Total synthesis of lipoteichoic acid of streptococcus pneumoniae

Article abstract of DOI:10.1002/anie.200906163

"Chemical Equation Preseted" Mixed signals: The glycophospholipid 1, consisting of two cholinylphosphoCalNAc units, two 2-acetamino-4-amino2,4,6- trideoxygalactose rings, three glucose residues each with different linkages to other sugar units, and a ribitolphosphate residue, has been synthesized. Target 1 is recognized by the immune system, but not by the TLR-2 signaling receptor as previously postulated.

Full text of DOI:10.1002/anie.200906163

Angewandte
Chemie
DOI: 10.1002/anie.200906163
Total Synthesis
Total Synthesis of Lipoteichoic Acid of Streptococcus pneumoniae**
Christian Marcus Pedersen, Ignacio Figueroa-Perez, Buko Lindner, Artur J. Ulmer,
Ulrich Zꢀhringer, and Richard R. Schmidt*
Dedicated to Professor Hans Paulsen
Streptococcus pneumoniae is one of the most common Gram-
positive pathogens. Upon colonizing the upper respiratory
tract it causes severe infections and it causes life-threatening
diseases like pneumonia, bacteremia, and meningitis when it
reaches the lower respiratory tract or the bloodstream,[1]
thereby resulting in a high mortality rate.[2,3]
As for all Gram-positive bacteria, the cell wall of
S. pneumoniae consists of several layers of peptidoglycans,
to which teichoic acids are covalently linked, and of lip-
oteichoic acids (LTAs) which are anchored in the cell
membrane. Structural analysis of pneumococcal LTA
revealed quite a different chemical composition compared
to that of LTA from Staphylococcus aureus.[4] The polyglycer-
ophosphate backbone of staphylococcal LTA is replaced by a
pentameric repeating unit which consists of a ribitolphos-
phate having in some cases an attached GalNAc residue and a
tetrasaccharide moiety connected to two attached phospho-
choline residues (Scheme 1, 1).[4,5]
The pneumococcal LTA is recognized by the innate
immune system, thereby stimulating the release of pro-
inflammatory cytokines, but with reduced potency compared
to staphylococcal LTA.[6] Activation is supposed to occur
through the Toll-like receptor 2 (TLR-2) with CD14 as a co-
receptor.[7] However, as TLR-2 appears to recognize a broad
range of structurally different bacterial compounds, specific
stimulation of the immune system through a TLR-2–LTA
interaction has been recently questioned.[8–11] In addition,
different strains of Gram-positive bacteria produce LTAs
having structural variations. Therefore, pneumococcal LTA
may have galactose instead of glucose in the repeating unit,[12]
ribitol with d-alanine residues,[13,14] or heterogeneity in the
attached phosphocholines.[4] These structural changes may
alter LTA-mediated functions of the bacterial cell wall, such
as adhesion to host cells, growth, and pathogenicity.
To investigate the impact of LTA and the effect of its
structural modifications upon LTA function, we developed
the first, and modular, synthesis of streptococcal LTA from
the R6 strain.[4,13] Hence, the major structural isomer 1a,
+
having R = H, X = NH3 , and n = 1 (instead of about 2),[13]
was selected as a target molecule. In this way, the previous
structural assignments could also be confirmed.
The design of the synthesis of LTA 1a is outlined in
Scheme 1. For a convergent synthesis, disconnections at two
glycosidic linkages (ꢀ and ꢀ) and at the phosphate linkages
1
3
(ꢀ and ꢀ), were envisaged, which would lead to the A-DAG
2
4
(DAG = diacylglycerol), CB, ED, and HGF fragments. The
disconnections of each of the fragments results in nine
carbohydrate-derived building blocks (2–10). Compound 2
is required as a precursor for the DAG moiety and com-
pounds 3–10 are precursors for the sugar moieties A–H in the
target molecule. The stereoselective generation of the glyco-
sidic linkage will be performed with O-glycosyl trichloroace-
timidates as glycosyl donors.[15]
For the envisaged modular synthesis of structurally
heterogeneous streptococcal LTAs, the building blocks for
sugar moieties B, D, and G are particularly important. The 2-
acetamino-4-amino-2,4,6-trideoxygalactose B is frequently
found as a constituent of bacterial saccharides;[4] derivatives
of this compound have been synthesized and also used in the
synthesis of fragments related to LTA from S. pneumoniae.[16]
For the total synthesis of 1a, B requires orthogonal N pro-
tection, hence the benzyloxycarbonyl protecting group (Z) at
the 4-position was chosen to allow the generation of an amino
or an acetylamino group, and the Troc group at the 2-position
was chosen to facilitate formation of the b linkage in the
glycosylation step for the glycosyl donor 4. The donor 4 was
[*] Dr. C. M. Pedersen, Dr. I. Figueroa-Perez, Prof. Dr. R. R. Schmidt
Fachbereich Chemie, Universitꢀt Konstanz
Fach 725, 78457 Konstanz (Germany)
obtained from glucosamine by
a
demanding route
(Scheme 2).[17] After the transformation of glucosamine into
the 2-azido derivative 11[18] and subsequent O-deacetylation,
the decisive steps for an efficient synthesis of 4 were the
regioselective 6-O-tosylation, which permitted the straight-
forward generation of the 6-deoxy derivative 12, and then
regioselective 3-O-benzoylation. Subsequently, the 4-O-tri-
flate could be formed and then reacted with potassium
phthalimide to deliver the versatile intermediate 13. Replace-
ment of the phthalimido group by the Z group, reduction of
the azide, and then introduction of the Troc group afforded
14, which was readily transformed into the glycosyl donor 4.
The structurally closely related moiety G also requires
Fax: (+49)7531-883-135
E-mail: richard.schmidt@uni-konstanz.de
Priv.-Doz. Dr. B. Lindner, Prof. Dr. A. J. Ulmer, Prof. Dr. U. Zꢀhringer
Forschungszentrum Borstel, Leibniz-Zentrum fꢁr Medizin und
Biowissenschaften, 23845 Borstel (Germany)
[**] This work was supported by the Deutsche Forschungsgemeinschaft
and the Fonds der Chemischen Industrie. C.M.P. is particularly
grateful for a fellowship from the Danish Agency for Science,
Technology and Innovation. We wish to thank B. Wegner, H. Moll, H.
Kꢀßner, and Dr. N. Gisch for excellent LTA purification and help in
MS and NMR analyses.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200906163.
orthogonal N protection:
a phthalimido group at the
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Scheme 1. LTA of Streptococcus pneumoniae and the retrosynthetic strategy showing building blocks 2–10. All=allyl, Alloc=allyloxycarbonyl, Bn=benzyl,
TBDPS=tert-butyldiphenylsilyl, Troc=2,2,2-trichloroethoxycarbonyl.
4-position and a 2-azido group facilitate the a selec-
tivity in the glycosylation step and allow the installa-
tion of an amino or an acetylamino group at C4,
thereby leading to the requisite glycosyl donor 9.
Donor 9 was readily obtained in two steps from
intermediate 13.[17] For the ribitol residue D, introduc-
tion of an a-GalNAc residue or eventually a d-alanyl
residue at O3 has to be envisaged. Hence, in addition
to the known building block 6, obtained from d-ribose
in six steps,[19] the 3-O-naphthylmethyl-protected
derivative, which possesses an oxidatively labile
Scheme 2. Synthesis of glycosyl donors 4 and 9. Reagents and conditions:
a) TfN3; b) Ac2O, Pyr (73%); c) 4-MeOC6H4OH, TfOH, CH2Cl2, 08C (84%);
d) 1. NaOMe, MeOH, 2. TsCl, Pyr, 3. Ac2O, DMAP (94%); e) TBAI, MeCN, reflux
(98%); f) NaBH3CN, DMPU, 958C (86%); g) 1. NaOMe, MeOH, 2. BzCl, Pyr,
protecting group to permit the required orthogonal
protecting group removal,[20]was prepared.
The galactosamine derived sugars E and F carry-
ing the phosphocholine residues at O6 are b-1,3- and
À308C (61%); h) 1. Tf2O, Pyr, 2. PhthNK, DMF, RT (84%); i) (CH2NH2)2, BuOH
a-1,4-linked, respectively. Therefore, when starting
from galactosamine different strategies for the syn-
thesis of building blocks 7 and 8, requiring permanent
benzyl protection at O4 or at O3, respectively, were
required. Azido groups in the 2-position were chosen
for 7 and 8 (as for 9) to concomitantly generate the
desired acetamino groups. In this way the a linkage
required for 7 would arise from thermodynamic
control, and the b linkage required for 8 would arise
(68%); j) ZCl, NaHCO3, THF/H2O (4:1; 88%); k) 1. Raney-Ni, H2, EtOH,
2. Troc-Cl, NaHCO3 (71%); l) Alloc-Cl, Pyr (85%); m) 1. CAN, MeCN/H2O,
2. CCl3CN, DBU, CH2Cl2 (89%); n) PhI(O2C-CF3)2, BF3·OEt2, CH2Cl2 saturated
with H2O (87%); o) CCl3CN, DBU, CH2Cl2 (72%). Bz=benzoyl, CAN=ceric
ammonium nitrate, DBU=1,8-diazabicyclo[5.4.0]undec-7-ene, DMAP=4-(dime-
thylamino)pyridine, DMF=N,N-dimethylformamide, DMPU=N,N’-dimethyl-
N,N’-propylideneurea, MP=4-methoxyphenyl, Phth=phthaloyl, Pyr=pyridine,
TBAI=tetra-n-butylammonium iodide, Tf=trifluoromethanesulfonyl,
THF=tetrahydrofuran, Ts=4-toluenesulfonyl, Z=benzyloxycarbonyl (instead of
Z also Cbz is used as an abbreviation).
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from the (kinetic) nitrile effect.[21] Temporary and overall
orthogonal TBDPS protection at O6 was chosen for the
regioselective introduction of the two phosphocholine resi-
dues. Hence, galactosamine was transformed by azida-
tion,[18,22] thioglycoside formation, and 4,6-O-benzylidenation
into the valuable intermediate 15 (Scheme 3). Compound 15
tions afforded, after O-deallylation with palladium chloride in
the presence of sodium acetate and acetic acid in ethyl
acetate,[26] the 3-O-unprotected A-DAG intermediate 18 as
the acceptor. As the anticipated convergent synthesis of the
CBA-DAG intermediate caused unexpected problems,[17] this
compound was successfully synthesized in a linear fashion.
Therefore, the reaction of 18 with 4 as glycosyl donor
furnished, as a result of anchimeric assistance, the desired
b linkage in the resulting disaccharide in high yield. Removal
of the Alloc group using [Pd(PPh3)4] and p-toluenesulfinate as
the nucleophile[27] afforded the 3b-O-unprotected BA-DAG
intermediate 19 as an acceptor. Reaction of 19 with glycosyl
donor 5 was performed under TMSOTf catalysis at À458C in
propionitrile to obtain, with the help of the nitrile effect,[21]
mainly the desired b-linked trisaccharide intermediate. After
reaction of the intermediate with Zn in acetic anhydride, Troc
removal and N-acetylation ensued in high yield.[28] 6c-O-
Deallylation was performed as described above. Phosphity-
lation of this compound with bis(diisopropylamino)cyanoe-
thoxyphosphine in the presence of diisopropylammonium
tetrazolide[29] afforded the CBA-DAG core structure 20,
which was suited for chain extension and hydrogenolysis to
readily liberate all functional groups.[17]
Scheme 3. Synthesis of building blocks 7 and 8. Reagents and con-
ditions: a) 1. TfN3, 2. Ac2O, Pyr (77%); b) PhSH, BF3·OEt2, CH2Cl2
(84%); c) 1. NaOMe, MeOH, 2. PhCH(OMe)2, p-TsOH, 408C (93%);
d) BnBr, NaH, DMF (88%); e) CSA, CH2Cl2/MeOH (77%);
f) TBDPSCl, Imidazole, DMF (94%); g) 1. Ac2O, Pyr, 2. NaCNBH3,
Bu2BOTf, CH2Cl2 (74%); h) TBDPSCl, Imidazole, CH2Cl2 (92%);
i) NBS, Me2CO, À158C (86%); j) CCl3CN, DBU, CH2Cl2 (91%).
NBS=N-bromosuccinimide.
The HGFED repeating unit 24, a pseudopentasaccharide,
was constructed from trisaccharide 22 as the glycosyl donor
and pseudodisaccharide 23 as the acceptor (Scheme 5). To
this end, 4-O-unprotected acceptor 8 was glycosylated with
donor 9 under TMSOTf catalysis in CH2Cl2 at room temper-
ature to afford a-linked disaccharide 21 (GF) in high yield.
Replacement of the N-phthaloyl group, which was required
for high a selectivity in the glycosylation step, by the
benzyloxycarbonyl (Z) group was performed by using a
then afforded, after 3-O-benzylation, benzylidene removal,
and regioselective 6-O-silylation with TBDPSCl, the desired
4-O-unprotected 8 as the acceptor. 3-O-Acetylation of 15 and
then reductive opening of the benzylidene ring furnished the
4-O-benzylated intermediate, which upon 6-O-silylation gave
the desired orthogonally protected intermediate 16. This
intermediate was readily transformed into the desired donor
7.
As there are no further functional groups
attached to glucose residues A, C, and H, standard
chain extending building blocks having one tempo-
rary protecting group are required. For temporary
protection for A a 3-O-allyl group was chosen, which
resulted in a known glycosyl donor (3)[23] as a
precursor, and for C a 6-O-allyl group was chosen,
thereby resulting in the known glycosyl donor 5[24] as
a precursor. Similarly the anticipated a- and b-
linkage formation would depend upon the thermo-
dynamic anomeric effect and on the nitrile effect,
respectively. For chain termination at glucose residue
H the standard tetra-O-benzyl-glucosyl donor 10[15]
was sufficient as a precursor. For the ligation of more
than one repeating unit (n = 2, etc.) the introduction
of phenoxyacetyl at O6 was successfully investigated.
1,2-O-Cyclohexylidene-sn-glycerol (2) is readily
obtained from d-mannitol.[25]
With these building blocks the total synthesis of
target molecule 1a could be investigated. The
Scheme 4. Synthesis of CBA-DAG intermediate 20. Reagents and conditions:
a) TMSOTf (0.05 equiv), CH2Cl2, RT (89%, a/b=4:1); b) HOAc, 808C (87%);
c) C13H27CO2H, DMAP, DCC (75%); d) PdCl2, NaOAc, HOAc, EtOAc (73%);
e) TMSOTf, CH2Cl2, RT (93%, only b); f) [Pd(PPh3)4], TolSO2Na, MeOH, THF
(69%); g) TMSOTf, EtCN, À458C (83%, b/a=5:1); h) Zn, Ac2O, ultrasound
(86%); i) PdCl2, MeOH, CH2Cl2 (79%); (iPr2N)2P-OCH2CH2CN, tetrazole, iPr2NH
A-DAG precursor 17 (Scheme 4) was obtained
from 2 and 3 in high overall yield and acceptable
anomeric selectivity by using TMSOTf as a catalyst
in dichloromethane at room temperature. Removal
of the cyclohexylidene group and introduction of two
myristoyl residues under standard reaction condi- (86%). TMS=trimethylsilyl, DCC=dicyclohexylcarbodiimide.
Angew. Chem. Int. Ed. 2010, 49, 2585 –2590
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and then with acetic anhydride in pyridine.
6e,6f-O-Desilylation with hydrogen fluoride
in pyridine afforded the HGFED intermediate
24. The introduction of the phosphocholine
residues was performed with cholinoxy-cya-
noethoxy-diisopropylaminophosphine[31] using
tetrazole as the activator, and then oxidation
using tert-butyl hydroperoxide to deliver the
desired product in high yield. 1d-O-Deallyla-
tion was successfully achieved with the
[(Ph3P)2RuCl2] complex[32] as the catalyst,
thereby leading to double bond migration.
Final acid hydrolysis of the resulting enol ether
afforded the desired HGFED intermediate
25.
The ligation of intermediates 25 and 20
(Scheme 6) was performed with tetrazole to
afford a mixed phosphite triester which was
then oxidized with tert-butyl hydroperoxide to
the phosphate. Subsequent treatment with
dimethylamine led to removal of the cya-
noethyl group and formation of the desired
phosphodiester dimethylammonium salt 26 in
68% yield.
For the final removal of the seventeen
O-benzyl- and Z groups the Pearlman cata-
lyst[33] was used with H2 under high pressure
conditions in a THF/H2O (2:1) solvent mix-
ture. Target molecule 1a was isolated in highly
pure form after hydrophobic interaction chro-
matography with an ammonium acetate/n-
propanol gradient.[34] The charge deconvo-
luted ESI FT ICR (electron spray ionization
Fourier transformation ion cyclotron reso-
nance) mass spectrum obtained using the
negative ion mode revealed many molecular
mass peaks that were in perfect agreement
Scheme 5. Synthesis of HGFED intermediate 25. Reagents and conditions: a) TMSOTf
(0.1 equiv), CH2Cl2, RT (76%); b) NaOMe, MeOH; then H2NCH2CH2NH2, NaOMe,
BuOH; then ZCl, NaHCO3 (92%); c) TMSOTf (0.15 equiv), MeCN, À408C (94%);
d) NBS, Me2CO, H2O, À158C; then CCl3CN, DBU, CH2Cl2 (71%); e) TMSOTf (0.1 equiv),
MeCN, À408C (88%); f) NaOMe, MeOH (quant); g) TMSOTf, CH2Cl2 (89%); h) H2S,
Pyr, H2O; then Ac2O, Pyr (84%); i) HF, Pyr (91%); j) iPr2NP(OCH2CH2CN)-
OCH2CH2NMe3+TsOÀ, tetrazole, MeCN; then tBuO2H (89%); k) [(Ph3P)2RuCl2], DBU,
EtOH; then HCl, Me2CO (81%).
with
the
mass
calculated
for
1a
(C96H179O49N8P3; calcd: M = 2321.0974; found
method published by Hindsgaul and co-workers,[30] after
which the Z group was introduced. This procedure led to
concomitant loss of the 3 g-O-benzoyl group. Glycosylation
with the donor 10, taking again advantage of the nitrile
effect,[21] furnished the desired b linkage in the HGF trisac-
charide in very high yield. The phenylthio group was cleaved
with NBS in aqueous acetone affording the desired 1f-O-
unprotected intermediate, which was readily transformed into
the trichloroacetimidate 22 as the glycosyl donor. Glycosyla-
tion of the ribitol derivative 6, having a 3-O-benzyl group,
with glycosyl donor 7 afforded the b-linked pseudodisacchar-
ide (ED) in very high yield. This transformation proceeded by
taking advantage of the nitrile effect. After 3e-O-deacetyla-
tion with sodium methanolate in MeOH the desired inter-
mediate 23 was obtained. Glycosylation of acceptor 23 with
the donor 22 using a TMSOTf catalyst in CH2Cl2 at room
temperature furnished the desired a linkage to provide the
HGEFD intermediate in high yield. The three azido groups
could be concomitantly transformed into acetamino groups
by treatment first with hydrogen sulfide in aqueous pyridine
2321.1038). For NMR spectroscopy a solvent mixture of
[D4]MeOH and D2O (7:3, 310 K) provided the best spectral
resolution. High coupling constants (3J1,2 ꢁ 8 Hz) of the
anomeric H1 resonances for residues B,C,E, and H revealed
3
b linkages, whereas J1,2 ꢁ 3.7 Hz for sugars A, F, and G
indicated a-anomeric linkages (Table 1). In the ROESY
experiment all connectivities between the anomeric and
protons next to those of the adjacent sugar residues (H-
3a,b,e,g and H-4f) could be unambiguously assigned, thereby
allowing the determination of the correct linkage pattern of
1a. In addition, the NMR data are in good agreement with
those reported for the natural material,[4,13] thereby addition-
ally confirming the previous structural assignments.
The induction of innate immune response by synthetic 1a
was tested in human peripheral blood cells using a whole-
blood assay and isolated human mononuclear cells
(Figure 1).[35,36] Both tests revealed that 1a stimulated the
release of pro-inflammatory cytokines such as IL-8, but it was
much weaker as compared to lipopolysaccharide (LPS) and
synthetic lipopeptide Pam3CSK4. However, contrary to pre-
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Scheme 6. Synthesis of the target molecule. Reagents and conditions: a) Tetrazole, MeCN, M.S. (3 ꢂ); then tBuO2H; Me2NH, EtOH (68%); b) Pd(OH)2/C,
H2, 40 atm, THF/H2O (2:1); then HIC for purification. HIC=hydrophobic interaction chromatography, M.S.=molecular sieves.
31
Table 1: 1H (H1), 13C (C1), and P chemical shift assignments for 1a.[a]
1H
13C
31P
Position[b] Shift [ppm][c] Position[d] Shift [ppm] Position[e] Shift [ppm]
1a
1b
1c
1d
1e
1f
4.78 (3.7)
4.71 (8.5)
4.66 (7.8)
3.98/4.07
4.69 (8.3)
5.19 (3.8)
5.08 (3.8)
4.66 (7.8)
1a
1b
1c
1d
1e
1f
102.2
104.4
106.8
67.8
106.9
96.2
6c
1d
6e
6f
1.56
1.56
-0.25[f]
-0.61[f]
1g
1h
1g
1h
102.1
104.3
[a] 1H (600.3 MHz), 13C (90.6 MHz), 31P (243.1 MHz). NMR spectra were
recorded in [D4]MeOH/D2O=7:3 (v/v) at 310 K (TSP dH,dC =0.00 ppm;
dP =0.00 ppm, external 85% aq. H3PO4). [b] The number indicates the
carbon atom to which the proton is attached and the letter represents the
specific pyran ring. [c] Values in brackets indicate 3J1,2 coupling constants
(Hz) of the anomeric protons. [d] The number represents the carbon atom
and the letter represents the specific pyran ring. [e] The number represents
the carbon atom to which the OP group is attached and the letter
represents the specific pyran ring. [f] Assignment may have to be
interchanged. For the complete 1H and 13C NMR data see the Supporting
Information.
vious postulations,[6] TLR-2 is not the signaling receptor for
synthetic LTA 1a.[37]
Figure 1. Induction of IL-8 release by synthetic LTA from S. pneumo-
niae. Human MNC (A) or human whole-blood cells (B) were stimu-
lated for 16 h with synthetic LTA from S. pneumoniae at 1 mgmLÀ1 and
10 mgmLÀ1. Pam3CSK4 (100 nm) and LPS (100 ngmLÀ1) were used as
control stimulus. The IL-8 release in the culture supernatant was
determined by ELISA. Each value represents the mean of triplicate
cultures ÆSD, n.d.=not detectable amounts. For details, see the
Supporting Information.
In conclusion, LTA 1a is structurally a highly complex
glycophospholipid that was successfully synthesized for the
first time in 88 steps from the sugar precursors. The nine
building blocks could be either stereoselectively glycosidically
linked or linked by phosphodiester formation, respectively, in
a convergent manner. Modification of these building blocks
will permit a modular synthesis of S. pneumoniae LTAs of
other strains. The structure of 1a was determined and the
previous structural assignment of the natural material con-
firmed. Compound 1a exhibits immunological activity result-
ing in cytokine release, but so far it proceeds through an
unknown mechanism, as TLR-2 and TLR-4 are not the
signaling receptors for synthetic pneumococcal LTA.[36] Addi-
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better understanding of the immune response, which in turn
might lead to new treatment opportunities against S. pneumo-
niae and Gram-positive bacterial infections in general.
Received: November 2, 2009
Revised: January 14, 2010
Published online: March 9, 2010
Keywords: cytokines · glycolipids · glycosylation ·
.
Gram-positive bacteria · total synthesis
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