A coordinated synthesis and conjugation strategy for the preparation of homogeneous glycoconjugate vaccine candidates

Article abstract of DOI:10.1002/anie.201006327

A sweet solution: A strategy for the synthesis of well-defined carbohydrate-based vaccines is presented. The approach couples complex oligosaccharide synthesis to site-specific conjugation methodology to provide pure glycoprotein vaccine candidates (see scheme). Copyright

Full text of DOI:10.1002/anie.201006327

DOI: 10.1002/anie.201006327
Glycoprotein Vaccines
A Coordinated Synthesis and Conjugation Strategy for the Preparation
of Homogeneous Glycoconjugate Vaccine Candidates**
Elizabeth J. Grayson, Gonꢀalo J. L. Bernardes, Justin M. Chalker, Omar Boutureira,
Julia R. Koeppe, and Benjamin G. Davis*
Glycoconjugates are the center of many therapeutic strat-
egies[1–3] and carbohydrate-based vaccines in particular hold
great promise.[4–8] The development of glycovaccines, how-
ever, can be hindered by the limited access offered by natural
sources to homogeneous antigenic carbohydrates; efficient
chemical synthesis offers an attractive route to pure samples
of these carbohydrates. Furthermore, for an optimal immune
response, the carbohydrate antigen should be conjugated to
an immunogenic carrier, usually a protein.[9,10] The synthesis
and use of well-defined glycoprotein therapeutics and glyco-
vaccines—uniform in sugar, site, and level of protein attach-
ment—is rare and most constructs are prepared and admin-
istered as complex mixtures.[4,9,10] Even strategies that utilize
pure synthetic glycan may employ non-selective methods for
subsequent conjugation to a protein carrier.[4,6,7,11,12] Given
the unknown influence of conjugation site on immunogenic
response, it is remarkable that, to our knowledge, no
homogeneous glycovaccine has been studied. To fully eval-
uate the structure–activity relationships (SARs) between
glycoprotein and immunogenicity, we have initiated a pro-
gram for the construction of such “pure” or uniform
glycoconjugate vaccines. We report a coherent strategy for
homogenous glycoprotein construction that coordinates both
carbohydrate synthesis and conjugation methodology. This
approach features glycosyl disulfides as versatile donors in
complex carbohydrate synthesis, providing strategic access to
glycosyl thiols that can be site-specifically attached to a
protein carrier through a well-defined thioether linkage
(Scheme 1).
Scheme 1. a) Coordinated synthesis and conjugation strategy for the
construction of glycoconjugate vaccines. Non-reducing (Nterm) to
[*] Dr. G. J. L. Bernardes, J. M. Chalker, Dr. O. Boutureira,
Dr. J. R. Koeppe, Prof. B. G. Davis
reducing terminus (Rterm) segmental assembly allows flexible building
block use and ready attachment of “growing” carbohydrate antigen
fragments to carriers during “growth”. b) Retrosynthetic analysis of
target O-antigenic repeating motif of 22535 Klebsiella pneumonia.
Department of Chemistry, University of Oxford
Chemistry Research Laboratory
12 Mansfield Road, Oxford OX1 3TA (UK)
Fax: (+44)1865-285-002
E-mail: ben.davis@chem.ox.ac.uk
Homepage: http://users.ox.ac.uk/~dplb0149
Dr. E. J. Grayson
Department of Chemistry, University of Durham
South Road, Durham DH1 3LE (UK)
We have previously reported the use of glycosyl disulfides
as donors in the synthesis of mono- and disaccharides.[13,14]
These novel donors exhibit reactivity similar to their thio-
glycoside counterparts,[15] but with the advantage that the
disulfide linkage can be readily cleaved and exchanged at any
stage, thereby tuning reactivity[16] through aglycon alteration.
Competition experiments have demonstrated that “armed”[17]
(more reactive) disulfide donors, can be activated preferen-
tially over “disarmed” disulfides (Figure 1).[14]
[**] We gratefully acknowledge the Leverhulme Trust (E.J.G.), FCT and
Gulbenkian, Portugal (G.J.L.B.), The Rhodes Trust and the National
Science Foundation (J.M.C.), EC (Marie Curie IEF, O.B.), and The
International AIDS Vaccine Initiative (J.R.K., G.J.L.B.) for financial
support. We thank Prof. M. G. Finn for kindly providing p75m/Qb
plasmid. B.G.D. is a Royal Society Wolfson Research Merit Award
recipient.
Disarmed disulfides can be activated under more stren-
uous conditions (e.g. higher temperature) or through ready
conversion to an “armed” disulfide. The differential reactivity
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201006327.
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Block 3 was prepared from butane-2,3-diacetal (BDA)-
protected thioglycoside 11 (Scheme 3).[29] Silylation and
bromination provided protected a-bromide 12. Reaction
with sodium methanethiosulfonate in the presence of
Figure 1. Examples of armed and disarmed glycosyl disulfides.
of mixed disulfide donors has not yet been explored in
complex oligosaccharide synthesis. Since aglycon alteration of
disulfides is mild, flexible, and compatible with many
protecting groups, and so can be used to fine-tune a glycan
synthesis even mid-way, we anticipated a strategic advantage
in carbohydrate construction.[18] Also, the disulfide products
can be easily reduced to thiols and then chemospecifically
conjugated to a protein scaffold in a controlled manner,[19–22]
allowing glycoconjugation at all stages of glycan synthesis
(Scheme 1). The dual role of disulfides both as donors and as
masked precursors to glycosyl thiols is the centerpiece of a
unified approach to homogenous glycoprotein synthesis that
we disclose here.
Rhamnosyl pentasaccharide 1 (Scheme 1) constitutes the
repeating unit of the O-antigenic polysaccharide of the 22535
strain of the pathogen Klebsiella pneumonia[23,24] and there-
fore represents a model glycan moiety for the generation of
glycoprotein vaccines.[25] Rhamnosyl donors, as 6-deoxy
sugars, are generally more reactive than their glucose
(hexose) counterparts[26] and thus constituted a good initial
platform for examining donor reactivity tuning by aglycon
alteration. Preliminary investigations revealed that orthogo-
nal activation of the thiorhamnosides was best achieved with
5-nitro-2-pyridyl (pNPy)[27] as the deactivating aglycon. This
ability to alter aglycon reactivity highlights a strategic
advantage of disulfide donors: the aglycon can be exchanged
to attenuate or promote reactivity as required and does not
require an entirely new synthesis of the building block.
We initiated the synthesis of 1 from the building blocks
2–5. Compound 2 was prepared from thioglycoside 6[28]
(Scheme 2). After conversion of 6 to the anomeric bromide
7, reaction with sodium methanethiosulfonate in the presence
of Bu4NBr gave methanethiosulfonate 8 as an inseparable
mixture of anomers (a:b = 5:1). Treatment with nitropyridyl
thiol pNPySH gave 9 as a separable mixture of anomers (74%
9a, 15% 9b). Desilylation of 9a with excess Et3N·3HF in THF
gave the required building block 2 in 81% yield with 4%
formation of regioisomer 10, a product of acetyl migration.
Scheme 3. Synthesis of disulfide donor 3: a) TESOTf, Py, CH2Cl2, 91%;
b) Br2, CH2Cl2, 08C, 100%; c) NaSSO2Me, Bu4NBr, dioxane, 808C,
75%; d) pNPySH, Et3N, CH2Cl2, 73% then Et3N·3HF, THF, 100%.
Bu4NBr gave 13 as a separable anomeric mixture in which,
unlike 8, the b-anomer was predominant (b:a = 6.5:1).[30] This
difference in anomeric selectivity in the formation of 8 and 13
may be associated with the rigidity conferred to the rhamnose
ring in 13 by the BDA protecting group.[31] 13b was used in the
next step and reaction with 5-nitro-2-pyridylthiol (pNPySH)
followed by desilylation gave target building block
(Scheme 3).
3
The first key glycosylation paired thioglycoside donor 5[32]
with acceptor 2 (Scheme 4). Using the mild activator N-
phenyl thiocaprolactam/Tf2O[33] at À108C, the disaccharide
14a was isolated in 68% yield with exclusive a selectivity and,
as a result of reactivity tuning, without any concomitant
activation of 2. 14a was then “aglycon altered”: converted
into armed donor 14b by disulfide reduction followed by
reaction with ethyl methanethiosulfonate. 14b was then
reacted with acceptor 3 to give rhamnotrisaccharide 15a in
66% yield, again with exclusive a selectivity and again
without activation of 3 (Scheme 4).
Final fragment assembly was achieved by activation of this
trisaccharide. Thus, 15a was converted/altered into the active
donor 15b, and then coupled with the disaccharide acceptor
16b. The precursor to 16b, protected disaccharide building
block 16a, had itself been prepared by reaction of 3 with 4
using N-phenyl thiocaprolactam/Tf2O at À208C; desilylation
furnished disarmed disulfide acceptor 16b. [3+2] coupling of
16b with trisaccharide building block 15b gave pentasacchar-
ide 17 which was then partially deprotected and reduced to
18. Finally, debenzylation was accomplished by Birch reduc-
tion. For ease of handling, the crude thiol was globally
acetylated, purified, and then subjected to Zemplꢀn depro-
tection to give the pentasaccharide 1 (Scheme 4). Exclusive
a-configurations in all glycosidic linkages were consistent
1
with the absence of nOe interactions in the H NMR spectra
between the signals for H-1 and H-3 or H-5. Regiochemistry
was established through COSY, ROESY, nOe, and HMBC
analysis.[34]
Deprotected mono- and disaccharide thiols 19 and 20, as
smaller fragments of the antigen pentasaccharide 1, were also
synthesized from intermediate building blocks 13 and 16b,
respectively (Scheme 5). Their syntheses from intermediate
glycosyl disulfide donors that had been used to create 1
Scheme 2. Synthesis of disulfide donor 2: a) TESOTf, Py, CH2Cl2, 96%
then Br2, CH2Cl2, 08C, 100%; b) NaSSO2Me, Bu4NBr, dioxane, 708C,
79%; c) pNPySH, Et3N, CH2Cl2, 74% 9a; d) Et3N·3HF, THF, 81% 2,
4% 10. TES=triethylsilyl.
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Scheme 4. Synthesis of pentasaccharide 1: a) N-phenyl thiocaprolactam, Tf2O, TTBP, CH2Cl2, À20 to À108C, 68%; b) 1. PBu3, CHCl3/dioxane/H2O;
2. EtSO2Me, Et3N, CH2Cl2; 85% over 2 steps; c) N-phenyl thiocaprolactam, Tf2O, TTBP, CH2Cl2, 108C–RT, 66%; d) 1. PBu3, CHCl3/dioxane/H2O;
2. EtSO2Me, Et3N, CH2Cl2; 77% over 2 steps; e) N-phenyl thiocaprolactam, Tf2O, TTBP, CH2Cl2, À20 to À108C, 66%; f) 1. Et3N·3HF, THF; 2. N-phenyl-
thiocaprolactam, Tf2O, TTBP, CH2Cl2, 108C–RT; 45% over steps; g) 1. TFA/H2O (9:1), 79%; 2. PBu3, dioxane/H2O, 78%; h) 1.Na liq. NH3/THF then Ac2O,
Py, 69%; 2. NaOMe, MeOH, 100%. TTBP=2,4,6-tri-tert-butylpyrimidine, TFA=trifluoroacetic acid.
A single copy of a carbohydrate displayed on a synthetic
lipopeptide has proved successful in the generation of anti-
bodies,[36] but this construct is quite different from typical
protein glycoconjugates and in most cases multiple sugar
loading has been shown to influence (and be necessary for)
the efficacy of the vaccine.[37,38] Moreover, the method used in
Scheme 6 generates diastereomers in the protein backbone as
a result of the Michael-type addition to dehydroalanine. As a
result (and to stay consistent to our goals of creating pure
multivalent glycoconjugates as putative vaccines, see above),
we decided to extend our glycoprotein vaccine technology to
the construction of a pure, single glycoform of a larger protein
carrier bearing multiple antigen copies as single diastereo-
mers at well-defined sites. Virus-like bacteriophage particle
Qb was chosen as a known immunogenic carrier[39,40] and non-
natural amino acid homoallylglycine (Hag) was site-specifi-
cally introduced into these protein systems as a “tag” for
thiyl–ene conjugation[22] through expression of corresponding
gene sequences in an auxotrophic strain of E. coli (B834
(DE3)).[41] A pure glycoprotein vaccine candidate displaying
180 copies of antigen 20 at well-defined sites was created
(Scheme 7). The conjugation chemistry proceeded with
greater than 95% conversion at pH 4.0 through the combined
use of light and an initiator, Vazo44.[34] The synthesis of
multivalent, pure glycovaccines with defined number of
antigen copies at precise sites will enable the determination
of where and how many antigen copies are necessary for
improved vaccine efficacy.
Finally, to explore the potential for this method to be
applied to other oligosaccharides another sequential armed/
disarmed glycosyldisulfide glycosylation was explored. This is
important given that the inherent reactivities (as well as
reactivity differences) may be drastically different in other
systems (see above). In particular, fully oxygenated hexoses,
such as d-glucose, are typically less reactive as glycosyl donors
Scheme 5. Synthesis of 19 and 20: a) TFA/H2O (9:1) then Ac2O, Py;
b) 1. PBu3, CHCl3/dioxane/H2O; 2. NaOMe, MeOH.
illustrates strategic access mid-route to key segments of the
final carbohydrate that bear an anomeric thiol, the tag needed
for site-specific conjugation.
Next these glycan epitope fragments of increasing com-
plexity, 19, 20 and 1, were site-specifically conjugated to a
model Bacillus protein, as a potential immunogenic carrier,
by conjugate addition to dehydroalanine (Dha) residue. Dha
was chemically installed[21] by oxidative elimination of the
single cysteine of the subtilisin protein (SBL) mutant S156C
using Tamuraꢁs reagent,[35] O-mesitylenesulfonylhydroxyl-
amine (MSH).[21] Reaction of SBL-Dha as a divergent
intermediate with 1, 19, 20 gave the corresponding desired
protein–epitope conjugates with greater than 95% conver-
sion. The synthesis of these single-glycoform glycoprotein
antigen carriers creates opportunities for SAR studies of
glycan structure in candidate vaccines (Scheme 6).
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Scheme 6. Site-specific conjugation of 19, 20, and 1 to model protein.
Scheme 7. Synthesis of well-defined conjugate bearing 180 copies of
sugar antigen by thiyl–ene chemistry on immunogenic protein plat-
form. Modification was successfully performed on Qb multimer. The
mass spectrum insert shows analysis of monomer protein formed
after exhaustive reduction and denaturation.
than 6-deoxyhexoses, such as the l-rhamno units used above.
We were pleased to observe that model oligomer 21 could be
assembled using an essentially identical disulfide reactivity-
tuning approach (Scheme 8). Again, as for the 6-deoxyhexose
system under the appropriate reaction conditions disacchar-
ide 22 was isolated, as a result of reactivity tuning, without any
concomitant activation of the disarmed donor motif.
Scheme 8. Synthesis of hexose oligomer 21: a) DMTST, TTBP, CH2Cl2,
À108C, 68%; b) DMTST, TTBP, CH2Cl2, RT, 55%. DMTST=dimethyl-
sulfonium triflate, pNP=para-nitrophenyl.
In summary, a strategy has been developed for the
construction of glycoconjugate vaccines in which the site of
glycan attachment is well-defined, allowing preparation of
more precisely defined candidates. This strategy coordinates
oligosaccharide synthesis with site-specific protein conjuga-
tion. Glycosyl disulfides are demonstrated as useful donors
for the synthesis of oligosaccharides. Their aglycon flexibility
allows straightforward iterative assembly of complex carbo-
hydrates and protecting group freedom since the “armed” or
“disarmed” status is altered through the aglycon rather than
the protecting groups of the sugar. Moreover, the use of
glycosyl disulfides advantageously delivers glycosyl thiol
products that are suitable for site-specific protein ligation,
here applied using two different site-selective and comple-
mentary methods.[21,22] Finally the use of pure, well-defined
glycoproteins as potential immunogens is rare, if not unpre-
cedented, and will allow insight into the effect of epitope
positioning and immune response. For example, the role and
importance of linkers between sugar epitope and protein
carrier is a matter for debate, with some examples high-
lighting usefully increased accessibility, while others show
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that the linker can confound and modulate immune
response.[42,43] Valuably, through the two methods that we
demonstrate here, identical sugar epitopes (starting from the
same sugar thiol direct from the synthetic route) can be
installed at different distances to test this hypothesis (in one
case through a one-carbon side chain and in the other through
a four-carbon). The use of this coordinated strategy for
therapeutic glycovaccine candidates is currently underway.
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Experimental Section
General glycosylation:
A solution of thiorhamnoside acceptor
(0.45 mmol), disulfide donor (0.45 mmol), N-phenylthiocaprolactam
(0.47 mmol), and TTBP (2,4,6-tri-tert-butylpyridine) (0.50 mmol) in
dry dichloromethane (15 mL) was stirred with molecular sieves
(0.30 g) under argon for 1 h. The mixture was cooled to À208C using a
cryocooler. Trifluoromethanesulfonic anhydride (0.50 mmol) was
added. After TLC showed complete consumption of starting materi-
als, the reaction mixture was cooled in a dry ice/acetone bath and
diluted with dichloromethane, and passed through a silica plug which
was washed with dichloromethane and then ethyl acetate. The eluent
was evaporated and the residue purified by flash column chromatog-
raphy.
General aglycon alteration: Argon was bubbled through a stirred
mixture of the disulfide (0.45 mmol), dioxane (2.7 mL), chloroform
(5.4 mL), and water (0.9 mL) for 30 min. Tributylphosphine
(0.91 mmol) was added. A deep orange color was produced, which
rapidly faded to yellow. After 1 h TLC indicated the reaction was
complete. The reaction mixture was concentrated and purified by
flash column chromatography. The resulting thiol (0.45 mmol) was re-
dissolved in dichloromethane (50 mL) and the solution added
dropwise to a solution of ethyl methanethiosulfonate (0.49 mmol)
and triethylamine (0.45 mmol) in dichloromethane (20 mL) at 08C
over 1.5 h. The ice bath was then removed and after an additional
hour TLC showed complete comsumption of starting material. The
solvent was evaporated and the residue purified by flash column
chromatography.
General protein conjugation to Dha procedure: A 100 mL aliquot
of 0.3 mgmLÀ1 SBL-C156Dha (pH 8.0 sodium phosphate, 50 mm) was
prepared as previously described.[21] A 60 mL aliquot of a 40 mm
solution of the sugar thiol in sodium phosphate buffer (50 mm,
pH 8.0) was added to SBL-C156Dha and vortexed to homogenize.
After shaking at room temperature or 378C for 1 to 3 h, LC-MS
analysis of the reaction mixture revealed complete conversion to the
corresponding glycoprotein.
Typical thiyl–ene addition procedure:[22] Di-rhamno-SH 20
(1.38 mg, 4.22 mmol) and Vazo44 (0.28 mg, 0.84 mmol) were added
to a solution of Qb-M16Hag protein (100 mL of 1.19 mgmLÀ1
,
8.44 nmol) in 250 mm ammonium acetate buffer (pH 4.0). The
reaction mixture was placed in a cuvette and irradiated with a
medium pressure 125 W Hg-lamp with borosilicate filter at room
temperature for 8 h. Small molecules were removed from a 50 mL
reaction mixture aliquot by loading the sample onto a PD minitrap
desalting column (GE Healthcare). An aliquot (20 mL) was mixed
with 1m DTT (dithiothreitol) in H2O (10 mL) and incubated at 608C
for 5 min to allow the protein to denature prior to analysis by LC-MS
which revealed complete conversion to the corresponding glycopro-
tein.
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Received: October 8, 2010
Revised: January 5, 2011
Published online: March 31, 2011
Keywords: disulfide donors · glycosylation · oligosaccharides ·
protein conjugation · vaccines
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