Synthesis of α-GalNAc thioconjugates from an α-GalNAc mercaptan

Article abstract of DOI:10.1021/jo0110909

A variety of functionalized thioglycosides and other derivatives (10-24) of 2-acetamido-2-deoxy-1-thio-α-D-galactopyranose have been prepared in good yields and with high anomeric purities by S-substitution reactions of the sulfide anion or sulfur-centered radical from mercaptan 6. Given the importance in nature of the α-GalNAc 1-O-linkage, and the greater chemical and biological stability of the corresponding 1-S-linkage, these thioconjugates may find application in studies of synthetic vaccines, enzyme inhibitors, glycomimetic scaffolds, and other complex carbohydrate systems.

Full text of DOI:10.1021/jo0110909

J . Org. Chem. 2002, 67, 2995-2999
2995
Syn th esis of r-Ga lNAc Th iocon ju ga tes fr om a n r-Ga lNAc
Mer ca p ta n
Spencer Knapp* and David S. Myers
Department of Chemistry & Chemical Biology, RutgerssThe State University of New J ersey,
6
10 Taylor Road, Piscataway, New J ersey 08854-8087
knapp@rutchem.rutgers.edu
Received November 20, 2001
A variety of functionalized thioglycosides and other derivatives (10-24) of 2-acetamido-2-deoxy-
-thio-R-D-galactopyranose have been prepared in good yields and with high anomeric purities by
1
S-substitution reactions of the sulfide anion or sulfur-centered radical from mercaptan 6. Given
the importance in nature of the R-GalNAc 1-O-linkage, and the greater chemical and biological
stability of the corresponding 1-S-linkage, these thioconjugates may find application in studies of
synthetic vaccines, enzyme inhibitors, glycomimetic scaffolds, and other complex carbohydrate
systems.
In tr od u ction
of R-GalNAc10 thioconjugates (1). Although this modifica-
tion is modest in chemical terms (only the stereochem-
istry at C-4′ is altered), it is of great significance in the
biological world. The Tn epitope (2), which features
N-acetylgalactosamine R-O-linked to a serine or threo-
nine residue, is one of the most common human tumor
cell surface motifs, and scarcely occurs at all in normal
cells. More elaborate tumor cell surface antigens (T,
sialyl-T, sialyl-Tn, and 2,3-sialyl-T) also feature this
linkage, and considerable effort has been expended in
synthesizing these motifs and various analogues and
linked versions in an effort to develop synthetic cancer
Replacement of the linking or anomeric oxygen in
oligosaccharides and other glycoconjugates with a sulfur
atom leads to an important class of synthetic carbohy-
1
dratessthe thioglycosides. These compounds have been
2
3
exploited as glycosyl donors, enzyme inhibitors, enzyme-
4
5
resistant scaffolds, and synthetic vaccines, and have
served as objects of study in their own right.6 The
versatility of organosulfur chemistry is reflected in the
preparation of thioglycosides by forming the S-bond to
7
the glycon or the aglycon or both, with sulfur able to
serve as a nucleophilic, electrophilic, or radical site, and
in the option of preparing several different oxidation
states at sulfur. The anomeric C-S bond is more stable
than C-O under some conditions (acidic hydrolysis,
1
1-16
vaccines.
The S-linked R-GalNAc-cysteine ana-
logues1 should exhibit enhanced resistance to chemical
or enzymatic transformation, and thus are attractive
synthetic targets. Indeed, a lipophilic thioconjugate of
R-GalNAc was prepared as a Tn vaccine candidate by
S-glycosylation of a protected cysteine, and was shown
to exhibit immunostimulating activity in mice spleno-
cytes at lower doses than the corresponding O-linked
serine conjugate, presumably because of its greater
stability toward R-glycosidases.5
7,18
8
enzymatic cleavage), but more reactive under others,
such as electrophilic activation leading to O-, N-, or
C-glycosylation. The anomeric mercaptan of 1-thiopyra-
noses anomerizes much more slowly than the pyranose
itself, particularly under basic conditions or when heated,
and this is a property that can be exploited for the
synthesis of thioglycosides.
Following our development of a method for generating
an R-GlcNAc mercaptan, and of reactions for linking it
with â-iodoalanine and phosphoramidite,9 we became
interested in extending this chemistry to the synthesis
*
To whom correspondence should be addressed.
(1) Horton, D.; Wander, J . D. In The Carbohydrates. Chemistry and
Biochemistry; Pigman, W., Horton, D., Eds.; Academic Press: New
York, 1980; Vol. IB, pp 799-842.
(2) Crich, D.; Smith, M. J . Am. Chem. Soc. 2001, 123, 9015-9020
and references therein.
3) Driguez, H. Chembiochem 2001, 2, 311-318. Driguez, H. Top.
Curr. Chem. 1997, 187, 85-116 and references therein.
4) Roy, R.; Hernandez-Mateo, F.; Santoyo-Gonzales, F. J . Org.
Chem. 2000, 65, 8743-8746 and references therein.
5) Bousquet, E.; Spadaro, A.; Pappalardo, M. S.; Bernardini, R.;
Romeo, R.; Panza, L.; Ronisvalle, G. J . Carbohydr. Chem. 2000, 19,
27-541.
6) Mu n˜ oz, J . L.; Garc ´ı a-Herrero, A.; Asensio, J . L.; Auzennaeau,
F.-I.; Ca n˜ ada, F. J .; J im e´ nez-Barbero, J . J . Chem. Soc., Perkin Trans.
2001, 867-872.
7) J obron, L.; Hummel, G. Org. Lett. 2000, 2, 2265-2267. Ibatullin,
F. M.; Shabalin, K. A.; J a¨ nis, J . V.; Selivanov, S. I. Tetrahedron Lett.
001, 42, 4565-4567. Xu, W. Z.; Springfield, S. A.; Koh, J . T.
Carbohydr. Res. 2000, 325, 169-176 and references therein.
8) Cohen, S. B.; Halcomb, R. L. J . Org. Chem. 2000, 65, 6145-
152,
(
(
(
5
(
1
(
(9) Knapp, S.; Myers, D. S. J . Org. Chem. 2001, 66, 3636-3638.
(10) R-GalNAc refers to 2-acetamido-2-deoxy-R-D-galactopyranosyl
as a component of glycoconjugates.
(11) Komba, S.; Meldal, M.; Werdelin, O.; J ensen, T.; Bock, K. J .
Chem. Soc., Perkin Trans. 1 1999, 415-419.
2
(
6
1
0.1021/jo0110909 CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/11/2002

2
996 J . Org. Chem., Vol. 67, No. 9, 2002
Knapp and Myers
Sch em e 2. P r ep a r a tion of
Sch em e 1. P r ep a r a tion of 2-Aceta m id o-2-d eoxy-
2
,3,4-tr i-O-a cetyl-1-th io-r-D-ga la ctop yr a n ose (6)
2-Aceta m id o-2-d eoxy-1-th io-r-D-ga la ctop yr a n ose (9)
â-anomerof 6 or 7 was detected. Mercaptan 6 repre-
sents an attactive building block for the synthesis of
R-GalNAc thioconjugates through linking reactions of the
R-situated -SH with a variety of aglycon precursors and
other spacers. For some linking reactions, such as those
run in aqueous solution, the deprotected mercaptan 9
The serine/threonine R-GalNAc linkage also appears
1
9
in a variety of other glycoproteins, while other linked
R-GalNAc units appear inter alia in the lipoteichoic acids
2
0
21
of certain bacterial cell walls, in pituitary hormones,
in the blood group A determinant,22 and in the biological
(Scheme 2) might be more appropriate. Therefore, 5 was
GalNAc donor species UDP-GalNAc (3).
deacetylated and then hydrolyzed to give 9, whose
identity was confirmed by reacetylation to generate 7.
Resu lts a n d Discu ssion
1
Although 9 appeared by H NMR analysis to be free of
the â-anomer, some anomerization of 9 may have oc-
curred during its acetylation, as the characteristic dou-
blet at 5.16 ppm (J ) 10 Hz),25 reflecting a trace impurity,
was observed in the spectrum of the crude reaction
mixture.
2
-Acetamido-2-deoxy-1,3,4,6-tetra-O-acetyl-â-D-galac-
topyranose (4; Scheme 1) was prepared as the pure
â-anomer according to the literature procedure.23 Treat-
ment of 4 with Lawesson’s reagent [2,4-bis(4-meth-
oxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide] in
hot toluene caused conversion of the amide to the
corresponding thioamide, and then cyclization with par-
ticipation of the thiocarbonyl sulfur atom at the
Mercaptan 6 was linked with a variety of coupling
partners, resulting in the efficient preparation of a
diverse family of potentially useful R-GalNAc thioconju-
gates (10-24) (Tables 1-3), each as single isomers. These
conjugation reactions can be divided into three types:
couplings under essentially neutral conditions (Table 1),
couplings under basic (anionic) conditions (Table 2), and
free radical couplings with alkenes (Table 3). They are
amoneric center produced the GalNAc-thiazoline 5. The
1
structure of 5 follows from the resemblance of its
H
and 1 C NMR spectra to those of the isomeric GlcNAc-
3
thiazoline,9
,24
including the five-bond coupling (J ) 1.5
. The thiazoline ring
Hz) between H-7a and 2-CH
3
5
the first nonglycosylative examples of R-GalNAc thio-
of 5 underwent hydrolysis with cleavage of the imine C-S
bond (rather than the anomeric C-S bond) to give
the acetamido â-mercaptan 6, which was further char-
acterized as the known S-acetate 7.25 No trace of the
conjugate synthesis.
Oxidative dimerization of 6 could be accomplished by
26
treatment with 10% aqueous hydrogen peroxide, but
disulfide 10 (Table 1) was obtained in better yield and
purity by treating a THF solution of 6 with iodine and
pyridine. Glycosyl disulfides have previously been ad-
(
12) Winterfeld, G. A.; Ito, Y.; Ogawa, T.; Schmidt, R. R. Eur. J .
Org. Chem. 1999, 5, 1167-1171.
13) George, S. K.; Schwientek, T.; Holm, B.; Reis, C. A.; Clausen,
(
26
vanced as useful glycoconjugates. Literature condi-
H.; Kihlberg, J . J . Am. Chem. Soc. 2001, 123, 11117-11125 and
references therein.
tions8 sufficed for conversion of 6 to the S-glycosyl
phosphothioate 11, a potential precursor to nucleoside
diphosphate analogues (compare 3) and other S-phos-
phorylated derivatives of 2-acetamido-2-deoxy-1-thio-R-
D-galactopyranose. Benzoquinone coupled smoothly to 1
equiv of 6 in methanol solution; subsequent mild oxida-
tion27 gave in quantitative yield the quinone 12, which
may be viewed as a glycosylated cyclitol mimic.
,9
(14) Allen, J . R.; Harris, C. R.; Danishefsky, S. J . J . Am. Chem. Soc.
2
001, 123, 1890-1897 and references therein.
(
(
Bioconjugate Chem. 2001, 12, 325-328.
(
7, 227-239.
(
15) Kihlberg, J .; Elofsson, M. Curr. Med. Chem. 1997, 4, 85-116.
16) Peri, F.; Cipolla, L.; Rescigno, M.; La Ferla, B.; Nicotra, F.
17) Connolly, D. T.; Roseman, S.; Lee, Y. C. Carbohydr. Res. 1980,
8
18) Zhu, Y.; van der Donk, W. A. Org. Lett. 2001, 3, 1189-1192.
19) Brockhausen I. In Glycoproteins; Montreuil, J ., Vliegenthart,
(
J . F. G., Schachter, H., Eds.; Elsevier: Amsterdam, 1995; Vol. 29a, pp
Mitsunobu reactions of 1-thiopyranoses have been
2
01-259.
2
8
(20) Navarre, W. W.; Schneewind, O. Microbiol. Mol. Biol. Rev. 1999,
previously demonstrated to lead to thioconjugates. In
the case of 6, coupling with 5 equiv of 2-butyne-1,4-diol
resulted in the mono-Mitsunobu product 13, a thiocon-
jugate with the potentially useful butyne spacer. 4-Iodo-
6
3, 174-229.
(21) Bielinska, M.; Boime, I. In Glycoproteins; Montreuil, J., Vliegent-
hart, J . F. G., Schachter, H., Eds.; Elsevier: Amsterdam, 1995; Vol.
9a, pp 565-587.
22) Watkins, W. M. In Glycoproteins; Montreuil, J ., Vliegenthart,
J . F. G., Schachter, H., Eds.; Elsevier: Amsterdam, 1995; Vol. 29a, pp
2
(
3
13-390.
(26) Szil a´ gyi, L.; Illy e´ s, T.-Z.; Herczegh, P. Tetrahedron Lett. 2001,
42, 3901-3903 and references therein.
(
23) Kim, T. Y.; Davidson, E. A. J . Org. Chem. 1963, 28, 2475-2476.
24) Knapp, S.; Vocadlo, D.; Gao, Z.; Kirk, B.; Lou, J .; Withers, S.
(
(27) Schnabelrauch, M.; Vasella, A.; Withers, S. G. Helv. Chim. Acta
1994, 77, 778-799.
G. J . Am. Chem. Soc. 1996, 118, 6804-6805.
25) Rakotomanomana N.; Lacombe, J .-M.; Pavia, A. A. Carbohydr.
Res. 1990, 197, 318-323.
(
(28) Falconer, R. A.; J ablonkai, I.; Toth, I. Tetrahedron Lett. 1999,
40, 8663-8666.

Synthesis of R-GalNAc Thioconjugates
J . Org. Chem., Vol. 67, No. 9, 2002 2997
Ta ble 1. r-Ga lNAc Th iocon ju ga tes P r ep a r ed fr om 6
Ta ble 2. r-Ga lNAc Th iocon ju ga tes P r ep a r ed fr om 6
u n d er Neu tr a l Con d ition s
u n d er Ba sic (An ion ic) Con d ition s
Ta ble 3. r-Ga lNAc Th iocon ju ga tes P r ep a r ed fr om 6 by
Ra d ica l Ad d ition to Alk en es
benzyl alcohol also coupled smoothly with 6 to afford 14,
a possible substrate for palladium-mediated reactions.
The sodium salt of 6 could be efficiently generated in
DMF solution, and this mercaptide was coupled to several
alkylating agents (Table 2). Alkylation with a â-iodoala-
nine29 gave S-glycosylated cysteine derivative 15 without
apparent epimerization at the R-carbon. This thioconju-
gate mimics the important Tn antigen (compare 2).
Reaction of sodio-6 with â-propiolactone led to the 3-thio-
propionic acid derivative 16, the first product of a
â-lactone/1-thiopyranose coupling of which we are aware.
Aryl substitution with 1-fluoro-4-nitrobenzene gave the
nitrophenyl thioglycoside 17 as expected,30 and alkylation
with 5-bromopentene and 4-nitrobenzyl bromide to give
1
8 and 19, respectively, was also straightforward. The
nitro groups of 17 and 19 could be reduced to amino
groups for peptide coupling or other further transforma-
tion.
3
1
Radical additions of mercaptans (even anomeric
3
2
-
SH ) to alkenes can be an efficient process for linking
the two components under mild, neutral conditions, pro-
vided one of them is used in excess. Mercaptan 6 reacted
smoothly with several commercially available alkenes to
give the corresponding adducts (Table 3). The alkene was
used in excess, with chloroform as a cosolvent, and the
additions were carried out at reflux with initiation by
azo(bis)isobutyronitrile. The electrophilic acceptor ac-
rolein gave the fastest addition (15 min), whereas the
more hindered cyclohexene required 3 h. The products
20-24 were isolated as single isomers. Three of them
(29) Stocking, E. M.; Schwarz, J . N.; Senn, H.; Salzmann, M.; Silks,
L. A. J . Chem. Soc., Perkin Trans. 1 1997, 2443-2447.
(
(
(
30) Driguez, H.; Szeja, W. Synthesis 1994, 1413-1414.
(
20-22) possess useful linker functionality (carboxalde-
31) Stacy, F. W.; Harris, J . F., J r. Org. React. 1963, 13, 150-376.
hyde, carboxylate, and hydroxyl, respectively) for further
transformation.
32) Lacombe, J . M.; Rakotomanomana, N.; Pavia, A. A. Tetrahedron
Lett. 1988, 29, 4293-4296.

2
998 J . Org. Chem., Vol. 67, No. 9, 2002
Knapp and Myers
Con clu sion
romethane/methanol). The mercapto triol 9 was characterized
by conversion to the known peracetate 7 (93% isolated yield)
by treatment with acetic anhydride/pyridine as for 6.
Mercaptan 6 is a versatile coupling partner for gener-
ating R-GalNAc thioconjugates by a variety of coupling
reactions, as exemplified by 10-24. Presumably other
functionalized thioglycosides, including thiodisaccharides,
could also be derived from 6 by using well-precedented
mercaptan S-alkylation methods, such as triflate alky-
Bis-S-(2-Acet a m id o-2-d eoxy-3,4,6-t r i-O-a cet yl-r-D-ga -
la ctop yr a n osyl) Disu lfid e (10). Iodine (66 µL of a 0.79 M
solution in THF, 0.052 mmol) was added in aliquots over a 15
min period to a solution of 32 mg (0.088 mmol) of mercaptan
6
in 0.75 mL of THF and 14 µL (0.176 mmol) of pyridine at 0
°C, whereupon a white precipitate formed and an orange color
3
33
lation, or conjugate addition to levoglucosenones. Se-
lective oxidation at sulfur or other modifications else-
where in the aglycon could further extend this family.
The availability of 6, and for that matter the R-GlcNAc
persisted. The reaction mixture was concentrated and then
chromatographed on silica with 97:3 dichloromethane/metha-
nol as the eluant to afford 32 mg (100%) of the disulfide 10 as
f
a white solid: mp 115-116 °C; R 0.25 (19:1 dichloromethane/
9
+
version as well, allows for the preparation of hundred-
methanol); FAB-MS m/z 731.3 (M + Li) .
milligram quantities of thioconjugates if required for
applications involving subsequent synthetic steps, and
further scaleup seems feasible. The R-GalNAc and
R-GlcNAc thioconjugates can thus join other thioglyco-
sides as useful probes of biological systems and as
enzyme-stable components in biomimetic constructs.
S-(2-Aceta m id o-2-d eoxy-3,4,6-tr i-O-a cetyl-r-D-ga la cto-
p yr a n osyl) O,O-Dia llyl P h osp h oth ioa te (11). Diallyl N,N-
diiosopropylphosphoramidite (37 µL, 0.140 mmol) was added
by syringe to a stirred solution of 30 mg (0.083 mmol) of the
mercaptan 6 and 17 mg of 1H-tetrazole in 0.5 mL of acetoni-
trile at -40 °C. After 2 h, 114 µL (0.83 mmol) of tert-butyl
hydroperoxide was added, and the reaction was allowed to stir
for an additional 2 h at -40 °C. The reaction was concentrated
and then chromatographed on silica with 1:1 ethyl acetate/
dichloromethane as the eluant to give 27 mg (63%) of the
Exp er im en ta l Section
(
3a R,5R,6R,7R,7a R)-5-(Acetoxym eth yl-6,7-d ia cetoxy-2-
f
phosphothioester 11 as a colorless oil: R 0.20 (19:1 dichlo-
romethane/methanol); FAB-MS m/z 530.0 (M + Li) .
+
m eth yl-5,6,7,7a-tetr ah ydr o-3aH-pyr an o[3,2-d]th iazole (5).
A stirred suspension of 360 mg (0.925 mmol) of 2-acetamido-
S-(2-Aceta m id o-2-d eoxy-3,4,6-tr i-O-a cetyl-r-D-ga la cto-
p yr a n osyl)-2-th ioben zoqu in on e (12). A solution of freshly
sublimed benzoquinone (12 mg, 0.110 mmol) in 0.65 mL of
methanol was added to a stirred solution of 40 mg of mercap-
tan 6 (0.110 mmol) in 0.65 mL of methanol. After 30 min, the
yellow color faded and the reaction was complete according to
2
-deoxy-1,3,4,6-tetra-O-acetyl-â-D-galactopyranose (4) and 318
mg of Lawesson’s reagent (0.785 mmol, 0.85 equiv) in 3.0 mL
of toluene was heated at 80 °C for 1.5 h. The red, homogeneous
reaction mixture was neutralized by the addition of 30 mg of
sodium bicarbonate and then chromatographed directly on
silica gel with 3:7 ethyl acetate/dichloromethane as the eluant
to provide 319 mg (100%) of the thiazoline triacetate 5 as a
f
TLC analysis. The R of the presumed intermediate hydro-
quinone is 0.22 (19:1 dichloromethane/methanol). Iodoben-
zenediacetate (53 mg, 0.165 mmol) was added, and the reaction
was stirred for an additional 30 min, during which time the
yellow color returned. The reaction was concentrated and then
chromatographed on silica with 1:1 ethyl acetate/dichlo-
romethane as the eluant to give 52 mg (100%) of 12 as a yellow
yellow syrup:
FAB-MS m/z 352.1 (M + Li) , 346.1 (M + H) .
-Acet a m id o-2-d eoxy-1-t h io-3,4,6-t r i-O-a cet yl-r-D-ga -
la ctop yr a n ose (6). A solution of 319 mg of thiazoline 5 in
.0 mL of methanol was stirred at 0 °C. Five drops of TFA
f
R 0.51 (1:1 ethyl acetate/dichloromethane);
+
+
2
3
and 5 drops of water were added, and the reaction was allowed
to warm to room temperature and stir for 2 h. The reaction
was concentrated and then chromatographed with 8:2 ethyl
acetate/dichloromethane as the eluant to provide 335 mg
f
film: R 0.40 (19:1 dichloromethane/methanol); FAB-MS m/z
+
478.1 (M + 2H + Li) .
1-Hyd r oxy-2-bu t yn -4-yl 2-Acet a m id o-2-d eoxy-1-t h io-
3,4,6-tr i-O-a cetyl-r-D-ga la ctop yr a n osid e (13). A 40% solu-
tion of dimethyl azodicarboxylate in toluene (65 µL, 0.172
mmol) was added by syringe to a stirred solution of 42 mg
(0.115 mmol) of mercaptan 6, 50 mg (0.190 mmol) of triphen-
ylphosphine, and 50 mg (0.575 mmol, 5 equiv) of 2-butyne-
1,4-diol in 1.2 mL of a 5:1 dichloromethane/DMF mixture at 0
°C. After 24 h the reaction was concentrated and then
chromatographed on silica with 3:2 ethyl acetate/dichlo-
romethane as the eluant to provide 42 mg (84%) of 13 as a
(
100%) of mercaptan 6 as a white foam:
dichloromethane/methanol); FAB-MS m/z 731.4 (2M + Li) .
-Aceta m id o-2-d eoxy-1,3,4,6-tetr a -O-a cetyl-1-th io-r-D-
f
R 0.22 (19:1
+
2
ga la ctop yr a n ose (7). A solution of 28 mg (0.0771 mmol) of 6
in 1.25 mL of a 3:2 pyridine/dichloromethane mixture was
treated with 73 µL of acetic anhydride (0.771 mmol) and one
crystal of DMAP and then stirred for 2 h. The reaction mixture
was concentrated and then chromatographed on silica with
1
(
°
4
:2 ethyl acetate/dichloromethane as the eluant to give 31 mg
99%) of the pentaacetate 7 as a white solid: mp 194-194.5
C; R 0.38 (19:1 dichloromethane/methanol); FAB-MS m/z
colorless oil: R
MS m/z 438.1 (M + Li) .
4-Iodoph en yl)m eth yl 2-Acetam ido-2-deoxy-1-th io-3,4,6-
f
0.21 (19:1 dichloromethane/methanol); FAB-
+
f
(
+
12.1 (M + Li) .
3a R,5R,6R,7R,7a R)-(6,7-Dih yd r oxy-5-(h yd r oxym eth yl-
-m e t h yl-5,6,7,7a -t e t r a h yd r o-3a H -p yr a n o[3,2-d ]t h ia z-
tr i-O-a cetyl-r-D-ga la ctop yr a n osid e (14). A solution of 50
mg (0.14 mmol) of 6 in 1 mL of dichloromethane was coupled
to 42 mg (0.18 mmol) of iodobenzyl alcohol by using the
Mitsunobu procedure described for 13. Chromatography as for
f
(
2
ole (8). A stirred solution of 48 mg of 5 was dissolved in
methanol and cooled to 0 °C. One drop of a 25% solution of
sodium methoxide in methanol was added, and the reaction
was allowed to warm to room temperature. The reaction was
complete in 2 h according to TLC analysis. The reaction
mixture was concentrated and then chromatographed on silica
with 19:1 dichloromethane/methanol as the eluant to give 31
mg of 8 as a white solid: mp 157-158 °C; R
dichloromethane/methanol); FAB-MS m/z 226.1 (M + Li) .
-Aceta m id o-2-d eoxy-1-th io-r-D-ga la ctop yr a n ose (9). A
13 provided 70 mg (88%) of 14 as a colorless oil: R 0.26 (19:1
+
dichloromethane/methanol); FAB-MS m/z 586.0 (M + Li) .
S-(2-Aceta m id o-2-d eoxy-3,4,6-tr i-O-a cetyl-r-D-ga la cto-
p yr a n osyl)-N-(t er t -bu t oxyca r bon yl)-L-cyst ein e Met h yl
Ester (15). A stirred solution of 56 mg (0.154 mmol) of GalNAc
mercaptan 6 in 1 mL of DMF was quickly cooled with a dry
ice/acetone bath and then treated with 141 µL (0.154 mmol)
of a 1.09 M solution of bis(trimethylsilyl)amide in DMF
followed by 44 mg (0.185 mmol, 1.2 equiv) of N-Boc-3-
iodoalanine methyl ester in 0.1 mL of DMF. The solution was
allowed to warm to room temperature and then stirred for 24
h. The reaction mixture was concentrated and then chromato-
graphed on silica with 3:7 ethyl acetate/dichloromethane as
f
0.19 (9:1
+
2
stirred solution of 8 in 1.0 mL of methanol was cooled to 0 °C,
treated with 2 drops of TFA and 2 drops of water, and allowed
to react for 6 h. The crude reaction mixture was concentrated
f
to give 33 mg (100% crude yield) of 9: R 0.28 (3:1 dichlo-
f
the eluant to give 63 mg (87%) of 15 as a colorless oil: R 0.45
(
33) Witczak, Z. J .; Chhabra, R.; Boryczewski, D. Carbohydr. Chem.
(19:1 dichloromethane/methanol); FAB-MS m/z 571.1 (M +
+
2
000, 19, 543-553, and references therein.
Li) .

Synthesis of R-GalNAc Thioconjugates
J . Org. Chem., Vol. 67, No. 9, 2002 2999
S-(2-Aceta m id o-2-d eoxy-3,4,6-tr i-O-a cetyl-r-D-ga la cto-
p yr a n osyl)-3-th iop r op ion ic Acid (16). A solution of 40 mg
S-(2-Aceta m id o-2-d eoxy-3,4,6-tr i-O-a cetyl-r-D-ga la cto-
p yr a n osyl)-5-th iop en ta n oic Acid (21). A solution of 40 mg
(0.110 mmol) of 6 and 10 mg of AIBN in 0.75 mL of a 1:2
4-pentenoic acid/chloroform mixture was heated at reflux for
1 h. The reaction mixture was cooled, concentrated, and then
chromatographed on silica with 19:1 dichloromethane/metha-
nol as the eluant to give 43 mg (92%) of 21 as a colorless oil:
(
0.110 mmol) of 6 in 0.75 mL of DMF was converted to its
sodium mercaptide as described for 15, and then 9 µL (0.143
mmol, 1.3 equiv) of â-propiolactone (caution: highly toxic,
cancer suspect agent) was added. The reaction mixture was
allowed to stir at room temperature for 24 h, concentrated,
and then chromatographed on silica with 19:1 dichloromethane/
methanol as the eluant to give 42 mg (88%) of 16 as a colorless
f
R 0.20 (19:1 dichloromethane/methanol); FAB-MS m/z 476.1
+
(M + 2Li - H) .
oil:
48.7 (M + Li) .
-Nitr op h en yl 2-Aceta m id o-2-d eoxy-1-th io-3,4,6-tr i-O-
R
f
0.31 (9:1 dichloromethane/methanol); FAB-MS m/z
S-(2-Aceta m id o-2-d eoxy-3,4,6-tr i-O-a cetyl-r-D-ga la cto-
p yr a n osyl)-5-th iop en ta n ol (22). A solution of 50 mg (0.137
mmol) of 6 and 15 mg of AIBN in 2.4 mL of a 1:1 4-pentenol/
chloroform mixture was heated at reflux for 1 h. The reaction
mixture was cooled, concentrated, and then chromatographed
on silica with 39:1 dichloromethane/methanol as the eluant
+
4
4
a cetyl-r-D-ga la ctop yr a n osid e (17). A stirred solution of 37
mg (0.110 mmol) of mercaptan 6 in 0.75 mL of DMF was
converted to its sodium salt as described for 15 and then
treated with 14 µL (0.133 mmol) of 1-fluoro-4-nitrobenzene.
The solution was allowed to stir at room temperature for 24
h, concentrated, and then chromatographed on silica with 1:2
ethyl acetate/dichloromethane as the eluant to give 47 mg
f
to give 55 mg (90%) of 22 as a colorless oil: R 0.3 (19:1
+
dichloromethane/methanol); FAB-MS m/z 456.7 (M + Li) .
Cycloh exyl 2-Aceta m id o-2-d eoxy-1-th io-3,4,6-tr i-O-a ce-
tyl-r-D-ga la ctop yr a n osid e (23). A solution of 50 mg (0.137
mmol) of 6 and 5 mg of AIBN in 2.2 mL of a 10:1 cyclohexene/
chloroform mixture was heated at reflux. Additional 5 mg
aliquots of AIBN were added after 1 and 2 h. After 3 h of
total reaction time, the mixture was cooled, concentrated,
and then chromatographed on silica with 1:3 ethyl acetate/
dichloromethane as the eluant to give 52 mg (81%) of 23
(
89%) of 17 as a white foam:
dichloromethane); FAB-MS m/z 491.1 (M + Li) .
-P en ten yl 2-Aceta m id o-2-d eoxy-1-th io-3,4,6-tr i-O-a ce-
tyl-r-D-ga la ctop yr a n osid e (18). A stirred solution of 29 mg
0.080 mmol) of 6 in 0.50 mL of DMF was converted to its
mercaptide as described for 15 and then treated with 12 µL
0.104 mmol, 1.2 equiv) of 4-pentenyl bromide. The solution
f
R 0.59 (1:1 ethyl acetate/
+
4
(
(
f
as a colorless oil: R 0.50 (7:3 dichloromethane/ethyl acetate);
+
was allowed to stir at room temperature for 24 h, concentrated,
and then chromatographed on silica with 1:4 ethyl acetate/
dichloromethane as the eluant to give 32 mg (93%) of 18
FAB-MS m/z 452.1 (M + Li) .
Cycloh exylm eth yl 2-Aceta m id o-2-d eoxy-1-th io-3,4,6-
tr i-O-a cetyl-r-D-ga la ctop yr a n osid e (24). A solution of 55
mg (0.151 mmol) of 6 and 2 mg of AIBN in 1.6 mL of a 3:1
methylenecyclohexane/chloroform mixture was heated at re-
flux. Additional 2 mg aliquots of AIBN were added after 30
min and 1 h. After 1.5 h of total reaction time, the reaction
mixture was cooled, concentrated, and then chromatographed
on silica with 2:1 dichloromethane/ethyl acetate as the eluant
as a colorless oil: R
FAB-MS m/z 438.1 (M + Li) .
4-Nit r op h en yl)m et h yl 2-Acet a m id o-2-d eoxy-1-t h io-
,4,6-tr i-O-a cetyl-r-D-ga la ctop yr a n osid e (19). A stirred
f
0.43 (7:3 dichloromethane/ethyl acetate);
+
(
3
solution of 40 mg (0.110 mmol) of 6 in 0.75 mL of DMF was
converted to its mercaptide as described above and then
treated with 29 mg (0.132 mmol, 1.2 equiv) of p-nitrobenzyl
bromide. The solution was allowed to stir at room temperature
for 24 h, concentrated, and then chromatographed on silica
with 1:2 ethyl acetate/dichloromethane as the eluant to give
f
to give 56 mg (81%) of 24 as a colorless oil: R 0.33 (19:1
+
dichloromethane/methanol); FAB-MS m/z 466.2 (M + Li) .
Ack n ow led gm en t. This work was supported by the
SynChem Research Institute and Wyeth-Ayerst. We are
grateful to Silvia Gonzalez for preparative assistance.
D.S.M. thanks Rutgers University for a graduate fel-
lowship.
4
f
3 mg (78%) of 19 as a colorless oil: R 0.41 (1:1 ethyl acetate/
+
dichloromethane); FAB-MS m/z 505.7 (M + Li) .
S-(2-Aceta m id o-2-d eoxy-3,4,6-tr i-O-a cetyl-r-D-ga la cto-
p yr a n osyl)-3-th iop r op ion a ld eh yd e (20). A solution of 46
mg (0.127 mmol) of 6 and 2 mg of AIBN in 2.4 mL of a 5:1
acrolein/chloroform mixture was heated at reflux for 15 min.
The reaction mixture was cooled, concentrated, and then
chromatographed on silica with 39:1 dichloromethane/metha-
nol as the eluant to give 43 mg (81%) of 20 as a colorless oil:
Su p p or t in g In for m a t ion Ava ila b le: Structures and
1
13
1
numbering, tabulated H and C NMR data, and printed H
13
and C NMR spectra for compounds 5-24. This material is
available free of charge via the Internet at http://pubs.acs.org.
f
R 0.29 (19:1 dichloromethane/methanol); FAB-MS m/z 426.1
+
(
M + Li) .
J O0110909