Highly efficient preparation of tumor antigen-containing glycopeptide building blocks from novel pentenyl glycosides

Article abstract of DOI:10.1016/S0008-6215(03)00323-9

O-Glycosylated amino acids containing the tumor-associated T(T _f)-antigen (β-D-Gal-(1→3)-α-D-GalNAc) disaccharide unit were conveniently synthesized in seven steps starting from D-galactose via an n-pentenyl glycoside (NPG) building block. Azidonitration of 3,4,6-tri-O-acetyl-D-galactal, followed by nitrate displacement with simultaneous acetate hydrolysis with sodium 4-penten-1-oxide, afforded n-pentenyl 2-deoxy-2-azidogalactoside (3) in near quantitative yield. Subsequent high-yielding transformations resulted in the synthesis of the key glycosyl donor n-pentenyl β-disaccharide 5 that was employed for the stereospecific preparation of glycosyl amino acids via NIS-promoted glycosylations with serine or threonine acceptors. The surprising utility of the reaction of sodium 4-penten-1-oxide with anomeric nitrates encouraged the detailed exploration of the action of a variety of nucleophiles on anomeric nitrates for the synthesis of useful 2-azido glycosyl donors directly from the 'classic' Lemieux azidonitration product of protected galactals. This expedient synthesis (28% overall yield from 1 to 7a) that makes use of heretofore rarely exploited pentenyl 2′-azidoglycosides, should be a valuable entry in the armamentarium of routes to biologically relevant glycopeptides in both mono- and multivalent forms.

Full text of DOI:10.1016/S0008-6215(03)00323-9

Carbohydrate Research 338 (2003) 1925ꢀ1935
/
www.elsevier.com/locate/carres
Highly efficient preparation of tumor antigen-containing glycopeptide
building blocks from novel pentenyl glycosides
Serge A. Svarovsky, Joseph J. Barchi, Jr.*
Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute, Frederick, National Institutes of Health, 376 Boyles Street,
Frederick, MD 21702, USA
Received 22 April 2003; accepted 13 June 2003
Abstract
O-Glycosylated amino acids containing the tumor-associated T(Tf)-antigen (b-
conveniently synthesized in seven steps starting from -galactose via an n-pentenyl glycoside (NPG) building block. Azidonitration
of 3,4,6-tri-O-acetyl- -galactal, followed by nitrate displacement with simultaneous acetate hydrolysis with sodium 4-penten-1-
D
-Gal-(10
/
3)-a-D-GalNAc) disaccharide unit were
D
D
oxide, afforded n-pentenyl 2-deoxy-2-azidogalactoside (3) in near quantitative yield. Subsequent high-yielding transformations
resulted in the synthesis of the key glycosyl donor n-pentenyl b-disaccharide 5 that was employed for the stereospecific preparation
of glycosyl amino acids via NIS-promoted glycosylations with serine or threonine acceptors. The surprising utility of the reaction of
sodium 4-penten-1-oxide with anomeric nitrates encouraged the detailed exploration of the action of a variety of nucleophiles on
anomeric nitrates for the synthesis of useful 2-azido glycosyl donors directly from the ‘classic’ Lemieux azidonitration product of
protected galactals. This expedient synthesis (28% overall yield from 1 to 7a) that makes use of heretofore rarely exploited pentenyl
2?-azidoglycosides, should be a valuable entry in the armamentarium of routes to biologically relevant glycopeptides in both mono-
and multivalent forms.
Published by Elsevier Ltd.
Keywords: T-Antigen; O-Glycopeptide; Stereoselective; Pentenylation
1. Introduction
(hereafter referred to as T antigen) (Gal-b(10
/
3)-a-
D
-
-
GalNac-Ser/Thr) and its biological precursor Tn (a-
D
Neoplastic transformation is generally accompanied by
structural alterations in cell-surface oligosaccharides.1
These changes are due to the altered expression of
specific glycoprocessing enzymes that are characteristic
of a particular tumorigenic phenotype. This results in
the accumulation of unusual glycosidic structures that
are recognized as non-self by the host immune system.
These tumor-associated antigens (TAA) are often used
as markers of the tumorigenic process. Several abnormal
glycans are found attached to cell-surface mucin pro-
teoglycans as truncated O-linked oligosaccharides. Two
of the more prevalent TAAs that are often found on
carcinoma-derived mucins, but much less frequently in
GalNAc-Ser/Thr) antigen (as well as sialylated ana-
logs).2 Specific immuno-based therapies and vaccine
constructs have been based on these structures.3
Due to the difficulty in isolating distinct, homoge-
neous and immunogenic glycopeptides from tumor
tissue, the majority of research aimed at carbohydrate-
based immunotherapy has relied on the use of semisyn-
thetic or fully synthetic immunogens.4 Since some recent
results5 in this field have shown clinical promise, it is
highly desirable to develop quick, inexpensive, and,
most importantly, highly stereoselective syntheses of
tumor-associated carbohydrate structures as well as the
glycosylated protein units (glycopeptides) from which
they are derived. Numerous chemical approaches to
these building blocks have been developed over the
years.6 Most of these procedures rely heavily on con-
ventional glycosyl donors (e.g., anomeric bromides,
trichloroacetimidates6g and thioglycosides6a) that do
normal tissues are the ThomsenꢀFriedenreich antigen
/
* Corresponding author. Fax: ꢁ1-301-8466033.
/
E-mail address: barchi@helix.nih.gov (J.J. Barchi, Jr.).
0008-6215/03/$ - see front matter. Published by Elsevier Ltd.
doi:10.1016/S0008-6215(03)00323-9

1926
S.A. Svarovsky, J.J. Barchi, Jr. / Carbohydrate Research 338 (2003) 1925ꢀ1935
/
not offer orthogonality or access to conjugation of the
saccharide to multivalent scaffolds, thus hindering the
synthetic flexibility of these methods. In this paper we
report a new route to T antigen glycoamino acid
building blocks via hydrolytically stable n-pentenyl
glycosides (NPGs).7
ciated with each individual substrate anomer is more
variable and the reversal of stereochemistry observed
was coincidental. Further proof for the stereospecificity
of the reaction came when pure anomers were reacted
similarly (see Section 3), and only pentenyl anomers
with complete stereochemical inversion were observed.
Benzylidenation of the mixture 3a,b with benzaldehyde
dimethylacetal afforded glycosyl acceptors 4a,b in 70ꢀ
/
75% yield using either p-toluenesulfonic acid or cam-
2. Results and discussion
phorsulfonic acid as a catalyst. At this point, the a/b
anomeric mixture was easily separable (DRf(a/b)ꢀ
1:1 hexanesꢀEtOAc), and the synthesis was carried
forward independently with each anomer.
/0.3 in
Our approach involves a novel synthesis of precursors to
2-acetamido-NPGs and other glycosyl donors starting
from the ‘classic’ azidonitration products of protected
galactals developed by Lemieux.8 Galactal 1 was
synthesized most expediently in 75% overall yield in
the one-pot procedure of Kozikowski and Lee,9 starting
/
Encouraged by the efficiency of the reaction of
sodium 4-penten-1-oxide to produce the NPG 3, we
undertook a more comprehensive exploration of the
reaction of nitrate 2 with various nucleophiles (Fig. 1).
Our primary objective was to examine the utility of
preparing other glycosyl donors by nucleophilic nitrate
displacement. Lemieux had used both bromide and
chloride ion to prepare anomeric halides from the
azidonitration product.8 A survey of the literature
revealed that anomeric xanthates11d and phosphates,11b
two important classes of glycosyl donors, have been
synthesized directly from glycosyl nitrates. A report of a
C-glycosylation of the Lemieux’ nitrate with allyltri-
from
D-galactose. Azidonitration of 1 following Le-
mieux’ procedure afforded a mixture of a- and b-nitrates
2 (Scheme 1) whose anomeric ratios (between 2:1 and
1:1 a/b) depended on subtle changes in reaction condi-
tions. This mixture was then pentenylated and deacety-
lated in one convenient and nearly quantitative step
using a solution of sodium metal (1.5 equiv) in 4-penten-
1-ol as solvent/reactant to give the inseparable mixture
of pentenyl glycosides 3. Similar nitrate displacements
using sodium methoxide10 or other anions of heteroa-
toms have been previously described.11 To our knowl-
edge, this is a novel route for preparing pentenyl
glycosides. The reaction evidently proceeded with com-
plete inversion of configuration at the anomeric center:
It was readily shown by NMR (see Section 3) that the
ratio of anomeric configuration in the nitrate mixture
was exactly reversed after reaction with the alkoxide
(anomers separated after the next step, see below).
However, the observed product composition does not
rule out the possibility that the stereoselectivity asso-
methylsilane and BF3×
/
OEt2 has also appeared.12
We reasoned that the utility of the 2-azido-nitrate
would be enhanced if other glycosyl donors could be
prepared directly from the mixture of nitrate esters 2. A
series of nucleophiles were tested regarding their reac-
tivity toward the compound 2. We tested several
nitrogen, sulfur, and selenium anions, as well as fluoride
anions derived from different sources (TBAF, LiF, KF,
spray-dried KF). With a wide variety of nucleophiles
under a series of reaction conditions, the major product
was simple nitrate removal to afford hemiacetal 8. Sinay
¨
and co-workers11d had previously reported that,
although xanthic acid anions will displace an anomeric
nitrate, simple thiols and base readily effected their
reduction,13 a transfomation that we also observed with
all thiolate anions that we tested. The more potent
reducing power of selenium was evident by the rapid
(B1 min) and quantitative reduction of the nitrate
/
mixture after treatment with benzene selenol in the
presence of Hunig’s base at room temperature. This is
one of the more effective and rapid reductions of
anomeric nitrates reported to date. The only positive
result regarding nitrate displacement was with potas-
sium thiocyanate (KTC), which gave a moderate yield
(50%) of the anomeric TC 9. In the interest of efficiency,
we studied this reaction on the peracetylated material
direct from the galactal azidonitration, although the
presence of acetate esters was potentially a burden for
many of the reaction conditions. Similar reaction of the
Scheme 1. (i) NaN3, CAN, MeCN, 59ꢀ
1-ol, 95%; (iii) PhCH(OMe)2, PTSA, MeCN, 70ꢀ
/
65%; (ii) Na, 4-penten-
75%.
/

S.A. Svarovsky, J.J. Barchi, Jr. / Carbohydrate Research 338 (2003) 1925ꢀ
/1935
1927
Fig. 1. Reactions of anomeric nitrates with various nucleophiles. Known procedures are marked with the appropriate reference in
brackets over the reaction arrow.
nitrates when alternative protecting groups are em-
ployed is the subject of a future report.
Progression to a T-antigen-containing serine deriva-
tive proceeded smoothly from compound 4 (Scheme 2).
Both acceptors 4a,b coupled to 2,3,4,6-tetra-O-acetyl-a-
D-galactopyranose trichloroacetimidate (prepared in
two steps from pentaacetyl galactose in 90% yield)14
under standard glycosylation conditions15 in nearly
quantitative yield (95% recovered after chromatogra-
phy) and with exclusive b stereoselectivity. The resulting
pentenyl disaccharide donors 5a,b were reacted with the
protected amino acid acceptors 6a,b using the standard
NISꢀstoichiometric TESOTf activation protocol pio-
/
neered by Fraser-Reid16 to afford glycosyl amino acids
7a,b in 64 and 43% yields, respectively. The reaction was
only effective with the donor 5b (see below). It is
interesting to note that when catalytic amounts of
TESOTf were employed, the coupling reaction pro-
ceeded with significantly lower efficiency.
Several aspects of glycosylations of the n-pentenyl 2?-
azidodisaccharides with amino acid acceptors are note-
worthy. A review of the literature reveals very few
reports of glycosylations employing n-pentenyl 2?-azi-
doglycosyl donors of any sugar class.17 Many of these
reported poor yields or abject failure of reactions with
various donors. Thus, we were pleased that good yields
(consistent 60ꢀ65%) of glycosylation products were
/
obtained with the b pentenyl glycoside 5b with the
serine derivative 6a. Threonine 6b also coupled but, as
expected, in lower yield (43%). The reactions of 5b with
6a,b were essentially stereospecific: virtually no b-
Scheme 2. (i) TMSOTf, CH2Cl2, MS, 90ꢀ
/
95%; (ii) NIS,
TESOTf, CH2Cl2, MS, 65% (RꢂH), 43% (Rꢂ
/
/CH3).

1928
S.A. Svarovsky, J.J. Barchi, Jr. / Carbohydrate Research 338 (2003) 1925ꢀ1935
/
anomer of the glycosylated amino acids was detected
during purification. To our surprise, the a-anomer 5a
through similar trichloracetimidate intermediates to
both serine and threonine-coupled T antigen building
blocks. The ease and efficiency of these additional steps
highlights the adaptability of this route when faced with
specific ‘unresponsive’ NPGs.
We reasoned that the cis-1,2 disposition of the n-
pentenyl and azido groups in the a-pentenyl glycosides
may facilitate interaction of these two functionalities
during the activation step of the glycosylation.19 Ac-
cordingly, both anomers 5a,b were reduced to the 2?-
gave very poor yields (B10%) and much slower reaction
/
rates with both 6a and 6b. The unexpected resistance to
glycosylation of 5a can be overcome by removal of the
pentenyl group with CH3CNꢀ
/
H2Oꢀ
NBS18 to 10a,b,
/
followed by trichloroacetimidate formation to the donor
11a (this was also accomplished with 5b) and reaction
with the aforementioned acceptors to give good yields of
both serine and threonine T-antigen-containing amino
acids (Scheme 3). It should be noted that although the
yields of these glycosylations are superior to those with
the pentenyl donors, the stereoselectivity was not as high
(7% and 14% of the b-anomer for the serine and
threonine products, respectively) These results also
mirror those of Qui and co-workers6h who proceeded
acetamido sugars 12a,b with excess zinc in 10:5:1 THFꢀ
/
AcOHꢀ 88%) and subjected to glycosyla-
/
Ac2O20 (80ꢀ
/
tion reactions with the protected serine 6a (Scheme 3).
Surprisingly, even with the donor 12b, very poor yields,
if any, were realized under identical glycosylation
conditions. These results may highlight the ‘disarming’
effect of the 2?-acetamido or azido group for this system
under activating conditions.
Preparation of Tn-containing amino acids was rea-
lized from common intermediate 4 via NPG 13 (Scheme
4). Reaction under identical NISꢀstoichiometric TE-
/
SOTf conditions with 6a gave known chloroacetate 14 in
a yield similar to that obtained during the preparation of
7a. Similar to the reaction of NPG 5a with 6a, pentenyl
Scheme 3. (i) Znꢀ
H2O, NBS, 60%; (iii) CCl3CN, CH2Cl2, DBU, 95%; (iv) 6a,b,
TMSOTf, CH2Cl2, MS, 90ꢀ95%; (v) 6a, NIS, TESOTf,
CH2Cl2, MS.
/
AcOHꢀ
/
Ac2Oꢀ
/
THF, 80ꢀ
/
88%; (ii) CH3CN,
/
Scheme 4. (i) (ClCH2CO)2O, Pyr, CH2Cl2, 93ꢀ
/
97%; (ii) 6a,
NIS, TESOTf, CH2Cl2, MS, 51% (13b), B5% (13a).
/

S.A. Svarovsky, J.J. Barchi, Jr. / Carbohydrate Research 338 (2003) 1925ꢀ
/1935
1929
glycoside 13a gave very poor yields of 14. This
procedure could be thought of as an alternative
assembly strategy for construction of the T antigen
disaccharide and other higher order sugar derivatives.
While this route offers little advantage in overall yield
over other methods,6 the presence of the pentenyl
glycoside also facilitates further manipulations of this
intermediate (vide infra).
Pentenyl glycosides have been used as orthogonal
protecting groups that also may be used as ‘spacers’ to
attach pendant oligosaccharides to relevant biological
probes or proteins through modifications of the terminal
olefin.21 This is another advantage of the pentenyl
group, whereas preparation of Tn and T antigens
clusters may be elaborated via derivatives of compounds
4a and 5a, respectively (Scheme 5). Thus, using a wide
range of chemistries, the pentenyl group may be
transformed into various functional groups for further
manipulations or to synthesize multivalent Tn, T-con-
taining constructs. Progress toward the development of
multivalent T-antigen displays will be outlined in a
future report.
3.2. General methods
Melting points were determined on a Fisherꢀ
/
Johns
melting point apparatus and are uncorrected. Rf values
refer to TLC performed on Analtech Uniplates GF pre-
coated with Silica Gel 60 to a thickness of 0.25 mm. The
spots were visualized by charring with a solution of
ammonium molybdate(IV) tetrahydrate (12.5 g) and
cerium(IV) sulfate tetrahydrate (5.0 g) in 10% aq H2SO4
(500 mL). All purifications were performed by flash
chromatography (FC) under medium pressure using
Silica Gel 60 (230ꢀ400 mesh, E. Merck) unless stated
/
otherwise. These purifications usually employed a step-
wise solvent polarity gradient, correlated with TLC
mobility.
NMR spectra were recorded on Varian Inova 400
instrument with residual CHCl3 (7.26 ppm) as the
1
internal standard at frequencies of 399.74 MHz for H
and 100.51 MHz for 13C. Assignments were based on
gCOSY, ROESY, and 13C/DEPT experiments. 1H
NMR data are tabulated in the order of multiplicity
(s, singlet; d, doublet; dd, doublet of doublets; dt, double
of triplets; t, triplet; q, quartet; m, multiplet; brs, broad
signal), number of protons, and coupling constant(s) in
hertz. IR spectra were taken with a JASCO FT/IR-615
spectrometer. Specific optical rotations were determined
using a JASCO-P1010 polarimeter in 0.5-dm cuvettes at
589 nm in chloroform. Five consecutive measurements
were taken, and the average value is given. Positive-ion
fast-atom bombardment mass spectra (FABMS) were
obtained at an accelerating voltage of 6 kV and a
resolution of 2000. Glycerol was used as the sample
matrix, and ionization was effected by xenon atoms.
Elemental analyses were performed by Atlantic Micro-
lab, Inc., Norcross, GA.
In conclusion, the highly useful preparation of
pentenyl glycosides from anomeric nitrates allows
efficient construction of glycoamino acid building
blocks in high overall yield (28% from commercial
material to 7a). The stereoselectivity and adaptability
of this route should facilitate the synthesis of biologi-
cally relevant glycopeptides on a large scale.
3. Experimental
3.1. Abbreviations
CAN (ceric ammonium nitrate); DBU (1,8-diazabicy-
clo[5.4.0]undec-7-ene); DIPEA (N,N-diisopropylethyl-
amine); EtOAc (ethyl acetate); NBS (N-bromosuccini-
mide); TMSOTf (trimethylsilyl trifluoromethanesulfo-
nate); TESOTf (triethylsilyl trifluoromethanesulfonate);
Unless otherwise noted, all chemicals were purchased
from Aldrichꢀ
further purification. N-Iodosuccinimide (NIS) was re-
crystallized twice from dioxaneꢀcarbon tetrachloride
prior to use. Activated, powdered 4A molecular sieves
(average diameter, 2ꢀ
/Sigma (Milwaukee, WI) and used without
/
˚
a-Gal-TCA
(1-O-(2,3,4,6-tetra-O-acetyl-a-
D-galacto-
/
3 mm, CAS # 70955-01-0) were
pyranosyl) 2,2,2-trichloroacetimidate; TLC (thin-layer
chromatography); FC (flash chromatography); MS
(molecular sieves).
flame dried and used for sensitive glycosylation reac-
tions.
Scheme 5.

1930
S.A. Svarovsky, J.J. Barchi, Jr. / Carbohydrate Research 338 (2003) 1925ꢀ1935
/
3.3. 4-Pentenyl 2-azido-2-deoxy-b-
(3b)
D
-galactopyranoside
J 7.81 Hz, H-1), 4.12 (dd, 1 H, J 1.2, 3.7 Hz, H-4), 4.07
(dd, 1 H, J 1.95, 12.5 Hz, H-6b), 3.90 (dt, 1 H, J 6.2, 9.5
Hz, OCH2CH2CH2CHÄ
H-3), 3.54 (dt, 1 H, J 6.8, 9.5 Hz, OCH2CH2CH2CHÄ
CH2), 3.38 (m, 1 H, H-5), 2.59 (d, 1 H, J 8.5 Hz, OH),
2.11ꢀ2.20 (m, 2 H, ÃCH2CHÄCH2), 1.66ꢀ1.82 (m, 2 H,
CH2CH2CHÄ
CH2); 13C NMR (100 MHz, CDCl3): d
138.23 (ÃCHÄCH2); 137.52, 129.56, 128.50, 126.59
(Ph), 115.22 (ÃCHÄCH2), 102.35 (PhCH), 101.63 (C-
1), 74.79, 71.59, 69.62, 69.24, 66.70
(Ã
OCH2CH2CH2CHÄCH2), 64.35 (C-6), 30.25, 28.88 (Ã
/
CH2), 3.47ꢀ3.61 (m, 2 H, H-2,
/
Sodium metal (46 mg, 2 mmol) was dissolved in 5 mL of
4-penten-1-ol at room temperature (rt), and a solution
of 506 mg (1.33 mmol) of 2a in a minimum amount of 4-
/
/
/
/
/
penten-1-ol was added. After 5 min, TLC (1:1 hexanesꢀ
/
Ã
/
/
ethyl acetate) indicated complete consumption of 2a.
The reaction was stirred for an additional 30 min and
carefully neutralized with weakly acidic ion-exchange
resin (Amberlite IRC-50). The mixture was filtered, and
/
/
/
/
/
/
/
excess of 4-penten-1-ol was distilled at 50 8C (16ꢀ
mbar) and recycled. The residual yellow syrup was
purified by FC (5ꢀ10% MeOH in CH2Cl2) to afford
364 mg (99.2%) of the title compound as a amorphous
solid. Rf 0.2 (10% MeOH in CH2Cl2, v/v); [a]D 538 (c
/18
CH2CH2CHÄ
/
CH2). FABMS: m/z (relative intensity)
362.2 (MHꢁ). Anal. Calcd for C18H23N3O5: C, 59.82;
H, 6.41; N, 11.63. Found: C, 59.77; H, 6.39; N, 11.58.
/
ꢁ
/
3.5. 4-Pentenyl 2-azido-4,6-O-benzylidene-2-deoxy-a-
galactopyranoside (4a)
D-
1.13, CHCl3); IR (neat): 3321.78, 2977.55, 2916.81,
2866.67, 2111.67, 1642.09; 1H NMR (400 MHz,
CDCl3): d 5.73ꢀ
(m, 2 H, ÃCHÄCH2), 4.27 (d, 1 H, J 7.95 Hz, bH-1),
3.37ꢀ4.02 (m, 8 H, H-2, H-3, H-4, H-5, H-6, ÃOCH2-
CH2CH2CHÄCH2), 2.11ꢀ2.19 (m, 2 H, ÃCH2CHÄ
CH2), 1.67ꢀ1.79 (m, 2 H, ÃOCH2CH2CH2CHÄCH2);
13C NMR (100 MHz, CDCl3): d 138.09 (Ã
CHÄCH2),
115.29 (ÃCHÄCH2), 102.71 (C-1), 74.01, 72.34, 69.84,
69.27, 64.36 (ÃOCH2CH2CH2CHÄCH2), 63.12 (C-6),
30.19, 28.93 (Ã CH2). FABMS: m/z
/
5.85 (m, 1 H, Ã
/
CH Ä
/
CH2), 4.93ꢀ
/
5.05
The title compound was obtained from the mixture of
3a,b (a/bꢂ3:2) using the procedure above in 83% (33%
4aꢁ50% 4b) yield. Upon benzylidenation, 4a was easily
separated from 4b by FC and obtained as white crystals,
mp 67ꢀ69 8C. Rf 0.64 (1:1 hexanesꢀethyl acetate); [a]D
1538 (c 1.14, CHCl3); IR (neat): 3495.35, 2908.13,
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
ꢁ
/
1
/
/
2106.85, 1640.16; H NMR (400 MHz, CDCl3) d 7.36ꢀ
7.51 (m, 5 H, Ph), 5.76ꢀ5.87 (m, 1 H, ÃCH ÄCH2), 5.58
(s, 1 H, CHPh), 4.97ꢀ5.08 (m, 2 H, ÃCHÄCH2), 5.00 (d,
1 H, J 3.32 Hz, aH-1), 4.22ꢀ4.28 (m, 2 H, H-4, H-6a),
4.16 (td, 1 H, J 3.9, 10.5 Hz, H-3), 4.06 (dd, 1 H, J 1.95,
/
/
/
/
/
/
/
OCH2CH2CH2CHÄ
/
/
/
/
(relative intensity) 274.1 (MHꢁ), 547.2 (M2Hꢁ); Anal.
Calcd for C11H19N3O5: C, 48.35; H, 7.01; N, 15.38.
Found: C, 48.05; H, 7.00; N, 15.36.
/
12.88 Hz, H-6b), 3.67ꢀ
CH2CHÄCH2, H-5), 3.45ꢀ
CH2CHÄ
2.12ꢀ2.21 (m, 2 H, Ã
CH2CH2CHÄ
CH2); 13C NMR (100 MHz, CDCl3): d
138.06 (ÃCHÄCH2), 137.51, 129.56, 128.53, 126.41
(Ph), 115.32 (ÃCHÄCH2), 101.50, 98.92, 75.75, 69.48,
68.15, 67.56, 62.98, 60.90 (C-6), 30.40, 28.78 (Ã
/
3.74 (m, 2 H, Ã
/
OCH2CH2-
OCH2CH2-
When a mixture of nitrates 2 (2a/2bꢂ/1.45) was used
/
/
3.54 (m, 2 H, Ã
/
in the above procedure, n-pentenyl glycosides 3 were
obtained with nearly exact reversal of anomeric config-
/
CH2, H-2), 2.43 (d, 1 H, J 10.54 Hz, OH),
CH2CHÄCH2), 1.69ꢀ1.79 (m, 2 H,
/
/
/
/
uration (3b/3aꢂ/1.5). At this stage the anomers were
Ã
/
/
inseparable and were used as a mixture in the next step.
/
/
/
/
3.4. 4-Pentenyl 2-azido-4,6-O-benzylidene-2-deoxy-b-
galactopyranoside (4b)
D-
/
OCH2CH2CH2CHÄ
/
CH2); FABMS: m/z (relative in-
tensity) 362.2 (MHꢁ). Anal. Calcd for C18H23N3O5: C,
59.82; H, 6.41; N, 11.63. Found: C, 60.10; H, 6.44; N,
11.59.
Compound 3b (610 mg, 2.25 mmol) was dissolved in 15
mL of anhyd acetonitrile, and the solution was cooled to
0 8C. Dimethoxytoluene (0.5 mL, 0.74 g, 4.9 mmol) was
added, and the reaction was stirred for 5 min. p-
Toluenesulfonic acid (PTSA) (9.4 mg, 0.057 mmol)
was added to the reaction mixture at once, and the
reaction was stirred at 0 8C for 3 h. The solution was
diluted with EtOAc, quenched with a few drops of Et3N
and concentrated in vacuo. The resulting light-yellow
3.6. 4-Pentenyl 2-azido-4,6-O-benzylidene-2-deoxy-3-O-
(2,3,4,6-tetra-O-acetyl-b-
galactopyranoside (5b)
D-galactopyranosyl)-b-D-
Compound 4b (225 mg, 0.62 mmol) was combined with
437 mg (0.89 mmol) of a-Gal-TCA,14 evaporated twice
with dry toluene, and dried overnight under vacuum.
The dried reagents were dissolved in 5 mL of anhyd
CH2Cl2 and transferred via canula to a two-neck flask
syrup was purified by FC (2:10
acetate) to afford 646 mg (80%) of the title compound
as a white solid. Rf 0.28 (1:1 hexanesꢀethyl acetate);
[a]D 11.68 (c 1.0, CHCl3); IR (neat): 3447.13, 3070.12,
2872.45, 2109.74, 1640.16. 1H NMR (400 MHz, CDCl3):
d 7.35ꢀ7.53 (m, 5 H, Ph), 5.74ꢀ5.86 (m, 1 H, ÃCH Ä
CH2), 5.52 (s, 1 H, CHPh), 4.93ꢀ5.06 (m, 2 H, ÃCHÄ
CH2), 4.29 (dd, 1 H, J 1.56, 12.5 Hz, H-6a), 4.27 (d, 1 H,
/
1:1 hexanesꢀ/ethyl
/
˚
containing 500 mg of activated 4A molecular sieves
ꢃ
/
(flame-dried and flushed with argon immediately prior
to use). The reaction mixture was stirred at 0 8C for 30
min under argon. TMSOTf (10 mL, 0.06 mmol) was
carefully added to the reaction mixture. After 20 min,
/
/
/
/
/
/
/

S.A. Svarovsky, J.J. Barchi, Jr. / Carbohydrate Research 338 (2003) 1925ꢀ
/1935
1931
TLC (1:1 hexanesꢀ/ethyl acetate) showed only one spot
4.18 (dd, 1 H, J 6.24, 11.32 Hz, Hƒ-6a), 4.05ꢀ
/
4.13 (m, 2
corresponding to the product. The mixture was diluted
with CH2Cl2 and quenched with a few drops of Et3N.
The solution was filtered through a short pad of Celite†,
washed successively with satd NaHCO3, brine, and
water and dried over MgSO4. Purification by FC (2:3
H, Hƒ-6b, H?-3), 4.02 (dd, 1 H, J 1.56, 12.49 Hz, H?-6b),
3.77 (dd, 1 H, J 3.51, 10.54 Hz, H?-2), 3.67ꢀ3.74 (m, 1
H, ÃOCH2CH2CH2CHÄCH2), 3.65 (bs, 1 H, H?-5),
3.46ꢀ3.54 (m, 1 H, ÃOCH2CH2CH2CHÄCH2), 2.10ꢀ
2.17 (m, 2 H, ÃCH2CHÄCH2), 1.95, 2.01, 2.03, 2.13
(s, 12 H, Ac), 1.68ꢀ1.76 (m, 2 H, ÃOCH2CH2CH2CHÄ
/
/
/
/
/
/
/
/
/
hexanesꢀ
product as a white solid: mp 83ꢀ
(1:1, 2:3 hexanesꢀethyl acetate); [a]D
CHCl3); IR (neat): 2876.31, 2114.56, 1747.19, 1640.16,
1367.28, 1216.86; 1H NMR (400 MHz, CDCl3): d 7.48ꢀ
7.53 (m, 2 H, Ph), 7.28ꢀ7.37 (m, 3 H, Ph), 5.79 (m, 1 H,
CH ÄCH2), 5.36 (m, 1 H, Hƒ-4), 5.23 (m, 1 H, Hƒ-2),
4.92ꢀ5.04 (m, 3 H, ÃCHÄCH2, Hƒ-3), 4.79 (d, 1 H, J1,2
8.2 Hz, bHƒ-1), 4.25 (d, 1 H, J 7.8 Hz, H?-1), 4.00ꢀ4.32
(m, 5 H, H?-4, H?-6, Hƒ-6), 3.92ꢀ3.99 (m, 1 H, Ã
OCH2CH2CH2CHÄCH2), 3.86 (m, 1 H, Hƒ-5), 3.77
(dd, 1 H, J 7.81, 10.54 Hz, H?-2), 3.45ꢀ3.53 (m, 1 H, Ã
OCH2CH2CH2CHÄCH2), 3.42 (dd, 1 H, J 3.51, 10.54
Hz, H?-3), 3.34 (bs, 1 H, H?-5), 1.95, 2.01, 2.04, 2.13 (4s,
12 H, CH3), 2.08ꢀ2.17 (m, 2 H, ÃCH2CHÄCH2), 1.65ꢀ
1.78 (m, 2 H, ÃCH2CH2CHÄ
CH2), 13C NMR (100
MHz, CDCl3): d 170.53, 170.50, 170.38, 169.50, 138.23,
137.85, 129.05, 128.30, 126.41, 115.19 (ÃCHÄCH2);
/
EtOAc) afforded 422 mg (98.5%) of the
85 8C; Rf 0.15, 0.43
11.58 (c 1.53,
/
/
/
/
CH2), 13C NMR (100 MHz, CDCl3): d 170.48, 170.34,
169.61, 137.98, 137.86, 129.04, 128.31, 126.33, 115.34,
102.65, 100.84, 98.90, 76.06, 71.26, 69.39, 68.88, 68.13,
67.18, 63.28, 61.61, 59.05, 30.40, 28.77, 20.91, 20.76.
FABMS: m/z (relative intensity): 692.2 (MHꢁ). Anal.
/
ꢁ
/
/
/
Ã
/
/
Calcd for C32H41N3O14×
/
H2O: C, 54.16; H, 6.11; N, 5.92.
/
/
/
Found: C, 53.96; H, 5.78; N, 5.71
/
/
/
3.8. N-(9-Fluorenylmethoxycarbonyl)-O-[2-azido-4,6-O-
benzylidene-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-b-D-
galactopyranosyl)-a-galactopyranosyl]-L-serine allyl ester
(7a)
/
/
/
/
/
/
/
/
3.8.1. Method A. Disaccharide 5b (50 mg, 0.07 mmol)
was combined with 6a (24 mg, 0.065 mmol) and
/
/
evaporated with dry toluene (2ꢄ5 mL) before drying
/
/
/
under vacuum overnight. The mixture was dissolved in 3
mL of anhyd CH2Cl2 and transferred into 100 mg of
˚
flame-dried 4A molecular sieves. The solution was
treated with 18 mg (0.08 mmol) of freshly recrystallized
N-iodosuccinimide, followed by TESOTf (17 mL, 0.07
mmol). After 30 min the reaction mixture was diluted
with 10 mL of CH2Cl2, quenched with one drop of
Et3N, and filtered. The filtrate was washed consecutively
with 10% Na2S2O3, satd NaHCO3, and water, and dried
over MgSO4. The residue was purified by flash chroma-
102.55, 102.48, 102.18, 100.94, 78.65, 76.52, 75.24,
71.11, 71.02, 69.48, 69.18, 68.93, 67.16, 66.75, 62.59,
61.51, 30.23, 28.85, 20.91, 20.87, 20.77. FABMS: m/z
(relative intensity): 692.2 (MHꢁ). Anal. Calcd for
C32H41N3O14: C, 55.57; H, 5.97; N, 6.08. Found: C,
55.13; H, 6.12; N, 5.73.
3.7. 4-Pentenyl 2-azido-4,6-O-benzylidene-2-deoxy-3-O-
(2,3,4,6-tetra-O-acetyl-b-
galactopyranoside (5a)
D-galactopyranosyl)-a-D-
tography (2:1 petroleum etherꢀacetone) to give 40 mg
/
(64%) of 7a as an amorphous solid.
Compound 4a (550 mg, 1.52 mmol) and 970 mg (1.97
mmol) of a-Gal-TCA14 were dissolved in 12 mL of
anhyd CH2Cl2 containing 600 mg of 4A molecular sieves
˚
3.8.2. Method B. A mixture of 300 mg (0.39 mmol) of
11a with 240 mg (0.65 mmol, 1.67 equiv) of 6a was
dissolved in 5 mL of dry toluene and rotoevaporated to
dryness. This procedure was repeated twice. The dried
mixture was then dried under vacuum at 40 8C over-
night, dissolved in 6 mL of anhyd CH2Cl2 and
˚
transferred via canula to 500 mg of flame-dried 4A
molecular sieves. The solution was cooled to ꢃ15 8C
(thermostat), and 15 mL of TMSOTf was added. In 10
min TLC showed no disaccharide 11a. After 20 min the
reaction was diluted with CH2Cl2, quenched with two
drops of Et3N, filtered through a short pad of Celite†
and concentrated. The residue was flash-chromato-
under argon. The mixture was cooled to 0 8C and stirred
for 15 min prior to addition of 20 mL (0.12 mmol, 0.06
equiv) of TMSOTf. After 35 min, the reaction was
quenched with a few drops of Et3N, and the molecular
sieves were filtered on a short pad of Celite†. The
filtrate was successively washed with satd NaHCO3,
brine, and H2O and dried over MgSO4. After evapora-
tion, the residue was purified by flash chromatography
/
on silica gel with 3:2 hexanesꢀ
(91% yield) of white solid: mp 125ꢀ
EtOAcꢀhexanes); [a]D 54.78 (c 1.00, CHCl3); IR
(neat): 2108.78, 1747.19, 1367.28, 1216.86, 1044.26; H
NMR (400 MHz, CDCl3): d 7.28ꢀ7.38 (m, 3 H, Ph),
7.48ꢀ7.52 (m, 2 H, Ph), 5.72ꢀ5.84 (m, 1 H, ÃCH ÄCH2),
5.38 (dd, 1 H, J 1.17, 3.51 Hz, Hƒ-4), 5.52 (s, 1 H,
PhCH), 4.94ꢀ5.05 (m, 4 H, ÃCHÄCH2, aH?-1 (J1,2 3.51
/EtOAc to give 960 mg
/
127 8C; Rf 0.21 (3:2
/
ꢁ
/
1
/
graphed with 1:1 hexanesꢀ
(66%) of 7a as a white amorphous solid along with 27
mg (7%) of b-anomer (a/bꢂ9:1). Identification of the
/
EtOAc to afford 250 mg
/
/
/
/
/
1
/
/
/
product was ascertained by correlation of the H NMR
spectrum to literature reports22 and by elemental and
mass spectral analysis.
Hz), Hƒ-3), 4.76 (d, 1 H, J 8.20 Hz, bHƒ-1), 4.35 (d, 1 H,
J 2.73 Hz, H?-4), 4.22 (dd, 1 H, J 1.56, 12.49 Hz, H?-6a),

1932
S.A. Svarovsky, J.J. Barchi, Jr. / Carbohydrate Research 338 (2003) 1925ꢀ1935
/
3.9. N-(9-Fluorenylmethoxycarbonyl)-O-[2-azido-4,6-O-
benzylidene-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl)-a-galactopyranosyl]-L-threonine allyl
3.11. 3,4,6-Tri-O-acetyl-2-azido-2-deoxy-a,b-D-
galactopyranosyl thiocyanate (9)
D-
ester (7b)
A mixture of 50 mg (0.13 mmol) of nitrates 2 (a/bꢂ
/3:2)
was dissolved in 2 mL of anhyd CH3CN. Potassium
thiocyanate (58 mg, 0.6 mmol) was added, followed by
150 mg of 18-crown-6. The reaction was stirred at rt
overnight and diluted with ether. The organic layer was
washed with brine and water, dried over MgSO4 and
evaporated to dryness. The residue was purified by flash
3.9.1. Method A. Compound 5b (110 mg, 0.16 mmol)
and 73 mg (0.19 mmol) of 6b were combined, and the
mixture was azeotropically dried twice with toluene and
then dried at 40 8C under vacuum. The mixture was
dissolved in 5 mL of anhyd CH2Cl2, and the solution
˚
was transferred into 200 mg of flame-dried 4A molecular
chromatography with 3:1 hexanesꢀ
/
EtOAc to give 31 mg
(50%, incomplete reaction) of 9 as a clear oil (1:1
sieves. The resulting suspension was stirred under argon
for a few minutes, and 36 mg (0.16 mmol) of freshly
recrystallized N-iodosuccinimide was added, followed
by 40 mL (0.17 mmol) of TESOTf was added by syringe.
After 30 min, the reaction was diluted with CH2Cl2 and
quenched with a few drops of Et3N. The molecular
sieves were filtered through a short pad of Celite†. The
filtrate was washed with Na2S2O3, water, dried over
MgSO4 and concentrated. The residue was purified on
mixture of a- and b-anomers). Rf 0.50 (1:1 hexanesꢀ
EtOAc), IR (neat): 2161.81 (SCN), 2116.49 (N3),
1
1748.16 (Ac); H NMR (400 MHz, CDCl3): d 6.10 (d,
/
1 H, J1,2 5.08 Hz, aH-1), 5.47 (m, 1 H, aH-4), 5.39 (m, 1
H, bH-4), 5.04 (dd, 1 H, J 3.12, 10.93 Hz, aH-3), 4.94
(dd, 1 H, J 3.12, 10.15 Hz, bH-3), 4.51 (d, 1 H, J 9.76
Hz, bH-1), 4.43 (m, 1 H, H-5), 4.33 (dd, 1 H, J 5.08,
/
10.54 Hz, aH-2), 4.10ꢀ
/
4.21 (m, 4 H, bH-6, aH-6), 3.94ꢀ
silica gel with 3:1 petroleum etherꢀ/acetone to afford 66
4.04 (m, 2 H, bH-2, H-5), 2.03, 2.04, 2.05, 2.06, 2.15,
2.16 (s, 18 H, Ac), 13C NMR (100 MHz, CDCl3) (b-
anomer 9 from a-nitrate 2): d 170.49, 170.14, 169.70,
107.57 (SCN), 83.86, 76.05, 72.99, 66.24, 61.35, 60.58,
20.82, 20.74, 20.71; FABMS: m/z (relative intensity):
373.1 (MHꢁ). Anal. Calcd for C13H16N4O7S: C, 41.93;
H, 4.33; N, 15.05. Found: C, 42.32; H, 4.38; N, 14.68.
mg (43%) of the title compound as a white solid.
3.9.2. Method B. Compound 11a (100 mg, 0.13 mmol)
was combined with 60 mg (0.16 mmol, 1.2 equiv) of 6b,
azeotropically dried twice with dry toluene and dried
under vacuum at 40 8C for 12 h. The mixture was
dissolved in 6 mL of anhyd CH2Cl2 and transferred into
˚
200 mg of flame-dried 4A molecular sieves. The solution
was cooled to ꢃ
/
15 8C, and 6 mL of TMSOTf was added.
3.12. 2-Azido-4,6-O-benzylidene-2-deoxy-3-O-(2,3,4,6-
tetra-O-acetyl-b-D-galactopyranosyl)-a-D-
galactopyranosyl) 2,2,2,-trichloroacetimidate (11a)
After 20 min, the reaction was diluted with CH2Cl2,
quenched with two drops of Et3N, filtered through a
short pad of Celite† and concentrated. The residue was
purified by FC with 1.3:1 hexanesꢀ
/
EtOAc to afford 86
A mixture of free pyranoses 10a,b25 (770 mg, 1.23
mmol) was dissolved in 20 mL of anhyd CH2Cl2, the
solution was cooled to 0 8C, and 2 mL (2.88 g, 20 mmol)
of CCl3CN was added, followed by eight drops of DBU.
After 30 min, the solution was evaporated and purified
mg (67%) of the desired a-anomer and 18 mg (14%) of
the b-anomer. Identification of the product was ascer-
tained by correlation of the 1H NMR spectrum to
literature reports23 and by elemental and mass spectral
analysis.
by FC with 1:1 hexanesꢀ
a white solid (no b-anomer was detected). Rf 0.60 (2:1
EtOAcꢀhexanes); IR (neat): 3305.39, 2113.60, 1746.23,
1709.59; 1H NMR (400 MHz, CDCl3): d 8.72 (s, 1 H, Ä
NH), 7.47ꢀ7.52 (m, 2 H, Ph), 7.27ꢀ7.37 (m, 3 H, Ph),
/EtOAc to give 811 mg (85%) of
3.10. Hydrolysis of 2 with phenyl selenide
/
/
To a solution of 100 mg (0.63 mmol) of PhSeH in 3 mL
of anhyd CH3CN solution of a mixture of 2a,b 50 mg
(0.13 mmol) in minimum amount of CH3CN was added,
followed by one drop of DIPEA. The solution became
yellow in color. TLC taken immediately after addition
showed only one product corresponding to hemiacetal 8,
and no remaining 2a,b. The reaction was diluted with 10
mL of EtOAc, washed with satd NH4Cl and water, then
dried over MgSO4 and concentrated. Purification by
/
/
6.52 (d, 1 H, J 3.12 Hz, aH?-1), 5.52 (s, 1 H, PhCH),
5.37 (m, 1 H, Hƒ-4), 5.27 (dd, 1 H, J 8.20, 10.54 Hz, Hƒ-
2), 4.98ꢀ
bHƒ-1), 4.42 (m, 1 H, H?-4), 3.88ꢀ
/
5.03 (m, 1 H, Hƒ-3), 4.80 (d, 1 H, J 7.81 Hz,
/
4.25 (m, 6 H, H?-2, H?-
3, H?-6, Hƒ-6), 3.86 (m, 1 H, Hƒ-5), 3.82 (bs, 1 H, H?-5),
1.94, 2.00, 2.01, 2.13 (s, 12 H, Ac). 13C NMR (100 MHz,
CDCl3): d 170.46, 170.43, 170.28, 169.57, 160.62,
137.66, 129.19, 128.38, 126.31, 102.37, 100.88, 95.99,
75.99, 75.40, 71.27, 71.19, 68.91, 67.23, 65.60, 61.73,
58.75, 20.92, 20.89, 20.74; FABMS: m/z (relative
intensity): 606.2 (MHꢁ). Anal. Calcd for C34H45NO15:
C, 52.01; H, 5.33; N, 6.74. Found: C, 51.77; H, 5.43; N,
6.47
flash chromatography (1:1 hexanesꢀEtOAc) afforded 40
/
mg (93%) of hemiacetal 8 as a clear oil. Identification of
1
the product was ascertained by correlation of the H
NMR spectrum to literature reports24 and by elemental
and mass spectral analysis.

S.A. Svarovsky, J.J. Barchi, Jr. / Carbohydrate Research 338 (2003) 1925ꢀ
/1935
1933
3.13. 4-Pentenyl [2-acetamido-4,6-O-benzylidene-2-
deoxy-3-O-(2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl)]-a- -galactopyranoside (12a)
H?-2), 2.00 (m, 2 H, Ã
/
OCH2CH2CH2CHÄ
/
CH2), 1.87,
D-
1.90, 1.95, 1.96, 2.07 (s, 15 H, Ac, NHAc), 1.45ꢀ
2 H, ÃOCH2CH2CH2CHÄ
CH2); 13C NMR (100 MHz,
CDCl3): d 171.19, 170.49, 170.45, 170.23, 169.39,
138.20, 138.07, 128.97, 128.23, 126.36, 115.07 (CHÄ
/1.62 (m,
D
/
/
Compound 5a (625 mg, 0.90 mmol) in 15 mL of THF, 7
mL of AcOH, and 2.5 mL of Ac2O was treated with 5 g
of zinc dust at rt. The reaction mixture was stirred for 1
h, diluted with EtOAc and filtered through a short pad
of Celite†. The filtrate was washed consecutively with
water, satd NaHCO3 and brine, then dried over MgSO4
and concentrated. The residue was purified by FC using
/
CH2), 101.42, 100.77, 99.11, 76.15, 75.80, 71.19, 71.01,
69.47, 69.20, 69.07, 67.32, 66.57, 61.87, 54.61, 30.16,
28.83, 23.87 (CH3C(O)NH), 20.97, 20.85, 20.71
(CH3C(O)O); FABMS: m/z (relative intensity): 708.3
(MHꢁ). Anal. Calcd for C34H45NO15: C, 57.70; H, 6.41;
N, 1.98. Found: C, 57.53; H, 6.46; N, 2.17.
5:1 EtOAcꢀ
a white solid (7:3 mixture of rotamers). Rf 0.24 (4:1
EtOAcꢀhexanes); [a]D 1548 (c 0.52, CHCl3); IR
(neat): 2934.16, 1747.19, 1661.37, 1536.99, 1368.25; H
NMR (400 MHz, CDCl3): d 7.47ꢀ7.55 (m, 2 H, Ph),
7.26ꢀ7.39 (m, 3 H, Ph), 5.77 (m, 1 H, ÃCH ÄCH2), 5.53
(d, 1 H, J 8.98 Hz, NH), 5.51 (s, 1 H, PhCH), 5.34 (d, 1
H, J 2.34 Hz, Hƒ-4), 5.13ꢀ5.19 (m, 1 H, Hƒ-2), 4.86ꢀ
5.04 (m, 4 H, Hƒ-3, H?-1, ÃCHÄCH2), 4.75 (d, 1 H, J
7.81 Hz, bHƒ-1), 4.57ꢀ4.66 (m, 1 H, H?-2), 3.91ꢀ4.27
(m, 6 H, H?-6, Hƒ-6, H?-4, Hƒ-5), 3.86ꢀ3.91 (m, 1 H, H?-
3), 3.63ꢀ3.70 (m, 1 H, ÃOCH2CH2CH2CHÄCH2), 3.60
(bs, 1 H, H?-5), 3.39ꢀ3.47 (m, 1 H, ÃOCH2CH2-
CH2CHÄCH2), 2.10 (m, 2 H, ÃOCH2CH2CH2CHÄ
CH2), 1.94, 1.95, 2.00, 2.01, 2.11 (s, 15 H, Ac, NHAc),
1.62ꢀ1.72 (m, 2 H, ÃOCH2CH2CH2CHÄ
CH2); 13C
/
hexanes to give 540 mg (85% yield) of 12a as
Glycosylations of 6a,b with 12a,b were carried out
under exactly the same conditions as outlined in Section
3.9.1 (vide supra).
/
ꢁ
/
1
/
3.15. 4-Pentenyl 2-azido-3-O-4,6-O-benzylidene-3-O-
chloroacetyl-2-deoxy-b-D-galactopyranoside (13b)
/
/
/
/
/
To a solution of 4b (300 mg, 0.83 mmol) in 10 mL of
anhyd CH2Cl2 was added 0.1 mL of dry pyridine. The
reaction mixture was cooled to 0 8C, and 170 mg (1
mmol) of chloroacetic anhydride was slowly added.
After stirring overnight, the reaction was diluted with
EtOAc, washed with 1 M HCl, twice with brine and
water, and then dried over MgSO4 and evaporated.
/
/
/
/
/
/
/
/
/
/
/
/
/
Separation on silica gel with 4:1 hexanesꢀ
340 mg (93%) of 12b as a clear oil. Rf 0.10 (4:1 hexanesꢀ
EtOAc); [a]D 23.98 (c 0.92, CHCl3); IR (neat):
2114.56 (N3), 1763.58 (Ac); 1H NMR (400 MHz,
CDCl3): d 7.50ꢀ7.46 (m, 2 H, Ph), 7.38ꢀ7.32 (m, 3 H,
Ph), 5.85ꢀ5.74 (m, 1 H, ÃCH2CH ÄCH2), 5.49 (s, 1 H,
PhCH), 5.06ꢀ4.94 (m, 2 H, ÃCH2CHÄCH2), 4.73 (dd, 1
H, J 3.51, 10.93 Hz, H-3), 4.35 (d, 1 H, J 7.81 Hz, bH-
1), 4.33ꢀ4.27 (m, 2 H, H-6a, H-4), 4.13 (d, 2 H,
ClCH2CO), 4.03 (m, 1 H, H-6b), 3.98 (m, 1 H, Ã
OCH2CH2CH2CHÄCH2), 3.89 (dd, 1 H, J 7.81, 10.93
Hz, H-2), 3.50ꢀ3.49 (m, 1 H, ÃOCH2CH2CH2CHÄ
CH2), 3.44 (m, 1 H, H-5), 2.19ꢀ2.12 (m, 2 H, Ã
CH2CHÄCH2), 1.82ꢀ1.66 (m, 2 H, ÃCH2CH2CHÄ
/
EtOAc yielded
/
/
/
/
NMR (100 MHz, CDCl3): d 170.53, 170.42, 170.35,
169.67, 169.61, 137.98, 137.86, 129.02, 128.33, 126.41,
126.26, 115.35, 101.26, 100.92, 98.35, 75.84, 74.44,
71.14, 71.03, 69.08, 67.74, 67.15, 63.24, 61.57, 48.43,
30.55, 30.47, 28.77, 23.66, 20.91, 20.89, 20.75. FABMS:
m/z (relative intensity): 708.3 (MHꢁ). Anal. Calcd for
ꢁ
/
/
/
/
/
/
/
/
/
C34H45NO15×
/
H2O: C, 56.27; H, 6.53; N, 1.93. Found: C,
/
56.32; H, 6.44; N, 1.91
/
/
3.14. 4-Pentenyl [2-acetamido-4,6-O-benzylidene-2-
/
/
/
deoxy-3-O-(2,3,4,6-tetra-O-acetyl-b-
D-
/
/
galactopyranosyl)]-b-D-galactopyranoside (12b)
/
/
/
/
CH2); 13C NMR (100 MHz, CDCl3): d 167.20, 138.14,
137.53, 129.39, 128.44, 126.45, 115.29, 102.38, 101.14,
73.91, 72.60, 69.73, 69.11, 66.32, 60.48, 40.95, 40.67,
30.22, 28.84; FABMS: m/z (relative intensity): 438.1
(MHꢁ). Anal. Calcd for C20H24ClN3O6: C, 54.86; H,
5.52; Cl, 8.10; N, 9.60; Found: C, 55.64; H, 5.44; N,
9.30.
A solution of 5b (500 mg, 0.72 mmol) was treated
identically as described for 5a above. After purification
(4:10
was obtained as white solid. Rf 0.18 (4:1 EtOAcꢀ
hexanes); [a]D 32.28 (c 1.17, CHCl3); IR (neat):
1743.33, 1660.41, 1552.42, 1367.28, 1216.86; H NMR
(400 MHz, CDCl3): d 7.43ꢀ7.50 (m, 2 H, Ph), 7.24ꢀ7.32
(m, 3 H, Ph), 5.66ꢀ5.77 (m, 1 H, ÃCH ÄCH2), 6.02 (d, 1
H, J 7.03 Hz, NH), 5.47 (s, 1 H, PhCH), 5.29 (d, 1 H, J
3.51 Hz, Hƒ-4), 5.11ꢀ5.17 (m, 1 H, Hƒ-2), 5.03 (d, 1 H, J
8.20 Hz, bH?-1), 4.86ꢀ4.97 (m, 3 H, Hƒ-3, ÃCHÄCH2),
4.71 (d, 1 H, J 8.20 Hz, bHƒ-1), 4.61ꢀ4.67 (m, 1 H, H?-
3), 4.19ꢀ4.26 (m, 2 H, H?-6a, H?-4), 3.94ꢀ4.10 (m, 3 H,
H?-6b, Hƒ-6), 3.82 (bs, 1 H, Hƒ-5), 3.80ꢀ3.86 (m, 1 H, Ã
OCH2CH2CH2CHÄCH2), 3.42 (m, 1 H, ÃOCH2CH2-
CH2CHÄCH2), 3.39 (bs, 1 H, H?-5), 3.28ꢀ3.35 (m, 1 H,
/
5:1 EtOAcꢀhexanes) 440 mg (86% yield) of 12b
/
/
ꢁ
/
1
/
/
/
/
/
3.16. 4-Pentenyl 2-azido-4,6-O-benzylidene-3-O-
chloroacetyl-2-deoxy-a-D-galactopyranoside (13a)
/
/
/
/
To a solution of 4a (300 mg, 0.83 mmol) in 10 mL of
anhyd CH2Cl2 was added 0.1 mL of dry pyridine. The
reaction mixture was cooled to 0 8C, and 170 mg (1
mmol) of chloroacetic anhydride was slowly added.
After stirring overnight, the reaction was diluted with
EtOAc, washed with 1 M HCl, twice with brine and
/
/
/
/
/
/
/
/
/

1934
S.A. Svarovsky, J.J. Barchi, Jr. / Carbohydrate Research 338 (2003) 1925ꢀ1935
/
576;
(c) Barchi, J. J. Curr. Pharm. Des. 2000, 6, 485ꢀ
2. (a) Dahlenborg, K.; Hultman, L.; Carlsson, R.; Jansson,
B. Int. J. Cancer 1997, 70, 63ꢀ71;
(b) Yamashita, Y.; Chung, Y. S.; Horie, R.; Kannagi, R.;
Sowa, M. J. Natl. Cancer Inst. 1995, 87, 441ꢀ446;
(c) Jansson, B.; Borrebaeck, C. A. K. Cancer Immunol.
Immunother. 1992, 34, 294ꢀ298.
water, and dried over MgSO4 and evaporated. Separa-
/
501.
tion on silica gel with 4:1 hexanesꢀ
mg (97%) of 13a as a clear oil. Rf 0.30 (4:1 hexanesꢀ
EtOAc); [a]D 105.58 (c 0.37, CHCl3); IR (neat):
2108.78 (N3), 1761.65 (Ac); 1H NMR (400 MHz,
CDCl3): d 7.49ꢀ7.44 (m, 2 H, Ph), 7.38ꢀ7.31 (m, 3 H,
Ph), 5.85ꢀ5.73 (m, 1 H, ÃCH2CH ÄCH2), 5.50 (s, 1 H,
PhCH), 5.35 (dd, 1 H, J 3.51, 10.93 Hz, H-3), 5.04 (d, 1
/
EtOAc yielded 351
/
/
ꢁ
/
/
/
/
/
/
/
/
3. (a) Adluri, S.; Helling, F.; Ogata, S.; Zhang, S. L.;
Itzkowitz, S. H.; Lloyd, K. O.; Livingston, P. O. Cancer
H, J 3.51 Hz, aH-1), 5.06ꢀ
/
4.94 (m, 2 H, Ã
/
CH2CHÄ
/
CH2), 4.48 (m, 1 H, H-4), 4.23 (dd, 1 H, J 1.17, 12.88
Hz, H-6a), 4.12 (s, 2 H, ClCH2CO), 4.04 (dd, 1 H, J
Immunol. Immunother. 1995, 41, 185ꢀ
(b) Koganty, R. R.; Reddish, M. A.; Longenecker, B. M.
Drug Discovery Today 1996, 1, 190ꢀ198.
4. (a) Allen, J. R.; Harris, C. R.; Danishefsky, S. J. J. Am.
Chem. Soc. 2001, 123, 1890ꢀ1897;
(b) Deshpande, P. P.; Danishefsky, S. J. Nature 1997, 387,
164ꢀ166;
/192;
1.56, 12.49 Hz, H-6b), 3.92ꢀ
1 H, H-5), 3.76ꢀ3.69 (m, 1 H, Ã
CH2), 3.54ꢀ3.47 (m, 1 H, ÃOCH2CH2CH2CHÄ
2.18ꢀ2.11 (m, 2 H, ÃCH2CHÄCH2), 1.76ꢀ1.68 (m, 2 H,
CH2CH2CHÄ
CH2); 13C NMR (100 MHz, CDCl3): d
/
3.87 (m, 1 H, H-2), 3.74 (m,
OCH2CH2CH2CHÄ
CH2),
/
/
/
/
/
/
/
/
/
/
/
/
/
Ã
/
/
(c) Maclean, G. D.; Bowen-Yacyshyn, M. B.; Samuel, J.;
Meikle, A.; Stuart, G.; Nation, J.; Poppema, S.; Jerry, M.;
Koganty, R.; Wong, T.; Longenecker, B. M. J. Immun-
167.17, 137.99, 137.59, 129.35, 128.43, 126.31, 115.42,
100.93, 98.66, 73.36, 71.57, 69.29, 68.28, 62.62, 57.33,
40.93, 30.39, 28.73; FABMS: m/z (relative intensity):
438.1 (MHꢁ). Anal. Calcd for C20H24ClN3O6: C, 54.86;
H, 5.52; N, 9.60; Found: C, 55.20; H, 5.51; N, 9.36.
other. 1992, 11, 292ꢀ305;
/
(d) Ragupathi, G.; Howard, L.; Cappello, S.; Koganty, R.
R.; Qiu, D. X.; Longenecker, B. M.; Reddish, M. A.;
Lloyd, K. O.; Livingston, P. O. Cancer Immunol. Immun-
3.17. N-(9-Fluorenylmethoxycarbonyl)-O-[2-azido-4,6-
O-benzylidene-3-O-chloroacetyl-2-deoxy-a-
galactopyranosyl]-O-L-serine allyl ester (14)
other. 1999, 48, 1ꢀ
5. Lloyd, K. O. Drug News Perspect. 2000, 13, 463ꢀ
6. (a) Luning, B.; Norberg, T.; Tejbrant, J. Glycoconjugate J.
1989, 6, 5ꢀ19;
(b) Komba, S.; Meldal, M.; Werdelin, O.; Jensen, T.;
Bock, K. J. Chem. Soc., Perkin Trans. 1 1999, 415ꢀ
/
8.
D-
/
470.
/
A mixture of 88 mg (0.20 mmol) of compound 13 and
115 mg (0.31 mmol) of Fmoc-Ser-OAll was azeotropi-
cally dried with toluene three times. The mixture was
then dried under vacuum at 40 8C overnight, and then
dissolved in 4 mL of anhyd CH2Cl2. To this solution 35
mg (0.20 mmol) of freshly recrystallized NIS was added
under argon, and the reaction was stirred at rt for
several minutes before 67 mL (0.20 mmol) of TESOTf
was added via syringe. After 1 h the reaction mixture
was diluted with 10 mL of CH2Cl2, quenched with one
drop of Et3N, and filtered. The filtrate was washed
consecutively with 10% Na2S2O3, satd NaHCO3, and
water, and then dried over MgSO4. The residue was
purified by FCC to give 73 mg (51%) of compound 14 as
an amorphous solid. Identification of the product was
/
419;
(c) St. Hilaire, P. M.; Cipolla, L.; Franco, A.; Tedebark,
U.; Tilly, D. A.; Meldal, M. J. Chem. Soc., Perkin Trans. 1
1999, 3559ꢀ
(d) Paulsen, H.; Schleyer, A.; Mathieux, N.; Meldal, M.;
Bock, K. J. Chem. Soc. Perkin Trans. 1 1997, 281ꢀ293;
/3564;
/
(e) Glunz, P. W.; Hintermann, S.; Williams, L. J.;
Schwarz, J. B.; Kuduk, S. D.; Kudryashov, V.; Lloyd,
K. O.; Danishefsky, S. J. J. Am. Chem. Soc. 2000, 122,
7273ꢀ7279;
/
(f) George, S. K.; Holm, B.; Reis, C. A.; Schwientek, T.;
Clausen, H.; Kihlberg, J. J. Chem. Soc., Perkin Trans. 1
2001, 880ꢀ
(g) Rademann, J.; Schmidt, R. R. Carbohydr. Res. 1995,
269, 217ꢀ225;
(h) Qiu, D.; Gandhi, S. S.; Koganty, R. R. Tetrahedron
Lett. 1996, 37, 595ꢀ598;
(i) Jiaang, W. T.; Chang, M. Y.; Tseng, P. H.; Chen, S. T.
Tetrahedron Lett. 2000, 41, 3127ꢀ3130.
/885;
1
/
ascertained by correlation of the H NMR spectrum to
that reported in the literature.26
/
/
Acknowledgements
7. Fraser-Reid, B.; Udodong, U. E.; Wu, Z. F.; Ottosson, H.;
Merritt, J. R.; Rao, C. S.; Roberts, C.; Madsen, R. Synlett
We thanks Drs James Kelley and Cong Lai for mass
spectra and the Center for Cancer Research, National
Cancer Institute for an FTE loan for S.A.S.
1992, 927ꢀ
8. Lemieux, R. U.; Ratcliffe, R. M. Can. J. Chem. 1979, 57,
1244ꢀ1249.
9. Kozikowski, A. P.; Lee, J. J. Org. Chem. 1990, 55, 863ꢀ
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870.
10. (a) Paulsen, H.; Paal, M.; Schultz, M. Tetrahedron Lett.
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