(Automated) Synthesis of Well-defined Staphylococcus Aureus Wall Teichoic Acid Fragments

Article abstract of DOI:10.1002/chem.202101242

Wall teichoic acids (WTAs) are important components of the cell wall of the opportunistic Gram-positive bacterium Staphylococcus aureus. WTAs are composed of repeating ribitol phosphate (RboP) residues that are decorated with d-alanine and N-acetyl-d-glucosamine (GlcNAc) modifications, in a seemingly random manner. These WTA-modifications play an important role in shaping the interactions of WTA with the host immune system. Due to the structural heterogeneity of WTAs, it is impossible to isolate pure and well-defined WTA molecules from bacterial sources. Therefore, here synthetic chemistry to assemble a broad library of WTA-fragments, incorporating all possible glycosylation modifications (α-GlcNAc at the RboP C4; β-GlcNAc at the RboP C4; β-GlcNAc at the RboP C3) described for S. aureus WTAs, is reported. DNA-type chemistry, employing ribitol phosphoramidite building blocks, protected with a dimethoxy trityl group, was used to efficiently generate a library of WTA-hexamers. Automated solid phase syntheses were used to assemble a WTA-dodecamer and glycosylated WTA-hexamer. The synthetic fragments have been fully characterized and diagnostic signals were identified to discriminate the different glycosylation patterns. The different glycosylated WTA-fragments were used to probe binding of monoclonal antibodies using WTA-functionalized magnetic beads, revealing the binding specificity of these WTA-specific antibodies and the importance of the specific location of the GlcNAc modifications on the WTA-chains.

Full text of DOI:10.1002/chem.202101242

Accepted Article
Accepted Article
Title: (Automated) Synthesis of Well-defined Staphylococcus
Aureus Wall Teichoic Acid Fragments
Authors: Jeroen D. C. Codée, Sara Ali, Astrid Hendriks, Rob van
Dalen, Thomas Bruyning, Nico Meeuwenoord, Herman
Overkleeft, Dmitri Filippov, Gijs van der Marel, and Nina van
Sorge
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.202101242
Link to VoR: https://doi.org/10.1002/chem.202101242
01/2020

10.1002/chem.202101242
Chemistry - A European Journal
FULL PAPER
(Automated) Synthesis of Well-defined Staphylococcus Aureus
Wall Teichoic Acid Fragments
Sara Ali,[a] Astrid Hendriks,[b] Rob van Dalen,[b] Thomas Bruyning,[a] Nico Meeuwenoord,[a] Herman S.
Overkleeft,[a] Dmitri V. Filippov,[a] Gijs A. van der Marel,[a] Nina M. van Sorge,[b] Jeroen D.C. Codée*[a]
[a]
[b]
S. Ali, T. Bruyning, N. Meeuwenoord, Prof. dr. H.S. Overkleeft, Dr. D.V. Filippov, Prof. dr. G.A. van der Marel, Prof. dr. J.D.C. Codée.
Leiden Institute of Chemistry, Leiden University
Einsteinweg 55, 2333 CC Leiden (The Netherlands)
E-mail: jcodee@chem.leidenuniv.nl
A. Hendriks, R. van Dalen, dr. N.M. van Sorge
Medical Microbiology, University Medical Center Utrecht, Utrecht University
Heidelberglaan 100, 3584 CX Utrecht (The Netherlands)
Supporting information for this article is given via a link at the end of the document.
Abstract: Wall teichoic acids (WTAs) are important components of
the cell wall of the opportunistic Gram-positive bacterium
Staphylococcus aureus. WTAs are composed of repeating ribitol
phosphate (RboP) residues that are decorated with D-alanine and N-
acetyl-D-glucosamine (GlcNAc) modifications, in a seemingly random
manner. These WTA-modifications play an important role in shaping
the interactions of WTA with the host immune system. Due to the
structural heterogeneity of WTAs, it is impossible to isolate pure and
well-defined WTA molecules from bacterial sources. Therefore, we
here report synthetic chemistry to assemble a broad library of WTA-
fragments, incorporating all possible glycosylation modifications (a-
GlcNAc at the RboP C4; b-GlcNAc at the RboP C4; b-GlcNAc at the
RboP C3) described for S. aureus WTAs. We have used DNA-type
chemistry, employing ribitol phosphoramidite building blocks,
protected with a dimethoxy trityl group, to efficiently generate a library
of WTA-hexamers. Automated solid phase syntheses were used to
assemble a WTA-dodecamer and glycosylated WTA-hexamer. The
synthetic fragments have been fully characterized and diagnostic
signals were identified to discriminate the different glycosylation
patterns. The different glycosylated WTA-fragments were used to
probe binding of monoclonal antibodies using WTA-functionalized
magnetic beads, revealing the binding specificity of these WTA-
specific antibodies and the importance of the specific location of the
GlcNAc modifications on the WTA-chains.
can be substituted in a seemingly random manner with D-alanine
(D-Ala) on C2, a/b-N-acetyl-D-glucosamine (GlcNAc) on C-4 or a
b-GlcNAc on C-3. The GlcNAc residues are introduced by three
dedicated glycosyltransferases, TarS[6] (1,4-b-GlcNAc), TarM[7]
(1,4-a-GlcNAc), and the recently discovered TarP[8] (1,3-b-
GlcNAc), respectively (See Figure 1).
OZ OX
OY
OH
NHAc
O
HO
HO
O
O
O
O
O
O
O
H
P
O
P
HO
HO
O
P
O
O O
AcHN
n
m
O
OR
O
m = 1-3
n = 20-40
HO
HO
HO
H
O
O
HO
HO
or
or
or
X =
HO
AcHN
AcHN
HO
H
H
O
HO
Y =
Z =
HO
AcHN
Me
O
or
H3N
R = peptidoglycan
Figure 1. Schematic representation of S. aureus WTA structure.
Introduction
The Gram-positive bacterium Staphylococcus aureus (S. aureus)
is a common member of the human microbiome and is commonly
found on the skin and in the nares. When entering the blood
stream or internal tissues, S. aureus can cause serious infections,
for which immunocompromised patients are especially at risk.[1]
Extensive use of antibiotics has resulted in increased resistance
among S. aureus strains against commonly-used antibiotics
leading to infections that are difficult to treat. Currently, methicillin
-resistant S. aureus (MRSA) is the most commonly identified
antibiotic-resistant pathogen in clinical medicine worldwide.[2] The
spread of MRSA highlights the urgent need for alternative
therapies, such as passive or active vaccination.[3] Wall teichoic
acids (WTAs) are prime constituents of the Gram-positive cell wall
and can function as effective antigenic epitopes. WTAs are
To unravel the roles of WTAs in biology at the molecular level,
well-defined fragments are indispensable tools. Since isolation
from the bacteria leads to heterogenous mixtures of fragments
and bacterial contaminations, organic synthesis is the method of
choice to generate WTA-fragments with pre-defined substitution
pattern. Because the WTA fragments are built up from repeating
units interconnected through phosphodiester linkages, well-
established DNA chemistry can be used to assemble the target
compounds, also offering the possibility for automation. Over the
years, several approaches have been reported for the assembly
of both WTAs as well as the structurally-related lipoteichoic acids
(LTAs). We have reported on different strategies to assemble
glycerol phosphate-based LTA fragments, including solution
phase, fluorous-tag based and automated solid phase
syntheses.[9] The synthesis of RboP WTA fragments has been
described before by the groups of Pozsgay[10], Lee[11], and
therefore promising candidates for the development of
a
Driguez[4b]
, each building on the use of phosphoramidite
conjugate vaccine against S. aureus infections.[4] WTAs are
anionic poly(ribitol phosphate) (RboP) chains covalently attached
to the thick peptidoglycan layer of the bacterial cell wall. They are
involved in a plethora of vital bacterial functions including host
interaction, biofilm formation, autolysin activity and their
overexpression can increase bacterial virulence.[5] The ribitol
residues that are interconnected through phosphodiester linkages,
chemistry. Pozsgay and Lee however reported that automation of
their syntheses was unsuccessful. We here report on the
development of chemistry that allows for the generation of well-
defined unsubstituted RboP oligomers, using both solution and
automated solid phase synthesis (ASPS) techniques. All
fragments are equipped with an aminohexanol spacer for
1
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10.1002/chem.202101242
Chemistry - A European Journal
FULL PAPER
conjugation purposes. Taking into account that bacterial WTA is
covalently attached to peptidoglycan at the RboP C1-position, this
is also the attachment site for the linker in the synthetic fragments.
We have equipped the synthetic fragments with a biotin handle,
and we have used these to functionalize streptavidin coated,
magnetic beads to generate WTA beads that have been used to
characterize the binding of several monoclonal antibodies at the
molecular level.[12] These binding studies have cleanly revealed
the binding specificity and differences between these antibodies,
not only showing clear differences between monoclonal
carbohydrate appendage, we also generated two hexamers
having both an a-(1,4)-GlcNAc and a b-(1,4)-GlcNAc on the
RboP-chain, leading to the following set of target compounds:
a(1,4) 4, and aa(1,4) 5, ba(1,4) 6, b(1,4) 7, bb(1,4) 8, ab(1,4) 9,
b(1,3) 10 and bb(1,3) 11.
Synthesis of building blocks
antibodies
recognizing
the
a-
or
b-linked
GlcNAc
The building blocks that we designed for the assembly of the
target compounds are depicted in Scheme 1A. The ribitol
phosphate building blocks feature a temporary dimethoxytrityl
group to mask the primary alcohol that is to be elongated during
ribitolphosphates, but also selectivity between the regioisomeric
b-GlcNAc WTA fragments. The synthetic WTA oligomers thus
represent valuable tools in mapping binding specificity of host
receptor molecules at the molecular level.
the chain elongation and
a cyanoethyl phosphoramidite
functionality. Ribitol building blocks 13, 14 and 15 carry an a-(1,4),
b-(1,4) and b-(1,3)-linked N-acetyl glucosamine appendage,
respectively, and the carbohydrate moieties in these building
blocks only carry benzyl ether protecting groups to allow for the
global deprotection of the fragments using mild hydrogenation
conditions. We used phosphoramidite hexanolamine 16[9a] to
introduce the amino linker in the molecules.
The synthesis of ribitol phosphoramidite 12 was accomplished as
described by Hermans et al.[14] Scheme 1B depicts the synthesis
of the glycosylated building blocks 13, 14 and 15. The synthesis
of the required C4-OH ribitol 20, started from ribose 17 by
allylation of the primary alcohol, followed by isopropylidene
hydrolysis to yield 18. Benzylation and ensuing acidic hydrolysis
of the methyl acetal yielded the corresponding lactol intermediate,
which was reduced using sodium borohydride to provide primary
alcohol 19. Protection with a TBDPS group then gave acceptor 20.
Driguez and Lee previously described the use of a glycosylation
strategy in which a mixture of a- and b-N-acetyl glucosamine was
generated. We set out to develop stereoselective glycosylation
methodology to streamline the assembly of the glycosylated
building blocks.
Results and Discussion
The set of compounds, generated for this study is depicted in
Figure 2. All fragments are functionalized with an aminohexanol
spacer for conjugation purposes. We have previously described
that a glycosylated glycerol phosphate hexamer LTA fragment
can be used as an adequate synthetic antigen in the development
of vaccines against Enterococcus faecalis.[13] We therefore
decided to target a set of glycosylated ribitol phosphate WTA
hexamers. In addition, we aimed to assemble longer non-
substituted fragments through automated synthesis. The set thus
comprises RboP-WTA chains carrying no carbohydrate
substituents varying in length from a hexamer (1) to an octamer
(2) and a dodecamer (3). Different carbohydrate decoration
patterns have been introduced (a-GlcNAc or b-GlcNAc residues
can be mounted on the C4-hydroxy groups of the RboP-
monomers or the RboP C3-hydroxy can carry a b-GlcNAc) and
we decided to mount a single GlcNAc residue at the terminal
RboP residue, or two GlcNAc residues at the third and the sixth
RboP. Besides the hexamers carrying
a
single type of
OH
OH
HO
HO
HO
O
AcHN
HO
HO
O
AcHN
HO
HO HO
O
O
O
O
HO HO
OH
O
O
O
O
O
O
O
O
O
O
O
=
=
=
=
=
P
P
P
P
O
OH
O
O
OH
O
O
NHAc
HO
O
NH2
4
OH
HO
1
2
3
H
H
H
7
11
4 α(1,4)
7 β(1,4)
H
H
10 β(1,3)
H
H
5 αα(1,4)
6 βα(1,4)
8 ββ(1,4)
9 αβ(1,4)
H
H
H
H
11 ββ(1,3)
Figure 2. The library of RboP-WTA structures, generated for this study.
2
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10.1002/chem.202101242
Chemistry - A European Journal
FULL PAPER
Dimethoxytrityl (DMTr) group to afford 25. The removal of the allyl
ether was accomplished by the iridium catalyzed allyl
isomerization and iodine mediated enol ether hydrolysis, under
slightly basic conditions to deliver alcohol 26, which was
functionalized with a cyanoethyl phosphoramidite to give the
building block 13 in 81% yield.
The assembly of the a-linked GlcNAc building block was
accomplished using glucosazide donor 21, which was coupled
with acceptor 20 to yield 22 as a 7:1 a/b-mixture. The two
anomers could be separated after Zemplén deacetylation, leading
to the pure a-product 23 in 70% yield.[15] Benzylation of the
liberated alcohols, followed by Staudinger reduction and
subsequent acetylation of the amine yielded 24 in 89% yield over
3 steps. Next the TBDPS group was exchanged for a 4,4'-
OBn
OBn
A
BnO
OBn
BnO
BnO
O
AcHN
OBn OBn
OBn
O
DMTrO
O
OCNE
AcNH
OBn O
P
OBn OBn
O
OBn O
OBn
12
N(i-Pr)2
DMTrO
13
O
OCNE
DMTrO
O
OCNE
DMTrO
O
OCNE
P
P
P
N(i-Pr)2
N(i-Pr)2
OBn
N(i-Pr)2
14
15
CNEO
O
NHCbz
P
O
NHAc
4
BnO
16
N(i-Pr)2
OBn
BnO
B
O
O
OBn OH
OBn OH
AllylO
OMe
HO
OMe
a,b
c,d,e
f
HO
OAllyl
TBDPSO
OAllyl
OAc
HO
18
OH
O
O
OBn
OBn
19
17
20
OBn
O
OR
NH
BnO
BnO
O
RO
RO
O
AcO
AcO
O
CCl3
AcHN
BnO
N3
N3
O
BnO
O
21
l
i
R1O
OR2
13
TBDPSO
OAllyl
g
OBn
OBn
24: R1 = TBDPS, R2 = Allyl
25: R1 = DMTr, R2 = Allyl
26: R1 = DMTr, R2 = H
22: R=Ac
h
j
23: R=Bn
k
OBn
OBn
OBn
OBn
BnO
BnO
NH
BnO
BnO
BnO
O
N3
AcHN
O
m
CCl3
O
O
N3
27
l
n
14
OBn O
OBn O
TBDPSO
OAllyl
R1O
OR2
OBn
OBn
28
29: R1 = TBDPS, R2 = Allyl
30: R1 = DMTr, R2 = Allyl
31: R1 = DMTr, R2 = H
j
k
O
O
HO
HO
O
O
O
O
O
O
AllylO
NAPO
O
AllylO
OH
OH
o,p
q,r
t
s
HO
O
NAPO
O
O
NAPO
35
34
32
33
OR OR
ONAP
OBn OBn
OBn OBn
OR2
l
v
m
R1O
OR2
R1O
TBDPSO
u
OAllyl
15
OAllyl
O
38: R1 = TBDPS, R2 = H
39: R1 = H, R2 = H
40: R1 = TBDPS, R2 = H
36: R = H
37: R = Bn
w
x
R3
O
BnO
OBn
BnO
41: R1 = TBDPS, R2 = Allyl, R3 = N3
n
j
42: R1 = TBDPS, R2 = Allyl, R3 = NHAc
43: R1 = DMTr, R2 = Allyl, R3 = NHAc
44: R1 = DMTr, R2 = H, R3 = NHAc
k
Scheme 1. Assembly of building blocks. a) AllylBr, NaH, THF/DMF (7:1), 0 °C to rt; b) AcOH/H2O 1:1, 50 °C, 300 mbar, 62% over 2 steps; c) BnBr, NaH, THF/DMF
(7:1), 0 °C to rt; d) 4M HCl dioxane, 80 °C; e) NaBH4, MeOH, 0 °C, 50% over 3 steps; f) TBDPSCl, TEA, DCM 0 °C to rt, 95%. g) 21, TMSOTf, DCM, rt, 92%, 7:1
a/b; h) i. NaOMe, MeOH, rt, 70% a-anomer; ii) BnBr, NaH, THF/DMF (7:1), 0 °C to rt, 73%; i) i. PMe3, KOH, THF; ii. Ac2O, pyridine, 24: 89% over 2 steps; j) i. TBAF,
THF, rt; ii) DMTrCl, TEA, DCM, 25: 67%; 30: 36%; 43: 60%; k) i. Ir(COD)(Ph2MeP)2PF6, H2, THF, ii. I2, sat. aq. NaHCO3, THF, 26: 88%; 31: 79%; 44: 94%; l) 2-
cyanoethyl-N,N-diisopropylchlorophosphoramidite, DIPEA, DCM, 13: 81%; 14: 78%; 15: 85%; m) 27, TMSOTf, ACN, -40 °C to 0 °C, 28: 85%; 41: 80%; n)
propanedithiol, pyridine, H2O, TEA, rt; ii. Ac2O, pyridine, 29: 59% 2 steps; 42: 86%; o) i. DMSO, Ac2O; ii. NaBH4, EtOH/H2O, 69% over 2 steps; iii. NAPBr, NaH,
TBAI, THF; p) p-TsOH∙H2O, MeOH, 75% over 2 steps; q) i. 0.2M NaIO4 in H2O, ethylene glycol, MeOH; ii. NaBH4, MeOH; r) AllylBr, NaH, THF/DMF, 88%; s)
3
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10.1002/chem.202101242
Chemistry - A European Journal
FULL PAPER
THF/H2O/formic acid, 85%; t) i. NaBH4, MeOH; ii. TBDPSCl, TEA, DCM, 57% over 2 steps; u) BnBr, NaH, THF/DMF; v) DDQ, DCM/H2O; w) TBAF, THF, 62% over
2 steps; x) TBDPSCl, TEA, DCM, 98%.
incorporated the glycosylated building blocks in the synthetic
scheme, generating the hexamers with one or two GlcNAc
residues. All chain elongation events proceeded uneventfully and
the yields for all coupling cycles are summarized in Scheme 2.
The presence of the carbohydrate appendages did not seem to
influence the efficiency of the condensation chemistry, reaffirming
the power of phosphoramidite chemistry to assemble this type of
biopolymer fragments. Deprotection of the fragments was
accomplished by first removing the cyanoethyl groups to deliver
the corresponding phosphate diesters followed by hydrogenation
to remove all benzyl groups. All target compounds were obtained
after gel permeation size exclusion purification and sodium ion
exchange.
The corresponding b-GlcNAc building block was generated
through the union of tribenzyl glucosazide donor 27 and acceptor
20. The use of acetonitrile as a b-directing solvent in combination
with a low reaction temperature ensured the stereoselective
formation of the desired b-glucosamine linkage and 28 was
obtained in 85%.[16] Similar protecting group manipulations as
described for the a-GlcNAc ribitol then delivered the required b-
GlcNAc building block 14.
The synthesis of the b-(1,3)-GlcNAc-functionalized ribitol
phosphoramidite 15 required the generation of C3-OH ribitol 40.
To this end, diacetone-D-glucose 32 was transformed into the
corresponding allose, having the required ribose stereochemistry,
through a well-established[17] oxidation-reduction sequence in
69% yield. Naphthylation then gave the fully protected
allofuranose and regioselective removal of the 5,6-isopropylidene
group using p-TsOH in MeOH provided compound 33 in 75% over
2 steps. Next, oxidative cleavage of the 5,6-diol with NaIO4 gave
the aldehyde, which was reduced to form the primary alcohol.
Allylation of this alcohol, afforded ribose 34 with a yield of 88%
over 3 steps. Subsequently, the 1,2-isopropylidene was cleaved
under acidic conditions to give 35. Reductive opening of
hemiacetal 35, and TBDPS protection of the primary alcohol then
provided 36. Benzylation of the remaining alcohols led to fully
protected ribitol 37. During this alkylation step, a byproduct
formed, due to TBDPS migration and this product could not be
separated from the desired product at this stage. Therefore, the
naphthyl ether and TBDPS ethers were removed to provide 38,
which could be purified from the formed byproduct yielding
product 39 in 62% yield over 3 steps. Reinstallation of the TBDPS
group on the primary alcohol furnished the C3-OH ribitol building
block 40.
Automated solid phase synthesis of RboP WTA fragments
Finally, we investigated the possibility to assemble RboP-WTA
oligomers using an automated solid phase approach (Scheme 3).
To this end, we selected the commercially-available controlled
pore glass (CPG) resin 68 that carries a phthalimide protected
aminohexanol spacer. Previously, Pozsgay and co-workers
described that an intractable mixture was obtained in the
attempted automated solid phase synthesis of a RboP-octa- and
dodecamer.[10] As suggested by the authors, this could have been
caused by the high concentration of TCA used to remove the
DMTr groups. In our solution phase syntheses described above,
we used a milder acid, DCA, for the removal of the DMTr group
and these conditions were here translated to the solid phase. The
syntheses were performed on an Äkta oligopilot plusTM
synthesizer on 10 µmol scale (Scheme 2). The DMTr group was
cleaved from resin 68 using 3% DCA in toluene and the coupling
with cyanoethyl (CNE) amidite 12 under 5-(Benzylthio)-1H-
tetrazole (BTT) activation then provided the resin bound
phosphite. Oxidation using I2 in pyridine/H2O yielded the
phosphate triester after which a capping step took place to
prevent any unreacted alcohol functionalities to react in the next
step, which could lead to byproducts that are difficult to separate.
Removal of the DMTr group allowed a new cycle to start and the
coupling cycles were repeated 7 and 11 times to reach the target
octa- and dodecamer. Treatment of the resin with 3% DCA
unmasked the primary alcohol and subsequent treatment with
aqueous 25% NH3 cleaved the cyanoethyl groups and released
the oligomers from the resin. Scheme 3B depicts the LCMS
chromatogram of the crude products 70 and 71, indicating highly
efficient syntheses of these oligomers. Purification of the crude
oligomers by reversed phase HPLC and desalting afforded 70 and
71 in 15% and 11% yield respectively. Hydrogenation of the semi-
protected octa- and dodecamer yielded the targets 2 and 3 both
in quantitative yields. We also probed the use of GlcNAc carrying
building blocks in the automated solid phase synthesis and
assembled hexamers 10 and 11, having either one or two b-(1,3)-
GlcNAc appendages. Also these syntheses proceeded
uneventfully, as judged from the amount of released DMT-groups
and the benzyl protected target hexamers 72 and 73 were
obtained in 20% and 11% yield, after reversed HPLC and a
desalting step. Final hydrogenolysis of the semi-protected
hexamers gave the target structures 10 and 11 in 87% and
quantitative yield, respectively.
The glycosylation of 40 was accomplished using donor 27 and the
reaction conditions described above delivered the glucosamine
ribitol 41 as a single anomer in 80% yield. This synthon was
uneventfully transformed into the desired b-(1,3)-GlcNAc ribitol
phosphoramidite 15 using the protecting and functional group
manipulations described above.
Assembly of RboP WTA fragments
Solution phase synthesis of RboP WTA fragments
With all required building blocks in hand, we set out to generate
the set of target structures, using a solution phase synthesis
approach depicted in Scheme 2. First the coupling chemistry was
explored in the synthesis of non-substituted hexamer 1. The
RboP-chain
elongation
steps
using
phosphoramidite
condensations consisted of 3 steps. In the first step the amidite
was activated under agency of 4,5-dicyanoimidazole (DCI) to
enable attack by the primary ribitol alcohol to form the phosphite
intermediate, which was oxidized in the next step using (10-
camphorsulfonyl)oxaziridine (CSO). Detritylation using 3%
dichloroacetic acid (DCA) in DCM liberated the primary alcohol
and silica gel column chromatography yielded the pure ribitol
phosphate fragment, ready for the next elongation step. Starting
with ribitol 45[14] and spacer amidite 16, monomer 46 was
obtained in 85% yield. From alcohol 46 the coupling cycles were
repeated five times to yield 47-51, all in good yield. Next, we
4
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10.1002/chem.202101242
Chemistry - A European Journal
FULL PAPER
OBn
BnO
OBn
AcHN
BnO
O
BnO BnO
O
BnO BnO
O
CNEO
CNEO
CNEO
O
O
P
O
O
P
O
O
P
=
=
=
=
OBn
O
OBn
O
O
O
O
NHAc
BnO
BnO
BnO
BnO
O
NHCbz
AcHN
OBn
BnO
4
BnO
O
CNEO
O
O
P
=
OBn
O
OBn OBn
OBn
a, 16
85%
DMTrO
OH
46
H
45
a, 12 74%
H
a, 12
88%
80%
a, 13 53%
a, 14 88%
a, 15 83%
47
H
H
H
H
2
2
2
2
48
54
59
64
a, 12
a, 12 82%
a, 12 89%
a, 12 92%
H
H
H
H
2
2
2
3
49
55
60
65
a, 12
76%
a, 12 89%
a, 12 68%
a, 12 87%
H
H
H
H
2
2
50
56
61
66
4
2
a, 12 91%
a, 12 61%
a, 13 88%
a, 14 79%
a, 13 76%
a, 15 100%
H
H
H
H
H
2
2
51
57
62
67
5
2
2
2
a, 13
H
H
73%
5
52
2
2
2
2
58
63
a, 14
H
85%
5
53
b
1, 4 - 9, 11
Scheme 2. Assembly of the target WTA-oligomers. Reagents and conditions: a) i. DCI, ACN, phosphoramidite 12, 13, 14 or 15; ii. CSO; iii. 3% DCA in DCM. b) i.
NH3 (30-33% aqueous solution), dioxane; ii. Pd black, H2, AcOH, H2O/dioxane, 1: 87%; 4: 96%; 5: 55%; 6: 16%; 7: 68%; 8: 78%; 9: 88%; 11: 70%.
A
B
I2, pyr/H2O
O
O
O
O
12 or 15
BTT, ACN
DCA
toluene
OBn OBn
CNEO
70
N
N
HO
N
O
O
N
O
n
H
H
n times
DMTr
P
4
4
OR
O
O
O
68: R = DMTr
69: R = H
a
b
OBn OBn
OBn
O
c
70: n = 8
71: n = 12
O
O
O
O
O
n
NH3
H
2, 3
P
71
4
O
OBn
O
β-GlcNAc =
OBn OBn
OBn OBn
OBn OBn
OR1
OBn OBn
OBn
c
O
O
P
O
P
BnO
BnO
HO
O
O
O
O
O
2
NH3
10, 11
P
NHAc
4
OR2
OBn
O
O
O
2
72: R1 = Bn, R2 = β-GlcNAc
73: R = R = β-GlcNAc
1
2
Scheme 3. Automated solid phase assembly of WTA-fragments. Reagents and conditions: a) 3% DCA in toluene (3 min); 12 or 15 BTT, ACN (5 min); I2, pyr/H2O
(1 min); Ac2O, N-methylimidazole, 2,6-lutidine, ACN (0.2 min); b) 25% NH3 (aq) (60 min); 70: 15%; 71: 11%; 72: 20%; 73: 11%; c) Pd black, H2, dioxane H2O, AcOH,
2: 100%; 3: 100%; 10: 87%; 11: 100%.
5
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Characterization of glycosylated hexamers
1
We characterized the assembled oligomers by H, 13C and 31P
NMR and Figure 3 depicts the 1H and 13C spectra of the WTA
hexamers carrying two a-1-4-, b-1,4- or b-1,3-GlcNAc residues,
namely compounds 5, 8, and 11. The NMR spectra of these well-
defined WTAs can be very useful for the structure determination
of new WTA-species isolated from bacterial strains, in particular
the position and configuration of these modifications along the
chain. As shown in Figure 2A, the anomeric protons of the a-
linked GlcNAc (in 5) are present at a different chemical shift value
than the anomeric protons of the b-GlcNAc (in 8 and 11). The b-
1,3-GlcNAc anomeric protons appear at 4.62 ppm, slightly lower
than the b-1,4-GlcNAc anomeric protons with resonances at 4.70
ppm. These values are in accordance with those reported by
Driguez and co-workers[4b] for b-1,3-GlcNAc modified WTA,
isolated from strain ATC 55804 with an anomeric value of 4.65
and with b-1,4-GlcNAc WTA isolated from strain Wood 46
showing anomeric signals at 4.75 ppm. The reported anomeric
signals for a-1,4-GlcNAc WTA from Newman D2C 5.07 (at 5.04
ppm) also agree well with the values for the a-1,4-GlcNAc WTA
(5.03 ppm and 5.06 ppm). The values are also well in line with the
TarP and TarM modified WTAs described by Gerlach et al.[8]
Figure 2B shows the 13C NMR of the WTAs 5, 8 and 11. The
anomeric signals of the b-1,3-GlcNAc WTA 11 appear at 101.6
and the C3 glycosylated Rbo 11 shows a shift for the ribitol C3 at
80.8 and 81.0 which is in agreement with the previously reported
data, which described a chemical shift of 81.8 ppm for this carbon,
appearing at a higher ppm value than the non-glycosylated ribitol
positions.[8] The b-1,4-GlcNAc WTA 8 anomeric shifts can be
found at 101.4 ppm and 101.6 ppm, comparable to the b-1,3-
GlcNAc anomeric signals. The Rbo C4 of this b-1,4-glycosylated
hexamer appear around 79.4 - 79.9, which is in accordance with
the value previously reported (d = 80.8 ppm) for glycosylated C-4
RboP-carbon atoms. The anomeric signals corresponding to the
a-1-4-GlcNAc WTA 5 appear at 96.4 ppm and 96.5 ppm. This
smaller chemical shift is expected for a-glycosidic linkages and
the RboP C4 can be found at 77.6 - 77.8 ppm.
31P NMR spectra have previously not been described for different
glycosylated WTA oligomers. As depicted in Figure 2C,
comparing the 31P NMR spectra of the a-1,4-GlcNAc WTA, b-1,4-
GlcNAc WTA and b-1,3-GlcNAc WTA hexamers, it appears that
the 31P-chemical shift is diagnostic for the type of GlcNAc
appendage. The 31P signals are assigned P1 to P6, as shown in
the schematic structure diagrams next to the spectra. The
spectrum of the aa-1,4-GlcNAc hexamer 5 shows three types of
signals: three around 2 ppm, a single peak at 1.8 ppm and two
peaks around 1.7 ppm. Considering that the phosphate next to
the spacer will be different from the other phosphate diesters that
are all flanked by two ribitol residues the single peak likely
corresponds to the phosphate diester attached to the spacer.
When the spectra of the a-1,4-GlcNAc- and aa-1,4-GlcNAc
hexamers 4 and 5, respectively, are compared it becomes clear
that one peak has shifted to a lower ppm value. This phosphorous
resonance thus likely corresponds to P3. This analysis also holds
for the b-1,4-GlcNAc- and bb-1,4-GlcNAc hexamers 7 and 8,
which show a similar chemical shift pattern. The 31P-spectrum of
the b-1,3 glycosylated WTAs 10 and 11 shows similarity to the b-
1,4- and a-1,4-GlcNAc WTAs. The introduction of the second
GlcNAc substituent at the third ribitol residue causes the
resonance of the phosphate diesters P3 and P4 (which are equally
close to the GlcNAc residue in the middle of the RboP moiety) to
shift to a lower chemical shift: the peaks around 1.8-1.9,
corresponding to four P signals, belong to P1, P3, P4 and P6. The
phosphodiesters, flanked by two non-substituted ribitols are found
around 2 ppm. In all, this analysis shows that 31P-NMR chemical
shifts can be diagnostic for the substitution pattern along the
RboP chain, and the relative intensity of the signals indicative for
the degree of glycosylation.
Figure 3. Characterization of the synthetic WTA-fragments. A) 1H-NMR (500
MHz, D2O) of 5, 8 and 11. B) 13C-NMR (126 MHz, D2O) of 5, 8 and 11. C) 31P-
NMR (202 MHz, D2O, 10 mg.mL, pH 7) of 4, 5, 7, 8, 10 and 11.
Antibody binding
6
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With the synthetic WTA fragments in hand, we set out to develop
an assay to interrogate antibody binding. Recently, Lupardus and
co-workers have described the development of different
monoclonal antibodies, generated from B-cells of S. aureus
infected patients.[18] Structural studies were performed with these
mAbs using X-ray crystallography on a monomeric b-1,4-GlcNAc
ribitol phosphate fragment and binding studies were performed
with whole bacteria, using biosynthesis knock-out strains to
unravel the role of the WTA-glycosylation pattern.[19] The
availability of our synthetic fragments now allows for direct binding
studies at the molecular level. We have recently described the
development of a magnetic bead assay to study the deposition of
antibodies through binding of enzymatically glycosylated WTA
fragments.[12] We here used the same set up and equipped the
synthesized hexamers with a biotin handle, exploiting the amine
function of the hereto introduced amino spacer (Figure 4A). Next,
show deposition of the antibody. Interestingly the ab-1,4-GlcNAc
9 functionalized beads show less antibody binding than the
corresponding ba-1,4 (6) derivatized beads. This may indicate
that the mAb binds better to “internal” a-GlcNAc-residues than to
an a-GlcNAc at the terminus of the WTA chain. The analysis of
clone 4624 provides
a nearly identical picture: a-GlcNAc-
functionalized WTA fragments are selectively recognized with a
single sugar residue being the minimal requirement for binding
(Figure 4C). Also this mAb seems to have a preference for internal
a-GlcNAc-residues.
Clones 4497 and 6292 have been identified to recognize b-1,4-
GlcNAc-WTA and Figure 4D shows that 4497 binds to all WTA-
hexamers carrying a b-1,4-GlcNAc-residue. A single b-1,4-
GlcNAc-moiety seems to be sufficient for binding and there does
not seem to be a strong preference for the position of the b-1,4-
GlcNAc on the WTA chain. This mAb also binds to the
regioisomeric b-1,3-GlcNAc epitopes (11), indicating that this
mAb is cross-reactive to TarP-expressing S. aureus. The binding
to the b-1,3-GlcNAc-WTA fragment 11 is however, significantly
weaker than binding to its b-1,4-counterpart 8. The binding assays
using clone 6292 (Figure 4E) show that the synthetic fragments
are poorly recognized by this mAb. In contrast, the hexamers
glycosylated using the b-1,4-GlcNAc-transferase TarS do induce
antibody deposition on the beads. The hexamers that have been
enzymatically glycosylated have a higher density of b-1,4-
GlcNAc-residues (an average of 3-4 GlcNAc residues per
hexamer) indicating that this mAb either binds preferentially to
WTA fragments carrying multiple b-1,4-GlcNAc-residues in close
proximity or that the affinity of this mAb is lower and thus requires
the simultaneous presentation of multiple epitopes, within the
same WTA-fragment.
streptavidin-coated
magnetic
dynabeads
M280
were
functionalized with the biotinylated hexamers and then
interrogated using monoclonal antibodies, generated to recognize
a-GlcNAc functionalized RboP WTAs (clone 4461 and clone
4624) or b-1,4-GlcNAc carrying RboP WTAs (clones 4497 and
6292).
As can be seen in Figure 4B, mAb 4461 binds to the beads
functionalized with the WTA fragments carrying an a-GlcNAc
appendage 4 and 5. The non-glycosylated RboP-hexamers or the
hexamers carrying b-GlcNAc-residues are not recognized by this
antibody. A single a-GlcNAc-functionality (as in 4) seems to be
sufficient for binding, but the beads functionalized with two a-
GlcNAc-residues (as in 5) show increased binding. The presence
of a b-1,4-GlcNAc in the WTA fragments does not interfere with
binding as the ab-1,4 (9) and ba-1,4 (6) functionalized beads both
.
7
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10.1002/chem.202101242
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Figure 4. Magnetic WTA-beads for antibody binding assays. A) Workflow for the antibody binding assay: 1) Biotinylation of WTA fragments 4-11; 2) Adsorption on
streptavidin coated M280 Dynabeads; 3) binding of monoclonal antibodies; 4) Alexa 488-Protein G conjugation; 5) Readout of fluorescent beads. B-E) Binding of
monoclonal antibody B) 4461, C) 4624, D) 4497 and E) 6292 to the WTA-functionalized beads.
2017; c) R. Adamo, A. Nilo, B. Castagner, O. Boutureira, F. Berti, G.
J. Bernardes, Chem. Sci. 2013, 4, 2995.
Conclusion
[5]
[6]
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S. Sobhanifar, L. J. Worrall, D. T. King, G. A. Wasney, L. Baumann,
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PLoS Pathog. 2016, 12, e1006067.
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Blaukopf, L. Baumann, E. Lameignere, M. Solomonson, E. D.
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Marel, Bioorg. Med. Chem. 2010, 18, 3668; b) D. van der Es, W. F.
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J. Hogendorf, N. Meeuwenoord, D. Laverde, A. van Diepen, H. S.
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Laverde, H. S. Overkleeft, J. Huebner, G. A. van der Marel, J. D.
Codee, Bioorg. Med. Chem. 2016, 24, 3893.
We have here described synthetic chemistry to assemble all
currently known glycosylation types of S. aureus RboP WTA. We
have developed stereoselective glycosylation reactions to
generate building blocks that have been used to assemble a-1,4,
b-1,4 and b-1,3-GlcNAc functionalized RboP-WTAs. A strategy
was used employing phosphoramidite chemistry in combination
with DMTr-protected building blocks. This “DNA-type” chemistry
was successfully transposed to an automated solid phase format,
significantly streamlining the assembly of longer WTA fragments.
Detailed spectroscopic analysis of the fragments has revealed
diagnostic signals that can be used as reference for future WTA-
structure elucidation studies. Finally, we have shown the
applicability of the synthetic WTA-fragments in the development
of a binding assay to map binding interactions of anti-WTA
monoclonal antibodies at the molecular level. These interaction
studies have shown that antibodies that recognize b-1,4-GlcNAc
WTA can be cross-reactive with b-1,3-GlcNAc WTA. It is thus
likely that IgG in human serum is also capable of interacting with
both TarS-WTA and TarP-WTA, as described by Van Dalen et al.
[7]
[8]
[9]
[10]
[12]
More detailed binding studies are required to pinpoint the
differences in binding between the two different epitopes and the
(recombinantly expressed) monoclonal antibodies or human sera.
It is expected that raising antibodies against S. aureus TarP-WTA,
or a synthetic conjugate featuring this WTA-type will lead to
specific anti-b-1,3-GlcNAc-WTA antibodies. Alternatively,
structure-guided antibody engineering may open up avenues to
generate selective and tight binding WTA-glycosylation type
specific antibodies. Well-defined WTA fragments are valuable
tools to delineate clear structure-activity relationships and will be
used in the future to characterize antibody binding epitopes and
to probe binding to other receptors, such as C-type lectins and
unravel the mode of action of biosynthesis enzymes at the
molecular level.
[11]
[12]
[13]
[14]
[15]
[16]
Acknowledgements
Simone Nicolardi and Fabrizio Chiodo for MALDI measurements.
Carla de Haas and Piet Aerts for technical assistance with
recombinant antibody production. This work was supported by
Vidi (91713303) and Vici (09150181910001) grants from the
Netherlands Organization for Health Research and Development
(ZonMW) to N.M.v.S.
[17]
[18]
W. Sowa, G. H. S. Thomas, Can. J. Chem. 1966, 44, 836.
R. Fong, K. Kajihara, M. Chen, I. Hotzel, S. Mariathasan, W. L. W.
Hazenbos, P. J. Lupardus, mAbs 2018, 10, 979.
[19]
J.-H. Lee, N.-H. Kim, V. Winstel, K. Kurokawa, J. Larsen, J.-H. An,
A. Khan, M.-Y. Seong, M. J. Lee, P. S. Andersen, A. Peschel,
B.-L. Lee, Infect. Immun. 2015, 83, 4247.
Keywords: ribitol phosphate • automated synthesis • wall
teichoic acids • Gram-positive bacteria • antibodies
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8
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9
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Entry for the Table of Contents
Using tailor made teichoic acid building blocks a library of glycosylated ribitol phosphate Staphylococcus aureus wall teichoic acids
(WTA) has been assembled, ranging in length and varying in N-acetyl glucosamine substitution pattern. The library of well-defined
WTAs has been used to develop a magnetic bead binding assay to interrogate monoclonal antibody binding, revealing the preference
of the various antibodies for different glycosylation patterns.
10
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