Synthesis of 6-PEtN-a-D-GalpNAc-(1→6)-β-D-Galp-(1→4)-β- D-GlcpNAc-(1→3)-β-D-Galp-(1→4)-β-D-Glcp, a Haemophilus influenzae lipopolysacharide structure, and biotin and protein conjugates thereof

Article abstract of DOI:10.3762/bjoc.6.80

Background: In bacteria with truncated lipopolysaccharide structures, i.e., lacking the O-antigen polysaccharide part, core structures are exposed to the immune system upon infection and thus their use as carbohydrate surface antigens in glycoconjugate va

Full text of DOI:10.3762/bjoc.6.80

Synthesis of 6-PEtN-α-D-GalpNAc-(1→6)-β-D-Galp-
(1→4)-β-D-GlcpNAc-(1→3)-β-D-Galp-(1→4)-β-D-Glcp,
a Haemophilus influenzae lipopolysacharide struc-
ture, and biotin and protein conjugates thereof
Andreas Sundgren1, Martina Lahmann2 and Stefan Oscarson*3
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2010, 6, 704–708.
1Department of Chemistry, Göteborg University, S-412 96
Gothenburg, Sweden, 2The School of Chemistry, University of
Bangor, Alun Roberts Building, Deiniol Road, Bangor, Gwynedd LL57
2UW, U.K. and 3Centre for Synthesis and Chemical Biology,
University College Dublin, Belfield, Dublin 4, Ireland, Phone: +353
17162318
doi:10.3762/bjoc.6.80
Received: 25 January 2010
Accepted: 07 July 2010
Published: 26 July 2010
Guest Editor: T. K. Lindhorst
Email:
Andreas Sundgren - andreas@riktad.com; Martina Lahmann -
m.lahmann@bangor.ac.uk; Stefan Oscarson* -
stefan.oscarson@ucd.ie
© 2010 Sundgren et al; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
conjugate vaccines; glycoconjugates; Haemophilus influenzae;
lacto-N-neotetraose; oligosaccharide synthesis; thioglycosides
Abstract
Background: In bacteria with truncated lipopolysaccharide structures, i.e., lacking the O-antigen polysaccharide part, core struc-
tures are exposed to the immune system upon infection and thus their use as carbohydrate surface antigens in glycoconjugate
vaccines can be considered and investigated. One such suggested structure from Haemophilus influenzae LPS is the phos-
phorylated pentasaccharide 6-PEtN-α-D-GalpNAc-(1→6)-β-D-Galp-(1→4)-β-D-GlcpNAc-(1→3)-β-D-Galp-(1→4)-β-D-Glcp.
Results: Starting from a spacer-containing lactose derivative a suitably protected lacto-N-neotetraose tetrasaccharide structure was
constructed through subsequential couplings with two thioglycoside donors, a glucosamine residue followed by a galactose deriva-
tive, using NIS/AgOTf as promoter. Removal of a silyl protecting group at the primary position of the non-reducing end residue
afforded an acceptor to which the terminal α-galactosamine moiety was introduced using a 2-azido bromo sugar and halide assisted
coupling conditions. Global deprotection afforded the non-phosphorylated target pentasaccharide, whereas removal of a silyl group
from the primary position of the non-reducing end residue produced a free hydroxy group which was phosphorylated using H-phos-
phonate chemistry to yield the phosphoethanolamine-containing protected pentasaccharide. Partial deprotection afforded the phos-
phorylated target pentasaccharide with a free spacer amino group but with a protected phosphoethanolamino group. Conjugation of
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Beilstein J. Org. Chem. 2010, 6, 704–708.
the spacer amino group to biotin or dimethyl squarate followed by deprotection of the phosphoethanolamino group and, in the case
of the squarate derivative, further reaction with a protein then afforded the title conjugates.
Conclusion: An effective synthesis of a biologically interesting pentasaccharide structure has been accomplished. The target
pentasaccharide, an α-GalNAc substituted lacto-N-neotetraose structure, comprises a phosphoethanolamine motif and a spacer
aglycon. Through the spacer, biotin and protein conjugates of the title compound have been constructed to allow further use in bio-
logical experiments.
Introduction
Haemophilus influenzae are Gram-negative bacteria divided Herein we describe the synthesis of this structure and its
into six serotypes, a–f, related to the structure of the capsular conjugation to biotin and a carrier protein to form glycoconju-
polysaccharide usually surrounding the bacterium [1]. Among gates that can be used in biological experiments to evaluate the
these serotypes, type b is the cause of the most severe diseases, immunological properties of the title structure.
i.a. meningitis and pneumonia. However, there are now several
commercial vaccines against this serotype that have proven to Results and Discussion
be highly effective [2]. These vaccines are glycoconjugate The target structure is, as noted above, a substituted lacto-N-
vaccines, based on capsular poly- or oligosaccharide structures, neotetraose structure that we have experience of from, i.a.,
either native or synthetic [3,4], linked to a carrier protein. The former synthetic work related to Streptococcus pneumoniae
lipopolysaccharide (LPS) of H. influenzae shows a huge struc- type 14 CPS structures [11]. To allow for the introduction of the
tural variety and hence non-capsulated bacteria are referred to GalNAc residue at the 6IV-position and the subsequent phos-
as non-typable H. influenzae (NTHi) [5,6]. This structural phorylation of the 6V-hydroxy group, two new galactosyl
diversity is a good defence mechanism against the human donors were designed and synthesised (Scheme 1). Reductive
acquired immune system, and NTHi often cause repetitive ring opening, with BH3/Bu2BOTf [12], of the benzylidene
infections. Another way used by H. influenzae to avoid the acetal in the known ethyl thioglycoside 1 [13] gave the
human immune system seems to be molecular mimicry, i.e. the
bacteria express common human carbohydrate structures on
their surface that the host recognises as self-structures and do
not produce antibodies against. Construction of a glycocon-
jugate vaccine against NTHi is thus quite complex. Carbo-
hydrate structures that are both exposed on the surface of most
bacteria and also immunogenic in humans must be found and
produced. To tackle this problem we are presently pursuing two
possible routes. A common conserved LPS inner-core pentasac-
charide structure has been identified, and efforts to produce this
structure, i.a. through synthesis, are on-going [7-9]. Further-
more, analysis of the LPS of NTHi strains to find frequent non-
human outer-core structures is continuously performed [5,6].
Scheme 1: i. BH3, Bu2BOTf, THF/CH2Cl2, 85%; ii. TBDMSCl, pyridine,
CH2Cl2, 90%; iii. TBDMSCl, pyridine, 92%; iv. BnBr, NaH, DMF, 85%.
One candidate recently suggested is a lacto-N-neotetraose struc-
ture substituted with a PEtN-GalNAc residue (Figure 1) [10].
Figure 1: The H. influenzae outer core target structure.
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Beilstein J. Org. Chem. 2010, 6, 704–708.
6-hydroxy derivative 2 (85%), which was then silylated to The 4-hydroxy group in GlcNAc derivatives is known to be
afford donor 3 (90%). Regioselective silylation of the 2-azido- quite unreactive towards glycosylations, which, i.a., has led to
galactose ethyl thioglycoside 4 [14] yielded the 6-O-silylated development of new protecting group patterns to improve the
compound 5 (92%), benzylation of which gave donor 6 (85%). reactivity [20,21]. Here, however, NIS/AgOTf-promoted glyco-
A benzyl group in the 4-position was preferred to an ester group sylation of acceptor 13 with donor 3 proceeded without diffi-
to avoid the risk of acyl migration during subsequent reactions; culties to produce the tetrasaccharide 14 in high yield (77%,
desilylation, glycosidation and phosphorylation.
Scheme 3). Before the introduction of the azido-galactose
residue, the spacer azido function was reduced to an amino
3II,4II-Diols of lactose are often used as acceptors for regio- group, which was protected as a benzyl carbamate (→ 15,
selective glycosidations in the 3-position. However, the selec- 91%). Removal of the TBDMS group with TBAF then gave the
tivity is dependent on the donor, promoter and conditions tetrasaccharide acceptor 16 (89%). To obtain complete α-selec-
employed [15], e.g., it has been shown that donors containing a tivity with 2-azido-galactose donors is not trivial. A 4,6-silyl
4,6-O-benzylidene acetal can give a mixture of products [16]. acetal has been suggested as one way to improve the selectivity
Hence, the diol 7 [17] was transformed using benzaldehyde [22]. We have earlier tried halide-assisted conditions [23],
dimethyl acetal and camphorsulfonic acid stereo-selectively into which is not that common due to the incompatibility between
the corresponding 3,4-endo-benzylidene derivative 8 which was the mild coupling conditions and the low reactivity of 2-azido
in turn converted to the 3-hydroxy derivative 9 by NaBH3CN/ donors, with good results when simple spacer alcohols were
HCl-mediated reductive opening of the acetal ring [18] (80% used as aglycons [24]. This approach also worked well here,
overall yield from 7, Scheme 2). NIS/AgOTf-promoted glyco- although extended reaction time was necessary. A halide-
sidation of this acceptor with donor 10 [19] (1.4 equiv) then assisted coupling between acceptor 16 and the bromosugar,
efficiently gave the β-linked trisaccharide 11 (83%). At this
stage the phthalimido group was removed by aminolysis and the
resulting amino compound acetylated to yield 12 (93%) with
the target acetamido function in place. Once more reductive
opening of a benzylidene acetal (NaBH3CN/HCl) gave a new
mono-hydroxy compound, the acceptor 13 (81%).
Scheme 3: i. NIS/AgOTf, CH2Cl2, 77%; ii. a) H2S, pyridine, Et3N;
Scheme 2: i. PhCH(OMe)2, CSA; ii. NaBH3CN, HCl/Et2O, THF, 80%;
iii. NIS/AgOTf, CH2Cl2, 83%; iv. a) NaOMe, MeOH; b) NH2(CH2)2NH2,
EtOH; c) Ac2O, pyridine, 93%; v. NaBH3CN, HCl/Et2O, THF, 81%.
b) CbzCl, pyridine, CH2Cl2, 91%; iii. TBAF, THF, 89%; iv. Br2, CH2Cl2;
v. Et4NBr, DMF, CH2Cl2, 79%; vi. a) H2S, pyridine, Et3N; b) Ac2O, pyri-
dine, CH2Cl2, 91%; vii. TBAF, THF, 85%.
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Beilstein J. Org. Chem. 2010, 6, 704–708.
obtained from thioglycoside 6, gave (after 11 days) the α-linked
pentasaccharide 17 in 79% yield. Due to the excess of donor
used and the long reaction time required the acetamido carbonyl
oxygen also partly behaved as a nucleophile and gave a pseudo-
hexasaccharide acetimidate side product according to MALDI-
TOF and NMR. Similar side products have earlier been
described [25-28]. Mild acid treatment of the glycosylation
mixture prior to purification led to cleavage of the formed
imidate and afforded the desired pentasaccharide in the above
noted yield.
Reduction of the azido group in 17 and subsequent acetylation
afforded compound 18 (91%). Removal of the TBDMS group,
again using TBAF, gave derivative 19 (85%), ready for the
introduction of the phosphoethanolamine. Compound 19 was
also completely deprotected by sodium methoxide treatment
followed by catalytic hydrogenolysis to give the non-phos-
phorylated target structure 20 (66%, Scheme 4), to be used as a
reference in biological experiments. Earlier we used the Cbz-
protected ethanolamine H-phosphonate monoester as a reagent
in the formation of phosphoethanolamines [29]. Since the
amino group in the spacer was already Cbz-protected and we
wanted to be able to differentiate between the two amino groups
during conjugation, a Boc-protected H-phosphonate monoester
21 was synthesised and used in the phosphorylation step. Acti-
vation of 21 with pivaloyl chloride in the presence of 19
afforded the H-phosphonate diester, which was oxidised with
I2/pyridine in water to afford the phosphate diester 22 [30].
Deprotection with sodium methoxide followed by catalytic
hydrogenolysis then afforded the still Boc-protected phospho-
ethanolamine pentasaccharide 23 (44% from 19) ready for
conjugation via the free spacer amino group.
The protein conjugations were carried out using squarate ester
methodology [31,32]. Reaction of compound 23 with dimethyl
squarate at neutral pH afforded the monomethyl ester squarate
amide of 23, from which the Boc-group was removed by acid
hydrolysis to afford derivative 24. Protein conjugations were
performed by reaction of 24 (20 equiv) with human serum
albumin (HSA). Compound 20 was similarly activated with
dimethyl squarate and conjugated to HSA. MALDI-TOF MS of
the HSA-conjugates of compounds 20 and 24 showed a loading
of 16 oligosaccharides/protein molecule and 7 oligosaccharides/
protein molecule, respectively. Since derivative 24 contains a
free amino group there is a possibility of oligomerisation in
addition to protein conjugation. To what extent this happened
was not examined. Compound 23 was also biotinylated for use
Scheme 4: i. NaOMe, MeOH; ii. H2, Pd/C, MeOH/H2O; iii. 21, PivCl,
pyridine, MeCN; iv. I2, H2O, pyridine; v. Dimethyl squarate or NHS-
biotin, buffer pH 7; vi. TFA (10% aqueous).
Supporting Information
Supporting Information File 1
Experimental Section
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-6-80-S1.pdf]
in ELISA-screening of antibodies raised against native LPS. Acknowledgements
Reaction with the commercial NHS-activated ester of biotin We thank the Swedish Research Council and Science Founda-
followed by TFA-treatment to remove the Boc-group afforded tion Ireland (Grant Number 08/IN.1/B2067) for financial
derivative 25.
support.
707

Beilstein J. Org. Chem. 2010, 6, 704–708.
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