Application of serine- and threonine-derived cyclic sulfamidates for the preparation of S-linked glycosyl amino acids in solution- and solid-phase peptide synthesis

Article abstract of DOI:10.1021/ja011932l

Cyclic sulfamidates were synthesized in 60% yield from L-serine and allo-L-threonine, respectively. These sulfamidates reacted with a variety of unprotected 1-thio sugars in aqueous bicarbonate buffer (pH 8) to afford the corresponding S-linked serine- and threonine-glycosyl amino acids with good diastereoselectivity (≥97%) after hydrolysis of the N-sulfates. The serine-derived sulfamidate was incorporated into a simple dipeptide to generate a reactive dipeptide substrate that underwent chemoselective ligation with a 1-thio sugar to afford an S-linked glycopeptide. This sulfamidate was also incorporated into a peptide on a solid support in conjunction with solid-phase peptide synthesis. Chemoselective ligation of a 1-thio sugar with the cyclic sulfamidate was achieved on the solid support, followed by removal of the N-sulfate. Finally, the peptide chain of the resulting support-bound S-linked glycopeptide was extended using standard peptide synthesis procedures.

Full text of DOI:10.1021/ja011932l

Published on Web 02/23/2002
Application of Serine- and Threonine-Derived Cyclic
Sulfamidates for the Preparation of S-Linked Glycosyl Amino
Acids in Solution- and Solid-Phase Peptide Synthesis
Scott B. Cohen and Randall L. Halcomb*
Contribution from the Department of Chemistry and Biochemistry, UniVersity of Colorado,
Boulder, Colorado 80309-0215
Received August 9, 2001
Abstract: Cyclic sulfamidates were synthesized in 60% yield from L-serine and allo-L-threonine, respectively.
These sulfamidates reacted with a variety of unprotected 1-thio sugars in aqueous bicarbonate buffer (pH
8) to afford the corresponding S-linked serine- and threonine-glycosyl amino acids with good diastereo-
selectivity (g97%) after hydrolysis of the N-sulfates. The serine-derived sulfamidate was incorporated into
a simple dipeptide to generate a reactive dipeptide substrate that underwent chemoselective ligation with
a 1-thio sugar to afford an S-linked glycopeptide. This sulfamidate was also incorporated into a peptide on
a solid support in conjunction with solid-phase peptide synthesis. Chemoselective ligation of a 1-thio sugar
with the cyclic sulfamidate was achieved on the solid support, followed by removal of the N-sulfate. Finally,
the peptide chain of the resulting support-bound S-linked glycopeptide was extended using standard peptide
synthesis procedures.
Scheme 1
O-Linked glycoproteins constitute a major class of glyco-
conjugates found in mammalian cells.1 The corresponding
S-linked glycoproteins are desired synthesis targets as a result
of their greater chemical stability and enzymatic resistance.2 A
major goal of our research is to develop a convergent approach
for the synthesis of S-linked glycopeptides through chemose-
lective ligations of unprotected carbohydrates with unprotected
peptides in aqueous solution or on a solid support.3
An intuitive method for preparing S-linked glycosyl amino
acids is through a reaction of an anomeric thiolate with an
alanine derivative containing a leaving group on the â-carbon
(Scheme 1). Advantages of this method include avoiding the
need for Lewis-acid mediated glycosylation protocols, and the
problems inherent in such methods. A potential limitation of
this route is the propensity of these alanine derivatives to
eliminate HX, affording compound 1. Subsequent Michael
addition of the sulfur nucleophile to 1 results in a mixture of
We reported the efficient preparation of S-linked glycosyl
serine conjugates, employing the cyclic sulfamidate 5a (Scheme
2) as the electrophilic component in reactions with a variety of
unprotected 1-thio sugars in aqueous reaction solvents.6 A cyclic
sulfamidate was chosen as a useful “â-alanyl” equivalent with
diastereomers that differ in configuration at the R-carbon of the
the hypothesis that constraining the leaving group in a five-
membered ring would reduce the rate of elimination due to poor
overlap between the developing enolate and the leaving oxygen
amino acid.3h These competing pathways were in fact observed
under certain experimental conditions using â-iodoalanine
derivatives.4b
(4) S-Linked amino acid glycosides: (a) Jobron, L.; Hummel, G. Org. Lett.
2000, 2, 2265-2268. (b) Ohnishi, Y.; Ichikawa, M.; Ichikawa, Y. Bioorg.
Med. Chem. Lett. 2000, 10, 1289-1291. (c) Knapp, S.; Myers, D. S. J.
Org. Chem. 2001, 66, 3636-3638.
(1) Taylor, C. M. Tetrahedron 1998, 54, 11317-11362.
(2) Horton, D.; Wander, J. D. Carbohydrates: Chemistry and Biochemistry;
Pigman, W. W., Horton, D., Eds.; Academic Press: New York, 1990; Vol.
4B, pp 799-842.
(5) Thio-sugars: (a) Lu, P.; Hindsgaul, O.; Li, H.; Palcic, M. M. Can. J. Chem.
1997, 75, 790-800. (b) Witczk, Z. J.; Chhabra, R.; Chen, H.; Xie, X.
Carbohydr. Res. 1997, 301, 167-175. (c) Eisele, T.; Windmuller, R.;
Schmidt, R. R. Carbohydr. Res. 1998, 306, 81-91. (d) Fernandez, J. M.
G.; Mellet, C. O.; Defaye, J. J. Org. Chem. 1998, 63, 3572-3580. (e)
Auzanneau, F. I.; Bennis, K.; Fanton, E.; Prome, D.; Defaye, J.; Gelas, J.
J. Chem. Soc., Perkin Trans. 1 1998, 3629-3635. (f) Hummel, G.;
Hindsgaul, O. Angew. Chem., Int. Ed. 1999, 38, 1782-1784. (g) Johnston,
B. D.; Pinto, B. M. J. Org. Chem. 2000, 65, 4607-4617. (h) Xu, W.;
Springfield, S. A.; Koh, J. T. Carbohydr. Res. 2000, 325, 169-176.
(6) Cohen, S. B.; Halcomb, R. L. Org. Lett. 2001, 3, 405-407.
(3) Chemoselective ligations: (a) Davis, N. J.; Flitsch, S. L. Tetrahedron Lett.
1991, 32, 6739-6796. (b) Marcaurelle, L. A.; Rodriguez, E. C.; Bertozzi,
C. R. Tetrahedron Lett. 1998, 39, 8417-8420. (c) Rodriguez, E. C.;
Marcaurelle, L. A.; Bertozzi, C. R. J. Org. Chem. 1998, 63, 7134-7135.
(d) Marcaurelle, L. A.; Bertozzi, C. R. Chem. Eur. J. 1999, 5, 1384-1390.
(e) Macmillan: D.; Bill, R. M.; Sage, K. A.; Fern, D.; Flitsch, S. L. Chem.
Biol. 2001, 8, 133-145. (f) Marcaurelle, L. A.; Bertozzi, C. R. J. Am.
Chem. Soc. 2001, 123, 1587-1595. (g) Shin, I.; Jung, H.; Lee, M.
Tetrahedron Lett. 2001, 42, 1325-1328. (h) Zhu, Y.; Van der Donk, W.
A. Org. Lettt. 2001, 3, 1189-1192.
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2534 VOL. 124, NO. 11, 2002 J. AM. CHEM. SOC.
10.1021/ja011932l CCC: $22.00 © 2002 American Chemical Society

Sulfamidates for Preparation of Glycoconjugates
A R T I C L E S
Scheme 2
Scheme 3
the PMB-protecting group was effected with ceric ammonium
nitrate,11 and hydrogenolysis of the benzyl ester proceeded
quantitatively to provide the desired substrates 5a and 5b
(Scheme 2). The cheap availability of serine allowed preparation
of products 3a-5a on a multigram scale. Formation of the cyclic
sulfamidate required protection of the primary amine while still
retaining the sp3 character of the nitrogen. Attempts to form a
cyclic sulfamidate directly from serine benzyl ester or N-BOC-
serine benzyl ester were unsuccessful.
Before the reactivity of 5a and 5b with sulfur nucleophiles
was examined, control experiments were performed to evaluate
the stability of the cyclic sulfamidates toward hydrolysis in
aqueous buffer and to determine the eagerness of 5a/5b to
provide the undesired elimination product 6 (Scheme 3, il-
lustrated for 5a). Substrate 5a (0.2 M) was incubated in D2O
with sodium bicarbonate (0.5 M) at pH 8 (23 °C), and its
decomposition was followed by 1H NMR. Loss of 5a proceeded
slowly (k ) 0.034 h-1, t1/2 ) 20 h) to form a mixture of the
hydrolysis products 7a and 7b (Scheme 3). Elimination product
6 was not observed, suggesting that under these conditions (pH
8), epimerization of the R-carbon does not occur. The same
experiment with 5b afforded an identical rate of hydrolysis (k
) 0.034 h-1), consistent with hydrolysis occurring at the sulfur
atom rather than at the sulfamidate C-O bond. As with 5a, the
elimination product derived from 5b was not observed.
Synthesis of S-Linked Glycosyl Amino Acids. After the
stability of cyclic sulfamidates 5a/5b in aqueous buffer was
established, their reactivity with unprotected 1-thio sugars to
generate S-linked glycosyl serine and threonine conjugates was
explored (Scheme 4). A solution of substrate 5a (0.2 M) in D2O
with sodium bicarbonate (0.5 M, pH 8, 23 °C) was treated with
the sodium salt of 1-thio-â-D-glucose (8, 0.2 M), and the reaction
atom (this transition state for elimination is the same as for a
5-endo-trig cyclization).7 Additionally, the use of aqueous
reaction solvents avoids the need for complex protecting groups,
particularly on the carbohydrate component. Cyclic sulfamidates
had previously been utilized in the synthesis of unnatural amino
acids8 and, in a nonpeptidic system, were shown to react with
a protected 1-thio sugar.9
In this report we provide a full account of our earlier results
and report application of this method to the more difficult case
of S-linked glycosyl threonine conjugates. Compound 5a was
also incorporated into a simple peptide to generate an electro-
philic peptide substrate for chemoselective ligation with an
unprotected 1-thio sugar. Finally, this chemistry was performed
on a solid support in conjunction with solid-phase peptide
synthesis to prepare S-linked glycopeptides.
Synthesis and Characterization of Serine- and Threonine-
Derived Cyclic Sulfamidates. Cyclic sulfamidates 5a and 5b
were synthesized in 60% overall yield starting from L-serine
and allo-L-threonine, respectively (Scheme 2). The reactivity
of the cyclic sulfamidate required protection of the amino acid
as a benzyl ester such that the carboxylate could be liberated
under neutral conditions. The amino acid benzyl esters were
protected as the p-methoxybenzylamines by reductive amination
with p-anisaldehyde to provide 2a/2b. Protected amino acids
2a/2b reacted cleanly with thionyl chloride at -78 °C in the
presence of excess pyridine to afford the corresponding cyclic
sulfamidites, which were then oxidized to the sulfamidates 3a/
3b with catalytic Ru(III) and periodate.10 Oxidative removal of
1
was monitored by H NMR. Thiolate addition to the â-carbon
occurred with an initial half-life of e10 min, approximately 2
orders of magnitude faster than sulfamidate hydrolysis. The
reaction was complete in 2-3 h, affording the N-sulfatyl
glycoconjugate in g95% yield. The N-sulfate was hydrolyzed
by incubation of the crude mixture in aqueous HCl (5 M) at 37
°C to provide glycoconjugate 11a in 90% isolated yield after
purification by size-exclusion chromatography (Scheme 4).
Treatment of 5a with 1-thio-N-acetyl-â-D-glucosamine (9)12
proceeded equally as well, affording 12a also in 90% yield.
Reaction of 5a with 1-thio-R-D-glucose (10)13 proceeded 2-3-
(7) Baldwin, J. E. J. Chem. Soc., Chem. Commun. 1976, 734-736.
(8) (a) Baldwin, J. E.; Spivey, A. C.; Schofield, C. J. Tetrahedron: Asymmetry
1990, 1, 881-884. (b) Boulton, L. T.; Stock, H. T.; Raphy, J.; Horwell, D.
C. J. Chem. Soc., Perkin Trans. 1 1999, 1421-1429.
(11) Yamaura, M.; Suzuki, T.; Hashimoto, H.; Yoshimura, J.; Okamoto, T.;
Shin, C. Bull. Chem. Soc. Jpn. 1985, 58, 1413-1420.
(12) Obtained by deacylating the peracetylated derivative with sodium methoxide
in methanol: Horton, D.; Wolfrom, M. L. J. Org. Chem. 1962, 27, 1794-
1799.
(9) Aguilera, B.; Fernandez-Mayoralas, A. J. Org. Chem. 1998, 63, 2719-
2723.
(10) Gao, Y.; Sharpless, K. B. J. Am. Chem. Soc. 1988, 110, 7538-7539.
9
J. AM. CHEM. SOC. VOL. 124, NO. 11, 2002 2535

A R T I C L E S
Cohen and Halcomb
Scheme 4
Scheme 5
fold slower due to the lower reactivity of the more hindered
axial nucleophile. However, glyconjugate 13a was still obtained
in good yield (85%, Scheme 4).
Changing the â-carbon from primary (serine) to secondary
(threonine) slowed the rate of thiolate addition by 1000-fold.
Treatment of 5b (0.2 M) with 8 (0.2 M) at 23 °C (pH 8) did
not afford any detectable addition product after 1 h. Increasing
the temperature to 37 °C for 20 h provided a low yield (ca.
10%) of product 11b after removal of the N-sulfate. The major
product of the reaction sequence was allo-threonine, indicating
that much of 5b was lost to hydrolysis. Two changes were
necessary for the thiolate addition (second-order) to better
compete against hydrolysis (first-order). First, an excess (2
equiv) of 1-thio sugar was used. Second, the concentration of
reactants was increased. By changing the monovalent counterion
from sodium to cesium, concentrations up to 1 M could be
attained. Treatment of 5b (0.5 M) with the cesium thiolate salt
of 8 (1 M) in aqueous cesium bicarbonate (1.5 M, pH 8) at 37
°C for 20 h afforded the desired conjugate as the major product.
Conjugate 11b was obtained in 60% isolated yield after removal
of the N-sulfate (aq HCl, 37 °C) and purification by size-
exclusion chromatography (Scheme 4). The N-acetylglu-
cosamine conjugate 12b was also obtained in 60% yield. The
R-thio conjugate 13b could not be obtained in higher than 40%
yield.14 Apparently the steric interactions between the â-carbon
and the axial thiolate were too much for thiolate addition to
effectively compete against hydrolysis. All six conjugates were
obtained as a single diastereomer at the R-carbon as determined
sis of 3a (Scheme 5). Activation of 14 (0.5 M) with PyBOP
(0.5 M) in the presence of leucine benzyl ester (0.45 M), using
N-methylmorpholine as a base, led to rapid (5 min) and complete
consumption of the leucine (Scheme 6). Dipeptide 15 was
obtained in 90% yield and was stable under the reaction
conditions (t1/2 > 4 h). The PMB-protecting group was cleanly
removed with ceric ammonium nitrate, and hydrogenolysis of
the benzyl ester was quantitative to provide the water-soluble
dipeptide substrate 17. Compound 16 was prepared directly from
PyBOP-activation of 5a in the presence of leucine benzyl ester.
However, this reaction proceeded in poor and inconsistent yield
(20-50%). Peptide 17 (0.1 M) underwent chemoselective
ligation with 8 (0.11 M) in aqueous sodium bicarbonate (0.25
M, pH 8, 23 °C), affording the S-linked glycopeptide 18 in 80%
isolated yield after removal of the N-sulfate and purification
by size-exclusion chromatography (Scheme 6).
Extension of this chemistry to the synthesis of a peptide
containing the cyclic sulfamidate within the interior rather than
at the N terminus was investigated. Attempts to acylate the ring
nitrogen of the sulfamidate were unsuccessful, probably because
of the poor nucleophilicity of the nitrogen. In a model reaction,
activation of N-BOC-glycine (1.5 equiv) with PyBOP (1.5
equiv) in the presence of 4a (1 equiv) did not afford the desired
N-acylated product after 4 h, during which time decomposition
of 4a became significant. The cyclic sulfamidate was also
observed to be unstable under the basic conditions required for
Fmoc removal in peptide synthesis (20% piperidine in DMF,
20 min per cycle). Compounds 3a and 4a had half-lives of e5
min in piperidine (20% in DMF) or DBU (10% in DMF). These
results necessitate addition of the 1-thio sugar to the cyclic
sulfamidate immediately after incorporation into the peptide
1
by H NMR (g97%).
Incorporation of the Cyclic Sulfamidate into Peptides.
After a variety of S-linked glycosyl amino acids were success-
fully prepared, the cyclic sulfamidate was next incorporated into
a simple peptide to generate an electrophilic peptide substrate.
Carboxylic acid 14 was prepared quantitatively by hydrogenoly-
(13) Obtained by deacylating the peracetylated derivative with sodium methoxide
in methanol: Gadelle, A.; DeFaye, J.; Pedersen, C. Carbohydr. Res. 1990,
200, 497-498.
(14) The preparation of 10 provided material that typically contained 1-2% of
8. As a result of the greater reactivity of 8 relative to 10, and the use of
excess 10, product 13b was isolated with 4-5% 11b, which was not
separable by size-exclusion chromatography.
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Sulfamidates for Preparation of Glycoconjugates
A R T I C L E S
Scheme 6
Scheme 7
chain, followed by removal of the N-sulfate, to continue the
peptide synthesis. This sequence was achieved on a solid
support.
in resin loading. The initial target was compound 18, whose
solution-phase synthesis served as a standard.
The terminal hydroxyl of the PEG was esterified with Fmoc-
leucine using standard procedures.15 Following Fmoc removal
with piperidine, the resulting resin was treated with amino acid
14 (5 equiv, 0.3 M) and PyBOP (5 equiv, 0.3 M) in DMF with
N-methylmorpholine for 30 min to provide 19 (Scheme 7). The
PMB-protecting group was then removed with ceric ammonium
nitrate (0.5 M) in 9/1 acetonitrile/water for 1 h to afford 20.
The addition of the cesium thiolate salt of 8 (0.5 M) was
performed in 1/1 dioxane/water for 18 h. Initial runs evaluated
these three transformations on solid support; the N-sulfate was
Synthesis of S-Linked Glycopeptides with Solid-Phase
Peptide Synthesis. The efficiency of the cyclic sulfamidate
chemistry described above prompted an investigation into
employing a solid support in conjunction with Fmoc-based solid-
phase peptide synthesis (SPPS). For this work, a polystyrene
resin modified with poly(ethylene glycol) (3000-4000 MW
PEG) was chosen for its ability to swell in both organic and
aqueous solvents. The resin employed was NovaSyn TG-
hydroxy resin, purchased from NovaBiochem. The success of
the reactions on solid phase was evaluated in terms of the crude
purity of the product rather than absolute yield, due to variations
(15) Blankemeyer, B.; Nimtz, M.; Frank, R. Tetrahedron Lett. 1990, 31, 1701-
1704.
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A R T I C L E S
Cohen and Halcomb
Scheme 8
hydrolyzed in solution. Thus, following opening of the cyclic
sulfamidate with 8 to give 21, the product was removed from
the support with sodium hydroxide (0.2 M in 1/1 dioxane/water,
30 min) and the N-sulfate was then hydrolyzed in aqueous HCl.
The reaction mixture was concentrated and analyzed directly
1
by H NMR, without purification (Scheme 7). Product 18 was
obtained in 93% and 94% purity in two separate runs. This
represents an average yield of g99% for each of the six
transformations on the support.
The final issue that was addressed was removal of the
N-sulfate on the solid support. Because of the slow rate of acid-
catalyzed hydrolysis (t1/2 ca. 4-6 h, 37 °C), this method was
not an attractive one for solid phase, as the reaction times would
likely have had to be extended severalfold. The limited solubility
of the N-sulfatyl glycoconjugates in organic solvents necessitated
use of aqueous conditions for removal of the N-sulfate.
However, the use of a solid support allows for organic solvents
as well. As an alternative to aqueous HCl, a Lewis acid-
catalyzed removal of the N-sulfate with boron trifluoride and a
thiol nucleophile was employed.16 Support-bound intermediate
21 was treated with boron trifluoride etherate (1 M) and
n-butanethiol (1 M) in dichloromethane for 20 h (Scheme 7).
The product was then removed from the support with NaOH.
attained in a synthesis. â-Haloalanine derivatives are not a viable
solution to this problem. In early studies, we found that the
â-chloroalanine dipeptide 23 was transformed to the elimination
product 24 with a half-life of ca. 2 min, even faster than
elimination of 3a (Scheme 8). Derivatives with better leaving
groups are anticipated to be even less stable under such
conditions. Finding an electrophilic modification of an amino
acid that will react with an anomeric thiolate nucleophile and
yet withstand conditions of Fmoc removal and peptide synthesis
has indeed been the biggest challenge in this area. The only
example of this is the use of a dehydroalanine amino acid (e.g.,
1). However, the Michael addition of the 1-thio sugar was
reported to afford a 1/1 mixture of diastereomers at the
R-carbon.3h A powerful class of related electrophilic amino acids
that was briefly investigated in this study consists of serine-
and threonine-derived â-lactones.17 These react readily with thiol
nucleophiles, but we found cyclic sulfamidates to be more
compatible with peptide synthesis under the conditions that were
employed.
1
Analysis by H NMR revealed the crude purity of 18 at 90%.
The ability to remove the N-sulfate on the solid support
enhanced the utility of the chemistry, as the peptide chain could
now be extended past the site of glycosylation while still on
the support. To demonstrate this point, support-bound 18 was
treated with Fmoc-threonine and PyBOP. Subsequent Fmoc
removal with piperidine, and cleavage from the support with
NaOH, provided S-linked glycopeptide 22 in ca. 85% crude
purity (nine steps total).
Another limitation, and the subject of our current research,
is that the methods described herein are compatible with
monosaccharides and not di- or polysaccharides. The incompat-
ibility arises from the conditions required to remove the
N-sulfate. Protic and Lewis acidic reagents were observed to
cleave the glycosidic linkage between saccharide units. Current
work is directed at finding a milder method for removing the
N-sulfate, perhaps by using a more specific Lewis acid. Finally,
preparation of other S-linked glycopeptides with SPPS and a
wider array of amino acids is in progress, and optimization with
respect to protecting groups and deprotection schemes may be
necessary. Resolving these issues should allow application of
this chemistry to the synthesis of larger, more complex S-linked
glycopeptides.
Scope and Future Directions
In this work exploring the chemistry of serine- and threonine-
derived cyclic sulfamidates, we demonstrated their use for
efficient and stereochemically controlled preparation of a variety
of S-linked glycosyl amino acids using unprotected 1-thio sugars.
The method was also extended to the construction of simple
S-linked glycopeptides on a solid support in conjunction with
SPPS. The use of the cyclic sulfamidate avoids potential
drawbacks of previous methods, such as epimerization of the
R-carbon; however, challenges still remain.
The primary limitation is that the cyclic sulfamidate cannot
withstand the basic conditions of Fmoc removal used in peptide
synthesis. Therefore, this electrophilic amino acid cannot be
incorporated within the interior of a longer peptide and is
currently limited to the N terminus. The 1-thio sugar must be
incorporated immediately after the cyclic sulfamidate is installed.
This places limitations on the overall convergence that can be
Experimental Section
General Procedures. Proton NMR (1H NMR) spectra were recorded
at 500 MHz. Chemical shifts are expressed in parts per million (δ)
and are referenced to residual protium in the NMR solvent: CD3S-
(O)CD2H, δ 2.49; DOH, δ 4.80; C6D5H, δ 7.16; CHCl3, δ 7.27. Carbon
NMR (13C NMR) spectra were recorded at 125 MHz. Chemical shifts
(δ ppm) are referenced to the carbon signal for the solvent: DMSO-
(17) (a) Pansare, S. V.; Vederas, J. C. J. Org Chem. 1989, 54, 2311-2316. (b)
Arnold, L. D.; Kalantar, T. H.; Vederas, J. C. J. Am. Chem. Soc. 1985,
107, 7105-7109.
(16) Kim, B. M.; So, S. M. Tetrahedron Lett. 1998, 39, 5381-5384.
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Sulfamidates for Preparation of Glycoconjugates
A R T I C L E S
d6, δ 39.5; CDCl3, δ 77.3; C6D6, δ 128.4; carbon spectra recorded in
D2O are referenced to an external standard of DMSO-d6.
hexane/EtOAc to 6/4. The sulfamidate 3a was obtained as a white solid
(8.3 g, 90%). TLC (SiO2, 1/1 hexane/EtOAc): Rf ) 0.55. H NMR
1
(C6D6): δ 7.18-7.06 (m, 7 H), 6.69 (d, J ) 8.7 Hz, 2 H), 4.85 (d, J
) 12.8 Hz, 1 H), 4.81 (d, J ) 12.8 H, 1 H), 4.27 (d, J ) 14.1 Hz, 1
H), 4.20 (d, J ) 14.1 Hz, 1 H), 4.15 (dd, J ) 4.4, 9.0 Hz, 1 H), 3.74
(dd, J ) 7.5, 9.0 Hz, 1 H), 3.46 (dd, J ) 4.4, 7.5 Hz, 1 H), 3.30 (s, 3
H). 13C NMR (C6D6): δ 168.1, 160.6, 135.6, 131.4, 129.2, 129.0, 126.5,
114.7, 68.1, 67.3, 58.8, 55.2, 50.7. IR (cm-1): 2959, 1748, 1613, 1514.
HREIMS: calcd for (M)+, 377.0933; found, 377.0943. Anal. calcd for
C18H19NO6S: C, 57.28; H, 5.07; N, 3.71; S, 8.50. Found: C, 56.90;
H, 4.77; N, 3.76; S, 8.82.
N-(p-Methoxybenzyl)-L-serine Benzyl Ester (2a). L-Serine (5.0 g,
48 mmol, 1.0 equiv) and NaHCO3 (6.0 g, 72 mmol, 1.5 equiv) were
dissolved in H2O (96 mL). CH3OH (96 mL) was added, followed by
[(CH3)3CO2C]2O (15.7 g, 72 mmol, 1.5 equiv). The solution was stirred
at room temperature for ca. 8 h, after which TLC (SiO2, 50/25/25
n-BuOH/HOAc/H2O) showed complete conversion of serine (Rf ) 0.20)
to a less polar product (Rf ) 0.85). The solvents were removed by
rotary evaporation, and the product mixture was partitioned between
HCl (1%) and EtOAc. The aqueous layer was extracted with EtOAc,
and the combined organic extracts were dried (Na2SO4), filtered, and
concentrated. The product was dissolved in CH3OH (200 mL), and Cs2-
CO3 (8.6 g, 26 mmol, 0.55 equiv) was added. Upon dissolution, the
solution was concentrated. The product was dissolved in DMF (100
mL) and benzyl bromide (6.3 mL, 53 mmol, 1.1 equiv) was added.
The solution was stirred at room temperature for 18 h, and the solvent
was removed under reduced pressure at 40 °C. The crude mixture was
partitioned between H2O and CH2Cl2. The aqueous layer was extracted
with CH2Cl2, and the combined organic extracts were dried (Na2SO4),
filtered, and concentrated. The product was dissolved in CH2Cl2 (200
mL), and CF3CO2H (37 mL, 480 mmol, 10 equiv) was added. The
reaction was stirred at room temperature for ca. 2 h, after which TLC
(SiO2, 1/1 hexane/EtOAc) showed complete conversion to a polar
product (Rf ) 0.10). The solution was concentrated, and the product
was dissolved in CH3OH (200 mL). CH3CO2H (5.7 mL, 96 mmol, 2
equiv) was added, and the reaction flask was placed in a water bath.
NaBH3CN (4.5 g, 72 mmol, 1.5 equiv) was added, followed by
p-anisaldehyde (6.3 mL, 52 mmol, 1.1 equiv). The reaction was stirred
at room temperature for 12 h and was then quenched by the addition
of NaHCO3 (12 g, 144 mmol, 3 equiv). The suspension was concen-
trated, and the crude mixture was partitioned between H2O and CH2-
Cl2. The aqueous layer was extracted with CH2Cl2, and the combined
organic extracts were dried (Na2SO4), filtered, and concentrated. The
product was purified by chromatography (silical gel, 250 mL), eluting
with 1/1 hexane/EtOAc. The protected serine derivative 2a was obtained
as a clear, colorless oil (10.6 g, 70%). TLC (SiO2, 100% EtOAc): Rf
) 0.45. 1H NMR (DMSO-d6): δ 7.38-7.31 (m, 5 H), 7.19 (d, J ) 8.6
Hz, 2 H), 6.85 (d, J ) 8.6 Hz, 2 H), 5.15 (s, 2 H), 4.86 (t, J ) 5.8 Hz,
1 H), 3.71 (s, 3 H), 3.66 (d, J ) 13.0 Hz, 1 H), 3.62 (t, J ) 5.8 Hz, 2
H), 3.55 (d, J ) 13.0 Hz, 1 H), 3.31 (br t, J ) 5.0 Hz, 1 H), 2.35 (br
s, 1 H). 13C NMR (DMSO-d6): δ 173.1, 158.2, 136.3, 132.1, 129.2,
128.4, 127.9, 127.8, 113.5, 65.4, 62.6, 62.2, 55.0, 50.3. IR (cm-1): 3200,
3042, 2954, 1733. HRFABMS: Calcd for (M + H)+, 316.1549; found,
316.1536. Anal. calcd for C18H21NO4: C, 68.55; H, 6.71; N, 4.44.
Found: C, 68.32; H, 6.43; N, 4.62.
(4S)-2,2-Dioxo-1,2,3-oxathiazolidinone-4-carboxylic Acid Benzyl
Ester (4a). The protected sulfamidate 3a (8.1 g, 21.5 mmol, 1.0 equiv)
was dissolved in CH3CN (150 mL). H2O (50 mL) was added with
stirring, followed by (NH4)2Ce(NO3)6 (36 g, 65 mmol, 3 equiv). The
reaction was stirred at room temperature for ca. 20 min, after which
TLC (SiO2, 1/1 hexane/EtOAc) showed complete conversion to a more
polar product (Rf ) 0.45). The reaction solution was partitioned between
NaHCO3 and CH2Cl2. The aqueous layer was extracted with CH2Cl2,
and the combined organic extracts were dried (Na2SO4), filtered, and
concentrated. The product was purified by chromatography (silica gel,
200 mL), eluting with 9/1 hexane/EtOAc to 7/3. Sulfamidate 4a was
1
obtained as a clear, colorless oil (5.2 g, 95%). H NMR (DMSO-d6):
δ 8.59 (d, J ) 6.0 Hz, 1 H), 7.42-7.32 (m, 5 H), 5.22 (d, J ) 12.5
Hz, 1 H), 5.19 (d, J ) 12.5 Hz, 1 H), 4.81-4.70 (m, 3 H). 13C NMR
(DMSO-d6): δ 168.4, 135.4, 128.5, 128.3, 128.0, 70.2, 67.0, 55.6. IR
(cm-1): 3269, 2958, 1746, 1188. HREIMS: calcd for (M)+, 257.0358;
found, 257.0346. Anal. calcd for C10H11NO5S: C, 46.69; H, 4.31; N,
5.44; S, 12.46. Found: C, 46.74; H, 4.53; N, 5.38; S, 12.78.
(4S)-2,2-Dioxo-1,2,3-oxathiazolidinone Carboxylic Acid (5a). The
protected sulfamidate 4a (1.25 g, 4.86 mmol, 1.0 equiv) was dissolved
in EtOAc (50 mL). Palladium-on-carbon (10 wt %, 260 mg, 0.24 mmol,
0.05 equiv) was added, and the suspension was stirred under 1 atm of
hydrogen for ca. 30 min, after which TLC (SiO2, 1/1 hexane/EtOAc)
showed complete conversion to a product that did not migrate by TLC.
The suspension was filtered through Celite and concentrated. The
sulfamidate 5a was used without further purification (810 mg, 100%).
1H NMR (DMSO-d6): δ 8.40 (br s, 1 H), 4.72 (dd, J ) 7.8, 8.7 Hz, 1
H), 4.65 (dd, J ) 4.8, 8.6 Hz, 1 H), 4.60 (dd, J ) 4.9, 7.8 Hz, 1 H).
13C NMR (DMSO-d6): δ 170.0, 70.6, 55.7. HREIMS: calcd for (M +
H)+, 167.9967; found, 167.9959. Anal. calcd for C3H5NO5S: C, 21.56;
H, 3.02; N, 8.38; S, 19.18. Found: C, 21.52; H, 2.88; N, 8.33; S, 18.89.
N-(p-Methoxybenzyl)-L-allo-threonine Benzyl Ester (2b). allo-L-
Threonine (1.0 g, 8.4 mmol, 1.0 equiv) and NaHCO3 (1.1 g, 12.6 mmol,
1.5 equiv) were dissolved in H2O (17 mL). CH3OH (17 mL) was added,
followed by [(CH3)3CO2C]2O (2.7 g, 12.6 mmol, 1.5 equiv). The
solution was stirred at room temperature for ca. 8 h, after which TLC
(SiO2, 50/25/25 n-BuOH/HOAc/H2O) showed complete conversion of
allo-L-threonine (Rf ) 0.20) to a less polar product (Rf ) 0.85). The
solvents were removed by rotary evaporation, and the product mixture
was partitioned between HCl (1%) and EtOAc. The aqueous layer was
extracted with EtOAc, and the combined organic extracts were dried
(4S)-N-(p-Methoxybenzyl)-2,2-dioxo-1,2,3-oxathiazolidinone-4-
carboxylic Acid Benzyl Ester (3a). The protected serine derivative
2a (7.7 g, 24.4 mmol, 1.0 equiv) was dissolved in CH2Cl2 (240 mL).
Pyridine (9.8 mL, 122 mmol, 5 equiv) was added, and the solution
was cooled to -78 °C. SOCl2 (2.1 mL, 29.3 mmol, 1.2 equiv) was
added over a period of 5 min, and the solution was stirred at -78 °C
for 5 min. The dry-ice bath was removed and the reaction allowed to
warm to room temperature. The reaction was quenched by the addition
of HCl (1%). The aqueous layer was extracted with CH2Cl2, and the
combined organic extracts were washed with NaHCO3, dried (Na2SO4),
filtered, and concentrated. The sulfamidite was dissolved in CH3CN
(60 mL), and the solution was cooled to 0 °C. NaIO4 (5.75 g, 26.8
mmol, 1.1 equiv) was added followed by RuCl3‚xH2O (50 mg, 0.24
mmol, 0.01 equiv). The reaction was initiated by addition of H2O (60
mL), and the reaction was stirred at 0 °C for 5 min. The ice bath was
removed, and the reaction was stirred for an additional 10 min. The
reaction solution was partitioned between CH2Cl2 and NaHCO3. The
aqueous layer was extracted with CH2Cl2, and the combined organic
extracts were dried (Na2SO4), filtered, and concentrated. The product
was purified by chromatography (silica gel, 200 mL), eluting with 8/2
(Na2SO4), filtered, and concentrated. The product was dissolved in CH3
-
OH (50 mL), and Cs2CO3 (1.5 g, 4.6 mmol, 0.55 equiv) was added.
Upon dissolution, the solution was concentrated. The product was
dissolved in DMF (20 mL), and benzyl bromide (1.1 mL, 9.2 mmol,
1.1 equiv) was added. The solution was stirred at room temperature
for 18 h, and the solvent was removed under reduced pressure at 40
°C. The crude mixture was partitioned between H2O and CH2Cl2. The
aqueous layer was extracted with CH2Cl2, and the combined organic
extracts were dried (Na2SO4), filtered, and concentrated. The product
was dissolved in CH2Cl2 (50 mL), and CF3CO2H (6.5 mL, 84 mmol,
10 equiv) was added. The reaction was stirred at room temperature for
ca. 2 h, after which TLC (SiO2, 1/1 hexane/EtOAc) showed complete
conversion to a polar product (Rf ) 0.10). The solution was concen-
trated, and the product was dissolved in CH3OH (34 mL). CH3CO2H
9
J. AM. CHEM. SOC. VOL. 124, NO. 11, 2002 2539

A R T I C L E S
Cohen and Halcomb
(1.0 mL, 17 mmol, 2 equiv) was added, and the reaction flask was
placed in a water bath. NaBH3CN (795 mg, 12.6 mmol, 1.5 equiv)
was added, followed by p-anisaldehyde (1.1 mL, 9.2 mmol, 1.1 equiv).
The reaction was stirred at room temperature for 12 h and was then
quenched by the addition of NaHCO3 (2.1 g, 25 mmol, 3 equiv). The
suspension was concentrated, and the crude mixture was partitioned
between H2O and CH2Cl2. The aqueous layer was extracted with CH2-
Cl2, and the combined organic extracts were dried (Na2SO4), filtered,
and concentrated. The product was purified by chromatography (silical
gel, 100 mL), eluting with 8/2 hexane/EtOAc to 1/1. The protected
allo-threonine derivative 2b was obtained as a clear, colorless oil (2.0
g, 72%). TLC (SiO2, 1/1 EtOAc/hexane): Rf ) 0.25. 1H NMR (C6D6):
δ 7.20-7.02 (m, 7 H), 6.76 (d, J ) 8.7 Hz, 2 H), 4.98 (d, J ) 12.3
Hz, 1 H), 4.95 (d, J ) 12.3 Hz, 1 H), 4.00 (m, 1 H), 3.66 (d, J ) 12.6
Hz, 1 H), 3.45 (d, J ) 12.6 Hz, 1 H), 3.35 (d, J ) 5.1 Hz, 1 H), 3.30
(s, 3 H), 0.98 (d, J ) 6.4 Hz, 3 H). 13C NMR (C6D6): δ 173.1, 159.4,
136.2, 132.0, 129.8, 128.6, 128.4, 114.1, 67.6, 66.4, 65.8, 54.7, 52.2,
19.1. IR (cm-1): 3446, 2935, 1732, 1513. HREIMS: calcd for (M +
H)+, 330.1705; found, 330.1698. Anal. calcd for C19H23NO4: C, 69.28;
H, 7.04; N, 4.25. Found: C, 69.58; H, 7.38; N, 4.44.
(d, J ) 7.6 Hz, 3 H). 13C NMR (CDCl3): δ 166.9, 134.0, 129.5, 129.3,
129.1, 79.7, 69.1, 60.3, 15.8. IR (cm-1): 3243, 2989, 1727. HREIMS:
calcd for (M)+, 271.0514; found, 271.0505. Anal. calcd for C11H13-
NO5S: C, 48.70; H, 4.83; N, 5.16; S, 11.82. Found: C, 48.84; H, 4.91;
N, 5.28; S, 12.00.
(4S,5S)-2,2-Dioxo-1,2,3-oxathiazolidinone-5-methyl-4-carboxyl-
ic Acid (5b). The protected sulfamidate 4b (590 mg, 2.18 mmol, 1.0
equiv) was dissolved in EtOAc (30 mL). Palladium-on-carbon (10 wt
%, 230 mg, 0.22 mmol, 0.1 equiv) was added, and the suspension was
stirred under 1 atm of hydrogen for ca. 30 min, after which TLC (SiO2,
1/1 hexane/EtOAc) showed complete conversion to a product that did
not migrate by TLC. The suspension was filtered through Celite and
concentrated. The sulfamidate 5b was used without further purification
(390 mg, 100%). 1H NMR (DMSO-d6): δ 8.25 (br s, 1 H), 5.14 (m, 1
H), 4.53 (d, J ) 6.6 Hz, 1 H), 1.34 (d, J ) 6.5 Hz, 3 H). 13C NMR
(DMSO-d6): δ 168.4, 80.1, 60.3, 15.5. HREIMS: calcd for (M + H)+,
182.0123; found, 182.0111. Anal. calcd for C4H7NO5S: C, 26.52; H,
3.89; N, 7.73 S, 17.70. Found: C, 26.72; H, 3.61; N, 7.57; S, 17.91.
1-Deoxy-1-thio-â-D-glucose, Sodium Thiolate Salt (8). 2,3,4,6-
Tetra-O-acetyl-1-S-acetyl-1-thio-â-D-glucopyranose (670 mg, 1.65 mmol,
1.0 equiv) was dissolved in CH3OH (20 mL). Sodium methoxide (6.6
mL, 500 mM in CH3OH, 3.3 mmol, 2.0 equiv) was added, and the
reaction was stirred at room temperature for 2 h. The reaction was
quenched by the addition of NaHCO3 (280 mg, 3.3 mmol, 2.0 equiv).
The solvent was removed by rotary evaporation, and the product was
used without purification. (360 mg, 100%).
(4S,5S)-N-(p-Methoxybenzyl)-2,2-dioxo-1,2,3-oxathiazolidinone-
5-methyl-4-carboxylic Acid Benzyl Ester (3b). The protected allo-
threonine derivative 2b (2.0 g, 6.1 mmol, 1.0 equiv) was dissolved in
CH2Cl2 (60 mL). Pyridine (2.4 mL, 31 mmol, 5 equiv) was added, and
the solution was cooled to -78 °C. SOCl2 (0.53 mL, 7.3 mmol, 1.2
equiv) was added over a period of 5 min, and the solution was stirred
at -78 °C for 5 min. The dry ice bath was removed and the reaction
allowed to warm to room temperature. The reaction was quenched by
the addition of HCl (1%). The aqueous layer was extracted with CH2-
Cl2, and the combined organic extracts were washed with NaHCO3,
dried (Na2SO4), filtered, and concentrated. The sulfamidite was
dissolved in CH3CN (30 mL), and the solution was cooled to 0 °C.
NaIO4 (1.44 g, 6.7 mmol, 1.1 equiv) was added followed by RuCl3‚
xH2O (65 mg, 0.31 mmol, 0.05 equiv). The reaction was initiated by
addition of H2O (30 mL), and the reaction was stirred at 0 °C for 5
min. The ice bath was removed, and the reaction was stirred for an
additional 10 min. The reaction solution was partitioned between CH2-
Cl2 and NaHCO3. The aqueous layer was extracted with CH2Cl2, and
the combined organic extracts were dried (Na2SO4), filtered, and
concentrated. The product was purified by chromatography (silica gel,
50 mL), eluting with 8/2 hexane/EtOAc to 6/4. The sulfamidate 3a
was obtained as a clear, colorless oil (2.15 g, 90%). TLC (SiO2, 8/2
1-Deoxy-1-thio-â-D-glucose, Cesium Thiolate Salt (8). 2,3,4,6-
Tetra-O-acetyl-1-S-acetyl-1-thio-â-D-glucopyranose (940 mg, 2.32 mmol,
1.0 equiv) was dissolved in CH3OH (25 mL). Cs2CO3 (1.5 g, 4.6 mmol,
2.0 equiv) was added, and the solution was stirred at room temperature
for 18 h. The solvent was removed by rotary evaporation, and the
product was used without purification. (760 mg, 100%).
2-Acetamido-1,2-dideoxy-1-thio-â-D-glucose, Sodium Thiolate
Salt (9). 2-Acetamido-3,4,6-tri-O-acetyl-1-S-acetyl-2-deoxy-1-thio-â-
D-glucopyranose12 (655 mg, 1.62 mmol, 1.0 equiv) was dissolved in
CH3OH (25 mL). Sodium methoxide (6.5 mL, 500 mM in CH3OH,
3.25 mmol, 2.0 equiv) was added, and the reaction was stirred at room
temperature for 2 h. The reaction was quenched by the addition of
NaHCO3 (270 mg, 3.25 mmol, 2.0 equiv). The solvent was removed
by rotary evaporation, and the product was used without purification.
1
(420 mg, 100%). H NMR (D2O): δ 4.62 (d, J ) 9.6 Hz, 1 H), 3.80
(dd, J ) 2.1, 12.3 Hz, 1 H), 3.62 (dd, J ) 5.9, 12.3 Hz, 1 H), 3.48 (m,
1 H), 3.40-3.30 (m, 3 H), 1.97 (s, 3 H). 13C NMR (D2O): δ 174.2,
82.3, 80.0, 76.6, 70.8, 62.0, 61.6, 22.9. Mass spectrometry (EI, ESI,
FAB) afforded signals corresponding to the symmetric disulfide.
2-Acetamido-1,2-dideoxy-1-thio-â-D-glucose, Cesium Thiolate
Salt (9). 2-Acetamido-3,4,6-tri-O-acetyl-1-S-acetyl-2-deoxy-1-thio-â-
D-glucopyranose12 (810 mg, 2.0 mmol, 1.0 equiv) was dissolved in CH3-
OH (25 mL). Cs2CO3 (1.3 g, 4.0 mmol, 2.0 equiv) was added, and the
solution was stirred at room temperature for 18 h. The solvent was
removed by rotary evaporation, and the product was used without
purification. (740 mg, 100%).
1
hexane/EtOAc): Rf ) 0.20. H NMR (C6D6): δ 7.18-7.00 (m, 7 H),
6.65 (d, J ) 8.6 Hz, 2 H), 4.81 (d, J ) 12.1 Hz, 1 H), 4.77 (d, J )
12.1 Hz, 1 H), 4.27 (m, 1 H), 4.22 (d, J ) 14.3 Hz, 1 H), 4.10 (d, J )
14.3 Hz, 1 H), 3.55 (d, J ) 6.7 Hz, 1 H), 3.25 (s, 3 H), 0.84 (d, J )
6.5 Hz, 3 H). 13C NMR (C6D6): δ 167.1, 160.6, 135.7, 131.3, 129.3,
129.2, 126.6, 114.7, 76.6, 67.6, 64.0, 55.1, 49.4, 15.8. IR (cm-1): 2938,
1751, 1179. HREIMS: calcd for (M)+, 391.1090; found, 391.1101.
Anal. calcd for C19H21NO6S: C, 58.30; H, 5.41; N, 3.58; S, 8.19.
Found: C, 58.07; H, 5.55; N, 3.64; S, 8.12.
(4S,5S)-2,2-Dioxo-1,2,3-oxathiazolidinone-5-methyl-4-carboxyl-
ic Acid Benzyl Ester (4b). The protected sulfamidate 3b (2.15 g, 5.5
mmol, 1.0 equiv) was dissolved in CH3CN (40 mL). H2O (15 mL)
was added with stirring, followed by (NH4)2Ce(NO3)6 (9.0 g, 16.5 mmol,
3 equiv), thus affording the following concentrations of reactants at
the onset of the reaction: 3b, 0.1 M; (NH4)2Ce(NO3)6, 0.3 M. The
reaction was stirred at room temperature for 30 min, and the solution
was partitioned between NaHCO3 and CH2Cl2. The aqueous layer was
extracted with CH2Cl2, and the combined organic extracts were dried
(Na2SO4), filtered, and concentrated. The product was purified by
chromatography (silica gel, 100 mL), eluting with 9/1 hexane/EtOAc
to 7/3. Sulfamidate 4b was obtained as a white solid (1.4, 95%). TLC
1-Deoxy-1-thio-r-D-glucose, Sodium Thiolate Salt (10). 2,3,4,6-
Tetra-O-acetyl-1-S-acetyl-1-thio-R-D-glucopyranose13 (380 mg, 0.94
mmol, 1.0 equiv) was dissolved in CH3OH (20 mL). Sodium methoxide
(3.8 mL, 500 mM in CH3OH, 1.9 mmol, 2.0 equiv) was added, and
the reaction was stirred at room temperature for 2 h. The reaction was
quenched by the addition of NaHCO3 (160 mg, 1.9 mmol, 2.0 equiv).
The solvent was removed by rotary evaporation, and the product was
1
used without purification. (205 mg, 100%). H NMR (D2O): δ 5.56
(d, J ) 5.3 Hz, 1 H), 4.09 (dt, J ) 3.6, 9.9 Hz, 1 H), 3.75 (m, 3 H),
3.48 (dd, J ) 5.4, 9.2 Hz, 1 H), 3.32 (t, J ) 9.6 Hz, 1 H). 13C NMR
(D2O): δ 83.7, 74.1, 72.2, 70.3, 70.1, 61.0. Mass spectrometry (EI,
ESI, FAB) afforded signals corresponding to the symmetric disulfide.
1-Deoxy-1-thio-r-D-glucose, Cesium Thiolate Salt (10). 2,3,4,6-
Tetra-O-acetyl-1-S-acetyl-1-thio-R-D-glucopyranose13 (810 mg, 2.0
1
(SiO2, 8/2 hexane/EtOAc): Rf ) 0.20. H NMR (CDCl3): δ 7.40 (m,
5 H), 5.33 (d, J ) 11.7 Hz, 1 H), 5.29 (br d, J ) 7.2 Hz, 1 H), 5.25
(d, J ) 11.7 Hz, 1 H), 5.08 (m, 1 H), 4.58 (t, J ) 7.3 Hz, 1 H), 1.31
9
2540 J. AM. CHEM. SOC. VOL. 124, NO. 11, 2002

Sulfamidates for Preparation of Glycoconjugates
A R T I C L E S
mmol, 1.0 equiv) was dissolved in CH3OH (30 mL). Cs2CO3 (1.3 g,
4.0 mmol, 2.0 equiv) was added, and the solution was stirred at room
temperature for 18 h. The solvent was removed by rotary evaporation,
and the product was used without purification. (655 mg, 100%).
lyze the sulfamidate, the crude material was dissolved in HCl (5 M,
15 mL). The hydrolysis reaction was incubated at 37 °C for 12 h, then
concentrated by rotary evaporation under reduced pressure. The product
was dissolved in H2O (10 mL), and the pH was brought to neutral by
the addition of NaHCO3 (ca. 600 mg). The solution was passed through
a nitrocellulose filter (0.45 µm). Product 13a was purified over a column
of Biogel P-2 (fine, column dimensions 2.5 cm i.d. × 70 cm L) eluting
with H2O (20 mL/h). Fractions containing the desired product were
identified by TLC (SiO2, 50/25/25 n-BuOH/HOAc/H2O, Rf ) 0.25)
with ninhydrin staining. Product 13a was obtained as a white solid
S-â-D-Glucopyranosyl-L-cysteine (11a). The sodium salt of 1-thio-
â-D-glucose (8) (360 mg, 1.65 mmol, 1.0 equiv) was dissolved in H2O
(8 mL). In a second flask, NaHCO3 (345 mg, 4.1 mmol, 2.5 equiv)
was added to 5a (275 mg, 1.65 mmol, 1.0 equiv). The reaction was
initiated by addition of the sodium thiolate solution to the flask
containing 5a, thus affording the following concentrations of reactants
at the onset of the reaction: 5a, 0.2 M; 8, 0.2 M; NaHCO3, 0.5 M. The
reaction was stirred at room temperature for 4 h and then concentrated
by rotary evaporation under reduced pressure. To hydrolyze the
sulfamidate, the crude material was dissolved in HCl (5 M, 20 mL).
The hydrolysis reaction was incubated at 37 °C for 24 h and then
concentrated by rotary evaporation under reduced pressure. The product
was dissolved in H2O (10 mL), and the pH was brought to neutral by
the addition of NaHCO3 (ca. 600 mg). The solution was passed through
a nitrocellulose filter (0.45 µm). Product 11a was purified over a column
of Biogel P-2 (fine, column dimensions 2.5 cm i.d. × 70 cm L) eluting
with H2O (20 mL/h). Fractions containing the desired product were
identified by TLC (SiO2, 50/25/25 n-BuOH/HOAc/H2O, Rf ) 0.25)
with ninhydrin staining. Product 11a was obtained as a white solid
1
(225 mg, 85%). H NMR (D2O, HCl salt): δ 5.38 (d, J ) 5.6 Hz, 1
H), 4.37 (dd, J ) 4.0, 6.2 Hz, 1 H), 3.89 (m, 1 H), 3.80 (dd, J ) 2.2,
12.3 Hz, 1 H), 3.76 (dd, J ) 6.2, 12.4 Hz, 1 H), 3.66 (dd, J ) 6.1,
12.6 Hz, 1 H), 3.44 (t, J ) 9.6 Hz, 1 H), 3.30 (t, J ) 9.6 Hz, 1 H),
3.28 (dd, J ) 6.4, 15.3 Hz, 1 H), 3.17 (dd, J ) 4.1, 15.3 Hz, 1 H). 13
C
NMR (D2O, HCl salt): δ 170.1, 87.1, 73.5, 73.1, 70.9, 69.7, 60.7, 53.3,
31.0. HRFABMS: calcd for (M + Na)+, 306.0623; found, 306.0631.
S-â-D-Glucopyranosyl-â-deoxy-â-thio-L-threonine (11b). The ce-
sium salt of 1-thio-â-D-glucose (8) (760 mg, 2.3 mmol, 2.0 equiv) was
dissolved in H2O (2 mL). In a second flask, CsHCO3 (670 mg, 3.5
mmol, 3.0 equiv) was added to 5b (210 mg, 1.16 mmol, 1.0 equiv).
The reaction was initiated by addition of the cesium thiolate solution
to the flask containing 5b, thus affording the following concentrations
of reactants at the onset of the reaction: 5b, 0.5 M; 8, 1.0 M; CsHCO3,
1.5 M. The reaction was incubated at 37 °C for 20 h, then concentrated
by rotary evaporation under reduced pressure. To hydrolyze the
sulfamidate, the crude material was dissolved in HCl (5 M, 10 mL).
The hydrolysis reaction was incubated at 37 °C for 24 h and then
concentrated by rotary evaporation under reduced pressure. The product
was dissolved in H2O (10 mL), and the pH was brought to neutral by
the addition of NaHCO3 (ca. 600 mg). The solution was passed through
a nitrocellulose filter (0.45 µm). Product 11b was purified over a column
of Biogel P-2 (fine, column dimensions 2.5 cm i.d. × 70 cm L) eluting
with H2O (20 mL/h). Fractions containing the desired product were
identified by TLC (SiO2, 50/25/25 n-BuOH/HOAc/H2O, Rf ) 0.25)
with ninhydrin staining and were followed by fractions containing allo-
threonine (Rf ) 0.30). Fractions containing 11b were pooled and
concentrated. The product (ca. 90% purity) was dissolved in H2O (4
mL) and eluted a second time over the Biogel column, affording a
pure product. Product 11a was obtained as a white solid (205 mg, 60%).
1H NMR (D2O, HCl salt): δ 4.49 (d, J ) 10.0 Hz, 1 H), 4.13 (d, J )
4.3 Hz, 1 H), 3.72 (dd, J ) 1.2, 12.3 Hz, 1 H), 3.65 (m, 1 H), 3.53 (m,
1 H), 3.32-3.24 (m, 3 H), 3.10 (dd, J ) 8.7, 10.0 Hz, 1 H), 1.32 (d,
J ) 7.4 Hz, 3 H). 13C NMR (D2O, HCl salt): δ 169.8, 85.4, 79,6,
77.1, 72.5, 69.2, 60.7, 58.0, 41.0, 18.9. HRFABMS: calcd for (M +
H)+, 298.0960; found, 298.0962.
1
(430 mg, 90%). H NMR (D2O, HCl salt): δ 4.40 (d, J ) 9.9 Hz, 1
H), 4.23 (dd, J ) 4.2, 7.6 Hz, 1 H), 3.70 (dd, J ) 2.3, 12.5 Hz, 1 H),
3.52 (dd, J ) 5.6, 12.7 Hz, 1 H), 3.36-3.30 (m, 3 H), 3.28 (dd, J )
4.2, 15.6 Hz, 1 H), 3.21 (dd, J ) 9.2, 9.7 Hz, 1 H), 3.07 (dd, J ) 7.6,
15.7 Hz, 1 H). 13C NMR (D2O, HCl salt): δ 170.2, 85.3, 80.3, 77.4,
72.2, 68.9, 61.4, 53.6, 30.2. HRFABMS: calcd for (M + H)+, 284.0804;
found, 284.0805.
S-2-Acetamido-2-deoxy-â-D-glucopyranosyl-L-cysteine (12a). The
sodium salt of 1-thio-N-acetyl-â-D-glucosamine (9) (420 mg, 1.62
mmol, 1.0 equiv) was dissolved in H2O (8 mL). In a second flask,
NaHCO3 (340 mg, 4.1 mmol, 2.5 equiv) was added to 5a (270 mg,
1.62 mmol, 1.0 equiv). The reaction was initiated by addition of the
sodium thiolate solution to the flask containing 5a, thus affording the
following concentrations of reactants at the onset of the reaction: 5a,
0.2 M; 9, 0.2 M; NaHCO3, 0.5 M. The reaction was stirred at room
temperature for 4 h and then concentrated by rotary evaporation under
reduced pressure. To hydrolyze the sulfamidate, the crude material was
dissolved in HCl (5 M, 20 mL). The hydrolysis reaction was incubated
at 37 °C for 24 h and then concentrated by rotary evaporation under
reduced pressure. The product was dissolved in H2O (10 mL), and the
pH was brought to neutral by the addition of NaHCO3 (ca. 600 mg).
The solution was passed through a nitrocellulose filter (0.45 µm).
Product 12a was purified over a column of Biogel P-2 (fine, column
dimensions 2.5 cm i.d. × 70 cm L) eluting with H2O (20 mL/h).
Fractions containing the desired product were identified by TLC (SiO2,
50/25/25 n-BuOH/HOAc/H2O, Rf ) 0.25) with ninhydrin staining.
S-2-Acetamido-2-deoxy-â-D-glucopyranosyl-â-deoxy-â-thio-L-
threonine (12b). The cesium salt of 1-thio-N-acetyl-â-D-glucosamine
(9) (740 mg, 2.0 mmol, 2.0 equiv) was dissolved in H2O (2 mL). In a
second flask, CsHCO3 (580 mg, 3.0 mmol, 3.0 equiv) was added to 5b
(180 mg, 1.0 mmol, 1.0 equiv). The reaction was initiated by addition
of the cesium thiolate solution to the flask containing 5b, thus affording
the following concentrations of reactants at the onset of the reaction:
5b, 0.5 M; 9, 1.0 M; CsHCO3, 1.5 M. The reaction was incubated at
37 °C for 20 h, then concentrated by rotary evaporation under reduced
pressure. To hydrolyze the sulfamidate, the crude material was dissolved
in HCl (5 M, 10 mL). The hydrolysis reaction was incubated at 37 °C
for 24 h, then concentrated by rotary evaporation under reduced
pressure. The product was dissolved in H2O (10 mL), and the pH was
brought to neutral by the addition of NaHCO3 (ca. 600 mg). The
solution was passed through a nitrocellulose filter (0.45 µm). Product
12b was purified over a column of Biogel P-2 (fine, column dimensions
2.5 cm i.d. × 70 cm L) eluting with H2O (20 mL/h). Fractions
containing the desired product were identified by TLC (SiO2, 50/25/
25 n-BuOH/HOAc/H2O, Rf ) 0.25) with ninhydrin staining and were
followed by fractions containing allo-threonine (Rf ) 0.30). Fractions
1
Product 12a was obtained as a white solid (470 mg, 90%). H NMR
(D2O, HCl salt): δ 4.42 (d, J ) 10.4 Hz, 1 H), 4.14 (dd, J ) 4.3, 7.4
Hz, 1 H), 3.67 (dd, J ) 2.2, 12.4 Hz, 1 H), 3.58 (t, J ) 10.2 Hz, 1 H),
3.49 (dd, J ) 5.2, 12.4 Hz, 1 H), 3.35 (t, J ) 9.2 Hz, 1 H), 3.30-3.24
(m, 2 H), 3.21 (dd, J ) 4.3, 15.6 Hz, 1 H), 2.95 (dd, J ) 7.4, 15.6 Hz,
1 H), 1.79 (s, 3 H). 13C NMR (D2O, HCl salt): δ 174.8, 169.9, 83.9,
80.0, 74.8, 69.6, 60.8, 54.2, 53.1, 30.0, 22.3. HRFABMS: calcd for
(M + H)+, 325.1069; found, 325.1057.
S-r-D-Glucopyranosyl-L-cysteine (13a). The sodium salt of 1-thio-
R-D-glucose (10) (205 mg, 0.94 mmol, 1.0 equiv) was dissolved in
H2O (4.7 mL). In a second flask, NaHCO3 (200 mg, 2.4 mmol, 2.5
equiv) was added to 5a (157 mg, 0.94 mmol, 1.0 equiv). The reaction
was initiated by addition of the sodium thiolate solution to the flask
containing 5a, thus affording the following concentrations of reactants
at the onset of the reaction: 5a, 0.2 M; 10, 0.2 M; NaHCO3, 0.5 M.
The reaction was stirred at room temperature for 4 h and then
concentrated by rotary evaporation under reduced pressure. To hydro-
9
J. AM. CHEM. SOC. VOL. 124, NO. 11, 2002 2541

A R T I C L E S
Cohen and Halcomb
containing 12b were pooled and concentrated. The product (ca. 90%
purity) was dissolved in H2O (4 mL) and eluted a second time over
the Biogel column, affording a pure product. Product 12b was obtained
as a white solid (200 mg, 60%). 1H NMR (D2O, HCl salt): δ 4.41 (d,
J ) 10.5 Hz, 1 H), 3.92 (d, J ) 4.1 Hz, 1 H), 3.53 (dd, J ) 2.0, 12.6
Hz, 1 H), 3.41 (m, 1 H), 3.35 (dd, J ) 4.7, 12.4 Hz, 1 H), 3.33 (t, J )
10.1 Hz, 1 H), 3.20 (t, J ) 9.2 Hz, 1 H), 3.10 (m, 2 H), 1.66 (s, 3 H),
1.07 (d, J ) 7.4 Hz, 3 H). 13C NMR (D2O, HCl salt): δ 174.7, 169.6,
85.0, 79.5, 74.8, 69.5, 60.6, 57.8, 54.8, 41.0, 22.3, 18.9. HRFABMS:
calcd for (M + H)+, 339.1226; found, 339.1220.
clear, colorless oil (1.4 g, 90%). TLC (SiO2, 1/1 EtOAc/hexane): Rf
) 0.45. 1H NMR (C6D6): δ 7.25-7.00 (m, 7 H), 6.76 (d, J ) 8.6 Hz,
2 H), 5.02 (d, J ) 12.3 Hz, 1 H), 4.94 (d, J ) 12.3 Hz, 1 H), 4.74 (m,
1 H), 4.30 (dd, J ) 3.8, 9.0 Hz, 1 H), 4.28 (d, J ) 13.9 Hz, 1 H), 4.00
(t, J ) 8.6 Hz, 1 H), 3.88 (d, J ) 13.9 Hz, 1 H), 3.75 (dd, J ) 3.7, 8.1
Hz, 1 H), 3.32 (s, 3 H), 1.70-1.58 (m, 2 H), 1.48 (m, 1 H), 0.81 (d,
J ) 6.3 Hz, 3 H), 0.78 (d, J ) 6.3 Hz, 3 H). 13C NMR (C6D6): δ
172.5, 168.3, 160.8, 136.3, 131.7, 129.2, 128.9, 115.1, 69.2, 67.5, 62.1,
55.2, 53.8, 51.7, 41.6, 25.2, 23.2, 22.0. IR (cm-1): 3562, 2958, 1747,
1514. HRFABMS: calcd for (M + H)+, 491.1852; found, 491.1854.
Anal. calcd for C24H30N2O7S: C, 58.76; H, 6.16; N, 5.71; S, 6.54.
Found: C, 58.92; H, 6.36; N, 5.64; S, 6.86.
S-r-D-Glucopyranosyl-â-deoxy-â-thio-L-threonine (13b). The ce-
sium salt of 1-thio-R-D-glucose (10) (655 mg, 2.0 mmol, 2.0 equiv)
was dissolved in H2O (2 mL). In a second flask, CsHCO3 (580 mg,
3.0 mmol, 3.0 equiv) was added to 5b (180 mg, 1.0 mmol, 1.0 equiv).
The reaction was initiated by addition of the cesium thiolate solution
to the flask containing 5b, thus affording the following concentrations
of reactants at the onset of the reaction: 5b, 0.5 M; 10, 1 M; CsHCO3,
1.5 M. The reaction was incubated at 37 °C for 20 h and then
concentrated by rotary evaporation under reduced pressure. To hydro-
lyze the sulfamidate, the crude material was dissolved in HCl (5 M,
10 mL). The hydrolysis reaction was incubated at 37 °C for 12 h and
then concentrated by rotary evaporation under reduced pressure. The
product was dissolved in H2O (10 mL), and the pH was brought to
neutral by the addition of NaHCO3 (ca. 600 mg). The solution was
passed through a nitrocellulose filter (0.45 µm). Product 13b was
purified over a column of Biogel P-2 (fine, column dimensions 2.5 cm
i.d. × 70 cm L) eluting with H2O (20 mL/h). Fractions containing the
desired product were identified by TLC (SiO2, 50/25/25 n-BuOH/
HOAc/H2O, Rf ) 0.25) with ninhydrin staining and were followed by
fractions containing allo-threonine (Rf ) 0.30). Fractions containing
13b were pooled and concentrated. The product (ca. 90% purity) was
dissolved in H2O (4 mL) and eluted a second time over the Biogel
column, affording a pure product. Product 13b was obtained as a white
(4S)-2,2-Dioxo-1,2,3-oxathiazolidinone-4-carboxyl-leucine Benzyl
Ester (16). Dipeptide 15 (1.10 g, 2.24 mmol, 1.0 equiv) was dissolved
in CH3CN (20 mL). H2O (2 mL) was added with stirring, followed by
(NH4)2Ce(NO3)6 (3.7 g, 6.7 mmol, 3.0 equiv). The reaction was stirred
at room temperature for ca. 30 min, after which TLC (SiO2, 1/1 hexane/
EtOAc) showed complete conversion to a more polar product (Rf )
0.25). The reaction solution was partitioned between NaHCO3 and CH2-
Cl2. The aqueous layer was extracted with CH2Cl2, and the combined
organic extracts were dried (Na2SO4), filtered, and concentrated. The
product was purified by chromatography (silica gel, 50 mL), eluting
with 8/2 hexane/EtOAc to 1/1. Dipeptide 16 was obtained as a clear,
colorless oil (750 mg, 90%). 1H NMR (C6D6): δ 7.19-7.03 (m, 6 H),
5.05 (br d, J ) 7.9 Hz, 1 H), 5.03 (d, J ) 12.2 Hz, 1 H), 4.96 (d, J )
12.2 Hz, 1 H), 4.83 (m, 1 H), 4.42 (dd, J ) 4.0, 8.9 Hz, 1 H), 4.06 (t,
J ) 8.5 Hz, 1 H), 3.76 (m, 1 H), 1.73 (m, 1 H), 1.61 (m, 1 H), 1.50
(m, 1 H), 0.81 (d, J ) 6.4 Hz, 3 H), 0.78 (d, J ) 6.4 Hz, 3 H). 13C
NMR (C6D6): δ 173.0, 168.6, 136.2, 129.2, 129.0, 128.9, 71.6, 67.8,
57.5, 51.8, 41.3, 25.2, 23.2, 21.8. IR (cm-1): 3273, 2960, 1738, 1667.
HRFABMS: calcd for (M + H)+, 371.1277; found, 371.1278. Anal.
calcd for C16H22N2O6S: C, 51.88; H, 5.99; N, 7.56; S, 8.66. Found:
C, 51.58; H, 6.13; N, 7.85; S, 9.05.
1
solid (120 mg, 40%). H NMR (D2O, HCl salt): δ 5.40 (d, J ) 5.5
(4S)-2,2-Dioxo-1,2,3-oxathiazolidinone-4-carboxyl-leucine (17).
The protected dipeptide 16 (270 mg, 0.73 mmol, 1.0 equiv) was
dissolved in EtOAc (30 mL). Palladium-on-carbon (10 wt %, 75 mg,
0.07 mmol, 0.1 equiv) was added, and the suspension was stirred under
1 atm of hydrogen for ca. 30 min, after which TLC (SiO2, 1/1 hexane/
EtOAc) showed complete conversion to a product that did not migrate
by TLC. The suspension was filtered through Celite and concentrated.
The dipeptide 17 was used without further purification (205 mg, 100%).
1H NMR (DMSO-d6): δ 8.31 (d, J ) 6.9 Hz, 1 H), 8.24 (d, J ) 8.0
Hz, 1 H), 4.72 (t, J ) 7.5 Hz, 1 H), 4.55-4.48 (m, 2 H), 4.25 (m, 1
H), 1.68-1.58 (m, 2 H), 1.51 (m, 1 H), 0.87 (d, J ) 6.4 Hz, 3 H), 0.81
(d, J ) 6.4 Hz, 3 H). 13C NMR (DMSO-d6): δ 173.4, 167.7, 71.0,
56.0, 50.5, 39.6, 24.1, 22.9, 21.2. HRFABMS: calcd for (M + H)+,
281.0807; found, 281.0803.
Hz, 1 H), 4.25 (d, J ) 4.0 Hz, 1 H), 3.91 (m, 1 H), 3.78 (dd, J ) 2.2,
12.5 Hz, 1 H), 3.73 (dd, J ) 5.7, 10.0 Hz, 1 H), 3.66 (m, 2 H), 3.44
(t, J ) 9.6 Hz, 1 H), 3.30 (t, J ) 9.5 Hz, 1 H), 1.40 (d, J ) 7.3 Hz,
3 H). 13C NMR (D2O, HCl salt): δ 170.1, 85.1, 73.5, 73.0, 71.0, 69.7,
60.6, 58.1, 39.1, 19.2. HRFABMS: calcd for (M + H)+, 298.0960;
found, 298.0969.
(4S)-N-(p-Methoxybenzyl)-2,2-dioxo-1,2,3-oxathiazolidinone-4-
carboxylic Acid (14). The protected sulfamidate 3a (3.0 g, 8.0 mmol,
1.0 equiv) was dissolved in EtOAc (100 mL). Palladium-on-carbon
(10 wt %, 840 mg, 0.8 mmol, 0.1 equiv) was added, and the suspension
was stirred under 1 atm of hydrogen for ca. 3 h, after which TLC (SiO2,
1/1 hexane/EtOAc) showed complete conversion to a product that did
not migrate by TLC. The suspension was filtered through Celite and
concentrated. The sulfamidate 14 was used without further purification
S-â-D-Glucopyranosyl-L-cysteinyl-leucine (18). The sodium salt of
1-thio-â-D-glucose (8) (205 mg, 0.95 mmol, 1.1 equiv) was dissolved
in H2O (4.8 mL). In a second flask, NaHCO3 (180 mg, 2.15 mmol, 2.5
equiv) was added to 17 (240 mg, 0.86 mmol, 1.0 equiv). The reaction
was initiated by addition of the sodium thiolate solution to the flask
containing 17, thus affording the following concentrations of reactants
at the onset of the reaction: 17, 0.18 M; 8, 0.20 M; NaHCO3, 0.45 M.
The reaction was stirred at room temperature for 6 h and then
concentrated by rotary evaporation under reduced pressure. To hydro-
lyze the sulfamidate, the crude material was dissolved in HCl (5 M,
15 mL). The hydrolysis reaction was incubated at 37 °C for 40 h and
then concentrated by rotary evaporation under reduced pressure. The
product was dissolved in H2O (10 mL), and the pH was brought to
neutral by the addition of NaHCO3 (ca. 400 mg). The solution was
passed through a nitrocellulose filter (0.45 µm). Product 18 was purified
first over a column of Biogel P-2 (fine, column dimensions 2.5 cm i.d.
× 70 cm L) eluting with H2O (20 mL/h). Fractions containing the
desired product were identified by TLC (SiO2, 50/25/25 n-BuOH/
HOAc/H2O, Rf ) 0.45) with ninhydrin staining. The fractions were
1
(2.3 g, 100%). H NMR (DMSO-d6): δ 7.31 (d, J ) 8.7 Hz, 2 H),
6.92 (d, J ) 8.7 Hz, 2 H), 4.75 (dd, J ) 7.7, 9.1 Hz, 1 H), 4.67 (dd,
J ) 4.5, 9.1 Hz, 1 H), 4.39 (d, J ) 14.3 Hz, 1 H), 4.35 (dd, J ) 4.4,
7.7 Hz, 1 H), 4.30 (d, J ) 14.3 Hz, 1 H), 3.74 (s, 3 H). 13C NMR
(DMSO-d6): δ 169.7, 159.2, 130.4, 126.6, 113.9, 68.3, 59.2, 55.1, 49.9.
HRFABMS: calcd for (M)+, 287.0464; found, 287.0463. Anal. calcd
for C11H13NO6S: C, 45.99; H, 4.56; N, 4.88; S, 11.16. Found: C, 45.88;
H, 4.83; N, 5.05; S, 11.38.
(4S)-N-(p-Methoxybenzyl)-2,2-Dioxo-1,2,3-oxathiazolidinone-4-
carboxyl-leucine benzyl ester (15). Sulfamidate 14 (1.02 g, 3.55 mmol,
1.0 equiv) and PyBOP (1.85 g, 3.55 mmol, 1.0 equiv) were dissolved
in DMF (7 mL). Leucine benzyl ester (700 mg, 3.20 mmol, 0.9 equiv)
was added, and the reaction was initiated by the addition of N-
methylmorpholine (0.97 mL, 8.9 mmol, 2.5 equiv). The reaction was
stirred at room temperature for 5 min, and the DMF was then removed
by rotary evaporation under reduced pressure at 30 °C. The product
was immediately purified by chromatography (silica gel, 200 mL),
eluting with 8/2 hexane/EtOAc to 6/4. Dipeptide 15 was obtained as a
9
2542 J. AM. CHEM. SOC. VOL. 124, NO. 11, 2002

Sulfamidates for Preparation of Glycoconjugates
A R T I C L E S
pooled, concentrated, and the product (ca. 95% purity) was dissolved
in aqueous NH4HCO3 (50 mM, 5 mL). Final purification was achieved
with reverse-phase HPLC: aliquots (0.5 mL) of the product solution
were eluted through a Beckman Ultrasphere reverse-phase column (10
mm i.d. × 250 mm L, particle size 5 µm), eluting at 2.0 mL/min with
92/8 aqueous NH4HCO3 (50 mM)/CH3OH. Peaks were detected by UV
absorbance at 230 nm. Product 18 eluted with a retention time of
approximately 15 min. Product 18 was obtained as a white solid (270
room temperature for 18 h. The resin was washed first with H2O,
followed by 1/1 dioxane/H2O, and finally dioxane. (6) Removal of
N-sulfate: The resin was washed with dry CH2Cl2. A solution (10 mL)
of Et2OBF3 (1 M, 10 mmol) and n-butane thiol (1 M, 10 mmol) in
CH2Cl2 was prepared and cannulated to the resin. The large excess of
Lewis acid was necessary due to the Lewis basicity of the PEG. The
suspension was shaken at room temperature for 20 h. The resin was
washed with CH2Cl2 followed by 1/1 dioxane/H2O, and finally dioxane.
(7) Peptide coupling with Fmoc-threonine: The resin was swelled in
DMF. Fmoc-threonine (510 mg, 1.5 mmol, 5 equiv) and PyBOP (730
mg, 1.5 mmol, 5 equiv) were added, followed by DMF (5 mL). The
suspension was shaken briefly to dissolve the reagents. The reaction
was initiated by addition of N-methylmorpholine (0.50 mL, 4.5 mmol,
15 equiv). The suspension was shaken at room temperature for 30 min,
and then the resin was washed with DMF. (8) Fmoc removal: The
resin was shaken in piperidine/DMF (1/4 v/v, 15 mL) at room
temperature for 20 min, and the resin was washed with DMF. (9)
Removal from support: The resin was washed with 1/1 dioxane/H2O.
The resin was shaken in a solution of NaOH [0.2 M in 1/1 dioxane/
H2O (7.5 mL, 1.5 mmol, 5 equiv)] at room temperature for 30 min.
The filtrate was collected in a flask containing HCl (5 M, 3 mL). The
solution was concentrated by rotary evaporation under reduced pressure.
The product was dissolved in H2O (7 mL), and the pH was brought to
neutral by the addition of NaHCO3 (ca. 300 mg). The solution was
passed through a nitrocellulose filter (0.45 µm). Product 22 was purified
over a column of Biogel P-2 (fine, column dimensions 2.5 cm i.d. ×
70 cm L) eluting with H2O (20 mL/h). Fractions containing the desired
product were identified by TLC (SiO2, 50/25/25 n-BuOH/HOAc/H2O,
Rf ) 0.35) with ninhydrin staining. Product 22 was obtained as a white
1
mg, 80%). H NMR (D2O, HCl salt): δ 4.76 (d, J ) 9.8 Hz, 1 H),
4.49 (m, 1 H), 4.43 (dd, J ) 4.9, 8.3 Hz, 1 H), 4.01 (dd, J ) 2.3, 12.4
Hz, 1 H), 3.80 (dd, J ) 6.0, 12.4 Hz, 1 H), 3.66 (m, 1 H), 3.62 (t, J )
9.0 Hz, 1 H), 3.50-3.56 (m, 3 H), 3.24 (dd, J ) 8.3, 15.3 Hz, 1 H),
1.84-1.72 (m, 3 H), 1.01 (d, J ) 6.0 Hz, 3 H), 0.97 (d, J ) 6.0 Hz,
3 H). 13C NMR (D2O, HCl salt): δ 175.9, 168.5, 85.1, 80.2, 77.3, 72.2,
69.9, 61.3, 53.5, 52.1, 39.4, 31.3, 24.8, 22.7, 21.3. HRFABMS: calcd
for (M + H)+, 397.1645; found, 397.1640.
Solid-Phase Synthesis of Threoninyl-S-â-D-glucopyranosyl-L-
cysteinyl-leucine (22). The synthesis was performed in a 25-mL manual
peptide synthesis vessel. NovaSyn TG-hydroxy resin (1.0 g, 0.3 mmol/
g, purchased from NovaBiochem) was swelled in CH2Cl2 (20 mL)
overnight. (1) Esterification of the terminal hydroxyl: Fmoc-leucine
(530 mg, 1.5 mmol, 5 equiv) was suspended in CH2Cl2 (5 mL).
N-Methylimidazole (90 µL, 1.13 mmol, 3.75 equiv) was added to afford
a clear solution upon stirring. 2,4,6-mesitylene-sulfonyl-3-nitro-1,2,4-
triazolide (MSNT)15 (445 mg, 1.5 mmol, 5 equiv) was then added,
affording the following concentrations: Fmoc-leucine, 0.3 M; MSNT,
0.3 M. The solution was cannulated to the resin. The suspension was
shaken at room temperature for 2 h, and then the resin was washed
with CH2Cl2 followed by DMF. (2) Fmoc removal: The resin was
shaken in piperidine/DMF (1/4 v/v, 15 mL) at room temperature for
20 min and was washed with DMF. (3) Peptide coupling with 14:
Amino acid 14 (430 mg, 1.5 mmol, 5 equiv) and PyBOP (730 mg, 1.5
mmol, 5 equiv) were dissolved in DMF (5 mL),, affording the following
concentrations: 14, 0.3 M; PyBOP, 0.3 M. The solution was cannulated
to the resin. The reaction was initiated by addition of N-methylmor-
pholine (0.42 mL, 3.8 mmol, 12.5 equiv). The suspension was shaken
at room temperature for 30 min, and then the resin was washed with
DMF. (4) PMB-removal: The resin was swelled for 10 min in CH3-
CN/H2O (20 mL, 9/1 v/v). The resin was shaken in a solution of (NH4)2-
Ce(NO3)6 [0.5 M in 9/1 CH3CN/H2O (10 mL, 5 mmol, 16 equiv)] at
room temperature for 1 h. The resin was washed thoroughly with 9/1
CH3CN/H2O follwed by washing with 1/1 dioxane/H2O. (5) Addition
of 8: 2,3,4,6-Tetra-O-acetyl-1-S-acetyl-1-thio-â-D-glucopyranose (1015
mg, 2.5 mmol, 8 equiv) was dissolved in CH3OH (30 mL). Cs2CO3
(1.6 g, 5 mmol, 16 equiv) was added, and the solution was stirred at
room temperature for 18 h. The solvent was removed by rotary
evaporation, and the product was dissolved in H2O (2.5 mL). Solid
CO2 was added with stirring until the pH of the solution had lowered
to pH 8. Dioxane (2.5 mL) was added, affording [8] at 0.5 M. The
solution was transferred to the resin, and the suspension was shaken at
1
solid (70 mg). H NMR (D2O): δ 4.61 (dd, J ) 5.8, 8.6 Hz, 1 H),
4.53 (d, J ) 9.9 Hz, 1 H), 4.17 (m, 1 H), 3.92 (t, J ) 6.0 Hz, 1 H),
3.88 (dd, J ) 2.1, 10.4 Hz, 1 H), 3.68 (dd, J ) 6.0, 12.5 Hz, 1 H),
3.48-3.25 (m, 6 H), 2.94 (dd, J ) 8.6, 14.2 Hz, 1 H), 1.55 (m, 3 H),
1.15 (d, J ) 6.5 Hz, 3 H), 0.87 (d, J ) 6.2 Hz, 3 H), 0.82 (d, J ) 6.2
Hz, 3 H). HRFABMS: calcd for (M + H)+, 498.2121; found, 498.2137.
Acknowledgment. This research was supported by the NIH
(GM55852). R.L.H. also thanks the National Science Foundation
(Career Award), the Camille and Henry Dreyfus Foundation
(Camille Dreyfus Teacher-Scholar Award), Pfizer (Junior
Faculty Award), and Novartis (Young Investigator Award) for
support. This work was greatly facilitated by a 500 MHz NMR
spectrometer that was purchased partly with funds from an NSF
Shared Instrumentation Grant (CHE-9523034).
1
Supporting Information Available: Copies of H and 13C
NMR spectra for all products (PDF). This material is available
free of charge via the Internet at http://pubs.acs.org.
JA011932L
9
J. AM. CHEM. SOC. VOL. 124, NO. 11, 2002 2543