Total synthesis of the escherichia coli O111 O-specific polysaccharide repeating unit

Article abstract of DOI:10.1002/chem.201204394

The first total synthesis of the O-antigen pentasaccharide repeating unit from Gram-negative bacteria Escherichia coli O111 was achieved starting from four monosaccharide building blocks. Key to the synthetic approach was a bis-glycosylation reaction to combine trisaccharide 10 and colitose 5. The colitose building block (5) was obtained de novo from non-carbohydrate precursors. The pentasaccharide was equipped at the reducing end with an amino spacer to provide a handle for subsequent conjugation to a carrier protein in anticipation of immunological studies. Killing them sweetly: As a first step towards diagnostics and vaccines to detect and prevent serious infections with Escherichia coli bacteria, the pentasaccharide repeating unit from the O-antigen of E. coli O111 was synthesized. Copyright

Full text of DOI:10.1002/chem.201204394

FULL PAPER
DOI: 10.1002/chem.201204394
Total Synthesis of the Escherichia coli O111 O-Specific Polysaccharide
Repeating Unit
Oliviana Calin, Steffen Eller, Heung Sik Hahm, and Peter H. Seeberger*[a]
Abstract: The first total synthesis of the O-antigen pentasaccharide repeating unit
from Gram-negative bacteria Escherichia coli O111 was achieved starting from
four monosaccharide building blocks. Key to the synthetic approach was a bis-gly-
cosylation reaction to combine trisaccharide 10 and colitose 5. The colitose build-
ing block (5) was obtained de novo from non-carbohydrate precursors. The penta-
saccharide was equipped at the reducing end with an amino spacer to provide a
handle for subsequent conjugation to a carrier protein in anticipation of immuno-
logical studies.
Keywords:
carbohydrates · Escherichia coli ·
glycosylation · vaccines
antigens
·
Introduction
E. coli O111 would circumvent this multidrug-resistance
problem. Serum antibodies to the O-specific polysaccharide
of O111 lipopolysaccharide protect mice and dogs against
infection with this pathogen type, which suggests that the in-
duction of antibodies by a carbohydrate-conjugate vaccine
would be protective.[4] The structure of the O-specific poly-
saccharide of O111 E. coli contains a pentasaccharide re-
peating unit with two rare colitose (3,6-dideoxy-l-xylo-hexo-
pyranose) residues as a distinguishing feature (Figure 1).[5]
The O-specific polysaccharide from E. coli O111 is also
found on other enteric pathogens, such as Salmonella ade-
laide[6] and Salmonella enterica O35.[7]
The bacterial contamination of food can result in great con-
cern in the general population at a time when safe food is
often taken for granted in developed countries. Infections
caused by Escherichia coli bacteria are rather common, but
have received increased attention in recent years owing to
the emergence of new strains. A new pathogenic strain of
the bacteria Escherichia coli, serotype O111, has emerged as
a significant cause for enteropathogenic, enterotoxigenic,
and enterohemorrhagic diseases in humans. For example,
enteropathogenic E. coli O111 (EPEC) causes diarrhea in
children, particularly in the developing world. Enterotoxi-
genic E. coli (ETEC) O111 is responsible for watery diar-
rhea in infants and is associated with heat-labile (cholera-
like) toxin, whilst enterohemorrhagic E. coli (EHEC) O111,
which generally carries at least one Shinga-toxin gene, is
one of the most common non-O157 causes of bloody diar-
rhea and hemolytic-uremic syndrome in developed coun-
tries.[1] The largest outbreak of Shinga-toxin-producing
E. coli O111 in U.S. history occurred in 2008 and affected
341 persons of all age groups. One patient out of the 70 that
were hospitalized died.[2]
Figure 1. Pentasaccharide repeating unit in the O-specific polysaccharide
of E. coli O111.
The problem with antibiotic resistance of EHEC O111 is
evident due to their excessive use in the livestock industry.[3]
The prevention of bacterial diseases by vaccination against
Pure pentasaccharide 1 is required to evaluate the immu-
nological properties of the O-specific polysaccharide of
E. coli O111 and to explore the potential of an oligosacchar-
ide-conjugate vaccine (see Scheme 1). This pentasaccharide
constitutes a significant synthetic challenge, because it con-
tains two colitose units that are naturally not accessible and
are labile in the context of an oligosaccharide. Herein, we
report the first total synthesis of a pentasaccharide repeating
unit that contains an aminopentanol handle at its reducing
end for attachment to any surface or carrier proteins.
[a] O. Calin, Dr. S. Eller, H. S. Hahm, Prof. Dr. P. H. Seeberger
Department of Biomolecular Systems
Max Planck Institute of Colloids and Interfaces
Am Mꢀhlenberg 1, 14476 Potsdam (Germany)
and Institute for Chemistry and Biochemistry
Freie Universitꢁt Berlin
Arnimallee 22, 14195 Berlin (Germany)
Fax : (+49)331-567-9302
E-mail: peter.seeberger@mpikg.mpg.de
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201204394.
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Results and Discussion
Synthesis of the building blocks: Most of the building blocks
that are needed for the synthesis of pentasaccharide repeat-
ing unit 1 can be accessed by using modified literature pro-
cedures. The synthesis of colitose building block 5 benefits
from a de novo synthesis that is more efficient than known
procedures.[10] Fully functionalized l-colitose glycosylating
agent 5 was synthesized in 10 steps, starting from commer-
cially available (S)-ethyl lactate, which cements the C5 posi-
tion of l-colitose (Scheme 2).[11] Protection of the alcohol
group on (S)-ethyl lactate as a 2-naphthylmethyl (Nap)
ether, followed by reduction of the ester moiety with
DIBAL, afforded aldehyde 12 in 77% yield over two steps.
Cram chelated allylation[12] of aldehyde 12 stereoselectively
provided homoallylic alcohol 14 in 91% yield. Upjohn oxi-
dation[13] of alkene 14 resulted in a 1:1 diastereoisomeric
mixture affording triol 15 in 49% yield. Having set and de-
fined all of the necessary stereocenters in triol 15, the syn-
thesis of l-colitose 5 was completed. Strategically, a building
block with a non-participating benzyl (Bn) ether protecting
group at C2 position was targeted, as l-colitose is found in
nature only bearing a-linkages to other sugars. Furthermore,
an electron-withdrawing benzoyl (Bz) protecting group was
to be placed at the C4 position to stabilize the electron-rich
deoxysugar building block. Thus, triol 15 was converted se-
lectively into 5-membered benzylidene acetal 16, which was
Initial attempt to synthesize pentasaccharide 1: Pentasac-
charide 1 has several synthetic challenges, the most promi-
nent of which is the a-linkages of the colitoses. Our retro-
synthetic analysis of pentasaccharide 1 revealed a conver-
gent [2+3] approach to be attractive (Scheme 1). The reduc-
ing end aminopentanol linker will allow for straightforward
conjugation. Target structure 1 may be obtained from fully
protected pentasaccharide 2 through a three-step deprotec-
tion sequence, including conversion of the trichlo-
ACHTUNGTRENNUNGroacetamide into the corresponding acetamide, cleavage of
the ester groups, and hydrogenolysis. In turn, pentasacchar-
ide 2 will be assembled from disaccharide building block 4
and trisaccharide 3. Disaccharide 4 can be derived from N-
benzyl-benzyloxycarbonyl-5-aminopentanol 9, glucosamine
thioglycoside 8, and galactose thioglycoside 7. The synthesis
of a colitose-containing trisaccharide analogue of compound
1 has already been reported by Bundle and co-workers,[8]
who employed a halide-assisted glycosylation procedure.[9]
The lability of dideoxyglycosyl halides and the need for a re-
action time of 10 days prompted us to consider N-phenyltri-
fluoroacetimidate colitose 5 as a precursor of trisaccharide
3. Thus, we envisaged that we could access trisaccharide 3
from glucose 6 and colitose imidate 5.
Scheme 1. Retrosynthetic analysis of pentasaccharide 1.
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Escherichia coli O111 O-Specific Polysaccharide Repeating Unit
FULL PAPER
droxy group as a levulinoyl ester afforded galactose building
block 7 in 69% yield over two steps.
Finally, known glucose building block 6[20] (Scheme 1),
containing free hydroxy groups at the C3 and C6 positions,
served as a branching building block.
Synthesis of disaccharide acceptor 4: With all of the re-
quired building blocks in hand, the assembly of the penta-
saccharide repeating unit through a [2+3] strategy was ex-
plored, commencing from the reducing end (Scheme 4). Thi-
Scheme 2. New synthesis of colitose building block 5: a) i) NapBr, NaH,
DMF, 08C to RT; ii) DIBAL, CH2Cl2, ꢀ788C, 90 min, 77% yield
(2 steps); b) compound 13, SnCl4, CH2Cl2, ꢀ788C, 2 h, 91% yield;
c) NMO, OsO4 (cat.), THF/H2O (2:1 v/v), RT, 24 h, 49% yield; d) PhCH-
ACHTUNGTRENNUNG(OMe)2, CSA, CuSO4, MeCN/THF (1:1 v/v), RT, 10 min; e) i) BzCl, pyri-
dine, RT, 2 h; ii) BH3·THF, TMSOTf, CH2Cl2, 08C to RT, 2 h, 71% yield
(3 steps); f) Dess–Martin periodinane, pyridine, CH2Cl2, RT, 1 h, 92%
yield; g) i) DDQ, MeOH, CH2Cl2, 08C to RT, 2 h; ii) CF3CACTHUNGRTENNG(U =NPh)Cl,
Cs2CO3, CH2Cl2, RT, 15 h, 68% yield (2 steps). DIBAL=diisobutylalumi-
num hydride, NMO=N-methylmorpholine-N-oxide, CSA=(ꢁ)-10-cam-
phorsulfonic acid, TMSOTf=trimethylsilyl trifluoromethanesulfonate,
DDQ=2,3-dichloro-5,6-dicyano-1,4-benzoquinone.
Scheme 4. Initial attempts towards reducing end disaccharide 4: a) i) NIS,
TfOH, CH2Cl2, 4 ꢄ M.S., ꢀ208C, 30 min; ii) TrtCl, pyridine, overnight,
70% yield (2 steps); b) i) compound 7, DMF, NIS, TMSOTf, CH2Cl2, 4 ꢄ
M.S., ꢀ108C, 12 h; ii) NaOMe, MeOH, 14% yield (2 steps). NIS=N-io-
dosuccinimide, Trt=triphenylmethyl.
benzoylated and subjected to regioselective benzylidene-
opening to furnish primary alcohol 17 in 71% yield over
three steps. Following Dess–Martin oxidation,[14] aldehyde
18 was converted into the corresponding hemiacetal by re-
moval of the Nap ether protecting group with DDQ. This
time, we opted to convert the colitose hemiacetal into N-tri-
fluorophenylacetimidate[15] 5, which is an attractive alterna-
tive to the previously synthesized colitose trichloroacetimi-
date, owing to its more moderate reactivity[16] and its lower
tendency to undergo O!N isoamide-to-amide rearrange-
ment at the anomeric center.
oglycoside 8 was treated with an excess of linker 9 in the
presence of NIS and TfOH at ꢀ208C to afford target glu-
cosamine 20. The presence of the trichloroacetamide as a
participating group at the C2 position ensured the selective
formation of the b-glycosidic linkage.[18] Furthermore, at
ꢀ208C, only the primary alcohol acted as a nucleophile in
the coupling reaction. To aid in purification, any excess
linker was tritylated to afford glucosamine 20 in 70% yield
after flash column chromatography on silica gel. Galactopy-
Having established a synthetic route to colitose glycosy-
lating agent 5, the remaining building blocks were prepared.
N-Benzyl-benzyloxycarbonyl-5-aminopentanol 9 and glucos-
amine building block 8 (Scheme 1) were synthesized by fol-
lowing literature procedures.[17,18] Galactose building block 7
(Scheme 1), which was equip-
ACHTUNGTRENrNGNU anose 7 was appended onto b-glucosamine 20 by using a
DMF-modulated glycosylation procedure[21] to direct the
stereochemical course of the reaction. In our hands, the
DMF-modulated glycosylation, followed by removal of the
levulinoyl ester under Zemplꢃn conditions,[22] provided the
target disaccharide (4) in only 14% yield. This low yield was
explained by the long glycosylation time, which favored
cleavage of the benzylidene acetal on the target molecule
and the formation of byproducts.
ped with an orthogonal levulin-
ic ester at the C4 position in an-
ticipation of chain-elongation,
was obtained from known ben-
To circumvent the problems that were encountered in the
synthesis of disaccharide 4, glucosamine building block 8
was converted into glycosylating agent 21 presenting an
Fmoc protecting group at the C3 position for chain-elonga-
tion and stable benzyl ethers at the C4 and C6 positions
(Scheme 5). We expected that the use of building block 21
would facilitate the isolation of compound 23, which would
perform better as acceptor in the coupling reaction with gal-
actopyranose 7. Following the placement of a TBS silyl
ether as a protecting group on the hydroxy group at the C3
position of thioglycoside 8, regioselective reductive opening
zylidene galactoside 19,[19] pre-
senting non-participating benzyl
ethers at the C2 and C3 posi-
Scheme 3. Synthesis of galac-
tose
building
block
7:
tions (Scheme 3). Regioselec-
tive reductive opening of the
benzylidene group on com-
pound 19 with triethylsilane in
the presence of triflic acid and
triflic anhydride, followed by
the protection of the C-4 hy-
a) i) Et3SiH,
TfOH,
Tf2O,
CH2Cl2, 08C to RT, 3 h; ii) Le-
vOH, DCC, DMAP, CH2Cl2,
08C to RT, 15 h, 69% yield
(2 steps).
Lev=levulinoyl,
DCC=N,N’-dicyclohexylcar-
bodiimide, DMAP=4-dime-
thylaminopyridine.
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P. H. Seeberger et al.
Scheme 5. Synthesis of glucosamine building block 21: a) i) TBSCl, imida-
zole, CH2Cl2; ii) BH3·THF, TMSOTf, CH2Cl2; iii) BnBr, NaH, DMF, 77%
yield (3 steps); b) i) BF3·OEt2, MeCN; ii) FmocCl, pyridine, CH2Cl2, 92%
yield (2 steps). TBS=tert-butyldimethylsilyl, Fmoc=fluorenylmethyloxy-
carbonyl.
of the benzylidene acetal, and subsequent benzylation of the
hydroxy group at the C6 position provided glucosamine 22
in 77% yield over three steps. Removal of the silyl ether at
the C3 position in the presence of BF3·OEt2 and protection
of the free hydroxy group with Fmoc provided the new glu-
cosamine glycosylating agent 21 in 92% yield over two
steps.
Thioglycoside 21 was successfully coupled to N-benzyl-
benzyloxycarbonyl-5-aminopentanol 9 at ꢀ208C upon acti-
vation by NIS and TfOH in CH2Cl2 (Scheme 6). Fmoc cleav-
Scheme 7. Attempts towards trisaccharide 4: a) compound 5 (2.5 equiv),
TMSOTf, CH2Cl2, 4 ꢄ M.S., ꢀ508C.
25 at the expense of the desired trisaccharide (3). The deg-
radation of electron-rich colitose residues in acidic media
has also been reported by Oscarson et al.[10b] The instability
of 3,6-dideoxysugars in acidic media rendered the assembly
of the target pentasaccharide by using a [2+3] approach un-
feasible, because the colitose residues would be exposed to
acidic conditions twice.
Total synthesis of the Escherichia coli O111 O-antigen: To
circumvent the degradation pathways, an alternative retro-
synthetic analysis was designed (Scheme 1). In this ap-
proach, the colitose residues would be introduced at a late
stage of the synthesis through a bis-glycosylation reaction
with trisaccharide 10. Trisaccharide 10 was further discon-
nected to disaccharide 24 and thioglucoside building block
11, equipped with an acetate ester at the C6 position as a
remote protecting group favoring the formation of the a-
linkage.[23]
Scheme 6. Synthesis of “reducing end” disaccharide 24: a) i) NIS, TfOH,
CH2Cl2, 3 ꢄ M.S. AW, ꢀ208C, 1 h; ii) Et3N, CH2Cl2, RT, 3 h, 71% yield
(2 steps); b) i) compound 7, NIS, TfOH, Et2O/CH2Cl2 (3:1 v/v), 3 ꢄ M.S.
AW, 0 8C, 30 min; ii) NaOMe, MeOH, RT, overnight, 48% yield (2 steps).
AW =acid washed.
To install residue C on disaccharide 24 (Figure 1), glucose
building block 11 was synthesized starting from known thio-
glucoside 27[24] (Scheme 8). Building block 11 contains non-
age by treatment with triethylamine afforded b-glucosamine
1
nucleophile 23 in 71% yield over two steps. The JC1,H1 cou-
pling constant of 164.8 Hz was characteristic for the forma-
tion of a b-glycosidic linkage. The coupling of nucleophile
23 with thiogalactoside 7 by activation with NIS and TfOH
in Et2O/CH2Cl2, followed by cleavage of the levulinoyl
ester, afforded target disaccharide 24 in 48% yield over two
steps. The configuration of the newly formed glycosidic
bond was assigned based on the coupling constant between
the C1 and H1 atoms on the galactopyranose residue
(1JC1,H1=171.3 Hz).
Scheme 8. Synthesis of thioglucoside 11: a) i) BnBr, NaH, DMF;
ii) BH3·THF, TMSOTf, CH2Cl2; (iii) Ac2O, pyridine, 74% yield (3 steps).
participating ether groups at the C2, C3, and C4 position, as
well as a participating acetate ester group at the C6 position,
to favor the formation of an a-glycosidic linkage. The or-
thogonal Nap ether at the C3 position would allow for
branching at this position. To access fully functionalized glu-
cose 11, first, the free hydroxy group of glucose 27 was ben-
zylated. Regioselective opening of the benzylidene acetal
with BH3·THF and TMSOTf afforded the primary alcohol
as an intermediate, which was further acetylated to yield
glycosylating agent 11 in 74% over three steps.
Attempts to synthesize trisaccharide 3: With thioglucoside 6
and l-colitose N-trifluorophenylacetimidate 5 in hand, we
attempted the synthesis of trisaccharide 3 (Scheme 7).
Owing to the high reactivity of deoxysugars,[16] the reaction
was performed at ꢀ508C with 1.25 equivalents of glycosyla-
ACHTUNGTRENNUNGting agent 5 per nucleophile. After 1 h, the target trisacchar-
ide was mainly observed, along with deletion sequences. A
prolonged reaction time (2 h) produced a-(1,6)-disaccharide
The coupling of thioglucoside 11 with disaccharide 24 was
performed at ꢀ508C by activation with NIS in the presence
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Escherichia coli O111 O-Specific Polysaccharide Repeating Unit
FULL PAPER
of TfOH in Et2O/CH2Cl2 to promote a selectivity
(Scheme 9). This procedure afforded a-trisaccharide 28 in
97% yield. Analysis of the coupling constant of the append-
Scheme 10. Deprotection of pentasaccharide 2 to produce compound 1:
a) i) Bu3SnH, AIBN, toluene, 908C, 2 h; ii) NaOMe, MeOH, 408C, over-
night; iii) Pd(OH)2, THF/H2O (1:1 v/v), 24 h, 64% yield (3 steps).
AIBN=azobisisobutyronitrile.
Scheme 9. Synthesis of colitose-containing pentasaccharide 2: a) NIS,
TfOH, Et2O/CH2Cl2 (3:1 v/v), 4 ꢄ M.S., ꢀ508C, 1 h, 97% yield;
b) i) NaOMe, MeOH, RT, 3 h; ii) DDQ, CH2Cl2/MeOH (9:1 v/v), 08C,
3 h, 73% yield (2 steps); c) compound 5, TMSOTf, CH2Cl2, 4 ꢄ M.S.,
ꢀ508C, 15 min, 63% yield.
drogenolysis on Pd(OH)2 to provide target pentasaccharide
1 in 64% yield over three steps.
ed glucose moiety (1JC1,H1 =172.4 Hz) allowed for the assign-
ment of the a stereochemistry at the newly formed center.
Cleavage of the acetate protecting group at the C6 position
under Zemplꢃn conditions, followed by removal of the Nap
ether at the C3 position by treatment with DDQ, afforded
diol 10 in 73% yield over two steps. Finally, the conversion
of diol 10 into the corresponding fully protected pentasac-
charide (2) was achieved through a bis-glycosylation reac-
tion by using four equivalents of colitose N-phenyltrifluor-
oacetimidate 5 in the presence of TMSOTf at ꢀ508C.
Quenching of the reaction after 15 min resulted in the for-
mation of the target pentasaccharide (2) in 63% yield. The
formation of the two a-linkages was confirmed by analysis
Conclusion
The first total synthesis of the pathogenic E. coli 0111 O-an-
tigen repeating pentasaccharide unit, starting from inexpen-
sive (S)-ethyl lactate, has been achieved in 21 linear steps
and 1.5% overall yield. The key step of this approach is a
bis-glycosylation reaction to install the acid-labile l-colitose
residues (Scheme 9). The pentasaccharide repeating unit
contains a terminal amine linker at its reducing end for im-
mobilization on any surface or conjugation to carrier pro-
teins for subsequent immunological evaluation.
ꢀ
of the anomeric C H coupling constants (see the Supporting
Information).
Experimental Section
Final deprotection of the target pentasaccharide (2) was
achieved in three steps (Scheme 10). To decrease the
number of transformations, the conversion of the trichloroa-
cetamide group into the corresponding acetamide group by
hydrogenolysis (H2, Pd(OH)2, THF/water) was attempted;
however, this reaction resulted in incomplete conversion. To
convert the TCA protecting group into the corresponding
acetyl group, pentasaccharide 2 was heated at 908C with
Bu3SnH and AIBN. Removal of the benzoate esters on the
colitose residues proceeded slowly under Zemplꢃn condi-
tions, but heating the intermediary pentasaccharide at 408C
overnight drove the reaction to completion. Particular care
was taken during the final hydrogenolysis reaction to avoid
acidic conditions, which could result in the degradation of
the colitose residue. Cleavage of the benzyl ethers and the
Cbz carbamate (Cbz=carboxybenzyl) were achieved by hy-
General experimental details: All chemicals were of reagent grade and
used as supplied unless otherwise noted. Molecular sieves were activated
prior to use by heating and drying under high vacuum. All reactions
were performed in oven-dried glassware under an argon atmosphere
unless otherwise noted. DMF, CH2Cl2, toluene, and THF were purified
by using a Cycle-Tainer Solvent Delivery System unless otherwise noted.
Analytical thin layer chromatography (TLC) was performed on Merck
silica gel 60 F254 plates (0.25 mm). The TLC plates were visualized by
UV light or by dipping the plate in a solution of cerium sulfate ammoni-
um molybdate (CAM) or in a 1:1 mixture of H2SO4 (2m) and resorcine
monomethylether (0.2%) in EtOH. Flash column chromatography was
performed by using forced flow of the indicated solvent through Fluka
Kieselgel 60 (230–400 mesh). Preparative HPLC purification was per-
1
formed on an Agilent 1200 Series. H and 13C NMR spectra were record-
ed on Varian Mercury 400 (400 MHz) or 600 (600 MHz) spectrometers in
CDCl3 or D2O; chemical shifts are referenced to internal standards
(CDCl3: 1H d=7.26 ppm, 13C d=77.0 ppm; D2O with acetone as internal
standard: 1H d=2.05 ppm, 13C d=29.84 or 206.26 ppm) unless otherwise
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P. H. Seeberger et al.
stated. Splitting patterns are indicated as s singlet, d doublet, t triplet,
q quartet, m multiplet, bs broad singlet, as apparent singlet, ad apparent
doublet, at apparent triplet, and aq apparent quadruplet for 1H NMR
data. NMR chemical shifts (d) are reported in ppm and coupling con-
stants (J) are reported in Hz. HRMS was performed by the MS service
of the Department of Organic Chemistry, Free University Berlin, on an
Agilent 6210 ESI-TOF (Agilent Technologies, Santa Clara, CA, USA).
IR spectra were recorded on a Perkin–Elmer 1600 FTIR spectrometer.
H6bA, H6aB, H4A, H5A), 3.55 (d, J=9.2 Hz, 1H; H6bB), 3.39 (bs, 2H;
H2A, OCH2), 3.17–3.10 (m, 2H; NCH2), 2.49 (s, 1H; OH), 1.51–1.36 (m,
4H; CH2), 1.30–1.12 ppm (m, 2H; CH2); 13C NMR (100 MHz, CDCl3, ro-
tamers): d=161.4 (CO), 138.3 (2C), 138.1, 137.9, 137.8, 137.7, 137.4,
128.4 (3C), 128.4 (3C), 128.4 (3C), 128.4 (3C), 128.3 (3C), 128.2 (3C),
128.0 (3C), 127.9, 127.8 (3C), 127.7 (4C), 127.7 (4C), 127.5, 127.4, 98.6
(1JC1A,H1A =166.0 Hz, C1A-b), 97.7 (1JC1B,H1B =171.3 Hz, C1B-a), 92.7
(NHCOCl3), 80.5 (C3A), 77.1 (C4A), 76.7 (C2B), 75.9 (C3B), 74.4 (CH2),
74.0 (C5A), 73.5 (2C, CH2), 73.4 (2C; CH2), 72.5 (CH2), 70.0 (C6B), 69.6
(OCH2), 69.3 (C5B), 68.9 (C6A), 68.2 (C4B), 67.0 (CO2CH2Ph), 59.0
(C2A), 50.4 and 50.1 (NCH2Ph, rotamers), 47.0 and 46.1 (CH2, rotamers),
27.9 (CH2), 27.4 (CH2), 23.1 ppm (CH2); IR (thin film): n˜ =3484, 3340,
2923, 2856, 1701, 1523, 1468, 1454, 1091, 1028, 698 cmꢀ1; HRMS (ESI):
m/z calcd for C69H75Cl3N2O13Na: 1267.4232 [M+Na]+; found: 1267.4163.
Optical rotations were measured on
a UniPol L 1000 polarimeter
(Schmidt & Haensch, Berlin, Germany) with concentrations expressed in
g/100 mL.
N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl-4,6-di-O-benzyl-2-tri-
chloroacetamido-b-d-glucopyranoside (23): A mixture of glucosamine
building block 21 (0.60 g, 0.78 mmol), linker
9 (0.38 g, 1.17 mmol,
1.5 equiv), freshly activated molecular sieves (3 ꢄ AW, 1.8 g), and NIS
(0.35 g, 1.56 mmol, 2 equiv) in CH2Cl2 (16 mL) was stirred for 15 min
before being cooled to ꢀ208C. After the addition of TfOH (7 mL,
78 mmol, 0.1 equiv), the mixture was stirred at ꢀ208C for 1 h. After re-
moval of the molecular sieves by filtration through a pad of celite, the re-
action mixture was diluted with CH2Cl2 and washed with a saturated
aqueous solution of NaHCO3. The organic layer was washed with a satu-
rated aqueous solution of Na2S2O3 and water, dried over MgSO4, and
concentrated in vacuo. The crude product was purified by column chro-
matography on silica gel (hexanes/EtOAc, 80:20 to 50:50) to yield the
crude product (0.61 g). The crude material was dissolved in CH2Cl2
(12 mL) and treated with Et3N (1.6 mL, 11.7 mmol). The solution was
stirred for 3 h at RT and then concentrated under reduced pressure. The
crude product was purified by flash column chromatography on silica gel
(hexanes/EtOAc, 90:10 to 50:50) to give target compound 23 as an oil
(0.45 g, 0.55 mmol, 71% yield over 2 steps). Rf =0.25 (hexanes/EtOAc,
70:30); [a]2D0 =ꢀ2.4 (c=0.35, CHCl3); 1H NMR (400 MHz, CDCl3): d=
7.36–7.23 (m, 20H), 7.17 (s, 1H), 5.16 (s, 2H), 4.74 (d, J=11.2 Hz; 1H),
4.69–4.61 (m, 3H), 4.55 (d, J=12.0 Hz; 1H), 4.48 (bs, 2H), 4.13–4.04 (m,
1H), 3.85–3.71 (m, 3H), 3.63–3.47 (m, 3H), 3.43–3.37 (m, 1H), 3.23–3.17
(m, 2H), 2.99 (s, 1H), 1.56–1.49 (m, 4H), 1.43–1.14 ppm (m, 2H);
13C NMR (100 MHz, CDCl3): d=162.3, 138.06, 138.03, 137.8, 128.6 (3C),
128.5 (3C), 128.5, 128.4 (3C), 128.0 (3C), 127.9, 127.8 (4C), 127.7, 127.3,
127.2, 99.66 (1JC1A,H1A =164.8 Hz; C1A-b), 92.6, 92.0, 78.3, 74.9, 74.5, 73.5
(2C), 72.9, 69.5, 69.1, 67.2, 58.4, 50.6, 50.3, 47.1, 46.2, 29.1, 27.9, 27.3,
23.2 ppm; IR (thin film): n˜ =3436, 3327, 2924, 2862, 1697, 1101,
N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl-6-O-acetyl-2,4-di-O-
benzyl-3-O-2-(naphthylmethyl)-a-d-glucopyranosyl-(1!4)-2,3,6-tri-O-
benzyl-a-d-galactopyranosyl-(1!3)-4,6-di-O-benzyl-2-trichloroacetami-
do-b-d-glucopyranoside (28): Acceptor 24 (40 mg, 32 mmol) and thioglu-
coside 11 (28 mg, 48 mmol, 1.5 equiv) were azeotroped with toluene and
dried in vacuo. After the addition of NIS (14 mg, 64 mmol, 2.0 equiv) and
freshly activated molecular sieves (4 ꢄ, 100 mg), the reaction mixture
was dissolved in Et2O/CH2Cl2 (320 mL, 3/1 v/v), cooled to ꢀ508C, and
stirred for 15 min.
A solution of TfOH in CH2Cl2 (30 mL, 3 mmol,
0.1 equiv) was added dropwise and the mixture was stirred for 1 h at
ꢀ508C. After removal of the molecular sieves by filtration through a pad
of celite, the reaction mixture was diluted with Et2O and washed with a
saturated aqueous solution of Na2S2O3. Following separation of the
layers, the organic layer was washed with a saturated aqueous solution of
NaHCO3 and brine, dried over MgSO4, and concentrated in vacuo. The
crude product was purified by NP-HPLC (NP=normal phase) on YMC
Pack silica gel (EtOAc/hexanes, 5% (5 min) to 30% (30 min)) to afford
the target trisaccharide as a colorless oil (55 mg, 31 mmol, 97% yield).
Rf =0.36 (hexanes/EtOAc, 70:30); [a]2D0 =+18.3 (c=1.0, CHCl3);
1H NMR (400 MHz, CDCl3, rotamers): d=7.90 (d, J=7.6 Hz, 1H;
NHCOCl3), 7.86–7.76 (m, 2H), 7.73–7.70 (m, 2H), 7.51–7.41 (m, 3H),
7.36–7.04 (m, 45H), 5.14 (d, J=6.0 Hz, 2H; CO2CH2Ph), 5.09–5.07 (m,
2H; CH2Ph, H1B), 5.00–4.95 (m, 2H; CH2Ph), 4.88 (d, J=10.8 Hz, 1H;
CH2Ph), 4.85 (d, J=3.2 Hz, 1H; H1C), 4.84–4.77 (m, 2H; H1A, CH2Ph),
4.71–4.69 (m, 2H; CH2Ph), 4.65 (d, J=12.0 Hz, 1H), 4.63–4.46 (m, 4H),
4.46 (d, J=10.4 Hz, 1H), 4.42 (bs, 2H; NCH2Ph), 4.30–4.23 (m, 2H; H5B,
H3A), 4.20–4.15 (m, 2H; CH2Ph, H6aC), 4.12–4.09 (m, 1H; H5C), 4.05
(dd, J=9.2, 9.6 Hz, 1H; H3C), 3.98–3.87 (m, 4H; CH2Ph, H4B, H6bC,
H2B), 3.83 (dd, J=10.4, 2.8 Hz, 1H; H3B), 3.77–3.56 (m, 7H; OCH2,
H6aA, H6bA, H4A, H6aB, H5A, H4C), 3.53 (dd, J=10.0, 3.2 Hz, 1H; H2C),
3.51–3.40 (m, 2H; H6bB, H2A), 3.35–3.32 (m, 1H; OCH2), 3.16–3.08 (m,
2H; NCH2), 1.97 (s, 3H; COCH3), 1.50–1.33 (m, 4H; CH2), 1.25–
1.09 ppm (m, 2H; CH2); 13C NMR (100 MHz, CDCl3, rotamers): d=
170.7, 161.5, 138.4, 138.3, 138.2, 138.0, 138.0, 137.7, 136.1, 133.4, 133.0,
128.6, 128.61, 128.5, 128.5, 128.5, 128.4, 128.3, 128.2, 128.2, 128.0, 127.9,
127.9, 127.8, 127.8, 127.7, 127.7, 127.6, 127.5, 127.5, 127.2, 126.6, 126.1,
126.0, 126.0, 99.3 (1JC1C,H1C =172.4 Hz, C1C-a), 99.1 (1JC1A,H1A =164.4 Hz,
C1A-b), 97.8 (1JC1B,H1B =169.1 Hz, C1B-a), 92.9 (NHCOCl3), 81.9 (C3C),
80.77 (C2C), 79.9 and 79.7 (C3A, rotamers), 77.6 (C2B), 77.5 (C4A), 77.3
(C4C), 76.4 (C3B), 75.9 (C4B), 75.7 (CH2Ph), 75.1 (CH2Ph), 74.4 (CH2Ph),
74.2 (C5A), 73.9 (CH2Ph), 73.5 (CH2Ph), 73.5 (CH2Ph), 73.1 (CH2Ph),
72.7 (CH2Ph), 70.6 (C5C), 69.7 (OCH2), 69.5 (C6B), 69.4 (C5B), 69.0
(C6A), 67.2 (CO2CH2Ph), 62.8 (C6C), 58.6 (C2A), 50.6 and 50.2
(NCH2Ph, rotamers), 47.2 and 46.2 (NCH2, rotamers), 29.3 (CH2), 28.0
and 27.5 (CH2, rotamers), 23.2 (CH2), 21.0 ppm (COCH3); IR (thin film):
1062 cmꢀ1
; HRMS (ESI): m/z calcd for C42H47Cl3N2O8Na: 835.2296
[M+Na]+; found: 835.2271.
N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl-2,3,6-tri-O-benzyl-a-d-
galactopyranosyl-(1!3)-4,6-di-O-benzyl-2-trichloroacetamido-b-d-gluco-
pyranoside (24): A mixture of b-thiogalactoside 7 (0.40 g, 0.60 mmol,
1.3 equiv), glucosamine acceptor 23 (0.38 g, 0.47 mmol), NIS (0.16 g,
0.7 mmol, 1.5 equiv), and freshly activated molecular sieves (3 ꢄ AW,
1 g) in Et2O/CH2Cl2 (16 mL, 3/1 v/v) was stirred at RT for 30 min. Then,
the mixture was cooled to 08C and TfOH (4 mL, 47 mmol, 0.1 equiv) was
added. The mixture was stirred for 30 min at 08C. After removal of the
molecular sieves by filtration through a pad of celite, the reaction mix-
ture was diluted with Et2O and washed with a saturated aqueous solution
of Na2S2O3. The organic layer was washed with a saturated aqueous solu-
tion of NaHCO3 and brine, dried over MgSO4, and concentrated in
vacuo. The crude product was purified by column chromatography on
silica gel (hexanes/EtOAc, 90:10 to 70:30) to yield the crude disaccharide
(0.35 g). The crude material was then dissolved in MeOH (12 mL) and
treated with a 0.5m solution of NaOMe in MeOH (90 mL, 45 mmol,
0.2 equiv). The mixture was stirred at RT overnight and then acidified to
pH 6 with Amberlite IR 120-H+ resin. The solvents were concentrated in
vacuo and the crude material was purified by flash column chromatogra-
phy on silica gel (hexanes/EtOAc, 90:10 to 75:25) to give the target disac-
charide as an oil (280 mg, 0.22 mmol, 48% yield over 2 steps). Rf =0.35
(hexanes/EtOAc, 70:30); [a]2D0 =+27.8 (c=0.5, CHCl3); 1H NMR
(400 MHz, CDCl3, rotamers): d=8.02 (d, J=6.4 Hz, 1H; NHCOCl3),
7.31–7.22 (m, 30H), 7.16–7.09 (m, 5H), 5.14 (s, 2H; CO2CH2), 5.08–4.93
(m, 3H; CH, H1A, H1B), 4.77–4.67 (m, 3H; CH2), 4.63–4.35 (m, 8H;
CH2), 4.35–4.28 (m, 1H; H3A), 4.17–4.14 (m, 1H; H5B), 3.92 (bs, 1H;
H4B), 3.85–3.78 (m, 2H; H2B, H3B), 3.77–3.59 (m, 6H; OCH2, H6aA,
n˜ =3340, 3062, 3030, 2930, 2867, 1739, 1702, 1454, 1234, 1091, 1060 cmꢀ1
HRMS (ESI): m/z calcd for C102H107Cl3N2O19
;
:
1791.6431 [M+Na]+;
found: 1791.6380.
N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl-2,4-di-O-benzyl-a-d-glu-
copyranosyl-(1!4)-2,3,6-tri-O-benzyl- a-d-galactopyranosyl-(1!3)-4,6-
di-O-benzyl-2-trichloroacetamido- b-d-glucopyranoside (10): A solution
of trisaccharide 28 (50 mg, 28 mmol) in MeOH (0.3 mL) was treated with
a 0.5m solution of NaOMe in MeOH (6 mL, 3 mmol, 0.1 equiv). The solu-
tion was stirred at RT for 3 h, diluted with MeOH, and then acidified to
pH 6 with Amberlite IR 120-H+ resin. The resin was filtered off and
4000
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Chem. Eur. J. 2013, 19, 3995 – 4002

Escherichia coli O111 O-Specific Polysaccharide Repeating Unit
FULL PAPER
washed several times with MeOH. The filtrate was concentrated in vacuo
to give an oil, which was further dissolved in a mixture of CH2Cl2/MeOH
(3.1 mL, 9/1 v/v). The mixture was cooled at 08C and treated with DDQ
(21 mg, 84 mmol, 3 equiv). After stirring for 3 h at 08C, the reaction was
quenched by the addition of a saturated aqueous solution of Na2S2O3 and
a saturated aqueous solution of NaHCO3. The mixture was extracted
with CH2Cl2 and the organic layer was dried over MgSO4 and concentrat-
ed under reduced pressure. The crude product was purified by flash
column chromatography on silica gel (hexanes/EtOAc, 70:30) to give of
target diol 10 as a yellow oil (21 mmol, 33 mg, 73% yield over 2 steps).
Rf =0.46 (hexanes/EtOAc, 50:50); [a]1D8 =+70.9 (c=0.25, CHCl3);
1H NMR (600 MHz, CDCl3, rotamers): d=7.86 (d, J=6.6 Hz, 1H), 7.42–
7.04 (m, 45H), 5.15 (d, J=6.6 Hz, 2H; CO2CH2Ph), 5.02 (s, 1H; H1B),
4.96 (d, J=11.0 Hz, 1H; CH2), 4.84 (d, J=6.0 Hz, 1H; CH2), 4.81 (s, 2H;
H1C, H1A), 4.72–4.65 (m, 4H; CH2Ph), 4.65–4.58 (m, 2H; CH2Ph), 4.56
(d, J=12.2 Hz, 1H; CH2Ph), 4.50 (d, J=12.0 Hz, 1H; CH2Ph), 4.45–4.41
(m, 3H; CH2Ph, NCH2Ph), 4.31–4.27 (m, 1H; CH2Ph), 4.23 (dd, J=7.8,
7.8 Hz, 1H; H3A), 4.14–4.08 (m, 2H; H5B, H3C), 4.04–4.02 (m, 2H; H5C,
CH2Ph), 3.86 ꢀ3.81 (m, 3H; H4B, H3B, H2B), 3.77–3.44 (m, 10H; OCH2,
H6aA, H6bA, H6aB, H4A, H5A, H6aC, H6bC, H6bB, H2A), 3.41 (dd, J=9.0,
9.6 Hz, 1H; H4C), 3.37–3.25 (m, 2H; H2C, OCH2), 3.17–3.09 (m, 2H;
NCH2), 2.37 (s, 1H; OH), 1.45–1.40 (m, 4H; CH2), 1.22–1.15 ppm (m,
2H; CH2); 13C NMR (150 MHz, CDCl3, rotamers): d=161.6, 138.4, 138.3,
138.2, 138.1, 137.9, 128.7, 128.6, 128.6, 128.5, 128.5, 128.4, 128.4, 128.3,
128.2, 128.1, 128.0, 127.9, 127.9, 127.9, 127.8, 127.8, 127.8, 127.7, 127.6,
127.3, 99.2 (1JC1A,H1A =165.5 Hz, C1A-b), 99.1 (1JC1C,H1C =173.0 Hz, C1C-
a), 97.9 (1JC1B,H1B =173.5 Hz, C1B-a), 92.9 (NHCOCCl3), 80.5 (C2C), 79.7
(C3A), 78.5 (C2B), 78.0 (C4C), 77.5 (C4A), 77.1 (C3B), 76.2 (C4B), 74.8
(CH2Ph), 74.4 (CH2Ph), 74.3 (C5A), 73.8 (CH2Ph), 73.57 (CH2Ph), 73.56
(CH2Ph), 73.52 (C3C), 73.2 (CH2Ph), 73.0 (CH2Ph), 71.4 (C5C), 70.6
(C5B), 69.8 (C6B), 69.7 (OCH2), 69.1 (C6A), 67.2 (CO2CH2Ph), 61.9
(C6C), 58.7 and 58.5 (C2A, rotamers), 50.6 and 50.3 (NCH2Ph, rotamers),
47.2 and 46.3 (NCH2, rotamers), 29.4 (CH2), 28.0 and 27.6 (CH2, rotam-
ers), 23.3 ppm (CH2); IR (thin film): n˜ =3339, 2835, 1678, 1637, 1091,
1027 cmꢀ1; HRMS (ESI): m/z calcd for C89H97Cl3N2NaO18: 1609.5700
[M+Na]+; found: 1609.5709.
0.73 ppm (d, J=6.4 Hz, 3H; H6E); 13C NMR (150 MHz, CDCl3, rotam-
ers): d=166.1, 166.0, 161.7, 138.8, 138.6, 138.6, 138.3, 138.0, 137.8, 137.4,
133.2, 133.1, 130.3, 130.2, 129.8, 129.7, 128.8, 128.6, 128.5, 128.5, 128.5,
128.4, 128.4, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.9, 127.8, 127.7,
127.7, 127.6, 127.5, 127.5, 127.3, 126.2, 98.8 (1JC1A,H1A =166.9 Hz, C1A-b),
98.6 (1JC1C,H1C =170.8 Hz, C1C-a), 97.9 (1JC1B,H1B =171.5 Hz, C1B-a), 96.7
(1JC1E,H1E =171.3 Hz, C1E-a), 96.5 (1JC1D,H1D =173.6 Hz, C1D-a), 93.1
(NHCOCCl3), 82.8 (C2C), 80.9 (C3A), 77.6 (C4C), 77.3 (C4A), 76.8 (C2B),
76.4 (C4B), 75.8 (C3B), 74.8, 74.7, 74.1, 74.1, 73.5 (C5A), 73.4 (C3C), 73.0,
72.9, 72.6 (C6B), 72.1 (C4E), 72.0 (C4D), 71.3 (C2E), 71.0, 70.9 (C5B), 70.6
(C5C), 69.8 (C2D), 69.2, 69.1 (C6A), 67.2 (NHCO2CH2Ph), 65.9 (C6C),
65.0 (C5D), 64.8 (C5E), 59.4 (C2A), 50.6 and 50.3 (NCH2Ph, rotamers),
47.2 and 46.3 (NCH2, rotamers), 29.4 (CH2), 29.1 (C3E), 28.3 (C3D), 28.0
and 27.6 (CH2, rotamers), 23.2 (CH2), 16.5 (C6E), 16.1 ppm (C6D); IR
(thin film): n˜ =3347, 2925, 2855, 1716, 1454, 1270, 1093, 1061, 1027,
984 cmꢀ1; HRMS (ESI): m/z calcd for C129H137Cl3N2NaO26: 2257.8423
[M+Na]+; found: 2257.8471.
5-Amino-pentanyl
3,6-dideoxy-a-l-xylo-hexopyranosyl-(1!3)-[3,6-di-
deoxy-a-l-xylo-hexopyranosyl-(1!6)]-a-l-galactopyranosyl)-a-d-gluco-
pyranosyl-(1!4)-a-d-galactopyranosyl-(1!3)-2-N-acetyl-b-glucopyrano-
side (1): A degassed solution of AIBN (15 mg, 89 mmol, 10 equiv) in tol-
uene (2 mL) was added to a solution of pentasaccharide 2 (20 mg,
8.9 mmol) and Bu3SnH (24 mL, 89 mmol, 10 equiv) in toluene (2 mL). The
mixture was stirred at 908C for 2 h and then passed through a pad of
silica gel (hexanes/EtOAc, 90:10 to 50:50) to give the crude intermediate
N-acetyl pentasaccharide (17 mg). MS (MALDI): m/z calcd for
C129H140N2NaO26: 2155.95 [M+Na]+; found: 2155.89.
The crude N-acetyl pentasaccharide was dissolved in MeOH (4 mL) and
treated with a 0.5m solution of NaOMe in MeOH (18 mL, 9 mmol,
1 equiv). The mixture was stirred at 408C overnight and then acidified to
pH 6 with Amberlite IR 120-H+ resin. After filtration, the mixture was
concentrated in vacuo to give the intermediate diol as an oil. MS
(MALDI): m/z calcd for C115H132N2NaO24: 1947.90 [M+Na]+; found:
1947.92.
The oil was taken up in a mixture of THF/water (6 mL, 1/1 v/v).
Pd(OH)2 (20%, 20 mg) was added and the suspension was stirred under
a H2 atmosphere for 24 h. After removal of the solvents, the crude prod-
uct was dissolved in water and filtered on a cotton pad. Removal of the
water by lyophilization, followed by gel-filtration chromatography (Se-
phadex LH-20, water) afforded the target pentasaccharide (5 mg,
5.6 mmol, 64% yield over 3 steps). Rf =0.50 (iPrOH/1m aq. NH4OAc,
1:2); [a]2D0 =+29.3 (c=0.06, CHCl3); 1H NMR (600 MHz, D2O): d=5.45
(d, J=2.4 Hz, 1H), 5.15 (d, J=3.0 Hz, 1H), 4.95 (d, J=3.6 Hz, 1H), 4.82
(d, J=3.6 Hz, 1H), 4.54 (d, J=8.4 Hz, 1H), 4.36–4.28 (m, 2H), 4.09 (s,
1H), 4.05–3.98 (m, 3H), 3.92–3.89 (m, 6H), 3.84–3.58 (m, 13H), 3.47–
3.44 (m, 1H), 3.01–2.95 (t, J=5.2 Hz, 2H), 2.04 (s, 3H), 2.01–1.94 (m,
4H), 1.69–1.64 (m, 2H), 1.62–1.57 (m, 2H), 1.44–1.36 (m, 2H), 1.15 (d,
J=6.6 Hz, 3H), 1.12 ppm (d, J=6.6 Hz, 3H); 13C NMR (150 MHz,
CD6CO): d=174.5, 101.3, 100.5, 99.7, 99.1, 98.9, 80.3, 79.7, 78.4, 75.8,
72.6, 71.6, 71.5, 70.9, 70.4, 69.9, 69.1, 68.9, 68.1, 66.93, 66.90, 66.7, 63.76,
63.73, 60.8, 59.9, 54.5, 39.6, 33.27, 33.21, 28.4, 26.7, 22.6, 22.5, 15.6 ppm
N-(Benzyl)benzyloxycarbonyl-5-amino-pentanyl-3,6-dideoxy-4-O-benzo-
yl-2-O-benzyl-a-l-xylo-hexopyranosyl-(1!3)-[3,6-dideoxy-4-O-benzoyl-
2-O-benzyl-a-l-xylo-hexopyranosyl-(1!6)]-2,4-di-O-benzyl-a-d-gluco-
pyranosyl-(1!4)-2,3,6-tri-O-benzyl-a-d-galactopyranosyl-(1!3)-4,6-di-
O-benzyl-2-trichloroacetamido-b-d-glucopyranoside (2): Colitose imidate
5 (35 mg, 68 mmol, 4 equiv) and nucleophile trisaccharide 10 (27 mg,
17 mmol) were azeotroped three times with toluene and dried in vacuo.
After the addition of freshly activated molecular sieves (4 ꢄ, 80 mg), the
mixture was suspended in anhydrous CH2Cl2 (340 mL) under an Ar at-
mosphere. The mixture was cooled to ꢀ508C and stirred for 15 min.
After the dropwise addition of a solution of TMSOTf in CH2Cl2 (30 mL,
1.7 mmol, 0.1 equiv), the reaction was stirred for 15 min at ꢀ508C. After
removal of the molecular sieves by filtration through a pad of celite, the
reaction mixture was diluted with CH2Cl2 and washed with a saturated
aqueous solution of NaHCO3. The organic layer was washed with brine
and dried over MgSO4. After removal of solvents in vacuo, the crude
product was purified by NP-LCMS on YMC Pack silica gel (EtOAc/hex-
anes, 5% (5 min) to 30% (40 min)) to afford the target pentasaccharide
as an oil (24 mg, 11 mmol, 63% yield). Rf =0.20 (hexanes/EtOAc, 70:30);
[a]2D0 =+10.3 (c=0.30, CHCl3); 1H NMR (600 MHz, CDCl3, rotamers):
d=8.07 (d, J=8.0 Hz, 1H; NHCOCl3), 8.04–8.02 (m, 2H), 7.90–7.88 (m,
2H), 7.60–7.51 (m, 3H), 7.49–7.26 (m, 22H), 7.24–7.04 (m, 21H), 6.99–
6.94 (m, 4H), 6.80 (d, J=7.2 Hz, 2H), 5.65 (d, J=3.2 Hz, 1H; H1D),
5.26–5.22 (m, 1H; H4E), 5.13 (s, 2H; CO2CH2Ph), 5.05–5.00 (m, 2H;
H1B, CH2Ph), 4.97–4.91 (m, 3H; 2 CH2Ph, H1A), 4.86 (d, J=3.2 Hz, 1H;
H1C), 4.82–4.77 (m, 2H; H4D, H1E), 4.74–4.66 (m, 4H; CH2Ph), 4.65–
4.60 (m, 2H; CH2Ph), 4.56–4.47 (m, 4H; CH2Ph), 4.44–4.17 (m, 12H;
CH2Ph, NCH2Ph, H3C, H5B, H3A, H5D, H5E), 4.03–3.92 (m, 4H; H6aB,
H5C, H2B, H4B), 3.87–3.84 (m, 2H; H2E, H4C), 3.80 (dd, J=10.2, 3.2 Hz,
1H; H3B), 3.77–3.50 (m, 10H; H6aC, H2D, H6aA, H6bA, OCH2, H2C,
H4A, H6bC, H5A, H6bB), 3.37–3.22 (m, 2H; OCH2, H2A), 3.19–2.95 (m,
2H, NCH2), 2.27–2.20 (m, 2H; H3E), 2.14–2.04 (m, 2H; H3D), 1.41–1.35
(m, 4H; CH2), 1.18–1.12 (m, CH2), 1.06 (d, J=6.6 Hz, 3H; H6D),
(2C); IR (thin film): n˜ =3369, 1649, 1563, 1377, 1347, 1139, 1076 cmꢀ1
;
HRMS (ESI): m/z calcd for C37H67N2O22: 891.4185 [M+H]+; found:
891.4208.
Acknowledgements
The Max-Planck Society and the Marie Curie ITN FP7 project
CARMUSYS (PITN-GA-2008-213592) are gratefully acknowledged for
their generous financial support. The authors would like to thank Dr. C.
Lebev Pereira and Dr. K. Gilmore for critically editing this manuscript.
[1] a) J. T. Brooks, D. Bergmire-Sweat, M. Kennedy, K. Hendricks, M.
Garcia, L. Marengo, J. Wells, M. Ying, W. Bidd, P. M. Griffin, R. M.
Hockstra, C. R. Friedman, Clin. Infect. Dis. 2004, 38, 190–198; b) A.
Chem. Eur. J. 2013, 19, 3995 – 4002
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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