Oligosaccharide-Peptide Ligation of Glycosyl Thiolates with Dehydropeptides: Synthesis of S-Linked Mucin-Related Glycopeptide Conjugates

Article abstract of DOI:10.1002/chem.200305290

A chemoselective strategy for oligosaccharide-peptide ligation is described in which α-thio analogues of mucin-related glycoconjugates can be readily accessed through site-selective conjugate addition of complex oligosaccharide thiolates to dehydroalanine

Full text of DOI:10.1002/chem.200305290

FULL PAPER
Oligosaccharide Peptide Ligation of Glycosyl Thiolates with
Dehydropeptides: Synthesis of S-Linked Mucin-Related
Glycopeptide Conjugates
Danica P. Galonic¬, Wilfred A. van der Donk,* and David Y. Gin*[a]
Abstract: A chemoselective strategy for oligosaccharide peptide ligation is de-
scribed in which a-thio analogues of mucin-related glycoconjugates can be readily
accessed through site-selective conjugate addition of complex oligosaccharide thio-
lates to dehydroalanine-containing peptides. The efficiency of the ligation is high-
lighted by the rapid convergent assembly of thio-isosteres of the four tumor-associ-
ated carbohydrate antigens, TN, T, STN, and 2,6-ST, as a pair of diastereoisomers at
the newly formed cysteine stereocenter. The process proceeds in high yield and
with complete retention of the a-anomeric configuration.
Keywords: carbohydrates ¥ chemo-
selective ligation ¥ dehydropeptide ¥
glycopeptides ¥ mucin
Introduction
Post-translational glycosylation introduces enormous struc-
tural diversity into glycoproteins. Inflammation, immune re-
sponse, as well as protein expression and folding are among
the many biological events in which the carbohydrate seg-
ments play essential roles. Given the high degree of hetero-
geneity in natural glycoproteins and the difficulties associat-
ed with the isolation of well-defined glycopeptides from nat-
ural sources, the development of efficient chemical methods
for the synthesis of carbohydrate peptide conjugates re-
mains a key challenge.[1] Protein glycosylation, with few ex-
ceptions, can be divided into two general classes–those in
which a GlcNAc residue is b-N-linked to the side chain of
Asn, and those in which a GalNAc residue is a-O-linked to
the side chain of Ser or Thr. The latter is characteristic of
mucins, which are O-glycosylated proteins comprising an im-
portant class of tumor-associated antigens that have received
considerable attention in cancer vaccine therapies.[2] Repre-
sentative mucin-associated carbohydrates are illustrated by:
TN (1), T (2), STN (3), and 2,6-ST (4) tumor-associated anti-
gens.
Traditional strategies for the preparation of oligosacchar-
ide peptide conjugates have involved the synthesis of fully
protected, pre-formed oligosaccharide/amino acid intermedi-
ates, followed by multistep elaboration of the amino acid
aglycone into the requisite peptide chain.[1,3] An alternate
approach involves chemoselective ligation in which site-spe-
cific attachment of the carbohydrate component occurs di-
rectly to a pre-formed peptide chain. Recent elegant advan-
ces concerning the construction of b-N-linked glycopeptides
have been realized in this regard;[1,4] however, ligation strat-
egies as applied to mucin-like glycopeptides are scarce given
the inherent difficulties associated with construction of the
a-O-GalNAc glycosidic bond,[5] especially with polypeptide
glycosyl acceptors. In this context, notable accomplishments
have been made in the ligation of a-glycosyl aminoxy
GalNAc carbohydrates with ketone-functionalized pepti-
des[6] to afford mucin-like conjugates incorporating an
oxime functionality[7] as the linker.
¬
[a] Prof. W. A. van der Donk, Prof. D. Y. Gin, D. P. Galonic
Department of Chemistry
University of Illinois, Urbana, IL 61801 (USA)
Fax : (+1)217-244-8024
E-mail: vddonk@scs.uiuc.edu
E-mail: gin@scs.uiuc.edu
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DOI: 10.1002/chem.200305290
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

FULL PAPER
We now report a new strategy for the ligation of a-
GalNAc-thiolate oligosaccharides (5) [Eq. (1)] with dehy-
droalanine (Dha)-containing peptides 6 by 1,4-conjugate ad-
dition, resulting in the efficient formation of the S-linked an-
alogues of mucin-related a-GalNAc glycoconjugates 7. This
one-pot oligosaccharide peptide ligation is site-selective for
dehydroalanine, maintains the a-anomeric stereochemical
integrity of the carbohydrate donor, and proceeds under
mild conditions, providing fully deprotected oligosaccharide
isosteres of the naturally occurring tumor-associated anti-
gens. Moreover, S-linked glycopeptides have been reported
to have enhanced chemical stability as well as increased re-
sistance to enzymatic hydrolysis relative to their O-linked
counterparts.[8]
nylselenocysteine residue, an ideal precursor to Dha resi-
dues in complex peptide synthesis.[10] Thus, chemoselective
selenide oxidation followed by elimination proceeds in near
quantitative yield to afford the Dha-containing peptide 9. A
similar short synthetic sequence was employed to prepare
the tripeptide Ac-Ser-Dha-Ala-NHMe (8!10).[11] Notably,
this synthetic approach to Dha peptides is compatible with
all naturally occurring amino acids, including those that are
prone to oxidation.[10]
However, a critical challenge in establishing this ligation
approach is the preparation and conjugate addition of
stereo-defined GalNAc-derived a-oligosaccharyl thiols,
which have not been synthesized heretofore. Thus, anomeric
sulfur analogues of all four of the tumor-associated carbohy-
drate antigen segments (1 4) were synthesized to probe the
feasibility, scope, and efficiency of this carbohydrate peptide
ligation. The C1-thio-analogue of the TN-antigen 1 is pre-
pared from 3,4,6-triacetoxy galactosyl thiazoline 11 [Eq.
(2)].[12] Acid-mediated hydrolysis of the thiazoline 11 under
Results and Discussion
Dehydroalanine residues within peptides serve as versatile
functionalities for chemoselective ligation given the paucity
of electrophilic reactive sites on native amino acid resi-
dues.[9] Two tripeptides incorporating a central Dha frag-
ment were prepared as Michael acceptors to establish the
feasibility of this ligation approach (Scheme 1). N-Fmoc-
Protected phenylselenocysteine 8 is coupled with N-methyl
glycine by using the benzotriazol-1-yl-oxytris(dimethylami-
no)phosphonium hexafluorophosphate (BOP) coupling
agent to generate the corresponding selectively protected di-
peptide in 61% yield. Subsequent Fmoc deprotection and
amino acylation with N-acetyl glycine provides the corre-
sponding selectively protected tripeptide with a central phe-
carefully controlled conditions yields the C1-galactosyl thio-
pyranose 12 with complete retention of a-stereochemistry.
Preparation of the thio analogue of the disaccharide T-
antigen
2
commences with Schmidt glycosylation[13]
(Scheme 2) of triisopropylsilylthiol with 2-azido glycosyl tri-
chloroacetimidate 13,[14] providing the a-S-silyl thioglycoside
14 in good yield (85%). Saponification of the acetate esters
followed by selective benzoylation of the primary C6-hy-
droxyl affords the thiogalactopyranoside 16. Glycosylation
of this diol acceptor proceeds chemoselectively at the equa-
torial C3 hydroxyl by using 2,3,4,6-tetra-O-benzoylgalacto-
Scheme 1. a) H-Gly-NHMe, BOP, DIPEA, CHCl3, 238C, 61%; b)
(CH2)5NH, CH2Cl2, 238C, 91%; c) Ac-Gly-OH, BOP, DIPEA, CHCl3,
238C, 81%; d) H2O2, MeCN, H2O, 23 8C, >99%; e) H-Ala-NHMe, BOP,
DIPEA, CHCl3, 238C, 72%; f) (CH2)5NH, CH2Cl2, 238C, 75%; g) Ac-
Ser(tBu)-OH, BOP, DIPEA, CHCl3, 238C, 97%; h) TFA, 08C; H2O2,
MeOH, CH2Cl2, 238C, 98%.
Scheme 2. a) TIPS-SH, TMSOTf, CH2Cl2, Et2O, ꢀ20!238C, 85%; b)
NaOMe, MeOH, 238C, 85%; c) BzCl, Et3N, CH2Cl2, ꢀ208C, 75%; d)
Ph2SO, Tf2O, CH2Cl2, ꢀ78!ꢀ208C, 68%; e) PhSeH, Et3N, pyr, 408C;
Ac2O, 23 8C, 76%.
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Oligosaccharide Peptide Ligation
5997 6006
pyranose (17) as the donor with our dehydrative glycosyla-
tion protocol (Ph2SO, Tf2O)[15] to afford the b-1,3-disacchar-
ide 18 (68%). It is worth noting that the dehydrative glyco-
sylation of 16 with 17 is performed in the absence of a triflic
acid scavenger, allowing for in situ productive rearrange-
ment of undesired orthoester adducts, byproducts which
often plague glycosylation with C2-acyl donors.[16] Exposure
of disaccharide 18 to benzeneselenol effects reduction of the
C2-azide accompanied by silyl deprotection of the anomeric
thiol. Subsequent treatment of the reaction mixture with
excess acetic anhydride provides the fully acylated glycosyl
thioacetate disaccharide 19 (76%), the T-antigen precursor
for carbohydrate peptide ligation.
Synthesis of the thio analogue of the STN-antigen oligo-
saccharide 3 features sialylation of S-triisopropylsilyl-2-
azido-2-deoxygalacto-1-thiopyranoside (15, Scheme 3) with
Scheme 4. a) (4-NO2-C6H4)(Ph)SO, Tf2O, 17, CH2Cl2, ꢀ78!ꢀ458C,
70%; b) [PdCl2(PPh3)2] (cat.), Bu3SnH, CH2Cl2, H2O, 23 8C, >99%; c)
20, TMSOTf (cat.), CH2Cl2, ꢀ788C, 66% (4.4:1, a:b); d) NaOH, MeOH,
H2O, 23 8C; Ac2O, pyr, DMAP, 238C, 51%; e) PPh3, THF, 238C, 91%; f)
TFA, MeOH, H2O, 0 8C, >99%.
galactopyranose (17) to afford the disaccharide 26 (70%).
Subsequent removal of the C6-O-allyloxycarbonyl group re-
veals the appropriate disaccharide nucleophile 27 (>99%)
for sialylation. Coupling of 27 with the sialyl phosphite
donor 20 in the presence of catalytic TMSOTf proceeds
with good a-selectivity (66%; 4.4:1, a:b) and is followed by
global deprotection and per-acetylation to afford the disac-
charide sialyl conjugate 28 (51%, 2 steps). A 1-S-to-2-N re-
ductive acyl-transfer protocol, similar to that employed in
the final steps to prepare 24 (vide supra), proceeds in 91%
yield (2 steps) to afford the acetylated 2,6-ST-antigen C1-
thiol 29, which can be used directly for carbohydrate pep-
tide ligation.
Site-selective ligations of the C1-S-carbohydrate donors
with the tripeptides 9 and 10 proceed efficiently through
1,4-addition into the dehydroalanine residue (Table 1). For
example, the C1-thio analogue of the TN-antigen monosac-
charide 12 serves as an effective nucleophile with both 9
and 10, providing excellent yields of the a-linked TN-antigen
isosteres 30 and 31 (79%, 1.4:1 dr; 92%, 1.1:1 dr; respec-
tively), thereby establishing the feasibility of stereoselective
a-GalNAc Michael ligation.[19] Importantly, the ligation pro-
cedure is equally compatible with oligosaccharides. Disac-
charides 19 and 24 undergo efficient coupling with 9 and 10
to provide the T-antigen thio-mucin linkages 32 (90%) and
33 (84%), as well as the STN-antigen counterparts 34 (84%)
and 35 (90%). More complex oligosaccharides are also ame-
nable to this ligation, exemplified by the coupling of the
mature 2,6-ST-S-trisaccharide 29 with peptides 9 and 10 to
afford the 2,6-ST-antigen isosteres 36 (88%) and 37 (75%).
It is worth noting that both O-acyl-protected glycosyl thiols
(12, 24, and 29) as well as glycosyl thioacetates (19) can be
employed as carbohydrate donors, since the mild ligation
conditions (MeOH, NaOMe, 238C) also promote concomi-
tant O- and S-deacylation to afford fully deprotected glyco-
peptide isosteres in a one-pot procedure. To our knowledge,
Scheme 3. a) TMSOTf, CH2Cl2, ꢀ458C, 82%, 6.5:1 a:b; b) NaOH,
MeOH, H2O, 23 8C; Ac2O, DMAP, pyr, 238C, 66%; c) PPh3, THF, 238C,
>99%; d) TFA, MeOH, H2O, 0 8C, >99%.
sialyl phosphite donor 20, incorporating the C1-N,N-dimeth-
yl glycolamide auxiliary to enhance a-selectivity.[17] The cou-
pling proceeds efficiently to form the sialylconjugate 21
(82%; 6.5:1, a:b), which is then subjected to global depro-
tection followed by per-acetylation to afford the glycosyl thi-
oacetate disaccharide 22 (66%, 2 steps). An anomeric S-to-
N reductive acyl-transfer sequence is then performed involv-
ing the treatment of 22 with PPh3 to promote 1-S-2-N-thia-
zoline formation (23) by means of an aza-Wittig-like con-
densation.[18] Subsequent TFA-mediated (TFA=trifluoro-
acetic acid) thiazoline ring opening affords the a-glycosyl
thiol 24, incorporating the naturally occurring C2-acetamido
group, in near quantitative yield over both steps.
The preparation of the C1-thio derivative of the 2,6-ST-
trisaccharide antigen 4 initially involves chemoselective de-
hydrative glycosylation of the C3-hydroxyl within S-triiso-
propylsilyl 6-O-allyloxycarbonyl-2-azido-2-deoxygalacto-1-
thiopyranoside (25, Scheme 4) with 2,3,4,6-tetra-O-benzoyl-
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

¬
D. Y. Gin, W. A. van der Donk, and D. P. Galonic
FULL PAPER
Table 1. Site-selective ligations of the C1-S-carbohydrate donors with the
tripeptides 9 and 10.
1,4-conjugate addition of complex oligosaccharide thiolates
to dehydropeptides. The rapid convergent preparation of
thio-isosteres of the four tumor-associated antigens, TN, T,
STN, and 2,6-ST, underscores the efficiencies of not only the
syntheses of GalNAc-derived a-oligosaccharyl thiols, but
also of the ligation event, a process that should facilitate the
construction of a host of glycopeptide conjugates for biologi-
cal evaluation.
Experimental Section
General: All reactions were performed in dry modified Schlenk (Kjel-
dahl shape) flasks fitted with a glass stopper under a positive pressure of
argon, unless otherwise noted. Air- and moisture-sensitive liquids were
transferred by means of a syringe. Organic solutions were concentrated
by rotary evaporation below 308C at approximately 25 Torr. Flash
column chromatography was performed employing 230 400 mesh silica
gel. Thin-layer chromatography was performed by using glass plates pre-
coated to a depth of 0.25 mm with 230 400 mesh silica gel impregnated
with a fluorescent indicator (254 nm). When necessary, solvents were de-
gassed by the freeze-pump thaw method (>3 cycles).
Dichloromethane, toluene, acetonitrile, and tetrahydrofuran were puri-
fied by passage through two packed columns of neutral alumina under an
argon atmosphere. Chloroform, pyridine, diisopropylethylamine, and tri-
ethylamine were distilled from calcium hydride at 760 Torr. Methanol
was distilled from magnesium oxide at 760 Torr. Millipore water was
used for reactions in an aqueous solvent or co-solvent.
Infrared (IR) spectra were obtained by using a Perkin Elmer Spectrum
BX spectrophotometer referenced to polystyrene standard. Data are pre-
1
sented as frequency of absorption (cmꢀ1). H and 13C NMR spectra were
recorded on Varian 400, Varian 500, Varian 750, and Varian Inova 500
NMR spectrometers; chemical shifts are expressed in ppm (d scale)
downfield from tetramethylsilane and are referenced to residual protium
in the NMR solvent (CHCl3: d=7.27 ppm). Data are presented as fol-
lows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet, m=
multiplet and/or multiple resonances), integration, and coupling constants
in Hertz (Hz).
Fmoc-phenylselenocysteinylglycine-N-methyl amide: N,N-diisopropyl-
ethylamine (2.1 mL, 11.9 mmol, 2.5 equiv) was added dropwise to a sus-
pension of amino acid 8 (2.4 g, 5.3 mmol, 1.1 equiv), glycine-N-methyl
amide hydrochloride (0.63 g, 5.0 mmol, 1.0 equiv), and BOP (2.33 g, 5.3
mmol, 1.1 equiv) in chloroform (66 mL) at 238C. The resulting solution
was stirred at this temperature for 10 h, diluted with dichloromethane
(1.4 L), and washed subsequently with 1m aqueous hydrochloric acid
(150 mL), saturated aqueous sodium bicarbonate (150 mL), and water
(150 mL). The organic layer was dried (sodium sulfate), filtered, and con-
centrated. The residue was purified by silica-gel flash chromatography
(5% methanol in dichloromethane) to afford the dipeptide (1.68 g, 61%)
this is the first report of oligosaccharide peptide ligation for
the preparation of thio analogues of tumor-associated carbo-
hydrate antigens,[20,21] a process that occurs not only in high
yields, but also with complete retention of the a-anomeric
configuration. Moreover, the establishment of this ligation
method suggests the prospect of its application with confor-
mationally constrained dehydropeptides to stereoselectively
access either d- or l-cysteine-linked mucin glycopeptide
mimics.
as
a white solid. M.p. 1708C; Rf =0.28 (6% methanol in dichloro-
methane); 1H NMR (500 MHz, CDCl3): d=7.77 (d, J=7.6 Hz, 2H),
7.56 7.27 (m, 11H), 6.61 (brs, 1H), 6.31 (brs, 1H), 5.55 (brd, J=5.2 Hz,
1H), 4.42 (d, J=6.8 Hz, 2H), 4.29 (m, 1H), 4.19 (t, J=6.6 Hz, 1H), 3.83
(brs, 2H), 3.29 (m, 2H), 2.78 ppm (d, J=4.8 Hz, 3H); 13C NMR (126
MHz, CDCl3): d=170.7, 164.7, 143.8, 143.7, 141.5, 133.4, 129.7, 128.1,
127.3, 125.2, 120.3, 67.5, 47.3, 43.4, 37.1, 37.0, 26.4 ppm; FTIR (neat film):
n˜ =3286, 2928, 2371, 1655, 1532, 1452, 1296, 1258, 1194, 984, 842, 741
cmꢀ1; HRMS (FAB)+ : m/z calcd for C27H27N3O4Se [M+H]+ : 538.1245;
found: 538.1246.
Phenylselenocysteinylglycine-N-methyl amide: A solution of piperidine
in dichloromethane (5%, 200 mL, 2.00 mmol, 40 equiv) was added to a
solution of Fmoc-phenylselenocystylglycine-N-methyl amide (100 mg,
0.19 mmol, 1 equiv) in dichloromethane (3.8 mL) at 238C. The resulting
mixture was stirred at this temperature for 1 h, and then concentrated.
The residue was purified by silica-gel flash chromatography (5% to 10%
methanol in dichloromethane) to afford the amine (53 mg, 91%) as col-
orless oil. Rf =0.22 (7% methanol in dichloromethane); 1H NMR (500
MHz, CD3OD): d=7.54 7.52 (m, 2H), 7.28 7.22 (m, 3H), 3.74 (A of
Conclusion
In summary, a novel strategy for oligosaccharide peptide li-
gation is described in which a-thio analogues of mucin-relat-
ed structures can be readily accessed through site-selective
6000
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Chem. Eur. J. 2003, 9, 5997 6006

Oligosaccharide Peptide Ligation
5997 6006
AB, J=16.8 Hz, 1H), 3.68 (B of AB, J=17.0 Hz, 1H), 3.50 (X of ABX,
a solution of Fmoc-phenylselenocysteinylalanine-N-methyl amide (0.84 g,
1.52 mmol, 1 equiv) in dichloromethane (31 mL) at 238C. The resulting
solution was stirred at this temperature for 1.5 h. The reaction mixture
was concentrated, and the residue was purified by silica-gel flash chroma-
tography (7% methanol in dichloromethane) to give the amine (375 mg,
75%) as a white solid. M.p. 1308C; Rf =0.34 (10% methanol in dichloro-
methane); 1H NMR (400 MHz, CDCl3): d=7.76 (d, J=7.1 Hz, 1H),
7.55 7.53 (m, 2H), 7.30 7.26 (m, 3H), 6.39 (brs, 1H), 4.34 (pent, J=7.0
Hz, 1H), 3.50 3.42 (m, 2H), 3.03 (dd, J=12.4, 8.3 Hz, 1H), 2.77 (d, J=
4.8 Hz, 3H), 1.71 (s, 2H), 1.36 ppm (d, J=7.08 Hz, 3H); 13C NMR (126
MHz, CDCl3): d=173.7, 172.6, 133.3, 129.5, 128.9, 127.7, 54.6, 48.8, 33.8,
26.4, 17.8 ppm; FTIR (neat film): n˜ =3305, 1648, 1515, 1478, 1438, 691
cmꢀ1; HRMS (FAB)+ : m/z calcd for C13H20N3O2Se [M+H]+ : 330.0720;
found: 330.0721.
J
BX =7.1, JAX =5.7 Hz, 1H), 3.27 (A of ABX, JAB =12.6, JAX =5.7 Hz,
1H), 3.09 (B of ABX, JAB =12.6, JBX =7.1 Hz, 1H), 2.69 ppm (s, 3H);
13C NMR (126 MHz, CD3OD): d=176.4, 172.1, 134.2, 130.8, 130.5, 128.5,
56.2, 43.5, 33.8, 26.4 ppm; FTIR (neat film): n˜ =3304, 1656, 1530, 1478,
1411, 738, 691 cmꢀ1
; : m/z calcd for C12H18N3O2Se
HRMS (FAB)+
[M+H]+ : 314.0578; found: 314.0572.
Acetylglycylphenylselenocysteinylglycine-N-methyl amide: N,N-diisopro-
pylethylamine (435 mL, 2.5 mmol, 2.5 equiv) was added dropwise to a sol-
ution of phenylselenocysteinylglycine-N-methyl amide (314 mg, 1.0
mmol, 1.0 equiv), acetyl glycine (131 mg, 1.1 mmol, 1.1 equiv), and BOP
(499 mg, 1.1 mmol, 1.1 equiv) in chloroform (26 mL) at 238C. The solu-
tion was stirred at this temperature for 4 h, diluted with 5% methanol in
dichloromethane (500 mL) and washed sequentially with 1m aqueous hy-
drochloric acid (40 mL), saturated aqueous sodium bicarbonate solution
(40 mL), and saturated aqueous sodium chloride solution (40 mL). The
combined aqueous washes were saturated with sodium sulfate and ex-
tracted with 5% methanol in dichloromethane (6î100 mL). The com-
bined organic layers were dried (sodium sulfate), filtered, and concentrat-
ed. The residue was purified by silica-gel flash chromatography (7%
methanol in dichloromethane), and re-purified by the same method (dry-
loaded, 12% methanol in dichloromethane), to afford tripeptide (334 mg,
81%) as a white solid. M.p. 2148C; Rf =0.17 (10% methanol in dichloro-
methane); 1H NMR (500 MHz, CD3OD): d=7.57 7.55 (m, 2H), 7.31
7.27 (m, 3H), 4.46 (X of ABX, J=8.6, 5.3 Hz, 1H), 3.85 (A of AB, J=
16.8 Hz, 1H), 3.79 (A of AB, J=16.8 Hz, 1H), 3.77 (B of AB, J=16.4
Hz, 1H), 3.69 (B of AB, J=16.6 Hz, 1H), 3.40 (A of ABX, JAB =13.0,
Acetylseryl-(O-tert-butyl)-phenylselenocysteinylalanine-N-methyl amide:
Diisopropylethylamine (269 mL, 1.55 mmol, 1.5 equiv) was added drop-
wise to a suspension of acetylserine-tert-butyl ether (236 mg, 1.1 mmol,
1.1 equiv), phenylselenocysteinylalanine-N-methyl amide (339 mg, 1.0
mmol, 1.0 equiv), and BOP (507 mg, 1.14 mmol, 1.1 equiv) in chloroform
(15 mL) at 238C. The resulting solution was stirred at this temperature
for 6 h. The reaction mixture was diluted with chloroform (800 mL) and
washed subsequently with 10% aqueous citric acid solution (80 mL), sa-
turated aqueous sodium bicarbonate solution (80 mL), and saturated
aqueous sodium chloride solution (80 mL). The organic layer was dried
(sodium sulfate), filtered, and concentrated. The residue was purified by
silica-gel flash chromatography (dry loaded, 5% to 7% methanol in di-
chloromethane), to give the tripeptide (513 mg, 97%) as a white solid.
M.p. 2628C; Rf =0.55 (14% methanol in dichloromethane); 1H NMR
(400 MHz, CD3OD): d=7.55 7.53 (m, 2H), 7.30 7.26 (m, 3H), 4.53 (X
of ABX, JBX =5.7, JAX =5.2 Hz, 1H), 4.33 (t, X of ABX, J=5.3 Hz, 1H),
4.21 (q, J=7.4 Hz, 1H), 3.65 (A of ABX, JAB =9.3, JAX =5.0 Hz, 1H),
3.59 (B of ABX, JAB =9.3, JBX =5.7 Hz, 1H), 3.36 (A of ABX, JAB =13.7,
J
AX =5.3 Hz, 1H), 3.18 (B of ABX, JAB =13.0, JAX =8.6 Hz, 1H), 2.72 (s,
3H), 2.01 ppm (s, 3H); 13C NMR (126 MHz, CD3OD): d=174.4, 173.2,
172.4, 172.1, 134.4, 130.6, 130.5, 128.7, 55.5, 43.9, 43.7, 29.3, 26.4, 22.5;
FTIR (neat film): n˜ =3278, 2426, 1625, 1528, 1437 ppm; HRMS (FAB)+
m/z calcd for C16H23N4O4Se [M+H]+ : 415.0885; found: 415.0884.
:
J
AX =5.2 Hz, 1H), 3.19 (B of ABX, JAB =13.7, JBX =5.7 Hz, 1H), 2.70 (s,
Tripeptide 9: Hydrogen peroxide (30%, 64 mL, 0.52 mmol, 2 equiv) was
added to a solution of acetylglycylphenylselenocysteinylglycine-N-methyl
amide (107 mg, 0.26 mmol, 1 equiv) in a mixture of water (3.1 mL) and
acetonitrile (3.1 mL) at 238C. The solution was stirred at this tempera-
ture for 7 h. Excess H2O2 was neutralized with dimethyl sulfide (100 mL).
The reaction was diluted with toluene (~15 mL), then concentrated, and
residue was purified by silica-gel flash chromatography (5% to 14%
methanol in dichloromethane) to give the dehydroalanine-containing tri-
peptide 9 (66 mg, >99%) as a white solid. M.p. 152 1538C; Rf =0.31
(14% methanol in dichloromethane); 1H NMR (500 MHz, CD3OD): d=
5.85 (s, 1H), 5.58 (s, 1H), 3.94 (s, 2H), 3.89 (s, 2H), 2.75 (s, 3H), 2.03
ppm (s, 3H); 13C NMR (126 MHz, CD3OD): d=174.3, 172.3, 171.0,
167.1, 137.3, 108.4, 44.3, 44.0, 26.4, 22.5 ppm; FTIR (neat film): n˜ =3291,
3H), 2.02 (s, 3H), 1.30 (d, J=7.27 Hz, 3H), 1.20 ppm (s, 9H); 13C NMR
(126 MHz, CD3OD): d=175.4, 174.2, 173.3, 172.3, 134.2, 130.9, 130.6,
128.7, 75.2, 62.7, 55.1, 51.0, 50.9, 28.1, 27.9, 27.9, 26.6, 22.8, 16.7 ppm;
FTIR (neat film): n˜ =3287, 2433, 1626, 1451 cmꢀ1; HRMS (FAB)+ : m/z
calcd for C22H35N4O5Se [M+H]+ : 515.1773; found: 515.1773.
Tripeptide 10: Trifluoroacetic acid (1.7 mL) was added to acetylseryl-(O-
tert-butyl)phenylselenocysteinylalanine-N-methyl amide (89 mg, 0.17
mmol) at 08C, and the resulting solution was stirred at this temperature
for 11.5 h. The reaction mixture was concentrated to give acetylserylphe-
nylselenocysteinylalanine-N-methyl amide. Hydrogen peroxide (30%, 47
mL, 0.34 mmol, 2 equiv) was added to a solution of the unpurified acetyl-
serylphenylselenocysteinylalanine-N-methyl amide (79 mg, 0.17 mmol, 1
equiv) in a mixture of methanol (4.7 mL) and dichloromethane (4.7 mL)
at 238C, and the resulting solution was stirred at this temperature for 1 h.
The reaction mixture was neutralized by the addition of dimethylsulfide
(100 mL), and concentrated. The residue was purified by silica-gel flash
chromatography to give tripeptide 10 (51 mg, 98%) as a white solid. M.p.
2388C; Rf =0.40 (20% methanol in dichloromethane); 1H NMR (500
MHz, CD3OD): d=5.85 (d, J=0.8 Hz, 1H), 5.69 (d, J=0.5 Hz, 1H), 4.44
(t, X of ABX, J=5.5 Hz, 1H), 4.36 (q, J=7.1 Hz, 1H), 3.87 (A of ABX,
2476, 1649, 1517, 1405 cmꢀ1
;
HRMS (FAB)+
:
m/z calcd for
C10H16N4NaO4 [M+Na]+ : 279.1069; found: 279.1070.
Fmoc-phenylselenocysteinylalanine-N-methyl amide: Diisopropylethyl-
amine (0.9 mL, 5.2 mmol, 2.5 equiv) was added dropwise to a suspension
of amino acid 8 (1.30 g, 2.3 mmol, 1.1 equiv), alanine-N-methyl amide hy-
drochloride (0.29 g, 2.1 mmol, 1.0 equiv), and BOP (1.02 g, 2.3 mmol, 1.1
equiv) in chloroform (38 mL) at 238C. The resulting mixture was stirred
at this temperature for 24 h. The reaction mixture was diluted with
chloroform (1.4 L) and washed subsequently with 1m aqueous hydrochlo-
ric acid solution (150 mL), saturated aqueous sodium bicarbonate solu-
tion (150 mL), and water (150 mL). The organic layer was dried (sodium
sulfate), filtered, and concentrated. The residue was purified by silica-gel
flash chromatography (5% methanol in dichloromethane) to give the di-
peptide (0.82 g, 72%) as a white solid. M.p. 2298C; Rf =0.36 (7% metha-
nol in dichloromethane); 1H NMR (500 MHz, CDCl3): d=7.78 (d, J=
7.5 Hz, 2H), 7.58 7.27 (m, 11H), 6.56 (d, J=7.6 Hz, 1H), 6.29 (brs, 1H),
5.58 (d, J=4.6 Hz, 1H), 4.45 4.33 (m, 4H), 4.20 (t, X of ABX, J=6.8 Hz,
1H), 3.30 (A of ABX, J AB =12.2, J AX =6.0 Hz, 1H), 3.24 (B of ABX, J
AB =12.2, J BX =12.2, 6.4 Hz, 1H), 2.79 (d, J=4.3 Hz, 3H), 1.36 ppm (d,
3H, J=7.0 Hz); 13C NMR (126 MHz, CDCl3): d=172.1, 170.0, 143.8,
141.5, 135.5, 133.2, 129.6, 128.1, 127.3, 125.2, 120.3, 67.5, 55.3, 49.3, 47.2,
29.5, 26.5, 18.2 ppm; FTIR (neat film): n˜ =3291, 1642, 1538, 1262, 735
cmꢀ1; HRMS (FAB)+ : m/z calcd for C28H30N3O4Se [M+H]+ : 552.1402;
found: 552.1400.
J
AB =11.0, JAX =5.4 Hz, 1H), 3.79 (B of ABX, JAB =11.0, JBX =5.6 Hz,
1H), 2.74 (s, 3H), 2.04 (s, 3H), 1.38 ppm (d, J=7.4 Hz, 3H); 13C NMR
(126 MHz, CD3OD): d=175.7, 174.0, 172.3, 166.3, 137.1, 109.9, 62.9, 57.6,
51.2, 26.5, 22.6, 17.9 ppm; FTIR (neat film): n˜ =3294, 1654, 1508 cmꢀ1
;
HRMS (FAB)+ : m/z calcd for C12H21N4O5 [M+H]+ : 301.1512; found:
301.1513.
Thiol 12: Water (62 mL, 5.02 mmol, 38.6 equiv) was added to a solution
of thiazoline 11 ( 30 mg, 0.13 mmol, 1.0 equiv) in a mixture of methanol
(730 mL) and trifluoroacetic acid (20 mL, 0.38 mmol, 3.0 equiv). The reac-
tion mixture was deoxygenated and stirred at 08C for 5 h. The reaction
mixture was concentrated to afford thiol 12 (31 mg, >99%) as a color-
1
less oil. Rf =0.18 (50% diethyl ether in dichloromethane); H NMR (500
MHz, CDCl3): d=6.01 (brd, J=8.2 Hz, 1H), 5.88 (t, J=5.9 Hz, 1H), 5.41
(d, J=2.3 Hz, 1H), 5.09 (dd, J=11.8, 3.2 Hz, 1H), 4.75 (ddd, J=13.4, 8.4,
5.1 Hz, 1H), 4.54 (t, J=6.4 Hz, 1H), 4.17 (dd, J=11.34, 6.4 Hz, 1H), 4.06
(dd, J=11.2, 6.6 Hz, 1H), 2.17 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H), 2.03 (s,
3H), 1.99 ppm (d, J=6.54 Hz, 1H); 13C NMR (126 MHz, CDCl3): d=
171.7, 171.5, 170.7, 170.5, 79.6, 68.0, 68.0, 67.2, 61.8, 48.8, 23.3, 21.0, 20.9,
Phenylselenocysteinylalanine-N-methyl amide: Piperidine (50% solution
in dichloromethane, 1.7 mL, 30.5 mmol, 20 equiv) was added dropwise to
6001
Chem. Eur. J. 2003, 9, 5997 6006
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

¬
D. Y. Gin, W. A. van der Donk, and D. P. Galonic
FULL PAPER
20.9 ppm; FTIR (neat film): n˜ =1748, 1661, 1372, 1233, 1086 cmꢀ1
;
fied by silica-gel flash chromatography (2.4% acetone in benzene), and
repurified by the same method (1.2% ethylacetate in dichloromethane)
to afford the disaccharide 18 (35 mg, 68%) as a white solid. M.p. 2178C;
Rf =0.25 (5% acetonitrile in benzene); 1H NMR (500 MHz, CDCl3): d=
8.10 (m, 10H), 7.00 (m, 15H), 6.40 (dd, J=10.6, 8.1 Hz, 1H), 6.08 (d, J=
3.4 Hz, 1H), 5.74 (dd, J=10.5, 3.6 Hz, 1H), 5.28 (d, J=4.5 Hz, 1H), 4.73
(m, 2H), 4.50 (dd, J=11.9, 8.2 Hz, 1H), 4.44 (d, J=8. 0 Hz, 1H), 4.43
(dd, J=11.3, 3.3 Hz, 1H), 4.20 (dd, J=11.6, 7.3 Hz, 1H), 4.09 (brs, 1H),
4.04 (dd, J=10.7, 2.8 Hz, 1H), 4.00 (dd, J=7.6, 4.4 Hz, 1H), 3.68 (dd,
J=6.1, 4.3 Hz, 1H), 2.84 (dd, J=2.8, 1.6 Hz, 1H), 0.99 ppm (m, 21H);
13C NMR (126 MHz, CDCl3): d=166.6, 166.2, 166.2, 166.1, 166.1, 133.9,
133.8, 133.6, 133.5, 133.4, 131.2, 130.6, 130.5, 130.5, 130.5, 130.4, 130.4,
130.4, 129.7, 129.6, 129.3, 129.2, 128.9, 128.6, 128.8, 128.7, 102.5, 81.5,
79.7, 72.7, 72.5, 70.2, 70.0, 69.2, 69.2, 65.2, 62.9, 60.6, 18.7, 18.6, 13.3 ppm;
HRMS (FAB)+ : m/z calcd for C14H22N1O8S [M+H]+ : 364.1066; found:
364.1065.
Monosaccharide 14: Trimethylsilyltrifluoromethane sulfonate (210 mL,
1.13 mmol, 0.1 equiv) was added to a solution of trichloroacetimidate 13
(5.37 g, 11.3 mmol, 1.0 equiv) and tri-iso-propylsilane thiol (7.5 mL, 33.9
mmol, 3.0 equiv) in a mixture of dichloromethane (40 mL) and diethyl
ether (40 mL) at ꢀ208C. The resulting mixture was stirred at this temper-
ature for 3 h, then at 238C for 2 h. The reaction mixture was neutralized
with N,N-diisopropylethylamine (0.5 mL), diluted with dichloromethane
(650 mL), and washed with saturated aqueous sodium chloride solution
(250 mL). The organic layer was dried (sodium sulfate), filtered, and con-
centrated. The residue was purified by silica-gel flash chromatography
(20% ethyl acetate in hexane) to afford S-TIPS monosaccharide 14 (4.84
g, 85%) as a yellowish oil. Rf =0.45 (40% ethyl acetate in hexane); 1H
NMR (500 MHz, CDCl3): d=5.60 (d, J=5.1 Hz, 1H), 5.46 (d, J=3.2, 1.2
Hz, 1H), 5.36 (dd, J=11.1, 3.2 Hz, 1H), 4.73 (td, J=6.5, 1.2 Hz, 1H),
4.12 (dd, J=11.1, 5.0 Hz, 1H), 4.11 (dd, J=11.4, 6.4 Hz, 1H), 4.05 (dd,
J=11.3, 6.7 Hz, 1H), 2.15 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H), 1.30 (hept,
J=7.5 Hz, 3H), 1.14 ppm (d, J=7.3 Hz, 18H); 13C NMR (126 MHz,
CDCl3): d=170.7, 170.2, 169.9, 80.7, 69.5, 67.9, 67.6, 61.8, 59.1, 20.9, 20.8,
20.8, 18.5, 18.4, 13.0 ppm; FTIR (neat film): n˜ =2947, 2868, 2110, 1753,
FTIR (neat film): n˜ =3446, 2111, 1728, 1268, 1109, 1070, 710 cmꢀ1
;
HRMS (FAB)+ : m/z calcd for C56H61N3O14NaSiS [M+Na]+ : 1082.3541;
found: 1082.3541.
Disaccharide 19: Benzeneselenol (127 mL, 1.21 mmol, 14 equiv) was
added to a solution of disaccharide 18 (90 mg, 0.09 mmol, 1 equiv) in a
mixture of triethylamine (1.5 mL) and pyridine (1.5 mL), leading to the
formation of yellow precipitate. The reaction mixture was heated to 408C
for 1.5 h, and then cooled to 238C prior to the addition of acetic anhy-
dride (470 mL, 4.31 mmol, 50 equiv). The clear solution was stirred at
238C for 18 h, and then concentrated in vacuo. The residue was purified
by silica-gel flash chromatography (40% ethyl acetate in hexane) to
afford thioacetyl disaccharide 19 (64 mg, 76%) as a white solid. M.p.
1568C; Rf =0.28 (12% ethyl acetate in dichloromethane); 1H NMR (500
MHz, CDCl3): d=8.14 (m, 2H), 8.02 (m, 4H), 7.95 (m, 2H), 7.77 (m,
2H), 7.65 (m, 1H), 7.53 (m, 5H), 7.43 (m, 7H), 7.26 (m, 2H), 6.31 (d,
J=4.7 Hz, 1H), 5.98 (d, J=3.4 Hz, 1H), 5.77 (dd, J=10.2, 7.6 Hz, 1H),
5.74 (brs, 1H), 5.63 (dd, J=10.3, 3.4 Hz, 1H), 5.45 (d, J=6.9 Hz, 1H),
5.02 (d, J=7.6 Hz, 1H), 4.76 (m, 1H), 4.65 (dd, J=11.2, 6.1 Hz, 1H),
4.39 (m, 3H), 4.32 (dd, J=11.5, 5.6 Hz, 1H), 4.20 (t, J=6.4 Hz, 1H), 3.79
(dd, J=11.1, 3.1 Hz, 1H), 2.26 (s, 3H), 2.16 (s, 3H), 1.16 ppm (s, 3H);
13C NMR (126 MHz, CDCl3): d=170.4, 170.0, 166.4, 166.3, 165.8, 165.7,
165.7, 134.0, 134.0, 133.7, 133.6, 133.4, 130.4, 130.2, 130.1, 130.1, 130.0,
130.0, 129.6, 129.2, 129.2, 129.0, 128.9, 128.9, 128.8, 128.7, 128.6, 100.6,
75.8, 72.0, 71.6, 71.1, 70.6, 68.0, 67.7, 62.6, 62.1, 49.2, 31.7, 22.8, 20.9 ppm;
FTIR (neat film): n˜ =3435, 1640, 1274 cmꢀ1; HRMS (FAB)+ : m/z calcd
for C53H49NO17NaS [M+Na]+ : 1026.2619; found: 1026.2623.
1370, 1229, 1082, 1048 cmꢀ1
;
HRMS (FAB)+
:
m/z calcd for
C21H38N3O7SiS [M+H]+ : 504.2200; found: 504.2199.
Monosaccharide 15: Sodium methoxide (5 mg, 0.09 mmol, 0.4 equiv) was
added to a solution of monosaccharide 14 (118 mg, 0.23 mmol, 1.0 equiv)
in methanol (2.8 mL) at 238C. The solution was stirred at this tempera-
ture for 1 h, and then concentrated in vacuo. The residue was purified by
silica-gel flash chromatography (8% methanol in dichloromethane) to
afford triol 15 (75 mg, 85%) as a colorless oil. Rf =0.08 (7% methanol in
dichloromethane); 1H NMR (500 MHz, CD3OD): d=5.55 (d, J=5.0 Hz,
1H), 4.27 (ddd, J=6.7, 5.6, 0.9 Hz, 1H), 3.98 (dd, J=3.2, 1.3 Hz, 1H),
3.95 (dd, J=10.6, 3.0 Hz, 1H), 3.86 (dd, J=10.5, 5.0 Hz, 1H), 3.76 (dd,
J=11.0, 7.1 Hz, 1H), 3.61 (dd, J=11.0, 5.5 Hz, 1H), 1.31 (hept, J=7.4
Hz, 3H), 1.16 ppm (d, J=7.2 Hz, 18H); 13C NMR (126 MHz, CD3OD):
d=82.7, 73.0, 70.5, 70.1, 62.8, 61.8, 19.1, 18.9, 14.2 ppm; FTIR (neat
film): n˜ =3338, 2865, 2105, 1056 cmꢀ1; HRMS (FAB)+ : m/z calcd for
C15H31N3O4NaSiS [M+Na]+ : 400.1702; found: 400.1703.
Monosaccharide 16: Benzoyl chloride (74 mL, 0.64 mmol, 1.1 equiv) was
added to a solution of triol 15 (215 mg, 0.57 mmol, 1.0 equiv) and tri-
ethylamine (160 mL, 1.15 mmol, 2.0 equiv) in dichloromethane (2.5 mL)
at ꢀ208C. The resulting solution was stirred at this temperature for 4.5 h.
Additional benzoyl chloride (7 mL, 0.06 mmol, 0.11 equiv) was added,
and the reaction mixture was stirred at ꢀ208C for a further 1.5 h. The re-
action mixture was diluted with dichloromethane (150 mL) and washed
with water (70 mL). The organic layer was dried (sodium sulfate), fil-
tered, and concentrated. The residue was purified by silica-gel flash chro-
matography (20% to 40% ethyl acetate in hexanes) to afford diol 16
(206 mg, 75%) as a colorless oil. Rf =0.64 (50% acetone in toluene); 1H
NMR (500 MHz, CDCl3): d=8.05 (m, 2H), 7.61 (m, 1H), 7.47 (m, 2H),
5.58 (d, J=5.1 Hz, 1H), 4.75 (dd, J=11.2, 7.4 Hz, 1H), 4.62 (t, J=6.5 Hz,
1H), 4.34 (dd, J=11.2, 5.8 Hz, 1H), 4.10 (ddd, J=10.5, 7.4, 3.3 Hz, 1H),
4.02 (brs, 1H), 3.98 (dd, J=10.4, 5.0 Hz, 1H), 3.09 (d, J=3.3 Hz, 1H),
2.65 (d, J=7.2 Hz, 1H), 1.27 (hept, J=7.2 Hz, 3H), 1.12 ppm (d, J=7.3
Hz, 18H); 13C NMR (126 MHz, CDCl3): d=167.2, 133.7, 130.1, 129.6,
128.7, 80.6, 69.0, 68.8, 68.7, 63.0, 62.1, 18.6, 18.4, 13.0 ppm; FTIR (neat
Disaccharide 21: Trimethylsilyltrifluoromethanesulfonate (3 mL, 0.02
mmol, 0.1 equiv) was added to a solution of sialoside 20 (108 mg, 0.19
mmol, 1.0 equiv) and triol 15 (91 mg, 0.29 mmol, 1.5 equiv) in dichloro-
methane (2.0 mL) at ꢀ458C, and the resulting solution was stirred at this
temperature for 3.5 h. The reaction mixture was neutralized with triethyl-
amine (45 mL, 0.38 mmol, 2 equiv) followed by concentration in vacuo.
The residue was purified by silica-gel flash chromatography (2 to 3 to
6% methanol in dichloromethane) to afford disaccharide 21 (120 mg,
87:13 a:b, 82% total), as a colorless oil. a isomer: Rf =0.18 ( 6% metha-
1
nol in ethyl acetate); H NMR (500 MHz, CDCl3): d=5.62 (d, J=9.9 Hz,
1H), 5.55 (d, J=5.1 Hz, 1H), 5.35 (m, 2H), 5.18 (ddd, J=11.9, 10.2, 4.7
Hz, 1H), 4.92 (d, J=14.4 Hz, 1H), 4.78 (d, J=14.4 Hz, 1H), 4.42 (m,
2H), 4.21 (d, J=3.8 Hz, 1H), 4.15 (dd, J=10.8, 1.4 Hz, 1H), 4.07 (m,
4H), 3.92 (dd, J=10.5, 5.1 Hz, 1H), 3.85 (d, J=6.2 Hz, 1H), 3.00 (s, 3H),
2.99 (s, 3H), 2.82 (d, J=9.1 Hz, 1H), 2.69 (dd, J=12.7, 4.8 Hz, 1H), 2.16
(s, 3H), 2.07 (s, 3H), 2.04 (s, 3H), 2.04 (s, 3H), 1.96 (t, J=12.3 Hz, 1H),
1.90 (s, 3H), 1.32 (m, 3H), 1.14 ppm (dd, J=7.4, 2.6 Hz, 18H); 13C NMR
(126 MHz, CDCl3): d=171.2, 171.2, 170.6, 170.4, 170.3, 167.4, 165.6, 98.7,
80.8, 72.9, 69.9, 69.5, 69.4, 69.2, 68.4, 67.7, 62.6, 62.2, 49.3, 37.4, 36.1, 36.0,
23.4, 21.2, 21.1, 21.0, 18.7, 18.5, 13.0 ppm; FTIR (neat film): n˜ =3436,
film): n˜ =3368, 1945, 2867, 2108, 1722, 1272, 710 cmꢀ1; HRMS (FAB)+
m/z calcd for C22H35N3O5NaSiS [M+Na]+ : 504.1964; found: 504.1964.
:
Disaccharide 18: Trifluoromethanesulfonic anhydride (18 mL, 0.10 mmol,
2.1 equiv) was added to a solution of hemiacetal 17 (44 mg, 0.07 mmol,
1.5 equiv) and diphenyl sulfoxide (42 mg, 0.21 mmol, 4.2 equiv) in di-
chloromethane (3.2 mL) at ꢀ788C. The resulting mixture was stirred at
this temperature for 10 min, and then at ꢀ488C for 1 h. A precooled
(ꢀ488C) solution of diol 16 (24 mg, 0.05 mmol, 1 equiv) in dichlorome-
thane (1 mL) was then added through a cannula. The resulting solution
was stirred at this temperature for 1 h, followed by warming to ꢀ208C
for 7 h. The reaction mixture was neutralized with triethylamine (135
mL), diluted with dichloromethane (110 mL), and washed sequentially
with saturated aqueous sodium bicarbonate solution (2î75 mL), and sa-
turated aqueous sodium chloride solution (75 mL). The organic layer was
dried (sodium sulfate), filtered, and concentrated. The residue was puri-
2110, 1645 cmꢀ1
; : m/z calcd for C38H63N5O17NaSiS
HRMS (FAB)+
[M+Na]+ : 944.3607; found: 944.3607.
Disaccharide 22: A solution of sodium hydroxide in water (1m, 1.5 mL,
1.5 mmol, 16 equiv) was added to a solution of disaccharide 21 (86 mg,
0.09 mmol, 1 equiv) in methanol (3.8 mL). The solution was deoxygenat-
ed, and stirred at 238C for 3 h. The reaction mixture was cooled to 08C,
and neutralized by the addition of a solution of acetic acid in methanol
(1m, 1.6 mL, 1.6 mmol, 17 equiv). The reaction mixture was concentrated
with toluene, and the crude product was then suspended in pyridine (1.8
mL) and acetic anhydride (950 mL, 9.7 mmol, 31 equiv) and deoxygenat-
ed. A catalytic amount of DMAP (~1 mg) was added, and the resulting
6002
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 5997 6006

Oligosaccharide Peptide Ligation
5997 6006
solution stirred at 238C for 21 h. The reaction mixture was partitioned
between ethyl acetate (100 mL) and water (80 mL), and the aqueous
layer was further extracted with ethyl acetate (2î100 mL). The com-
bined organic layers were dried (sodium sulfate), filtered, and concentrat-
ed, and the residue was purified by silica-gel flash chromatography
(20:160:0.1 MeOH/CH2Cl2/AcOH) to afford the disaccharide 22 (50 mg,
66%) as a white solid. M.p. 188 1908C; Rf =0.54 (4:1:1 n-BuOH/AcOH/
H2O); 1H NMR (500 MHz, CD3OD): d=6.19 (d, J=5.3 Hz, 1H), 5.48
(dd, J=3.2, 1.1 Hz, 1H), 5.40 (m, 1H), 5.33 (dd, J=8.4, 1.8 Hz, 1H), 4.94
(m, 2H), 4.50 (dd, J=10.8, 1.7 Hz, 1H), 4.45 (dd, J=11.1, 5.1 Hz, 1H),
4.34 (dd, J=12.1, 2.6 Hz, 1H), 4.25 (td, J=6.6, 0.9 Hz, 1H), 4.20 (dd, J=
12.4, 5.2 Hz, 1H), 3.84 (m, 2H), 3.42 (dd, J=9.4, 4.3 Hz, 1H), 2.57 (dd,
J=12.3, 4.8 Hz, 1H), 2.44 (s, 3H), 2.16 (s, 3H), 2.16 (s, 3H), 2.08 (s, 3H),
2.02 (s, 3H), 2.02 (s, 3H), 1.96 (s, 3H), 1.84 (s, 3H), 1.62 ppm (t, J=12.0
Hz, 1H); 13C NMR (126 MHz, CD3OD): d=193.7, 173.0, 173.7, 172.6,
172.2, 172.2, 172.1, 171.8, 171.2, 101.6, 83.7, 73.5, 73.1, 72.5, 71.7, 70.0,
69.1, 68.9, 64.1, 63.7, 59.2, 50.8, 39.7, 31.6, 22.8, 21.4, 21.1, 21.0, 20.9, 20.9,
20.8 ppm; FTIR (neat film): n˜ =3369, 2113, 1744, 1618, 1370, 1230, 1040
cmꢀ1; HRMS (ESI)ꢀ: m/z calcd for C31H41N4O19S [MꢀH]ꢀ: 805.2086;
found: 805.2110.
orless oil. Rf =0.33 (50% ethyl acetate in hexanes); 1H NMR (500 MHz,
CDCl3): d=5.92 (m, 1H), 5.57 (d, J=5.0 Hz, 1H), 5.40 5.36 (m, 1H),
5.31 5.28 (m, 1H), 4.65 4.63 (m, 2H), 4.55 (t, J=6.6 Hz, 1H), 4.45 (dd,
J=11.3, 6.7 Hz, 1H), 4.24 (dd, J=11.3, 6.1 Hz, 1H), 4.08 (ddd, J=10.4,
7.0, 3.4 Hz, 1H), 4.00 (brm, 1H), 3.95 (dd, J=10.4, 5.0 Hz, 1H), 2.85 (d,
J=3.4 Hz, 1H), 2.62 (d, J=7.1 Hz, 1H), 1.30 (m, 3H), 1.14 ppm (d, 7.2
Hz, 18H); 13C NMR (126 MHz, CDCl3): d=155.4, 131.4, 119.4, 80.5,
77.6, 77.2, 76.9, 69.1, 66.0, 61.9, 18.6, 18.4, 13.0 ppm; FTIR (neat film):
n˜ =3369, 2946, 2109, 1749, 1257 cmꢀ1; HRMS (FAB)+ : m/z calcd for
C19H35N3O6NaSiS [M+Na]+ : 484.1914; found: 484.1912.
Disaccharide 26: Trifluoromethanesulfonic anhydride (47 mL, 0.28 mmol,
1.9 equiv) was added to a solution of hemiacetal 17 (125 mg, 0.21 mmol,
1.4 equiv) and 4-nitrophenylphenyl sulfoxide (139 mg, 0.56 mmol, 3.8
equiv) in dichloromethane (9 mL) at ꢀ788C. The resulting mixture was
stirred at this temperature for 10 min, and then at ꢀ488C for 1 h. A pre-
cooled (ꢀ488C) solution of diol 25 (65 mg, 0.15 mmol, 1 equiv) in di-
chloromethane (2.5 mL) was then added through a cannula. The solution
was stirred at this temperature for 6 h. The reaction mixture was neutral-
ized with triethylamine (380 mL), diluted with dichloromethane (200 mL),
and washed sequentially with saturated aqueous sodium bicarbonate sol-
ution (2î125 mL) and saturated aqueous sodium chloride solution (125
mL). The organic layer was dried (sodium sulfate), filtered, and concen-
trated. The residue was purified by silica-gel flash chromatography
(2.4% acetone in benzene), and repurified by the same method (22%
ethylacetate in petroleum ether) to afford the disaccharide 26 (102 mg,
70%) as a white solid. M.p. 182 1838C; Rf =0.19 (22% ethylacetate in
petroleum ether); 1H NMR (500 MHz, CDCl3): d=8.11 7.97 (m,
6H),7.78 (m, 2H), 7.65 (m, 1H), 7.56 7.23 (m, 11H), 6.01 (dd, J=3.5,
0.9 Hz, 1H), 5.96 5.87 (m, 2H), 5.61 (dd, J=10.5, 3.5 Hz, 1H), 5.51, (d,
J=4.3 Hz, 1H), 5.38 5.34 (m, 1H), 5.28 5.26 (m, 1H), 5.13 (d, J=8.0 Hz,
1H), 4.65 (dd, J=11.5, 7.4 Hz, 1H), 4.61 (dt, J=7.8, 1.3 Hz, 2H), 4.48
(dd, J=11.8, 5.5 Hz, 1H), 4.45 (t, J=7.3 Hz, 1H), 4.41 (td, J=6.0, 0.8 Hz,
1H), 4.32 (dd, J=11.6, 7.3 Hz, 1H), 4.19 (brs, 1H), 4.11 (dd, J=11.5, 4.4
Hz, 1H), 4.05 (m, 2H), 2.73 (brs, 1H), 1.27 (m, 3H), 1.10 ppm (dd, J=
7.4, 4.8 Hz, 18H); 13C NMR (126 MHz, CDCl3): d=166.2, 165.8, 165.7,
165.7, 155.0, 133.9, 133.6, 133.6, 133.5, 131.7, 130.2, 130.0, 130.0, 129.9,
129.4, 129.3, 129.0, 128.9, 128.8, 128.8, 128.5, 119.0, 102.4, 80.6, 78.5, 72.1,
71.7, 69.5, 68.8, 68.6, 68.2, 66.8, 62.3, 60.4, 18.5, 18.3, 12.9 ppm; FTIR
(neat film): n˜ =3505, 2946, 2111, 1730, 1264, 1109, 1026, 710 cmꢀ1; HRMS
(FAB)+ : m/z calcd for C53H61N3O15NaSiS [M+Na]+ : 1062.3490; found:
1062.3490.
Thiazoline 23: Triphenylphosphine (44 mg, 0.17 mmol, 1.4 equiv) was
added to a solution of disaccharide 22 (95 mg, 0.12 mmol, 1 equiv) in tet-
rahydrofuran (2.8 mL) at 238C. The solution was stirred at this tempera-
ture for 16 h, and then concentrated. The residue was purified by silica-
gel flash column chromatography (gradient elution: 50:300:0.1 MeOH/
CH2Cl2/AcOH to 50:250:0.1 MeOH/CH2Cl2/AcOH) to afford the thiazo-
line 23 (90 mg, >99%) as a white solid. M.p.=1898C; Rf =0.20 (17%
methanol in dichloromethane); 1H NMR (500 MHz, CD3OD): d=6.44
(d, J=6.1 Hz, 1H), 5.53 (t, J=3.3 Hz, 1H), 5.41 (ddd, J=8.7, 5.8, 2.7 Hz,
1H), 5.31 (dd, J=8.6, 2.0 Hz, 1H), 5.09 (dd, J=8.5, 3.1 Hz, 1H), 4.94 (m,
1H), 4.51 (dd, J=10.7, 1.8 Hz, 1H), 4.40 (m, 1H), 4.34 (m, 2H), 4.12 (dd,
J=12.5, 5.9 Hz, 1H), 3.94 (dd, J=11.3, 7.7 Hz, 1H), 3.89 (t, J=10.6 Hz,
1H), 3.47 (dd, J=11.2, 5.2 Hz, 1H), 2.62 (dd, J=12.2, 4.7 Hz, 1H), 2.26
(d, J=1.1 Hz, 3H), 2.16 (s, 3H), 2.12 (s, 3H), 2.09 (s, 3H), 2.04 (s, 3H),
2.00 (s, 3H), 1.97 (s, 3H), 1.84 (s, 3H), 1.64 ppm (t, J=12.4 Hz, 1H); 13C
NMR (126 MHz, CD3OD): d=174.8, 173.7, 172.9, 172.6, 172.2, 172.0,
171.9, 171.8, 101.8, 90.4, 75.1, 74.0, 73.0, 72.8, 72.0, 69.9, 69.2, 68.4, 63.9,
63.7, 50.8, 39.7, 22.8, 21.5, 21.4, 21.1, 21.0, 21.0, 20.9, 20.9 ppm; FTIR
(neat film): n˜ =3588, 1735, 1654, 1372, 1230 cmꢀ1; HRMS (ESI)ꢀ: m/z
calcd for C31H41N2O18S [MꢀH]ꢀ: 761.2085; found: 761.2086.
Thiol 24: Water (110 mL) was added to a solution of thiazoline 23 (26 mg,
0.03 mmol, 1 equiv) in methanol (1.3 mL) containing trifluoroacetic acid
(50 mL, 0.62 mmol, 19 equiv). The resulting solution was deoxygenated,
and stirred at 08C for 8 h. The reaction mixture was diluted with toluene
(15 mL) and then concentrated. Residual TFA was removed from the
product by azeotropic coevaporation (2î15 mL methanol; 1î15 mL
tolene), followed by lyophylization (30 mL water) to afford thiol 24 (27
mg, >99%), as a white solid. M.p. 1958C (decomp); Rf =0.18 (17%
methanol in dichloromethane); 1H NMR (500 MHz, CD3OD): d=5.85
(d, J=5.2 Hz, 1H), 5.49 (dd, J=3.0, 0.8 Hz, 1H), 5.43 (ddd, J=9.4, 5.3,
2.6 Hz, 1H), 5.32 (dd, J=9.4, 2.0 Hz, 1H), 5.12 (dd, J=12.0, 3.2 Hz, 1H),
4.90 (m, 1H), 4.58 (t, J=7.0 Hz, 1H), 4.55 (dd, J=11.8, 5.2 Hz, 1H), 4.27
(dd, J=9.4, 1.8 Hz, 1H), 4.24 (dd, J=12.5, 2.7 Hz, 1H), 4.09 (dd, J=12.5,
5.3 H, 1Hz), 3.96 (t, J=10.5 Hz, 1H), 3.84 (dd, J=10.0, 6.2 Hz, 1H), 3.40
(dd, J=10.0, 7.5 Hz, 1H), 2.58 (dd, J=12.6, 4.7 Hz, 1H), 2.14 (s, 3H),
2.13 (s, 3H), 2.09 (s, 3H), 2.00 (s, 3H), 1.98 (s, 3H), 1.96 (2 overlapping s,
6H), 1.84 (s, 3H), 1.75 ppm (t, J=12.4 H, 1Hz); 13C NMR (126 MHz,
CD3OD): d=180.8, 173.9, 173.7, 172.7, 172.4, 172.2, 172.0, 171.9, 171.8,
80.5, 73.2, 71.5, 70.0, 69.8, 69.4, 68.9, 68.9, 64.1, 63.8, 50.6, 49.8, 39.5, 22.8,
22.5, 21.4, 21.1, 20.9, 20.9, 20.8 ppm; FTIR (neat film): n˜ =3448, 1736,
1654, 1375, 12236, 1045 cmꢀ1; HRMS (ESI)ꢀ: m/z calcd for C31H43N2O19S
[MꢀH]ꢀ: 779.2181; found: 779.2175.
Disaccharide 27: Tri-n-butyltin hydride (265 mL, 0.98 mmol, 10 equiv) was
added to a deoxygenated solution of disaccharide 26 (102 mg, 0.10 mmol,
1 equiv) and trans-dichlorobis(triphenylphosphine)palladium(ii) (7 mg,
0.01 mmol, 0.09 equiv) in dichloromethane (12.5 mL) and water (245 mL).
The resulting brown solution was stirred at 238C for 14 min, and then
concentrated in vacuo. The residue was purified by silica-gel flash
column chromatography (8% ethylacetate in dichloromethane) to afford
disaccharide diol 27 (94 mg, >99%) as a yellowish solid. M.p. 112
1138C; Rf =0.29 (50% ethylacetate in hexanes); 1H NMR (500 MHz,
C6D6): d=8.22 8.20 (m, 4H), 8.15 8.13 (m, 2H), 8.00 7.98 (m, 2H),
7.16 6.70 (m, 12H), 6.38 (dd, J=10.5, 8.0 Hz, 1H), 6.05 (dd, J=3.3, 0.8
Hz, 1H), 5.71 (dd, J=10.6, 3.4 Hz, 1H), 5.30 (d, J=4.1 Hz, 1H), 4.46 (d,
J=8.2 Hz, 1H), 4.44 (dd, J=10.8, 7.6 Hz, 1H), 4.31 (t, J=5.4 Hz, 1H),
4.28 (dd, J=11.8, 4.1 Hz, 1H), 4.11 (brd, J=1.7 Hz, 1H), 4.01 (m, 2H),
3.83 (m, 1H), 3.76 (m, 1H), 3.63 (ddd, J=7.7, 3.6, 0.8 Hz, 1H), 2.78 (brs,
1H), 1.87 (dd, J=8.1, 4.2 Hz, 1H), 1.12 1.01 ppm (m, 21H); 13C NMR
(126 MHz, CDCl3): d=166.2, 165.9, 165.7, 165.7, 134.0, 133.8, 133.6,
133.5, 130.3, 130.0, 130.0, 129.9, 129.4, 129.3, 129.0, 128.9, 128.8, 128.8,
128.5, 128.5, 102.6, 80.9, 79.0, 72.3, 71.8, 70.4, 69.8, 69.5, 68.4, 63.0, 62.6,
60.3, 18.6, 18.4, 13.0 ppm; FTIR (neat film): n˜ =3580, 3472, 2949, 2110,
1729, 1267, 1108, 1070 cmꢀ1; HRMS (FAB)+ : m/z calcd for C49H58N3O13-
NaSiS (M+H+Na)+ : 979.3357; found: 979.3357.
Monosaccharide 25: Allylchloroformate (91 mL, 0.86 mmol, 1.3 equiv)
was added portionwise to a solution of monosaccharide 15 (252 mg, 0.66
mmol, 1.0 equiv) and triethylamine (185 mL, 1.32 mmol, 2 equiv) in di-
chloromethane (9 mL) at ꢀ208C, and the solution was stirred at this tem-
perature for 2 h. Water (5 mL) was added, and the reaction mixture was
partitioned between dichloromethane (150 mL) and water (60 mL). The
organic layer was dried (Na2SO4), filtered, and concentrated in vacuo.
The residue was purified by silica-gel flash column chromatography
(20% ethyl acetate in toluene) to afford diol 25 (141 mg, 46%) as a col-
Tri-iso-propylsilylthio-4,7,8,9-tetra-O-acetyl-5-N-acetylamino-1-N,N-di-
methylglycolamido-a-d-sialosyl-(2!6)-(2,3,4,6-tetra-O-benzoyl-b-d-gal-
actopyranosyl-(1!3)-2-azido-1,2-dideoxy-a-d-galactopyranoside): A sol-
ution of trimethylsilyltrifluoromethanesulfonate (1.8 mL, 0.01 mmol, 0.1
equiv) in dichloromethane (16 mL) was added to a solution of sialoside
20 (51 mg, 0.08 mmol, 1.0 equiv) and disaccharide 27 (99 mg, 0.10 mmol,
1.4 equiv) in dichloromethane (2.5 mL) at ꢀ788C, and the solution was
stirred at this temperature for 6 h. The reaction mixture was neutralized
6003
Chem. Eur. J. 2003, 9, 5997 6006
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

¬
D. Y. Gin, W. A. van der Donk, and D. P. Galonic
FULL PAPER
with triethylamine (50 mL), and concentrated in vacuo. The residue was
purified by silica-gel flash chromatography (33 to 50 to 67% acetonitrile
in toluene) to afford the trisaccharide as separable mixture of anomers
(73 mg, 81:19 a:b, 66% total). a-isomer: M.p. 1208C; Rf =0.45 ( 66%
acetonitrile in toluene); 1H NMR (500 MHz, CDCl3): d=8.11 8.09 (m,
2H), 8.01 7.97 (m, 4H), 7.79 7.77 (m, 2H), 7.65 7.34 (m, 10H), 7.24 (m,
2H), 6.02 (d, J=3.4 Hz, 1H), 5.89 (dd, J=10.4, 8.0 Hz, 1H), 5.85 (d, J=
10.2 Hz, 1H), 5.60 (dd, J=10.3, 3.4 Hz, 1H), 5.53 (d, J=4.8 Hz, 1H),
5.39 (td, J=6.5, 2.9 Hz, 1H), 5.34 (dd, J=6.4, 2.0 Hz, 1H), 5.24 (ddd, J=
12.0, 10.1, 4.6 Hz, 1H), 5.16 (d, J=7.8 Hz, 1H), 4.83 (q, J=14.4 Hz, 2H),
4.64 (dd, J=11.1, 6.5 Hz, 1H), 4.43 (dd, J=11.2, 6.9 Hz, 1H), 4.37 (m,
2H), 4.23 (m, 2H), 4.14 (m, 2H), 4.06 (dd, J=10.4, 4.8 Hz, 1H), 4.01 (dd,
J=10.6, 2.7 Hz, 1H), 3.91 (dd, J=9.8, 6.2 Hz, 1H), 3.78 (dd, J=9.8, 6.1
Hz, 1H), 2.99 (m, 1H), 2.96 (s, 3H), 2.95 (s, 3H), 2.70 (dd, J=12.6, 4.8
Hz, 1H), 2.14 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 2.01 (s, 3H), 1.90 (t, J=
12.2 Hz, 1H), 1.29 (m, 3H), 1.13 ppm (t, J=7.7 Hz, 18H); 13C NMR
(126 MHz, C6D6): d=170.7, 170.5, 170.4, 170.4, 169.9, 168.5, 166.3, 166.2,
166.2, 166.1, 165.6, 133.8, 133.7, 133.5, 133.5, 130.6, 130.6, 130.4, 130.4,
130.0, 129.8, 129.3, 129.1, 128.9, 128.8, 128.7, 128.5, 128.3, 103.3, 99.9,
81.6, 79.7, 74.2, 72.7, 72.3, 71.6, 70.8, 70.5, 70.3, 69.4, 69.2, 69.1, 64.3, 63.7,
62.7, 62.6, 61.3, 50.0, 38.9, 35.4, 34.9, 23.1, 21.3, 21.1, 20.9, 20.8, 19.0, 18.9,
13.5 ppm; FTIR (neat film): n˜ =3450, 2938, 2110, 1733, 1667, 1266, 1106,
MHz, CD3OD): d=174.9, 173.7, 172.7, 172.3, 172.2, 172.2, 172.0, 171.9,
171.7, 171.6, 169.9, 101.8, 101.7, 89.6, 79.6, 77.7, 75.0, 72.9, 72.4, 72.1, 72.0,
70.7, 70.7, 70.1, 69.4, 68.9, 63.9, 63.8, 62.6, 50.9, 39.7, 22.8, 21.5, 21.2, 21.1,
21.0, 20.9, 20.9, 20.7, 20.6 ppm; FTIR (neat film): n˜ =3436, 1749, 1637,
1371, 1227, 1047 cmꢀ1 HRMS (ESI)ꢀ: m/z calcd for C43H57N2O26S
;
[MꢀH]ꢀ: 1049.2920; found: 1049.2919.
Thiol 29: Water (52 mL) was added to a solution of trisaccharide thiazo-
line (17 mg, 0.02 mmol, 1 equiv) in methanol (0.6 mL) containing tri-
fluoroacetic acid (12 mL, 0.31 mmol, 19 equiv). The resulting solution was
deoxygenated, and stirred at 08C for 8.5 h. The reaction mixture was di-
luted with toluene (20 mL) and then concentrated. Residual TFA was re-
moved from the product by azeotropic coevaporation (5î10 mL of meth-
anol and 15 mL of toluene) to afford thiol 29 (17 mg, >99%), as a white
solid. M.p. 2208C (decomp); Rf =0.22 (17% methanol in dichlorometh-
ane); 1H NMR (500 MHz, CD3OD): d=5.69 (d, J=5.2 Hz, 1H), 5.46 (d,
J=3.2 Hz, 1H), 5.40 (ddd, J=8.2, 5.3, 2.3 Hz, 1H), 5.37 (dd, J=3.4, 1.2
Hz, 1H), 5.32 (dd, J=8.6, 2.2 Hz, 1H), 5.07 (dd, J=10.5, 3.4 Hz, 1H),
5.00 (dd, J=10.5, 7.7 Hz, 1H), 4.91 (m, 1H), 4.82 (d, J=7.8 Hz, 1H),
4.58 (m, 1H), 4.45 (t, J=5.5 Hz, 1H), 4.31 (m, 2H), 4.18 (d, J=6.7 Hz,
1H), 4.11 (dd, J=12.2, 5.4 Hz, 1H), 4.05 (td, J=6.4, 0.8 Hz, 1H), 4.02
(dd, J=11.1, 3.4 Hz, 1H), 3.95 (m, 1H), 3.82 (dd, J=10.4, 7.2 Hz, 1H),
3.42 (dd, J=10.3, 4.8 Hz, 1H), 2.61 (dd, J=13.0, 4.8 Hz, 1H), 2.14 (s,
3H), 2.13 (s, 3H), 2.11 (s, 3H), 2.08 (s, 3H), 2.05 (s, 3H), 2.02 (s, 3H),
2.01 (s, 3H), 2.00 (s, 3H), 1.98 (s, 3H), 1.94 (s, 3H), 1.83 (s, 3H), 1.75
ppm (t, J=12.4 Hz, 1H); 13C NMR (126 MHz, CD3OD): d=173.7, 173.4,
172.7, 172.3, 172.2, 172.1, 171.9, 171.7, 171.7, 171.6, 171.4, 102.3, 80.6,
73.7, 73.2, 72.2, 72.0, 71.4, 69.9, 69.1, 68.7, 64.8, 63.7, 62.5, 51.2, 50.5, 23.0,
22.8, 21.3, 21.2, 21.1, 20.9, 20.9, 20.9, 20.6, 20.6 ppm; FTIR (neat film):
n˜ =3451, 1744, 1645, 1374, 1228, 1052 cmꢀ1; HRMS (ESI)ꢀ: m/z calcd for
C43H59N2O27S [MꢀH]ꢀ: 1067.3026; found: 1067.2975.
1070, 708 cmꢀ1
; : m/z calcd for C72H89N5O26NaSiS
HRMS (FAB)+
[M+Na]+ : 1522.5183; found: 1522.5177.
Trisaccharide 28: A solution of sodium hydroxide in water (1m, 560 mL,
0.56 mmol, 30 equiv) was added to a solution of tri-iso-propylsilylthio-
4,7,8,9-tetra-O-acetyl-5-N-acetylamino-1-N,N-dimethylglycolamido-a-d-
sialosyl-(2!6)-(2,3,4,6-tetra-O-benzoyl-b-d-galactopyranosyl-(1!3)-2-
azido-1,2-dideoxy-a-d-galactopyranoside) (28 mg, 0.02 mmol, 1 equiv) in
methanol (1.8 mL). The resulting solution was deoxygenated, and stirred
at 238C for 3 h. The residue was diluted with toluene (~50 mL) and con-
centrated. The residue was suspended in pyridine (1.5 mL) and acetic an-
hydride (750 mL, 7.9 mmol, 427 equiv) with a catalytic amount of DMAP
(~0.2 mg), and reaction mixture deoxygenated and stirred at 238C for 15
h. The reaction was partitioned between ethyl acetate (80 mL) and 1m
hydrochloric acid (45 mL). The aqueous layer was further extracted with
ethyl acetate (2î40 mL), and the combined organic layers were dried
(sodium sulfate), filtered, and concentrated. The residue was purified by
silica-gel flash chromatography (20:160:0.1 methanol/dichloromethane/
acetic acid) to afford the trisaccharide 28 (10 mg, 51%) as a white solid.
M.p. 176 1778C; Rf =0.17 (11% methanol in dichloromethane); 1H
NMR (500 MHz, CD3OD): d=6.21 (d, J=5.4 Hz, 1H), 5.48 (dd, J=3.3,
1.0 Hz, 1H), 5.37 (m, 2H), 5.31 (dd, J=7.8, 2.3 Hz, 1H), 5.13 (dd, J=
10.3, 3.3 Hz, 1H), 5.07 (dd, J=10.6, 7.6 Hz, 1H), 4.95 (m, 1H), 4.55 (dd,
J=12.3, 2.1 Hz, 1H), 4.36 (dd, J=12.4, 2.7 Hz, 1H), 4.29 (dd, J=10.9,
5.5 Hz, 1H), 4.12 (m, 4H), 4.04 (dd, J=7.9, 3.0 Hz, 1H), 3.88 (t, J=10.3
Hz, 1H), 3.79 (dd, J=10.8, 3.5 Hz, 1H), 3.75 (dd, J=10.9, 7.6 Hz, 1H),
3.48 (dd, J=11.1, 3.5 Hz, 1H), 2.58 (dd, J=12.1, 4.8 Hz, 1H), 2.44 (s,
3H), 2.14 (s, 6H), 2.11 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H),
2.01 (s, 3H), 1.95 (s, 3H), 1.94 (s, 3H), 1.82 (s, 3H),1.64 ppm (t, J=12.1
Monosaccharide tripeptide conjugate 30: A deoxygenated solution of
sodium methoxide (4 mg, 0.08 mmol, 1 equiv) in methanol (760 mL) was
added to a mixture of thiol 12 (28 mg, 0.08 mmol, 1.0 equiv) and tripep-
tide 9 (21 mg, 0.083 mmol, 1.1 equiv) at 238C. A white precipitate was
observed after 2 h. The reaction mixture was stirred at this temperature
for a total of 4.5 h, and then neutralized with a solution of acetic acid in
methanol (1m, 91 mL, 0.09 mmol, 1.2 equiv) followed by concentration in
vacuo. The residue was purified by silica-gel flash chromatography (25%
to 50% methanol in dichloromethane), followed by LH 20 Sephadex
size-exclusion chromatography (methanol) to afford glycoconjugate 30
(30 mg, 79%, dr=1.4:1) as a white solid. Rf =0.42 ( 50% methanol in di-
chloromethane); 1H NMR (500 MHz, D2O): d=5.55 (d, J=5.6 Hz,
1.4H), 5.53 (d, J=5.6 Hz, 1H), 4.63 (dd, J=7.6, 5.1 Hz, 1.4H), 4.56 (dd,
J=8.5, 6.0 Hz, 1H), 4.36 3.74 (m, 28.8H), 3.16 3.05 (m, 3.8H), 2.89 (dd,
J=14.0, 8.5 Hz, 1H), 2.73 (s, 7.2H), 2.07 (s, 4.2H), 2.06 (s, 3H), 2.03 (s,
4.2H), 2.02 ppm (s, 3H); 13C NMR (126 MHz, D2O): d=175.1, 175.0,
174.7, 174.7, 172.9, 172.8, 172.2, 172.0, 171.7, 171.6, 85.2, 83.9, 72.0, 71.9,
68.6, 68.5, 67.6, 67.5, 61.4, 61.3, 54.2, 53.4, 50.2, 50.1, 49.0, 42.7, 42.7, 42.5,
32.1, 31.1, 25.9, 22.0, 22.0, 21.8, 21.8 ppm; FTIR (neat film): n˜ =3436,
2092, 1645 cmꢀ1; HRMS (FAB)+ : m/z calcd for C18H32N5O9S [M+H]+
:
Hz, 1H); FTIR (neat film): n˜ =3394, 2113, 1752, 1597, 1369, 1228 cmꢀ1
;
494.1921; found: 494.1921.
HRMS (ESI)ꢀ: m/z calcd for C43H57N4O27S [MꢀH]ꢀ: 1093.2931; found:
Monosaccharide tripeptide conjugate 31: A deoxygenated solution of
sodium methoxide (2 mg, 0.04 mmol, 1 equiv) in methanol (415 mL) was
added to the mixture of thiol 12 (15 mg, 0.05 mmol, 1.0 equiv) and tripep-
tide 10 (15 mg, 0.05 mmol, 1.2 equiv) at 238C. After 2 h, a white precipi-
tate was observed. The reaction mixture was stirred at this temperature
for a total of 4 h, and then neutralized with a solution of acetic acid in
methanol (1m, 50 mL, 0.05 mmol, 1.2 equiv) followed by concentration in
vacuo. The residue was purified by silica-gel flash chromatography (25%
to 50% methanol in dichloromethane), followed by LH 20 Sephadex
size-exclusion chromatography (methanol) to afford glycoconjugate 31
(20 mg, 92%, dr=1.1:1) as a white solid. Rf =0.07 ( 25% methanol in di-
chloromethane); 1H NMR (500 MHz, CD3OD): d=5.62 (d, J=5.4 Hz,
1H), 5.59 (d, J=5.4 Hz, 1H), 4.61 (dd, J=7.9, 4.8 Hz, 1H), 4.53 (t, J=
7.4 Hz, 1.1H), 4.42 (m, 4.3H), 4.29 (m, 2.1H), 4.19 (m, 2.2H), 3.88 3.64
(m, 15.2H), 3.09 (m, 3.2H), 2.85 (dd, J=13.9, 7.4 Hz, 1.1H), 2.73 (s, 3H),
2.72 (s, 3.3H), 2.04 (s, 3H), 2.03 (s, 3.3H), 1.99 (s, 3H), 1.98 (s, 3.3H),
1.37 (d, J=7.4 Hz, 3.3H), 1.34 ppm (d, J=7.1 Hz, 3H); 13C NMR (126
MHz, D2O): d=175.2, 175.2, 174.7, 174.6, 172.4, 172.3, 172.1, 171.8, 84.9,
83.6, 71.9, 71.8, 68.6, 68.5, 67.6, 67.6, 61.4, 61.2, 61.1, 55.9, 55.7, 53.8, 53.1,
50.2, 50.0, 49.0, 31.9, 31.0, 26.0, 25.9, 22.0, 22.0, 21.8, 16.6 ppm; FTIR
1093.2880.
4,7,8,9-Tetra-O-acetyl-5-N-acetylamino-a-d-sialosyl-(2!6)-(2,3,4,6-tetra-
O-acetyl-b-d-galactopyranosyl-(1!3)-4-O-acetyl-2-amino-1,2-dideoxy-1-
S-2-N-(ethan-1-yl-1-ylidene)-a-d-galactopyranoside: Triphenylphosphine
(4 mg, 0.02 mmol, 1.7 equiv) was added to a solution of trisaccharide 28
(10 mg, 0.01 mmol, 1.0 equiv) in tetrahydrofuran (600 mL) at 238C. The
solution was stirred at this temperature for 26.5 h, and concentrated. The
residue was purified by silica-gel flash column chromatography
(50:300:0.1 methanol/dichloromethane/acetic acid) to afford the thiazo-
line (9 mg, 91%) as a white solid. M.p.=195 1978C; Rf =0.20 (14%
methanol in dichloromethane); 1H NMR (500 MHz, CD3OD): d=6.30
(d, J=6.0 Hz, 1H), 5.54 (t, J=3.5 Hz, 1H), 5.41 (ddd, J=8.4, 5.8, 2.6 Hz,
1H), 5.38 (dd, J=3.4, 1.2 Hz, 1H), 5.31 (dd, J=9.0, 2.3 Hz, 1H), 5.14
(dd, J=10.0, 3.4 Hz, 1H), 5.06 (m, 2H), 4.94 (m, 1H), 4.51 (dd, J=10.8,
1.6 Hz, 1H), 4.34 (dd, J=12.4, 2.7 Hz, 1H), 4.27 (m, 1H), 4.21 4.08 (m,
6H), 3.92 (m, 2H), 3.44 (dd, J=11.1, 3.4 Hz, 1H), 2.61 (dd, J=12.2, 4.8
Hz, 1H), 2.28 (d, J=1.6 Hz, 3H), 2.15 (2 overlapping s, 6H), 2.13 (s,
3H), 2.10 (s, 3H), 2.08 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H), 1.97 (s, 3H),
1.95 (s, 3H), 1.84 (s, 3H), 1.68 ppm (t, J=12.1 Hz, 1H); 13C NMR (126
6004
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 5997 6006

Oligosaccharide Peptide Ligation
5997 6006
(neat film): n˜ =3294, 1654, 1420, 1067 cmꢀ1; HRMS (FAB)+ : m/z calcd
for C20H35N5O10NaS [M+Na]+ : 560.2002; found: 560.2001.
ppm; FTIR (neat film): n˜ =3294, 1654, 1560, 1376, 1035 cmꢀ1; HRMS
(ESI)ꢀ: m/z calcd for C29H47N6O17S [MꢀH]ꢀ: 783.2718; found: 783.2738.
Disaccharide tripeptide conjugate 35:
A deoxygenated solution of
Disaccharide tripeptide conjugate 32:
A deoxygenated solution of
sodium methoxide (1.1 mg, 0.02 mmol, 2 equiv) in methanol (400 mL) was
added to a mixture of thiol 24 (8 mg, 0.01 mmol, 1 equiv) and tripetide 10
(4 mg, 0.01 mmol, 1.2 equiv) at 238C. The reaction mixture was stirred at
228C for 14 h, when another portion of sodium methoxide (1.1 mg, 0.02
mmol, 2 equiv) in methanol (200 mL) was added, and the resulting solu-
tion was stirred for an additional 7 h. The reaction mixture was neutral-
ized by the addition of a solution of acetic acid in methanol (1m, 46 mL,
0.05 mmol, 4.2 equiv), followed by concentration in vacuo. The residue
was purified by silica-gel flash chromatography (50% to 67% methanol
in dichloromethane), followed by LH 20 Sephadex size-exclusion chro-
matography (methanol) to afford glycoconjugate 35 (8 mg, 90%, dr=
1.2:1.0) as a white solid. Rf =0.28 (4:4:1:1 MeOH/EtOAc/AcOH/H2O);
1H NMR (500 MHz, CD3OD): d=5.59 (d, J=5.4 Hz, 1H), 5.58 (d, J=
5.5 Hz, 1.2H), 4.50 (dd, J=8.1, 6.2 Hz, 1.2H), 4.41 (m, 5.7H), 4.3 (m,
4.6H), 4.00 3.51 (m, 36.5H), 3.26 (dd, J=14.2, 9.8 Hz, 1.2H), 3.12 (m,
2H), 2.83 (m, 3.7 Hz), 2.73 (2 overlapped singlets, 6.6H), 2.10 (s, 3H),
2.04 (s, 3.6H), 2.02 (s, 7.2H), 1.97 (s, 3H), 1.96 (s, 3H), 1.67 (m, 2.2H),
1.39 (d, J=7.2 Hz, 3H), 1.34 ppm (d, J=3.6 Hz, 3.6H); 13C NMR (126
MHz, CD3OD): d=175.8, 175.7, 175.7, 175.5, 174.1, 174.1, 174.0, 173.9,
173.7, 172.7, 172.3, 170.5, 102.3, 102.1, 74.9, 74.7, 73.1, 73.0, 71.9, 71.8,
70.4, 70.4, 70.2, 69.7, 69.7, 69.4, 65.1, 64.7, 63.5, 63.2, 57.8, 57.7, 57.5, 56.3,
55.3, 54.2, 54.1, 52.0, 51.9, 51.2, 51.1, 50.0, 49.8, 26.6, 26.6, 23.1, 22.9, 22.8,
sodium methoxide (1.0 mg, 0.02 mmol, 1 equiv) in methanol (180 mL) was
added to a mixture of disaccharide 19 (20 mg, 0.02 mmol, 1.0 equiv) and
peptide 9 (6 mg, 0.02 mmol, 1.1 equiv) at 238C. After 3 h, a white precipi-
tate was observed. The reaction mixture was stirred at 238C for a total of
6.5 h and then neutralized by the addition of a solution of acetic acid in
methanol (1m, 23 mL, 0.02 mmol, 1.2 equiv), followed by concentration in
vacuo. The residue was purified by silica-gel flash chromatography (50%
to 67% methanol in dichloromethane), followed by LH 20 Sephadex
size-exclusion chromatography (methanol) to afford glycoconjugate 32
(12 mg, 90%, dr=1.2:1.0) as a white solid. Rf =0.33 (33% dichloro-
1
methane in methanol); H NMR (500 MHz, D2O): d=5.50 (d, J=5.6 Hz,
1.2H), 5.48 (d, J=5.6 Hz, 1H), 4.62 (dd, J=7.8, 5.1 Hz, 1.2H), 4.56 4.51
(m, 3.3H), 4.48 (d, J=7.8 Hz, 2H), 4.30 4.24 (m, 5H), 3.94 3.69 (m,
25.2H), 4.64 3.59 (m, 5.3H), 3.49 (dd, J=9.8, 7.7 Hz, 2H), 3.13 (dd, J=
14.1, 6.0 Hz, 1H), 3.11 (dd, J=14.5, 7.9 Hz, 1.2H), 3.06 (dd, J=14.5, 5.1
Hz, 1.2H), 2.88 (dd, J=14.1, 8.5 Hz, 1H), 2.71 (s, 6.6H), 2.04 (s, 3.5H),
2.04 (s, 3H), 2.00 (s, 3.6H), 1.99 ppm (s, 3H); 13C NMR (126 MHz,
CD3OD): d=174.4, 174.2, 174.2, 174.2, 173.4, 173.2, 172.6, 173.3, 172.2,
172.2, 106.2, 87.4, 86.7, 79.0, 78.9, 77.0, 75.0, 73.4, 72.7, 70.4, 70.4, 70.0,
70.0, 63.4, 63.2, 62.8, 55.8, 55.4, 50.8, 50.8, 50.0, 44.1, 43.7, 43.7, 43.7, 34.2,
33.3, 26.5, 26.5, 22.9, 22.9, 22.6, 22.6 ppm; FTIR (neat film): n˜ =3307,
1654, 1560, 1412, 1077 cmꢀ1
C24H41N5O14NaS [M+Na]+ : 678.2268; found: 678.2265.
Disaccharide tripeptide conjugate 33: deoxygenated solution of
;
HRMS (FAB)+
:
m/z calcd for
22.7, 22.7, 18.1 ppm; FTIR (neat film): n˜ =3307, 1654, 1560, 1034 cmꢀ1
;
HRMS (ESI)ꢀ: m/z calcd for C31H51N6O18S [MꢀH]ꢀ: 827.2981; found:
A
827.3016.
sodium methoxide (1.8 mg, 0.03 mmol, 1 equiv) in methanol (530 mL) was
added to a mixture of disaccharide 19 (33 mg, 0.03 mmol, 1.0 equiv) and
tripeptide 10 (12 mg, 0.04 mmol, 1.2 equiv) at 238C. After 2 h, white pre-
cipitate was observed. The reaction mixture was stirred at 238C for a
total of 4 h and then neutralized by the addition of a solution of acetic
acid in methanol (1m, 40 mL, 0.04 mmol, 1.2 equiv), followed by concen-
tration in vacuo. The residue was purified by silica-gel flash chromatogra-
phy (33% to 67% methanol in dichloromethane), followed by LH 20 Se-
phadex size-exclusion chromatography (methanol) to afford glycoconju-
gate 33 (19 mg, 84%, dr=1.1:1.0) as a white solid. Rf =0.22 (50% di-
chloromethane in methanol); 1H NMR (500 MHz, CD3OD): d=5.65 (d,
J=5.5 Hz, 1.1H), 5.62 (d, J=5.6 Hz, 1H), 4.63 4.52 (m, 3.5H), 4.43 (m,
3.6H), 4.32 4.17 (m, 5.2H), 3.90 3.68 (m, 15.2H), 3.54 (m, 3.5H), 3.46
(dd, J=9.9, 3.4 Hz, 2.2H), 3.15 3.04 (m, 3.2H), 2.87 (dd, J=13.8, 7.5 Hz,
1H), 2.73 (s, 3.3H), 2.72 (s, 3H), 2.04 (s, 3.3H), 2.03 (s, 3H), 1.98 (s, 3H),
1.97 (s, 3.3H), 1.38 (d, J=7.1 Hz, 3H), 1.34 ppm (d, J=7.4 Hz, 3.3H);
13C NMR (126 MHz, D2O): d=179.4, 179.0, 175.2, 175.2, 174.7, 172.4,
172.3, 172.1, 171.7, 104.6, 104.5, 85.1, 83.7, 77.0, 76.9, 75.1, 72.6, 71.6, 71.6,
70.7, 68.7, 68.6, 61.4, 61.3, 61.2, 61.1, 55.9, 55.7, 53.9, 53.1, 50.2, 50.1, 48.9,
48.8, 32.0, 30.9, 26.0, 25.9, 22.1, 21.8, 16.6, 16.6 ppm; FTIR (neat film):
n˜ =3307, 1654, 1560, 1375, 1076 cmꢀ1; HRMS (FAB)+ : m/z calcd for
C26H45N5O15NaS [M+Na]+ : 722.2531; found: 722.2531.
Trisaccharide tripeptide conjugate 36:
A deoxygenated solution of
sodium methoxide (1.3 mg, 0.02 mmol, 4 equiv) in methanol (300 mL) was
added to a mixture of thiol 29 (6 mg, 0.006 mmol, 1.0 equiv) and tripep-
tide 9 (3 mg, 0.01 mmol, 1.9 equiv) at 238C. After 1.5 h, a white precipi-
tate was observed. The reaction mixture was stirred at 238C for a total of
16.5 h, and then neutralized by the addition of a solution of acetic acid in
methanol (1m, 25 mL, 0.02 mmol, 4.2 equiv) followed by concentration in
vacuo. The residue was purified by silica-gel flash chromatography (33%
to 75% methanol in dichloromethane), followed by LH 20 Sephadex
size-exclusion chromatography (methanol) to afford glycoconjugate 36
(5 mg, 88%, dr=1.7:1.0) as a white solid. Rf =0.08 (4:4:1:1 MeOH/
EtOAc/AcOH/H2O); 1H NMR (500 MHz, CD3OD): d=5.64 (d, J=5.4
Hz, 1H), 5.56 (d, J=5.6 Hz, 1.7H), 4.58 (m, 6.6H), 4.44 (d, J=7.6 Hz,
2.7H), 4.39 (dd, J=8.4, 3.5 Hz, 1.7H), 4.23 (m, 2.7H), 4.10 3.38 (m,
82.2H), 3.27 (dd, J=14.4, 8.2 Hz, 1.7H), 3.17 (dd, J=14.0, 6.8 Hz, 1H),
3.01 (dd, J=14.5, 4.2 Hz, 1H), 2.82 (m, 4.4H), 2.76 (s, 3H), 2.74 (s,
5.1H), 2.08 (s, 5.1 Hz), 2.05 (s, 3H), 2.02 (s, 3H), 2.01 (s, 5.1H), 1.95 (s,
8.1H), 1.58 ppm (m, 2.7H); 13C NMR (188 MHz, CD3OD): d=176.1,
175.5, 175.4, 174.6, 174.5, 174.1, 173.9, 173.9, 173.2, 173.0, 172.3, 172.2,
172.0, 106.0, 102.4, 87.3, 78.7, 78.6, 76.8, 74.9 (multiple resonances of pyr-
anoside carbons), 74.7, 74.5, 72.9 (multiple resonances of pyranoside car-
bons), 72.5, 71.8, 70.4, 70.3, 70.3, 70.1 (multiple resonances of pyranoside
carbons), 69.7, 69.6, 69.5, 65.5, 64.5 (multiple resonances of pyranoside
carbons), 62.5 (multiple resonances of pyranoside carbons), 55.8, 54.1,
50.5, 44.2, 43.8, 42.2, 41.6, 34.3, 30.8 (multiple resonances of methyl car-
bons), 26.3 (multiple resonances of methyl carbons), 22.7 ppm (multiple
resonances of methyl carbons); FTIR (neat film): n˜ =3307, 1654, 1594,
1077, 1033 cmꢀ1; HRMS (ESI)ꢀ: m/z calcd for C35H57N6O22S [MꢀH]ꢀ:
945.3247; found: 945.3234.
Disaccharide tripeptide conjugate 34:
A deoxygenated solution of
sodium methoxide (2.6 mg, 0.05 mmol, 4 equiv) in methanol (570 mL) was
added to a mixture of thiol 24 (10 mg, 0.01 mmol, 1 equiv) and tripeptide
9 (4 mg, 0.01 mmol, 1.2 equiv) at 238C. The reaction mixture was stirred
at 238C for 14 h and then neutralized by the addition of a solution of
acetic acid in methanol (1m, 52 mL, 0.05 mmol, 4.2 equiv) followed by
concentration in vacuo. The residue was purified by silica-gel flash chro-
matography (33% to 67% methanol in dichloromethane), followed by
LH 20 Sephadex size-exclusion chromatography (methanol) to afford gly-
Trisaccharide tripeptide conjugate 37:
A deoxygenated solution of
sodium methoxide (1.4 mg, 0.026 mmol, 4 equiv) in methanol (345 mL)
was added to a mixture of thiol 29 (7 mg, 0.007 mmol, 1.0 equiv) and tri-
peptide 10 (3 mg, 0.01 mmol, 1.7 equiv) at 238C. The reaction mixture
was stirred at 238C for a total of 17 h, during which time a white precipi-
tate was formed. The reaction mixture was neutralized by the addition of
a solution of acetic acid in methanol (1m, 28 mL, 0.03 mmol, 4.2 equiv)
followed by concentration in vacuo. The residue was purified by silica-gel
flash chromatography (33% to 75% methanol in dichloromethane), fol-
lowed by LH 20 Sephadex size-exclusion chromatography (methanol) to
afford glycoconjugate 37 (5 mg, 75%, dr=1.2:1.0) as a white solid. Rf =
0.10 (4:4:1:1 MeOH/EtOAc/AcOH/H2O); 1H NMR (500 MHz, CD3OD):
d=5.62 (d, J=5.6 Hz, 2.2H), 4.56 3.44 (m, 53H), 3.14 (m, 2H), 2.83 (m,
4.6 Hz), 2.73 (s, 6.6H), 2.11 (s, 3H), 2.04 (s, 3.6H), 2.01 (s, 6.6H), 1.96
coconjugate 34 (8 mg, 84%, dr=1.3:1.0) as
a white solid. Rf =0.15
(4:4:1:1 MeOH/EtOAc/AcOH/H2O); 1H NMR (500 MHz, CD3OD): d=
5.61 (d, J=5.5 Hz, 1H), 5.53 (d, J=1.3 Hz, 1.3H), 4.67 (t, J=7.1 Hz,
1H), 4.57 (dd, J=8.0, 3.9 Hz, 1.3H), 4.41 (m, 3.9H), 4.34 (dd, J=7.4, 4.4
Hz, 1.3H), 4.06 3.51 (m, 40H), 3.22 (dd, J=14.4, 8.3 Hz, 1.3H), 3.14 (dd,
J=13.8, 6.9 Hz, 1H), 3.00 (dd, J=14.4, 4.2 Hz, 1H), 2.82 (m, 3.7H), 2.75
(s, 3H), 2.74 (s, 3.9H), 2.08 (s, 3.9H), 2.04 (s, 3H), 2.02 (s, 7H), 1.96 (s,
7H), 1.57 ppm (m, 2.3H); 13C NMR (126 MHz, CD3OD): d=175.8,
175.8, 175.7, 174.7, 174.7, 174.3, 174.1, 174.1, 173.8, 173.4, 173.4, 173.2,
173.2, 172.4, 172.3, 87.7, 87.1, 74.9, 74.7, 73.1, 73.0, 72.3, 72.0, 70.4, 70.3,
69.8, 69.7, 69.5, 69.4, 65.5, 65.4, 64.8, 64.7, 56.1, 56.0, 54.3, 54.1, 52.0, 50.1,
49.8, 44.4, 44.1, 44.0, 43.9, 42.5, 42.0, 34.6, 26.5, 23.1, 22.9, 22.8, 22.8, 22.8
6005
Chem. Eur. J. 2003, 9, 5997 6006
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

¬
D. Y. Gin, W. A. van der Donk, and D. P. Galonic
FULL PAPER
(m, 6.6H), 1.72 (t, J=11.3 Hz, 1H), 1.66 (t, J=12.0 Hz, 1H), 1.39 (d, J=
7.2 Hz, 3H), 1.35 ppm (d, J=7.3 Hz, 3.6H); 13C NMR (188 MHz,
CD3OD): d=175.7, 175.5, 175.5, 175.4, 174.5, 174.0, 173.9, 173.9, 173.7,
173.5, 173.2, 172.5, 172.2, 106.0, 102.2, 102.0, 85.8, 78.8 (multiple resonan-
ces of pyranoside carbons), 76.8, 74.9, 74.8, 74.6, 74.5, 73.0 (multiple reso-
nances of pyranoside carbons), 72.8 (multiple resonances of pyranoside
carbons), 72.5, 71.4, 71.3, 70.2 (multiple resonances of pyranoside car-
bons), 69.9, 69.6 (multiple resonances of pyranoside carbons), 65.1, 64.5
(multiple resonances of pyranoside carbons), 63.4, 63.1, 62.5, 62.5, 57.6,
57.3, 56.2, 54.0, 53.9, 51.1, 51.0, 50.5, 42.3, 40.9, 32.8, 30.8 (multiple reso-
nances of methyl carbons), 26.5 (multiple resonances of methyl carbons),
22.7 (multiple resonances of methyl carbons), 22.6 (multiple resonances
of methyl carbons), 17.9 ppm; FTIR (neat film): n˜ =3306, 1638, 1377,
[7] For other examples of chemical ligations via oxime linkers, see:
a) S. E. Cervigni, P. Dumy, M. Mutter, Angew. Chem. 1996, 108,
1325 1328; Angew. Chem. Int. Ed. Engl. 1996, 35, 1230 1232; b) Y.
Zhao, S. B. H. Kent, B. T. Chait, Proc. Natl. Acad. Sci. USA 1997,
94, 1629 1633; c) E. C. Rodriguez, K. A. Winans, D. S. King, C. R.
Bertozzi, J. Am. Chem. Soc. 1997, 119, 9905 9906; d) H. Liu, L.
Wang, A. Brock, C.-H. Wong, P. G. Schultz, J. Am. Chem. Soc. 2003,
125, 1702 1703.
[8] a) E. Baran, S. Drabarek, Pol. J. Chem. 1978, 52, 941 946; b) M.
Gerz, H. Matter, H. Kessler, Angew. Chem. 1993, 105, 311 313;
Angew. Chem. Int. Ed. Engl. 1993, 32, 269 271; c) L. A. Marcaur-
elle, C. R. Bertozzi, Chem. Eur. J. 1999, 5, 1384 1390; d) E. Bous-
quet, A. Spadaro, M. S. Pappalardo, R. Bernardini, R. Romeo, L.
Panza, G. Ronsisvalle, J. Carbohydr. Chem. 2000, 19, 527 541.
[9] Y. Zhu, W. A. van der Donk, Org. Lett. 2001, 3, 1189 1192.
[10] N. M. Okeley, Y. Zhu, W. A. van der Donk, Org. Lett. 2000, 2,
3603 3606.
1074 cmꢀ1
;
HRMS (ESI)ꢀ: m/z calcd for C37H61N6O23S [MꢀH]ꢀ:
989.3509; found: 989.3499.
[11] For a related glycosylated STA peptide sequence in MUC1 see: P.
L. Devine, I. F. C. McKenzie, BioEssays 1992, 14, 619 625, and ref-
erence [1a].
Acknowledgement
[12] S. Knapp, D. S. Myers, J. Org. Chem. 2002, 67, 2995 2999.
[13] R. R. Schmidt, W. Kinzy, Adv. Carbohydr. Chem. Biochem. 1994, 50,
21 123.
[14] G. Grundler, R. R. Schmidt, Liebigs Ann. Chem. 1984, 1826 1847.
[15] a) B. A. Garcia, J. L. Poole, D. Y. Gin, J. Am. Chem. Soc. 1997, 119,
7597 7598; b) B. A. Garcia, D. Y. Gin, J. Am. Chem. Soc. 2000, 122,
4269 4279.
[16] See, for example: X.-S. Ye, C.-H. Wong, J. Org. Chem. 2000, 65,
2410 2436.
[17] J. M. Haberman, D. Y. Gin, Org. Lett. 2001, 3, 1665 1668.
[18] a) R. Brossmer, H. Mack, Tetrahedron Lett. 1981, 22, 933 936;
b) G. B. Kok, M. Campbell, B. Mackey, M. von Itzstein, J. Chem.
Soc. Perkin Trans. 1 1996, 2811 2815.
[19] The successful a-GalNHAc ligation complements our previous ini-
tial report of b-GlcNHAc ligations with monosaccharides (ref. [9]).
[20] For examples of the synthesis of C1-S-monosaccharide-amino acid/
dipeptide conjugates, see: a) S. B. Cohen, R. L. Halcomb, Org. Lett.
2001, 3, 405 407; b) S. Knapp, D. S. Myers, J. Org. Chem. 2001, 66,
3636 3638; c) S. B. Cohen, R. L. Halcomb, J. Am. Chem. Soc. 2002,
124, 2534 2543.
[21] For the synthesis of b-thio mono- and disaccharide peptide conju-
gates via the glycosylation of cysteine- and homocysteine-containing
peptides with glycosyl halides, see: X. Zhu, K. Pachamuthu, R. R.
Schmidt, J. Org. Chem. 2003, 68, 5641 5651.
This research was supported by the National Institutes of Health
(GM58833 and GM58822). UIUC Lycan and Perel Predoctoral Fellow-
ships to DPG are gratefully acknowledged.
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[5] For approaches to the synthesis of the a-GalNHAc-Ser/Thr linkage,
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Received: June 30, 2003 [F5290]
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Chem. Eur. J. 2003, 9, 5997 6006