DOI: 10.1002/chem.201404336

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Total Synthesis of Five Lipoteichoic acids of Clostridium difficile Wouter F. J. Hogendorf,[a] Nicolas Gisch,[b] Dominik Schwudke,[b] Holger Heine,[c] Mikael Bols,[a] and Christian Marcus Pedersen*[a] Dedicated to Professor Richard R. Schmidt the problem from elderly and immune compromised patients to the formerly low-risk patients.[2] The treatment of the new hypervirulent strains is costly and limited to only few antibiotics, such as vancomycin and fidaxomicin[3] and recurrence remains a problem. With the increasing morbidity and mortality caused by C. difficile infections and the highly increased resistance to antibiotics, the interest in alternative treatments and prevention has never been bigger. Vaccines could to a large extent diminish the problems associated with C. difficile infections, especially with recurrence.[4] Cell-surface glycans are potential candidates for vaccine development and the structures of two polysaccharides (PS) from C. difficile (PS-I and PS-II)[5,7] have been elucidated. Synthetic fragments of these polysaccharides have been synthesized and evaluated as vaccine candidates.[6] A third cell-surface glycan has also been discovered and was initially termed as PS-III. The structure has recently been characterized and identified as a lipoteichoic acid (LTA).[7] Conjugates of purified deacylated LTA isolates with carrier proteins were tested on rabbits and mice resulting in the formation of protective antibodies against C. difficile strains.[8] LTA from C. difficile was therefore considered to be a good candidate vaccine component. With these promising results and the LTA structure clarified, the synthesis of pure homogeneous materials to investigate the immunological mechanism became highly interesting. During our efforts Seeberger and co-workers published the synthesis of the repeating unit and a dimer thereof.[9] Microarrays revealed that the serum from patients suffering from C. difficile infections contained antibodies that interacted with the synthetic LTA repeating unit. The natural LTA, however, occurs as a conjugate of repeating units (up to 10 in length) and a glycolipid anchor, which potentially has a considerable immunological impact as well. Our interest in the LTA from C. difficile originates from studies on LTAs from different bacteria and their interaction with the innate immune system, in which synthetic compounds as well as their derivatives have provided new insights into the biological pathways.[10] Several synthetic LTAs have been prepared,[11] but only a few have shown biological activity and the studies have been limited to either innate or adaptive immunologic studies. The first fully active LTA to be synthesized was from S. aureus and the importance of d-alanyl residues for its activity has been claimed.[12] Several unnatural derivatives were synthesized and probed in innate immunological studies to clarify the biological pathway.[13] Recently the biological active type 1 LTA from Streptococcus species DSM 8747 has been synthesized by Qiao et al.[14] and Sauvageau et al.[15] Total synthesis of

Abstract: The emergence of hypervirulent resistant strains have made Clostridium difficile a notorious nosocomial pathogen and has resulted in a renewed interest in preventive strategies, such as vaccines based on (synthetic) cell wall antigens. Recently, the structure of the lipoteichoic acid (LTA) of this species has been elucidated. Additionally, this LTA was found to induce the formation of protective antibodies against C. difficile in rabbits and mice. The LTA from C. difficile is isolated as a microheterogenous mixture, differing in size and composition, impeding any structure–activity relationship studies. To ensure reliable biological results, pure and well-defined synthetic samples are required. In this work the total synthesis of LTAs from C. difficile with defined chain length is described and the initial biological results are presented.

Introduction Clostridium difficile is a Gram-positive pathogen colonizing the intestinal tract in ~ 60 % of newborns and around 3 % of adults. The appearance of hypervirulent strains has become a rapidly growing problem in healthcare in the 21st century.[1] Infection with C. difficile occurs mainly as a result of using broad spectra antibiotics in the treatment of bacterial infections, which besides targeting the pathogen also effects the normal gut microbiota. This results in an overgrowth of toxin producing C. difficile leading to antibiotic-associated diarrhea. The emergence of hypervirulent strains (e.g. BI/NAP1/027) has spread [a] Dr. W. F. J. Hogendorf, Prof. M. Bols, Prof. C. M. Pedersen Department of Chemistry, University of Copenhagen Universitetsparken 5, 2100, Copenhagen Ø (Denmark) E-mail: [email protected] [b] Dr. N. Gisch, Dr. D. Schwudke+ Division of Bioanalytical Chemistry, Research Center Borstel Leibniz-Center for Medicine and Biosciences Parkallee 1–40, 23845 Borstel (Germany) [c] Prof. Dr. H. Heine+ Division of Innate Immunity, Research Center Borstel Leibniz-Center for Medicine and Biosciences Parkallee 1–40, 23845 Borstel (Germany) [+] Members of the Airway Research Center North (ARCN) and the German Center for Lung Research (DZL) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201404336. Chem. Eur. J. 2014, 20, 1 – 7

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Communication a derivative of the more complex LTA from S. pneumoniae has zylation of the resulting diol, to give the fully protected donor also been achieved[16] and its interaction with the host immune 4. system revealed that not TLR-2 but the complement pathway Glycosylation of the 2-deoxy-2-azidoglucosyl acceptor 5 with was activated.[17] Since the structural motive is very different donor 4 relied on selective activation of the thiophenyl in 4, without affecting the thiophenyl moiety in the acceptor. Preacbetween the families of LTAs, and dependent on the origin, tivation using diphenyl sulfoxide, Tf2O, and TTBP at low tema different biological pathway is not a surprise, but what about other LTAs from other Gram-positive bacteria? Will they interperature,[19] followed by addition of the acceptor 5, gave the act with the immune system and how? In this communication disaccharide donor 11 in 65 % yield as an anomeric mixture we present our synthesis of five LTAs from C. difficile—from the (6:1). As the yield and selectivity were moderate, the donor 4 monomer with one repeating unit to the pentamer with five. was transformed into the trichloroacetimidate 8 in two steps. In addition, initial biological studies in relation to innate imTMSOTf-catalyzed glycosylation in diethyl ether[20] gave the demunity are presented. sired a-product 11 in excellent yield and selectivity. Ph2SO/ The structure of LTA from C. difficile 1 is presented in Tf2O/TTBP-mediated glycosylation with the glycerate acceptor Figure 1.[7] The LTA consists of a lipid anchor part containing 6, or glycosylation by using the corresponding trichloroacetimidate gave anomeric mixtures, which could not be separated. a trisaccharide and a diacylglycerol (DAG) part, in which the Alternatively, regioselective opening of the benzylidene moiety acyl group can be C14, C16, or C18 monounsaturated or satuof compound 11 turned out to be complicated by giving mulrated. Natural isolates of the LTA 1 have variations in the tiple products and only 32 % of the desired mono-ol 12. Dimedegree of N-acetylation with ~ 30 % of the amino groups unthoxytritylation (not shown) of the mono-ol 12 was furtheracetylated. The terminal repeating unit is generally N-acetylated and nonphosphorylated on O-6. In a retrosynthetic analysis, the obvious disconnection in a modular synthesis would be the 6–6’ phosphodiester bridges giving the lipid anchor and a disaccharide glycosylated with glyceric acid (Figure 2). For the construction of the LTA, benzyl groups were chosen as persistent protective groups and hence glycolipid anchor derivative 2 was required. The syntheFigure 1. Structure of LTA from C. difficile. sis of this gentiotriosyl glycolipid has recently been achieved.[18] For the synthesis of the repeating unit, an orthogonal protective group strategy was needed to allow sequential prolongation of the LTA by using phosphoramidite chemistry. Initially it was proposed to use the 4,6-O-benzylidene to differentiate the 4- and 6-positions in 5 by regioselective reductive opening in the synthesis towards disaccharide repeating unit 3. The required building blocks were therefore a benzylideneprotected acceptor 5 and a glycosyl donor with a temporary protected 6-OH (4). The building blocks could be synthesized from the common intermediate triol 7, which was either benzylidenated to give the acceptor 5 or selectively 6-O-protected by using TBDPS-Cl followed by ben- Figure 2. Retrosynthetic analysis. &

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Communication more unsuccessful and the protective strategy had to be rethought. As an alternative pair of orthogonal protective groups Fmoc and silylethers were chosen[21] and it was decided to build-up the repeating unit from the reducing end starting with glycosylation of the glycerate 6.[9] TBS protection of 5 followed by reductive opening of the benzylidene afforded 9, which was Fmoc-protected and transformed into the trichloroacetimidate 10 (Scheme 1) followed by glycosylation with benzyl-protected

tilled thioacetic acid in pyridine; subsequent removal of the TBDPS, using HF·Pyr. in THF, gave the mono-ol 15, which was protected with DMTr-Cl in pyridine. DBU-mediated removal of the Fmoc and treatment with bis(diisopropylamino)cyanoethoxyphosphine in the presence of diisopropylammonium tetrazolide gave phosphoramidite 3 (Scheme 3). With the lipid anchor mono-ol 2 in hand the assembly of the LTA could begin. Dicyanoimidazole (DCI) mediated ligation of 2 and 3 gave the coupled product as a phosphite triester, which was oxidized by iodine in aqueous pyridine/THF to the corresponding phosphate. Removal of the DMTr-protection was mediated by dichloroacetic acid and triethylsilane in CH2Cl2 giving the mono-ol 16, in 62 % over 3 steps, ready for chain elongation or global deprotection to give 1 a. Chain elongation was performed by repeating the three steps, that is, catalyzed ligation, Scheme 1. Synthesis of glycosyl donors for the construction of the repeating unit. DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene; Fmoc = 9-fluorenylmethoxycarbonyl; NBS = N-bromosuccinimide. phosphite oxidation, and DMTr (2R)-glyceric acid 6 to give the pseudodisaccharide in excellent yield and selectivity. It should be mentioned that glycosylation with the 6-O-DMTr analogue of 10 (not shown) gave a lower yield and moderate selectivity (68 %, a/b ~ 6:1), which prohibit- Scheme 3. Synthesis of the repeating unit module. DMTr = 4,4’-dimethoxytrityl. ed this route and therefore the oligomerization from this site of the repeating unit. Removal of the 3-O-TBS group of the pseuremoval, and the first repetition gave protected LTA 17 with dodisaccharide by HF·pyridine (Scheme 2) gave the acceptor two repeating units in 74 %. The following elongations were 13 ready for glycosylation with the trichloroacetimidate donor less straightforward and the reactions resulted in mixtures of 8. Glycosylation between the donor 8 and the acceptor 13 was LTA with different chain lengths in decreasing yields conducted in Et2O to promote a-selectivity (a/b ~ 15:1). The (Scheme 4). These mixtures could not be separated at this point and with this approach five repeating units seems to be yield of 14 was modest (60 %), but acceptable when taking rethe limit. Longer teichoic acid oligomers might be accessible covered starting material (13) into account (79 %). using solid or fluorous phase chemistry as illustrated by Code With a solid synthesis of the building blocks and the repeatand co-workers.[22] ing unit in place, the protective group manipulations to get the phosphoramidite 3, could begin. The two azido groups in Deprotection of the five fully protected LTAs (16–20) was 14 were transformed into NHAc by reduction with freshly disperformed by a two-step procedure. The cyanoethyl protective groups were removed by treatment with DBU in CH2Cl2 followed by column chromatography to give 21–25. Compounds with different chain lengths could, however, still not be separated. Removal of the benzyl protective groups, up to 34 (in 25), were carried out by hydrogenolysis with palladium black and hydrogen (20 bar) in slightly acidic THF water mixtures. The Scheme 2. Glycosylations to construct the repeating unit. Chem. Eur. J. 2014, 20, 1 – 7

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Scheme 4. Assembly of the fully protected LTA.

solvent composition is crucial to maintain the intermediates in solution and to minimize aggregation. The progress was analyzed by 1H NMR spectroscopy and three to five subsequent rounds of hydrogenolysis were needed for full deprotection (justified by the disappearance of aromatic signals in the 1 H NMR spectrum). The crude LTAs (1 a–e) were purified by hydrophobic interaction chromatography (HIC) with an ammonium acetate/n-propanol gradient as used for isolation of natural LTA,[23] resulting in highly pure LTAs. The before-mentioned mixtures of LTAs with different numbers of repeating units were additionally purified by C18 reverse-phase chromatography using complex solvent mixtures containing methanol, chloroform, water, and NH4OAc, originally developed for the purification of lipopolysaccharides (LPS),[24] to give highly pure and homogeneous samples of 1 c and 1 d. Compound 1 e was obtained after HIC as a mixture with 1 d as the major components as well as 1 b and 1c as minor components. However, this mixture might very well reflect the heterogeneous mixture present in the C. difficile cell wall.[7] As an example the 1H, 13CHSQC NMR spectrum of 1 b including signal assignment is shown in Figure 3. The repeating unit 15 was deprotected (Scheme 5) in two steps; first removal of the Fmoc protective group by using DBU in CH2Cl2, followed by hydrogenolysis over palladium black to give the fully deprotected repeating unit 26. The lipid anchor 27 was debenzylated as previously described.[18]

The immunomodulating properties of the five LTAs (1 a–e), the repeating disaccharide unit 26, and the gentiotriosyl glycolipid 27 were investigated in human mononuclear cells (hMNCs, Figure 4A) as well as in a whole blood assay (Figure 4B) by using 0.4 or 4 mm of the stimuli, respectively. These concentrations are comparable to those of synthetic S. pneumoniae LTA that showed stimulating activity in these assays.[16] Here, both assays clearly revealed that none of the investigated compounds 1 a–e, 26, and 27 activates the innate immune system. Increasing the concentration of the stimuli to 40 mm

Figure 3. 1H, 13C-HSQC NMR spectrum (700 MHz) of 1 b including signal assignment.

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Scheme 5. Two-step global deprotection to give the LTA targets (1 a–e), the repeating unit (26), and the lipid anchor (27).

In conclusion we have, for the first time, synthesized LTA from Clostridium difficile with a defined chain length. These synthetic LTAs represent the main components found in the heterogeneous natural isolates. The modular synthesis allows modification in each building block to synthesize more of the natural products with other substitution patterns as illustrated in Figure 1. The synthetic LTA structures have been carefully analyzed by NMR spectroscopy and/or high-resolution mass spectrometry. Initial innate immunological studies revealed that the synthesized LTA, the lipid anchor and the repeating unit do not release any cytokines similar to the LTAs from Streptococcus pneumoniae and Staphylococcus aureus used in comparable concentrations. More biological studies are required to clarify the biological role in adaptive immunity.

Acknowledgements We gratefully acknowledge S. Thomsen and U. Schombel for excellent LTA purification, H. Kßner (NMR), Theis Brock-Nannestad, B. Kunz, and V. Scholz (MS) for their analytical measurements, as well as S. Al-Badri for performing the immune assays. Keywords: glycolipids · Clostridium difficile · total synthesis · lipoteichoic acid · Gram-positive bacteria [1] Reviewed in: C. Ghose, EMI, 2013, 2, e62. [2] a) V. G. Loo, L. Poirier, M. A. Miller, M. Oughton, M. D. Libman, S. Michaud, A.-M. Bourgault, T. Nguyen, C. Frenette, M. Kelly, A. Vibien, P. Brassard, S. Fenn, K. Dewar, T. J. Hudson, R. Horn, P. Ren, Y. Monczak, A. Dascal, New Engl. J. Med. 2005, 353, 2442 – 2449; b) L. C. MacDonald, Infect. Control Hosp. Epidemiol. 2005, 26, 672 – 675. [3] F. Babakhani, A. Gomez, N. Robert, P. Sears, J. Med. Microbiol. 2011, 60, 1213 – 1217. [4] L. V. McFarland, C. M. Surawicz, M. Rubin, G. W. Elmer, R. M. Greenburg, Infect. Control. Hosp. Epidemiol. 1999, 20, 43 – 50. [5] a) L. Bertolo, A. G. Boncheff, Z. Ma, Y.-H. Chen, T. Wakeford, R. M. Friendship, J. Rosseau, J. S. Wesse, M. Chu, M. Mallozzi, G. Vedantam, M. A. Monteiro, Carbohydr. Res. 2012, 354, 79 – 86; b) R. Adamo, M. R. Romano, F. Berti, R. Leuzzi, M. Tontini, E. Danieli, E. Cappelletti, O. S. Cakici, E. Swennen, V. Pinto, B. Brogioni, D. Proietti, C. L. Galeotti, L. Lay, M. A. Monteiro, M. Scarselli, P. Costantino, ACS Chem. Biol. 2012, 7,

Figure 4. Induction of IL-6 release by LTAs 1 a–e, repeating unit 26 and glycolipid 27. Human mononuclear cells (A) or human whole-blood cells (B) were stimulated with 0.4 and 4 mm of indicated stimuli. Compound 1 e represents a mixture of 1 e and 1 d as major compounds containing small amounts of 1 c and 1 b as well. LPS (1 and 10 mg ml 1) and synthetic lipoprotein Pam3C-SK4. (10 and 100 nm) were used as control stimuli. The IL-6 release in the culture supernatant was determined by ELISA. Each value represents the mean of triplicate cultures  SD. For details, see the Supporting Information.

did not change this (data not shown). Pam3C-SK4 (10 and 100 nm) and LPS (1 and 10 mg ml 1) were used as control stimuli in both assays. Chem. Eur. J. 2014, 20, 1 – 7

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[8] [9] [10] [11]

[12] [13]

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1420 – 1428; c) M. A. Monteiro, J. Ganeshapillai: WO033268A1 (2009); US0330125A1 (2010); NZ583750 (2010). a) M. A. Monteiro, Z. Ma, L. Bertolo, Y. Jiao, L. Arroyo, D. Hodgins, M. Mallozzi, G. Vedantam, M. Sagermann, J. Sundsmo, H. Chow, Expert Rev. Vaccines 2013, 12, 421 – 431; b) C. E. Martin, F. Broecker, M. A. Oberli, J. Komor, J. Mattner, C. Anish, P. H. Seeberger, J. Am. Chem. Soc. 2013, 135, 9713 – 9722; c) C. E. Martin, M. W. Weishaupt, P. H. Seeberger, Chem. Commun. 2011, 47, 10260 – 10262; d) Y. Jiao, Z. Ma, D. Hodgins, B. Pequegnat, L. Bertolo, L. Arroyo, M. A. Monteiro, Carbohydr. Res. 2013, 378, 15 – 25; e) E. Danieli, L. Lay, D. Proietti, F. Berti, P. Costantino, R. Adamo, Org. Lett. 2011, 13, 378 – 381; f) M. A. Oberli, M.-L. Hecht, P. Bindschdler, A. Adibekian, T. Adam, P. H. Seeberger, Chem. Biol. 2011, 18, 580 – 588. a) J. Ganeshapillai, E. Vinogradov, J. Rousseau, Carbohydr. Res. 2008, 343, 703 – 710; b) C. W. Reid, E. Vinogradov, J. Li, H. C. Jarrell, S. M. Logan, J. R. Brisson, Carbohydr. Res. 2012, 354, 65 – 73. A. D. Cox, F. St. Michael, A. Aubry, C. M. Cairns, P. C. R. Strong, A. C. Hayes, S. M. Logan, Glycoconjugate J. 2013, 30, 843 – 855. C. E. Martin, F. Broecker, S. Eller, M. A. Oberli, C. Anish, C. L. Pereira, P. H. Seeberger, Chem. Commun. 2013, 49, 7159 – 7161. R. R. Schmidt, C. M. Pedersen, Y. Qiao, U. Zhringer, Org. Biomol. Chem. 2011, 9, 2040 – 2051. Lipoteichoic acid synthesis has been reviewed in: a) C. M. Pedersen, M. Bols, Y. Qiao, ARKIVOC 2013, 249 – 275; b) C. M. Pedersen, R. R. Schmidt, in Microbial Glycobiology (Ed.: A. P. Moran), Elsevier, London, UK, 2009, pp. 455 – 476; c) S. Kusumoto, M. Oikawa, in Glycoscience, Vol. 3 (Eds.: B. O. Fraser-Reid, K. Tatsuta, J. Thiem), Springer, Berlin, Germany, 2001, 2107 – 2148. A. Stadelmaier, S. Morath, T. Hartung, R. R. Schmidt, Angew. Chem. 2003, 115, 945 – 949; Angew. Chem. Int. Ed. 2003, 42, 916 – 920. a) S. Morath, A. Stadelmaier, A. Geyer, R. R. Schmidt, T. Hartung, J. Exp. Med. 2002, 195, 1635 – 1640; b) I. Figueroa-Perez, A. Stadelmaier, S. Deininger, S. von Aulock, T. Hartung, R. R. Schmidt, Carbohydr. Res. 2006, 341, 2901 – 2911.

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[14] Y. Qiao, B. Lindner, U. Zhringer, P. Truog, R. R. Schmidt, Bioorg. Med. Chem. 2010, 18, 3696 – 3702. [15] J. Sauvageau, A. J. Foster, A. A. Khan, S. H. Chee, I. M. Sims, M. S. M. Timmer, B. L. Stocker, ChemBioChem 2012, 13, 2416 – 2424. [16] C. M. Pedersen, I. Figueroa-Perez, B. Lindner, A. J. Ulmer, U. Zhringer, R. R. Schmidt, Angew. Chem. 2010, 122, 2639 – 2644; Angew. Chem. Int. Ed. 2010, 49, 2585 – 2590. [17] a) E. Stermann, M. Lacroix, E. Gout, E. Laffly, C. M. Pedersen, R. R. Schmidt, T. Vernet, C. Gaboriaud, A.-M. Di Guilmi, N. M. Thielens, Immunobiology 2012, 217, 1188 – 1189; b) E. Stermann, M. Lacroix, E. Gout, E. Lafly, C. M. Pedersen, L. Martin, A. Amoroso, R. R. Schmidt, U. Zhringer, C. Gaboriaud, A. M. Di Guilmi, N. M. Thielens (unpublished results). [18] W. F. J. Hogendorf, V. Jagalski, T. G. Pomorski, M. Bols, M. Cardenas, C. M. Pedersen, Molecules 2013, 18, 13546 – 13573. [19] J. D. C. Codee, R. E. J. N. Litjens, R. Den Heeten, H. S. Overkleeft, J. H. Van Boom, G. A. van der Marel, Org. Lett. 2003, 5, 1519 – 1522. [20] R. U. Lemieux, Pure Appl. Chem. 1971, 25, 527 – 548. [21] S. D. Markad, R. R. Schmidt, Eur. J. Org. Chem. 2009, 5002 – 5011. [22] a) W. F. J. Hogendorf, L. N. Lameijer, T. J. M. Beenakker, H. S. Overkleeft, D. V. Filippov, J. D. C. Code, G. A. van der Marel, Org. Lett. 2012, 14, 848 – 851; b) W. F. J. Hogendorf, N. Meeuwenoord, H. S. Overkleeft, D. V. Filippov, D. Laverde, A. Kropec, J. Huebner, G. A. van der Marel, J. D. C. Code, Chem. Commun. 2011, 47, 8961 – 8963; c) W. F. J. Hogendorf, A. Kropec, D. V. Filippov, H. S. Overkleeft, J. Huebner, G. A. van der Marel, J. D. C. Code, Carbohydr. Res. 2012, 356, 142 – 151. [23] N. Gisch, T. Kohler, A. J. Ulmer, J. Mthing, T. Pribyl, K. Fischer, B. Lindner, S. Hammerschmidt, U. Zhringer, J. Biol. Chem. 2013, 288, 15654 – 15667. [24] See the Supporting Information for details.

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COMMUNICATION & Total Synthesis W. F. J. Hogendorf, N. Gisch, D. Schwudke, H. Heine, M. Bols, C. M. Pedersen* && – && Total Synthesis of Five Lipoteichoic acids of Clostridium difficile Access granted! Access has been granted to five lipoteichoic acids from the nosocomial pathogen Clostridium difficile. An efficient modular synthesis of this lipoteichoic acid (LTA) has been developed and used to synthesize pure

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compounds for immunological studies. The yields for the chain elongation remains consistently high for n = 1 to 4 (see scheme), which seems to be the limit for efficient solution-phase synthesis in this system.

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Total Synthesis of Five Lipoteichoic acids of Clostridium difficile.

The emergence of hypervirulent resistant strains have made Clostridium difficile a notorious nosocomial pathogen and has resulted in a renewed interes...
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