A stereochemical surprise at the late stage of the synthesis of fully N-differentiated heparin oligosaccharides containing amino, acetamido, and N-sulfonate groups

Article abstract of DOI:10.1021/jo035732z

The glucosamine residues in heparin-like glycosaminoglycans have been found to exist as amines, acetamides, and N-sulfonates. To develop a completely general, modular synthesis of heparin, three degrees of orthogonal nitrogen protection are required. Repo

Full text of DOI:10.1021/jo035732z

A Ster eoch em ica l Su r p r ise a t th e La te Sta ge of th e Syn th esis of
F u lly N-Differ en tia ted Hep a r in Oligosa cch a r id es Con ta in in g
Am in o, Aceta m id o, a n d N-Su lfon a te Gr ou p s
Gregory J . S. Lohman and Peter H. Seeberger*,†
Massachusetts Institute of Technology, 77 Massachusetts Avenue 18-292,
Cambridge, Massachusetts 02139
seeberger@org.chem.ethz.ch
Received November 25, 2003
The glucosamine residues in heparin-like glycosaminoglycans have been found to exist as amines,
acetamides, and N-sulfonates. To develop a completely general, modular synthesis of heparin, three
degrees of orthogonal nitrogen protection are required. Reported herein is a strategy for the synthesis
of fully N-differentiated heparin oligosaccharides in the context of target octasaccharide 1, which
contains an N-acetate, N-sulfonates, and a free amine. The protecting group scheme used in the
synthesis blocked the N-acetate as a N-diacetate, the N-sulfonates as azido groups, and the amine
as a N-CBz; free hydroxyls were masked as benzyl ethers and O-sulfonates as acetate esters.
Disaccharide and tetrasaccharide modules were synthesized using this strategy; however, the union
of tetrasaccharide trichloroacetimidate 4 with disaccharide acceptor 5 unexpectedly formed the
undesired â-linked glycoside in addition to the R-linkage anticipated for iduronic acid nucleophiles,
resulting in an inseparable 6:1 R/â mixture of products. Detailed studies into the basis for this
unexpected result were conducted and are also reported.
Heparin-like glycosaminoglycans (HLGAGs) are a class
of polysaccharides found protein-conjugated in the ex-
tracellular matrix and free in the circulatory system.
HLGAGs are involved in an array of signaling functions,
including regulation of the coagulation cascade, growth
factor interactions, and viral entry into cells.1 Heparin
fractions isolated from animal sources are used clinically
as anticoagulants,2,3 and several synthetic analogues of
heparin have been prepared as anticoagulant drug
candidates.4-7
HLGAGs consist of alternating 1,2-anti-(1f4) linked
uronic acid residues and R-(1f4)-linked glucosamine
residues. The polysaccharides exist in a wide variety of
O-and N-sulfonated as well as N-acetylated states with
a high sequence variability. Due to both the high struc-
tural complexity and the difficulty of isolating homoge-
neous structures from natural sources, heparin is an ideal
target for a modular synthetic method. Defined sequences
of heparin are needed for biological study, and the ability
to generate diverse libraries of heparin would greatly
facilitate studies into the structure-activity relationship
of HLGAGs. The direct study of heparin biochemistry and
the development of HLGAG analytical techniques would
benefit from the availability of defined sequences. No
completely general method for the production of heparin
oligosaccharides exists, and substantial synthetic chal-
lenges remain to be overcome.8
Recent reports describe fully differentiated monosac-
charide building blocks for HLGAG synthesis.9-15 Several
sequences have been produced, including oligomers of the
fibroblast growth factor (FGF) disaccharide repeat16-22
† Current Address: Laboratory for Organic Chemistry, ETH Ho¨ng-
gerberg/HCI
Switzerland.
F
315 Wolfgang-Pauli-Strasse 10, CH-8093 Zurich,
(8) For a review see: Poletti, L.; Lay, L. Eur. J . Org. Chem. 2003,
2999-3024.
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609-652. Bernfield, M.; Go¨tte, M.; Park, P. W.; Reizes, O.; Fitzgerald,
M. L.; Lincecum, J .; Zako, M. Annu. Rev. Biochem. 1999, 68, 729-
777. Conrad, H. E. Heparin Binding Proteins; Academic Press: New
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10.1021/jo035732z CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/13/2004
J . Org. Chem. 2004, 69, 4081-4093
4081

Lohman and Seeberger
F IGUR E 1. Target structure as reported from sequencing studies.33 The stereochemistry at the indicated site is unknown.
Counterions after oligosaccharide purification are primarily sodium.
and several variants and analogues of the antithrombin
III (ATIII) binding structure.12,23-28 The amine group has
been masked as an azide in most heparin syntheses to
date, allowing access to only poly-N-sulfonated struc-
tures. One synthesis of the ATIII binding sequence
included a nonreducing end GlcNAc residue, but the
method used was not general and the use of the N-acetate
as the protecting group resulted in poor coupling yields.27
2-Azidoglucosyl trichloroacetimidates or halides fre-
quently serve as glycosylating agents to install R-glu-
cosamine linkages. These reagents have shown high
R-selectivity when coupled to the C4 hydroxyl group of
iduronic acid but afford mixtures of products when
coupled to glucuronic acid.24,26,29 In our recent synthesis
of a ATIII-binding hexasaccharide,12 all R-glucosamine
linkages were formed with complete selectivity during
the assembly of disaccharide modules via glycosylation
of conformationally constrained 1,2-O-isopropylidene-
iduronic or glucuronic acids with 2-azidoglucosyl trichlo-
roacetimidates.30
1) have been proposed for the herpes-binding sequence.33
The isolated structures contained 4,5-dehydrouronic acids
at the nonreducing end and at the indicated site as a
result of the degradation techniques used. The structure
contains N-sulfonates, an N-acetate, and a free amine.
This report describes a strategy for the synthesis of
N-differentiated heparin oligosaccharides en route to fully
protected target 2 (Figure 2). Iduronic acid was selected
arbitrarily in the two uronic acid positions of unknown
stereochemistry to reduce the complexity of the synthetic
target. As in previous heparin syntheses, O-sulfonates
were masked as O-acetates, free hydroxyls as O-benzyl
ethers, carboxylic acids as methyl esters, and N-sul-
fonates as azide groups.12,16-27 An N-diacetate marked the
position of N-acetate34,35 while the free amine was masked
as the benzyl carbamate (CBz). These protecting groups
would allow for the deprotection and elaboration of 2
through a modification of previously established heparin
deprotection protocols.12,16,18,20,21,24-26,36 All O-acetates and
methyl esters, as well as one of the N-diacetate amides,
will be removed using LiOOH/NaOH. Selective reduction
of the azides could be achieved by Staudinger, thiol, SmI2,
or a variety of other selective reduction chemistries.37
Sulfonation of all hydroxyls and amines with SO3‚NEt3,
followed by Pd/C reductive removal of benzyl ethers and
the CBz carbamate, would reveal the fully deprotected
and elaborated structure.
The HLGAG octasaccharide 1 is known to bind viral
coat proteins on herpes simplex virus 1 (HSV-1) and is
thought to be involved in viral cell entry.31,32 On the basis
of sequencing efforts that combine enzymatic degradation
and mass spectrometry, two possible structures (Figure
(18) Tabeur, C.; Mallet, J .-M.; Bono, F.; Herbert, J .-M.; Petitou, M.;
Sinay¨, P. Bioorg. Med. Chem. 1999, 7, 2003-2012.
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P.; Herbert, J .-M.; Petitou, M.; Sinay¨, P. Bioorg. Med. Chem. 1999, 7,
1567-1580.
Resu lts a n d Discu ssion
Fully protected octasaccharide 2 was the proposed
target en route to the HSV-1 binding octasaccharide 1.
Retrosynthetic analysis of 2 revealed that two disaccha-
rides (3, 5) and one tetrasaccharide (4) would provide the
key modules based on the assumption that 2-azido
glucosamines can be coupled to iduronic acid residues
with complete R-stereoselectivity.7,12,16,17,30,38-42 Interme-
diates 3-5 can be readily assembled from five previously
reported monosaccharide modules: iduronic acid deriva-
tives 6, 8, and 914 and GlcN3 derivatives 712 and 10.
Syn t h esis of Non r ed u cin g E n d Disa cca h r id e
Tr ich lor oa cetim id a te 3. Disaccharide 3 was synthe-
(20) de Paz, J .-L.; Angulo, J .; Lassaletta, J .-M.; Nieto, P. M.;
Ridondo-Horcajo, M.; Lozano, R. M.; Gime´nez-Gallego, G.; Martin-
Lomas, M. ChemBioChem 2001, 2, 673-685.
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L. Eur. J . Org. Chem. 2001, 2727-2734.
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J . Org. Chem. 2003, 3308-3324.
(23) Sinay¨, P.; J acquinet, J .-C. Carbohydr. Res. 1984, 132, C5-C9.
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P.; J acquinet, J .-C.; Torri, D. Carbohydr. Res. 1986, 147, 221-236.
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van Aelst, S. F.; van den Bosch, R. H.; Mertens, J . M. R.; van der Vlugt,
F. A. J . Carbohydr. Chem. 1985, 4, 293-321.
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1993, 32, 1671-1690.
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Copeland, R. J .; Raska, C. S.; Yoshida, K.; Eisenberg, R. J .; Cohen,
G.; Linhardt, R. J .; Sasisekharan, R. J . Biol. Chem. 2002, 277, 33456-
33467.
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R. D. Nature: Biotechnol. 2003, 21, 1343-1346.
(29) Petitou, M.; Duchaussoy, P.; Lederman, I.; Choay, J .; J acquinet,
J .-C.; Sinay¨, P. Carbohydr. Res. 1987, 167, 67-75.
(30) Orgueira, H. A.; Bartolozzi, A.; Schell, P.; Seeberger, P. H.
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36, 6871-6874.
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3065-3068.
(31) For Reviews, see: Liu, J .; Thorp, S. C. Med. Res. Rev. 2002,
22, 1-25. Spear, P. G.; Longnecker, R. J . Virol. 2003, 77, 10179-10185.
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J . D.; Cohen, G. H.; Eisenberg, R. J .; Rosenberg, R. D.; Spear, P. G.
Cell 1999, 99, 13-22.
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Proced. Int. 2002, 34, 109-147.
4082 J . Org. Chem., Vol. 69, No. 12, 2004

Synthesis of Fully N-Differentiated Heparin Oligosaccharides
F IGURE 2. Retrosynthetic analysis.
SCHEME 1
sized by coupling iduronic acid trichloroacetimidate 6 and
glucosamine azide 7 to yield the desired disaccharide 11
in 74% yield (Scheme 1). Removal of the levulinic (Lev)
ester with hydrazine proceeded in 92% yield to give 12.
This product was converted to its benzyl ether 13 in 76%
yield using benzyl trichloroacetimidate and trifluo-
romethanesulfonic acid (TfOH) activator. Cleavage of the
anomeric silyl ether with TBAF/AcOH, followed by reac-
tion with DBU/Cl3CCN, yielded 78% of trichloroacetimi-
date disaccharide building block 3.
Syn th esis of Tetr a sa cch a r id e Tr ich lor oa cetim i-
d a te 4. Synthesis of the central tetrasaccharide 4 pre-
sented more of a challenge (Scheme 2). Iduronic acid
donor 9 and GlcN3 derivative 7 were coupled through
TMSOTf activation in 88% yield. Conversion of the
product disaccharide 14 into glycosyl trichloroacetimidate
15 was accomplished by cleavage of the TBDMS group
followed by reaction with DBU/Cl3CCN (96% yield for two
steps). Union of 15 with iduronic acid derivative 8
afforded trisaccharide 16 with complete R-selectivity in
83% yield.
Conversion of the azide moiety in trisaccharide 16 to
an N-acetate was accomplished in one pot and 84% yield
by treatment with thiolacetic acid in pyridine.43 Removal
(38) Ojeda, R.; de Paz, J . L.; Martin-Lomas, M.; Lassaletta, J . M.
Synlett 1999, 8, 1316-1318.
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253-264.
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Bioorg. Med. Chem. Lett. 1999, 9, 3127-3132.
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Fucase, K.; Sobel, M.; Kusumoto, S. Tetrahedron Lett. 1996, 37, 1053-
1056.
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Kusumoto, S. Tetrahedron Lett. 1999, 40, 5725-5728.
J . Org. Chem, Vol. 69, No. 12, 2004 4083

Lohman and Seeberger
SCHEME 2
SCHEME 3
of the isopropylidene group from product trisaccharide
17 with trifluoroacetic acid followed by selective silyl
ether formation at the anomeric hydroxyl group38 fur-
nished 18 in a 93% yield. Microwave irradiation of 18 in
isopropenyl acetate with catalytic p-toluenesulfonic acid35
simultaneously installed the desired N-diacetate and
acetylated the free hydroxyl, forming 19 in 86% yield.
This transformation could not be achieved under a
variety of thermal conditions, with only poor yields of 19
achieved even after several days. Conversion of 19 to
trisaccharide trichloroacetimidate building block 20 was
achieved in 84% yield by cleavage of the TBDMS ether
and reaction with DBU/Cl3CCN.
conversion to 21; most unreacted 7 could be recovered
and recycled. Tetrasaccharide 21 was converted to gly-
cosyl trichloroacetimidate 4 in 89% yield.
Syn th esis of Red u cin g En d Disa cch a r id es 5 a n d
30. The reducing end disaccharide 5 was synthesized
from known 2-N-CBz glucosamine 22 (Scheme 3).44
Selective acetylation of the primary hydroxyl with acetyl
chloride in 2,4,6-collidine afforded acceptor 10 in 82%
yield.45 Glucosamine 10 reacted with iduronic acid donor
9 under TMSOTf activation to form disaccharide 23 in
highly variable yields. Close monitoring by TLC revealed
that 10 was rapidly consumed to form ortho ester 24 that
1
was identified by H NMR (Figure 3). When left under
reaction conditions for several hours, 24 rearranged to
the desired disaccharide 23. Rearrangement and hy-
drolysis of the ortho ester intermediate was highly
Glycosylating agent 20 coupled only poorly with 2-azido-
glucose 7 in the presence of catalytic TMSOTf, with
mainly hydrolyzed trisaccharide lactol formed instead.
Use of a large excess (5 equiv) of 7 resulted in a 71%
(44) Fukuda, Y. Sasai, H. Suami, T. Bull. Chem. Soc. J pn. 1982,1574-
1578.
(43) Schwartz, J . B.; Kuduk, S. D.; Chen, X.; Sames, D.; Glunz, P.
W.; Danishefsky, S. J . J . Am. Chem. Soc. 1999, 121, 2662-2673.
(45) Ishihara, K.; Kurihara, H.; Yamamoto, H. J . Org. Chem. 1993,
58, 3791-3793.
4084 J . Org. Chem., Vol. 69, No. 12, 2004

Synthesis of Fully N-Differentiated Heparin Oligosaccharides
F IGURE 4. Glucosamine CBz acceptor.
F IGURE 3. Ortho ester byproduct.
formation. Use of a C2 benzoate protecting group can
overcome this effect.
SCHEME 4
Attem p ted Block Cou p lin g of Tetr a sa cch a r id e 4
w ith Red u cin g En d Disa cch a r id es 5 a n d 30. With
compounds 3, 4, and 5 as well as alternate reducing end
acceptor 30 in hand, assembly of the fully protected
target 2 was attempted (Scheme 5). Coupling of 4 and 5
under TMSOTf activation proceeded in 59% yield when
reacted at -40 °C in the presence of molecular sieves.
Although the desired R-linked hexasaccharide 32 was
obtained as the major product, it was contaminated with
17% of an inseparable impurity. Even after purification
via normal- and reversed-phase silica gel HPLC and size-
exclusion chromatography (Bio-rad Bio-Beads), some of
the contaminant always remained.
Varying the reaction conditions for coupling 4 and 5
did not prevent formation of the minor product. Variation
of reaction temperature (from -78 to 0 °C) and activator
(TMSOTf and TBSOTf) affected the coupling yields, but
the isolated product always contained the minor product
(data not shown). Varying the polarity of the reaction
solvent through addition of heptanes had no effect on the
ratio of the products.
Removal of the allyl protecting group through treat-
ment of the mixture with PdCl2/NaOAc in acetic acid and
water and further coupling with disaccharide 3 to form
the octasaccharide 2 did not allow for the separation of
the minor product at this later stage of the synthesis.
Spectral data indicated that the major isomer did react
as desired, but a minor product copurified at each step,
suggesting that the contaminant reacted similarly. Fi-
nally, coupling of tetrasaccharide 4 with disaccharide 30
under the best conditions found for the coupling with 5
resulted in the formation of a 1:1 mixture of inseparable
products (data not shown).
To identify the minor product contaminating 32, pos-
sible reaction byproducts were systematically eliminated.
Size-exclusion purification ruled out any decomposition
products of 5. Small amounts of hydrolyzed 4 were
detected and separated from the products through silica
gel chromatography. Tetrasaccharide 33, formed by rear-
rangement of 4, was also observed in the crude reaction
mixture but could be readily separated from product 2
(Figure 5). Acetylation, N-diacetylation, and silylation to
sensitive to the reaction conditions and resulted in the
variability in yield of desired disaccharide 23.
While sufficient quantities of 5 could be produced via
PdCl2-catalyzed allyl ether removal from disaccharide 23,
an alternate reducing end disaccharide was also prepared
(Scheme 4). Synthesis of iduronic acid 28 followed
established procedures,14 with the exception of the use
of a C-2 benzoate ester, which was included to reduce
ortho ester formation. Glycosylating agent 28 reacted
cleanly with the CBz glucosamine derivative 10 to give
29 in excellent yield. No ortho ester intermediate was
observed. This disaccharide was treated with PdCl2/
NaOAc in acetic acid and water to afford disaccharide
30 in 83% yield.
The replacement of a C2 acetate to a C2 benzoate group
dramatically improved the yield of this coupling reaction.
Interestingly, glycosylating agent 9 containing a C2
acetate and the N-CBz glucosamine n-pentenyl glycoside
31 (Figure 4) coupled in high yield without the formation
of ortho ester (data not shown). All other couplings of
iduronic acid donors reported here proceed in good yields
to 1-O-TBDMS ether â-glucosamines. On the basis of
these and previous12 observations, it appears that the
stereoelectronics of R-glucosamine acceptors negatively
influence the outcome of coupling reactions with uronic
acid trichloroacetimidates, greatly favoring ortho ester
SCHEME 5
J . Org. Chem, Vol. 69, No. 12, 2004 4085

Lohman and Seeberger
F IGURE 5. Rearranged glycosyl donors.
SCHEME 6
cap potentially rearranged tetrasaccharides containing
a free hydroxyl group or an N-acetamide resulted in
reisolated 32 that contained the same contaminant in an
unchanged ratio.
troscopy (HSQC, Figure 6) showed that one anomeric
cross-peak of the minor isomer was consistent with a
â-GlcN3 anomeric cross-peak.46-48
These observation indicate that the erosion of anomeric
selectivity is dependent both on the size of the glycosy-
lating agent and on the exact nature of the nucleophile.
The conformation of uronic acid acceptors has a profound
influence on the anomeric selectivity of couplings involv-
ing such monomers,30 whereby the 1C4 conformation
normally adopted by iduronic acid derivatives overwhelm-
ingly favors R-products. This selectivity has also been
observed for oligosaccharides containing an iduronic acid
at the nonreducing end.
On the basis of these observations, the minor product
was likely an isomer of the desired hexasaccharide. Two
major possibilities exist: either acceptor 5 rearranged
under the reaction conditions to result in 1f2-linked
hexasaccharide or an R/â mixture of 1f4-linked isomers
was being produced. Treatment of acceptors 5 and 30
under coupling conditions in the absence of a glycosylat-
ing agent resulted exclusively in the reisolation of
unchanged starting materials. Thus, the formation of
1f2-linked regioisomer hexasaccharide could be ruled
out. In direct contrast to previous reports,7,12,16-17,30,38-42
the coupling of a 2-azido glucosamine donor and the C4
hydroxyl group of iduronic acid seemed to produce an
anomeric mixture of products.
In the case described here, we hypothesize the selectiv-
ity is eroded due to an altered conformation of the
iduronic acid. The 2-N-CBz glucosamine residue con-
nected to the anomeric position of the iduronic acid is
the major difference between acceptor 5 and previously
described, R-selective iduronic acid disaccharide accep-
tors. The nitrogen protecting group used to differentiate
the glucosamine may be responsible for loss of selectivity
in the union of 4 and 5. Presumably the iduronic acid
acceptor is forced out of the 1C4 conformation that
appears to allow 2-azido glucosyl trichloroacetimidates
to react with complete R-selectivity. The loss of selectivity
was apparently exacerbated by the change of the C2
protecting group from acetate to the sterically bulkier
benzoate. The C2 substituent is placed axial when the
Exa m in a tion of Mod el Tr isa cch a r id es 36 a n d 37.
Since a detailed NMR analysis of the hexasaccharide
mixture was difficult due to the large number of overlap-
ping signals, a simplified model system was devised
(Scheme 6). Allylation of 7 using silver oxide and allyl
bromide furnished glucosamine 34 in 75% yield. Conver-
sion of 34 to trichloroacetimidate 35 was achieved in 89%
yield. Reaction of monosaccharide trichloroacetimidate
35 with 5 and 30 yielded trisaccharides 36 and 37,
respectively. The structure of rearranged glycosylating
agent 38 (Figure 5) was confirmed by 2-D NMR experi-
ments. Trisaccharide 36 contained less than 5% of a
contaminating product, while trisaccharide 37 was iso-
lated as a 5:1 mixture. Detailed analysis of 37 of by NMR
and mass spectrometry identified an apparent mixture
of trisaccharide isomers. Carbon-proton correlation spec-
(46) Tai, V. W.-F.; Imperiali, B. J . Org. Chem. 2001, 66, 6217-6228.
(47) Westman, J .; Nilsson, M.; Ornitz, D. M.; Svahnm C.-M. J .
Carbohydr. Chem. 1995, 14, 95-114.
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Carbohydr. Chem. 2000, 19, 939-958.
4086 J . Org. Chem., Vol. 69, No. 12, 2004

Synthesis of Fully N-Differentiated Heparin Oligosaccharides
F IGURE 6. HSQC spectrum of the anomeric region of trisaccharide 37. The indicated cross-peak arises from the minor isomer
and is consistent with a â-glucosamine H1-C1 cross-peak.
sugar ring is in the 1C4 conformation; thus, larger
0.9; IR (thin film, NaCl plates) 2928, 2110, 1745, 1739, 1721
1
cm-1; H NMR (500 MHz, CDCl3) δ 7.22-7.39 (m, 20H), 5.31
4
susbstituents likely favor the C1 conformation.
(d, J ) 3.6 Hz, 1H), 4.91 (t, J ) 3.6 Hz, 1H), 4.81 (d, J ) 3.6
Due to the loss of selectivity in this block coupling, and
the difficulties encountered with purification, it was
judged that the proposed strategy was not useful for
producing the target octasaccharide. It is becoming
Hz, 1H), 4.69-4.75 (m, 3H), 4.43-4.60 (m, 6H), 4.02 (t, J )
9.3 Hz, 1H), 3.87 (t, J ) 4.1, 1H), 3.81 (t, J ) 3.9 Hz, 1H),
3.70-3.71 (m, 2H), 3.44 (s, 3H), 3.38 (m, 1H), 3.35 (d2, J ) 7.5
Hz, 10 Hz, 1H), 3.26 (t, J ) 4.5 Hz, 1H), 2.63 (m, 2H), 2.46
increasingly clear that the coupling of larger oligosac-
charide building blocks in the synthesis of heparin-like
glycosaminoglycans is greatly influenced by the confor-
mation of the nucleophile. The loss of selectivity in these
couplings is a significant obstacle to the general synthesis
of HLGAGs. A systematic reevaluation of the protecting
group and anomeric leaving group chemistries used in
heparin synthesis will greatly facilitate further work on
the assembly of larger structures using the modular
approach.
(m, 2H), 2.12 (s, 3H), 0.94 (s, 9H), 0.17 (s, 3H), 0.15 (s, 3H);
13C NMR (125 MHz, CDCl3) δ 206.4, 172.1, 169.8, 138.5, 138.4,
137.9, 137.8, 128.6, 128.5, 128.4, 128.3, 128.1, 128.07, 128.04,
128.0, 127.7, 127.6, 127.4, 97.8, 97.4, 81.0, 75.4, 75.0, 74.9, 74.7,
73.6, 73.4, 73.6, 73.4, 72.9, 72.7, 69.6, 69.3, 68.8, 68.4, 51.9,
37.9, 30.0, 28.1, 25.8, 10.2, -4.0, -5.0; ESI MS (C52H65N3O13
-
Si) m/z (M + Na+) calcd 990.4179, obsd 990.4145.
ter t-Bu tyld im eth ylsilyl (Meth yl 3,4-d i-O-ben zyl-R-L-
id op yr a n osid u r on a t e)-(1f4)-2-a zid o-3,6-d i-O-b en zyl-2-
d eoxy-â-D-glu cop yr a n osid e (12). Disaccharide 11 (176 mg,
0.182 mmol) was dissolved in pyridine (550 µL) and acetic acid
(360 µL). Hydrazine hydrate (44 µL, 0.91 mmol) was added,
and the reaction mixture was stirred for 1 h. The reaction was
quenched by addition of acetone (5 mL) and the mixture stirred
for 30 min. The solution was poured into ethyl acetate (50 mL)
and washed with 1 N HCl, brine, and saturated NaHCO3. The
organic phase was dried over MgSO4, and solvent was removed
under reduced pressure. Flash chromatography on silica
(hexanes/ethyl acetate 17:3) afforded 12 (145 mg, 0.167 mmol,
Exp er im en ta l Section
ter t-Bu tyld im eth ylsilyl (Meth yl 3,4-d i-O-ben zyl-2-O-
levon u yl-R-L-id op yr a n osid u r on a te)-(1f4)-2-a zid o-3,6-d i-
O-b en zyl-2-d eoxy-â-D-glu cop yr a n osid e (11). Trichloro-
acetimidate 614 (220 mg, 0.349 mmol) and monosaccharide 712
(145 mg, 0.29 mmol) were coevaporated three times with
anhydrous toluene. The compounds were dissolved in anhy-
drous CH2Cl2 (3 mL) under N2 and cooled to -15 °C. Tri-
methylsilyl trifluoromethanesulfonate (7.5 µL, 0.042 mmol)
was added, and the reaction mixture was stirred for 45 min.
The reaction was quenched by addition of NEt3 (0.2 mL), and
the solvent was removed under reduced pressure. Flash
chromatography on silica (hexanes/ethyl acetate 17:3) afforded
recovered 7 (13 mg, 0.026 mmol, 9%). Further elution (hexanes/
ethyl acetate 3:1) yielded 11 (208 mg, 0.215 mmol, 74%) as a
clear oil: Rf 0.13 (hexanes/ethyl acetate 3:1); [R]24D -28.3, c )
92%) as a clear oil: Rf 0.21 (hexanes/ethyl acetate 3:1); [R]24
D
-47.6, c ) 0.8; IR (thin film, NaCl plates) 3495, 2929, 2857,
2109, 1776, 1742, 839 cm-1 1H NMR (500 MHz, CDCl3) δ
;
7.21-7.4 (m, 20H), 5.23 (d, J ) 1.5 Hz, 1H), 4.90 (d, J ) 2.3
Hz, 1H), 4.45-4.71 (m, 9H), 3.98 (ψt, J ) 9.4 1H), 3.83-3.87
(m, 2H), 3.67-3.72 (m, 3H), 3.43 (d, J ) 10.9 Hz, 1H), 3.35-
3.4 (m, 2H), 3.36 (s, 3H), 3.22 (ψt, J ) 9.5 Hz, 1H), 0.94 (s,
9H), 0.17 (s, 3H), 0.15 (s, 3H); 13C NMR (125 MHz, CDCl3) δ
169.9, 138.5, 138.1, 137.9, 136.9, 128.7, 128.6, 128.4, 128.3,
128.2, 128.1, 127.9, 127.4, 101.1, 97.6, 81.2, 75.5, 75.0, 74.3,
J . Org. Chem, Vol. 69, No. 12, 2004 4087

Lohman and Seeberger
73.3, 73.2, 72.3, 68.9, 68.2, 67.2, 52.0, 25.7, 18.2, -4.0, -5.0;
ESI MS (C47H59N3O11Si) m/z (M + Na+) calcd 892.3811, obsd
892.3829.
4.67 (m, 2H), 4.51-4.6 (m, 2H), 4.47 (d, J ) 12.2 Hz, 0.4H),
4.42 (d, J ) 12.0 Hz, 0.6H), 4.24 (t, J ) 9.6 Hz, 0.6H), 4.13 (t,
J ) 9.3 Hz, 0.4 H), 3.91-4.04 (m, 3H), 3.76-3.84 (m, 2H),
3.65-3.71 (m, 1H), 3.35-3.56 (m 6H); 13C NMR (125 MHz,
CDCl3) δ 170.6, 170.5, 161.8, 161.3, 139.0, 138.9, 138.85, 138.8,
138.75, 138.7, 138.6, 138.35, 138.30, 129.6, 129.4, 129.15,
129.11, 129.1, 129.07, 129.05, 129.03, 128.9, 128.89, 128.87,
128.85, 128.65, 128.63, 128.60, 128.58, 128.57, 128.55, 128.4,
128.35, 128.1, 100.6, 100.5, 97.4, 15.5, 91.7, 81.75, 81.7, 81.0,
79.9, 79.6, 78.8, 78.1, 77.0, 76.4, 75.9, 75.85, 75.8, 75.7, 75.4,
75.35, 75.1, 74.4, 74.0, 73.9, 73.8, 72.7, 68.0, 67.8, 65.9, 63.1,
52.5, 52.4; MS (C50H51Cl3N4O11) m/z (M + Na+) calcd 1011.2518,
obsd 1011.2507.
ter t-Bu tyld im eth ylsilyl (Meth yl 2,3,4-tr i-O-ben zyl-R-L-
id op yr a n osid u r on a t e)-(1f4)-2-a zid o-3,6-d i-O-b en zyl-2-
d eoxy-â-D-glu cop yr a n osid e (13). Disaccharide 12 (125 mg,
0.144 mmol) was coevaporated three times with anhydrous
toluene and dissolved in anhydrous hexanes (1.4 mL) and a
minimum amount of anhydrous CH2Cl2 to dissolve the com-
pound. Benzyl trichloroacetimidate (54 µL, 0.288 mmol) was
added, followed by one drop (∼1 µL) of triflic acid. After 90
min, the solution was filtered to remove the precipitated
trichloroacetamide, and the solvent was removed under re-
duced pressure. Flash chromatography on silica (hexanes/ethyl
acetate 9:1) yielded 13 (105 mg, 0.109 mmol, 76%) as a clear
ter t-Bu tyld im eth ylsilyl (Meth yl 2-O-a cetyl-4-O-a llyl-3-
O-ben zyl-R-L-id op yr a n osid u r on a te)-(1f4)-2-a zid o-3,6-d i-
O-ben zyl-2-d eoxy-â-D-glu cop yr a n osid e (14). Trichloroacet-
imidate 914 (62 mg, 0.118 mmol) and monosaccharide 712 (40
mg, 0.079 mmol) were combined and coevaporated three times
with anhydrous toluene. The mixture was dissolved in anhy-
drous CH2Cl2 (1 mL) under N2 and cooled to -15 °C. Tri-
methylsilyl trifluoromethanesulfonate (130 µL of a 0.1 M
solution in CH2Cl2) was added and the reaction mixture stirred
for 30 min at reduced temperature and then allowed to warm
to room temperature over 30 min. The reaction was quenched
with NEt3 (0.2 mL), and the solvent was removed under
reduced pressure. Flash chromatography on silica (hexanes/
ethyl acetate 17:3) afforded 14 (60 mg, 0.070 mmol, 88%) as a
clear oil: Rf 0.28 (hexanes/ethyl acetate 3:1); [R]24D -35.3, c )
oil: Rf 0.53 (hexanes/ethyl acetate 3:1); [R]24 -20.1, c ) 0.7;
D
IR (thin film, NaCl plates) 2928, 2110, 1741 cm-1; H NMR
1
(500 MHz, CDCl3) δ 7.24-7.37 (m, 25H, aryl H), 5.40 (d,
2
3J H1-H2 ) 6.4, 1H, IdoA H1), 4.85 (d, J ) 10.7 Hz, 1H, benzyl
CH2), 4.73 (d, 2J ) 11.2 Hz, 1H, benzyl CH2), 4.72 (m, 1H,
benzyl CH2), 4.71 (m, 1H, benzyl CH2), 4.70 (m, 1H, benzyl
CH2), 4.68 (d, 2J ) 11.5 Hz, 1H, benzyl CH2), 4.64 (d, 2J )
11.6 Hz, 1H, benzyl CH2), 4.60 (d, 2J ) 11.6 Hz, 1H, benzyl
3
2
CH2), 4.58 (d, J H4-H5 ) 5.8, 1H, IdoA H4), 4.54 (d, J ) 12.4
Hz, 1H, benzyl CH2), 4.47 (d, 3J H1-H2 ) 7.4 Hz, 1H, GlcN3 H1),
4.45 (d, 2J ) 12.4 Hz, 1H, benzyl CH2), 3.98 (m, 1H, GlcN3
H4), 3.96 (m, 1H, IdoA H3), 3.81 (d2, J H3-H4 ) 7.4, J H4-H5
)
3
3
5.8, 1H, IdoA H4), 3.71 (d2, 2J ) 10.7 Hz, J H5-H6 ) 4.3 Hz,
3
2
3
1H, GlcN3 H6), 3.66 (d2, J ) 10.7 Hz, J H5-H6′ ) 1.7 Hz, 1H,
GlcN3 H6′), 3.53 (s, 3H, OCH3), 3.37 (d2, 3J H1-H2 ) 6.4, 3J H2-H3
) 7.5, 1H, IdoA H2), 3.34 (m, 1H, GlcN3 H5), 3.31 (m, 1H GlcN3
H2), 3.29 (m, 1H, GlcN3 H3), 0.95 (s, 9H, TBS tert-butyl), 0.16
(s, 3H, TBS methyl), 0.14 (s, 3H, TBS methyl); 13C NMR (125
MHz, CDCl3) δ 170.0 (IdoA C6), 138.6 (benzyl quat), 138.5
(benzyl quat), 138.4 (benzyl quat), 137.9 (benzyl quat), 128.6
(aromatic C), 128.55 (aromatic C), 128.5 (aromatic C), 128.4
(aromatic C), 128.3 (aromatic C), 128.25 (aromatic C), 128.15
(aromatic C), 128.1 (aromatic C), 128.05 (aromatic C), 128.0
(aromatic C), 127.9 (aromatic C), 127.8 (aromatic C), 127.7
(aromatic C), 127.65 (aromatic C), 127.5 (aromatic C), 100.0
(IdoA C1), 97.3 (GlcN3 C1), 81.1 (GlcN3 C2), 80.6 (IdoA C2),
79.0 (IdoA C3), 77.2 (IdoA C4), 75.9 (GlcN3 C4), 75.5 (GlcN3
C3), 75.0 (benzyl CH2), 74.9 (benzyl CH2), 74.6 (benzyl CH2),
73.4 (benzyl CH2), 73.35 (benzyl CH2), 72.0 (IdoA C5), 68.6
(GlcN3 C51), 68.3 (GlcN3 C6), 52.0 (OCH3), 25.8 (TBS tert-butyl
CH3), 18.2 (TBS tert-butyl quat), -4.0 (TBS methyl CH3), -5.0
(TBS methyl CH3); ESI MS (C54H65N3O11Si) m/z (M + Na+)
calcd 982.4281, obsd 982.4250.
0.62; IR (thin film, NaCl plates) 2926, 2110, 1764, 1741 cm-1
;
1H NMR (500 MHz, CDCl3) δ 7.24-7.39 (m, 15H, aromatics),
3
5.76 (m, 1H, allyl OCH2CHdCH2), 5.30 (d, J H1-H2 ) 3.5 Hz,
4
1H, IdoA H1), 5.17 (d3, J ) 17.4, 3 Hz, J ) 1.6 Hz, 1H, allyl
OCH2HCHdCHH), 5.13 (d3, J ) 10.4, 3 Hz, 4J ) 1.1 Hz), 4.89
3
(ψt, J ) 3.8 Hz, 1H, IdoA H2), 4.79 (d, J H5-H4 ) 3.8 Hz, 1H,
IdoA H5), 4.76 (d, 2J ) 11.1 Hz, 1H, GlcN3O3 benzyl CH2),
2
2
4.73 (d, J ) 11.7 Hz, 1H, IdoA O3 benzyl CH2), 4.71 (d, J )
2
11.1 Hz, 1H, GlcN3O3 benzyl CH2), 4.68 (d, J ) 11.7 Hz, 1H,
IdoA O3 benzyl CH2), 4.60 (d, 2J ) 12.3 Hz, 1H, GlcN3O6
2
benzyl CH2), 4.55 (d, J ) 12.3 Hz, 1H, GlcN3O6 benzyl CH2),
3
4.49 (d, J H1-H2 ) 7.5 Hz, 1H GlcN3 H1), 3.99 (m, 1H, allyl
OCHHCHdCH2), 3.98 (m, 1H, GlcN3 H4), 3.93 (d2ψt, J ) 1.4
Hz, 5.8 Hz, 12.8 Hz, 1H, allyl OCHHCHdCH2), 3.84 (ψt, J )
4.6 Hz, 1H, IdoA H3), 3.74 (ψt, J ) 4.2, 1H, IdoA H4), 3.68
(m, 2H, GlcN3 H6), 3.51 (s, 3H, -OCH3), 3.37 (m, 1H, GlcN3
H5), 3.35 (m, 1H GlcN3 H2), 3.27 (d2, 3J ) 9 Hz, 9.8 Hz, GlcN3
H3), 1.99 (s, 3H, acetate CH3), 0.96 (s, 9H, TBDMS tert-butyl),
0.16 (s, 3H, TBDMS methyl), 0.14 (s, 3H, TBDMS methyl);
13C NMR (125 MHz, CDCl3) δ 170.3 (acetate carbonyl), 169.8
(IdoA C6), 138.5 (GlcN3 O3 benzyl CH2), 138.3 (GlcN3O6 benzyl
quat), 137.9 (IdoA O3 benzyl quat), 134.4 (allyl OCH2CHd
CH2), 128.6 (aromatic C), 128.5 (aromatic C), 128.3 (aromatic
C), 128.1 (aromatic C), 128.05 (aromatic C), 127.8 (aromatic
C), 127.7 (aromatic C), 127.6 (aromatic C), 127.5 (aromatic C),
117.6 (allyl OCH2CHdCH2), 98.0 (IdoA C1), 97.4 (GlcN3 C1),
81.1 (GlcN3 C3), 75.4 (GlcN3 C5), 75.1 (GlcN3 C4), 75.1 (IdoA
C4), 74.8 (GlcN3 O3 benzyl CH2), 73.9 (IdoA C3), 73.5 (GlcN3-
O6 benzyl CH2), 73.1 (IdoA O3 benzyl CH2), 71.8 (allyl OCH2-
CHdCH2), 69.7 (IdoA C5), 69.4 (IdoA C2), 68.8 (GlcN3 C2),
68.5 (GlcN3 C6), 52.0 (OCH3), 25.8 (TBDMS tert-butyl CH3),
21.2 (acetate CH3), 18.2 (TBDMS t-butyl quat), -4.0 (TBDMS
methyl), -5.0 (TBDMS methyl); ESI MS (C45H59N3O12Si) m/z
(M + Na+) calcd 884.3760, obsd 884.3756.
Meth yl (2-O-Acetyl-4-O-a llyl-3-O-ben zyl-R-L-id op yr a -
n osid )u r on a te-(1f4)-2-a zid o-3,6-d i-O-ben zyl-2-d eoxy-D-
glu cop yr a n osid e Tr ich lor oa cetim id a te (15). Disaccharide
14 (960 mg, 1.11 mmol) was coevaporated three times with
anhydrous toluene and dissolved in anhydrous THF (10 mL).
The solution was cooled to 0 °C, and acetic acid (71 µL, 1.23
mmol) was added followed by TBAF (1.0 M in THF, 1.23 mL).
After 45 min, the reaction was poured into saturated NH4Cl,
and the aqueous phase was extracted with ethyl acetate (3 ×
Met h yl 2,3,4-Tr i-O-b en zyl-R-L-id op yr a n osid u r on a t e-
(1f4)-2-a zid o-3,6-d i-O-b en zyl-2-d eoxy-D-glu cop yr a n o-
sid e Tr ich lor oa cetim id a te (3). Disaccharide 13 (30 mg, 31
µmol) was coevaporated three times with anhydrous toluene
and dissolved in anhydrous THF (500 µL). The solution was
cooled to 0 °C, and acetic acid (2 µL, 35 µmol) was added
followed by TBAF (34 µL, 1.0 M in THF). After 1 h, the reaction
was poured into ethyl acetate (100 mL) and washed twice with
brine. The organic phase was dried over MgSO4, and the
solvent was removed under reduced pressure.
The residue was coevaporated three times with anhydrous
toluene and dissolved in anhydrous CH2Cl2 (500 µL) under N2.
The solution was cooled to 0 °C, and trichloroacetonitrile (47
µL, 0.47 mmol) and DBU (0.5 µL, 3 µmol) were added. The
reaction mixture was stirred for 30 min and then allowed to
warm to room temperature. The solvent was evaporated, and
the residue was purified by flash chromatography on silica
(toluene/ethyl acetate 9:1) afforded 3 (24 mg, 24 µmol, 78%,
1.5:1 R/â) as a yellow oil: Rf 0.57, 0.63 (hexanes/ethyl acetate
2:1); IR (thin film, NaCl plates) 3030, 2918, 2112, 1761, 1739,
1
1674 cm-1; H NMR (500 MHz, CDCl3) δ 8.70 (s, 0.4H), 8.69
(s, 0.6H), 7.12-7.39 (m, 25H), 6.38 (d, J ) 3.5 Hz, 0.6H), 5.6
(d, J ) 8.4 Hz, 0.4H), 5.44-5.50 (m, 1H), 5.03 (d, J ) 10.3 Hz,
0.6H), 4.92 (d, J ) 10.7 Hz, 0.4 H), 4.70-4.78 (m, 5H), 4.63-
4088 J . Org. Chem., Vol. 69, No. 12, 2004

Synthesis of Fully N-Differentiated Heparin Oligosaccharides
150 mL). The organic phases were combined and dried over
MgSO4, and the solvent was removed under reduced pressure.
mmol, 84%): Rf 0.31 (hexanes/ethyl acetate 1:2); [R]24 -1.3,
D
c ) 1.03; IR (thin film, NaCl plates) 3030, 2931, 1767, 1739,
1682 cm-1; 1H NMR (500 MHz, CDCl3) δ 7.16-7.41 (m, 20H),
6.53 (d, J ) 9.7 Hz, 1H), 5.74 (m, 1H), 5.36 (d, J ) 2.3 Hz,
1H), 5.30 (d, J ) 3.6 Hz, 1H), 5.16 (d3, J ) 1.6, 3.3, 17.2 Hz,
1H), 5.10 (d3, J ) 1.3, 3.3, 10.4 Hz, 1H), 4.89 (t, J ) 4 Hz, 1H),
4.66-4.74 (m, 6H), 4.57-4.6 (m, 3H), 4.48 (d, J ) 1.4 Hz, 1H),
4.38 (d, J ) 11.5 Hz, 1H), 4.36 (dt, J ) 3.7, 10.5 Hz, 1H), 4.08
(t, J ) 9.5 Hz, 2H), 3.93-3.99 (m, 4H), 3.88 (t, J ) 2.2 Hz,
1H), 3.83 (t, J ) 4.8 Hz, 1H), 3.76 (m, 1H), 3.68 (m, 1H), 3.67
(s, 3H), 3.60 (d2, J ) 2.3, 11 Hz, 1H), 3.52-3.56 (m, 1H), 3.47
(s, 3H), 3.43 (d2, J ) 9.3, 10.5 Hz), 1.93 (s, 3H), 1.8 (s, 3H),
1.55 (s, 3H), 1.36 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 170.2,
170.15, 169.8, 169.1, 138.9, 138.2, 138.0, 136.6, 134.5, 129.0,
128.7, 128.65, 128.5, 128.3, 128.2, 128.1, 128.0, 127.8, 127.7,
127.6, 127.0, 117.5, 112.3, 98.0, 97.7, 96.6, 79.4, 75.3, 75.25,
75.2, 74.7, 74.0, 73.6, 73.25, 73.2, 72.2, 71.9, 71.5, 71.4, 71.35,
69.6, 69.4, 68.1, 52.7, 52.3, 52.0, 28.6, 26.4, 23.1, 21.2; ESI MS
(C58H69NO19) m/z (M + Na+) calcd 1106.4356, obsd 1106.4328.
The residue was coevaporated three times with anhydrous
toluene and dissolved in anhydrous CH2Cl2 (10 mL) under N2.
The solution was cooled to 0 °C and trichloroacetonitrile (1.7
mL, 16.7 mmol) and DBU (32 µL, 0.22 mmol) was added. The
reaction mixture was stirred for 30 min and then allowed to
warm to room temperature. The solvent was evaporated, and
the residue was purified by flash chromatography on silica
(hexanes/ethyl acetate 3:1) to afford 15 (950 mg, 1.07 mmol,
96%, 2:1 â:R) as a yellow oil: Rf 0.14 (hexanes/ethyl acetate
3:1); IR (thin film, NaCl plates) 3338, 3031, 2876, 2113, 1745,
1739, 1675 cm-1; 1H NMR (500 MHz, CDCl3) δ 8.70 (s, 0.67H),
8.69 (s, 0.33H), 7.24-7.38 (m, 15H), 6.40 (d, J ) 3.5 Hz, 0.33H),
5.74-5.79 (m, 1H), 5.60 (d, J ) 8.3 Hz, 0.67H), 5.35-5.37 (m,
1H), 5.17-5.21 (m, 2H), 4.51-4.94 (m, 8H), 4.2 (t, J ) 4.4,
0.33 H), 4.12 (t, 0.66H), 3.83-4.02 (m, 4H), 3.63-3.78 (m, 4H),
3.41-3.59 (m, 2H), 3.52 (s, 2H), 3.51 (s, 1H), 1.97 (s, 2H), 1.96
(s, 1H); 13C NMR (125 MHz, CDCl3) δ 170.2, 170.19, 169.8,
161.2, 160.9, 138.2, 138.1, 138.0, 137.98, 137.95, 137.92, 134.4,
134.3, 128.66, 128.65, 128.57, 128.48, 128.39, 128.33, 128.18,
128.15, 128.1, 127.98, 127.9, 127.87, 127.77, 127.71, 127.63,
127.6, 117.73, 117.67, 97.9, 96.9, 95.0, 90.7, 78.5, 76.3, 75.3,
75.17, 75.15, 75.0, 75.9, 74.5, 74.2, 74.0, 73.6, 73.5, 73.3, 73.2,
71.95, 71.9, 70.2, 69.9, 69.7, 69.5, 67.8, 67.7, 65.7, 63.1, 52.1,
52.0, 21.22, 21.20; ESI MS (C41H45Cl3N4O12) m/z (M + Na+)
calcd 913.1992, obsd 913.1996.
ter t-Bu t yld im et h ysilyl Met h yl [Met h yl (2-O-a cet yl-
4-O-a llyl-3-O-b en zyl-R-L-id op yr a n osid )u r on a t e-(1f4)-2-
a ceta m id o-3,6-d i-O-ben zyl-2-d eoxy-R-D-glu cop yr a n osid e-
(1f4)-3-O-ben zyl-â-L-idopyr an osid]u r on ate (18). Trisaccha-
ride 17 (1.66 g, 1.53 mmol) was dissolved in TFA (90% aq, 30
mL) and stirred for 20 min. The solvent was removed under
reduced pressure and the residue coevaporated five times with
anhydrous toluene. The crude material was dissolved in
anhydrous CH2Cl2 (4 mL) and cooled to -25 °C under N2.
TBDMSCl (347 mg, 2.3 mmol) and imidazole (417 mg, 6.12
mmol) were added, and the reaction mixture was stirred for
16 h at -5 °C. Methanol (1 mL) was added and the mixture
stirred for 15 min and poured into ethyl acetate (300 mL). The
organic phase was washed with 1 N HCl, brine, and saturated
NaHCO3. The organic phase was dried over MgSO4, and
solvent was removed under reduced pressure. Flash chroma-
tography on silica (hexanes/ethyl acetate 1:1) afforded 18 (1.65
g, 1.43 mmol, 93%) as a clear oil (15:1 â/R). Characterization
data is reported for the â isomer only: Rf 0.28 (hexanes/ethyl
acetate 1:2); IR (thin film, NaCl plates) 3336, 3031, 2858, 1767,
1739, 1674 cm-1; 1H NMR (500 MHz, CDCl3) δ 7.19-7.41 (m,
20H), 6.71 (d, J ) 9.6 Hz, 1H); 5.7-5.78 (m, 1H), 5.26 (d, J )
3.5 Hz, 1H), 5.14 (d3, J ) 17.1, 3.2, 1.6 Hz, 1H), 5.09 (d3, J )
10.3, 2.9, 1.2 Hz, 1H), 5.02 (d, J ) 1.3 Hz, 1H), 4.85 (t, J ) 4
Hz, 1H), 4.76 (d, J ) 3.8 Hz, 1H), 4.67-4.73 (m, 4H), 4.62 (d,
J ) 11.6 Hz, 1H), 4.51-4.57 (m, 4H), 4.45 (d, J ) 11.6 Hz,
1H), 4.27-4.32 (m, 1H), 4.05 (t, J ) 9.5 Hz, 1H), 3.88-3.93
(m, 3H), 3.80 (t, J ) 4.8 Hz, 1H), 3.74-3.77 (m, 2H), 3.70 (t,
J ) 4.3 Hz, 1H), 3.67 (s, 3H), 3.53-3.67 (m, 4H), 3.48 (s, 3H),
2.68 (s, 1H), 1.94 (s, 3H), 1.73 (s, 3H), 0.96 (s, 9H), 0.24 (s,
3H), 0.19 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 170.5, 170.1,
169.8, 168.9, 138.9, 138.2, 138.0, 136.9, 134.4, 128.9, 128.65,
128.6, 128.4, 128.16, 128.15, 128.0, 127.9, 127.85, 127.75,
127.6, 127.1, 117.5, 98.0, 97.7, 93.8, 78.3, 75.3, 75.1, 74.4, 74.0,
73.7, 73.5, 73.4, 73.1, 73.0, 72.1, 71.8, 71.7, 69.5, 69.4, 68.7,
68.2, 52.5, 52.2, 51.9, 25.9, 23.1, 21.1, 18.4, -3.9, -5.2; ESI
MS (C61H79NO14Si) m/z (M + Na+) calcd 1180.4908, obsd
1180.4883.
Meth yl [Meth yl (2-O-a cetyl-4-O-a llyl-3-O-ben zyl-R-L-
id op yr a n osid )u r on a t e-(1f4)-2-a zid o-3,6-d i-O-b en zyl-2-
d eoxy-R-D-glu cop yr a n osid e-(1f4)-3-O-ben zyl-1,2-isop r o-
p ylid in e-â-L-id op yr a n osid ]u r on a te (16). Trichloroacetimi-
date 15 (950 mg, 1.07 mmol) and monosaccharide 812,30 (433
mg, 1.28 mmol) were combined and coevaporated three times
with anhydrous toluene. The mixture was dissolved in anhy-
drous CH2Cl2 (10 mL) under N2 and cooled to -25 °C, freshly
activated 4 Å molecular sieves (1 g) were added, and the
reaction mixture was stirred for 30 min. Trimethylsilyl tri-
fluoromethanesulfonate (25 µL, 0.107 mmol) was added, and
the reaction mixture was stirred for 90 min at reduced
temperature. The reaction was quenched with NEt3 (1 mL),
and the solvent was removed under reduced pressure. Flash
chromatography on silica (toluene/ethyl acetate 10:1 f 20:3)
afforded 16 (940 mg, 0.88 mmol, 83%) as a clear oil: Rf 0.23
(hexanes/ethyl acetate 2:1); [R]24D -12.5, c ) 1.2; IR (thin film,
NaCl plates) 2932, 2108, 1765, 1741 cm-1; 1H NMR (500 MHz,
CDCl3) δ 7.24-7.29 (m, 20H), 5.75-5.81 (m, 1H), 5.35 (d, J )
4.9 Hz, 1H), 5.32 (d, J ) 2.5 Hz, 1H), 5.19 (d3, J ) 1.6, 3.2,
17.2 Hz, 1H), 5.13 (d3, J ) 1.2, 2.8, 10.4 Hz, 1H), 4.86-4.89
(m, 3H), 4.65-4.74 (m, 6H), 4.61 (d, J ) 12.2 Hz, 1H), 4.51 (d,
J ) 12 Hz, 1H), 4.40 d, J ) 1.4 Hz, 1H), 4.19 (t, J ) 2.3 Hz,
1H), 3.95-4.08 (m, 5H), 3.86 (t, J ) 6 Hz, 1H), 3.70-3.77 (m,
4H), 3.64 (s, 3H), 3.61 (d2 J ) 1.9, 11.1 Hz, 1H), 3.51 (s, 3H),
,
3.43 (d2, J ) 3.3, 10.3 Hz, 1H), 1.96 (s, 3H), 1.58 (s, 3H), 1.37
(s, 3H); 13C NMR (125 MHz, CDCl3) δ 169.9, 161.2, 138.3,
138.15, 138.1, 137.2, 134.4, 128.8, 128.7, 128.5, 128.4, 128.3,
128.2, 128.1, 128.05, 128.0, 127.9, 127.8, 127.5, 117.7, 112.3,
98.3, 97.9, 97.1, 78.3, 75.9, 75.45, 75.4, 75.2, 74.9, 73.6, 73.4,
73.0, 72.8, 72.1, 71.8, 71.4, 70.8, 70.6, 67.5, 63.7, 52.5, 52.0,
28.2, 26.3, 21.2; ESI MS (C56H65N3O18) m/z (M + Na+) calcd
1090.4155, obsd 1090.4123.
ter t-Bu t yld im et h ysilyl Met h yl [Met h yl (2-O-a cet yl-
4-O-a llyl-3-O-b en zyl-R-L-id op yr a n osid )u r on a t e-(1f4)-2-
N-a cetyla ceta m id o-3,6-d i-O-ben zyl-2-d eoxy-R-D-glu cop y-
r a n osid e-(1f4)-2-O-a cetyl-3-O-ben zyl-â-L-id op yr a n osid ]-
u r on a te (19). Trisaccharide 18 (1.66 g, 1.42 mmol) was
coevaporated three times with anhydrous toluene and dis-
solved in isopropenyl acetate (15 mL) under N2. p-Toluene-
sulfonic acid (700 µL, 0.1 M in DMF) was added and the
mixture irradiated in a CEM Discover Series Microwave for 5
h at 30 W, 90 °C max. The reaction was poured into ethyl
acetate (100 mL) and washed with saturated NaHCO3. The
organic phase was dried over MgSO4, and the solvent was
removed under reduced pressure. Flash chromatography on
silica (hexanes/ethyl acetate 7:3) afforded 19 (1.52 g, 1.22
mmol, 86%) as a clear oil: Rf 0.38 (hexanes/ethyl acetate 1:1);
Meth yl [Meth yl (2-O-a cetyl-4-O-a llyl-3-O-ben zyl-R-L-
idopyr an osid)u r on ate-(1f4)-2-acetam ido-3,6-di-O-ben zyl-
2-d eoxy-R-D-glu cop yr a n osid e-(1f4)-3-O-b en zyl-1,2-iso-
p r op ylid in e-â-L-id op yr a n osid ]u r on a te (17). Trisaccharide
16 (940 mg, 0.88 mmol) was coevaporated three times with
anhydrous toluene. The compound was dissolved in anhydrous
pyridine (9 mL) under N2 and cooled to 0 °C. Thiolacetic acid
(9 mL) was added and the cooling bath removed. The reaction
mixture was stirred for 12 h, and the solvent was removed
under reduced pressure. Flash chromatography on silica
(hexanes/ethyl acetate 7:3) eluted the side products. Further
elution (hexanes/ethyl acetate 1:1) afforded 17 (800 mg, 0.74
J . Org. Chem, Vol. 69, No. 12, 2004 4089

Lohman and Seeberger
[R]24 +52.9, c ) 0.91; IR (thin film, NaCl plates) 2952, 1741,
68.4, 67.7, 58.9, 52.1, 52.0, 26.7, 25.8, 21.1, 21.0, 18.1, -4.0,
-5.0; ESI MS (C85H104N4O25Si) m/z (M + Na+) calcd 1631.6651,
obsd 1631.6701.
D
1675 cm-1; 1H NMR (500 MHz, CDCl3) δ 7.12-7.39 (m, 20H),
5.74 (m, 1H), 5.34 (d, J ) 3.4 Hz, 1H), 5.16 (m, 1H), 5.10 (m,
1H), 5.02 (d, J ) 1.9 Hz, 1H), 5.01 (d, J ) 3.8 Hz, 1H), 4.99
(m, 1H), 4.92 (ψt, 1H), 4.78 (d, J ) 11.5 Hz, 1H), 4.67-4.75
(m, 4H), 4.52-4.62 (m, 5H), 4.45 (d, J ) 1.8, 1H), 4.33 (d2, J
) 4.0, 11 Hz, 1H), 3.91-4.03 (m, 6H), 3.85 (ψt, 1H), 3.75 (s,
3H), 3.67-3.74 (m, 3H), 3.49 (s, 3H), 2.20 (s, 6H), 2.03 (s, 3H),
1.98 (s, 3H), 0.87 (s, 9H), 0.14 (s, 3H), 0.09 (s, 3H); 13C NMR
(125 MHz, CDCl3) δ 175.9, 170.3, 170.0, 169.9, 168.8, 139.2,
138.03, 137.97, 137.2, 134.4, 128.8, 128.7, 128.5, 128.4, 128.24,
128.19, 128.06, 127.95, 127.87, 127.2, 127.1, 117.7, 98.0, 95.7,
93.6, 77.5, 76.3, 75.5, 74.7, 73.7, 73.4, 73.1, 72.9, 72.4, 72.3,
71.9, 70.4, 70.2, 70.0, 67.9, 67.7, 59.8, 52.4, 52.0, 26.8, 25.7,
21.2, 21.0, 18.0, -3.9, -5.4; ESI MS (C65H83NO21Si) m/z (M +
Na+) calcd 1264.5119, obsd 1264.5082.
Meth yl [Meth yl (2-O-a cetyl-4-O-a llyl-3-O-ben zyl-R-L-
id op yr a n osid )u r on a te-(1f4)-2-N-a cetyla ceta m id o-3,6-d i-
O-ben zyl-2-d eoxy-R-D-glu cop yr a n osid e-(1f4)-2-O-a cetyl-
3-O-ben zyl-R-L-id op yr a n osid ]u r on a te-(1f4))-2-a zid o-3,6-
di-O-ben zyl-2-deoxy-D-glu copyr an oside Tr ich lor oacetim i-
d a te (4). Tetrasaccharide 21 (251 mg, 0.156 mmol) was
coevaporated three times with anhydrous toluene and dis-
solved in anhydrous THF (1.5 mL). The solution was cooled
to 0 °C, and acetic acid (10.2 µL, 0.18 mmol) was added
followed by TBAF (1.0 M in THF, 172 µL). After 30 min, the
reaction was poured into saturated NH4Cl and extracted three
times with ethyl acetate. The organic phase was dried over
MgSO4, and the solvent was removed under reduced pressure.
Meth yl [Meth yl (2-O-a cetyl-4-O-a llyl-3-O-ben zyl-R-L-
id op yr a n osid )u r on a te-(1f4)-2-N-a cetyla ceta m id o-3,6-d i-
O-ben zyl-2-d eoxy-R-D-glu cop yr a n osid e-(1f4)-2-O-a cetyl-
3-O-ben zyl-L-idopyr an osid]u r on ate Tr ich lor oacetim idate
(20). Trisaccharide 19 (325 mg, 0.262 mmol) was coevaporated
three times with anhydrous toluene and dissolved in anhy-
drous THF (2.5 mL). The solution was cooled to 0 °C, and acetic
acid (22.5 µL, 0.39 mmol) was added followed by TBAF (1.0 M
in THF, 290 µL). After 80 min, the reaction was poured into
saturated NH4Cl and extracted with ethyl acetate (3 × 200
mL). The organic phase was dried over MgSO4, and the solvent
was removed under reduced pressure.
The residue was coevaporated three times with anhydrous
toluene and dissolved in anhydrous CH2Cl2 (1.5 mL) under N2.
The solution was cooled to 0 °C, and trichloroacetonitrile (226
µL, 2.25 mmol) and DBU (4.3 µL, 0.03 mmol) were added. The
reaction mixture was stirred for 45 min and then allowed to
warm to room temperature. The solvent was evaporated, and
the residue was purified by flash chromatography on silica
(elutant toluene/ethyl acetate 7:3) to afford 4 (228 mg, 0.139
mmol, 89%, 6:1 mixture of isomers) as a yellow oil: Rf 0.3,
0.375 (hexanes/ethyl acetate 3:2); IR (thin film, NaCl plates)
2926, 2112, 1739, 1675 cm-1; ESI MS (C81H90Cl3N5O25) m/z (M
+ Na+) calcd 1660.4883, obsd 1660.4913.
The residue was coevaporated three times with anhydrous
toluene and dissolved in anhydrous CH2Cl2 (2.5 mL) under N2.
The solution was cooled to 0 °C and trichloroacetonitrile (394
µL, 3.93 mmol) and DBU (7.4 µL, 0.052 mmol) added. The
reaction mixture was stirred for 60 min and then allowed to
warm to room temperature. The solvent was evaporated, and
the residue was purified by flash chromatography on silica
(elutant toluene/ethyl acetate 3:1) to afford 20 (281 mg, 0.22
mmol, 84%, 4:1 mixture of isomers) as a yellow oil: Rf 0.27,
0.33 (hexanes/ethyl acetate 1:1); IR (thin film, NaCl plates)
3447, 1739, 1674, 1650 cm-1; ESI MS (C61H69Cl3N2O21) m/z (M
+ Na+) calcd 1293.3351, obsd 1293.3347.
Meth yl 2-Ben zyloxyca r bon yla m in o-2-d eoxy-3,6-d i-O-
a cetyl-R-D-glu cop yr a n osid e (10). Compound 2243 was co-
evaporated twice with anhydrous toluene and dissolved in
2,4,6-collidine (7 mL, distilled from CaH). The mixture was
cooled to -40 °C, and acetyl chloride (270 µL, 3.78 mmol) was
added. The reaction mixture was stirred overnight under N2
at -40 °C. Water (1 mL) was added, and the reaction warmed
to room temperature and poured into ethyl acetate (600 mL).
The organic phase was washed with 1 N HCl, brine, and
saturated NaHCO3 and dried over MgSO4, and the solvent was
removed under reduced pressure. Flash chromatography on
silica (hexanes/ethyl acetate7:3) afforded 17 (1.17 g, 82%) as
a glassy solid: Rf 0.19 (hexanes/ethyl acetate 1:1); [R]24D +55.2,
c ) 1.26; mp ) 98.0-99.5 °C; IR (thin film, NaCl plates) 3434,
ter t-Bu tyld im eth ylsilyl (Meth yl [m eth yl (2-O-a cetyl-
4-O-a llyl-3-O-ben zyl-R-L-id op yr a n osid )u r on a t e-(1f4)-2-
N-a cet yla cet a m id o-3,6-d i-O-b en zyl-2-d eoxy-R-D-glu co-
p yr a n osid e-(1f4)-2-O-a cetyl-3-O-ben zyl-R-L-id op yr a n o-
sid]u r on ate-(1f4))-2-azido-3,6-di-O-ben zyl-2-deoxy-â-D-glu -
cop yr a n osid e (21). Trichloroacetimidate 20 (288 mg, 0.226
mmol) and 7 (565 mg, 1.13 mmol) were combined and coevapo-
rated three times with anhydrous toluene. The mixture was
dissolved in CH2Cl2 (2.25 mL) under N2 and cooled to -25 °C.
Trimethylsilyl trifluoromethanesulfonate (4 µL, 0.023 mmol)
was added and the reaction mixture stirred for 1.5 h at reduced
temperature. The reaction was quenched with NEt3 (0.1 mL),
and the solvent was removed under reduced pressure. Flash
chromatography on silica (hexanes/ethyl acetate 9:1) eluted
unreacted 7 (480 mg, 0.96 mmol). Further elution (hexanes/
ethyl acetate 7:3) afforded 21 (259 mg, 0.16 mmol, 71%) as a
clear oil: Rf 0.37 (hexanes/ethyl acetate 1:1); [R]24D +6.22, c )
1.05; IR (thin film, NaCl plates) 2952, 2111, 1756, 1741, 1675
cm-1; 1H NMR (500 MHz, CDCl3) δ 7.13-7.4 (m, 30H), 5.72-
5.79 (m, 1H), 5.36 (d, J ) 4.8 Hz, 1H), 5.16-5.2 (m, 2H), 5.12
(d2, J ) 1.5, 10.5 Hz, 1H), 5.05 (d, J ) 3.8 Hz, 1H), 4.88-4.93
(m, 2H), 4.47-4.78 (m, 10H), 4.37 (d2, J ) 3.7, 11.1 Hz, 1H),
3.93-4.06 (m, 5H), 3.86-3.90 (m, 2H), 3.77-3.80 (m, 1H),
3.63-3.74 (m, 5H), 3.50 (s, 3H), 3.47 (s, 3H), 3.32-3.37 (m,
2H), 3.24 (ψt, 1H), 2.14 (s, 6H), 1.97 (s, 3H), 1.94 (s, 3H), 0.94
(s, 9H), 0.17 (s, 3H), 0.15 (s, 3H); 13C NMR (125 MHz, CDCl3)
δ 175.6, 170.0, 169.91, 169.9, 169.2, 138.9, 138.4, 138.3, 138.08,
138.03, 137.6, 134.3, 128.7, 128.6, 128.55, 128.5, 128.3, 128.2,
128.15, 128.08, 128.05, 127.9, 127.85, 127.8, 127.7, 127.67,
127.66, 127.5, 127.2, 127.15, 117.75, 98.13, 98.1, 97.4, 96.7,
81.1, 76.7, 76.3, 75.7, 75.4, 75.0, 74.8, 74.7, 73.7, 73.5, 73.4,
73.2, 72.8, 72.0, 71.8, 71.7, 71.2, 70.7, 70.6, 69.0, 68.9, 68.8,
2956, 1739, 1710, 1520 cm-1
;
1H NMR (500 MHz, CDCl3) δ
3
7.30-7.36 (m, 5H, CBz aromatic), 5.17 (d, J H2-NH )10 Hz,
2
1H, carbamate NH), 5.13 (d, J ) 12.3 Hz, 1H Cbz CH2), 5.06
3
3
2
(d2, J H3-H4 ) 9.4 Hz, J H2-H3 ) 10.5 Hz, 1H, H3), 5.02 (d, J
3
) 12.3 Hz, 1H, Cbz CH2), 4.71 (d, J H1-H2 ) 3.6 Hz, 1H, H1),
4.44 (d2, J H5-H6 ) 4.4 Hz, J H6)H6′ ) 12.2 Hz, 1H, H6), 4.29
3
2
3
2
(d2, J H5-H6′ ) 2.2 Hz, J H6)H6′ ) 12.2 Hz, 1H, H6′), 3.95 (dψt,
3J H1-H2 ) 3.6 Hz, J ) 10.3 Hz, 1H, H2), 3.77 (d3 J H5-H6′ ) 2.2
3
,
3
3
Hz, J H5-H6 ) 4.4 Hz, J H4-H5 ) 10 Hz, 1H, H5), 3.57 (ψt, J )
9.4 Hz, 1H, H4), 3.38 (s, 3H, OCH3), 3.17 (s (b), 1H, OH), 2.11
(s, 3H, 3-O-acetate CH3), 1.94 (s, 6H, 3-O-acetate CH3); 13C
NMR (125 MHz, CDCl3) δ 172.2 (6-O-acetate carbonyl), 171.6
(3-O-acetate carbonyl), 156.1 (CBz carbonyl), 136.4 (CBz
phenyl quaternary), 128.7 (aromatic C), 128.3 (aromatic C),
128.2 (aromatic C), 98.8 (C1), 74.1 (C3), 70.0 (C5), 68.8 (C4),
67.0 (CBz CH2), 63.1 (C6), 55.4 (OCH3), 53.7 (C2), 21.0 (3-O-
acetate CH3), 20.9 (6-O-acetate CH3); ESI MS (C19H25NO9) m/z
(M + Na+) calcd 434.1422, obsd 434.1420.
Met h yl Met h yl 2-O-Acet yl-4-O-a llyl-3-O-b en zyl-R-L-
id op yr a n osid u r on a te-(1f4)-3,6-d i-O-a cetyl-2-ben zyloxy-
ca r b on yla m in o-2-d eoxy-R-D-glu cop yr a n osid e (23). Gly-
cosyl trichloroacetimidate 9 (243 mg, 0.46 mmol) and monosac-
charide 10 (136 mg, 0.33 mmol) were combined and coevapo-
rated three times with anhydrous toluene. The mixture was
dissolved in anhydrous CH2Cl2 (5 mL) under N2 and cooled to
-15 °C. Trimethylsilyl trifluoromethanesulfonate (8 µL, 46
µmol) was added, and the mixture was stirred for 3 h at low
temperature. The cooling bath was removed, and the reaction
mixture was stirred for an additional 3 h at room temperature,
followed by quenching with NEt3 (0.5 mL). The solvent was
4090 J . Org. Chem., Vol. 69, No. 12, 2004

Synthesis of Fully N-Differentiated Heparin Oligosaccharides
removed under reduced pressure. Flash chromatography on
silica (hexanes/ethyl acetate 7:3) afforded 23 (143 mg, 0.195
mmol, 59%): Rf 0.26 (hexanes/ethyl acetate 1:1); [R]24D +5.75,
c ) 1.4; IR (thin film, NaCl plates) 1739, 1516 cm-1; 1H NMR
(500 MHz, CDCl3) δ 72.7-7.37 (m, 10H), 5.69-5.76 (m, 1H),
5.19 (d2, J ) 9 Hz, 10.6 Hz, 1H), 5.10-5.15 (m, 3H), 4.99-
5.04 (m, 3H), 4.75-4.77 (m, 2H), 4.72 (d, J ) 12.2 Hz, 1H),
4.70 (d, J ) 3.6 Hz, 1H), 4.62 (d J ) 12.2 Hz, 1H), 4.28 (d, J
) 2.8 Hz, 2H), 3.79-3.99 (m, 5H), 3.78 (s, 3H), 3.74 (m, 2H),
3.36 (s, 3H), 2.13 (s, 3H), 2.06 (s, 3H), 1.92 (s, 3H); 13C NMR
(125 MHz, CDCl3) δ 171.5, 171.1, 170.3, 169.8, 156.1, 137.8,
136.5,134.2, 128.7, 128.6, 128.4, 128.3, 128.0, 127.8, 118.0,
99.6, 98.7, 76.2, 74.5, 72.7, 72.5, 72.1, 71.8, 69.8, 68.8, 68.7,
acetate 3:1); IR (thin film, NaCl plate) 3519, 2929, 2858, 1770,
1
1739 cm-1; H NMR (500 MHz, CDCl3) major isomer δ 7.31-
7.40 (m, 5H), 5.72-5.80 (m, 1H), 5.14-5.18 (m, 2H), 5.03 (d,
J ) 0.8 Hz, 1H), 4.64 (d, J ) 12 Hz, 1H), 4.60 (d, J ) 12 Hz,
1H), 4.52 (d, J ) 1.7 Hz, 1H), 4.04 (d2, J ) 5.9, 12.6 Hz, 1H),
3.89-3.93 (m, 2H), 3.78 (s, 3H), 3.76 (s, 1H), 3.63-3.65 (m,
1H), 3.04 (d, J ) 10.1 Hz, 1H), 0.93 (s, 9H), 0.19 (s, 3H), 0.16
(s, 3H), minor isomer δ 5.32 (s, 1H), 4.92 (d, J ) 1.8 Hz, 1H),
3.81 (s, 3H), 3.40 (d, J ) 10 Hz, 1H), 0.87 (s, 9H), 0.14 (s, 3H),
0.12 (s, 3H); 13C NMR (125 MHz, CDCl3) major isomer δ 169.4,
137.5, 133.9, 128.8, 128.3, 127.9, 116.6, 94.7, 74.3, 74.0, 73.7,
72.5, 72.0, 68.8, 52.3, 26.0, 18.4, -3.9, -4.8, minor isomer δ
170.5, 138.0, 133.6, 128.7, 128.3, 127.6, 118.5, 96.4, 74.4, 72.8,
72.1, 71.7, 67.4, 67.3, 52.4, 25.7, 18.0; ESI MS (C23H36O7Si)
m/z (M + Na+) calcd 475.2123, obsd 475.2143.
67.1, 62.4, 55.5, 54.2, 52.3, 21.3, 21.1, 21.0; ESI MS (C38H47
-
NO16) m/z (M + Na+) calcd 796.2793, obsd 796.2750.
Meth yl (ter t-Bu tyld im eth ylsilyl 4-O-a llyl-2-O-ben zoyl-
3-O-ben zyl-L-id op yr a n osid )u r on a te (27). Compound 26
(488 mg, 1.08 mmol) was coevaporated three times with
toluene and dissolved in CH2Cl2 (10 mL). DMAP (13.5 mg, 0.11
mmol) and benzoyl chloride (250 µL, 2.16 mmol) were added,
and the reaction mixture was stirred overnight under N2 with
exclusion of light. The reaction mixture was poured into ethyl
acetate (200 mL) and washed with 1 N HCl, brine, and
saturated aq NaHCO3. The organic phase was dried over
MgSO4 and, filtered, and the solvent was removed under
reduced pressure. Flash silica gel column chromatography
(hexanes/ethyl acetate 9:1) afforded 27 (464 mg, 0.833 mmol,
77%, 3:1 mixture of isomers) as a clear oil: Rf 0.33 (hexanes/
ethyl acetate 85:15); IR (thin film, NaCl plate) 2929, 2857,
Meth yl (Meth yl 2-O-a cetyl-3-O-ben zyl-R-L-id op yr a n o-
sid u r on a te)-(1f4)-3,6-d i-O-a cetyl-2-ben zyloxyca r bon yl-
a m in o-2-d eoxy-R-D-glu cop yr a n osid e (5). Disaccharide 23
(140 mg, 0.19 mmol) and sodium acetate (600 mg) were
combined and dissolved in acetic acid (2.6 mL) and water (150
µL). PdCl2 (202 mg, 1.14 mmol) was added, and the reaction
mixture was stirred for 9 h. Palladium black precipitated from
solution over the course of the reaction. The reaction mixture
was poured into saturated NaHCO3 (200 mL), and the aqueous
layer was extracted with ethyl acetate (3 × 200 mL). The
combined organic phases were dried over MgSO4, and the
solvent was removed under reduced pressure. Flash chroma-
tography on silica (1:1 hexanes/ethyl acetate) afforded 5 (113
mg, 0.16 mmol, 86%) as a clear oil: Rf 0.38 (hexanes/ethyl
acetate 1:3); [R]24D +17.5, c ) 0.99; IR (thin film, NaCl plates)
1
1772, 1723 cm-1; H NMR (500 MHz, CDCl3) δ 8.11 (d, J )
3447, 2955, 1743, 1521 cm-1
;
1H NMR (500 MHz, CDCl3) δ
7.9 Hz, 1.5H), 8.06 (s, J ) 8.1 Hz, 0.5H), 7.53-7.58 (m, 1H),
7.30-7.43 (m, 7H), 5.64-5.80 (m, 1H), 5.48 (s, 0.25H), 5.01-
5.23 (m, 4H), 4.93 (d, J ) 2.9 Hz, 0.25H), 4.80-4.82 (m, 1H),
4.67-4.71 (m, 1H), 4.58 (s, 0.75H), 4.04 (m, 1H), 3.89-3.95
(m, 2H), 3.75-3.80 (m, 4.5 H), 0.87 (s, 2.25H), 0.82 (s, 6.75H),
0.13 (s, 3H), 0.10 (s, 3H); 13C NMR (125 MHz, CDCl3) major
isomer δ 169.4, 166.5, 137.5, 134.5, 133.1, 130.3, 128.7, 128.45,
128.4, 128.3, 128.1, 117.6, 93.4, 74.3, 73.6, 73.0, 72.9, 71.6, 68.2,
52.3, 25.8, 18.1, -3.7, -5.0; minor isomer δ 170.6, 165.9, 138.0,
134.4, 133.4, 130.4, 130.2, 129.9, 128.5, 128.35, 127.9, 117.4,
93.5, 75.1, 73.2, 72.6, 71.7, 70.0, 69.1, 52.35, 25.7, 18.04, -4.4,
-5.4; ESI MS (C30H40O8Si) m/z (M + Na+) calcd 579.2385, obsd
579.2369.
3
7.29-7.38 (m, 10H, aromatic H), 5.21 (d2, J ) 9.0, 10.6 Hz,
1H, GlcN H3), 5.13 (d, 2J ) 12.2 Hz, 1H, CBz CH2), 5.03 (d, 2J
3
) 12.2 Hz, 1H, CBz CH2), 4.98 (d, J H2-NH ) 10 Hz, 1H, CBz
NH), 4.94 (s, 1H, IdoA H1), 4.83 (d, 3J H5-H4 ) 1.9 Hz, 1H, IdoA
2
H5), 4.81, (s, 1H, IdoA H2), 4.71 (d, J ) 12.6, 1H 3-O-benzyl
3
CH2), 4.70 (d, J H1-H2 ) 3.9 Hz, 1H, GlcN H1), 4.62 (d, 2J )
12.2, 1H 3-O-benzyl CH2), 4.31 (d2, 2J H6-H6′ ) 12.3 Hz, 3J H6-H5
2
3
) 3.3 Hz, 1H, GlcN H6), 4.25 (d2, J H6-H6′ ) 12.3 Hz, J H6′-H5
) 1.8 Hz, 1H, GlcN H6′), 3.96 (m, 1H, IdoA H4), 3.92 (m, 1H,
GlcN H2), 3.80 (s, 3H, methyl ester CH3), 3.82 (m, 1H, GlcN
H4), 3.75 (m, 1H, GlcN H5), 3.68 (ψt, 1H), 3.36 (s, 3H, methyl
3
glycoside CH3), 2.65 (d, J H4-OH ) 11.7 Hz), 2.13 (s, 3H, 3-O-
Meth yl 4-O-Allyl-2-O-ben zoyl-3-O-ben zyl-L-id op yr a n o-
sid u r on a te Tr ich lor oa cetim id a te (28). Monosaccharide 27
(303 mg, 0.544 mmol) was coevaporated three times with
toluene and dissolved in THF (5 mL). The solution was cooled
to 0 °C, and acetic acid (37.5 µL, 0.653 mmol) was added
followed by TBAF (1.0 M in THF, 600 µL). After 30 min, the
reaction was poured into ethyl acetate (100 mL) and washed
twice with brine. The organic phase was dried over MgSO4
and filtered, and the solvent was removed under reduced
pressure.
The residue was coevaporated three times with toluene and
dissolved in CH2Cl2 (4 mL) under N2. The solution was cooled
to 0 °C, and trichloroacetonitrile (820 µL, 8.16 mmol) and DBU
(15 µL, 0.1 mmol) were added. The reaction mixture was
stirred for 30 min, allowed to warm to room temperature, and
stirred for an additional 1.5 h. The solvent was evaporated,
and the residue was purified by flash silica gel column
chromatography (silica quenched with 1% NEt3 in toluene,
eluted with toluene/ethyl acetate 19:1) afforded 28 (210 mg,
0.36 mmol, 66%, 3:1 mixture of isomers) as a yellow oil: Rf
0.3 (hexanes/ethyl acetate 85:15); IR (thin film, NaCl plate)
1723, 1621 cm-1; ESI MS (C26H26Cl3NO8) m/z (M + Na+) calcd
608.0616, obsd 608.0717.
acetate CH3), 2.10 (s, 3H, 2-O-acetate CH3), 1.93 (s, 3H, 3-O-
acetate CH3); 13C NMR (125 MHz, CDCl3) δ 171.5 (3-O-acetate
carbonyl), 171.1 (6-O-acetate carbonyl), 169.6 (IdoA C6), 169.3
(2-O-acetate carbonyl), 156.0 (CBz carbonyl), 137.5 (benzyl
quat), 136.5 (CBz quat), 128.7 (aromatic C), 128.6 (aromatic
C), 128.4 (aromatic C), 128.3 (aromatic C), 128.1 (aromatic C),
127.6 (aromatic C), 99.8 (IdoA C1), 98.7 (GlcN C1), 77.0 (GlcN
C4), 74.0 (IdoA C3), 72.3 (GlcN C3), 71.9 (benzyl CH2), 68.8
(IdoA C5), 68.7 (GlcN C5), 68.0 (IdoA C4), 67.3 (IdoA C2), 67.1
(CBz CH2), 62.3 (GlcN C6), 55.6 (methyl glycoside CH3), 54.2
(GlcN C2), 52.5 (methyl ester CH3), 21.2 (2-O-acetate CH3),
21.05 (6-O-acetate CH3), 20.9 (3-O-acetate CH3); ESI MS
(C35H43NO16) m/z (M + Na+) calcd 756.2474, obsd 756.2443.
Meth yl (ter t-Bu tyld im eth ylsilyl 4-O-a llyl-3-O-ben zyl-
L-id op yr a n osid )u r on a te (26). Monosaccharide 2514 (200 mg,
0.529 mmol) was dissolved in TFA (90% aq, 5 mL) and stirred
for 30 min. The solvent was removed under reduced pressure
and the residue coevaporated five times with toluene. The
crude material was dissolved in CH2Cl2 (500 µL) and cooled
to -25 °C under N2. TBDMS-Cl (120 mg, 0.794 mmol) and
imidazole (144 mg, 2.12 mmol) were added, and the reaction
mixture was stirred for 16 h at -25 °C. Methanol (1 mL) was
added, and the mixture was stirred for 30 min and then diluted
into ethyl acetate. The organic phase was washed with 1 N
HCl, brine, and saturated aq NaHCO3. The organic phase was
dried over MgSO4, and the solvent was removed under reduced
pressure. Flash silica gel column chromatography (hexanes/
ethyl acetate 85:15) afforded 26 (170 mg, 0.376 mmol, 71%)
as a clear oil (6:1 mixture of isomers): Rf 0.37 (hexanes/ethyl
Meth yl Meth yl 2-O-Ben zoyl-3-O-ben zyl-R-L-id op yr a n o-
sid u r on a t e-(1f4)-3,6-d i-O-a cet yl-2-b en zyloxyca r b on yl-
a m in o-2-d eoxy-R-D-glu cop yr a n osid e (30). Trichloroacetim-
idate 28 (210 mg, 0.358 mmol) and monosaccharide 10 (98 mg,
0.238 mmol) were combined and coevaporated three times with
anhydrous toluene. The sugars were dissolved in anhydrous
J . Org. Chem, Vol. 69, No. 12, 2004 4091

Lohman and Seeberger
CH2Cl2 (2.5 mL) under N2 and cooled to -15 °C. Trimethylsilyl
trifluoromethanesulfonate (6.5 µL, 36 µmol) was added, and
the mixture was stirred for 1 h, allowed to warm to room
temperature, and stirred for an additional 30 min. The reaction
was quenched by addition of NEt3 (0.5 mL), and the solvent
was removed under reduced pressure. Flash chromatography
on silica (hexanes/ethyl acetate 65:35) afforded 29 (185 mg,
0.22 mmol, 93%).
30 min. Silver(I) oxide (227 mg, 0.98 mmol) was added, light
was excluded, and the reaction mixture was stirred for 40 h.
The mixture was filtered through Celite, concentrated, and
purified via flash column chromatography on silica (elutant
50:1 hexanes/ethyl acetate) to yield 34 (114 mg, 0.21 mmol,
75%) as a clear oil: Rf 0.38 (hexanes/ethyl acetate 10:1); [R]24
D
-3.57, c ) 1.15; IR (thin film, NaCl plates) 2929, 2858, 2110
cm-1; 1H NMR (500 MHz, CDCl3) δ 7.28-7.42 (m, 10H), 5.82-
5.90 (m, 1H), 5.21 (d3, J ) 1.6, 3.3, 17.2 Hz, 1H), 5.14 (dψt, J
) 10.4, 1.7 Hz, 1H), 4.86 (d, J ) 10.9 Hz, 1H), 4.79 (d, J )
10.9 Hz, 1H), 4.64 (d, J ) 12 Hz, 1H), 4.58 (d, J ) 12 Hz, 1H),
4.51-4.52 (m, 1H), 4.27-2.31 (m, 1H), 4.07-4.11 (m, 1H), 3.70
(m, 2H), 3.49-3.52 (m, 1H), 3.38-3.41 (m, 1H), 3.31-3.36 (m,
2H), 0.97 (s, 9H), 0.20 (s, 3H), 0.18 (s, 3H); 13C NMR (125 MHz,
CDCl3) δ 138.4, 138.3, 134.8, 128.6, 128.5, 128.3, 128.0, 127.8,
127.79, 117.2, 97.4, 83.0, 77.8, 75.6, 75.3, 74.0, 73.7, 69.0, 68.8,
25.8, 18.2, -4.0, -5.0; ESI MS (C29H41Cl3N3O5Si) m/z (M +
Na+) calcd 562.2708, obsd 562.2681.
4-O-Allyl-2-a zid o-3,6-d i-O-ben zyl-2-d eoxy-D-glu cop yr a -
n osid e Tr ich lor oa cetim id a te (35). Monosaccharide 34 (155
mg, 0.287 mmol) was coevaporated three times with toluene
and dissolved in THF (2.5 mL). The solution was cooled to 0
°C, and acetic acid (18 µL, 0.313 mmol) was added followed
by TBAF (1.0 M in THF, 299 µL). After 30 min, the reaction
was poured into saturated NH4Cl and extracted four times
with CH2Cl2. The organic phases were combined, dried over
MgSO4, and filtered, and the solvent was removed under
reduced pressure.
Disaccharide 29 (34 mg, 40.7 µmol) and sodium acetate (40
mg) were combined and dissolved in acetic acid (455 µL) and
water (25 µL). PdCl2 (42 mg, 0.24 mmol) was added, and the
reaction mixture was stirred for 8 h. Palladium black precipi-
tated from the solution over the course of the reaction. The
reaction mixture was poured into saturated NaHCO3 (50 mL),
and the aqueous layer was extracted with ethyl acetate (3 ×
50 mL). The combined organic phases were dried over MgSO4,
and the solvent was removed under reduced pressure. Flash
chromatography on silica (4:1 toluene/ethyl acetate) afforded
30 (27 mg, 33.9 µmol, 83%) as a clear oil: Rf 0.19 (hexanes/
ethyl acetate 1:1); [R]24 +26.0, c ) 0.75; IR (thin film, NaCl
D
plates) 3421, 1740 cm-1; 1H NMR (500 MHz, CDCl3) δ 7.97 (d,
J ) 7.4 Hz, 2H), 7.61 (t, J ) 7.5 Hz, 1H), 7.45 (t, J ) 8.0 Hz,
1H), 7.27-7.38 (m, 10H), 5.24 (d2, J ) 9.3, 10.4 Hz, 1H), 5.13
(ψt, 2H), 4.99-5.04 (m, 3H), 4.90 (d, J ) 1.6 Hz, 1H), 4.77 (d,
J ) 12.1 Hz, 1H), 4.70 (d, J ) 3.5 Hz, 1H), 4.66 (d, J ) 12.1
Hz, 1H), 4.38 (d2, J ) 3.3, 12.3 Hz, 1H), 4.31 (d, J ) 10.0 Hz,
1H), 4.04 (d, J ) 11.4 Hz, 1H), 3.79-3.87 (m, 4H), 3.81 (s,
3H), 3.37 (s, 3H), 2.70 (d, J ) 11.6 Hz, 1H), 2.13 (s, 3H), 1.92
(s, 3H); 13C NMR (125 MHz, CDCl3) δ 171.5, 171.0, 169.6,
165.1, 156.0, 137.6, 136.4, 134.0, 129.9, 128.9, 128.8, 128.75,
128.7, 128.6, 128.3, 128.2, 128.0, 127.6, 99.8, 98.6, 77.5, 77.0,
74.4, 72.3, 72.1, 68.8, 68.6, 68.1, 68.0, 67.5, 62.3, 55.5, 54.2,
52.4, 21.0, 20.8; ESI MS (C40H45NO8) m/z (M + Na+) calcd
818.2636, obsd 818.2561.
The residue was coevaporated three times with toluene and
dissolved in CH2Cl2 (2.5 mL) under N2. The solution was cooled
to 0 °C, and trichloroacetonitrile (409 µL, 4.08 mmol) and DBU
(7.5 µL, 0.054 mmol) were added. The reaction mixture was
stirred for 30 min, allowed to warm to room temperature, and
stirred for an additional 30 min. The solvent was evaporated,
and the residue was purified by flash silica gel column
chromatography (hexanes/ethyl acetate 9:1) afforded 35 (133
mg, 0.23 mmol, 81%,∼3:2 R/â mixture) as a yellow oil: Rf 0.2,
0.3 (hexanes/ethyl acetate 85:15); IR (thin film, NaCl plates)
Meth yl [Meth yl (m eth yl [m eth yl (2-O-a cetyl-4-O-a llyl-
3-O-b en zyl-R-L-id op yr a n osid )u r on a t e-(1f4)-2-N-a cet yl-
a ceta m id o-3,6-d i-O-ben zyl-2-d eoxy-R-D-glu cop yr a n osid e-
(1f4)-2-O-a cet yl-3-O-b en zyl-R-L-id op yr a n osid ]u r on a t e-
(1f4))-2-azido-3,6-di-O-ben zyl-2-deoxy-D-glu copyr an oside-
(1f4)-2-O-a cetyl-3-O-ben zyl-R-L-id op yr a n osid )u r on a te]-
(1f4)-3,6-di-O-acetyl-2-ben zyloxycar bon ylam in o-2-deoxy-
R-D-glu cop yr a n osid e (32). In a typical trial, tetrasaccharide
4 (145 mg, 88 µmol) and disaccharide 5 (71 mg, 97 µmol) were
combined and coevaporated three times with anhydrous
toluene. The sugars were dissolved in anhydrous CH2Cl2 (1
mL) under N2 and cooled to -40 °C, 4 Å molecular sieves (300
mg) were added, and the reaction mixture was stirred for 30
min. Trimethylsilyl trifluoromethanesulfonate (1.6 µL, 9 µmol)
was added, and the mixture was stirred for 1 h. The reaction
was quenched by addition of NEt3 (0.1 mL), and the solvent
was removed under reduced pressure. Flash chromatography
on silica (hexanes/ethyl acetate 60:40) eluted first a low
polarity fraction (61 mg) which contained primarily 33 as a
1:1 R/â mixture, followed by a fraction containing 32. Further
elution (hexanes/ethyl acetate 40:60) afforded recovered 5 (37
mg). The product-containing fraction was further purified by
size-exclusion chromatography on Biorad Biobeads, 1x cross-
linking (40 cm × 2.5 cm, toluene elutant), to yield 32 (104 mg,
47 µmol, 53%) as a 6:1 R/â mixture: 1H NMR (500 MHz,
CDCl3) major isomer characteristic peaks δ 5.72-5.79 (m, 1H,
allyl CH)CH2), 3.62 (s, 3H, methyl ester), 3.50 (s, 3H, methyl
ester), 3.49 (s, 3H, methyl ester), 3.36 (s, 3H, methyl glycoside),
2.13 (s, 3H, acetate), 2.12 (s, 6H, NAc2), 2.07 (s, 3H, acetate),
1.94 (s, 3H, acetate), 1.92 (s, 3H, acetate), 1.87 (s, 3H, acetate);
ESI MS (C114H131N5O40) m/z (M + Na+) calcd 2232.8263, obsd
2232.8263.
3340, 2868, 2112, 1675 cm-1 1H NMR (500 MHz, CDCl3) δ
;
8.75 (s, 0.4H), 8.72 (s, 0.6H), 7.27-7.43 (m, 10H), 6.42 (d, J )
3.4 Hz, 0.6H), 5.83-5.86 (m, 1H), 5.62 (d, J ) 8.4 Hz, 0.4H),
5.14-5.23 (m, 2H), 4.87-4.94 (m, 2H), 4.64 (d, J ) 11.4 Hz,
1H), 4.50-4.57 (m, 1H), 4.27-4.30 (m, 1H), 3.95-4.09 (m,
2.4H), 3.50-3.79 (m, 5.6H); 13C NMR (125 MHz, CDCl3) δ
161.3, 160.9, 138.1, 137.95, 137.8, 134.5, 134.4, 128.7, 128.65,
128.6, 128.5, 128.4, 128.3, 128.2, 128.13, 128.1, 128.05, 128.0,
127.9, 117.6, 117.4, 96.9, 95.1, 91.1, 89.5, 83.0, 80.0, 77.6, 77.0,
76.3, 75.8, 75.7, 74.2, 74.0, 73.75, 73.7, 73.6, 68.0, 67.5, 65.8,
63.0; ESI MS (C25H27Cl3N4O5) m/z (M + Na+) calcd 591.0945,
obsd 591.0939.
Met h yl [Met h yl (4-O-a llyl-2-a zid o-3,6-d i-O-b en zyl-2-
deoxy-D-glu copyr an oside 2-O-acetyl-3-O-ben zyl-R-L-idopy-
r a n osid )u r on a te]-(1f4)-3,6-d i-O-a cetyl-2-ben zyloxyca r -
bon yla m in o-2-d eoxy-R-D-glu cop yr a n osid e (36) Trichloro-
acetimidate 35 (30 mg, 52 µmol) and disaccharide 5 (19 mg,
26 µmol) were combined and coevaporated three times with
anhydrous toluene. The sugars were dissolved in anhydrous
CH2Cl2 (1 mL) under N2 and cooled to -40 °C, 4 Å molecular
sieves (90 mg) were added, and the reaction mixture was
stirred for 30 min. Trimethylsilyl trifluoromethanesulfonate
(1 µL) was added, and the mixture was stirred for 2 h. The
reaction was quenched by addition of NEt3 (0.1 mL), and the
solvent was removed under reduced pressure. Flash chroma-
tography on silica (hexanes/ethyl acetate 90:10) eluted 38 (22.6
mg, 39 µmol) as a 1:1 R/â mixture. Further elution (hexanes/
ethyl acetate 70:30) afforded 36 (9 mg) as a ∼20:1 R/â mixture.
Characterization data is reported for the major isomer only:
IR (thin film, NaCl plates) cm-1 2936, 2108, 1741.5, 15.14; 1H
NMR (500 MHz, CDCl3) δ 7.31-7.42 (m, 20H), 5.73-5.82 (m,
1H), 4.99-5.24 (m, 8H), 4.93 (d, J ) 2.3 Hz, 1H), 4.62-4.86
(m, 10H), 4.51 (d, J ) 12.1 Hz, 1H), 4.20-4.33 (m, 4H), 3.56-
4.05 (m, 15H), 3.71 (s, 3H), 3.34 (s, 3H), 3.24 (m, 2H), 2.12 (s,
ter t-Bu tyld im eth ylsilyl 4-O-Allyl-2-a zid o-3,6-d i-O-ben -
zyl-2-d eoxy-â-D-glu cop yr a n osid e (34). Compound 712 (140
mg, 0.28 mmol) was coevaporated three times with anhydrous
toluene and dissolved in CH2Cl2 (1.5 mL). Freshly activated 4
Å molecular sieves (300 mg) and allyl bromide (114 µL, 1.4
mmol) were added, and the reaction mixture was stirred for
4092 J . Org. Chem., Vol. 69, No. 12, 2004

Synthesis of Fully N-Differentiated Heparin Oligosaccharides
3H), 2.08 (s, 3H), 1.90 (s, 3H); 13C NMR (125 MHz, CDCl3) δ
171.5, 171.0, 170.2, 169.5, 150.1, 137.94, 137.93, 137.6, 136.5,
134.8, 128.7, 128.68, 128.64, 128.6, 128.5, 128.4, 128.3, 128.14,
128.11, 128.05, 128.0, 127.8, 127.7, 116.5, 99.5, 98.8, 97.3, 79.9,
77.8, 76.4, 75.5, 73.7, 73.69, 73.0, 72.4, 72.2, 72.1, 71.6, 69.0,
68.7, 68.2, 67.9, 67.1, 63.3, 62.3, 55.6, 54.3, 52.3, 21.1, 21.0,
20.9; ESI MS (C61H69Cl3N2O21) m/z (M + Na+) calcd 1163.4325,
obsd 1163.4331.
N-(4-O-Allyl-2-azido-3,6-di-O-ben zyl-2-deoxy-D-glu copy-
r a n osid e) Tr ich lor oa ceta m id e (38) This compound was
isolated as a byproduct from the synthesis of 36 and 37 and
isolated as a 3:2 mixture of anomers: Rf 0.23, 0.29 (hexanes/
ethyl acetate 85:15); IR (thin film, NaCl plates) 3333, 3065,
2917, 2866, 2713, 1709, 1705 cm-1; 1H NMR (500 MHz, CDCl3)
δ 7.29-7.41 (m, 10H), 7.13 (d, J ) 9.1 Hz, 0.6H), 7.01 (d, J )
6.5 Hz, 0.4H), 5.75-5.87 (m, 1H), 5.64 (ψt, 0.4H), 5.13-5.23
(m, 2H), 4.85-4.98 (m, 2.6H), 4.63-4.67 (m, 1H), 4.50-4.53
(m, 1H), 4.22-4.27 (m, 1H), 4.01-4.09 (m, 1H), 3.87-3.90 (m,
0.4H), 3.42-3.73 (m, 5.6H); 13C NMR (125 MHz, CDCl3) δ
162.1, 161.9, 137.8, 137.6, 137.3, 134.4, 134.2, 128.8, 128.7,
128.61, 128.6, 128.5, 128.45, 128.4, 128.3, 128.2, 128.15, 128.1,
128.0, 117.9, 117.3, 83.9, 81.2, 80.5, 77.5, 77.1, 70.0, 76.0, 75.9,
Met h yl [Met h yl (4-O-a llyl-2-a zid o-3,6-d i-O-b en zyl-2-
deoxy-D-glu copyr an oside 2-O-ben zoyl-3-O-ben zyl-R-L-ido-
p yr a n osid )u r on a t e]-(1f4)-3,6-d i-O-a cet yl-2-b en zyloxy-
ca r bon yla m in o-2-d eoxy-R-D-glu cop yr a n osid e (37) Trichlo-
roacetimidate 35 (13 mg, 23 µmol) and disaccharide 30 (9 mg,
11 µmol) were combined and coevaporated three times with
anhydrous toluene. The sugars were dissolved in anhydrous
CH2Cl2 (500 µL) under N2 and cooled to -40 °C, 4 Å molecular
sieves (40 mg) were added, and the reaction mixture was
stirred for 30 min. Trimethylsilyl trifluoromethanesulfonate
(1 µL) was added, and the mixture was stirred for 2 h. The
reaction was quenched by addition of NEt3 (0.1 mL), and the
solvent was removed under reduced pressure. Flash chroma-
tography on silica (hexanes/ethyl acetate 90:10) eluted 38 (8
mg) as a 1:1 R/â mixture. Further elution (hexanes/ethyl
acetate 70:30) afforded 37 (9 mg, 68%). Characterization data
is reported for major isomer only: IR (thin film, NaCl plates)
3032, 2928, 2110, 1767, 1742, 1723 cm-1; 1H NMR (500 MHz,
CDCl3) characteristic peaks δ 8.13 (d, J ) 8.1 Hz, 2H,
benzoate), 7.52 (ψt, 1H, benzoate), 7.45 (ψt, 2H, benzoate),
5.77-5.85 (m, 1H, allyl CHdCH2), 3.75 (s, 3H, methyl ester),
3.35 (s, 3H, methyl glycoside), 2.06 (s, 3H, acetate), 1.90 (s,
3H, acetate); 13C NMR (125 MHz, CDCl3) δ 171.4, 170.8, 169.5,
165.7, 156.1, 138.2, 137.9, 137.7, 136.5, 134.9, 133.6, 133.5,
130.2, 129.9, 129.6, 128.9, 128.7, 128.6, 128.5, 128.41, 128.28,
128.23, 128.13, 128.0, 127.92, 127.84, 127.6, 116.6, 99.9, 99.1,
98.6, 80.2, 77.5, 76.6, 75.2, 74.7, 73.8, 73.7, 72.9, 72.1, 69.4,
69.3, 68.8, 68.0, 67.1 63.6, 63.4, 55.6, 54.3, 52.3, 21.0, 20.9;
ESI MS (C61H69Cl3N2O21) m/z (M + Na+) calcd 1225.4481, obsd
1225.4477.
74.0, 73.95, 73.8, 72.7, 67.9, 67.8, 65.8, 61.7; ESI MS (C25H27
-
Cl3N4O5) m/z (M + Na+) calcd 591.0945, obsd 591.0942.
Ack n ow led gm en t . Financial support from the
National Institutes of Health (HL-64799), the Hu-
man Frontiers Science Program (HFSP RGY72-2002),
CaPCURE (Research Awards to P.H.S.), the American
Society for Engineering Education (National Defense
Science and Engineering Graduate Fellowship for
G.J .S.L.), Merck (Academic Development Award to
P.H.S.), the Alfred P. Sloan Foundation (Research
Fellowship to P.H.S.), and GlaxoSmithKline (Fellowship
to P.H.S.), is gratefully acknowledged. Funding for the
MIT-DCIF Varian 500 MHz NMR was provided by the
NSF (Award CHE-9808061). Funding for the MIT-DCIF
Varian 300 MHz NMR was provided by the NSF (Award
DBI-9729592). The CEM Discover Series Microwave
was provided by CEM Corp. We thank Dr. Emma
Palmacci, Dr. Herna´n Orgueria, and Daniel Snyder for
helpful discussions.
Su p p or t in g In for m a t ion Ava ila b le: 1H NMR and 13C
NMR spectra for all compounds not previously reported. This
material is available free of charge via the Internet at
http://pubs.acs.org.
J O035732Z
J . Org. Chem, Vol. 69, No. 12, 2004 4093