 °C (from n-hexanes-EtOAc); Rf 0.65 (2:1
toluene-EtOAc); [R]D ) +3.96 (c 0.91, CHCl3); 1H NMR
(CDCl3) δ 7.70-7.67 (m, 4H, Ar H), 7.66-7.33 (m, 6H, Ar H),
6.98-6.95 (m, 2H, Ar H), 6.76-6.73 (m, 2H, Ar H), 5.92-5.83
(m, 1H, CHdCH2), 5.39 (dd, 1H, J 1,2 ) 8.05 Hz, J 2,3 ) 10.00
Hz, H-2), 5.31-5.20 (m, 2H, dCH2), 4.81 (d, 1H, H-1), 4.20-
4.15 (m, 1H, 1/2 ) CH2), 4.11 (brs, 1H, H-4), 4.08-4.03 (m,
1H, 1/2 ) CH2), 3.98, 3.97 (2s, 2H, H-6), 3.74 (s, 3H, OMe),
3.60 (brt, 1H, H-5), 3.51 (dd, 1H, J 3,4 ) 3.17 Hz, H-3), 2.56 (d,
1H, J ) 1.22 Hz, OH), 2.11 (s, 3H, MeCO), 1.07 (s, 9H, t-Bu).
Anal. Calcd for C34H42SiO8: C, 67.29; H, 6.99. Found: C, 67.41;
H, 7.02.
27R: Rf 0.41 (1:2 toluene-EtOAc); [R]D ) +34.0 (c 1.20,
CHCl3); 1H NMR (CDCl3) δ 7.47-7.19 (m, 25H, Ar H), 7.11 (d,
1H, J ) 9.87 Hz, NH), 5.45 (d, 1H, J 3,4 ) 3.42 Hz, H-43), 5.43
(d, 1H, J 1,2 ) 4.39 Hz, H-13), 5.42 (s, 1H, PhCH), 5.22 (dd, 1H,
J 1,2 ) 8.29 Hz, J 2,3 ) 10.00 Hz, H-22), 5.19, 5.15 (ABq, 2H, J
) 12.44 Hz, PhCH2), 5.15 (dd, 1H, J 2,3 ) 8.78 Hz, J 3,4 ) 11.22
Hz, H-31), 5.15 (dd, 1H, J 2,3 ) 10.49 Hz, H-33), 5.10 (s, 2H,
PhCH2), 4.93-4.88 (m, 4H, 2PhCH2), 4.90 (m, 1H, H-23), 4.60
(m, 1H, SerR), 4.46 (d, 1H, H-12), 4.44 (m, 1H, Serâa), 4.35 (d,
1H, J 1,2 ) 7.56 Hz, H-11), 4.26 (d, 1H, J gem ) 12.44 Hz, H-6a2),
4.23 (d, 1H, J 3,4 ) 3.66 Hz, H-42), 4.16-4.03 (m, 2H, H-6a,b3),
4.15 (m, 1H, H-21), 4.12 (s, 1H, H-53), 4.05 (dd, 1H, H-6b2),
3.90 (dd, 1H, J 4,5eq ) 5.12 Hz, J gem ) 11.47 Hz, H-5eq1), 3.81
(dd, 1H, H-32), 3.81 (m, 1H, H-41), 3.69 (dd, 1H, J R,âb ) 2.93
Hz, J gem ) 9.51 Hz, Serâb), 3.39 (s, 1H, H-52), 3.26 (dd, 1H,
J 4,5ax ) 10.25 Hz, H-5ax1), 2.12, 2.09, 2.05, 1.94, 1.90, 1.39 (6s,
3H × 6, 6Me). Anal. Calcd for C68H76NPO28‚H2O: C, 58.15; H,
5.61; N, 1.00. Found: C, 58.40; H, 5.75; N, 0.84.
27â: Rf 0.34 (1:2 toluene-EtOAc); [R]D ) -7.8 (c 1.59,
CHCl3); 1H NMR (CDCl3) δ 7.49-7.19 (m, 25H, Ar H), 7.11 (d,
1H, J ) 9.03 Hz, NH), 5.52 (s, 1H, PhCH), 5.37 (d, 1H, J 3,4
3.41 Hz, H-43), 5.20 (m, 1H, H-22), 5.19, 5.15 (ABq, 2H, J )
12.44 Hz, PhCH2), 5.18 (m, 1H, H-23), 5.12 (t, 1H, J 2,3 ) J 3,4
)
)
9.03 Hz, H-31), 5.12, 5.10 (2s, 2H × 2, 2PhCH2), 4.95 (dd, 1H,
J 2,3 ) 10.49 Hz, H-33), 4.92-4.85 (m, 4H, 2PhCH2), 4.64 (d,
1H, J 1,2 ) 8.05 Hz, H-13), 4.60 (m, 1H, SerR), 4.43 (d, 1H, J 1,2
) 7.81 Hz, H-12), 4.43 (m, 1H, Serâa), 4.34 (d, 1H, J 1,2 ) 7.32
Hz, H-11), 4.25 (d, 1H, J gem ) 11.22 Hz, H-6a2), 4.23 (d, 1H,
J 3,4 ) 3.17 Hz, H-42), 4.21 (dd, 1H, J 5,6a ) 5.83 Hz, J gem ) 11.23
Hz, H-6a3), 4.14 (m, 1H, H-21), 4.10 (dd, 1H, J 5,6b ) 7.31 Hz,
H-6b3), 4.05 (d, 1H, H-6b2), 3.90 (dd, 1H, J 4,5eq ) 5.37 Hz, J gem
) 11.46 Hz, H-5eq1), 3.88 (brt, 1H, J ) 6.83 Hz, H-53), 3.80
(m, 1H, H-41), 3.79 (dd, 1H, J 2,3 ) 10.14 Hz, H-32), 3.68 (dd,
1H, J R,âb ) 3.17 Hz, J gem ) 9.51 Hz, Serâb), 3.40 (s, 1H, H-52),
3.23 (dd, 1H, J 4,5ax ) 10.00 Hz, H-5ax1), 2.15, 2.06, 2.05, 1.99,
1.96, 1.89 (6s, 3H × 6, 6Me). Anal. Calcd for C68H76NPO28
‚
0.5H2O: C, 58.53; H, 5.57; N, 1.00. Found: C, 58.85; H, 5.93;
N, 1.00.
O-[O-â-D-Ga la ctop yr a n osyl-(1f3)-O-â-D-ga la ctop yr a n -
osyl-(1f4)-(2-O-p h osp h on o-â-D-xylop yr a n osyl)]-L-ser in e,
Tr isod iu m Sa lt (4). A solution of 26â (7.7 mg, 4.9 µmol) in
MeOH (2 mL) was exposed to hydrogenolytic conditions
overnight as described for the synthesis of 2. The residue was
diluted with MeOH (1 mL), H2O (0.5 mL), and Et3N (0.5 mL)
and was stirred for 5 d. The volatiles were evaporated, and
the crude materials were eluted from a column of LH-20 (1%
AcOH) to give 4 (1.7 mg, 49%): Rf 0.20 (1:1:1 n-BuOH-AcOH-
H2O); [R]D ) -14.1 (c 0.17, H2O). For 1H NMR data see Table
1. FABMS of 4 eluted from AG500W-X8 (Na+): m/z 686.3
(calcd for C20H35NPO20Na2 686.13, [M - Na + 2H]+), 708.3
(calcd for C20H34NPO20Na3 708.11, [M + H]+), 730.2 (calcd for
30: Rf 0.39 (2:1 toluene-EtOAc); [R]D ) -13.2 (c 1.15,
1
CHCl3); H NMR (CDCl3) δ 7.69-7.65 (m, 4H, Ar H), 7.44-
7.32 (m, 6H, Ar H), 7.05-7.01 (m, 2H, Ar H), 6.79-6.74 (m,
2H, Ar H), 6.03-5.93 (m, 1H, CHdCH2), 5.37-5.23 (m, 2H,
dCH2), 4.73 (d, 1H, J 1,2 ) 7.80 Hz, H-1), 4.28-4.19 (m, 2H,
dCH2), 4.11 (brs, 1H, H-4), 4.03-3.92 (m, 3H, H-2,6a,6b), 3.75
(s, 3H, OMe), 3.59 (brt, 1H, H-5), 3.43 (dd, 1H, J 2,3 ) 9.51 Hz,
J 3,4 ) 3.07 Hz, H-3), 2.54 (d, 1H, J ) 1.95 Hz, OH-4), 2.50 (d,
1H, J ) 2.20 Hz, OH-2), 1.04 (s, 9H, t-Bu). Anal. Calcd for
C
32H40SiO7: C, 68.04; H, 7.15. Found: C, 67.77; H, 7.18.
4-Meth oxyp h en yl 2-O-Acetyl-3-O-a llyl-6-O-ter t-bu tyl-
d ip h en ylsilyl-4-O-(4-m eth ylben zoyl)-â-D-ga la ctop yr a n o-
sid e (31). To a solution of 29 (497.3 mg, 0.819 mmol) in
pyridine (5 mL) were added 4-methylbenzoyl chloride (0.32 mL,
2.42 mmol) and a catalytic amount of DMAP. After stirring
overnight, MeOH (1.0 mL) was added. The crude material
obtained by the usual workup was eluted from a column of
silica gel (n-hexane-100:1-50:1-30:1-15:1-8:1-5:1-3:1-2:1
n-hexanes-EtOAc) to give 31 (576.4 mg, 97%) as a syrup: Rf
0.49 (2:1 n-hexanes-EtOAc); [R]D ) +33.5 (c 1.55, CHCl3); 1H
NMR (CDCl3) δ 7.96-7.94 (m, 2H, Ar H), 7.64-7.62 (m, 2H,
Ar H), 7.54-7.52 (m, 2H, Ar H), 7.42-7.30 (m, 4H, Ar H),
7.26-7.13 (m, 4H, Ar H), 6.98-6.96 (m, 2H, Ar H), 6.77-6.74
(m, 2H, Ar H), 5.85-5.75 (m, 1H, CHdCH2), 5.79 (d, 1H, J 3,4
) 3.65 Hz, H-4), 5.39 (dd, 1H, J 1,2 ) 8.05 Hz, J 2,3 ) 10.00 Hz,
H-2), 5.26-5.13 (m, 2H, dCH2), 4.90 (d, 1H, H-1), 4.25-4.20
(m, 1H, 1/2 ) CH2), 4.03-3.92 (m, 1H, 1/2 ) CH2), 3.85-3.80
(m, 3H, H-5,6a,6b), 3.74 (s, 3H, OMe), 3.67 (dd, 1H, H-3), 2.42
(s, 3H, PhMe), 2.09 (s, 3H, MeCO), 1.02 (s, 9H, t-Bu). Anal.
Calcd for C48H52SiO9: C, 69.58; H, 6.69. Found: C, 69.66; H,
6.69.
C
20H33NPO20Na4 730.09, [M + Na]+).
O-[O-(6-O-Su lfo-â-D-ga la ctop yr a n osyl)-(1f3)-O-â-D-ga -
lactopyr an osyl-(1f4)-(2-O-ph osph on o-â-D-xylopyr an osyl)]-
L-ser in e, Tetr a sod iu m Sa lt (5). To a solution of 26â (12.2
mg, 7.8 µmol) in DMF (1 mL) was added SO3‚NMe3 (29.2 mg,
0.210 mmol). The reaction mixture was stirred for 6 h at 50-
60 °Cand was subjected to columns of LH-20 (1:1 CHCl3-
MeOH) and AG50W-X8 (Na+) (8:1 MeOH-H2O) to give the
sulfate quantitatively, which was used for deprotection without
further purification. A solution of the sulfate in MeOH (2 mL)
was exposed to hydrogenolytic conditions for 2 d as described
for the synthesis of 2. Methanolic 0.1 M NaOMe (0.25 mL)
was added to the solution of the residue in MeOH (2 mL). The
reaction mixture was stirred for 1 d. After neutralization with
50% AcOH, the volatiles were evaporated. The crude materials
were eluted from columns of LH-20 (1% AcOH) and AG500W-
X8 (Na+) to give 5 (3.7 mg, 53%): Rf 0.47 (1:1:1 EtOAc-
MeOH-H2O); [R]D ) -13.8 (c 0.13, H2O). For 1H NMR data
see Table 1. FABMS: m/z 788.2 (calcd for C20H34NPSO23Na3

3082 J . Org. Chem., Vol. 66, No. 9, 2001
Tamura and Nishihara
4-Meth oxyp h en yl 2-O-Acetyl-6-O-ter t-bu tyld ip h en yl-
silyl-4-O-(4-m eth ylben zoyl)-â-D-ga la ctop yr a n osid e (32).
A solution of 31 (346.2 mg, 0.478 mmol) in THF (6 mL) was
added to a solution (5 mL) containing the iridium complex as
described for the synthesis of 24. After stirring overnight, H2O
(7.2 mL), NaHCO3 (1.52 g, 18.1 mmol), and I2 (242 mg, 0.95
mmol) were added to the reaction mixture and the solution
stirred for 15 min. The crude material obtained in the same
manner as described for the synthesis of 24 was eluted from
a column of silica gel (30:1-10:1-5:1-2:1-1:1 n-hexanes-
EtOAc) to give 32 (322.7 mg, 99%) as a syrup: Rf 0.48 (1:1
n-hexanes-EtOAc); [R]D ) -0.23 (c 1.72, CHCl3); 1H NMR
(CDCl3) δ 7.97-7.95 (m, 2H, Ar H), 7.624-7.621 (m, 2H, Ar
H), 7.61-7.60 (m, 2H, Ar H), 7.52-7.50 (m, 3H, Ar H), 7.42-
7.23 (m, 3H, Ar H), 7.19-7.16 (m, 3H, Ar H), 6.99-6.97 (m,
2H, Ar H), 6.79-6.76 (m, 2H, Ar H), 5.70 (d, 1H, J 3,4 ) 3.66
Hz, H-4), 5.28 (dd, 1H, J 1,2 ) 8.05 Hz, J 2,3 ) 9.76 Hz, H-2),
4.92 (d, 1H, H-1), 4.02 (m, 1H, H-5), 3.90-3.77 (m, 3H, H-3,-
6a,6b), 3.75 (s, 3H, OMe), 2.66 (d, 1H, J ) 5.85 Hz, OH-3),
2.44 (s, 3H, PhMe), 2.15 (s, 3H, MeCO), 1.02 (s, 9H, t-Bu). Anal.
Calcd for C39H44SiO9‚0.1H2O:C, 67.89; H, 6.47. Found: C,
67.89; H, 6.45.
4-Meth oxyp h en yl O-(2,3,4,6-Tetr a -O-a cetyl-R,â-D-ga la c-
t op yr a n osyl)-(1f3)-2-O-a cet yl-6-O-ter t-b u t yld ip h en yl-
silyl-4-O-(4-m eth ylben zoyl)-â-D-ga la ctop yr a n osid e (35R
a n d 35â) a n d 4-Meth oxyp h en yl 2,3-Di-O-a cetyl-6-O-ter t-
b u t yld ip h en ylsilyl-4-O-(4-m et h ylb en zoyl)-â-D-ga la ct o-
p yr a n osid e (33). Meth od A. To a solution of the known
2,3,4,6-tetra-O-acetyl-R-D-galactopyranosyl trichloroacetimi-
date14 (34, 195.9 mg, 0.398 mmol) and 32 (169.5 mg, 0.247
mmol) in CH2Cl2 (10 mL) was added MS AW300 (1.2 g). After
stirring at room temperature for 30 min, TMSOTf (50 µL, 0.28
mmol) was added at -20 °C and the solution stirred for 2 h
with the temperature gradually being raised to 8 °C. Et3N,
aqueous NaHCO3, and CHCl3 were added to the reaction
mixture, and insoluble materials were filtered on Celite. Crude
material obtained in the usual manner was eluted from
columns of S-X1 (toluene) and silica gel (4:1-3:1-2:1-3:2-
1:1-1:3 n-hexanes-EtOAc) to give 35R (15.5 mg, 6%), 35â
(108.8 mg, 43%), and 33 (43.1 mg, 24%) as syrups. Meth od
B. To a solution of 25 (97.6 mg, 0.258 mmol) and 32 (112.7
mg, 0.165 mmol) in DCE (7 mL) was added MS AW300 (1.2
g). After stirring at room temperature for 90 min, a solution
of NIS (227 mg, 1.01 mmol) and TfOH (14 µL, 0.16 mmol) in
a mixture of DCE (3 mL) and Et2O (3 mL) was added at -20
°C and the solution stirred for 45 min with the temperature
gradually being raised to -10 °C. The reaction mixture was
worked up as described for the synthesis of 19. The crude
material obtained was purified as described in method A to
give 35R (11.0 mg, 7%), 35â (59.5 mg, 36%), and 33 (9.2 mg,
8%).
H-31), 3.87-3.83 (m, 2H, H-51,52,6a1), 3.77 (dd, 1H, J 5,6b ) 6.84
Hz, J gem ) 11.95 Hz, H-6b1), 3.73 (s, 3H, OMe), 2.41 (s, 3H,
PhMe), 2.13, 2.08, 2.03, 1.94, 1.92 (5s, 3H × 5, 5MeCO), 1.04
(s, 9H, t-Bu). Anal. Calcd for C53H62SiO18‚0.5H2O: C, 62.15;
H, 6.21. Found: C, 62.24; H, 6.16.
33: Rf 0.58 (1:1 n-hexanes-EtOAc); [R]D ) +27.3 (c 0.85,
1
CHCl3); H NMR (CDCl3) δ 7.97-7.95 (m, 2H, Ar H), 7.62-
7.61 (m, 2H, Ar H), 7.49-7.47 (m, 2H, Ar H), 7.43-7.25 (m,
6H, Ar H), 7.18-7.13 (m, 2H, Ar H), 6.98-6.95 (m, 2H, Ar H),
6.79-6.76 (m, 2H, Ar H), 5.80 (d, 1H, J 3,4 ) 3.41 Hz, H-4),
5.48 (dd, 1H, J 1,2 ) 8.05 Hz, J 2,3 ) 10.25 Hz, H-2), 5.19 (dd,
1H, H-3), 4.95 (d, 1H, H-1), 3.97 (brt, 1H, H-5), 3.83-3.77 (m,
2H, H-6a,b), 3.75 (s, 3H, OMe), 2.44 (s, 3H, PhMe), 2.07, 1.98
(2s, 3H × 2, 2MeCO), 0.99 (s, 9H, t-Bu).
O-(2,3,4,6-Tetr a -O-a cetyl-â-D-ga la ctop yr a n osyl)-(1f3)-
2-O-a cetyl-6-O-ter t-bu tyld ip h en ylsilyl-4-O-(4-m eth ylben -
zoyl)-R,â-D-ga la ctop yr a n osyl Tr ich lor oa cetim id a te (36).
To a solution of 35â (312.0 mg, 0.307 mmol) in CH3CN (20
mL) and H2O (5 mL) was added CAN (843 mg, 1.54 mmol) at
0 °C. After stirring for 50 min, the reaction mixture was diluted
with brine and extracted with CHCl3. The organic phase was
washed with brine. The crude material obtained in the usual
manner was eluted from a column of silica gel (4:1-3:1-2:1-
1:1-2:3-1:5 n-hexanes-EtOAc) to give a hemiacetal (260.5
mg, 93%) as a syrup, which was used for the next reaction
without further purification: Rf 0.18 (1:1 n-hexanes-EtOAc).
To a solution of the hemiacetal (260.5 mg, 0.287 mmol) and
CCl3CN (1.6 mL) in CH2Cl2 (6 mL) was added 2 drops of DBU
at 0 °C. After stirring for 30 min at 0 °C and for 20 min more
at room temperature, the reaction mixture was directly eluted
from a column of silica gel (10:1-6:1-4:1-2:1-3:2-1:1 n-hex-
anes-EtOAc) to give 36 (279.3 mg, 93%) as an anomeric
mixture. The product was used for the next reaction without
further purification: Rf 0.39 and 0.29 (1:1 n-hexanes-EtOAc);
1H NMR (CDCl3) δ 8.68 (s, 1H, NHâ), 8.65 (s, 1H, NHR), 7.90,
7.88 (2s, 2H, Ar H), 7.61-7.54 (m, 4H, Ar H), 7.40-7.23 (m,
6H, Ar H), 6.62 (d, 1H, J 1,2 ) 3.66 Hz, HR-11), 5.87 (d, 1H, J 1,2
) 8.29 Hz, Hâ-11), 5.88 (d, 1H, J 3,4 ) 3.42 Hz, HR-41), 5.75 (d,
1H, J 3,4 ) 3.17 Hz, Hâ-41), 5.60 (dd, 1H, J 2,3 ) 10.00 Hz, Hâ-
21), 5.44 (dd, 1H, J 2,3 ) 10.49 Hz, HR-21), 5.33 (d, 1H, J 3,4
)
3.42 Hz, HR-42), 5.32 (d, 1H, J 3,4 ) 3.42 Hz, Hâ-42), 5.09 (dd,
1H, J 1,2 ) 7.81 Hz, J 2,3 ) 10.49 Hz, HR-22), 5.03 (dd, 1H, J 1,2
) 7.81 Hz, J 2,3 ) 10.49 Hz, Hâ-22), 4.96 (dd, 1H, HR-32), 4.92
(dd, 1H, Hâ-32), 4.72 (d, 1H, HR-12), 4.65 (d, 1H, Hâ-12), 4.38
(dd, 1H, HR-31), 4.34 (brt, 1H, J ) 6.17 Hz, HR-51), 4.19-4.04
(m, 2H, H-6a,b2), 4.08 (dd, 1H, Hâ-31), 3.99 (brt, 1H, J ) 6.34
Hz, Hâ-51), 3.91 (brt, 1H, J ) 7.20 Hz, HR-52), 3.87 (brt, 1H,
J ) 7.57 Hz, Hâ-52), 3.83 (dd, 1H, J 5,6a ) 5.61 Hz, J gem ) 10.73
Hz, Hâ-6a1), 3.77 (dd, 1H, J 5,6a ) 6.10 Hz, J gem ) 10.73 Hz,
HR-6a1), 3.74 (dd, 1H, J 5,6b ) 6.59 Hz, Hâ-6b1), 3.68 (dd, 1H,
J 5,6b ) 6.83 Hz, HR-6b1), 2.42 (s, 3H, PhMe), 2.11, 2.10, 2.08
× 3, 2.05 × 2, 1.94, 1.93, 1.92 (10s, 15H, 5MeCO), 0.99 (s, 9H,
t-Bu).
35R: Rf 0.35 (1:1 n-hexanes-EtOAc); [R]D ) +56.5 (c 1.08,
1
CHCl3); H NMR (CDCl3) δ 7.95-7.94 (m, 2H, Ar H), 7.61-
7.59 (m, 2H, Ar H), 7.51-7.47 (m, 2 H, Ar H), 7.41-7.24 (m,
6 H, Ar H), 7.20-7.12 (m, 2 H, Ar H), 7.01-6.94 (m, 2H, Ar
H), 6.80-6.74 (m, 2H, Ar H), 5.79 (d, 1H, J 3,4 ) 2.93 Hz, H-41),
5.58 (dd, 1H, J 1,2 ) 8.05 Hz, J 2,3 ) 10.24 Hz, H-21), 5.47 (d,
1H, J 1,2 ) 3.66 Hz, H-12), 5.45 (d, 1H, J 3,4 ) 3.17 Hz, H-42),
5.28 (dd, 1H, J 2,3 ) 10.73 Hz, H-22), 5.08 (dd, 1H, H-32), 4.88
(d, 1H, H-11), 4.27 (brt, 1H, J ) 6.59 Hz, H-52), 4.20 (dd, 1H,
N-Ben zyloxyca r bon yl-O-{O-(2,3,4,6-tetr a -O-a cetyl-R,â-
D-ga la ctop yr a n osyl)-(1f3)-O-[2-O-a cetyl-6-O-ter t-bu tyl-
d ip h en ylsilyl-4-O-(4-m eth ylben zoyl)-â-D-ga la ctop yr a n o-
syl]-(1f4)-(3-O-a cet yl-2-O-d ib en zyloxyp h osp h in yl-â-D-
xylop yr a n osyl)}-L-ser in e Ben zyl Ester (37R a n d 37â). To
a solution of 36 (190.5 mg, 0.181 mmol) and 16 (199.3 mg,
0.261 mmol) in CH2Cl2 (8 mL) was added MS AW300 (600 mg).
After stirring at room temperature for 2 h, TMSOTf (23 µL,
0.13 mmol) was added at -20 °C and the reaction mixture
was stirred for 1.5 h with the temperature gradually being
raised to -10 °C. The workup was executed as described for
the synthesis of 35, and the crude material was eluted from
columns of S-X1 (toluene) and silica gel (7:1-6:1-5:1-4:1-
3:1-5:2 n-hexanes-EtOAc) to give 37R (68.6 mg, 23%) and
37â (53.9 mg, 18%) as syrups. The â-anomer [Rf 0.41 (1:1
toluene-EtOAc)] was used for the next reaction without
further purification. 37R: Rf 0.57 (1:1 toluene-EtOAc); [R]D
) +16.7 (c 1.70, CHCl3); 1H NMR (CDCl3) δ 7.87-7.80 (m, 3H,
Ar H), 7.65-7.52 (m, 6H, Ar H), 7.39-7.16 (m, 25H, Ar H),
5.64 (d, 1H, J 3,4 ) 3.66 Hz, H-42), 5.31 (d, 1H, J 3,4 ) 2.58 Hz,
H-43), 5.22-5.13 (m, 5H, H-12,2PhCH2), 5.20 (m, 1H, H-31),
5.12 (m, 1H, H-22), 5.01 (dd, 1H, J 1,2 ) 7.80 Hz, J 2,3 ) 10.49
J 5,6a ) 6.83 Hz, J gem ) 10.98 Hz, H-6a2), 4.06 (dd, 1H, J 5,6b
)
6.83 Hz, H-6b2), 4.05 (dd, 1H, H-31), 3.85 (brt, 1H, J ) 6.47
Hz, H-51), 3.79-3.71(m, 2H, H-6a,b1), 3.75 (s, 3H, OMe), 2.43
(s, 3H, PhMe), 2.17, 2.13, 2.04, 1.90, 1.60 (5s, 3H × 5, 5MeCO),
1.00 (s, 9H, t-Bu). Anal. Calcd for C53H62SiO18‚0.5H2O: C,
62.15; H, 6.21. Found: C, 62.29; H, 6.18.
35â: Rf 0.26 (1:1 n-hexanes-EtOAc); [R]D ) +26.4 (c 1.12,
1
CHCl3); H NMR (CDCl3) δ 7.91-7.89 (m, 2H, Ar H), 7.63-
7.58 (m, 4H, Ar H), 7.38-7.21 (m, 8H, Ar H), 7.02-7.00 (m,
2H, Ar H), 6.74-6.72 (m, 2H, Ar H), 5.64 (d, 1H, J 3,4 ) 3.41
Hz, H-41), 5.55 (dd, 1H, J 1,2 ) 8.05 Hz, J 2,3 ) 10.00 Hz, H-21),
5.31 (d, 1H, J 3,4 ) 3.41 Hz, H-42), 5.03 (dd, 1H, J 1,2 ) 7.81 Hz,
J 2,3 ) 10.48 Hz, H-22), 4.92 (dd, 1H, H-32), 4.89 (d, 1H, H-11),
4.63 (d, 1H, H-12), 4.16 (dd, 1H, J 5,6a ) 6.10 Hz, J gem ) 11.22
Hz, H-6a2), 4.03 (dd, 1H, J 5,6b ) 7.56 Hz, H-6b2), 4.00 (dd, 1H,

Phosphorylated and Sulfated Glycosyl Serines of GAG
J . Org. Chem., Vol. 66, No. 9, 2001 3083
Hz, H-23), 4.93-4.87 (m, 5H, H-33,2PhCH2), 4.65 (d, 1H, H-13),
O-[O-â-D-Ga la ctop yr a n osyl-(1f3)-O-(6-O-su lfo-â-D-ga -
la ctop yr a n osyl)-(1f4)-(2-O-p h osp h on o-â-D-xylop yr a n o-
syl)]-L-ser in e, Tr isod iu m Sa lt (6). To a solution of 38 (23.9
mg, 16.8 µmol) in DMF (3 mL) was added SO3‚NMe3 (73.0 mg,
0.524 mmol). The reaction mixture was stirred overnight at
50-60 °C and eluted from columns of LH-20 (1:1 CHCl3-
MeOH) and AG50W-X8 (Na+) (8:1 MeOH-H2O) to give the
sulfate in 93% yield (23.9 mg), which was used for deprotection
without further purification. To a solution of the sulfate in
MeOH (6 mL) was added a catalytic amount of Pd-C. The
reaction mixture was stirred under an H2 atmosphere over-
night and filtered on Celite, and the volatiles were removed
under diminished pressure. The residue was diluted with
MeOH (3 mL), H2O (1.5 mL), and Et3N (1.5 mL), and the
reaction mixture was stirred for 3 d. Then, methanolic 0.1 M
NaOMe (0.8 mL) was added to the reaction mixture with
stirring overnight. After neutralization with 50% AcOH, the
volatiles were evaporated. The crude materials were eluted
4.64 (m, 1H, SerR), 4.46 (m, 1H, Serâa), 4.32 (d, 1H, J 1,2
)
7.56 Hz, H-11), 4.19 (dd, 1H, J 2,3 ) 10.00 Hz, H-32), 4.12 (m,
1H, H-6a3), 4.07 (m, 1H, H-5eq1), 4.02 (m, 2H, H-52,6b3), 4.01
(m, 1H, H-21), 3.86 (brt, 1H, J ) 7.20 Hz, H-53), 3.76 (dd, 1H,
J 5,6a ) 4.40 Hz, J gem ) 10.97 Hz, H-6a2), 3.75 (m, 1H, H-41),
3.66 (m, 1H, Serâb), 3.62 (dd, 1H, J 5,6b ) 7.56 Hz, H-6b2), 3.26
(t, 1H, J 4,5ax ) J gem ) 10.28 Hz, H-5ax1), 2.40 (s, 3H, PhMe),
2.10, 2.06, 2.03, 1.92, 1.89 (5s, 15H, 5MeCO), 1.00 (s, 9H, t-Bu).
N-Ben zyloxyca r b on yl-O-{O-(2,3,4,6-t et r a -O-a cet yl-â-
D-ga la ct op yr a n osyl)-(1f3)-O-[2-O-a cet yl-4-O-(4-m et h yl-
b en zoyl)-â-D-ga la ct op yr a n osyl]-(1f4)-(3-O-a cet yl-2-O-
d ib e n zyloxyp h osp h in yl-â-D-xylop yr a n osyl)}-L-se r in e
Ben zyl Ester (38). To a solution of 37â (53.9 mg, 32.6 µmol)
in THF (2 mL) were added AcOH (36 µL) and 1 M TBAF (0.31
mL). The reaction mixture was stirred for 22 h and was worked
up as described for the synthesis of 20. The crude materials
obtained were eluted from a column of LH-20 (1:1 MeOH-
CHCl3) and purified by PTLC (1:3 × 2 toluene-EtOAc) to give
38 (34.8 mg, 75%): Rf 0.37 (1:3 toluene-EtOAc); [R]D ) +5.05
from
a column of LH-20 (1% AcOH), and the fractions
1
containing triaosyl serines were condensed. The residue was
diluted with H2O (3 mL) and 0.5 M NaOH (0.3 mL) was added
with stirring for 3 d. The same workup and purification as
above afforded 6 (8.2 mg, 65% from 38): Rf 0.18 (1:1:1
(c 1.03, CHCl3); H NMR (CDCl3) δ 7.93-7.91 (m, 2H, Ar H),
7.52-7.16 (m, 22H, Ar H), 7.02 (d, 1H, J ) 8.78 Hz, NH), 5.46
(d, 1H, J 3,4 ) 3.17 Hz, H-42), 5.27 (d, 1H, J 3,4 ) 3.66 Hz, H-43),
5.25 (dd, 1H, J 1,2 ) 8.05 Hz, J 2,3 ) 10.02 Hz, H-22), 5.20, 5.16
(ABq, 2H, J ) 12.44 Hz, PhCH2), 5.10 (m, 1H, H-31), 5.10 (s,
2H, PhCH2), 5.02 (dd, 1H, J 1,2 ) 7.81 Hz, J 2,3 ) 10.49 Hz, H-23),
4.95-4.86 (m, 4H, 2 PhCH2), 4.91 (m, 1H, H-33), 4.61 (m, 1H,
SerR), 4.58 (d, 1H, H-13), 4.45 (d, 1H, H-12), 4.42 (dd, 1H, J R,âa
) 2.20 Hz, J gem ) 9.27 Hz, Serâa), 4.35 (d, 1H, J 1,2 ) 7.07 Hz,
H-11), 4.14-4.06 (m, 2H, H-21,6a3), 3.93 (dd, 1H, J 2,3 ) 10.00
Hz, H-32), 3.92 (dd, 1H, J 5,6b ) 5.61 Hz, J gem ) 10.00 Hz, H-6b3),
3.84 (brt, 1H, J ) 7.20 Hz, H-53), 3.83 (dd, 1H, J 4,5eq ) 5.61
Hz, H-5eq1), 3.75 (m, 1H, H-41), 3.73-3.60 (m, 2H, H-52,6a2),
3.65 (m, 1H, Serâb), 3.54 (m, 1H, OH-62), 3.47 (m, 1H, H-6b2),
3.23 (dd, 1H, J 4,5ax ) 9.52 Hz, J gem ) 11.46 Hz, H-5ax1), 2.41
(s, 3H, PhMe), 2.10, 2.05, 2.02, 1.96, 1.92, 1.80 (6s, 3H × 6,
6MeCO). Anal. Calcd for C69H78NPO29‚1.5H2O: C, 57.41; H,
5.67; N, 0.97. Found: C, 57.79; H, 6.11; N, 1.09.
1
n-BuOH-AcOH-H2O); [R]D ) -8.4 (c 0.67, H2O). For H NMR
data see Table 1. FABMS: m/z 788.0 (calcd for C20H34NPSO23
Na3 788.07, [M - Na + 2H]+), 810.0 (calcd for C20H33NPSO23
Na4 810.05, [M + H]+).
-
-
Ack n ow led gm en t. A part of this project was sup-
ported by the Kato Memorial Bioscience Foundation. We
are grateful to Dr. S. Kurono, Dr. H. Koshino, and Dr.
S. Manabe (RIKEN) for the FABMS and 1H NMR
measurements. The authors also thank Ms. M. Yoshida
and her staff (RIKEN) for the elemental analyses.
J O001540H