α-glycosylation by d -glucosamine-derived donors: Synthesis of heparosan and heparin analogues that interact with mycobacterial heparin-binding hemagglutinin

Article abstract of DOI:10.1021/ja302640p

Numerous biomolecules possess α-d-glucosamine as structural component. However, chemical glycosylations aimed at this backbone are usually not easily attained without generating the unwanted β-isomer. We report herein a versatile approach in affording full α-stereoselectivity built upon a carefully selected set of orthogonal protecting groups on a d-glucosaminyl donor. The excellent stereoselectivity provided by the protecting group combination was found independent of leaving groups and activators. With the trichloroacetimidate as the optimum donor leaving group, core skeletons of glycosylphosphatidyl inositol anchors, heparosan, heparan sulfate, and heparin were efficiently assembled. The orthogonal protecting groups were successfully manipulated to further carry out the total syntheses of heparosan tri- and pentasaccharides and heparin di-, tetra-, hexa-, and octasaccharide analogues. Using the heparin analogues, heparin-binding hemagglutinin, a virulence factor of Mycobacterium tuberculosis, was found to bind at least six sugar units with the interaction notably being entropically driven.

Full text of DOI:10.1021/ja302640p

Article
pubs.acs.org/JACS
α-Glycosylation by D-Glucosamine-Derived Donors: Synthesis of
Heparosan and Heparin Analogues That Interact with Mycobacterial
Heparin-Binding Hemagglutinin
Medel Manuel L. Zulueta,†,‡,# Shu-Yi Lin,†,§,# Ya-Ting Lin,§ Ching-Jui Huang,§ Chun-Chih Wang,§
Chiao-Chu Ku,† Zhonghao Shi,† Chia-Lin Chyan,*,∥ Deli Irene,∥ Liang-Hin Lim,∥ Tsung-I Tsai,†
Yu-Peng Hu,†,§ Susan D. Arco,‡ Chi-Huey Wong,† and Shang-Cheng Hung†,*,⊥
†Genomics Research Center, Academia Sinica, No. 128, Section 2, Academia Road, Taipei 115, Taiwan
‡Institute of Chemistry, University of the Philippines, Diliman, Quezon City 1101, Philippines
§Department of Chemistry, National Tsing Hua University, No. 101, Section 2, Kuang-Fu Road, Hsinchu 300, Taiwan
∥Department of Chemistry, National Dong Hwa University, No. 1, Sec. 2, Ta-Hsueh Road, Shoufeng, Hualien 974, Taiwan
⊥Department of Applied Chemistry, National Chiao Tung University, No. 1001, Ta-Hsueh Road, Hsinchu 300, Taiwan
S
* Supporting Information
ABSTRACT: Numerous biomolecules possess α-D-glucos-
amine as structural component. However, chemical glyco-
sylations aimed at this backbone are usually not easily attained
without generating the unwanted β-isomer. We report herein a
versatile approach in affording full α-stereoselectivity built
upon a carefully selected set of orthogonal protecting groups
on a D-glucosaminyl donor. The excellent stereoselectivity
provided by the protecting group combination was found
independent of leaving groups and activators. With the
trichloroacetimidate as the optimum donor leaving group, core skeletons of glycosylphosphatidyl inositol anchors, heparosan,
heparan sulfate, and heparin were efficiently assembled. The orthogonal protecting groups were successfully manipulated to
further carry out the total syntheses of heparosan tri- and pentasaccharides and heparin di-, tetra-, hexa-, and octasaccharide
analogues. Using the heparin analogues, heparin-binding hemagglutinin, a virulence factor of Mycobacterium tuberculosis, was
found to bind at least six sugar units with the interaction notably being entropically driven.
inositol (GPI)-anchors, which tether proteins of various
functions to the cell surface, contain D-glucosamine α1→6-
linked to myo-inositol (Figure 1).5 α-D-Glucosamine is also an
INTRODUCTION
■
The numerous important roles of carbohydrates in living
systems are underscored by their high abundance and
molecular diversity. Once regarded as merely structural support
and energy storage molecules, sugars are now acknowledged as
vital players in physiological and pathophysiological processes
making them potential drug development targets.1 They exist as
glycoconjugates at the surface of every known cell, interacting
and coordinating with various extracellular matrix elements.2
Several natural products also carry sugar moieties that influence
their biological potency.3 With fewer monomer types than
proteins, the exquisite complexity of carbohydrate polymers
primarily arises from the multiple potential linkage points
within the pyranosyl or furanosyl ring of each residue and the
chirality of the anomeric center generated on assembly.
Appreciation of the structure−function relationship of these
biopolymers requires well-defined compounds, which are,
unfortunately, difficult to isolate in sufficient quantity and
purity from Nature. Thus, synthetic oligosaccharide prepara-
tions offer a dependable solution to vast demands.4
Figure 1. Structures of the α-D-glucosamine-containing disaccharide
units found in GPI anchors, heparan sulfate, and heparin.
important feature, together with D-glucuronic and L-iduronic
acid, in the repeating disaccharide of the structurally related
heparan sulfate and heparin. Heparan sulfate is a linear
polysulfated polysaccharide ubiquitously distributed on the
Several important biomolecules carry D-glucosamine in α-
form as core component. For example, glycosylphosphatidyl-
Received: March 18, 2012
Published: May 15, 2012
© 2012 American Chemical Society
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cell surface as a component of proteoglycans. It is a known
mediator of several biologically important events including viral
and bacterial infection, cell growth, inflammation, wound
healing, tumor metastasis, lipid metabolism, and diseases of
the nervous system.6 Found mainly in mast cells, heparin is a
widely used clinical anticoagulant through its tight binding and
consequent activation of antithrombin III.7 On the basis of this
interaction, the synthetic pentasaccharide fondaparinux is now
effectively utilized for the prevention of venous thrombolitic
events following surgery.8 Alternating N-acetyl-α-D-glucos-
amine (GlcNAcα) and β-D-glucuronic acid all with 1→4
linkages are present in the capsular polysaccharide of
Escherichia coli strain K5.9 Termed heparosan, it is actively
being employed in the preparation of the more complex
heparan sulfate/heparin saccharides through chemoenzymatic
transformations.10 The highly immunogenic O-antigen of
several serotypes of Shigella contains GlcNAcα in their
repeating structure.11 GlcNAcα is also present in the cell-wall
teichoic acid of some Staphylococcus species.12 Aside from these
biopolymers, α-D-glucosamine is found in several natural
products such as antibiotics (e.g., tunicamycins and neo-
mycin)13 and bacterial antioxidants (e.g., mycothiol and
bacillithiol).14
including heparosan and heparin, which were both synthesized
in various lengths. With analogues of heparin at hand, we
further examined their interaction with heparin-binding
hemagglutinin (HBHA), a virulence factor of Mycobacterium
tuberculosis.25
RESULTS AND DISCUSSION
■
α-Stereoselective Glucosaminylation. Besides the famil-
iar 2-C neighboring group assistance, other functional groups
also influence the stereoselectivity of glycosylation. For
example, remote acyl moieties, bulky substituents, and fused
diol protecting groups all demonstrated potent stereodirecting
effects in a variety of glycosyl donors.26 Because the D-
glucosamine residue of heparin/heparan sulfateour motiva-
tion for this explorationmay be modified at the 2-N, 3-O, and
6-O positions and must be amenable to further coupling at 4-O,
some degree of orthogonality among the protecting groups is
needed. For the 2-C functionality, we chose the azido group for
it imparts low steric hindrance and does not complicate NMR
analysis of sugar derivatives.27 It is also stable in many synthetic
transformations, but is readily converted to the amine by
reduction (e.g., hydrogenation or Staudinger reaction)28 or
directly to the acetamide by thioacetic acid treatment,29
properties that nicely fit the D-glucosamine residues found in
Nature. At 4-O, we required a blocking group that may both be
a temporary and permanent protection. To this end, the 2-
naphthylmethyl (2-NAP) group is suitable because it can be
chemoselectively cleaved by 2,3-dichloro-5,6-dicyano-1,4-ben-
zoquinone (DDQ) or simultaneously removed together with
the other permanent arylmethyl groups by hydrogenolysis in
later synthetic steps. With these primary requirements aside, we
examined the effect of several protecting groups at the 3-O and
6-O positions on the stereoselectivity of glucosaminylation in
CH2Cl2 using the trichloroacetimidate as leaving group and the
known 1,6-anhydro-β-L-idopyranosyl 4-alcohol 824f as glycosyl
acceptor (Table 1). The α-products of such couplings possess
the backbone of the major repeating component of heparin.
Employing catalytic trimethylsilyl triflate (TMSOTf) as
promoter, the markedly higher yield and α/β ratio were
notable when benzoyl (Bz) group was utilized instead of benzyl
(Bn) (entries 1−4). This increase in stereoselectivity probably
resulted from the remote assistance by the Bz group. The
installation of tert-butyldiphenylsilyl (TBDPS) moiety at the 6-
O position further gave better results (entries 5 and 6). Finally,
when the p-bromobenzyl (p-BrBn) group was positioned at 3-
O while TBDPS blocks 6-O, complete stereoselectivity was
afforded generating the α-disaccharide 15 in 84% yield (entry
7). Both situated at the β-face of the donor, these large groups
may have prevented the approach of the acceptor at the zone
that would generate the unwanted stereoisomer. Notably,
methods are available for the chemoselective removal of
TBDPS and p-BrBn groups,28b rendering donor 7 with a
protecting group pattern that holds a high level of
orthogonality. Following the successful stereoselective glyco-
sylation of acceptor 8 using donor 7 and TMSOTf, other
typical trichloroacetimidate promoters were further examined.
The imidate activation by silver(I) triflate (AgOTf) and triflic
acid (TfOH) exclusively furnished the α-adduct 15 in 63%
(entry 8) and 66% (entry 9) yields, respectively. As for
BF3·Et2O, the yield was a meager 26%; 49% of 8 was recovered
(entry 10).
Because of the 1,2-cis nature of the α-D-glucosamine
glycosidic bond, control of its stereoselective formation without
the β-isomer is challenging.15 Typically, such couplings take
advantage of the anomeric effectthe primary requirement of
which is a nonparticipating group masking the 2-amine moiety.
For this purpose, 2,3-trans-oxazolidinone, p-methoxybenzylide-
neamino group, and azide are the most prominent in recent
literature. Initially applied by Kerns to D-glucosamine in 2001,16
the ring-fused oxazolidinone provided good α-stereoselectivity;
but its use was curtailed by high promoter loading and several
side reactions. Later modifications by acetylation17 or
benzylation18 at the nitrogen position, together with the proper
selection of activators and solvent, gave better stereocontrol
and yield. Nguyen and co-workers, on the other hand, found
that nickel catalysts, through coordination with the approaching
acceptor and the p-methoxybenzylideneamino group at the 2-C
position, could deliver the stereoselective α-glycosidation of
trichloroacetimidate donors.19 The most commonly used 2-
amino protecting group is the azide, first utilized for its
nonparticipating property in glycosylation by Paulsen20 more
than 30 years ago. Nevertheless, it provided varying levels of α-
stereoselectivity, often rendering anomeric mixtures that led to
tedious purification. Drawing from the observation that the
axially oriented 4-hydroxyl of L-iduronate acceptors provided
full α-stereoselectivity on glycosylation with 2-azido-2-deoxy-D-
glucosyl donors, Seeberger suggested that forcing the hydroxyl
nucleophile of the acceptor to assume the axial position would
enable excellent stereoselectivity.21 Glycosylations of inositol-
type acceptors following this concept, however, did not lead to
the same outcome.22 Alternatively, Boons and co-workers
reported that an added thioether to the reaction mixture assists
in the α-selective glycosidation of D-glucosaminyl trichloroace-
timidate donors.23
Our work on heparin and heparan sulfate synthesis24
elevated the desire to find a highly useful strategy for
stereoselective α-glucosaminylation. Accordingly, we present
herein a versatile approach in generating the α-glycosidic bond
built upon a carefully selected set of orthogonal protecting
groups on a D-glucosaminyl donor. Using this donor, backbones
of some naturally occurring sugars were efficiently generated
We then focused on the effect of various leaving groups on
the stereoselectivity of the 4-alcohol 8 glycosylation. The results
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assembly of some important sugar skeletons (Scheme 1).
Table 1. Coupling of the 4-Alcohol 8 and Variously
Protected D-Glucosamine-Derived Trichloroacetimidate
Donors
Glycosylation of the myo-inositol 6-alcohol 2132 afforded the α-
Scheme 1. Glycosylation of Various Acceptors Using the D-
Glucosaminyl Donor 7
a
b
entry
donor
promoter
time (h)
product (α/β )
yield (%)
1
2
3
4
5
6
7
1
2
3
4
5
6
7
7
7
7
TMSOTf
TMSOTf
TMSOTf
TMSOTf
TMSOTf
TMSOTf
TMSOTf
AgOTf
1.5
1.5
1.5
1.5
2
9 (3.2/1)
63
71
72
73
89
76
84
10 (4.5/1)
11 (5.5/1)
12 (5.6/1)
13 (7.1/1)
14 (8.5/1)
15 (α only)
15 (α only)
15 (α only)
15 (α only)
2.5
2
c
d
8
2.5
2
63
d
9
TfOH
66
d
10
BF3·Et2O
1.5
26
a
0.2 equiv TMSOTf, 5 equiv AgOTf, 0.2 equiv TfOH, and 1.2 equiv
b
BF3·Et2O were used. Based on isolated yields; the α-isomer has J1′,2′ of
c
3.7−3.8 Hz, whereas the β-isomer has 7.6−8.0 Hz. The reaction was
d
carried out from −40 °C to rt. Recovered acceptor 8: 13% (entry 8),
17% (entry 9), and 49% (entry 10). MS: molecular sieves.
pseudodisaccharide 22 in 71% yield as a single isomer.
Compound 22 holds the core skeleton found in GPI anchors.
The respective couplings of 7 with the 4-alcohols 2324d and
2533 both led to the disaccharides 24 and 26, which are also
component backbones of heparin and heparan sulfate, in
complete α-stereoselectivity and satisfactory yields. Addition-
ally, 26 may be directly used for the synthesis of heparosan
oligosaccharides. The preparation of compound 26 deserves
more comment. Here, we utilized a trichloroacetimidate donor,
a thioglycoside acceptor, and AgOTf as promoter. We found
that promoting the reaction using TMSOTf, TfOH, or
BF3·Et2O furnished the desired product in poor yields
(>13%), a likely consequence of the commonly observed
sulfide aglycon transfer.34
of the reaction leading to the disaccharide 15 are outlined in
Table 2. Dehydrative glycosylation30 (entry 1), glycosyl
Table 2. Coupling of the D-Glucosaminyl Donors 16−20 and
the 4-Alcohol 8
Synthesis of Heparosan Oligosaccharides. The enzy-
matic degradation of the E. coli strain K5 capsular
polysaccharide supplies heparosan oligomers of various
lengths.10 However, the isolation of sufficiently pure compound
of defined size from this mixture is very difficult. With
compound 26 in hand, we could generate synthetic heparosan
oligosaccharides with precise structure and molecular weight.
Described in Scheme 2 is the preparation of heparosan tri- and
pentasaccharides, which may both act as substrates in
chemoenzymatic transformations toward heparin/heparan
sulfate structures. Condensation of the thioglycoside 26 and
the acceptor 2735 mediated by NIS and TMSOTf supplied the
trisaccharide 28 in 86% yield. Here, the Bz group situated at the
2-O position of the donor assisted in generating the exclusive β-
linkage (J1′,2′ = 8.6 Hz). Cleavage of 2-NAP in 28 by DDQ
supplied the 4″-alcohol (78%), which upon further glycosyla-
tion by 26 may form the pentasaccharide skeleton 29.
Nonetheless, the glycosylation yield employing NIS and
TMSOTf was only 12%, possibly because of the trisaccharide
4″-O-TMS formation, which may hinder effective coupling.
entry
donor
promoter
temp (°C)
rt
time (h)
yield (%)
a
1
2
3
4
5
16
17
18
19
20
Me2S/Tf2O
NIS/TfOH
TMSOTf
TMSOTf
AgOTf
16
4
42
−78 to −20
−40 to 0
−40 to 0
−20 to 0
79
73
2
a
3
48
a
2.5
30
a
Recovered acceptor 8: 16% (entry 1), 11% (entry 4), and 23% (entry
5). NIS: N-iodosuccinimide.
phosphate (entry 4), and glycosyl chloride (entry 5) gave
modest results whereas thioglycoside (entry 2) and N-phenyl
trifluoroacetimidate31 (entry 3) provided good yields. In all
these cases, precedence toward α-selectivity was observed.
Thus, the importance of proper selection of protecting groups
in achieving α-stereoselectivity is highlighted. Our protecting
group combination exclusively afforded the α-isomer amidst the
use of various leaving groups and promoters.
After establishing the effectiveness of our system with the 4-
alcohol 8, the trichloroacetimidate 7 was utilized in the
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a
Scheme 2. Synthesis of the Heparosan Tri- and Pentasaccharide
a
Reagents and conditions: (a) NIS, TMSOTf, 4 Å MS, CH2Cl2, −78 °C to rt, 3 h; 86%. (b) (1) DDQ, CH2Cl2/H2O = 18/1, 2 h; 78%; (2) 26, NIS,
TfOH, 4 Å MS, CH2Cl2, −78 °C to rt, 2 h; 64%. (c) (1) AcSH, Pyr, CHCl3, 36 h; (2) NaOMe, MeOH, 16 h; 30: 74%, 31: 72%. (d) TEMPO, BAIB,
CH2Cl2/H2O = 2/1, 2 h; 32: 90%, 33: 77%. (e) (1) TBAF, AcOH, THF, 40 °C, 3 d; (2) H2, Pd(OH)2/C, phosphate buffer (pH 7), 2 d; 34: 91%,
35: 95%. Pyr: pyridine; TBAF: tetra-n-butylammonium fluoride.
a
Scheme 3. Synthesis of Heparin Analogues
a
Reagents and conditions: (a) DDQ, CH2Cl2/H2O = 18/1, rt, 4 h; 36: 92%, 40: 93%. (b) Ac2O, cat. Cu(OTf)2, 0 °C; 37: 99%, 41: 98%. (c) (1) satd
NH3, MeOH/THF = 1/4, 0 °C, 6 h; (2) CCl3CN, K2CO3; 38: 90%, 42: 85%. (d) cat. TMSOTf, 4 Å MS, CH2Cl2, −40 to 0 °C, 2 h; 39: 98%, 43:
82%, 44: 84%. (e) NaOMe, CH2Cl2/MeOH = 1/1; 45: 98%, 46: 98%, 47: 91%, 48: 92%. (f) TEMPO, BAIB, CH2Cl2, 24 h; 49: 93%, 50: 78%, 51:
86%. (g) (1) TBAF, AcOH, THF, rt, 5 h; 95%; (2) SO3·Et3N, DMF, 60 °C, 3 d; 92%. (h) (1) TBAF, THF, 50 °C, 24 h, then, LiOH, rt, 3 h; (2)
SO3·Et3N, DMF, 60 °C, 3 d; 53: 75%, 54: 73%; 55: 62%. (i) (1) PMe3, NaOH, THF/H2O = 9/1, rt, 5 h; (2) SO3·Pyr, Et3N, Pyr/DMF = 3/1, rt, 24
h; 56: 86%, 57: 75%, 58: 79%; 59: 69%. (j) H2, Pd(OH)2/C, phosphate buffer (pH 7), rt, 2 d; 60: 94%, 61: 90%, 62: 94%, 63: 87%.
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Conversely, when TfOH was used in place of TMSOTf, the
target compound 29 was isolated in 64% yield.
the cyclic ester was identified through the correlation between
the 2-H and 6-C of the internal L-iduronyl residue in the
HMBC spectra (Figure 2) and corroborated by the relatively
After constructing the oligosaccharide skeletons, functional
group transformations were next carried out to achieve the
desired materials. First, the azido groups of the trisaccharide 28
and pentasaccharide 29 underwent conversion to the acetamide
using thioacetic acid. Subsequent treatment with NaOMe in
MeOH resulted to deacylation delivering compounds 30 (74%
from 28) and 31 (72% from 29). The primary hydroxyl groups
in 30 and 31 were next oxidized to the carboxyls by employing
a catalytic amount of 2,2,6,6-tetramethyl-1-piperidinyloxyl free
radical (TEMPO) with excess [bis(acetoxy)iodo]benzene
(BAIB) as co-oxidant to afford the acids 32 and 33 in 90%
and 77% yields, respectively. Desilylation followed by global
hydrogenolysis provided the target compounds 34 (91%) and
35 (95%), the structures of which were verified using NMR and
mass spectroscopy (see the Supporting Information).
Figure 2. The HMBC spectrum of compound 49 identifying the
lactone structure.
Synthesis of Heparin Analogues. Heparin, being more
readily accessible, is often used as a model in probing the
character of heparan sulfate-mediated interactions the cell
surface. Indeed, over 100 heparin-binding proteins have been
identified.36 The major repeating component of heparin, among
48 disaccharide possibilities, is the N- and 6-O-sulfonated α-D-
glucosamine 1→4-linked to 2-O-sulfonated α-L-iduronic acid.
Acquisition of specific structurally defined heparin oligosac-
charides from natural sources is a technical hurdle. Con-
sequently, carbohydrate chemists developed several method-
ologies for their chemical and chemoenzymatic prepara-
tions.24,37
Scheme 3 illustrates our synthesis of the di-, tetra-, hexa-, and
octasaccharide analogues of heparin starting from the
disaccharide 15. The 2-NAP group and the easily transformed
anhydroidose moiety allow conversion into either a glycosyl
acceptor or donor. Accordingly, DDQ treatment of the fully
protected skeletons 15 and 39 gave the acceptors 36 (92%) and
40 (93%), respectively. On the other hand, copper(II) triflate
[Cu(OTf)2]-mediated acetolysis38 of the same starting
materials provided the diacetate 37 (99%) and the triacetate
41 (98%), which were both converted into the respective
imidates 38 and 42 by successive exposures to a saturated NH3
solution in MeOH/THF and CCl3CN in the presence of
K2CO3. With the use of the acceptors 36 and 40 and the
donors 38 and 42, the assembly of the heparin backbones 39
(98%), 43 (82%), and 44 (84%) under TMSOTf activation was
achieved in a convergent and highly stereoselective manner.
The W-coupling between the anomeric proton and 3-H, the
small-to-zero coupling constant for 1-H, and the lack of NOE
correlation between 1-H and 5-H of the involved L-idose
residue are evidence that the newly formed glycosidic bond is
α-configured and primarily exists in 1C4 conformation. Here, we
chose to keep the bicyclic skeleton of the reducing end sugar to
eliminate the installation of a noncarbohydrate aglycone that
often results in a mixture of isomers, causing purification
problems. A possible advantage of a rigid ring system at the
reducing end is the relative ease of co-crystallization with a
binding protein due to the reduced motion of the terminal
group.
high IR CO stretching band observed around 1790 cm−1.
Desilylation of 45 was accomplished using TBAF/AcOH
reagent system to furnish the 2,6′-diol (95%), which was then
treated with SO3·Et3N to afford the disulfate 52 in 92% yield.
For 49−51, the basic TBAF affected not only the removal of
TBDPS groups, but also the lactone ring opening as observed
using TLC. LiOH was subsequently added to the solution to
ascertain the complete cleavage of the lactone. O-Sulfonation of
the partially purified materials gave the polysulfated compounds
53 (75% from 49), 54 (73% from 50), and 55 (62% from 51).
Here, O-sulfonations were verified by the downfield shifts (>0.5
ppm) in the resonances of the respective geminal protons. The
smooth azido-to-amino conversion was successfully imple-
mented using PMe3 in THF/H2O with added NaOH to avoid
the interference of the free carboxylic acid during the course of
the reaction.39 Treatment with SO3·Pyr resulted in the amine
sulfonation delivering the products 56 (86% from 52), 57 (75%
from 53), 58 (79% from 54), and 59 (69% from 55). While O-
1
sulfonation could be detected from H NMR downfield shifts,
no such trend occurs during N-sulfonation. Thankfully, we
found that this functionalization can be identified using the
highly indicative downfield shifts (4−5 ppm) in the 13C
resonance of the nitrogen-bonded carbon (Figure 3). We also
figured, upon comparison with the spectra of 32 and 33, that
N-acetylated and N-sulfonated glucosamine residues can be
differentiated by inspection of their 13C chemical shifts. Finally,
the compounds were subjected to hydrogenolysis to remove all
arylmethyl groups and supply the heparin analogues 60, 61, 62,
and 63 in 94%, 90%, 94%, and 87% yields, respectively. The
structures of these final compounds were confirmed by NMR
and mass spectroscopic analysis (see the Supporting
Information).
Interaction of the Heparin Analogues and
HBHA60−199. The invasion of alveolar macrophages by M.
tuberculosis has an outstanding role in the pathogenesis of
tuberculosis, a devastating disease that leaves millions dead each
year.40 Besides this interaction, the bacteria also infect
respiratory epithelial cells initiated upon adherence to heparan
sulfate proteoglycans. The responsible bacterial adhesin is
HBHA, a 199-amino acid protein, the absence of which severely
impairs the extrapulmonary dissemination of the pathogen.41
HBHA has a coiled coil N-terminal domain associated with the
agglutination property of the protein and a lysine-rich C-
terminal region predisposed for binding with the polyanionic
sugars of epithelial cells.42
Compounds 15, 39, 43, and 44 were next subjected to
multifunctional group transformations leading to the target
heparin analogues. Deacylation was conveniently carried out
under Zemplen conditions to give the alcohols 45−48 in
́
excellent yields. TEMPO oxidation of 46−48 resulted to the
lactones 49 (93%), 50 (78%), and 51 (86%). The presence of
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Figure 3. Comparisons of carbons attached to azido, amino, sulfonatamido, and acetamido groups in DEPT-135 spectra (solvent: CD3OD).
We examined the interaction of the heparin analogues 60−
63 and a truncated form of HBHA (amino acid 60−199) using
isothermal titration calorimetry (ITC) (Table 3). A commer-
Table 3. Thermodynamic Parameters of Sugar-Binding with
a
HBHA60−199 Obtained Using ITC
c
KD
ΔH
ΔS
ΔG
b
sugar
n
(μM)
(cal·mol−1
)
(cal·mol−1·K−1
)
(cal·mol−1
)
d
63
1.0
1.0
19
4.2
( 0.4)
3500
33.3
−6430
−7330
Figure 4. Compound 63−HBHA60−199 ITC titration profile showing
an endothermic interaction. Panel A shows the raw titration data and
panel B shows the integrated heats of binding generated from the raw
data after subtracting the heats of dilution.
63
62
4370
( 150)
39.3 ( 0.3)
( 0.1)
1.1
19 ( 7) 2400
( 90)
29.7 ( 1.0)
−6450
( 0.2)
e
61
−
−
−
−
−
−
−
−
−
−
e
for hexasaccharide 62. These data suggests a length of at least 6
sugar units for effective HBHA binding to occur.
60
3 kDa
heparin
1.2
( 0.1)
5.3
( 1.1)
4220
( 1140)
38.3 ( 4.2)
−7200
a
CONCLUSIONS
The values reported are averages of results from three experiments
■
b
with standard deviations indicated in parentheses. Sugar-to-protein
binding ratio. The actual parameter measured is association constant,
the inverse of dissociation constant (KD). The protein utilized has an
We have successfully developed a powerful donor for the
formation of 2-deoxy-2-amino-α-D-glucosides in high stereo-
selectivity and good yields. The α-isomer was exclusively
generated regardless of promoter and leaving group. Moreover,
excellent α-stereoselectivities were afforded upon coupling with
selected glycosyl acceptors to form the core structures of
important sugars including GPI anchors, heparosan, heparan
sulfate, and heparin. The orthogonal protecting groups built
within our donor were effectively transformed and manipulated
to furnish heparosan oligosaccharides and heparin analogues.
Examination of the interaction of the prepared heparin
analogues and HBHA60−199 revealed an entropically driven
binding for the octasaccharide and hexasaccharide. Our
disaccharide and tetrasaccharide heparin analogues did not
bind to HBHA. From these examples, we have shown the
flexibility of our donor in oligosaccharide synthesis. Further
pursuit of other relevant bioactive compounds is currently in
progress.
c
d
N-terminal hexahistidine tag; the values reported came from a single
experiment. No binding was detected.
e
cially acquired 3 kDa heparin was also tested for comparison.
Aggregation problems were incurred during the purification of
the full-length HBHA expressed in E. coli; hence, we settled
with its slightly shortened form. Moreover, the hexahistidine tag
was cleaved from our recombinant protein prior to ITC
measurement because it caused measurable decrease in binding
affinity. Here, the protein solution in Tris buffer (pH 7.6) was
titrated with the sugar dissolved in the same solvent at 25 °C.
To account for the contribution of heat of dilution, the buffer
without the protein was also titrated with the sugar solution.
No HBHA60−199 binding was detected for the disaccharide 60
and tetrasaccharide 61. On the other hand, the associations of
HBHA60−199 with the hexasaccharide 62, the octasaccharide 63,
and the commercial heparin were all found to be endothermic
with the sugar−protein binding stabilized by entropic factors. A
representative titration isotherm is shown in Figure 4. The
release of the ordered water molecules from the interface
between HBHA60−199 and the sugar molecules are quite
possibly the major driving force for the adhesion process.
Compound 63 has a slightly better binding than the 3 kDa
heparin with both sugars having nearly 1:1 binding
stoichiometry with HBHA60−199. The binding affinity is lower
EXPERIMENTAL SECTION
■
General Procedure for the Synthesis of Compounds 9−15. A
solution of the trichloroacetimidate donor (1−7, 1.2 equiv) and the 4-
alcohol 8 (1.0 equiv) in CH2Cl2 (10 mL per total gram of donor,
acceptor, and molecular sieves) with freshly dried 4 Å molecular sieves
(1.5 g per total gram of the donor and the acceptor) was stirred at
room temperature for 1 h under N2 atmosphere. The mixture was
cooled to −78 °C, TMSOTf (0.2 equiv) was added to the solution,
and the mixture was gradually warmed up to 0 °C. After stirring for
1.5−2.5 h, Et3N (0.3 equiv) was added to quench the reaction and the
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Journal of the American Chemical Society
Article
mixture was filtered through Celite. The filtrate was concentrated
under reduced pressure to furnish a residue, which was purified by
flash column chromatography to give the expected disaccharide. The
yields of compounds 9−15 are listed in Table 1.
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ASSOCIATED CONTENT
■
S
* Supporting Information
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9461−9462. (b) Kerns, R. J.; Zha, C.; Benakli, K.; Liang, Y.-Z.
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Detailed experimental procedures and 1H and 13C NMR
spectra for relevant compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
schung@gate.sinica.edu.tw; chyan@mail.ndhu.edu.tw
Author Contributions
#These authors contributed equally.
Notes
The authors declare no competing financial interest.
̌
́ ̌
, C.; Stenzel, W. Angew. Chem., Int. Ed. Engl.
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1976, 15, 440−441.
ACKNOWLEDGMENTS
■
(21) Orgueira, H. A.; Bartolozzi, A.; Schell, P.; Seeberger, P. H.
Angew. Chem., Int. Ed. 2002, 41, 2128−2131.
(22) Cid, M. B.; Alfonso, F.; Martín-Lomas, M. Chem.Eur. J. 2005,
We dedicate this paper to Prof. Chun-Chen Liao on the
occasion of his 70th birthday. This work was supported by the
National Science Council (NSC 97-2113-M-001-033-MY3,
NSC 98-2119-M-001-008-MY2) and Academia Sinica. The
authors thank Dr. Wei-Juin Su for providing the DNA of
HBHA from M. tuberculosis H37Rv.
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