Galacturonic acid lactones in the synthesis of all trisaccharide repeating units of the zwitterionic polysaccharide Sp1

Article abstract of DOI:10.1021/jo102363d

A modular approach toward the synthesis of all possible trimer repeating units of the type 1 capsular polysaccharide of Streptococcus pneumonia, Sp1, is described. This zwitterionic polysaccharide is built up from trisaccharide repeats, which in turn are composed of two galacturonic acid monomers and a 2,4,6-trideoxy-4-amino-2-acetamido-D-galactose moiety. All monomeric constituents are linked through cis-glycosidic bonds. To overcome the difficulty associated with the efficient stereoselective introduction of the R-galacturonic acid bonds, we have employed galacturonic acid-[3,6]-lactone building blocks. Not only did these building blocks perform well when used as donor galactosides, they were also shown to be reactive acceptor glycosides when equipped with a free hydroxyl function. All three frame-shifted trimer repeats were constructed via highly stereoselective glycosylation reactions, with one exception. The epimeric mixture of trisaccharides, formed in the nonselective glycosylation event, could be readily separated after global deprotection using high performance anion exchange chromatography (HPEAC).

Full text of DOI:10.1021/jo102363d

ARTICLE
pubs.acs.org/joc
Galacturonic Acid Lactones in the Synthesis of All Trisaccharide
Repeating Units of the Zwitterionic Polysaccharide Sp1
Alphert E. Christina, Leendert J. van den Bos, Herman S. Overkleeft, Gijsbert A. van der Marel,* and
Jeroen D. C. Codꢀee*
Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands
S Supporting Information
b
ABSTRACT:
A modular approach toward the synthesis of all possible trimer repeating units of the type 1 capsular polysaccharide of Streptococcus
pneumonia, Sp1, is described. This zwitterionic polysaccharide is built up from trisaccharide repeats, which in turn are composed of
two galacturonic acid monomers and a 2,4,6-trideoxy-4-amino-2-acetamido-D-galactose moiety. All monomeric constituents are
linked through cis-glycosidic bonds. To overcome the difficulty associated with the efficient stereoselective introduction of the
R-galacturonic acid bonds, we have employed galacturonic acid-[3,6]-lactone building blocks. Not only did these building blocks
perform well when used as donor galactosides, they were also shown to be reactive acceptor glycosides when equipped with a free
hydroxyl function. All three frame-shifted trimer repeats were constructed via highly stereoselective glycosylation reactions, with one
exception. The epimeric mixture of trisaccharides, formed in the nonselective glycosylation event, could be readily separated after
global deprotection using high performance anion exchange chromatography (HPEAC).
’ INTRODUCTION
stimulating CD-4þ T-cell proliferation, after being presented via
the MHC-II processing pathway.2,4,5 In addition, ZPs have also
been shown to stimulate the innate immune system through
interaction with the Toll-like receptor 2 (TLR2).6 To elucidate
the mode of action of ZPs at the molecular level, well-defined ZPs
fragments can serve as valuable tools.7 We therefore set out to
develop a strategy to synthesize fragments of the S. pneumonia
zwitterionic polysaccharide. S. pneumonia is the causative agent of
pneumonia, bacteremia, otitis media, meningitis, and peritonitis,8
and one of its capsular polysaccharides, Sp1, is a prominent
member of the ZPs family.4,9 Sp1 is an overall anionic polysac-
charide built up from nonbranching [f3)-R-2,4,6-trideoxy-4-
amino-D-GalNAc-(1f4)-R-D-GalAp-(1f3)-R-D-GalAp-(1f]10
trisaccharide repeats, as depicted in Figure 1. The trimer repeat
contains two R-D-galacturonic acids in addition to the rare R-2,4,
Zwitterionic polysaccharides (ZPs) present an unique class of
polysaccharides from both a structural and a biological perspec-
tive.1,2 These bacterial polysaccharides contain both basic amino
functions and acidic carboxylate groups, accommodating them
with a zwitterionic character at physiological pH. This not only
distinguishes them from other naturally occurring polysaccharides
but also bestows them with distinctive biological activity. ZPs are
the only class of polysaccharides that is capable of eliciting a T-cell-
dependent immune response, a mode of action that was long
thought to be confined to peptides.1,2 Indeed, the sole manner in
which regular polysaccharides could be applied in effective vaccine
formulations has been through conjugation to immunostimulatory
carrier proteins.3 Without these proteins, (capsular) polysacchar-
ides are processed by antigen presenting cells but not presented by
MHC class II proteins to T-cells, a key step in the realization of
adaptive immune responses. In contrast, ZPs are capable of
Received: November 29, 2010
Published: February 22, 2011
r
2011 American Chemical Society
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ARTICLE
Figure 1. Sp1 polysaccharide and our retrosynthetic strategy toward the three frame-shifted trimer repeats 1-3.
6-trideoxy-4-amino-2-acetamido-D-galactose moiety. We here re-
port the assembly of all three possible spacers containing Sp1
repeating units 1-3 (Figure 1).
postglycosylation oxidation approach.15 For the synthesis of all
three frame-shifted repeating units of the Sp1 saccharide, we
opted for a modular strategy in which monomeric building blocks
can be combined in a flexible manner, as retrosynthetically
depicted in Figure 1. To limit the amount of synthetic transfor-
mations at the oligosaccharide stage, and especially avoid the late-
stage multiple oxidation step, we explored the use of C-6 oxidized
galactosyl building blocks. We have previously described that
conformationally locked 1-thiogalacturonic acid lactone donors
show excellent reactivity17,18 as well as anomeric selectivity in
glycosidations to provide R-linked galacturonides in excellent
yield.19 This makes them attractive donor glycosides in the
assembly of the target Sp1-oligomers. Additionally, the inverted
1C4-chair conformation of the galacturonic acid-[3,6]-lactones
positions the C4-OH equatorially, as opposed to the less
’ RESULTS AND DISCUSSION
The synthesis of (fragments of) the Sp1 oligosaccharide is
complicated by the presence of the uronic acid moieties and the
rare 2,4,6-trideoxy-4-amino-D-GalNAc monosaccharide,7,11,12
which are interconnected through 1,2-cis-glycosidic bonds. Dif-
ferent approaches have been pursued for the introduction of
uronic acids in oligosaccharide chains.13 These can be introduced
at the monosaccharide level in a pre-glycosylation oxidation
strategy, which uses glycuronic acid building blocks as donor
and acceptor in the construction of the target oligomer. Alter-
natively, a post-glycosylation-oxidation approach can be fol-
lowed in which the oligosaccharide backbone is built up prior to
the installment of the carboxylate functions. Galacturonic acids
are generally considered to be relatively poor glycosyl donors14,15
because of the electron-withdrawing effect of the C-5 carboxylic
acid ester. Similarly, the C-5 carboxylate exerts a deactivating
effect on the nucleophilicity of the proximal hydroxyl functions,
which explains why the C-4 hydroxyl group in galacturonic acid
acceptors has been regarded as a poor nucleophile.16 In the first
synthesis of two Sp1-oligomers, Bundle and co-workers therefore
resorted to the use of nonoxidized galactose building blocks in a
4
accessible axial orientation in the normal C1-chair.20 In the
synthesis of L-guluronate alginate oligomers, Hung and co-
workers have shown that changing the orientation of the C4-
OH of a gulosyl acceptor from an axial to an equatorial position,
by locking the L-gulosyl ring in a 4C1-conformation with an 1,6-
anhydro bridge, increases the nucleophilicity of the C4-OH.21
We reasoned that the inversion of the galacturonic acid chair
conformation in the lactone synthons can have a similar bene-
ficial effect on the galacturonic acid C4-hydroxyl. Thus, for our
assembly of the Sp1-repeating units we envisaged two galacturonic
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Scheme 1a
a Reagents and conditions: (a) TBDMSCl, imidazole, DMF; (b) BnBr, NaH, DMF, 0 °C; (c) TBAF, THF, 80% (over three steps); (d) TEMPO, BAIB,
DCM, H2O, 75%; (e) TEMPO, BAIB, DCM, H2O; (f) AcOH/H2O (4/1 v/v), 60 °C; (g) ethyl chloroformate, DiPEA, THF, 34% (over three steps);
(h) (ClAc)2O, pyridine, DCM, quant; (i) triethylamine 3HF, THF, 98%; (j) ClC(dNPh)CF3, Cs2CO3, H2O, acetone, 83% (R/β 1:3); (k) acceptor 9,
3
cat. TfOH, DCM/Et2O (1/1 v/v), 0 °C, quant (R/β 4:1); (l) thiourea, EtOH, pyridine, 65°C, 78% for 20, 16% for the β-anomer.
acid lactones: fully protected lactone thioglycoside 4 and acceptor
lactone 5. These will be combined with suitably protected 2,4,6-
trideoxy-4-amino-D-galactosamine building blocks 6-8 bearing a
nonparticipating azidefunctionalityatC2anda benzyloxycarbonyl
(Z)-protected amine at C4 (Figure 1). In our synthetic plan, we
capped the reducing ends of the trimer repeating units with a
glycerol spacer.22
Synthesis of the Building Blocks. The synthesis of the
required building blocks is depicted in Scheme 1. The assembly
of the galacturonic acid lactone building blocks 4 and 5 started
from known β-1-thiogalactoside 10 (Scheme 1A).23 Selective
silylation of the C-3 and C-6 hydroxyls in 10 led to diol 11,24
which was used without purification in the ensuing benzylation
step to provide the fully protected galactoside 12. Desilylation
of crude 12 using tetrabutylammonium fluoride in THF led
to the isolation of diol 13, which was isolated in 80% yield over
the three steps. 2,2,6,6-Tetramethyl-1-piperidinyloxy(TEMPO)/
[bis(acetoxy)iodo]benzene (BAIB)-mediated oxidation25,26 of
the primary alcohol in 13 was followed by in situ lactone formation
to provide the target galacturonic acid lactone 4 in one step, as we
described previously.19
Figure 2. Schematic representation of observed crosspeaks from 2D
NMR experiments with lactone building blocks 4 (a1, H2-CH2 Bn
(HMBC); b1, H4-CH2 Bn (HMBC); c1, H3-H5 (COSY)) and 5 (a2,
H2-CH2 Bn (HMBC); b2, H4-OH (COSY); c2, H3-H5 (COSY))
whereupon the assignment of peaks was based.
product, providing an indication that the equatorially oriented
hydroxyl function in lactone acceptor 5 is a reactive nucleophile.
The structure of the lactone building blocks 4 and 5 was fully
ascertained by NMR spectroscopy using 1H, 13C, 1H-1H COSY,
and 1H-13C HSQC data (Figure 2). A vicinal coupling of H-4
with 4-OH in 5 led to spectral allocation of the H-4 proton signal
in the spectrum of 5. The resonance of H-3 was assigned on the
basis of its relatively large chemical shift.30 Because the 1H NMR
spectrum of 5 showed a coupling of H-3 with both H-2 and
H-5,31 the latter two protons were distinguished by a long-range
1H-13C HMBC NMR experiment, in which a clear cross peak
between H-2 and the benzylic carbon was revealed. In this
experiment, a crosspeak between C-6 and H-3 was also observed.
Chemical shift similarities in the spectra of 4 and 5, in combina-
tion with a 1H-13C HMBC NMR experiment, which revealed a
crosspeak between H-4 of 4 and a benzylic carbon, led to the full
assignment of the resonance sets belonging to lactone 4.
To construct galacturonic acid lactone acceptor 5, β-1-thio-
galactoside 10 was transformed into partially protected 14
::
following an one-pot procedure reported by Sinay and co-
workers.27 TEMPO/BAIB-mediated oxidation of the primary
hydroxyl and subsequent acidic hydrolysis of the acetonide gave
crude acid 16.28 Lactonization of the acid was accomplished
using ethyl chloroformate to generate the mixed anhydride,
which cyclized to give 5 in 34% yield over the last three steps.29
The concentration at which this lactonization step was per-
formed turned out to be of vital importance to the outcome of the
reaction. Insufficiently diluted conditions afforded 17 as a side
The construction of the required 2,4,6-trideoxy-4-amino-D-
galactosamine building blocks7,11,18,32 is outlined in Scheme 1B.
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Scheme 2a
a Reagents and conditions: (a) 4, Ph2SO, Tf2O, DCM, TTBP, -60 °C then acceptor 6, 75%; (b) BnOH, AcCl, 50°C, overnight (quant); (c) (ClAc)2O,
pyridine, 93%; (d) triethylamine 3HF, THF, 84% (R/β 1:4); (e) ClC(dNPh)CF3, Cs2CO3, H2O, acetone, 85%; (f) acceptor 5, cat. TfOH, DCM, 81%
3
(R/β 8:1); (g) Ph2SO, Tf2O, DCM, TTBP, -60°C then acceptor 9 (81%); (h) thiourea, EtOH, pyridine, 65 °C, 76%; (i) AcSH/pyridine (1/1 v/v),
94%; (j) TMSONa, DCM, then H2/Pd(C), t-BuOH, H2O, HCl (52% over two steps).
We have recently reported the synthesis 2-azido-4-(benzyl-
oxycarbonyl)amino-2,4,6-trideoxy-D-galactose building blocks in
the context of the synthesis of the tetrasaccharide repeating unit of
the Bacteroides fragilis ZPs,7 and building block 6 was obtained
from D-glucosamine using very similar procedures.33 Lactol 7 was
obtained from 6 by chloroacetylation of the C-3 OH, followed by
anomeric desilylation. From 7, donor 8 was readily obtained by
installment of the N-phenyltrifluoroimidate function.34 Imidate 8
was condensed with glycerol acceptor 9, accessible from solketal
following literature procedures,35 under the agency of a catalytic
amount of triflic acid (TfOH) in dichloromethane to provide 19
as a 1:1 mixture of inseparable anomers. Although the use of a
DCM/Et2O solvent system in this glycosylation event led to the
preferential formation of the R-anomer,36 the anomers remained
inseparable at this stage. Fortunately, after dechloroacetylation of
19, the epimers were separable by flash column chromatography,
and 2,4,6-trideoxy-4-amino-D-galactosamine building block 20
was obtained in 78% from 19, alongside 16% of its C1-epimer.
Assembly of the Trimer Repeats. With all monomeric
building blocks in hand, we set out to assemble the three
frame-shifted trimer repeats 1-3. The synthesis of the first
trisaccharide started with the coupling of lactone 4 and 2,4,6-
trideoxy-4-amino-D-galactosamine 6 (Scheme 2). To this end,
donor 4 was preactivated using in situ generated diphenylsulfo-
nium bistriflate37 and subsequently treated with acceptor 6 to
give disaccharide 21 in good yield and excellent stereoselectivity.
Opening of the lactone ring using benzyl alcohol under acidic
conditions19 afforded benzyl ester 22 quantitatively. Chloroace-
tylation of the liberated hydroxyl functionality, anomeric desily-
lation, and installment of the N-phenyltrifluoroimidate function
then led to dimeric glycosyl donor 25. Coupling of this donor
with lactone acceptor 5 employing TfOH as a promoter gave S-
phenyl trimer 26 in 81% yield and 8:1 R/β selectivity, showcas-
ing the apt nucleophilicity of lactone acceptor 5. The R-epimer of
thioglycoside 26 could be isolated through column chromatogh-
raphy (64%) and was subsequently condensed with acceptor 9 in
a Ph2SO/Tf2O-mediated preactivation glycosylation event fur-
nishing the fully protected glycerol capped trisaccharide 27 as the
sole anomer. Global deprotection started with removal of the
chloroacetylgroup to give alcohol 28. Reduction of the azide in
28 with either PMe3 or dithiothreitol and ensuing acetylation of
the amine and free C300-OH gave only low yields of the desired
product. The use of freshly distilled thiolacetic acid and pyridine
on the other hand, gave acetamide 29 in 94% yield,38 with the
alcohol functionality still intact. Studies to open the lactone ring
in compound 30 (vide infra) prompted us to use TMSONa as
nucleophilic reagent to hydrolyze the lactone functionality in
29.39 Hydrogenolysis of the remaining benzyl ester, benzyloxy
carbamate, and benzyl ethers then furnished the first target
trisaccharide 1 in 52% yield over the last two steps.
For the construction of trisaccharide 2, lactone donor 4 was
converted into glycerol-capped galacturonic acid ester acceptor
31 (Scheme 3). To this end, lactone donor 4 was coupled with
acceptor 9 in a Ph2SO/Tf2O-mediated glycosylation to yield 30
with excellent anomeric selectivity (R/β = 10:1). Unfortunately,
the acid-catalyzed opening of lactone 30 in benzyl alcohol as
described above did not lead to the anticipated product. Instead,
we observed a product resulting from the endocyclic opening of
the galactopyranosyl core (32). Use of Bu2SnO40 in BnOH did
give the desired product, but only in low yields. Finally, we
succeeded in opening the lactone ring using TMSONa to afford
the free acid, which was subsequently treated with benzyl
bromide and Cs2CO3 providing the reducing end galacturonic
acid acceptor 31 in 75% yield. Next, hemiacetal 7 and key lactone
acceptor 5 were coupled under the agency of Ph2SO and Tf2O to
stereoselectively form the R-linked dimer 33 in 84% yield. The
generated thioglycoside 33 was engaged next in a glycosylation
event with spacer-capped galacturonic acid ester acceptor 31 to
produce the fully protected trisaccharide 34, again with complete
R-selectivity. To deliver target compound 2, the same global
deprotection strategy was envisioned as used previously for the
fully protected trisaccharide 27. Thus, dechloroacetylation with
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Scheme 3a
a Reagents and conditions: (a) 4, Ph2SO, Tf2O, DCM, TTBP, -60 °C then acceptor 9, 93% (R/β 10:1); (b) (1) TMSONa, DCM, (2) BnBr, Cs2CO3,
DMF, 75% (two steps); (c) 7, Ph2SO, Tf2O, DCM, TTBP, -60 °C then acceptor 5, 84%; (d) 33, Ph2SO, Tf2O, DCM, TTBP, -60°C then acceptor 31,
60%; (e) thiourea, EtOH, pyridine, 65 °C, 62%; (f) thiolacetic acid/pyridine (1/1 v/v), 66%; (g) TMSONa, DCM, then H2/Pd(C), t-BuOH, H2O, HCl
(38%); (h) H2/Pd(C), t-BuOH, H2O, HCl, then H2O, HCl (38%).
Scheme 4a
a Reagents and conditions: (a) 4, Ph2SO, Tf2O, DCM, TTBP, -60 °C then acceptor 20, 70%; (b) (1) TMSONa, DCM, (2) BnBr, Cs2CO3, DMF, 88%
(over two steps); (c) 4, Ph2SO, Tf2O, DCM, TTBP, -60 °C then acceptor 39, 78%; (d) AcSH, pyridine, 40%; (e) TMSONa, DCM, then H2/Pd(C),
t-BuOH, H2O, HCl, 3a: 28%, 3b: 17%.
thiourea was followed by reduction of the azide group using AcSH
and simultaneous N-acetylation to produce acetamide 36. Un-
fortunately, treatment of 36 with TMSONa in dichloromethane
and ensuing hydrogenolysis did not afford the anticipated trisac-
charide 2. Instead, oxazolidinone 37 was obtained as a result from
intramolecular nucleophilic attack of the 300-OH onto the nearby
benzyl carbamate during the TMSONa treatment. The formation
of the oxazolidinone could be circumvented by reversal of the
lactone ring-opening and reduction steps. Hydrogenolysis of
acetamide 36 under mildly acidic conditions provided the crude
donor 4 and acceptor 20. As depicted in Scheme 4, disaccharide
38 was produced in a completely R-selective fashion in 70% yield.
TMSONa opening of the lactone functionality in 38 and sub-
sequent benzyl ester installment set the stage for the next
glycosylation event, in which key lactone donor 4 was employed
again. The coupling of 4 and 39, proceeded without any
selectivity, and trisaccharide 40 was isolated as an inseparable
1:1 mixture in 78% yield. Changing the solvent system to either
toluene or acetonitrile in dichloromethane did not significantly
alter the stereochemical outcome of the glycosylation. In light of
the excellent R-selectivity of all previous condensations using the
galacturonic acid-[3,6]-lactone donors, and especially the coup-
ling of 33 with galacturonic acid acceptor 31, the lack of
selectivity in the condensation of 4 and 39 is surprising, and
we have no adequate explanation for this result at this moment.
Global deprotection of the epimeric mixture was accomplished
by reduction of the azide in 40 and concomitant acetylation to
1
trimer lactone. The H NMR spectrum of the crude lactone
recorded directly after hydrogenolysis already showed partial
hydrolysis of the lactone ring, and therefore, the crude lactone
was further subjected to mild acidic hydrolysis conditions to
provide the target trimer 2 in 38% over the last two steps.
The assembly of the third and last trisaccharide 3 commenced
with the sulfonium bistriflate-mediated condensation of lactone
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’ EXPERIMENTAL SECTION
General Procedure for Glycosylations Using Ph2SO/Tf2O.
A solution of donor (1 equiv), diphenyl sulfoxide (1.1 equiv), and tri-tert-
butylpyrimidine (1.5 equiv) in DCM (0.05M) was stirred over activated
ꢀ
molecular sieves (3 Å) for 30 min. The mixture was cooled to
-60 °C before triflic acid anhydride (1.1 equiv) was added. The mixture
was allowed to warm to -45 °C and was subsequently recooled to -60 °C
ꢀ
before a mixture (dried over 3 Å molecular sieves) of acceptor (1.5 equiv)
and tri-tert-butylpyrimidine (1.0 equiv) in a small amount of DCM was
added. Stirring was continued, and the reaction mixture was allowed to warm
to -10 °C. The reaction mixture was quenched with triethylamine (5.0
equiv), diluted with DCM, and washed with satd aq NaHCO3. The aqueous
phase was extracted with DCM, and the combined organic phases were dried
(MgSO4), filtered, and concentrated under reduced pressure. Flash column
chromatography and removal of the eluent afforded the coupled product.
Phenyl 2,4-Di-O-benzyl-1-thio-β-D-galactopyranoside (13).
To a mixture of 10.0 g of phenyl-1-thiogalactopyranoside in 150 mL of
DMF (36.8 mmol, 1 equiv) was added 8.77 g of imidazole (128.8 mmol, 3.5
equiv) and 16.64 g of TBSCl (110.4 mmol, 3 equiv). After 2 h of stirring,
TLC analysis showed complete consumption of the starting material. The
reaction was quenched by the addition of 3 mL of MeOH. The mixture was
partitioned between H2O and Et2O, and the aqueous layer was extracted.
The combined organic phases were washed with aq 1 M HCl, satd aq
NaHCO3, and brine, dried over MgSO4, filtered, and evaporated. The
crude product was dissolved in 150 mL of DMF, and to this solution were
added 13.2 mL of BnBr (110.4 mmol, 3 equiv) and 4.42 g of NaH (60% in
mineral oil, 110.4 mmol, 3 equiv) at 0 °C. After being stirred at ambient
temperature overnight, the reaction was quenched with MeOH at 0 °C,
taken up in Et2O, and washed with 5% aq LiCl and brine. After drying over
MgSO4, filtration, and concentration under reduced pressure, the residue
was dissolved in 40 mL of THF and treated with 146.8 mL of 1.0 M TBAF
(in THF, 146.8 mmol, 4 equiv). The mixture was stirred for 3 h and
subsequently taken up in EtOAc and H2O. The water layer was further
extracted with EtOAc, and the combined organic layers were dried
(MgSO4), filtered, and evaporated. Purification by flash column chroma-
tography using EtOAc/PE (7/13 f 9/11) afforded the target compound
13 (13.4 g, 29.6 mmol, 80% over three steps): Rf 0.27 (EtOAc/PE, 1/1,
v/v); [R]22D þ1 (c 1.4, CHCl3); IR (neat, cm-1) 1311, 1049, 1018, 871,
732, 640, 694; 1H NMR (400 MHz, CDCl3, HH-COSY, HSQC) δ 7.53
(m, 2H, Harom), 7.43-7.09 (m, 13H, Harom), 4.90 (d, J = 10.8 Hz, 1H, CH2
Bn), 4.76 (d, J = 11.6 Hz, 1H, CH2 Bn), 4.68-4.54 (m, 3H, CH2 Bn, H-1),
3.84 (dd, J = 11.0, 7.4 Hz, 1H, H-6), 3.75 (s, 1H, H-4), 3.74-3.65 (m, 2H,
H-2, H-3), 3.56 (dd, J = 11.2, 4.5 Hz, 1H, H-6), 3.49-3.42 (m, 1H, H-5),
2.41 (bs, 1H, OH), 2.03 (bs, 1H, OH); 13C NMR (100 MHz, CDCl3, HH-
COSY, HSQC) δ 138.0, 137.9, 133.9 (Cq Ph), 131.2, 128.9, 128.5, 128.4,
128.3, 128.0, 127.9, 127.2 (CHarom), 87.1 (C-1), 79.0 (C-5), 78.1 (C-2),
75.8, 75.7 (C-3, C-4), 75.2, 74.7 (CH2 Bn), 62.1 (C-6); HRMS [M þ
Na]þ calcd for C26H28N3O5SNa 475.15497, found 475.15567.
Figure 3. Dionex HPAEC (high performance an-ion exchange chro-
matography) trace of the epimeric mixture obtained after hydrogenation
of 41 under acidic conditions, showing a retention time difference of
12.32 min between both epimers. Recovery: 28% for 3a and 17% for 3b
(over two steps from 41).
give acetamide 41 in moderate yield. Then, TMSONa-mediated
lactone hydrolysis and ensuing removal of the remaining protec-
tive groups left us with a crude epimeric mixture of fully
deprotected trisaccharides. The two anomers could be separated
by high performance anion exchange chromatography (HP-
AEC).41 The use of a gradient of 30-80 mM NaOAc in
100 mM NaOH led to the elution of the R- and β-epimers with
a retention time difference of over 10 min, as depicted in Figure 3.
Preparative ion-exchange chromatography gave us target C-100-
R-configured trisaccharide 3a (28% over the last two steps) and
its β-epimer 3b (17% over the last two steps) in pure form. The
structures of the trisaccharides were corroborated by NMR
spectroscopy. The heteronuclear one-bond coupling constant
of C-100 and H-100 in 3a (1JC-H = 170.3 Hz) and 3b (1JC-H
=
161.5 Hz) unambiguously confirmed the R-anomeric configura-
tion for the former and the β-anomeric configuration for the
latter trisaccharide.
’ CONCLUSION
In summary, we have described the synthesis of all three
frame-shifted trisaccharide repeats of the zwitterionic polysac-
charide Sp1 of S. pneumonia exploiting the use of 1-thio
galacturonic acid lactones as key donor and acceptor building
blocks. The galacturonic acid-[3,6]-lactones proved to be effi-
cient donor galactosides for the construction of the galacturonic
acid-containing target compounds. The yields of the glycosyla-
tions using the lactone donors were good to excellent and in all
but one of the galacturonylations very high R-selectivities were
observed. For the unexpected lack of R-selectivity in the con-
densation of key lactone donor 4 and dimer acceptor 39 there
currently is no satisfactory explanation. In addition to being
adequate donor glycosides, the galacturonic acid lactones were
also shown to be excellent nucleophiles when equipped with a
Phenyl 2,4-Di-O-benzyl-1-thio-β-D-galactopyranosiduro-
no-3,6-lactone (4). The thioglycoside lactone was synthesized as
reported previously, and all physical and spectroscopic data were in
accordance with the published data:25 Previously, assignments of H-4
and H-5 as well as C-4 and C-5 were interchanged. See ref 25. 1H NMR
(400 MHz, CDCl3, HH-COSY, HSQC) δ 7.63-7.09 (m, 15H, Harom),
5.41 (s, 1H, H-1), 4.80 (dd, J = 4.7, 1.3 Hz, 1H, H-3), 4.65 (d, J = 11.8 Hz,
1H, CH2 Bn), 4.59 (2s, 2H, CH2 Bn), 4.54 (d, J = 11.8 Hz, 1H, CH2 Bn),
4.39 (br s, 1H, H-4), 4.27 (d, J = 4.7 Hz, 1H, H-2), 4.04 (br s, 1H, H-5);
13C NMR (100 MHz, CDCl3, HH-COSY, HSQC): δ 172.6 (CdO),
136.7, 136.5, 133.6 (Cq Ph), 132.7, 129.0, 128.7, 128.6, 128.5, 128.3,
128.1, 128.0, 127.8 (CHarom), 85.8 (C-1), 78.9 (C-3), 78.6 (C-2), 76.0
(C-4), 73.0 (CH2 Bn), 71.5 (CH2 Bn), 70.8 (C-5).
1
free C4-hydroxyl function. The C4-chair conformation of lac-
tone 5 places the C4-hydroxyl in an equatorial position which
makes it significantly more reactive toward incoming electro-
philes. It is envisaged that this strategy can also be applied to
other glycuronic acid acceptors. Finally, HPAEC proved to be a
powerful purification technique for this class of compounds as
the difference in one stereochemical center led to a significant
difference in retention time, allowing the separation of the two
epimers.
Phenyl 2-O-Benzyl-1-thio-β-D-galactopyranosidurono-
3,6-lactone (5). To a vigorously stirred solution of 3.59 g of
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phenyl 2-O-benzyl-3,4-O-isopropylidene-1-thio-β-galactopyrano-
side (8.93 mmol, 1 equiv) in 30 mL of DCM and 15 mL of H2O
was added 279 mg of TEMPO (1.79 mmol, 0.2 equiv) and 7.19 g of
BAIB (22.3 mmol, 2.5 equiv). After 2 h of stirring at room tempera-
ture, Na2S2O3 solution (10% in H2O) was added, and the mixture was
extracted with EtOAc. The combined organic extracts were washed
with brine, dried (MgSO4), filtered, and evaporated. The crude acid
was stirred at 65 °C in 20 mL of AcOH/H2O (4/1 v/v) until TLC
analysis showed disappearance of the starting material. The mixture
was concentrated in vacuo and coevaporated with toluene. The crude
product was dissolved in 350 mL of anhydrous DCM followed by
addition of 1.77 mL of Dipea (10.7 mmol, 1.2 equiv) and 939 μL of
ethyl chloroformate (9.82 mmol, 1.1 equiv). After 3 h of stirring at
ambient temperature, the mixture was evaporated, and lactone 5 was
obtained in pure form after flash column chromatography using
EtOAc/PE (1/4 f 1/3) (1.08 g, 3.0 mmol, 34% over three steps): Rf
0.26 (EtOAc/PE, 3/7, v/v); [R]22D -223 (c 1.0, CH2Cl2); IR (neat,
t-Bu), 0.97 (d, J = 6.3 Hz, 3H, H-6); 13C NMR (100 MHz, CDCl3, HH-
COSY, HSQC) δ 166.5, 156.45 (CdO), 136.2 (Cq Ph), 135.7
(CHarom), 132.9, 132.4 (Cq Ph),, 129.9, 128.5, 128.3, 128.1, 127.5,
127.4 (CHarom), 97.0 (C-1), 74.6 (C-3), 68.9 (C-5), 67.1 (CH2 Cbz),
63.5 (C-2), 51.6 (C-4), 40.5 (CH2 ClAc), 26.7 (CH3 t-Bu), 19.0,
(Cq t-Bu), 16.0 (C-6); HRMS [M þ Na]þ calcd for C32H37ClN4O6-
SiNa 659.20631, found 659.20672.
4-(N-Benzyloxycarbonylamino)-2-azido-3-O-chloroacetyl-
2,4,6-trideoxy-D-galactopyranose (7). Galactosazide 18 (1.03 g,
1.62 mmol, 1 equiv) in 10 mL of THF was treated with 527 μL of
N3Et 3HF (3.23 mmol, 2 equiv), and the mixture was stirred at 70 °C
3
for 30 min. When the reaction mixture had cooled to ambient
temperature, EtOAc was added, and the organic mixture was washed
with satd aq NaHCO3. The aqueous layer was extracted with DCM, and
the combined organic layers were dried over MgSO4, filtered, and
evaporated. Purification by flash column chromatography using
EtOAc/PE (1/3 f 3/7) yielded galactopyranose 7 (632 mg, 1.58
mmol, 98%, R/β 1:2) with a minor unidentified side product: Rf 0.42
(EtOAc/PE, 2/3, v/v); IR (neat, cm-1) 3356 (br), 2361, 2114, 1701,
1526, 1061; 1H NMR (400 MHz, CDCl3, HH-COSY, HSQC) δ 7.42-
7.29 (m, 5H, Harom), 5.44 (d, J = 9.6 Hz, 0.7H, NH-R), 5.35-5.28 (m,
0.6H, H-1R, H-3R), 5.17-5.04 (m, 2H, CH2 Cbz-R, CH2 Cbz-β), 4.75
(dt, J = 13.2, 6.6 Hz, 0.7H, H-3β), 4.63 (d, J = 8.0 Hz, 0.7H, H-1β),
4.52-4.47 (m, 0.3H, H-5R), 4.33 (s, 0.7H, OH-β), 4.26-4.22 (m, 0.3H,
H-4R), 4.18-4.12 (m, 0.7H, H-4β), 3.96-3.86 (m, 2H, CH2 ClAc),
3.81-3.74 (m, 0.7H, H-5β), 3.56 (dd, J = 11.1, 3.7 Hz, 0.3H, H-2R),
3.50 (dd, J = 10.8, 8.0 Hz, 0.7H, H-2β), 3.42 (s, 0.3H, OH-R), 1.24 (d,
J = 6.4 Hz, 0.7H, H-6β), 1.18 (d, J = 6.5 Hz, 0.3H, H-6R); 13C NMR
(100 MHz, CDCl3, HH-COSY, HSQC) δ 166.9, 157.1, 157.0 (CdO),
136.1, 136.0 (Cq Ph), 128.6, 128.5, 128.3, 128.1, 127.9 (CHarom), 96.2
(C-1β), 91.8 (C-1R), 74.6 (C-3β), 72.2 (C-3R), 69.2 (C-5β), 67.3, 67.2
(CH2 Cbz), 64.1 (C-5R), 61.8 (C-2β), 58.0 (C-2R), 52.5 (C-4R), 51.8
(C-4β), 40.6, 40.5 (CH2 ClAc), 16.4 (C-6β), 16.3 (C-6R); HRMS
[M þ H]þ calcd for C16H20ClN4O6 399.10659, found 399.10647.
4-(N-Benzyloxycarbonylamino)-2-azido-3-O-chloroace-
tyl-2,4,6-trideoxy-r/β-D-galactopyranosyl (N-Phenyl)trifluoro-
acetimidate (8). To a solution of 511 mg of hemiacetal 7 (1.28
mmol, 1 equiv) in 6.1 mL of acetone and 0.3 mL of H2O were added 460
mg of CsCO3 (1.41 mmol, 1.1 equiv) and 532 mg of ClC(CdNPh)CF3
(2.56 mmol, 2 equiv). When TLC analysis showed complete consump-
tion of the starting material, the mixture was coevaporated with toluene.
Purification by flash column chromatography using EtOAc/PE (1/9 f
3/7) yielded 606 mg of imidate 8 (1.06 mmol, 83%, anomers R/β 1:3):
Rf 0.54 (EtOAc/PE, 1/3, v/v); IR (neat, cm-1) 2116, 1717, 1524, 1211,
1163, 1072, 696; 1H NMR (400 MHz, CDCl3, HH-COSY, HSQC) (T =
333 K) δ 7.41-7.23 (m, 9.3H, Harom), 7.09 (m, 1.4H, Harom), 6.83 (m,
2.7H, Harom), 6.35 (s, 0.3H, H-1R), 5.49 (d, J = 7.5 Hz, 1H, H-1β), 5.28
(dd, J = 11.1, 3.5 Hz, 0.3H, H-3R), 5.20-4.96 (m, 4H, CH2 Cbz, NH),
4.81 (dd, J = 10.7, 3.9 Hz, 1H, H-3β), 4.38-4.26 (m, 0.7H, H-4R,
H-5R), 4.16 (dd, J = 9.7, 3.1 Hz, 1H, H-4β), 3.89 (s, 2.7H, CH2 ClAc),
3.81 (dd, J = 10.9, 3.9 Hz, 0.3H, H-2R), 3.75-3.59 (m, 2H, H-2β,
H-5β), 1.20 (m, 4H, H-6); 13C NMR (100 MHz, CDCl3, HH-COSY,
HSQC) (T=333K) δ 166.4, 156.7 (CdO), 143.1, 143.0, 136.3 (Cq Ph),
128.8, 128.6, 128.3, 128.0, 124.7, 124.6, 119.3, 119.2 (CHarom), 95.9 (C-
1β), 93.7 (C-1R), 74.6 (C-3β), 72.2 (C-3R), 70.6 (C-5β), 67.5 (C-5R),
67.4 (CH2 Cbz), 60.2 (C-2β), 57.2 (C-2R), 52.4 (C-4R), 51.8 (C-4β),
40.3 (CH2 ClAc), 16.2 (C-6); HRMS [M - (C(NdPh)CF3) þ H þ
Na]þ calcd for C16H19ClN4O6 421.08853, found 421.08845.
1
cm-1) 3440, 1794, 1095, 1055, 694; H NMR (400 MHz, CDCl3,
HH-COSY, HSQC) δ 7.45-7.23 (m, 10H, Harom), 5.39 (s, 1H, H-1),
4.77 (dd, J = 4.7, 1.3 Hz, 1H, H-3), 4.62-4.50 (m, 3H, H-4, CH2 Bn),
4.22 (d, J = 4.7 Hz, 1H, H-2), 3.98 (t, J = 1.3 Hz, 1H, H-5), 3.29 (s, 1H,
OH); 13C NMR (100 MHz, CDCl3, HH-COSY, HSQC) δ 174.1
(CdO), 136.4, 133.4 (Cq Ph), 132.5, 129.0, 128.6, 128.3, 128.1,
128.0 (CHarom), 85.3 (C-1), 81.2 (C-3), 78.3 (C-2), 72.9 (CH2 Bn),
72.5 (C-5), 69.5 (C-4); HRMS [M þ Na]þ calcd for C19H18O5SNa
381.07672, found 381.07675. When lactonization of the crude acid
diol 16 was executed at a 0.21 M concentration, lactone 5 was isolated
in 26% along with 19% of 2-O-benzyl-1-thio-β-D-galactopyranosi-
duronyl-3,6-lactone) 2-O-benzyl-1-thio-β-D-galactopyranosylur-
onate (17): Rf 0.41 (EtOAc/PE, 2/3, v/v); [R]22 -158 (c 2.0,
D
CH2Cl2); IR (neat, cm-1) 3470, 1806, 1774, 1101, 741, 692; “a”
designates signals belonging to the galacturonic acid ester, and “b” is
1
used for signals stemming from the galacturonic acid lactone; H
NMR (400 MHz, CDCl3, HH-COSY, HSQC) 1H NMR (400 MHz,
CDCl3) δ 7.60 (d, J = 7.0 Hz, 2H, Harom), 7.48-7.20 (m, 18H,
Harom), 5.59 (s, 1H, H-4b), 5.44 (s, 1H, H-1b), 5.08 (d, J = 4.6 Hz, 1H,
H-3b), 4.93 (d, J = 10.8 Hz, 1H, CH2 Bn), 4.70-4.65 (m, 2H, CH2
Bn), 4.62-4.54 (m, 2H, CH2 Bn, H-1a), 4.35 (d, J = 4.8 Hz, 1H,
H-2b), 4.20-4.16 (m, 2H, H-4a, H-5b), 4.09 (s, 1H, H-5a), 3.73-
3.62 (m, 2H, H-3a, H-2a), 3.50 (d, J = 4.1 Hz, 1H, OH), 3.25 (d, J =
4.1 Hz, 1H, OH); 13C NMR (100 MHz, CDCl3, HH-COSY, HSQC)
13C NMR (101 MHz, CDCl3) δ 171.9, 166.3 (CdO), 137.9, 136.3,
133.3 (Cq Ph), 133.0, 132.6, 132.4, 129.0, 128.6, 128.5, 128.3, 128.2,
128.1, 128.0, 127.9 (CHarom), 87.6 (C-1a), 86.3 (C-1b), 78.4 (C-3b),
78.1 (C-2b), 77.2 (C-2a), 76.4 (C-5a), 75.4 (CH2 Bn), 74.0 (C-3a),
72.9 (CH2 Bn), 72.6 (C-4b), 70.0, 69.8 (C-4a, C-5b); HRMS [M þ
Na]þ calcd for C38H36O10S2Na 739.16421, found 739.16431.
tert-Butyldiphenylsilyl 4-(N-Benzyloxycarbonylamino)-2-
azido-3-O-chloroacetyl-2,4,6-trideoxy-β-D-galactopyra-
noside (18). To a mixture of alcohol 6 (860 mg, 1.54 mmol, 1 equiv),
5 mL of DCM, and 607 μL of pyridine (7.68 mmol, 5 equiv) was added
525 mg of chloroacetic anhydride (3.07 mmol, 2 equiv). After 1 h, 500
μL of H2O was added, and the mixture was stirred for another 15 min.
After evaporation, the residue was taken up in EtOAc and washed with
aq 1 M HCl, satd aq NaHCO3, and brine. The organic phase was dried
over MgSO4, filtered, and evaporated to dryness yielding title compound
18 (984 mg, 1.54 mmol, quant): Rf 0.79 (EtOAc/PE, 1/3, v/v); [R]22
D
-9 (c 1.0, CH2Cl2); IR (neat, cm-1) 2114, 1713, 1504, 1165, 1057, 733;
1H NMR (400 MHz, CDCl3, HH-COSY, HSQC) δ 7.71-7.68 (m, 4H,
Harom), 7.46-7.25 (m, 11H, Harom), 5.14 (d, J = 12.2 Hz, 1H, CH2 Cbz),
5.02 (d, J = 12.2 Hz, 1H, CH2 Cbz), 4.93 (d, J = 9.5 Hz, 1H, NH), 4.64
(dd, J = 10.7, 3.7 Hz, 1H, H-3), 4.44 (d, J = 7.7 Hz, 1H, H-1), 4.00 (dd,
J = 9.5, 3.4 Hz, 1H, H-4), 3.95-3.81 (m, 2H, CH2, ClAc), 3.48 (dd, J =
10.4, 8.0 Hz, 1H, H-2), 3.36 (q, J = 6.2 Hz, 1H, H-5), 1.11 (s, 9H, CH3
4-(N-Benzyloxycarbonylamino)-2-azido-3-O-chloroacetyl-
2,4,6-trideoxy-r/β-D-galactopyranosyl-(1f3)-1,2-di-O-ben-
zyl-sn-glycerol (19). A catalytic amount of triflic acid was added
under anhydrous conditions to a mixture of 403 mg of imidate 8 (707
μmol, 1 equiv) and 578 mg of alcohol 9 (2.21 mmol, 3 equiv) in 7 mL of
DCM/Et2O (1/1 v/v) at 0 °C. After 20 min of stirring, TLC analysis
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showed complete conversion of the starting material. The reaction was
quenched by adding triethylamine, and the solvents were removed in
vacuo. Purification by size-exclusion chromatography (DCM/MeOH 1/
1 v/v) yielded the title compound as an anomeric mixture (460 mg, 704
2,4-Di-O-benzyl-r-D-galactopyranosiduronyl-3,6-lacto-
ne-(1f3)-tert-butyldiphenylsilyl 4-(N-Benzyloxycarbonyla-
mino)-2-azido-2,4,6-trideoxy-β-D-galactopyranoside (21).
Lactone 4 (338 mg) was coupled to alcohol 6 (0.77 equiv instead of 1.5
equiv) according to the general procedure for glycosidations using
Ph2SO/Tf2O. The reaction was quenched using triethylamine (5 equiv),
and the title compound 21 was obtained in 75% yield (391 mg, 435
μmol): flash column chromatography eluent:EtOAc/toluene (0/1 f
1/49); Rf 0.78 (EtOAc/toluene, 1/4, v/v); [R]22D 27 (c 1.0, CH2Cl2); IR
(neat, cm-1) 2860, 2361, 2114, 1800, 1717, 1506, 1061; 1H NMR (400
MHz, CDCl3, HH-COSY, HSQC) δ 7.79-7.08 (m, 25H, Harom),
5.13-5.00 (m, 3H, CH2 Cbz, H-10), 4.92 (d, J = 9.7 Hz, 1H, NH),
4.79 (d, J = 11.6 Hz, 1H, CH2 Bn), 4.71 (d, J = 3.8 Hz, 1H, H-30), 4.62-
4.52 (m, 2H, CH2 Bn), 4.50 (s, 1H, H-40), 4.36 (d, J = 7.8 Hz, 1H, H-1),
4.27 (d, J = 11.6 Hz, 1H, CH2 Bn), 4.18 (s, 1H, H-50), 4.05 (dd, J = 4.9,
2.1 Hz, 1H, H-20), 3.92 (dd, J = 10.0, 3.7 Hz, 1H, H-4), 3.68 (dd, J = 10.5,
4.0 Hz, 1H, H-3), 3.30-3.23 (m, 2H, H-2, H-5), 1.10 (s, 9H, CH3 t-Bu),
1.00 (d, J = 6.3 Hz, 3H, H-6); 13C NMR (100 MHz, CDCl3, HH-COSY,
HSQC) δ 171.8 (CdO), 156.5 (CdO Cbz), 138.0, 136.8 (Cq Ph),
135.8 (CHarom), 133.1, 132.5 (Cq Ph), 129.9, 129.8, 128.6, 128.5, 128.4,
128.3, 128.1, 127.8, 127.7, 127.5, 127.4 (CHarom), 96.9 (C-1), 95.6
(C-10), 80.4 (C-30), 77.1 (C-3), 75.8 (C-40), 75.0 (C-20), 74.3 (CH2 Bn),
72.2 (C-50), 71.5 (CH2 Bn), 69.0 (C-5), 67.2 (CH2 Cbz), 64.8 (C-2),
50.9 (C-4), 26.8 (CH3 t-Bu), 19.1 (Cq t-Bu), 16.3 (C-6); HRMS [M þ
Na]þ calcd for C50H54N4O10SiNa 921.35014, found 921.35091.
Benzyl 2,4-Di-O-benzyl-r-D-galactopyranosyluronate-(1f3)-
tert-butyldiphenylsilyl 4-(N-benzyloxycarbonylamino)-2-
azido-2,4,6-trideoxy-β-D-galactopyranoside (22). After addi-
tion of a catalytic amount of AcCl to a solution of 412 mg of lactone 21
(367 μmol) in 2 mL of BnOH, the mixture was allowed to stir overnight
at 50 °C. Following neutralization using triethylamine, the mixture was
diluted with EtOAc and washed with satd aq NaHCO3 and brine. The
organic phase was dried (MgSO4), filtered, and concentrated in vacuo.
Size-exclusion chromatography (DCM/MeOH 1/1 v/v) followed by
flash column chromatography using EtOAc/toluene (1/19f1/9) gave
370 mg (367 μmol, quant) of the title compound 22: Rf 0.51 (EtOAc/
toluene, 1/4, v/v); [R]22D þ34 (c 0.39, CH2Cl2); IR (neat, cm-1) 3500,
2932, 2112, 1761, 1719, 1508, 1107, 1059; 1H NMR (400 MHz, CDCl3,
HH-COSY, HSQC) δ 7.75-7.60 (m, 4H, Harom), 7.49-7.16 (m, 24H,
μmol, R/β 4:1, quant): Rf 0.81 (EtOAc/PE, 3/7, v/v); IR (neat, cm-1
)
2936, 2111, 1768, 1718, 1520, 1041, 698; 1H NMR (400 MHz, CDCl3,
HH-COSY, HSQC) δ 7.42-7.23 (m, 18.8H, Harom), 5.25 (dd, J = 11.1,
3.7 Hz, 1H, H-3R), 5.13 (dd, J = 10.6, 7.5 Hz, 2.5H, CH2 Cbz, NH), 5.01
(d, J = 12.3 Hz, 1,25H, CH2 Cbz), 4.89 (d, J = 3.7 Hz, 1H, H-1R), 4.72-
4.60 (m, 2.8H, CH2 Bn, H-3β), 4.58-4.48 (m, 2.5H, CH2 Bn), 4.28 (d, J
= 8.0 Hz, 0.25H, H-1β), 4.18-4.04 (m, 2.25H, H-4R, H-5R, H-4β),
3.99-3.73 (m, 4.8H, CH2 Gro, CH Gro, CH2 ClAc), 3.63-3.57 (m,
4H, CH2 Gro, H-5β), 3.45-3.35 (m, 1.3H, H-2β, H-2R), 1.19 (d, J = 6.3
Hz, 0.8H, H-6β), 1.04 (d, J = 6.4 Hz, 3H, H-6R); 13C NMR (100 MHz,
CDCl3, HH-COSY, HSQC) δ 166.6, 156.6, 156.5 (CdO), 138.2, 137.9,
136.2 (Cq Ph), 128.5, 128.3, 128.2, 127.9, 127.6, 127.5 (CHarom), 102.2
(C-1β), 97.9 (C-1R), 76.8 (CH Gro-β), 76.6 (CH Gro-R), 74.3 (C-3β),
73.3 (CH2 Bn), 72.1, 72.0 (CH2 Bn), 71.6 (C-3R), 69.5, 69.4, 69.2 (CH2
Gro), 68.9 (C-5β), 67.9 (CH2 Gro), 67.0 (CH2 Cbz), 64.0 (C-5R), 60.8
(C-2β), 57.2 (C-2R), 52.3 (C-4R), 51.6 (C-4β), 40.5 (CH2 ClAc), 16.3
(C-6β), 16.1 (C-6R); HRMS [M þ Na]þ calcd for C33H37ClN4O8Na
675.21921, found 675.21973.
4-(N-Benzyloxycarbonylamino)-2-azido-2,4,6-trideoxy-r-
D-galactopyranosyl-(1f3)-1,2-di-O-benzyl-sn-glycerol
(20r) and 4-(N-Benzyloxycarbonylamino)-2-azido-2,4,6-tri-
deoxy-β-D-galactopyranosyl-(1f3)-1,2-di-O-benzyl-sn-gly-
cerol (20β). A mixture of 460 mg of azide 19 (704 μmol, 1 equiv), 454
μL of pyridine (5.63 mmol, 8 equiv), 161 mg of thiourea (2.11 mmol, 3
equiv), and 7 mL of ethanol was stirred for 3 h at 65 °C. The mixture was
concentrated, and the crude residue was taken up in EtOAc. The organic
phase was washed with aq 1 M HCl, satd aq NaHCO3, and brine,
dried over MgSO4, filtered, and evaporated. Purification by flash
column chromatography using EtOAc/PE (1/4 f 3/7) yielded 317
mg of 20r (549 μmol, 78%) and 66 mg of 20β (114 μmol, 16%). 20r:
Rf 0.3 (EtOAc/PE, 1/3, v/v); [R]22D þ91 (c 1.0, CH2Cl2); IR (neat,
cm-1) 3418, 2916, 2106, 1697, 1026, 725; 1H NMR (400 MHz,
CDCl3, HH-COSY, HSQC) δ 7.40-7.23 (m, 15H, Harom), 5.17-
5.06 (m, 3H, NH, CH2 Cbz), 4.81 (d, J = 3.7 Hz, 1H, H-1), 4.71-4.62
(m, 2H, CH2 Bn), 4.54 (dd, J = 12.0, 12.0 Hz, 2H, CH2 Bn), 4.14 (dd,
J = 10.7, 3.5 Hz, 1H, H-3), 4.08 (q, J = 6.5 Hz, 1H, H-5), 3.96 (dd, J =
8.8, 2.5 Hz, 1H, H-4), 3.83-3.76 (m, 2H, CH Gro, CH2 Gro), 3.63
(d, J = 4.7 Hz, 2H, CH2 Gro), 3.57 (dd, J = 9.4, 4.3 Hz, 1H, CH2 Gro),
3.24 (s, 1H, OH), 3.12 (dd, J = 10.7, 3.6 Hz, 1H, H-2), 1.07 (d, J = 6.5
Hz, 3H, H-6); 13C NMR (100 MHz, CDCl3, HH-COSY, HSQC) δ
158.1 (CdO), 138.3, 138.0, 135.8 (Cq Ph), 128.6, 128.3, 128.1,
127.7, 127.6, 127.6 (CHarom), 98.2 (C-1), 76.8 (CH Gro), 73.4 (CH2
Bn), 72.1 (CH2 Bn), 69.4 (CH2 Gro), 68.5 (C-3), 67.6 (CH2 Gro),
67.5 (CH2 Cbz), 64.5 (C-5), 60.4 (C-2), 55.8 (C-4), 16.4 (C-6);
HRMS [M þ Na]þ calcd for C31H36N4O7Na 599.24762, found
599.24731. 20β: Rf 0.16 (EtOAc/PE, 1/3, v/v); [R]22D -13 (c 1.0,
H
arom), 7.14-7.03 (m, 2H, Harom), 5.54 (d, J = 3.3 Hz, 1H, H-10), 5.14
(d, J = 12.2 Hz, 1H, CH2 Bn), 5.06 (m, 2H, CH2 Bn), 4.91 (d, J = 10.4
Hz, 1H, NH), 4.76 (d, J = 12.2 Hz, 1H, CH2 Bn), 4.68-4.61 (m, 3H,
H-50, CH2 Bn), 4.42-4.36 (m, 3H, H-1, CH2 Bn), 4.30 (s, 1H, H-40),
4.21 (dd, J = 10.0, 3.2 Hz, 1H, H-30), 4.09 (dd, J = 10.1, 3.5 Hz, 1H, H-4),
3.90 (dd, J = 10.1, 3.3 Hz, 1H, H-20), 3.57 (dd, J = 10.8, 4.2 Hz, 1H, H-3),
3.38 (dd, J = 10.9, 7.7 Hz, 1H, H-2), 3.23 (q, J = 6.1 Hz, 1H, H-5), 2.12 (s,
1H, OH), 1.09 (s, 9H, CH3 t-Bu), 0.92 (d, J = 6.3 Hz, 3H, H-6); 13
C
NMR (100 MHz, CDCl3, HH-COSY, HSQC) δ 168.3 (CdO), 156.7
(CdO Cbz), 138.2, 137.8, 136.0 (Cq Ph), 135.8, 135.7 (CHarom), 135.1,
133.2, 132.8 (Cq Ph), 129.9, 129.8, 128.5, 128.4, 128.2, 128.0, 127.9,
127.6, 127.5, 127.4 (CHarom), 97.2 (C-1), 92.1 (C-10), 77.6 (C-40), 74.9
(CH2 Bn), 74.8 (C-20), 72.3 (C-3), 72.0 (CH2 Bn), 70.6 (C-50), 69.8
(C-5), 69.3 (C-30), 67.0 (CH2 Cbz, CH2 Bn), 65.3 (C-2), 49.9 (C-4),
26.8 (CH3 t-Bu), 19.1 (Cq t-Bu), 16.0 (C-6); HRMS [M þ Na]þ calcd
for C57H62N4O11SiNa 1029.40766, found 1029.40828.
Benzyl 2,4-Di-O-benzyl-3-O-chloroacetyl-r-D-galactopy-
ranosyluronate-(1f3)-tert-butyldiphenylsilyl 4-(N-Benzylo-
xycarbonylamino)-2-azido-2,4,6-trideoxy-β-D-galactopyr-
anoside (23). Alcohol 22 (2.10 g, 2.09 mmol, 1 equiv) was coevapo-
rated with toluene and dissolved in 10 mL of DCM. A 1.68 mL (20.9
mmol, 10 equiv) portion of pyridine and 1.07 g of (ClAc)2O (6.3 mmol,
3 equiv) were added at 0 °C, and the mixture was stirred at ambient
temperature for 2.5 h. Next, H2O and EtOAc were added, and the
organic phase was washed with aq 1 M HCl, satd aq NaHCO3 and brine.
1
CH2Cl2); IR (neat, cm-1) 3348, 2870, 2106, 1705, 1049, 741; H
NMR (400 MHz, CDCl3, HH-COSY, HSQC) δ 7.47-7.17 (m, 15H,
Harom), 5.29-5.03 (m, 3H, NH, CH2 Cbz), 4.67 (s, 2H, CH2 Bn),
4.54 (s, 2H, CH2 Bn), 4.22 (d, J = 8.0 Hz, 1H, H-1), 3.99-3.91 (m,
2H, CH2 Gro, H-4), 3.83-3.78 (m, 1H, CH Gro), 3.72 (dd, J = 10.2,
5.2 Hz, 1H, CH2 Gro), 3.63 (dd, J = 4.9, 1.5 Hz, 2H, CH2 Gro), 3.60-
3.53 (m, 2H, H-3, H-5), 3.22 (dd, J = 10.0, 8.2 Hz, 1H, H-2), 3.16 (d,
J = 2.3 Hz, 1H, OH), 1.19 (d, J = 6.2 Hz, 3H, H-6); 13C NMR (100
MHz, CDCl3, HH-COSY, HSQC) δ 158.0 (CdO), 138.6, 138.2,
136.0 (Cq Ph), 128.6, -128.4, 128.3, 128.1, 127.7, 127.6 (CHarom),
102.5 (C-1), 77.0 (CH Gro), 73.5 (CH2 Bn), 72.5 (C-3), 72.3 (CH2
Bn), 69.8 (CH2 Gro), 69.5 (C-5) (CH2 Gro), 67.5 (CH2 Cbz), 64.5
(C-2), 54.9 (C-4), 16.6 (C-6); HRMS [M þ H]þ calcd for
C31H36N4O7 577.26568, found 577.26583.
1699
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ARTICLE
The organic layer was dried (MgSO4), filtered, and evaporated. Pur-
ification by flash column chromatography using EtOAc/Tol (0/1 f 1/
19) yielded 2.11 g of chloroacetate 23 (1.94 mmol, 93%): Rf 0.81
H
arom), 7.13 (t, J = 7.5 Hz, 1H, Harom), 7.08-7.00 (m, 2H, Harom), 6.84
(d, J = 7.8 Hz, 2H, Harom), 5.57 (d, J = 2.8 Hz, 1H, H-10), 5.41 (dd, J =
10.6, 2.9 Hz, 1H, H-30), 5.23-5.03 (m, 4H, NH, CH2 Cbz or CH2
CO2Bn), 4.79 (s, 1H, H-50), 4.74-4.64 (m, 2H, CH2 Cbz or CH2
CO2Bn, CH2 Bn), 4.46-4.38 (m, 2H, CH2 Bn, H-40), 4.37-4.22
(m, 3H, CH2 Bn, H-4), 4.13 (dd, J = 10.5, 3.4 Hz, 1H, H-20), 3.82-3.73
(m, 1H, H-5), 3.73-3.66 (m, 1H, H-2), 3.63 (d, J = 14.9 Hz, 1H, CH2
ClAc), 3.52 (d, J = 14.9 Hz, 1H, CH2 ClAc), 1.22 (d, J = 6.9 Hz, 3H,
H-6); 13C NMR (100 MHz, CDCl3, HH-COSY, HSQC) δ 167.6, 166.4
(CdO), 156.6 (CdO Cbz), 142.9, 137.8, 137.6, 135.8, 134.8 (Cq Ph),
128.8, 128.8, 128.7, 128.6, 128.4, 128.3, 128.2, 128.1, 127.9, 127.8, 127.7,
127.6, 124.6, 119.2 (CHarom), 92.9 (C-10), 76.8 (C-40), 75.1 (CH2 Cbz
or CH2 CO2Bn), 72.8 (C-30), 72.7 (CH2 Cbz or CH2 CO2Bn), 72.6
(C-3 or C-5), 72.1 (C-20), 71.2 (C-3 or C-5), 70.3 (C-50), 67.3, 67.0
(CH2 Bn), 61.3 (C-2), 49.7 (C-4), 40.3 (CH2 ClAc), 16.3 (C-6); HRMS
[M - (C(NdPh)CF3) þ H þ Na]þ calcd for C43H45ClN4O12Na
867.26147, found 867.26164.
(EtOAc/toluene, 1/4, v/v); [R]22 þ78 (c 1.0, CH2Cl2); IR (neat,
D
cm-1) 2936, 2112, 1760, 1718, 1508, 1063, 696; 1H NMR (400 MHz,
CDCl3, HH-COSY, HSQC) δ 7.68 (dd, J = 7.0, 3.4 Hz, 4H, Harom),
7.46-7.12 (m, 24H, Harom), 7.08-6.97 (m, 2H, Harom), 5.53 (d, J = 3.3
Hz, 1H, H-10), 5.41 (dd, J = 10.5, 2.9 Hz, 1H, 30), 5.15 (dd, J = 12.1, 12.1
Hz, 2H, CH2 Bn), 5.03 (d, J = 12.2 Hz, 1H, CH2 Bn), 4.89 (d, J = 10.4
Hz, 1H, NH), 4.78 (s, 1H, 50), 4.67 (dd, J = 12.4, 12.4 Hz, 2H, CH2 Bn),
4.44-4.37 (m, 3H, H-40, H-1, CH2 Bn), 4.31 (dd, J = 11.8, 11.8 Hz, 2H,
CH2 Bn), 4.14-4.02 (m, 2H, H-20, H-4), 3.63 (d, J = 14.9 Hz, 1H, CH2
ClAc), 3.58 (dd, J = 10.8, 4.1 Hz, 1H, H-3), 3.51 (d, J = 14.9 Hz, 1H, CH2
ClAc), 3.42 (dd, J = 10.7, 7.7 Hz, 1H, H-2), 3.23 (q, J = 6.1 Hz, 1H, H-5),
1.10 (s, 9H, CH3 t-Bu), 0.91 (d, J = 6.3 Hz, 3H, H-6); 13C NMR (100
MHz, CDCl3, HH-COSY, HSQC) δ 167.8, 166.4 (CdO), 156.6 (CdO
Cbz), 138.0, 137.7, 136.0 (Cq Ph), 135.8, 135.7 (CHarom), 134.9, 133.2,
132.8 (Cq Ph), 129.9, 129.8, 128.8, 128.6, 128.4, 128.3, 128.2, 128.1,
128.0, 127.8, 127.7, 127.6, 127.5, 127.4 (CHarom), 97.3 (C-1), 92.7
(C-10), 76.8 (C-40), 75.0 (CH2 Bn), 72.9 (C-30), 72.6 (C-3, CH2 Bn),
72.2 (C-20), 70.2 (C-50), 69.7 (C-5), 67.2, 66.9 (CH2 Bn), 65.1 (C-2),
49.9 (C-4), 40.3 (CH2 ClAc), 26.8 (CH3 t-Bu), 19.1 (Cq t-Bu), 16.0
(C-6); HRMS [M þ Na]þ calcd for C59H63ClN4O12SiNa 1105.37925,
found 1105.38018.
Benzyl 2,4-Di-O-benzyl-3-O-chloroacetyl-r-D-galactopy-
ranosyluronate-(1f3)-4-(N-benzyloxycarbonylamino)-2-
azido-2,4,6-trideoxy-D-galactopyranosyl-(1f4)-phenyl 2-O-
benzyl-1-thio-β-D-galactopyranosidurono-3,6-lactone (26).
Imidate donor (25) (600 mg, 590 μmol, 1 equiv) and 391 mg of lactone
acceptor 5 (1.09 mmol, 1.85 equiv) were coevaporated with toluene and
stirred over activated 3 Å molecular sieves in 6 mL of DCM for 30 min.
The mixture was cooled to 0 °C before 5 μL of triflic acid (59 μmol, 0.1
equiv) was added. After 30 min of stirring, the reaction was quenched by
the addition of 41 μL of triethylamine (295 μmol, 0.5 equiv). The
mixture was diluted with DCM and washed with satd aq NaHCO3. The
aqueous phase was extracted with DCM, and the combined organic
extracts were dried (MgSO4), filtered, and concentrated under reduced
pressure. Size-exclusion chromatography (DCM/MeOH 1/1 v/v) gave
565 mg (476 μmol, 81%) of the two possible epimers. Partial separation
of this mixture by flash column chromatography (eluent: EtOAc/PE 1/3
f 9/11) afforded the pure R-coupled product 26 (450 mg, 379 μmol,
64%): Rf 0.57 (EtOAc/toluene, 1/4, v/v); [R]22D þ62 (c 1.0, CH2Cl2);
Benzyl 2,4-Di-O-benzyl-3-O-chloroacetyl-r-D-galactopy-
ranosyluronate-(1f3)-4-(N-benzyloxycarbonylamino)-2-
azido-2,4,6-trideoxy-D-galactopyranose (24). A mixture of
2.05 g of dimer 23 (1.89 mmol, 1 equiv), 20 mL of THF and 2.47 mL
of triethylamine 3HF (15.13 mmol, 8 equiv) was stirred overnight at
3
70 °C. The solvent was removed using a rotary evaporator, and the crude
anomers were purified by flash column chromatography using EtOAc/
PE (7/13f9/11). The title compound 24 (1.35 g, 1.60 mmol, 84%, R/β
1:4) was obtained. Data of major anomer (β): Rf 0.24 (EtOAc/PE, 2/3,
1
v/v); IR (neat, cm-1) 3425, 2108, 1763, 1717, 1541, 1244, 1036; H
NMR (400 MHz, CDCl3, HH-COSY, HSQC) δ 7.38-7.15 (m, 18H,
Harom), 7.08-7.00 (m, 2H, Harom), 5.54 (d, J = 3.3 Hz, 1H, H-10), 5.43-
5.36 (m, 2H, H-30, NH), 5.21-5.10 (m, 2H, CH2 Bn), 5.06 (d, J = 12.3
Hz, 1H, CH2 Cbz or CH2 CO2Bn), 4.85 (s, 1H, H-50), 4.67 (m, 2H, CH2
Bn), 4.55 (d, J = 7.0 Hz, 1H, H-1), 4.44-4.37 (m, 2H, CH2 Bn, H-40),
4.36-4.28 (m, 2H, CH2 Bn), 4.20 (dd, J = 10.4, 4.3 Hz, 1H, H-4), 4.11
(dd, J = 10.5, 3.3 Hz, 1H, H-20), 3.70 (dd, J = 10.8, 4.2 Hz, 1H, H-3),
3.66-3.44 (m, 4H, CH2 ClAc, H-5, H-2), 1.19 (d, J = 6.3 Hz, 3H, H-6);
13C NMR (100 MHz, CDCl3, HH-COSY, HSQC) δ 167.8, 166.3
(CdO), 156.9 (CdO Cbz), 137.7, 137.6, 136.0, 134.7 (Cq Ph), 128.9,
128.7, 128.5, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5,
127.4 (CHarom), 96.5 (C-1), 92.5 (C-10), 76.7 (C-40), 74.9 (CH2 Cbz or
CH2 CO2Bn), 72.8, (C-30), 72.5 (CH2 Cbz or CH2 CO2Bn), 72.4
(C-3), 72.2 (C-20), 70.1 (C-50), 69.8 (C-5), 67.2, 66.7 (CH2 Bn), 63.1
(C-2), 49.9 (C-4), 40.2 (CH2 ClAc), 16.4 (C-6); HRMS [M þ Na]þ
calcd for C16H19ClN4O6Na 867.26147, found 867.26183.
Benzyl 2,4-Di-O-benzyl-3-O-chloroacetyl-r-D-galactopy-
ranosyluronate-(1f3)-4-(N-benzyloxycarbonylamino)-2-
azido-2,4,6-trideoxy-D-galactopyranosyl N-Phenyltrifluor-
oacetimidate (25). To a solution of 666 mg of hemiacetal 24
(0.787 mmol, 1 equiv) in 15 mLof acetone/H2O (19/1) were added
282 mg of Cs2CO3 (0.866 mmol, 1.1 equiv) and 327 mg of ClC-
(CdNPh)CF3 (1.57 mmol, 2.0 equiv). The mixture was stirred over-
night at ambient temperature and evaporated to dryness after the
addition of 328 μL of triethylamine (2.36 mmol, 3.0 equiv). Flash
column chromatography of the crude product using EtOAc/PE (3/7)
with 1% triethyamine as eluent afforded 680 mg (669 μmol, 85%) of the
title imidate as one of the two possible epimers: Rf 0.64 (EtOAc/toluene,
1/4, v/v); IR (neat, cm-1) 2112, 1759, 1717, 1516, 1209, 1078, 696; 1H
NMR (400 MHz, CDCl3, HH-COSY, HSQC) δ 7.39-7.18 (m, 20H,
IR (neat, cm-1) 2106, 1805, 1759, 1720, 1520, 1242, 1034; H NMR
1
(400 MHz, CDCl3, HH-COSY, HSQC) δ 7.50-7.43 (m, 2H, Harom),
7.41-7.14 (m, 26H, Harom), 7.07-7.00 (m, 2H, Harom), 5.54 (s, 1H,
H-100), 5.42 (s, 1H, H-1), 5.39 (dd, J = 10.7, 2.7 Hz, 1H, H-300), 5.18 (s,
2H, CH2), 5.09-4.96 (m, 3H, CH2, H-10, NH), 4.86 (d, J = 4.7 Hz, 1H,
H-2), 4.69-4.63 (m, 4H, CH2 Bn, H-500), 4.58-4.55 (m, 2H, H-4,
CH2), 4.40-4.37 (m, 2H, H-400, CH2 Bn), 4.35-4.25 (m, 4H, H-40,
H-3, CH2 Bn), 4.18 (dd, J = 10.6, 3.9 Hz, 1H, H-30), 4.14-4.05 (m, 3H,
H-200, H-50, H-5), 3.56 (d, J = 14.9 Hz, 1H, CH2 ClAc), 3.46 (d, J = 14.9
Hz, 1H, CH2 ClAc), 3.39 (dd, J = 11.0, 3.9 Hz, 1H, H-20), 1.13 (d, J = 6.2
Hz, 3H, H-60); 13C NMR (100 MHz, CDCl3, HH-COSY, HSQC) δ
171.7, 167.5, 166.4 (CdO), 156.7 (CdO Cbz), 138.0, 137.7, 136.3,
135.9, 134.9, 133.3 (Cq Ph), 132.8, 129.0, 128.8, 128.6, 128.4, 128.3,
128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6 (CHarom), 97.7 (C-10),
93.1 (C-100), 85.7 (C-1), 79.1 (C-2), 78.4 (C-3), 76.8 (C-400), 76.3
(C-4), 75.1, 73.2 (CH2 Bn), 72.8 (C-300, CH2 Bn), 72.3 (C-200), 70.4
(C-500), 70.2 (C-50), 69.9 (C-30), 67.4, 67.0 (CH2), 66.5 (C-5), 58.8
(C-20), 50.7 (C-40), 40.3 (CH2 ClAc), 16.3 (C-60);
Benzyl 2,4-Di-O-benzyl-3-O-chloroacetyl-r-D-galactopy-
ranosyluronate-(1f3)-4-(N-benzyloxycarbonylamino)-2-
azido-2,4,6-trideoxy-D-galactopyranosyl-(1f4)-2-O-ben-
zyl-r-D-galactopyranosiduronyl-3,6-lactone-(1f3)-sn-gly-
cerol (27). Thioglycoside 26 (328 mg) was coupled to glycerol
acceptor 9 (4.0 equiv instead of 1.5 equiv) according to the general
procedure for glycosylations using Ph2SO/Tf2O. The reaction was
quenched with triethylamine (5 equiv), and the title compound 27
was purified using size-exclusion chromatography: yield 81% (302 mg,
224 μmol); Rf 0.59 (EtOAc/PE, 2/3, v/v); [R]22D þ99 (c 1.0, CH2Cl2);
IR (neat, cm-1) 2874, 2108, 1803, 1761, 1719, 1028, 696; H NMR
1
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The Journal of Organic Chemistry
ARTICLE
(400 MHz, CDCl3, HH-COSY, HSQC) δ 7.39-7.16 (m, 33H, Harom),
7.06-6.99 (m, 2H, Harom), 5.53 (d, J = 2.5 Hz, 1H, H-100), 5.38 (dd, J =
10.6, 2.7 Hz, 1H, H-300), 5.25-5.12 (m, 2H, CH2 Bn), 5.05 (d, J = 12.2
Hz, 1H, CH2 Bn), 4.99-4.97 (m, 2H, NH, H-10), 4.90-4.86 (m, 2H,
H-1, CH2 Bn), 4.74-4.61 (m, 7H, H-4, H-3, H-500, CH2 Bn), 4.58-4.45
(m, 3H, CH2 Bn), 4.40-4.37 (m, 2H, CH2 Bn, H-400), 4.34-4.26 (m,
3H, H-40, CH2 Bn), 4.21-4.14 (m, 2H, H-5, H-30), 4.13-4.08 (m, 2H,
CH2 Gro, H-200), 4.02 (q, J = 6.3 Hz, 1H, H-50), 3.89 (dd, J = 5.0, 2.2 Hz,
1H, H-2), 3.87-3.82 (m, 1H, CH Gro), 3.70 (dd, J = 10.7, 6.9 Hz, 1H,
CH2 Gro), 3.59-3.52 (m, 3H, CH2 Gro, CH2 ClAc), 3.45 (d, J = 14.9
Hz, 1H, CH2 ClAc), 3.38 (dd, J = 10.9, 3.9 Hz, 1H, H-20), 1.11 (d, J = 6.3
Hz, 3H, H-60); 13C NMR (100 MHz, CDCl3, HH-COSY, HSQC) δ
170.7, 167.5, 166.3 (CdO), 156.7 (CdO Cbz), 138.3, 137.9, 137.6,
137.4, 135.9, 134.9 (Cq Ph), 128.9, 128.6, 128.5, 128.4, 128.3, 128.2,
128.1, 127.9, 127.8, 127.7, 127.6 (CHarom), 99.3 (C-1), 97.5 (C-10), 93.0
(C-100), 80.6 (C-3), 77.2 (CH Gro), 76.8 (C-400), 75.5 (C-4), 75.1 (CH2
Bn), 74.5 (CH2 Bn), 74.3 (C-2), 73.4 (CH2 Bn), 72.8 (CH2 Bn, C-3),
72.3 (CH2 Bn, C-200), 71.4 (C-5), 71.1 (CH2 Gro), 70.4 (C-500), 69.8
(C-30), 69.5 (CH2 Gro), 67.4, 67.0 (CH2), 66.2 (C-50), 58.7 (C-20), 50.6
(C-40), 40.3 (CH2 ClAc), 16.2 (C-60); HRMS [M þ Na]þ calcd for
C73H75ClN4O19Na 1369.46062, found 1369.46265.
the title acetamide (89 mg, 69 μmol, 94%): Rf 0.41 (EtOAc/PE, 3/2, v/
v); [R]22 þ97 (c 0.7, CH2Cl2); IR (neat, cm-1) 3368, 2924, 1801,
D
1718, 1668, 1028, 697; 1H NMR (400 MHz, CDCl3, HH-COSY,
HSQC) δ 7.40-7.19 (m, 33H, Harom), 7.13 (d, J = 6.7 Hz, 2H, Harom),
5.43 (d, J = 8.9 Hz, 1H, NH), 5.24 (d, J = 9.9 Hz, 1H, NH), 5.19-5.12
(m, 2H, CH2, H-100), 5.02 (d, J = 12.4 Hz, 1H, CH2 Bn), 4.94 (d, J = 12.0
Hz, 1H, CH2 Bn), 4.91-4.77 (m, 4H, CH2 Bn, H-10, H-1), 4.73-4.59
(m, 6H, CH2 Bn, H-4, H-3), 4.53-4.37 (m, 6H, CH2 Bn, H-500), 4.20-
4.05 (m, 5H, H-40, H-400, H-20, H-5, CH2 Gro), 3.98 (dd, J = 9.9, 2.7 Hz,
1H, H-300), 3.93-3.80 (m, 4H, H-2, H-50, CH Gro, H-200), 3.75-3.65
(m, 2H, H-30, CH2 Gro), 3.55 (dd, J = 5.0, 1.7 Hz, 2H, CH2 Gro), 1.71
(s, 3H, CH3 NHAc), 1.12 (d, J = 6.4 Hz, 3H, H-60); 13C NMR (100
MHz, CDCl3, HH-COSY, HSQC) δ 171.2, 170.4, 168.3 (CdO), 156.9
(CdO Cbz), 138.3, 138.1, 137.9, 137.3, 136.4, 134.8 (Cq Ph), 128.8,
128.7, 128.6, 128.5, 128.4, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6,
127.5 (CHarom), 99.2 (C-1), 96.4 (C-10), 96.0 (C-100), 80.9 (C-3), 77.2
(C-400, CH Gro), 75.4 (C-200), 74.7, 74.5 (CH2 Bn), 74.2 (C-2), 73.9
(C-4), 73.4 (CH2 Bn), 73.3 (C-30), 72.7, 72.3 (CH2 Bn), 71.2 (C-5 or
C-500), 71.1 (CH2 Gro, C-5 or C-500), 69.4 (CH2 Gro), 69.2 (C-300),
67.2, 66.6 (CH2 Bn), 66.4 (C-50), 52.1 (C-40), 48.3 (C-20), 22.8 (CH3
NHAc), 16.4 (C-60); HRMS [M þ Na]þ calcd for C73H78N2O19Na
1309.50910, found 1309.50940.
Benzyl 2,4-Di-O-benzyl-r-D-galactopyranosyluronate-
(1f3)-4-(N-benzyloxycarbonylamino)-2-azido-2,4,6-tri-
deoxy-D-galactopyranosyl-(1f4)-2-O-benzyl-r-D-galac-
topyranosiduronyl-3,6-lactone-(1f3)-1,2-di-O-benzyl-sn-
glycerol (28). A solution of 302 mg of compound 27 (224 μmol, 1
equiv), 145 μL of pyridine (1.79 mmol, 8 equiv), and 51 mg of thiourea
(672 μmol, 3 equiv) in 4 mL of EtOH was stirred at 65 °C for 3 h. The
mixture was concentrated under reduced pressure, diluted with EtOAc,
and washed with aq 1 M HCl, satd aq NaHCO3, and brine. The organic
phase was dried (MgSO4), filtered, and concentrated under reduced
pressure. Flash column chromatography using EtOAc/toluene (1/
4f1/3) gave 218 mg (171 μmol, 76%) of the title compound 28: Rf
r-D-Galactopyranosyluronate-(1f3)-2-acetamido-4-ami-
no-2,4,6-trideoxy-r-D-galactopyranosyl-(1f4)-r-D-galacto-
pyranosyluronate-(1f3)-sn-glycerol (1). To a solution of 21
mg (16 μmol, 1 equiv) of compound 29 in 1.5 mL of DCM was added
8.0 mg of TMSONa (71 μmol, 4.5 equiv). The mixture was stirred for
2.5 h, followed by the addition of 20 μL of AcOH (355 μmol, 22.5
equiv), evaporation, and elution over a plug of silica (eluent: EtOAc,
then EtOAc/MeOH/H2O/AcOH 88/10/1/1). After removal of the
eluent, the crude product was dissolved in 7 mL of t-BuOH/H2O (5/2
v/v) and stirred under argon atmosphere. A catalytic amount of
palladium on activated charcoal and 125 μL of 1 M aq HCl were added,
and the mixture was allowed to stir for 2 days under hydrogen atmo-
sphere. Filtration over Celite, gel filtration (HW-40, 0.15 M Et3NHOAc
in H2O), and subsequent lyophilization afforded 5.2 mg of the pure title
0.62 (EtOAc/PE, 2/3, v/v); [R]22 þ95 (c 1.0, CH2Cl2); IR (neat,
D
cm-1) 2924, 2106, 1805, 1759, 1713, 1535, 1034; 1H NMR (400 MHz,
CDCl3, HH-COSY, HSQC) δ 7.39-7.12 (m, 33H, Harom), 7.11-7.05
(m, 2H, Harom), 5.55 (d, J = 2.2 Hz, 1H, H-100), 5.15 (m, 2H, CH2 Bn),
5.09-5.05 (m, 2H, NH, CH2 Bn), 4.92 (d, J = 4.2 Hz, 1H, H-10), 4.89-
4.83 (m, 2H, H-1, CH2 Bn), 4.74-4.58 (m, 7H, H-4, H-3, CH2 Bn,
CH2), 4.55 (s, 1H, H-500), 4.54-4.44 (m, 3H, CH2 Bn), 4.43-4.28 (m,
3H, CH2 Bn, H-40), 4.24 (s, 1H, H-400), 4.20-4.11 (m, 3H, H-5, H-300,
H-30), 4.09 (dd, J = 10.7, 3.5 Hz, 1H, CH2 Gro), 4.00 (q, J = 6.2 Hz, 1H,
H-50), 3.92-3.81 (m, 3H, H-200, H-2, CH Gro), 3.68 (dd, J = 10.7, 6.9
Hz, 1H, CH2 Gro), 3.54 (d, J = 5.3 Hz, 2H, CH2 Gro), 3.36 (dd, J = 11.0,
3.9 Hz, 1H, H-20), 2.04 (s, 1H, OH), 1.10 (d, J = 6.2 Hz, 3H, H-60); 13C
NMR (100 MHz, CDCl3, HH-COSY, HSQC) δ 170.6, 168.0 (CdO),
156.7 (CdO Cbz), 138.2, 138.0, 137.8, 137.7, 137.2, 135.8, 134.9 (Cq
Ph), 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6,
127.5, 127.4 (CHarom), 99.2 (C-1), 97.3 (C-10), 92.3 (C-100), 80.5 (C-3),
77.6 (C-400), 77.1 (CH Gro), 75.1 (C-4), 74.8 (CH2 Bn), 74.6 (C-200),
74.3 (CH2 Bn), 74.2 (C-2), 73.3 (CH2 Bn), 72.2 (CH2 Bn), 72.0 (CH2
Bn), 71.2 (C-5), 71.0 (CH2 Gro), 70.6 (C-500), 69.3 (CH2 Gro, C-30),
69.0 (C-300), 67.0, 66.9 (CH2), 66.2 (C-50), 58.6 (C-20), 50.5 (C-40),
16.1 (C-60); HRMS [M þ Na]þ calcd for C71H74N4O18Na 1293.48903,
found 1293.49043.
1
compound 1 (8.3 μmol, 52% over two steps): H NMR (600 MHz,
D2O, HH-COSY, HSQC, HMBC, TOCSY, T= 290 K) δ 5.07 (d, J = 2.4
Hz, 1H, H-100), 5.04 (d, J = 3.7 Hz, 1H, H-1), 4.98 (d, J = 4.0 Hz, 1H,
H-10), 4.79 (q, J = 6.7 Hz, 1H, H-50), 4.37 (d, J = 2.7 Hz, 1H, H-4), 4.34
(s, 1H, H-5), 4.27-4.25 (m, 2H, H-400, H-30), 4.14 (s, 1H, H-500), 4.11
(dd, J = 11.4, 4.0 Hz, 1H, H-20), 4.07 (dd, J = 10.6, 3.1 Hz, 1H, H-3),
3.96-3.91 (m, 1H, CH Gro), 3.89 (dd, J = 10.6, 3.8 Hz, 1H, H-2), 3.85
(m, 2H, H-200, H-300), 3.84-3.78 (m, 2H, H-40, CH2 Gro), 3.66 (dd, J =
11.8, 4.6 Hz, 1H, CH2 Gro), 3.58 (dd, J = 11.8, 6.2 Hz, 1H, CH2 Gro),
3.53 (dd, J = 10.6, 7.0 Hz, 1H, CH2 Gro), 2.02 (s, 3H, CH3 NHAc), 1.26
(d, J = 6.7 Hz, 3H, H-60); 13C NMR (151 MHz, D2O, HH-COSY,
HSQC, HMBC, TOCSY, T = 290 K) δ 175.6, 175.5, 174.7 (CdO), 99.6
(C-1), 99.1 (C-10), 98.9 (C-100), 80.0 (C-4), 73.3 (C-30), 73.2 (C-500),
71.5 (CH Gro), 71.3 (C-400, C-5), 70.1 (C-300, CH2 Gro), 69.4 (C-3),
68.8 (C-2), 68.0 (C-20), 63.7 (C-50), 63.2 (CH2 Gro), 53.5 (C-40), 48.4
(C-20), 23.0 (CH3 NHAc), 16.3 (C-60); 13C-HMBC NMR (151 MHz,
0
0
D2O, T= 290 K) δ 99.6 (JC1-H1 = 171.1 Hz, C-1), 99.1 (JC1 -H1 = 174.4
þ
00
Hz, C-10), 98.9 (JC1 -H1 = 171.1 Hz, C-1 ); HRMS [M þ H] calcd for
00
00
C23H39N2O18 631.21924, found 631.21934.
2,4-Di-O-benzyl-r-D-galactopyranosiduronyl-3,6-lactone-
(1f3)-1,2-di-O-benzyl-sn-glycerol (30). 448 mg of lactone 4
was coupled to alcohol 9 according to the general procedure for
glycosidations using Ph2SO/Tf2O, yielding 567 mg of the title com-
pound 30 (928 μmol, R/β 10:1, 93%): flash column chromatography
gradient EtOAc/PE (1/9 f 1/4); Rf 0.73 (EtOAc/toluene, 1/5, v/v);
IR (neat, cm-1) 3030, 2868, 1798, 1454, 1057, 696; NMR assignment of
the major anomer (R), 1H NMR (400 MHz, CDCl3, HH-COSY,
HSQC) δ 7.37-7.25 (m, 18H, Harom), 7.19 (dd, J = 6.7, 2.7 Hz, 2H,
Benzyl 2,4-Di-O-benzyl-r-D-galactopyranosyluronate-
(1f3)-2-acetamido-4-(N-benzyloxycarbonylamino)-2,4,6-
trideoxy-r-D-galactopyranosyl-(1f4)-2-O-benzyl-r-D-ga-
lactopyranosiduronyl-3,6-lactone-(1f3)-1,2-di-O-ben-
zyl-sn-glycerol (29). To an ice-cooled solution of 94 mg of azide 28
(74 μmol) in 1 mL of pyridine was added 1 mL of freshly distilled
thiolacetic acid. The mixture was stirred at room temperature for 4 h,
concentrated under reduced pressure, and coevaporated with toluene.
Flash column chromatography using EtOAc/PE (1/1f3/2) afforded
1701
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The Journal of Organic Chemistry
ARTICLE
Harom), 4.86-4.83 (m, 2H, H-1, CH2 Bn), 4.71-4.61 (m, 3H, H-3, CH2
Bn), 4.56 (s, 2H, CH2 Bn), 4.51 (d, J = 4.6 Hz, 2H, CH2 Bn), 4.48-4.43
(m, 2H, H-4, CH2 Bn), 4.14 (t, J = 1.6 Hz, 1H, H-5), 4.08 (dd, J = 10.7,
3.6 Hz, 1H, CH2 Gro), 3.88 (dd, J = 5.0, 2.4 Hz, 1H, H-2), 3.86-3.81
(m, 1H, CH Gro), 3.67 (dd, J = 10.7, 6.9 Hz, 1H, CH2 Gro), 3.54 (dd, J =
5.7, 4.7 Hz, 2H, CH2 Gro); 13C NMR (100 MHz, CDCl3, HH-COSY,
HSQC) δ 171.5 (CdO), 138.3, 137.8, 137.4, 136.6 (Cq Ph), 128.5,
128.4, 128.3, 128.2, 128.1, 127.9, 127.7, 127.6, 127.5 (CHarom), 99.2
(C-1), 80.1 (C-3), 77.1 (CH Gro), 75.4 (C-5), 74.3 (C-2), 74.2 (CH2
Bn), 73.2 (CH2 Bn), 72.1 (CH2 Bn), 71.8 (C-4), 71.3 (CH2 Bn), 71.0
(CH2 Gro), 69.4 (CH2 Gro); HRMS [M þ Na]þ calcd for
C37H38O8Na 633.24589, found 633.24698.
Gro), 2.85 (s, 1H, OH); 13C NMR (100 MHz, CDCl3, HH-COSY,
HSQC, galacturonic acid based numbering, major epimer) δ 174.4
(CdO), 138.0, 137.3, 137.2, 137.0 (Cq Ph), 128.5, 128.4, 128.3, 128.1,
127.9, 127.8, 127.7, 127.6, 127.5 (CHarom), 103.4 (C-1), 79.5 (C-3,
C-4), 77.1 (CH Gro), 76.3 (C-2), 74.4 (CH2 Bn), 74.3 (C-5), 73.4 (CH2
Bn), 72.2 (CH2 Bn), 71.9 (CH2 Bn), 70.5 (CH2 Bn), 69.7 (CH2 Gro),
69.3 (CH2 Gro); HRMS [M þ Na]þ calcd for C44H46O9Na 741.30340,
found 741.30347.
4-(N-Benzyloxycarbonylamino)-2-azido-3-O-chloroa-
cetyl-2,4,6-trideoxy-D-galactopyranosyl-(1f4)-phenyl 2-O-
Benzyl-1-thio-β-D-galactopyranosidurono-3,6-lactone
(33). A mixture of 441 mg of hemiacetal 7 (1.11 mmol, 1 equiv), 559
mg of diphenyl sulfoxide (2.76 mmol, 2.5 equiv), and 330 mg of tri-tert-
butylpyrimidine (1.33 mmol, 1.2 equiv) was coevaporated with toluene
Benzyl 2,4-Di-O-benzyl-r-D-galactopyranosyluronate-
(1f3)-1,2-di-O-benzyl-sn-glycerol (31). To a solution of 831
mg of lactone 30 (1.36 mmol, 1 equiv) in 14 mL of DCM was added 160
mg of TMSONa (1.43 mmol, 1.05 equiv). After 30 min, TLC indicated
complete consumption of the starting material, and the mixture was
evaporated and filtered through a plug of silica gel using EtOAc/
toluene/AcOH (20/79/1) as eluent. After removal of the eluent, the
crude acid was dissolved in 14 mL of DMF. Next, 154 μL of BnBr (1.28
mmol, 1.1 equiv) and 418 mg of Cs2CO3 (1.28 mmol, 1.1 equiv) were
added, and the reaction was stirred for 2 h. The mixture was diluted with
EtOAc and washed with H2O and brine. Drying over MgSO4, filtration,
and concentration under reduced pressure gave the crude product,
which was purified by flash column chromatography using EtOAc/PE
(1/4 f 1/3) to give ester 31 (729 mg, 1.01 mmol, 75% over two steps):
ꢀ
and stirred over activated molecular sieves (3 Å) for 30 min in 10 mL of
DCM. The mixture was cooled to -60 °C before 202 μL of triflic acid
anhydride (1.22 mmol, 1.1 equiv) was added. The mixture was allowed
ꢀ
to warm to -40 °C before a mixture (dried over 3 Å molecular sieves) of
792 mg of acceptor 5 (2.21 mmol, 2 equiv) and 330 mg of tri-tert-
butylpyrimidine (1.33 mmol, 1.2 equiv) in 2 mL of DCM was added.
Stirring was continued, and the reaction mixture was allowed to warm to
4 °C overnight. The reaction mixture was quenched with 769 μL of
triethylamine (5.53 mmol, 5.0 equiv), diluted with DCM, and washed
with satd aq NaHCO3. The aqueous phase was extracted with DCM, and
the combined organic phases were dried (MgSO4), filtered, and con-
centrated under reduced pressure. Purification by size-exclusion chro-
matography (DCM/MeOH 1/1 v/v) and flash column chromatography
(eluent: EtOAc/PE 1/4 f 3/7) gave the title compound 33 as a white
Rf 0.52 (EtOAc/toluene, 1/3, v/v); [R]22 þ44 (c 1.0, CH2Cl2); IR
D
(neat, cm-1) 3030, 2870, 1759, 1454, 1107, 696; 1H NMR (400 MHz,
CDCl3, HH-COSY, HSQC) δ 7.36-7.15 (m, 25H, Harom), 5.09-4.95
(m, 3H, CH2 Bn, H-1), 4.71 (d, J = 11.7 Hz, 1H, CH2 Bn), 4.65-4.55
(m, 4H, CH2 Bn), 4.49 (s, 3H, CH2 Bn, H-5), 4.45 (d, J = 11.7 Hz, 1H,
CH2 Bn), 4.22 (dd, J = 3.1, 1.5 Hz, 1H, H-4), 4.11-4.05 (m, 1H, H-3),
3.88-3.80 (m, 2H, H-2, CH2 Gro), 3.77-3.70 (m, 1H, CH Gro), 3.58
(d, J = 5.0 Hz, 2H, CH2 Gro), 3.53 (dd, J = 10.4, 5.5 Hz, 1H, CH2 Gro),
2.28 (d, J = 4.6 Hz, 1H, OH); 13C NMR (100 MHz, CDCl3, HH-COSY,
HSQC) δ 168.5 (CdO), 138.4, 138.3, 138.1, 138.0, 135.0 (Cq Ph),
128.6, 128.5, 128.4, 128.3, 128.3, 128.2, 127.9, 127.6, 127.5 (CHarom),
97.4 (C-1), 78.0 (C-4), 76.7 (CH Gro), 76.6 (C-2), 74.9 (CH2 Bn), 73.4
(CH2 Bn), 72.6 (CH2 Bn), 71.9 (CH2 Bn), 70.4 (C-5), 69.7 (CH2 Bn,
C-3), 68.4 (CH2 Gro), 66.9 (CH2 COOBn); HRMS [M þ Na]þ calcd
for C44H46O9Na 741.30340, found 741.30352.
foam (690 mg, 933 μmol, 84%): Rf 0.68 (EtOAc/PE, 2/3, v/v); [R]22
D
-38 (c 1.0, CH2Cl2); IR (neat, cm-1) 2112, 1805, 1717, 1514, 1042; 1H
NMR (400 MHz, CDCl3, HH-COSY, HSQC) δ 7.48-7.27 (m, 15H,
Harom), 5.42 (s, 1H, H-1), 5.24-5.12 (m, 2H, H-30, CH2 Cbz), 5.08-
4.96 (m, 3H, CH2 Cbz, NH, H-10), 4.89 (dd, J = 4.9, 1.5 Hz, 1H, H-3),
4.69 (d, J = 11.8 Hz, 1H, CH2 Bn), 4.63-4.54 (m, 2H, CH2 Bn, H-4),
4.30 (d, J = 4.8 Hz, 1H, H-2), 4.27-4.20 (m, 2H, H-50, H-40), 4.11
(s, 1H, H-5), 3.98-3.83 (m, 2H, CH2 ClAc), 3.48 (dd, J = 11.1, 3.8 Hz,
1H, H-20), 1.17 (d, J = 6.4 Hz, 3H, H-60); 13C NMR (100 MHz, CDCl3,
HH-COSY, HSQC) δ 171.7, 166.5 (CdO), 156.6 (CdO Cbz), 136.3,
136.1, 133.2 (Cq Ph), 132.8, 129.0, 128.8, 128.6, 128.4, 128.3, 128.1,
128.0 (CHarom), 97.9 (C-10), 85.7 (C-1), 79.1 (C-3), 78.4 (C-2), 76.6
(C-4), 73.2 (CH2 Bn), 71.6 (C-30), 70.2 (C-5), 67.3 (CH2 Cbz), 65.7
(C-50), 57.1 (C-20), 52.2 (C-40), 40.5 (CH2 ClAc), 16.3 (C-60); HRMS
[M þ Na]þ calcd for C35H35ClN4O10SNa 761.16546, found
761.16560.
1,2,4-Tri-O-benzyl-1-(1,2-di-O-benzyl-sn-glycerol)-D-ga-
lactopyranosiduronic Acid 3,6-Lactone (32). To a mixture of
57 mg of lactone 30 (93 μmol, 1 equiv), 96 μL of benzyl alcohol (933
μmol, 10 equiv), and 9.5 μL of thiophenol (93 μmol, 1 equiv) in 2 mL of
dichloromethane was added 11 μL of SnCl4 (93 μmol, 1 equiv). After
being stirred for 2 days at ambient temperature, the mixture was
neutralized with triethylamine, and 1 mL of satd aq NaHCO3 was
added. After rigorous stirring for 1 hr, the mixture was filtrated through a
plug of Celite, and the solid material was rinsed with dichloromethane.
The filtrate was washed with satd aq NaHCO3 and brine, dried
(MgSO4), filtered, and concentrated under reduced pressure. Purifica-
tion by size-exclusion chromatography (DCM/MeOH 1/1 v/v) and
flash column chromatography (eluent: EtOAc/PE 1/3) gave the title
compound 32 (34 mg, 47 μmol, 51%) as an epimeric mixture: Rf 0.42
(EtOAc/toluene, 1/4, v/v); IR (neat, cm-1) 3420, 2875, 1791, 1454,
4-(N-Benzyloxycarbonylamino)-2-azido-3-O-chloroa-
cetyl-2,4,6-trideoxy-D-galactopyranosyl-(1f4)-2-O-benzyl-
r-D-galactopyranosiduronyl-3,6-lactone-(1f4)-benzyl 2,4-
di-O-benzyl-r-D-galactopyranosyluronate-(1f3)-1,2-di-O-
benzyl-sn-glycerol (34). Lactone 33 (538 mg) was coupled to
alcohol 31 (2 equiv instead of 1.5 equiv) according to the general
procedure for glycosylations using Ph2SO/Tf2O. The reaction was
quenched with triethylamine (5 equiv), and the title compound 34
was obtained in 60% yield (590 mg, 438 μmol) after size-exclusion
chromatography (DCM/MeOH 1/1 v/v): Rf 0.58 (EtOAc/toluene, 1/
3, v/v); [R]22 þ101 (c 1.0, CH2Cl2); IR (neat, cm-1) 2110, 1803,
D
1761, 1720, 1454, 1043; 1H NMR (400 MHz, CDCl3, HH-COSY,
HSQC) δ 7.43-7.01 (m, 35H, Harom), 5.17-5.14 (m, 2H, H-300, CH2
Bn), 5.08-4.94 (m, 6H, CH2 Bn, H-10, H-1, NH), 4.89-4.80 (m, 3H,
H-100, CH2 Bn), 4.73-4.69 (m, 2H, H-40, H-30), 4.66-4.59 (m, 2H,
CH2 Bn), 4.57-4.48 (m, 4H, CH2 Bn), 4.46 (s, 1H, H-5), 4.41-4.33
(m, 3H, H-3, CH2 Bn), 4.25-4.21 (m, 3H, H-4, H-50, H-400), 4.18 (q, J =
6.6 Hz, 1H, H-500), 3.99 (dd, J = 10.1, 3.5 Hz, 1H, H-2), 3.89 (d, J = 4.6
Hz, 2H, CH2 ClAc), 3.88-3.80 (m, 2H, CH2 Gro, H-20), 3.80-3.74 (m,
1H, CH Gro), 3.65-3.57 (m, 3H, CH2 Gro), 3.42 (dd, J = 11.2, 3.8 Hz,
1
1100, 1061, 698; H NMR (400 MHz, CDCl3, HH-COSY, HSQC,
galacturonic acid based numbering, major epimer) δ 7.36-7.10 (m,
25H, Harom), 4.81 (d, J = 7.2 Hz, 1H, H-1), 4.80-4.73 (m, 2H, CH2 Bn),
4.65 (d, J = 2.2 Hz, 2H, CH2 Bn), 4.62-4.57 (m, 2H, CH2 Bn), 4.53 (dd,
J = 6.8, 1.7 Hz, 1H, H-3), 4.49 (s, 2H, CH2 Bn), 4.43 (dd, J = 6.8, 4.5 Hz,
1H, H-5), 4.33 (d, J = 11.7 Hz, 1H, CH2 Bn), 4.28 (d, J = 11.5 Hz, 1H,
CH2 Bn), 4.11 (t, J = 6.8 Hz, 1H, H-4), 3.88-3.81 (m, 1H, CH2 Gro),
3.81-3.74 (m, 2H, CH2 Gro, CH Gro), 3.63-3.56 (m, 3H, H-2, CH2
1702
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The Journal of Organic Chemistry
ARTICLE
1H, H-200), 1.14 (d, J = 6.4 Hz, 3H, H-600); 13C NMR (100 MHz, CDCl3,
HH-COSY, HSQC) δ 170.7, 168.2, 166.5 (CdO), 156.5 (CH2 Cbz),
138.6, 138.5, 138.2, 137.9, 137.1, 136.0, 134.8 (Cq Ph), 128.7, 128.6,
128.5, 128.5, 128.4, 128.3, 128.2, 128.1, 127.9, 127.8, 127.6, 127.5, 127.4,
127.2, 126.9 (CHarom), 98.4 (C-1), 97.7 (C-100), 94.9 (C-10), 80.5 (C-
30), 76.5 (CH Gro), 75.7 (C-4), 75.5 (C-40), 75.4 (C-3), 74.9 (C-20),
74.7 (CH2 Bn), 74.2 (CH2 Bn), 73.9 (C-2), 73.3 (2 CH2 Bn), 71.9 (CH2
Bn), 71.5 (C-300), 71.2 (C-50), 70.1 (C-5), 69.7 (CH2 Gro), 68.6 (CH2
Gro), 67.3 (CH2), 67.1 (CH2), 65.4 (C-500), 56.9 (C-200), 52.1 (C-400),
40.5 (CH2 ClAc), 16.2 (C-600); HRMS [M þ Na]þ calcd for
C73H75ClN4O19Na 1369.46062, found 1369.46158.
(100 MHz, CDCl3, HH-COSY, HSQC) δ 171.5, 168.2 (CdO), 157.7
(CH2 Cbz), 138.4, 138.3, 138.2, 137.8, 137.1, 136.0, 134.8 (Cq Ph),
128.6, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.6, 127.4, 127.3,
127.0, 126.7 (CHarom), 98.4 (C-1), 97.5 (C-100), 94.9 (C-10), 80.9 (C-
30), 76.4 (CH Gro), 75.7 (C-4), 75.6 (C-3), 74.7, 74.6 (C-20, C-40, CH2
Bn), 74.0 (CH2 Bn), 73.7 (C-2), 73.4, 73.1, 71.7 (CH2 Bn), 71.3 (C-
50), 70.0 (C-5), 69.6, 68.4 (CH2 Gro), 67.7 (C-300), 67.0, 66.9 (CH2),
66.1 (C-500), 55.2 (C-400), 50.2 (C-200), 22.9 (CH3 NHAc), 16.4
(C-600); HRMS [M þ Na]þ calcd for C73H78N2O19Na 1309.50910,
found 1309.50986.
2-Acetamido-4-amino-2,4,6-trideoxy-r-D-galactopyrano-
syl-(1f4)-r-D-galactopyranosyluronate-(1f3)-r-D-galac-
topyranosyluronate-(1f3)-sn-glycerol (2). Compound 36 (54
mg, 42 μmol, 1 equiv) was dissolved in 7 mL of t-BuOH/H2O (5/2 v/v)
and stirred under argon atmosphere. A catalytic amount of palladium on
activated charcoal and 105 μL of 1 M aq HCl were added, and the
mixture was allowed to stir for 2 days under hydrogen atmosphere.
Following filtration over Celite and removal of the eluent, the crude
product was allowed to stir in 0.04 M aq HCl for an additional 2 days.
Next, the mixture was concentrated in vacuo and purified by ion-
exchange column chromatography (30 mM NaOAc (aq)/100 mM
NaOH (aq) f 80 mM NaOAc (aq)/100 mM NaOH (aq)) and
filtration (HW-40, 0.15 M Et3NHOAc in H2O) to afford 10 mg of the
4-(N-Benzyloxycarbonylamino)-2-azido-2,4,6-trideoxy-D-
galactopyranosyl-(1f4)-2-O-benzyl-r-D-galactopyrano-
siduronyl-3,6-lactone-(1f4)-benzyl 2,4-di-O-benzyl-r-D-
galactopyranosyluronate-(1f3)-1,2-di-O-benzyl-sn-glycer-
ol (35). A solution of 76 mg of compound 34 (56 μmol, 1 equiv), 36 μL
of pyridine (451 μmol, 8 equiv), and 13 mg (169 μmol, 3 equiv) of
thiourea in 1 mL of EtOH was stirred at 65 °C for 6 h. The mixture was
concentrated under reduced pressure, diluted with EtOAc, and washed
with aq 1 M HCl, satd aq NaHCO3, and brine. The organic phase was
dried (MgSO4), filtered, and concentrated in vacuo. Flash column
chromatography using EtOAc/PE (7/13f9/11) gave 44 mg (34 μmol,
62%) of the title compound 35: Rf 0.48 (EtOAc/PE, 3/7, v/v); [R]22
D
1
þ97 (c 1.0, CH2Cl2); IR (neat, cm-1) 3400, 2874, 2110, 1081, 1717,
1705, 1028; 1H NMR (400 MHz, CDCl3, HH-COSY, HSQC) δ 7.45-
6.99 (m, 35H, Harom), 5.12 (s, 2H, CH2 Bn), 5.08-4.98 (m, 4H, NH,
CH2 Bn, H-10), 4.96 (d, J = 3.6 Hz, 1H, H-1), 4.87-4.78 (m, 3H, CH2,
H-100), 4.71 (dd, J = 4.9, 1.8 Hz, 1H, H-30), 4.69 (s, 1H, H-40), 4.67-4.59
(m, 2H, CH2 Bn), 4.58-4.48 (m, 4H, CH2 Bn), 4.46 (s, 1H, H-5),
4.42-4.32 (m, 3H, CH2 Bn, H-3), 4.23 (s, 2H, H-4, H-50), 4.09 (m, 2H,
H-300, H-500), 4.03-3.96 (m, 2H, H-400, H-2), 3.85 (dd, J = 10.4, 4.5 Hz,
1H, CH2 Gro), 3.81 (dd, J = 4.6, 2.5 Hz, 1H, H-20), 3.79-3.73 (m, 1H,
CH Gro), 3.65-3.55 (m, 3H, CH Gro), 3.14 (dd, J = 10.7, 3.8 Hz, 1H,
H-200), 1.13 (d, J = 6.4 Hz, 3H, H-600); 13C NMR (100 MHz, CDCl3,
HH-COSY, HSQC) δ 171.1, 168.3 (CdO), 158.0 (CH2 Cbz), 138.6,
138.5, 138.2, 137.9, 137.2, 135.7, 134.9 (Cq Ph), 128.7, 128.6, 128.5,
128.5, 128.5, 128.4, 128.3, 128.2, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5,
127.4, 127.40, 127.2, 126.9 (CHarom), 98.5 (C-1), 98.4 (C-100), 94.9
(C-10), 80.6 (C-30), 76.6 (CH Gro), 75.9 (C-40), 75.8 (C-4), 75.5 (C-3),
74.9 (C-20), 74.7, 74.2 (CH2 Bn), 73.9 (C-2), 73.4, 73.3, 71.9 (CH2 Bn),
71.4 (C-50), 70.1 (C-5), 69.7, 68.6 (CH2 Gro), 68.4 (C-300), 67.6, 67.1
(CH2), 65.8 (C-500), 59.9 (C-200), 55.5 (C-400), 16.4 (C-600); HRMS [M
þ Na]þ calcd for C71H74N4O18Na 1293.48903, found 1293.48964.
4-(N-Benzyloxycarbonylamino)-2-acetamido-2,4,6-tri-
deoxy-D-galactopyranosyl-(1f4)-2-O-benzyl-r-D-galac-
topyranosiduronyl-3,6-lactone-(1f4)-benzyl 2,4-di-O-
benzyl-r-D-galactopyranosyluronate-(1f3)-1,2-di-O-
benzyl-sn-glycerol (36). To an ice-cooled solution of 281 mg
of azide 35 (221 μmol) in 5 mL of pyridine was added 5 mL of freshly
distilled thiolacetic acid. The mixture was stirred at room temperature
for 2.5 h, concentrated under reduced pressure, and coevaporated with
toluene. Flash column chromatography using EtOAc/PE (7/3f1/0)
afforded the title acetamide (188 mg, 146 μmol, 66%): Rf 0.30 (EtOAc/
PE, 3/1, v/v); [R]22D þ83 (c 0.8, CH2Cl2); IR (neat, cm-1) 3300, 2924,
1801, 1759, 1717, 1661, 1540, 1028; 1H NMR (400 MHz, CDCl3, HH-
COSY, HSQC) δ 7.51-6.91 (m, 35H, Harom), 5.67 (d, J = 8.4 Hz, 1H,
NH), 5.40 (d, J = 9.7 Hz, 1H, NH), 5.18-4.99 (m, 5H, CH2 Bn, H-10),
4.95 (d, J = 3.5 Hz, 1H, H-1), 4.91 (d, J = 3.7 Hz, 1H, H-100), 4.84-
4.80 (m, 2H, CH2 Bn), 4.69 (dd, J = 4.9, 1.5 Hz, 1H, H-30), 4.66 (s,
1H, H-40), 4.62 (m, 2H, CH2 Bn), 4.59-4.48 (m, 4H, CH2 Bn), 4.45
(s, 1H, H-5), 4.42-4.31 (m, 3H, CH2 Bn, H-3), 4.22 (s, 1H, H-4), 4.12
(s, 1H, H-50), 4.06-3.96 (m, 4H, H-200, H-400, H-500, H-2), 3.86-3.73
(m, 4H, CH2 Gro, H-300, H-20, CH Gro), 3.65-3.56 (m, 3H, CH2 Gro),
1.92 (s, 3H, CH3 NHAc), 1.12 (d, J = 6.2 Hz, 3H, H-600); 13C NMR
pure title compound 2 (16 μmol, 38%) after lyophilization: H NMR
(600 MHz, D2O, HH-COSY, HSQC, HMBC, TOCSY, T = 293 K) δ
5.32 (d, J = 3.8 Hz, 1H, H-10), 5.08 (d, J = 3.8 Hz, 1H, H-1), 5.03 (d, J =
3.8 Hz, 1H, H-100), 4.85 (q, J = 6.5 Hz, 1H, H-500), 4.68 (s, 1H, H-50),
4.60 (d, J = 1.9 Hz, 1H, H-4), 4.44 (d, J = 2.4 Hz, 1H, H-40), 4.39 (s, 1H,
H-5), 4.27 (dd, J = 11.3, 4.3 Hz, 1H, H-300), 4.21-4.18 (m, 2H, H-30,
H-3), 4.09 (dd, J = 11.3, 3.8 Hz, 1H, H-200), 4.04-3.95 (m, 3H, H-2, CH
Gro, H-20), 3.88 (dd, J = 10.6, 3.6 Hz, 1H, CH2 Gro), 3.74 (dd, J = 11.8,
4.5 Hz, 1H, CH2 Gro), 3.70 (d, J = 3.7 Hz, 1H, H-400), 3.66 (dd, J = 11.8,
6.2 Hz, 1H, CH2 Gro), 3.57 (dd, J = 10.5, 7.1 Hz, 1H, CH2 Gro), 2.17 (s,
3H, CH3 NHAc), 1.33 (d, J = 6.7 Hz, 3H); 13C NMR (151 MHz, D2O,
HH-COSY, HSQC, HMBC, TOCSY, T = 293 K) δ 176.1, 176.0, 175.3
(CdO), 99.7 (C-100, C-1), 96.9 (C-10), 81.0 (C-40), 76.2 (C-3), 72.1,
72.0 (C-5, C-50), 71.5 (CH Gro), 70.0 (CH2 Gro), 69.4 (C-30), 68.8
(C-20), 68.5 (C-4), 67.5 (C-2), 65.5 (C-300), 64.2 (C-5), 63.2 (CH2
Gro), 56.2 (C-400), 50.2 (C-200), 23.2 (CH3 NHAc), 16.3 (C-600); 13C-
00
00
HMBC NMR (151 MHz, D2O, T = 293 K) δ 99.7 (JC1 -H1 = 173.31
Hz, JC1-H1 = 170.6 Hz, C-100, C-1), 96.9 (JC1 -H1 = 170.3 Hz,
C-10); HRMS [M þ H]þ calcd for C23H39N2O18 631.21924, found
631.21928.
0
0
(1,3-Oxazolidino-2-one)[5,4-c]-2-acetamido-4-amino-2,4,6-
trideoxy-r-D-galactopyranosyl-(1f4)-r-D-galactopyrano-
syluronate-(1f3)-r-D-galactopyranosyluronate-(1f3)-sn-
glycerol (37). To a solution of 40.0 mg (31 μmol, 1 equiv) of
compound 36 in 1.5 mL of DCM was added 14 mg of TMSONa (124
μmol, 4.0 equiv). The mixture was stirred for 1 h, followed by
evaporation of the solvent and elution of the crude product over a
plug of silica (eluent: EtOAc, then EtOAc/MeOH/H2O/AcOH 93/5/
1/1, then EtOAc/MeOH/H2O/AcOH 88/10/1/1). After removal of
the eluent, the crude product was dissolved in 5 mL of t-BuOH/H2O
(4/1 v/v) and stirred under argon atmosphere. A catalytic amount of
palladium on activated charcoal and 190 μL of 1 M aq HCl were added,
and the mixture was allowed to stir overnight under hydrogen atmo-
sphere. Filtration over Celite, followed by gel filtration (HW-40,
0.15 M Et3NHOAc in H2O) and subsequent lyophilization, afforded
8.95 mg of the title compound 37 (11.8 μmol, 38% over two steps): 1H
NMR (600 MHz, D2O, HH-COSY, HSQC, T = 306 K) δ 5.22 (d, J =
3.8 Hz, 1H, H-10), 4.99 (d, J = 3.8 Hz, 1H, H-1), 4.95 (d, J = 3.8 Hz, 1H,
H-100), 4.79 (dd, J = 9.0, 7.2 Hz, 1H, H-300), 4.70-4.61 (m, 2H, H-50,
H-500), 4.52 (d, J = 2.0 Hz, 1H, H-4), 4.40-4.33 (m, 2H, H-5, H-40),
4.16-4.06 (m, 4H, H-400, H-30, H-200, H-3), 3.94-3.86 (m, 3H, H-2,
1703
dx.doi.org/10.1021/jo102363d |J. Org. Chem. 2011, 76, 1692–1706

The Journal of Organic Chemistry
ARTICLE
CH Gro, H-20), 3.78 (dd, J = 10.5, 3.7 Hz, 1H, CH2 Gro), 3.64 (dd, J =
11.8, 4.6 Hz, 1H, CH2 Gro), 3.56 (dd, J = 11.8, 6.2 Hz, 1H, CH2 Gro),
3.49 (dd, J = 10.6, 7.0 Hz, 1H, CH2 Gro), 3.19 (q, J = 7.3 Hz, 10H, CH2
Et3NHOAc), 2.07 (s, 3H, CH3 NHAc), 1.28-1.24 (m, 18H, H-600,
CH3 Et3NHOAc); 13C NMR (151 MHz, D2O, HH-COSY, HSQC,
T = 306 K) δ 175.5, 175.3, 174.8, 162.7 (CdO), 99.8 (C-1), 99.2
(C-100), 96.9 (C-10), 80.9 (C-40), 76.7 (C-300), 76.1 (C-3), 71.9, 71.8
(C-5, C-50), 71.6 (CH Gro), 70.1 (CH2 Gro), 69.5 (C-30), 68.8 (C-20),
68.4 (C-4), 67.5 (C-2), 63.3 (CH2 Gro), 63.1 (C-500), 56.9 (C-400),
50.7 (C-200), 47.6 (CH2 Et3NHOAc), 23.2 (CH3 NHAc), 17.0 (C-600),
H-3, H-30), 3.97-3.90 (m, 2H, H-20, H-5), 3.81-3.70 (m, 2H, CH2
Gro, CH Gro), 3.61-3.53 (m, 3H, CH2 Gro), 3.30 (dd, J = 10.5, 3.9
Hz, 1H, H-2), 2.18 (d, J = 4.0 Hz, 1H, OH), 1.03 (d, J = 6.4 Hz, 3H,
H-6); 13C NMR (100 MHz, CDCl3, HH-COSY, HSQC) δ 168.1
(CdO COOBn), 156.8 (CdO Cbz), 138.3, 138.1, 138.0, 137.8,
135.9, 134.9 (Cq Ph), 128.4, 128.3, 128.1, 128.0, 127.9, 127.8,
127.6, 127.4 (CHarom), 97.7 (C-1), 92.3 (C-10), 77.6 (C-40), 76.8
(CH Gro), 74.8 (CH2 Bn), 74.8 (C-20), 73.3 (CH2 Bn), 72.1, 72.0
(CH2 Bn), 70.6 (C-50), 69.5 (C-3), 69.3 (CH2 Gro), 69.1 (C-30),
67.9 (CH2 Gro), 66.9, 66.9 (CH2 Cbz, CH2 COOBn), 65.0 (C-5),
59.2 (C-2), 50.8 (C-4), 16.2 (C-6); HRMS [M þ Na]þ calcd for
C58H62N4O13Na 1045.42056, found 1045.42095.
2,4-Di-O-benzyl-r/β-D-galactopyranosiduronyl-3,6-lact-
one-(1f3)-benzyl 2,4-Di-O-benzyl-r-D-galactopyranosy-
luronate-(1f3)-4-(N-benzyloxycarbonylamino)-2-azido-
2,4,6-trideoxy-r-D-galactopyranosyl-(1f3)-1,2-di-O-ben-
zyl-sn-glycerol (40). Lactone 4 (62 mg, 139 μmol) was coupled to
95 mg of alcohol 39 (93 μmol, 0.67 equiv) according to the general
procedure for glycosidations using Ph2SO/Tf2O, yielding 99 mg of the
title epimers (73 μmol, 78%, R/β 5:4) after size-exclusion chromatog-
raphy (DCM/MeOH 1/1 v/v) and flash column chromatography
(eluent: EtOAc/PE 1/4 f 3/7): Rf 0.42 (EtOAc/PE, 2/3, v/v); IR
(neat, cm-1) 2870, 2106, 1801, 1761, 1717, 1497, 1454, 1028, 696; 1H
NMR (400 MHz, CDCl3, HH-COSY, HSQC) δ 7.42-6.81 (m, 72H,
Harom), 5.67 (d, J = 2.9 Hz, 0.8H, H-10β), 5.49 (d, J = 2.5 Hz, 1H, H-10R),
5.45 (s, 0.8H, H-100β), 5.14-4.58 (m, 22H), 4.57-4.35 (m, 14.4H),
4.35-4.00 (m, 12.4H), 4.00-3.84 (m, 3.4H), 3.85-3.69 (m, 4.6H),
3.58-3.56 (m, 5.4H), 3.37 (dd, J = 10.6, 3.9 Hz, 0.8H, H-2β), 3.31 (dd,
J = 10.6, 3.9 Hz, 1H, H-2R), 1.03-0.98 (m, 5.4H, H-6R, H-6β); 13C
NMR (100 MHz, CDCl3, HH-COSY, HSQC) δ 173.5, 171.6, 168.0,
167.9, 156.7, 139.3, 138.5, 138.3, 138.1, 137.8, 137.3, 136.9, 136.8, 136.7,
136.2, 136.0, 135.1, 134.8, 128.8, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1,
128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.2, 127.1, 126.9, 99.9
(C-100β), 97.7 (C-1R, C-1β), 95.2 (C-100R), 93.4 (C-10R), 91.9 (C-10β),
80.1, 79.0, 78.5, 77.2, 76.9, 76.4, 75.9, 75.8, 75.7, 75.4, 75.1, 74.7, 74.3,
74.0, 73.4, 73.0, 72.7, 72.4, 72.2, 72.1, 71.7, 71.4, 71.3, 71.2, 70.6, 70.3,
69.8 (C-3β), 69.6 (C-3R), 69.5, 68.1, 68.0, 67.1, 66.8, 66.7, 66.6, 65.1
(C-5β, C-5R), 59.5 (C-2β), 59.1 (C-2R), 50.8 (C-4R), 50.7 (C-4β),
16.3 (C-6R), 16.2 (C-6β); HRMS [M þ Na]þ calcd for C78H80N4O18-
Na 1383.53598, found 1383.53760.
9.2 (CH3 Et3NHOAc); 13C-HMBC NMR (151 MHz, D2O, T = 306
00
00
00
K) δ 99.9 (JC1-H1 = 170.9 Hz, C-1), 98.8 (JC1 -H1 = 172.5 Hz, C-1 ),
þ
97.1 (JC1 -H1 = 172.0 Hz, C-10); HRMS [M þ H] calcd for
0
0
C24H37N2O19 657.19850, found 657.19866.
2,4-Di-O-benzyl-r-D-galactopyranosiduronyl-3,6-lactone-
(1f3)-4-(N-benzyloxycarbonylamino)-2-azido-2,4,6-trideo-
xy-r-D-galactopyranosyl-(1f3)-1,2-di-O-benzyl-sn-glycerol
(38). Lactone 4 (257 mg) was coupled to 220 mg of alcohol 20 (382
μmol, 0.66 equiv) according to the general procedure for glycosidations
using Ph2SO/Tf2O, yielding 244 mg of the title compound 38 (267
μmol, 70%) after size-exclusion chromatography (DCM/MeOH 1/1
v/v) and flash column chromatography (eluent: EtOAc/PE 1/3 f
3/7): Rf 0.68 (EtOAc/toluene, 1/4, v/v); [R]22D þ106 (c 1.0, CH2Cl2);
1
IR (neat, cm-1) 2872, 2110, 1798, 1717, 1454, 1026, 696; H NMR
(400 MHz, CDCl3, HH-COSY, HSQC); δ 7.55-7.11 (m, 25H, Harom),
5.14 (d, J = 2.3 Hz, 1H, H-10), 5.13-4.99 (m, 3H, NH, CH2 Cbz), 4.82
(d, J = 3.8 Hz, 1H, H-1), 4.79 (d, J = 11.7 Hz, 1H, CH2 Bn), 4.73-4.61
(m, 3H, CH2 Bn, H-30), 4.58-4.47 (m, 5H, CH2 Bn, H-40), 4.29-4.17
(m, 3H, CH2 Bn, H-50, H-3), 4.10 (dd, J = 5.0, 2.3 Hz, 1H, H-20), 4.01
(dd, J = 9.6, 2.9 Hz, 1H, H-4), 3.94 (q, J = 6.1 Hz, 1H, H-5), 3.82-3.73
(m, 2H, CH Gro, CH2 Gro), 3.62 (d, J = 4.5 Hz, 2H, CH2 Gro), 3.60-
3.53 (m, 1H, CH2 Gro), 3.15 (dd, J = 10.8, 3.8 Hz, 1H, H-2), 1.05 (d, J =
6.4 Hz, 3H, H-6); 13C NMR (100 MHz, CDCl3, HH-COSY, HSQC) δ
171.9 (CdO), 156.6 (CdO Cbz), 138.3, 138.0, 136.8, 135.8 (Cq Ph),
128.7, 128.5, 128.5, 128.3, 128.0, 127.7, 127.6, 127.4 (CHarom), 98.2
(C-1), 95.6 (C-10), 80.4 (C-30), 76.7 (CH Gro), 75.8 (C-40), 75.1
(C-20), 74.3 (CH2 Bn), 73.5 (C-3), 73.4 (CH2 Bn), 72.0 (CH2 Bn,
C-50), 71.4 (CH2 Bn), 69.3 (CH2 Bn), 67.9 (CH2 Bn), 67.0 (CH2 Cbz),
64.2 (C-5), 58.3 (C-2), 51.8 (C-4), 16.4 (C-6); HRMS [M þ Na]þ
calcd for C51H54N4O12Na 937.36304, found 937.36340.
2,4-Di-O-benzyl-r/β-D-galactopyranosiduronyl-3,6-lacto-
ne-(1f3)-benzyl 2,4-Di-O-benzyl-r-D-galactopyranosyluro-
nate-(1f3)-2-acetamido-4-(N-benzyloxycarbonylamino)-
2,4,6-trideoxy-r-D-galactopyranosyl-(1f3)-1,2-di-O-ben-
zyl-sn-glycerol (41). To an ice-cooled solution of 61 mg of epimeric
azides 40 (45 μmol) in 1.5 mL of pyridine was added 1.5 mL of freshly
distilled thiolacetic acid. The mixture was stirred at room temperature
for 3 h, concentrated under reduced pressure, and coevaporated with
toluene. Flash column chromatography using EtOAc/PE (2/3) afforded
title epimers 41 (25 mg, 18 μmol, R/β 1:1, 40%): Rf 0.67 (EtOAc/PE,
3/2, v/v); IR (neat, cm-1) 2930, 1802, 1718, 1668, 1497, 1454, 1027,
Benzyl 2,4-Di-O-benzyl-r-D-galactopyranosylurona-
te-(1f3)-4-(N-benzyloxycarbonylamino)-2-azido-2,4,6-tri-
deoxy-r-D-galactopyranosyl-(1f3)-1,2-di-O-benzyl-sn-
glycerol (39). To a solution of 88 mg of lactone 38 (96 μmmol, 1
equiv) in 2 mL of DCM was added 13 mg of TMSONa (115 μmol,
1.2 equiv). After 50 min, TLC indicated complete consumption of
the starting material, and the reaction was quenched with 28 μL of
AcOH (481 μmol, 5.0 equiv), after which the mixture was coeva-
porated with toluene and subsequently dissolved in 2 mL of DMF.
Next, 17 μL of BnBr (144 μmol, 1.5 equiv) and 39 mg of Cs2CO3
(120 μmol, 1.25 equiv) were added, and the reaction was stirred
until TLC analysis showed complete consumption of the starting
material. The mixture was diluted with EtOAc and washed with
H2O and brine. Drying over MgSO4, filtration, and concentration
under reduced pressure gave the crude product. Purification by
flash column chromatography using EtOAc/toluene (1/4) gave
the title compound 39 in pure form (86 mg, 84 μmol, 88% over two
1
731, 695; H NMR (400 MHz, CDCl3, HH-COSY, HSQC) δ 7.48-
6.84 (m, 80H, Harom), 5.73 (dd, J = 15.8, 9.2 Hz, 2H, NH), 5.45 (s, 1H,
H-100β), 5.29 (d, J = 2.9 Hz, 1H, H-10R or H-10β), 5.22-4.99 (m, 8H),
4.95 (d, J = 12.4 Hz, 1H), 4.87 (m, 2H), 4.79 (d, J = 12.6 Hz, 1H), 4.75-
4.26 (m, 34H), 4.26-4.07 (m, 7H), 4.05-3.65 (m, 13H), 3.62-3.39
(m, 6H), 1.72 (s, 3H, CH3 NHAc), 1.71 (s, 3H, CH3 NHAc), 1.06 (m,
6H, H-6R, H-6β); 13C NMR (100 MHz, CDCl3, HH-COSY, HSQC) δ
173.7, 171.7, 170.2, 170.1, 168.4, 168.2, 156.8, 139.3, 138.7, 138.2, 138.1,
138.0, 137.9, 137.8, 137.4, 136.9, 136.8, 136.4, 135.7, 135.0, 134.8, 128.8,
128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 127.9, 127.8, 127.7, 127.6, 127.5,
127.4, 127.3, 127.0, 126.9, 99.8 (C-100β), 98.1 (C-1R or C1-β), 97.9
(C-1R or C1-β), 97.6 (C-10R or C10-β), 95.5 (C-100R), 95.4 (C-10R or
C10-β), 80.0, 79.2, 77.3, 77.1, 76.8, 76.6, 76.0, 75.7, 75.5, 75.4, 75.0, 74.5,
steps): Rf 0.46 (EtOAc/toluene, 1/3, v/v); [R]22 þ122 (c 1.0,
D
CH2Cl2); IR (neat, cm-1) 2880, 2104, 1761, 1719, 1094, 1026,
1
696; H NMR (400 MHz, CDCl3, HH-COSY, HSQC) δ 7.37-
7.07 (m, 30H, Harom), 5.59 (d, J = 3.0 Hz, 1H, H-10), 5.11-4.96 (m,
4H, NH, CH2 Bn), 4.88 (d, J = 3.8 Hz, 1H, H1), 4.75 (d, J = 12.2 Hz,
1H, CH2 Bn), 4.70-4.58 (m, 5H, H-50, CH2 Bn), 4.55-4.45 (m,
2H, CH2 Bn), 4.40 (m, 2H, CH2 Bn), 4.28-4.20 (m, 4H, H-40, H-4,
1704
dx.doi.org/10.1021/jo102363d |J. Org. Chem. 2011, 76, 1692–1706

The Journal of Organic Chemistry
ARTICLE
74.2, 73.8, 73.7, 73.6, 73.5, 73.1, 72.4, 72.0, 71.9, 71.7, 71.6, 71.4, 71.2,
71.0, 70.6, 69.4, 69.3, 68.2, 67.1, 66.8, 66.5, 66.4, 65.7 (C-5R or C-5β),
65.5 (C-5R or C-5β), 52.5 (C-4R or C-4β), 52.1 (C-4R or C-4β), 48.8
(C-2R or C-2β), 48.5 (C-2R or C-2β), 22.9 (2 ꢀ CH3 NHAc), 16.5 (C-
6R, C-6β); HRMS [M þ Na]þ calcd for C80H84N2O19Na 1399.55605
C29H35N3O7S, found 1399.55602.
C-10), 98.3 (JC1-H1 = 173.5 Hz, C-1); HRMS [M þ H]þ calcd for
C23H39N2O18 631.21924, found 631.21923.
’ ASSOCIATED CONTENT
S
Supporting Information. Spectroscopic data of the re-
b
r-D-Galactopyranosyluronate-(1f3)-r-D-galactopyrano-
syluronate-(1f3)-2-acetamido-4-amino-2,4,6-trideoxy-r-
D-galactopyranosyl-(1f3)-sn-glycerol (3a) and β-D-Galacto-
pyranosyluronate-(1f3)-R-D-galactopyranosyluronate-(1f3)-
2-acetamido-4-amino-2,4,6-trideoxy-R-D-galactopyranosyl-
(1f3)-sn-glycerol (3b). To a solution of 35 mg (25 μmol, 1 equiv)
of epimeric mixture 41 in 1.5 mL of DCM was added 11.4 mg of
TMSONa (102 μmol, 4 equiv). The mixture was stirred for 1 h, followed
by evaporation and elution over a plug of silica (eluent: EtOAc, then
EtOAc/MeOH/H2O/AcOH 88/10/1/1). After removal of the eluent,
the crude product was dissolved in t-BuOH/H2O (4/1 v/v) and stirred
under argon atmosphere. A catalytic amount of palladium on activated
charcoal and 170 μL of 1 M aq HCl were added, and the mixture was
allowed to stir for 2 days under hydrogen atmosphere. Filtration over
Celite, gel filtration (HW-40, 0.15 M Et3NHOAc in H2O), and ion-
exchange column chromatography (30 mM NaOAc (aq)/100 mM
NaOH (aq) f 80 mM NaOAc (aq)/100 mM NaOH (aq)) yielded
two fractions that were both subjected to another gel filtration step
(HW-40, 0.15 M Et3NHOAc in H2O) to give the two title epimers in
pure form after lyophilization (R-epimer 3a: 4.4 mg, 7.0 μmol 28% over
two steps, β-epimer 3b: 2.7 mg, 4.3 μmol, 17% over two steps). R-
ported compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: marel_g@chem.leidenuniv.nl; jcodee@chem.leidenuniv.nl.
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1
Epimer 3a: H NMR (600 MHz, D2O, HH-COSY, HSQC, HMBC,
TOCSY, T= 298 K) δ 5.17 (d, J = 3.1 Hz, 1H, H-100), 5.07 (d, J = 2.6 Hz,
1H, H-10), 4.89 (d, J = 3.8 Hz, 1H, H-1), 4.66 (s, 1H, H-50), 4.46 (s, 1H,
H-40), 4.35 (q, J = 6.4 Hz, 1H, H-5), 4.31-4.25 (m, 2H, H-400, H-3),
4.19-4.13 (m, 2H, H-2, H-500), 3.99-3.93 (m, 3H, H-30, H-300, H-20),
3.93-3.89 (m, 1H, CH Gro), 3.87 (dd, J = 10.5, 3.2 Hz, 1H, H-200), 3.82
(d, J = 4.0 Hz, 1H, H-4), 3.77 (dd, J = 10.6, 3.5 Hz, 1H, CH2 Gro), 3.65
(dd, J = 11.7, 4.7 Hz, 1H, CH2 Gro), 3.58 (dd, J = 11.7, 6.2 Hz, 1H, CH2
Gro), 3.46 (dd, J = 10.5, 6.6 Hz, 1H, CH2 Gro), 3.18 (q, J = 7.3 Hz, 1.2H,
CH2 Et3NHOAc), 1.97 (s, 3H, CH3 NHAc), 1.29 (d, J = 6.7 Hz, 3H,
H-6), 1.26 (t, J = 7.3 Hz, 1.9H, CH3 Et3NHOAc); 13C NMR (151 MHz,
D2O, HH-COSY, HSQC, HMBC, TOCSY, T= 298 K) δ 176.3, 175.5,
175.4 (CdO), 99.7 (C-10), 98.3 (C-1), 97.4 (C-100), 76.3 (C-30),
73.8(C-3), 73.0 (C-500), 72.6 (C-5), 71.6 (C-400), 71.4 (CH Gro),
70.4 (C-300), 70.0 (CH2 Gro), 68.8 (C-200), 68.5 (C-40), 66.7 (C-20),
63.4 (C-5), 63.2 (CH2 Gro), 53.7 (C-4), 48.7 (C-2), 47.6 (CH2
Et3NHOAc), 22.7 (CH3 NHAc), 16.5 (C-6), 9.2 (CH3 Et3NHOAc);
13C-HMBC NMR (151 MHz, D2O, T = 298 K) δ 99.7 (JC1 -H1 = 170.3
0
0
Hz, C-10), 98.3 (JC1-H1 = 173.6 Hz, C-1), 97.4 (JC1 -H1 = 170.3 Hz,C-
100); HRMS [M þ H]þ calcd for C23H39N2O18 631.21924, found
631.21927. β-Epimer 3b: 1H NMR (600 MHz, D2O, HH-COSY,
HSQC, HMBC, TOCSY, T= 298 K) δ 5.09 (d, J = 3.6 Hz, 1H, H-10),
4.90 (d, J = 3.8 Hz, 1H, H-1), 4.61 (d, J = 7.8 Hz, 1H, H-100), 4.58 (s, 1H,
H-40), 4.36 (q, J = 6.5 Hz, 1H, H-5), 4.29 (dd, J = 11.3, 4.4 Hz, 1H, H-3),
4.20-4.15 (m, 2H, H-400, H-2), 4.14 (s, 1H, H-50), 4.06 (s, 1H, H-500),
4.05-3.98 (m, 2H, H-20, H-30), 3.91 (dd, J = 9.6, 5.3 Hz, 1H, CH Gro),
3.84 (d, J = 3.7 Hz, 1H, H-4), 3.77 (dd, J = 10.6, 3.6 Hz, 1H, CH2 Gro),
3.70 (dd, J = 10.0, 3.5 Hz, 1H, H-300), 3.65 (dd, J = 11.7, 4.7 Hz, 1H, CH2
Gro), 3.63-3.56 (m, 2H, CH2 Gro, H-200), 3.46 (dd, J = 10.6, 6.6 Hz,
1H, CH2 Gro), 1.97 (s, 3H, CH3 NHAc), 1.89 (s, 1H, CH3 AcOH), 1.31
(t, J = 6.7 Hz, 3H, H-6); 13C NMR (151 MHz, D2O, HH-COSY, HSQC,
HMBC, TOCSY, T= 298 K) δ 176.1, 176.0, 175.5 (CdO), 104.7
(C-100), 99.0 (C-10), 98.3 (C-1), 80.3 (C-30), 76.5 (C-500), 73.6 (C-300),
73.3 (C-3), 73.2 (C-50), 71.5 (C-200), 71.4 (CH Gro), 71.2 (C-400), 70.7
(C-40), 70.0 (CH Gro), 67.5 (C-20), 63.2 (C-5), 53.7 (C-4), 48.7 (C-2),
00
00
22.7 (CH3 NHAc), 16.5 (C-6); 13C-HMBC NMR (151 MHz, D2O, T =
(15) Wu, X. Y.; Cui, L. N.; Lipinski, T.; Bundle, D. R. Chem.—Eur. J.
2010, 16, 3476–3488.
00
00
00
0
0
298 K) δ 104.7 (JC1 -H1 = 161.5 Hz, C-1 ), 99.0 (JC1 -H1 = 170.0 Hz,
1705
dx.doi.org/10.1021/jo102363d |J. Org. Chem. 2011, 76, 1692–1706

The Journal of Organic Chemistry
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