Diversity-oriented synthesis of inner core oligosaccharides of the lipopolysaccharide of pathogenic gram-negative bacteria

Article abstract of DOI:10.1021/ja401164s

Lipopolysaccharide (LPS) is a potent virulence factor of pathogenic Gram-negative bacteria. To better understand the role of LPS in host-pathogen interactions and to elucidate the antigenic and immunogenic properties of LPS inner core region, a collection of well-defined l-glycero-d-manno-heptose (Hep) and 3-deoxy-α-d-manno-oct-2-ulosonic acid (Kdo)-containing inner core oligosaccharides is required. To address this need, we developed a diversity-oriented approach based on a common orthogonal protected disaccharide Hep-Kdo. Utilizing this new approach, we synthesized a range of LPS inner core oligosaccharides from a variety of pathogenic bacteria including Y. pestis, H. influenzae, and Proteus that cause plague, meningitis, and severe wound infections, respectively. Rapid access to these highly branched core oligosaccharides relied on elaboration of the disaccharide Hep-Kdo core as basis for the elongation with various flexible modules including unique Hep and 4-amino-4-deoxy-β-l-arabinose (Ara4N) monosaccharides and branched Hep-Hep disaccharides. A regio- and stereoselective glycosylation of Kdo 7,8-diol was key to selective installation of the Ara4N moiety at the 8-hydroxyl group of Kdo moiety of the Hep-Kdo disaccharide. The structure of the LPS inner core oligosaccharides was confirmed by comparison of ¹H NMR spectra of synthetic antigens and isolated fragments. These synthetic LPS core oligosaccharides can be covalently bound to carrier proteins via the reducing end pentyl amine linker, to explore their antigenic and immunogenic properties as well as potential applications such as diagnostic tools and vaccines.

Full text of DOI:10.1021/ja401164s

Article
pubs.acs.org/JACS
Diversity-oriented Synthesis of Inner Core Oligosaccharides of the
Lipopolysaccharide of Pathogenic Gram-negative Bacteria
You Yang,†,‡ Shunsuke Oishi,†,‡ Christopher E. Martin,†,‡ and Peter H. Seeberger*,†,‡
†Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Am Muhlenberg 1, 14476 Potsdam, Germany
̈
‡Institute of Chemistry and Biochemistry, Freie Universitat Berlin, Arnimallee 22, 14195 Berlin, Germany
̈
S
* Supporting Information
ABSTRACT: Lipopolysaccharide (LPS) is a potent virulence factor of
pathogenic Gram-negative bacteria. To better understand the role of LPS in
host−pathogen interactions and to elucidate the antigenic and immunogenic
properties of LPS inner core region, a collection of well-defined L-glycero-D-
manno-heptose (Hep) and 3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo)-
containing inner core oligosaccharides is required. To address this need, we
developed a diversity-oriented approach based on a common orthogonal
protected disaccharide Hep-Kdo. Utilizing this new approach, we synthesized a
range of LPS inner core oligosaccharides from a variety of pathogenic bacteria
including Y. pestis, H. influenzae, and Proteus that cause plague, meningitis, and
severe wound infections, respectively. Rapid access to these highly branched
core oligosaccharides relied on elaboration of the disaccharide Hep-Kdo core as basis for the elongation with various flexible
modules including unique Hep and 4-amino-4-deoxy-β-L-arabinose (Ara4N) monosaccharides and branched Hep-Hep
disaccharides. A regio- and stereoselective glycosylation of Kdo 7,8-diol was key to selective installation of the Ara4N moiety at
the 8-hydroxyl group of Kdo moiety of the Hep-Kdo disaccharide. The structure of the LPS inner core oligosaccharides was
1
confirmed by comparison of H NMR spectra of synthetic antigens and isolated fragments. These synthetic LPS core
oligosaccharides can be covalently bound to carrier proteins via the reducing end pentyl amine linker, to explore their antigenic
and immunogenic properties as well as potential applications such as diagnostic tools and vaccines.
(Kdo) and L-glycero-D-manno-heptose (Hep) are ubiquitously
present in most LPS structures (Figure 1). Another frequently
detected sugar unit in the LPS is 4-amino-4-deoxy-β-L-
arabinose (Ara4N).6 Attachment of Ara4N to the LPS, via a
glycosidic linkage at the C-8 position of Kdo, has been
INTRODUCTION
■
Many Gram-negative bacteria are notorious pathogens that can
lead to a variety of infectious diseases, such as meningitis,
pneumonia and plague.1 The pathogenicity and virulence of
Gram-negative bacteria are often associated with the lip-
opolysaccharide (LPS) coat.2,3 LPS, a highly complex
glycolipid, is the major component the outer membrane of
Gram-negative bacteria.3 Elucidation of the role of LPS is
essential for understanding the host−pathogen interactions
during the infection of Gram-negative bacteria. As a potent
virulence factor, LPS also serves as a surface pathogen-
associated antigen for recognition by the host immune system.3
Therefore, LPS has attracted much interest for the develop-
ment of diagnostic tools, therapeutic reagents and vaccine
candidates.
LPS is comprised of the lipid A moiety, the core
oligosaccharide, and the O-specific polysaccharide. The core
oligosaccharide, connecting lipid A with the outer O-antigen, is
present in every natural LPS structure. The O-antigen in turn
may be missing in many pathogenic bacteria such as N.
meningitidis, H. influenzae and Y. pestis.4,5
Structurally, the core region of LPS can be further subdivided
into inner core and outer core. The main structural motifs of
the inner core are highly conserved throughout the Gram-
negative bacteria while the sugars of the outer core vary greatly
between strains.4 3-Deoxy-α-D-manno-oct-2-ulosonic acid
Figure 1. Conserved inner core oligosaccharides of LPS from Y. pestis,
H. influenzae and Proteus. A, 3-Deoxy-α-D-manno-oct-2-ulosonic acid
(Kdo); B−E, L-glycero-D-manno-heptose (Hep); F, 4-amino-4-deoxy-β-
L-arabinose (Ara4N).
Received: February 1, 2013
Published: March 24, 2013
© 2013 American Chemical Society
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Figure 2. Synthetic LPS antigens for immunological studies.
implicated as a major factor of bacterial resistance against
cationic antimicrobials, such as polymyxin B (PmB).7
(Figure 2). Monosaccharides 1, 2 as well as disaccharide 3 are
basic structural elements of the LPS core region, and
trisaccharide 4 is highly conserved in most Gram-negative
bacteria. Tetrasaccharides 5, 6, and 7, incorporating different
branching points at the core trisaccharide 4, are conserved in Y.
pestis, H. influenzae, and Proteus bacteria. These well-defined
synthetic LPS inner core oligosaccharides can be conjugated to
carrier proteins for immunization studies. The resulting
glycoconjugates help elucidate the antigenic and immunogenic
properties as diagnostic markers and vaccine candidates.
Furthermore, the structural basis for the interaction of LPS-
specific antibodies directed against synthetic antigens (epitope
mapping) can be enabled.14 In addition, synthetic core antigens
may serve as substrates for enzymes that add immunorelevant
decorating groups onto the core region.7,15
Most Gram-negative bacteria share the trisaccharide L,D-Hep-
(1→3)-L,D-Hep-(1→5)-Kdo as a common conserved inner
core structure (ABC in Figure 1).8 This core can be decorated
with other glycans, phosphates, or occasionally acetyl groups.
Elongation of the core trisaccharide ABC at the C-7 position of
the Hep moiety C with Hep residue D, gives rise to the
conserved core tetrasaccharide L,D-Hep-(1→7)-L,D-Hep-(1→
3)-L,D-Hep-(1→5)-Kdo (ABCD) in Y. pestis.9,10 Extension of
the core trisaccharide ABC at the C-2 position of Hep C with
Hep residue E, generates the conserved core tetrasaccharide
ABCE L,D-Hep-(1→2)-L,D-Hep-(1→3)-L,D-Hep-(1→5)-Kdo in
H. influenza.11 Affixing Ara4N residue F at the C-8 position of
Kdo A within the core trisaccharide, furnishes the conserved
core tetrasaccharide A(F)BC L,D-Hep-(1→3)-L,D-Hep-(1→5)-
[L-Ara4N-(1→8)]-Kdo common to most Proteus strains.6
Synthetic approaches toward the LPS inner core region
require the production of multigram quantities of the higher
carbon sugars Hep and Kdo followed by transformation into
suitable building blocks. Previous synthetic efforts toward LPS
inner core oligosaccharides covered the basic structural motifs
of the inner core region.12,13 The LPS inner core structures are
highly branched, and target-oriented synthesis is often not
flexible enough to accommodate the synthesis of mutiple
branched oligosaccharides. A general strategy for the diversity-
oriented synthesis of a library of LPS inner core oligosacchar-
ides would be ideal to rapidly access these molecules.
RESULTS AND DISCUSSION
■
The chemical synthesis of several LPS oligosaccharides is
challenging as it requires the formation of various glycosidic
linkages within the steric constraints of the highly branched
inner core region. To date, target-oriented strategies have been
employed for the synthesis of structurally closely related
molecules.12,13 The efficient synthesis of diverse LPS inner core
oligosaccharides necessitates a general, diversity-oriented
strategy relying on common modules.16
It was envisaged for oligosaccharides 4−7 to be prepared
from common disaccharide 8 that is decorated with the
orthogonal protecting groups levulinate (Lev)17 and isopropy-
lidene acetal,18 respectively (Scheme 1). Disaccharide 8 can be
Here, we report the synthesis of LPS inner core
oligosaccharides 1-7 using a general and flexible approach
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Scheme 1. Retrosynthetic Analysis of LPS Core Oligosaccharides 4−7
synthesized in gram scale by coupling heptose trichloroaceti-
midate19 9 with Kdo building block 10. Thus, a foundation for
the extension with various monosaccharide and branched
disaccharide residues will be laid. The Kdo moiety of
disaccharide 8 possesses a N-benzyl-N-benzyloxycarbonyl
pentyl linker20 for further conjugation to a carrier protein.
Flexible building blocks to install highly branched sugar
residues of LPS include Hep building blocks 11−16, and
Ara4N building block 1713d (Scheme 1). All required Hep
building blocks 11−16 were to be accessed by modification of
Hep intermediates 18 and 19 obtained by a de novo synthesis
we developed recently.13e,21 Based on common disaccharide 8,
installation of the terminal α-linked Hep residue of 4 should be
straightforward by using building blocks 11 or 12. Removal of
all protecting groups of the newly formed trisaccharide is
expected to give target trisaccharide 4. The α-(1→7) linked
diheptose moiety of 5 will be constructed by employing Hep
building blocks 11 and 15. The α-(1→2) linked diheptose
moiety of 6 will be prepared by glycosylation of Hep building
blocks 13 or 14 with Hep 16. The resulting α-(1→7) and α-
(1→2) linked diheptoses are converted to the glycosyl N-
phenyl trifluoroacetimidate22 for [2 + 2] couplings with the
building block produced by removal of the Lev group in 8. The
[2 + 2] couplings will rely on the anomeric effect23 of Hep
assisted by solvent effects24 to ensure stereoselectivity and will
give access to target tetrasaccharides 5 and 6 after global
deprotection. The β-linked Ara4N moiety of 7 will be regio-
and stereoselectively installed at C-8 position of the Kdo 7,8-
vicinal diol derived from 8 and Ara4N building block 17. The
required β-(1→8) linked trisaccharide containing Ara4N will be
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Scheme 2. Synthesis of the Conserved Inner Core Trisaccharide 4
Scheme 3. Synthesis of the Inner Core Tetrasaccharide 5 of Y. pestis
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Scheme 4. Synthesis of the Inner Core Tetrasaccharide 6 of H. influenzae
transformed into a trisaccharide building block with a C-3
hydroxyl group after Lev cleavage. Heptosylation of the
resulting trisaccharide with 12 will provide target tetrasacchar-
ide 7 following global deprotection.
The synthesis of tetrasaccharide 5 of Y. pestis was based on
known intermediate 19.21 Selective benzylation of the C-2
position of 19 with benzyl bromide and silver(I) oxide gave
lactone 22 in 70% yield (Scheme 3). Reduction of 22 with
lithium tri-tert-butoxyaluminium hydride28 followed by levuli-
noylation of both hydroxyl groups provided heptose 23 in 53%
yield over two steps. Analysis of the NMR spectra of 23
confirmed the location of the benzyl and levulinoyl groups.26
The silyl ether in 23 was cleaved using HF·pyridine to afford
heptose 15. Coupling of Hep 11 with Hep 15 using NIS and
TMSOTf as promoters29 proceeded smoothly to afford α-(1→
7)-linked disaccharide 24 in 79% yield. Selective cleavage of the
anomeric levulinate in 24 with ammonia,30 followed by N-
phenyl trifluoroacetimidate formation produced disaccharide
building block 25. A TMSOTf catalyzed [2 + 2] coupling of N-
phenyl trifluoroacetimidate 25 and 20 furnished tetrasaccharide
26 in 67% yield. The desired configuration of tetrasaccharide
26 was confirmed by the coupling constants between C-1 and
H-1 of the corresponding heptoses (1JC−H = 174.0, 179.4 and
175.8 Hz, respectively).26 Final deprotection of 26 involved
acidic isopropylidene acetal cleavage, saponification, and
hydrogenolysis to provide target tetrasaccharide 5 in 71%
yield over three steps.
The synthesis of the α-(1→2)-linked diheptose moiety in
tetrasaccharide 6 of H. influenza started from Hep 2721 that can
be accessed by reduction of Hep intermediate 18 with lithium
tri-tert-butoxyaluminium hydride. Cleavage of the silyl ethers in
27 with HF·pyridine and subsequent acetylation gave 28 in
85% yield (Scheme 4). Ester 28 was deacetylated selectively to
afford the heptose hemiacetal that was treated with
trichloroacetonitrile and N-phenyl trifluoroacetimidoyl chloride
respectively to furnish Hep building blocks 13 and 14.
Coupling of Hep trichloroacetimidate 13 and Hep 16 produced
Executing on this plan, disaccharide 8 was prepared using
Hep trichloroacetimidate 9 and Kdo building block 10,
employing an efficient glycosylation protocol that was
developed specifically to meet the challenge.13e For that
purpose, protecting groups, anomeric leaving groups, and
glycosylation conditions were fine-tuned for the construction of
this α-(1→5) linked Hep-Kdo linkage. The robust glycosylation
finally produced gram amounts of disaccharide 8. Removal of
the Lev group in 8 using hydrazine in a mixture of acetic acid,
pyridine and dichloromethane gave disaccharide 20 in 82%
yield (Scheme 2). The solvent mixture acts as a buffer such that
all other ester groups remained and no side products were
formed.
Hep thioglycoside 1113e was transformed to the correspond-
ing N-phenyl trifluoroacetimidate glycosylating agent 12 in 74%
yield over two steps (Scheme 2). Condensation of Hep 12 with
disaccharide 20 was catalyzed by TMSOTf and produced
trisaccharide 21 in 73% yield as the α-anomer. The
configuration of trisaccharide 21 was confirmed by the coupling
constants between C-1 and H-1 of the corresponding heptoses
(1JC−H = 176.4 and 176.0 Hz, respectively).25,26 Global
deprotection of 21 was accomplished via a three-step procedure
to afford trisaccharide 4. Acidic cleavage of the isopropylidene
acetal group with aqueous acetic acid at 70 °C was followed by
saponification of all esters by reaction with aqueous sodium
hydroxide. Finally, hydrogenolysis of the benzyl, p-bromoben-
zyl (PBB)27 and benzyloxycarbonyl groups over Pd/C in a
mixture of methanol and water with a catalytic amount of acetic
acid produced 4 (75% over three steps).
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Scheme 5. Synthesis of the Proteus Inner Core Tetrasaccharide 7
Figure 3. Confirmation of structural integrity of LPS core oligosaccharides 4−7 by NMR.
a disappointingly low yield of the α-(1→2)-linked disaccharide
due to extensive hydrolysis of 13. Coupling of Hep N-phenyl
trifluoroacetimidate 14 and Hep 16 was more productive in the
presence of TMSOTf. Treatment of the newly formed
disaccharide with NBS gave disaccharide 29 in 56% yield
over two steps, allowing for the separation of disaccharide 29
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from the remaining derivatives of monosaccharide 16 at this
step. Disaccharide hemiacetal 29 was converted into N-phenyl
trifluoroacetimidate 30 that engaged in a [2 + 2] coupling with
disaccharide 20. TMSOTf activation of 30 in CH2Cl2/Et2O
provided the desired α-linked tetrasaccharide 31 in 65% yield.
The three step global deprotection of 31 gave target
tetrasaccharide 6 in 75% yield over three steps. The
configuration of tetrasaccharide 6 was confirmed by the
coupling constants between C-1 and H-1 of the corresponding
heptoses (1JC−H = 173.4, 172.2 and 175.8 Hz, respectively).26
Synthetic work toward the tetrasaccharide 7 of Proteus
commenced with the removal of the isopropylidene acetal
group of disaccharide 8 by treatment with aqueous acetic acid
at 70 °C, without affecting levulinoyl, acetyl, benzyl, p-
bromobenzyl, benzyloxycarbonyl and methyl carboxylate
groups, to afford the corresponding 7,8-vicinal diol 32 (Scheme
5). Regio- and stereoselective glycosylation of diol 32 with
Ara4N building block to trisaccharide 33 proved challenging.
Thus, leaving group, catalyst, temperature and solvent effects
were optimized to obtain a satisfactory selectivity. When 1.6
equiv. Ara4N N-phenyl trifluoroacetimidate 1713d was used,
glycosylation with disaccharide 32 activated by TMSOTf in a
mixture of CH2Cl2/Et2O at −78 °C provided the desired β-
(1→8) linked trisaccharide 33 in 58% yield as the major
product, albeit accompanied with traces of the anomeric
stereoisomer and regioisomers as well as the 7,8-di-Ara4N
byproduct. The anomeric configuration of the Ara4N residue in
33 was assigned as a 1,2-cis-β-glycosidic linkage based on the
1−7 of LPS, equipped with a pentyl amine linker at the
reducing end, were obtained for immunological studies.
CONCLUSION
■
Here, we describe a flexible synthetic strategy for the diversity-
oriented synthesis of branched LPS core oligosaccharides. This
novel approach provided access to densely sterically crowded
oligosaccharides such as common core trisaccharide 4 found in
most Gram-negative bacteria and the conserved tetrasaccharide
5−7 in Y. pestis, H. influenzae and Proteus. The diversity-
oriented approach relied on the recruitment of a common
orthogonal protected disaccharide 8 that was extended with
various flexible modules including unique Hep and Ara4N
monosaccharides and branched Hep-Hep disaccharides. The
regio- and stereoselective extension of the Kdo 7,8-diol with
Ara4N proved challenging but was accomplished by identi-
fication of appropriate reaction conditions. This synthetic
strategy is modular and flexible so as to access other LPS
oligosaccharides consisting of Hep and Kdo rapidly. These
diverse LPS core oligosaccharides, with a pentyl amine linker at
the reducing end, are currently undergoing immunological
assessment.
EXPERIMENTAL SECTION
■
Reagents and General Procedures. Commercial reagents were
used without further purification except where noted. Solvents were
dried and redistilled prior to use in the usual way. All reactions were
performed in oven-dried glassware with magnetic stirring under an
inert atmosphere unless noted otherwise. Analytical thin layer
chromatography (TLC) was performed on Kieselgel 60 F254 glass
plates precoated with a 0.25 mm thickness of silica gel. The TLC
plates were visualized with UV light and by staining with Hanessian
solution (ceric sulfate and ammonium molybdate in aqueous sulfuric
acid) or sulfuric acid-ethanol solution. Column chromatography was
performed on Fluka Kieselgel 60 (230−400 mesh). Optical rotations
(OR) were measured with a Schmidt & Haensch UniPol L1000
coupling constant between H-1′ and H-2′ of Ara4N (JH‑1′,H‑2′
=
3.2 Hz, δH‑1′ = 4.88 ppm).26 Acetylation of the 7-hydroxyl
group in 33 with acetic anhydride in CH2Cl2 gave trisaccharide
34 in 83% yield. The attachment site of Ara4N in 33 was
confirmed by the low-field chemical shift of 7″-H of Kdo
residue in 34 due to the O-7″ acetyl moiety (δH‑7″ = 5.03
ppm).26 Unmasking of 34 afforded trisaccharide 35 in 82%
yield using hydrazine and acetic acid. A TMSOTf-catalyzed
heptosylation of trisaccharide 35 with heptose building block
12 afforded tetrasaccharide 36 in 81% yield as the α-anomer.
Removal of all ester groups in 36 with aqueous sodium
hydroxide, followed by hydrogenolysis of the azide, benzyl, PBB
and benzyloxycarbonyl groups over Pd/C in a mixture of
methanol, water and acetic acid, furnished target tetrasaccharide
7 in 80% yield over two steps. The configuration of
tetrasaccharide 7 was confirmed by the coupling constants
between C-1 and H-1 of the corresponding heptoses and
Ara4N (1JC−H = 174.6, 171.0 and 171.0 Hz, respectively).26 The
amine of the aminopentyl linker in 7 is more reactive than the
amine of 4-amino-4-deoxy-L-Ara4N moiety,31 therefore the
amine of the artificial linker of 7 will be selectively conjugated
to a carrier protein.
polarimeter at a concentration (c) expressed in g/100 mL. 1H and 13
C
NMR spectra were measured with a Varian 400-MR or Varian 600-MR
spectrometer with Me4Si as the internal standard. Multiplicities are
quoted as singlet (s), broad singlet (br s), doublet (d), doublet of
doublets (dd), triplet (t), or multiplet (m). Spectra were assigned
using COSY and HSQC. All NMR chemical shifts (δ) were recorded
in ppm and coupling constants (J) were reported in Hz. High-
resolution ESI and MALDI mass spectra were recorded on an IonSpec
Ultra instrument. MALDI-TOF spectra were recorded on a Bruker
Daltonics Autoflex Speed, using 2,4,6-trihydroxyacetophenone
(THAP) as the matrix.
Methyl [N-Benzyl−benzyloxycarbonyl-5-aminopentyl (2-O-
acetyl-3,7-di-O-benzoyl-4-O-para-bromobenzyl-6-O-benzyl-L-
glycero-α-D-manno-heptopyranosyl)-(1→7)-(2,6-di-O-benzyl-3-
O-levulinoyl-4-O-para-bromobenzyl-L-glycero-α-D-manno-hep-
topyranosyl)-(1→3)-(2,7-di-O-acetyl-4-O-para-bromobenzyl-6-
O-benzyl-L-glycero-α-D-manno-heptopyranosyl)-(1→5)-4-O-
benzyl-7,8-O-isopropylidene-3-deoxy-α-D-manno-oct-2-
ulopyranosid]onate (26). To a stirred mixture of building block 25
(42 mg, 27 μmol), building block 20 (23 mg, 19 μmol), and freshly
activated 4 Å MS in anhydrous diethyl ether and dichloromethane (1/
1, v/v, 2 mL) at 0 °C, was added TMSOTf in CH2Cl2 (0.05 M, 56 μL)
under nitrogen. The temperarture was allowed to warm to room
temperature and the stirring continued for 1 h. The mixture was
quenched with Et3N, and filtered. The filtrate was concentrated in
vacuo to give a residue that was purified by silica gel column
Monosaccharide 1 and disaccharide 3 were prepared by
standard global deprotection of the corresponding mono- and
disaccharide skeleton (see Supporting Information for details,
SI). Monosaccharide 2 was prepared by attachment of Hep 11
to the N-benzyl-N-benzyloxycarbonyl pentyl linker followed by
global deprotection (see SI for details).
1
The H NMR spectra, especially the chemical shifts of the
chromatography (toluene/EtOAc: 7/1) to afford 26 (28 mg, 67%) as a
anomeric protons, of synthetic antigens 4−7 were found to be
nearly identical to those reported for isolated LPS fragments
(Figure 3).32−34 In addition, assignment of the NMR signals of
1−7 confirmed the structural integrity of the desired
oligosaccharides (see SI for details). The synthetic antigens
1
white solid: [α]20 = +15.9 (c 0.25, CHCl3); H NMR (600 MHz,
D
Pyridine-d5) δ 8.25−7.21 (m, 57 H, Ar), 6.34 (dd, J = 3.0, 10.2 Hz, 1
H, H-3), 6.11 (br s, 1 H, H-2), 5.92 (br s, 1 H, H-1″), 5.88 (br s, 1 H,
H-1), 5.81 (br s, 1 H, H-2″), 5.76 (dd, J = 3.0, 7.2 Hz, 1 H, H-3′), 5.63
(br s, 1 H, H-1′), 5.62 (d-like, J = 12.0 Hz, 1 H), 5.41−5.31 (m, 3 H),
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5.17 (m, 1 H), 5.08−4.68 (m, 27 H), 4.64−4.54 (m, 5 H), 4.45 (m, 1
H), 4.36 (d-like, J = 11.4 Hz, 1 H), 4.25 (m, 2 H), 4.20 (t, J = 3.0 Hz, 1
H), 4.00 (d-like, J = 8.4 Hz, 1 H), 3.82 (s, 3 H, C(O)OCH3), 3.60 (m,
2 H), 3.43 (m, 1 H), 3.32 (m, 1 H), 2.69 (m, 2 H, C(O)CH2CH2C-
(O)), 2.63 (m, 2 H, C(O)CH2CH2C(O)), 2.52 (m, 1 H, H-3‴e), 2.35
(t, J = 12.0 Hz, 1 H, H-3‴a), 2.27 (s, 3 H, C(O)CH3), 2.01 (s, 3 H,
C(O)CH3), 1.98 (s, 3 H, C(O)CH3), 1.96 (s, 3 H, C(O)CH3), 1.65
(m, 4 H, CCH2C), 1.42 (s, 3 H, C(CH3)2), 1.39 (s, 3 H, C(CH3)2),
1.29 (m, 2 H, CCH2C); 13C NMR (150 MHz, Pyridine-d5) δ 206.8,
173.2, 171.3, 171.1, 170.2, 169.6, 166.9, 166.6, 150.9, 140.7, 140.5,
140.1, 139.6, 139.5, 139.3, 139.1, 138.9, 138.7, 136.4, 135.8, 134.3,
134.0, 132.4, 132.3, 131.4, 130.9, 130.7, 130.6, 130.5, 130.2, 129.8,
129.7, 129.6, 129.5, 129.4, 129.3, 129.0, 128.9, 128.8, 128.6, 128.5,
128.4, 128.3, 128.2, 124.4, 123.8, 122.3, 122.0, 110.7 (C(CH3)2), 102.1
(C-1′, JC,H = 174.0 Hz), 100.9 (C-2‴), 98.1 (C-1, JC,H = 179.4 Hz),
97.9 (C-1″, JC,H = 175.8 Hz), 79.7, 77.9, 77.3, 76.8, 76.2, 76.0, 75.7,
75.3, 74.9, 74.8, 74.5, 74.2, 74.1, 74.0, 73.9, 73.8, 73.7, 73.6, 73.5, 73.3,
72.4, 72.1, 71.7, 71.3, 71.2, 68.7, 67.8, 67.6, 67.5, 64.7, 53.4, 38.5, 33.4
(C-3‴), 30.6, 30.4, 30.1, 29.2, 27.8, 25.7, 24.4, 21.5, 21.4, 21.3; HRMS
(ESI) m/z calcd for C134H144Br3NO35Na [M + Na]+ 2589.6980, found
2589.7470.
Methyl [N-Benzyl−benzyloxycarbonyl-5-aminopentyl
(2,3,7-tri-O-acetyl-4-O-para-bromobenzyl-6-O-benzyl-L-glyc-
ero-α-D-manno-heptopyranosyl)-(1→2)-(3,7-di-O-benzoyl-4-O-
para-bromo-benzyl-6-O-benzyl-L-glycero-α-D-manno-hepto-
pyranosyl)-(1→3)-(2,7-di-O-acetyl-4-O-para-bromobenzyl-6-O-
benzyl-L-glycero-α-D-manno-heptopyranosyl)-(1→5)-4-O-ben-
zyl-7,8-O-isopropylidene-3-deoxy-α-D-manno-oct-2-
ulopyranosid]onate (31). To a stirred mixture of building block 30
(45 mg, 0.032 mmol), building block 20 (23 mg, 0.019 mmol), and
freshly activated 4 Å MS in anhydrous diethyl ether and dichloro-
methane (1/1, v/v, 2.8 mL) at 0 °C, was added TMSOTf in CH2Cl2
(0.05 M, 64 μL) under nitrogen. The temperature was allowed to
warm to room temperature and the stirring continued for 1 h. The
mixture was quenched with Et3N, and filtered. The filtrate was
concentrated in vacuo to give a residue that was purified by silica gel
Et3N, and filtered. The filtrate was concentrated in vacuo to give a
residue that was purified by silica gel column chromatography
(hexane/EtOAc: 6/5 to 1/1) to afford 33 (138 mg, 58%) as a white
foam: [α]20D = +59.6 (c 0.4, CHCl3); 1H NMR (400 MHz, CDCl3) δ
7.45−7.07 (m, 34 H), 5.38 (dd, J = 3.2, 9.6 Hz, 1 H, H-3), 5.30 (br s, 1
H, H-2), 5.24 (br s, 1 H, H-1), 5.16 (m, 2 H, BnCH2OC(O)N), 4.88
(d, J = 3.2 Hz, 1 H, H-1′), 4.80 (m, 2 H), 4.72−4.61 (m, 4 H), 4.54−
4.37 (m, 7 H), 4.30 (m, 1 H), 4.20 (d-like, J = 11.6 Hz, 1 H), 4.12 (m,
1 H), 4.05−3.94 (m, 4 H), 3.87−3.80 (m, 4 H), 3.75 (s, 3 H,
C(O)OCH3), 3.71 (m, 1 H), 3.57 (m, 2 H), 3.39 (m, 2 H), 3.21 (m, 3
H), 2.73−2.57 (m, 2 H, C(O)CH2CH2C(O)), 2.43 (m, 2 H,
C(O)CH2CH2C(O)), 2.27 (m, 1 H, H-3″e), 2.13 (s, 3 H), 2.08 (s, 3
H), 2.00 (m, 1 H, H-3″a), 1.97 (s, 3 H), 1.46 (m, 4 H), 1.21 (m, 2 H);
HRMS (ESI) m/z calcd for C85H97BrN4O23Na [M + Na]+ 1643.5625,
found 1643.5727. To a solution of trisaccharide 33 (100 mg, 0.062
mmol) and DMAP (60 mg, 0.491 mmol) in CH2Cl2 (9 mL), was
added Ac2O (0.6 mL). After being stirred at room temperature
overnight, the mixture was washed with saturated aqueous NaHCO3,
and brine. The organic layer was dried over Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by silica gel column
chromatography (hexane/EtOAc: 1/1) to give 34 (85 mg, 83%) as a
1
colorless syrup: [α]20 = +56.5 (c 0.4, CHCl3); H NMR (400 MHz,
D
CDCl3) δ 7.43−7.04 (m, 34 H), 5.37 (dd, J = 3.2, 9.6 Hz, 1 H, H-3),
5.23 (br s, 1 H, H-2), 5.14 (m, 2 H, BnCH2OC(O)N), 5.03 (m, 1 H,
H-7″), 4.86 (d, J = 3.2 Hz, 1 H, H-1′), 4.81 (d, J = 1.2 Hz, 1 H, H-1),
4.75 (d-like, J = 12.0 Hz, 1 H, OCH2Ar), 4.69−4.60 (m, 4 H), 4.53−
4.33 (m, 7 H), 4.22−3.87 (m, 11 H), 3.76 (m, 3 H), 3.74 (s, 3 H,
C(O)OCH3), 3.54 (m, 1 H), 3.47 (m, 1 H), 3.19−3.12 (m, 3 H), 2.76
(m, 1 H, C(O)CH2CH2C(O)), 2.60 (m, 1 H, C(O)CH2CH2C(O)),
2.43 (m, 2 H, C(O)CH2CH2C(O)), 2.26 (m, 1 H, H-3″e), 2.13 (s, 3
H), 2.10 (s, 3 H), 2.00 (m, 1 H, H-3″a), 1.98 (s, 3 H), 1.94 (s, 3 H),
1.42 (m, 4 H), 1.21 (m, 2 H); 13C NMR (100 MHz, CDCl3) δ 206.4,
171.5, 170.6, 170.0, 169.7, 168.3, 156.6, 156.1, 138.4, 137.9, 137.7,
137.4, 136.7, 131.3, 129.1, 129.0, 128.8, 128.5, 128.4, 128.3, 128.2,
128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.2, 127.1, 121.3, 98.8
(C-2″), 98.3 (C-1), 98.1 (C-1′), 77.3, 75.9, 73.3, 73.2, 72.9, 72.8, 72.7,
72.4, 72.1, 71.9, 70.4, 70.3, 70.0, 67.1, 65.3, 65.1, 63.5, 60.7, 59.6, 52.4,
50.3, 50.0, 47.0, 46.1, 37.8, 31.9 (C-3″), 29.8, 29.7, 29.3, 27.9, 27.5,
23.3, 21.1, 21.0; HRMS (ESI) m/z calcd for C87H99BrN4O24Na [M +
Na]+ 1685.5730, found 1685.5767.
column chromatography (toluene/EtOAc: 10/1 to 8/1) to afford 31
1
(30 mg, 65%) as a white foam: [α]20 = +10.4 (c 1.0, CHCl3); H
D
NMR (600 MHz, Pyridine-d5) δ 8.54−6.87 (m, 52 H, Ar), 6.15 (br s,
1 H, H-1′), 6.10 (dd, J = 3.0, 9.6 Hz, 1 H), 6.05 (m, 2 H), 5.82 (br s, 1
H, H-1), 5.69 (t-like, J = 2.4 Hz, 1 H), 5.46 (dd, J = 4.2, 10.8 Hz, 1 H),
5.39 (m, 2 H), 5.11−4.86 (m, 24 H), 4.81−4.68 (m, 4 H), 4.62−4.48
(m, 5 H), 4.34 (m, 2 H), 4.23 (m, 1 H), 4.00 (m, 1 H), 3.96 (s, 3 H,
C(O)OCH3), 3.82 (dd, J = 6.0, 8.4 Hz, 1 H), 3.74 (m, 1 H), 3.50−
3.44 (m, 3 H), 3.30 (m, 1 H), 2.76 (m, 1 H, H-3″e), 2.53 (t, J = 10.8
Hz, 1 H, H-3″a), 2.23 (s, 3 H, C(O)CH3), 2.14 (s, 6 H, C(O)CH3),
2.13 (s, 3 H, C(O)CH3), 1.98 (s, 3 H, C(O)CH3), 1.58 (m, 4 H,
CCH2C), 1.41 (s, 3 H, C(CH3)2), 1.34 (s, 3 H, C(CH3)2), 1.29 (m, 2
H, CCH2C); 13C NMR (150 MHz, Pyridine-d5) δ171.9, 171.8, 171.7,
171.6, 171.2, 170.0, 168.0, 167.0, 151.5, 150.7, 141.1, 141.0, 140.2,
140.1, 139.9, 139.6, 139.5, 139.2, 137.0, 136.4, 134.9, 134.7, 133.1,
133.0, 132.5, 132.1, 131.7, 131.6, 131.4, 130.9, 130.6, 130.5, 130.4,
130.3, 130.2, 130.1, 130.0, 129.9, 129.6, 129.5, 129.4, 129.3, 129.2,
129.0, 128.9, 128.8, 125.0, 124.4, 122.9, 122.7, 122.3, 111.1
(C(CH3)2), 100.9 (C-1′, JC,H = 176.0 Hz), 100.7 (C-2″), 98.9 (C-1,
JC,H = 176.4 Hz), 80.3, 77.4, 76.8, 76.5, 76.0, 75.6, 75.3, 75.1, 74.9,
74.8, 74.3, 74.2, 74.1, 74.0, 73.9, 73.7, 73.4, 73.1, 72.9, 71.7, 71.6, 69.3,
68.4, 68.2, 66.6, 65.1, 64.2, 53.8, 33.4 (C-3″), 31.2, 30.8, 28.2, 26.5,
24.9, 22.3, 22.2, 22.1, 22.0, 21.8; HRMS (MALDI) m/z calcd for
C126H136Br3NO35Na [M + Na]+ 2485.6354, found 2485.5990.
Methyl 2,7-Di-O-acetyl-3-O-levulinoyl-4-O-para-bromoben-
zyl-6-O-benzyl-L-glycero-α-D-manno-heptopyranosyl-(1→5)-[4-
azido-2,3-di-O-benzyl-4-deoxy-β-L-arabinopyranosyl-(1→8)]-
(N-benzyl−benzyloxycarbonyl-5-aminopentyl 7-O-acetyl-4-O-
benzyl-3-deoxy-α-D-manno-oct-2-ulopyranoside)onate (34).
To a stirred mixture of building block 17 (130 mg, 0.247 mmol),
building block 32 (190 mg, 0.148 mmol), and freshly activated 4 Å MS
in anhydrous diethyl ether and dichloromethane (1/1, v/v, 20 mL) at
−78 °C, was added TMSOTf in CH2Cl2 (0.05 M, 164 μL) under
nitrogen. The temperarture was allowed to warm to room temperature
and the stirring continued for 2 h. The mixture was quenched with
Methyl 2-O-Acetyl-3,7-di-O-benzoyl-4-O-para-bromoben-
zyl-6-O-benzyl-L-glycero-α-D-manno-heptopyranosyl-(1→3)-
2,7-di-O-acetyl-4-O-para-bromobenzyl-6-O-benzyl-L-glycero-
α-D-manno-heptopyranosyl-(1→5)-[4-azido-2,3-di-O-benzyl-4-
deoxy-β-L-arabinopyranosyl-(1→8)]-(N-benzyl−benzyloxycar-
bonyl-5-aminopentyl 7-O-acetyl-4-O-benzyl-3-deoxy-α-D-
manno-oct-2-ulopyranoside)onate (36). To a stirred mixture of
building block 12 (34 mg, 38 μmol), building block 35 (30 mg, 19
μmol), and freshly activated 4 Å MS in anhydrous dichloromethane (3
mL) at 0 °C, was added TMSOTf in CH2Cl2 (0.05 M, 62 μL) under
nitrogen. The temperature was allowed to warm to room temperature
and the stirring continued for 1 h. The mixture was quenched with
Et3N, and filtered. The filtrate was concentrated in vacuo to give a
residue that was purified by silica gel column chromatography
(hexane/EtOAc: 5/2 to 2/1) to afford 36 (35 mg, 81%) as a white
1
foam: [α]20 = +24.5 (c 0.3, CHCl3); H NMR (400 MHz, Pyridine-
d5) δ 8.46−D7.10 (m, 53 H), 6.15 (dd, J = 3.2, 9.6 Hz, 1 H, H-3), 5.97
(br s, 1 H, H-2′), 5.93 (m, 1 H, H-2), 5.69 (s, 1 H, H-1), 5.50 (m, 1 H,
H-7‴), 5.48 (m, 1 H), 5.42 (s, 1 H, H-1′), 5.36 (m, 2 H,
BnCH2OC(O)N), 5.29 (d-like, J = 11.6 Hz, 1 H), 5.26 (d, J = 3.2 Hz,
1 H, H-1″), 5.13 (m, 1 H), 5.05−4.96 (m, 10 H), 4.86−4.72 (m, 8 H),
4.70−4.59 (m, 5 H), 4.53 (m, 1 H), 4.47 (m, 2 H), 4.38 (m, 1 H), 4.24
(m, 2 H), 4.15−4.04 (m, 3 H), 3.93 (m, 1 H), 3.84 (m, 1 H), 3.79 (s, 3
H, C(O)OCH3), 3.68 (m, 1 H), 3.49 (m, 1 H), 3.37 (m, 1 H), 3.27
(m, 1 H), 2.72 (d-like, J = 10.0 Hz, 1 H, H-3‴e), 2.50 (t, J = 12.4 Hz, 1
H, H-3‴a), 2.17 (s, 3 H), 2.14 (s, 3 H), 2.05 (s, 3 H), 1.99 (s, 3 H),
1.60 (m, 4 H), 1.36 (m, 2 H); 13C NMR (100 MHz, Pyridine-d5) δ
170.8, 170.5, 170.3, 170.0, 168.8, 166.9, 165.9, 139.6, 139.4, 139.3,
139.1, 138.9, 138.7, 138.3, 138.1, 136.1, 135.1, 133.8, 133.5, 131.8,
131.6, 130.9, 130.4, 130.3, 130.0, 129.8, 129.7, 129.5, 129.3, 129.0,
6269
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Journal of the American Chemical Society
Article
128.9, 128.8, 128.5, 128.3, 128.2, 128.1, 127.9, 127.6, 125.8, 124.1,
123.7, 123.0, 121.6, 121.5, 99.8 (C-2‴), 99.6 (C-1, JC,H = 176.0 Hz),
98.9 (C-1′, JC,H = 174.8 Hz), 98.7 (C-1″, JC,H = 170.4 Hz), 79.9, 77.8,
77.1, 76.7, 76.2, 75.9, 75.5, 74.2, 74.0, 73.9, 73.3, 73.1, 72.9, 72.8, 72.4,
72.3, 72.2, 71.3, 70.8, 70.7, 70.4, 67.2, 66.0, 64.8, 64.7, 64.1, 61.0, 60.4,
52.5, 50.7, 50.3, 47.5, 46.7, 32.7 (C-3‴), 30.1, 29.9, 28.6, 28.0, 23.8,
21.2, 21.0, 20.8, 20.5; HRMS (ESI) m/z calcd for
C119H126Br2N4O31Na [M + Na]+ 2290.6684, found 2290.6683.
L-Glycero-α-D-manno-heptopyranosyl-(1→7)-L-glycero-α-D-
manno-heptopyranosyl-(1→3)-L-glycero-α-D-manno-heptopyr-
anosyl-(1→5)-2-(5-amino)pentyl-3-deoxy-α-D-manno-oct-2-
ulopyra nosidonic acid (5). A solution of tetrasaccharide 26 (20 mg,
7.79 μmol) in acetic acid/water (8/1, v/v, 2.7 mL) was stirred at 70 °C
overnight until TLC indicated complete conversion of starting
material. The mixture was coevaporated with toluene and dried in
vacuo to give the corresponding diol as a white solid. The resulting diol
was dissolved in a mixture of dioxane, methanol and 1 M aq NaOH
(3/1/1, v/v/v, 2.00 mL). After stirring at room temperature overnight,
the reaction mixture was diluted with methanol and neutralized with
Amberlite IR120 H+ resin. After filtration, the filtrate was concentrated
in vacuo and eluted through Sephadex LH-20 column (CH2Cl2/
MeOH: 1/1) to give the corresponding tetrasaccharide as a white
solid. A mixture of the resulting tetrasaccharide and Pd/C (100 mg,
10%) in methanol, water and acetic acid (65/13/1, v/v/v, 4.86 mL)
was stirred under an atmosphere of H2 at room temperature for 24 h.
Filtration, concentration in vacuo and elution through Sephadex LH-20
column (H2O) provided 5 (5 mg, 71% over three steps) as a white
solid: [α]20D = +83.4 (c 0.3, H2O); 1H NMR (600 MHz, D2O) δ 5.13
(s, 1 H, H-1″), 5.11 (s, 1 H, H-1′), 4.98 (s, 1 H, H-1), 4.17−4.12 (m, 4
H, H-4‴), 4.06−3.95 (m, 8 H), 3.91−3.62 (m, 15 H), 3.41 (m, 1 H,
OCH2), 3.29 (m, 1 H, OCH2), 3.00 (t, J = 7.8 Hz, 2 H, NCH2), 2.07
(dd, J = 4.2, 12.6 Hz, 1 H, H-3‴e), 1.83 (t, J = 12.6 Hz, 1 H, H-3‴a),
1.68 (m, 2 H, CCH2C), 1.61 (m, 2 H, CCH2C), 1.42 (m, 2 H,
CCH2C); 13C NMR (150 MHz, D2O) δ 177.7 (C(O)), 105.2 (C-1″,
42.0 (NCH2), 37.5 (C-3‴), 30.8, 29.1, 25.0; HRMS (MALDI) m/z
calcd for C34H61NO26Na [M + Na]+ 922.3380, found 922.3330.
L-Glycero-α-D-manno-heptopyranosyl-(1→3)-L-glycero-α-D-
manno-heptopyranosyl-(1→5)-[4-amino-4-deoxy-β-L-arabino-
pyranosyl-(1→8)]-2-(5-amino)pentyl-3-deoxy-α-D-manno-oct-
2-ulopy ranosidonic acid (7). Tetrasaccharide 36 (33 mg, 0.015
mmol) was dissolved in a mixture of dioxane, methanol and 1 M aq
NaOH (3/1/1, v/v/v, 2.0 mL). After stirring at room temperature
overnight, the reaction mixture was diluted with methanol and
neutralized with Amberlite IR120 H+ resin. After filtration, the filtrate
was concentrated in vacuo and eluted through Sephadex LH-20
column (CH2Cl2/MeOH: 1/1) to give the corresponding tetrasac-
charide as a white solid. A mixture of the resulting tetrasaccharide and
Pd/C (100 mg, 10%) in methanol, water and acetic acid (85/17/1, v/
v/v, 6.06 mL) was stirred under an atmosphere of H2 at room
temperature overnight. Filtration, concentration in vacuo and elution
through Sephadex LH-20 column (H2O) provided 7 (10 mg, 80%
1
over two steps) as a white solid: [α]20 = +87.4 (c 0.5, H2O); H
D
NMR (600 MHz, D2O) δ 5.24 (s, 1 H, H-1), 5.10 (s, 1 H, H-1′), 5.08
(s, 1 H, H-1″), 4.17 (m, 2 H, H-4‴), 4.13−3.89 (m, 13 H), 3.81−3.68
(m, 9 H), 3.60 (d-like, J = 9.6 Hz, 1 H), 3.39 (m, 1 H, OCH2), 3.33
(m, 1 H, OCH2), 3.03 (t, J = 7.8 Hz, 2 H, NCH2), 2.11 (dd, J = 4.2,
13.2 Hz, 1 H, H-3‴e), 1.87 (t, J = 12.6 Hz, 1 H, H-3‴a), 1.70 (m, 2 H,
CCH2C), 1.62 (m, 2 H, CCH2C), 1.44 (m, 2 H, CCH2C); 13C NMR
(150 MHz, D2O) δ 177.6 (C(O)), 104.3 (C-1, JC,H = 174.6 Hz), 104.0
(C-1′, JC,H = 171.0 Hz), 102.5 (C-2‴), 101.4 (C-1″, JC,H = 171.0 Hz),
79.0, 77.4, 74.6, 74.4, 73.3, 72.7, 71.5, 71.2, 70.4, 68.7, 68.2, 65.8, 60.8,
54.4, 42.0 (NCH2), 37.4 (C-3‴), 30.9, 29.2, 25.2; HRMS (ESI) m/z
calcd for C32H57N2O23 [M − H]− 837.3352, found 837.3374.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details, characterization data and copies of NMR
spectra for new compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
JC,H = 175.8 Hz), 103.9 (C-1′, JC,H = 173.4 Hz), 103.1 (C-1, JC,H
=
172.2 Hz), 102.5 (C-2‴), 82.7, 77.7, 75.2, 74.7, 74.1, 73.9, 73.4, 73.1,
72.7, 72.6, 72.5, 72.1, 71.9, 71.5, 71.4, 71.0, 69.0, 68.8, 68.4, 67.8, 66.0,
65.7, 65.6, 65.5, 42.0 (NCH2), 37.6 (C-3‴), 30.8, 29.1, 25.0; HRMS
(ESI) m/z calcd for C34H61NO26Na [M + Na]+ 922.3380, found
922.3325.
AUTHOR INFORMATION
■
Corresponding Author
peter.seeberger@mpikg.mpg.de
L-Glycero-α-D-manno-heptopyranosyl-(1→2)-L-glycero-α-D-
manno-heptopyranosyl-(1→3)-L-glycero-α-D-manno-heptopyr-
anosyl-(1→5)-2-(5-amino)pentyl-3-deoxy-α-D-manno-oct-2-
ulopyra nosidonic acid (6). A solution of tetrasaccharide 31 (11 mg,
4.46 μmol) in acetic acid/water (8/1, v/v, 1.3 mL) was stirred at 70 °C
for 7 h until TLC indicated complete conversion of starting material.
The mixture was coevaporated with toluene and dried in vacuo to give
the corresponding diol as a white solid. The resulting diol was
dissolved in a mixture of dioxane, methanol and 1 M aq NaOH (3/1/
1, v/v/v, 2.0 mL). After stirring at room temperature overnight, the
reaction mixture was diluted with methanol and neutralized with
Amberlite IR120 H+ resin. After filtration, the filtrate was concentrated
in vacuo and eluted through Sephadex LH-20 column (CH2Cl2/
MeOH: 1/1) to give the corresponding tetrasaccharide as a white
solid. A mixture of the resulting tetrasaccharide and Pd/C (100 mg,
10%) in methanol, water and acetic acid (68/17/1, v/v/v, 5.06 mL)
was stirred under an atmosphere of H2 at room temperature for 24 h.
Filtration, concentration in vacuo and elution through Sephadex LH-20
column (H2O) provided 6 (3 mg, 75% over three steps) as a white
solid: [α]20D = +47.2 (c 0.3, H2O); 1H NMR (600 MHz, D2O) δ 5.40
(d, J = 1.2 Hz, 1 H, H-1′), 5.10 (d, J = 1.2 Hz, H-1), 5.08 (d, J = 1.8
Hz, H-1″), 4.19−4.15 (m, 2 H, H-4‴), 4.11−3.94 (m, 12 H), 3.91−
3.85 (m, 2 H), 3.82 (m, 1 H), 3.78−3.63 (m, 10 H), 3.44 (m, 1 H,
OCH2), 3.30 (m, 1 H, OCH2), 3.02 (t, J = 7.2 Hz, 2 H, NCH2), 2.12
(dd, J = 4.2, 12.6 Hz, 1 H, H-3‴e), 1.86 (t, J = 12.6 Hz, 1 H, H-3‴a),
1.70 (m, 2 H, CCH2C), 1.63 (m, 2 H, CCH2C), 1.45 (m, 2 H,
CCH2C); 13C NMR (150 MHz, D2O) δ 177.7 (C(O)), 104.7 (C-1″,
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support from the Max Planck Society and the Korber
Foundation is gratefully acknowledged. We thank Dr. Claney L.
Pereira for critically editing this manuscript.
̈
REFERENCES
■
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JC,H = 173.4 Hz), 103.9 (C-1, JC,H = 172.2 Hz), 102.8 (C-1′, JC,H
=
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6270
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Journal of the American Chemical Society
Article
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