We report the automated glycan assembly (AGA) of different oligosaccharide fragments of the bacterial peptidoglycan (PGN) backbone. Iterative addition on a solid support of an acetyl glucosamine and a new muramic acid building block is followed by cleavage from the solid support and final deprotection providing 10 oligosaccharides up to six units

Benazzi, Valentina

Dallabernardina, Pietro

Laman, Jon D.

Loeffler, Felix F.

Seeberger, Peter H.

English

We report the automated glycan assembly (AGA) of different oligosaccharide fragments of the bacterial peptidoglycan (PGN) backbone. Iterative addition on a solid support of an acetyl glucosamine and a new muramic acid building block is followed by cleavage from the solid support and final deprotection providing 10 oligosaccharides up to six units.</p

ARTS repository - University of Groningen

   University of GroningenAutomated glycan assembly of peptidoglycan backbone fragmentsDallabernardina, Pietro; Benazzi, Valentina; Laman, Jon D.; Seeberger, Peter H.; Loeffler,Felix F.Published in:Organic & Biomolecular ChemistryDOI:10.1039/d1ob01987bIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2021Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Dallabernardina, P., Benazzi, V., Laman, J. D., Seeberger, P. H., & Loeffler, F. F. (2021). Automatedglycan assembly of peptidoglycan backbone fragments. Organic & Biomolecular Chemistry, 45, 9829-9832.Advance online publication. https://doi.org/10.1039/d1ob01987bCopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 10-09-2023Organic &Biomolecular ChemistryCOMMUNICATIONCite this: DOI: 10.1039/d1ob01987bReceived 10th October 2021,Accepted 22nd October 2021DOI: 10.1039/d1ob01987brsc.li/obcAutomated glycan assembly of peptidoglycanbackbone fragments†Pietro Dallabernardina,a Valentina Benazzi,a,b Jon D. Laman, cPeter H. Seebergera,d and Felix F. Loeffler *aWe report the automated glycan assembly (AGA) of different oligo-saccharide fragments of the bacterial peptidoglycan (PGN) back-bone. Iterative addition on a solid support of an acetyl glucos-amine and a new muramic acid building block is followed by clea-vage from the solid support and final deprotection providing 10oligosaccharides up to six units.Peptidoglycans (PGN) are an essential component of the cellwall of both Gram-positive and Gram-negative bacteria. PGNserve a structural function, counteracting osmotic pressureand maintaining cell shape, and protect against externalthreats.1,2 The main PGN structure is made of linear glycanstrands cross-linked by short peptide chains (Fig. 1b).3 Severalstructural modifications at both the glycan part, such as acetyl-ation, and the stem peptide are possible in order to give rise tohundreds of structural variants.4 Nevertheless, the structure ofthe glycan backbone is generally conserved and it is composedof two alternating amino sugars (Fig. 1a), N-acetylglucosamine(GlcNAc) and N-acetylmuramic acid (MurNAc), linked by aβ-1–4 bond. The D-lactoyl group of each MurNAc residue is con-nected through an amide bond to a stem peptide that can beup to five amino acid residues long.5 Many modifications canoccur at the peptide chain,2 in particular at the third aminoacid. The majority of Gram-positive bacteria have an L-Lys atposition three, while the majority of Gram-negative bacteriahave a non-proteinogenic amino acid A2pm (2,6-diaminopime-lic acid). The final dipeptide might present some modifi-cations due to acquired resistance to antibiotics.6 Stem pep-tides from different glycan strands may be cross-linked eitherdirectly or by an interpeptide bridge consisting of one to sevenamino acid residues.2In the past two decades, PGNs drew interest not only as apossible target for antibiotics,7 but also for their role as immu-nostimulatory molecules,8–11 as well as in mouse brain devel-opment and behavior.12 It was suggested that PGN fragmentshave many diverse roles as signalling molecules, includingcommunication between bacteria and bacteria with the host,as well as pathogenesis in animals and plants.5 PGNs havealso been correlated with some autoimmune and chronicinflammatory diseases.13–16 In particular, some studies14,17–19have demonstrated that peptidoglycan fragments are presentwithin phagocytes in the brain of multiple sclerosis patientsand that anti-PGN antibodies are present in cerebrospinalfluid during active disease, suggesting a possible link betweenFig. 1 (a) Symbolic representation and structures of the two aminosugars, N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid(MurNAc). (b) Schematic representation of the peptidoglycan structure.The different glycan strands are branched on the MurNAc residues witha sequence of amino acids (dark green circles) called stem peptide. Aninterpeptide bridge (light green box) may connect the two stempeptides.†Electronic supplementary information (ESI) available. See DOI: 10.1039/d1ob01987baDepartment of Biomolecular Systems, Max Planck Institute of Colloids andInterfaces, Am Muehlenberg 1, 14476 Potsdam, Germany.E-mail: felix.loeffler@mpikg.mpg.debUniversity of Pavia, Department of Organic Chemistry, V.le Torquato Taramelli, 10,27100 Pavia, ItalycDepartment of Pathology & Medical Biology, University Medical Center Groningen,Groningen, The NetherlandsdFreie Universität Berlin, Institute of Chemistry and Biochemistry, Arnimallee 22,14195 Berlin, GermanyThis journal is © The Royal Society of Chemistry 2021 Org. Biomol. Chem.Open Access Article. Published on 25 October 2021. Downloaded on 11/25/2021 10:00:31 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journalthese molecules and disease. Therefore, the level and identityof PGNs in the blood, urine, and tissues, might serve as apotential marker for progression of autoimmune and inflam-matory diseases, and, hence, as a tool in therapy monitoring.13Given the biological relevance of these molecules, insightinto PGN structure is essential to understand the role of PGNsin pathogenesis, discovering the mechanisms involved in theirrelease, and how they are detected by bacterial versus humanhost cells.5 Even though different proteins with PGN bindingactivity have been identified, the exact epitopes they recognizeremain poorly characterized.20 A major drawback in thecharacterization of the binding motif is the complexity in thepurification of the PGN fragments from biological sources.20Therefore, to obtain pure compounds it is necessary to resortto chemical synthesis. The chemical synthesis of such oligo-saccharide libraries is not trivial and requires an immense syn-thetic effort.21–24 Automated glycan assembly (AGA)25 hasalready proven its versatility in the generation of oligosacchar-ide collections.26,27 Iterative coupling of different buildingblocks (BB) on a solid support allows for the synthesis of well-defined oligo- and polysaccharide structures in a fast manner.Here, we report the AGA of ten fragments related to theglycan strands of PGNs using two different monosaccharideBBs. To prepare the compound collection, we designed BBs 1and 2 (Scheme 1) that can be synthesized starting from anunprotected glucosamine. The amine at C2 was equipped witha trichloroacetyl (TCA) as participating protecting group, toensure β selectivity during glycosylation. The TCA can be con-verted into the desired acetyl group upon reduction during thefinal deprotection.Fluorenylmethoxycarbonyl (Fmoc) was installed at C4 andused as a temporary protecting group for β1–4 chain elonga-tion. A lactic ester was installed in position three of the BB 2as precursor for the lactic acid moiety of the muramic acid. Allother positions were protected with benzyl (Bn) permanentprotecting groups. The phosphate leaving group was preferredto other leaving groups due to observed higher couplingefficiency in AGA for glucosamine BBs.28 The two desired BBs1 and 2 are accessible starting from the unprotected glucos-amine with a divergent synthetic pathway from glucosaminethioglycoside 3.29 Glycosyl phosphate building block 1 was pre-pared in nine steps, following published procedures.28–30BB 2 was synthesized starting from thiogycoside 329(Scheme 2) that was treated with NaH and (–)-ethyl (S)-2-trifluro-methanesulfonyl propionate, previously prepared in situ fromethyl L-(–)-lactate and triflic anhydride,31 to obtain the monosac-charide 4 with the ethyl propionate at C3. Different solventswere tested. Initially, DMF was used, but only starting materialwas recovered. In THF the reaction appeared to proceed to com-pletion, but the desired compound was obtained in an insepar-able mixture with different side products. Compound 3 provedinsoluble in dichloromethane. Therefore, we added some dropsof DMF, to completely dissolve the starting material. Thismixture gave the best result, affording 4 in 53% yield. The regio-selective opening of the benzylidene was attempted using theBroensted acid trifluoroacetic acid and triethylsilane32 ashydride donor. These conditions provided a low yield since aside product originated from the condensation of the freehydroxyl group on position 4 and the ethyl ester. This sideproduct was previously reported when Kinzy et al. used aBroensted acid to hydrolyze the benzylidene acetal.33Therefore, another strategy for the selective benzylideneopening, involving Me3N·BH3 and the Lewis acid BF3·OEt2 inacetonitrile was adopted.34 Under these conditions, the for-mation of the lactone side product was not observed. Finally,the hydroxyl group on C4 was protected with Fmoc to give 6and subsequently, the thioglycoside was transformed into thecorresponding α-glycosyl phosphate 2.With BB 1 and 2 in hand, we moved forward with the syn-thesis of our collection of oligosaccharides using AGA(Scheme 3). Merrifield resin functionalized with a photocleavablelinker was used as solid support.35 The BBs were coupled to thesolid support using TMSOTf-promoted glycosylation, employingone cycle and five equivalents of BB. Then, an acidic cappingsolution was used to acetylate the unreacted hydroxyl groupsand, thereby, minimizing deletion sequences.36Subsequently, the Fmoc carbonate was cleaved from thestructure, preparing the monosaccharide for the installation ofScheme 1 Retrosynthetic approach to obtain the glycan backbone ofPGN and the two BB required for the synthesis.Scheme 2 Synthesis of the MurNAc BB 2. Reagents and conditions: (a)(–)-ethyl (S)-2-trifluoromethylsulfonyloxy propionate, NaH, DMF/CH2Cl2, 0 °C → rt, 53%; (b) Me3N·BH3, BF3·OEt2, CH3CN, 0 °C → rt, 65%;(c) FmoCl, pyridine, CH2Cl2, rt, 93%; (d) (BuO)2P(O)OH, NIS, TfOH, 0 °C→ rt, 83%.Communication Organic & Biomolecular ChemistryOrg. Biomol. Chem. This journal is © The Royal Society of Chemistry 2021Open Access Article. Published on 25 October 2021. Downloaded on 11/25/2021 10:00:31 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinethe next unit. This cycle of coupling, capping, and deprotectionwas repeated until the desired sequence was achieved. OnceAGA was completed, the oligosaccharides were cleaved from theresin using UV light (365 nm) in a continuous flow photo-reactor.35 After purification of the desired oligosaccharides fromthe deletion sequences, the basic hydrolysis of the lactoyl estersto carboxylic acids was performed using LiOH in a solution ofTHF/dioxane/H2O.31 The remaining protecting groups werecleaved by hydrogenolysis with Pd/C and H2. Under these con-ditions, the TCA group was reduced to acetamide. This last stepwas more problematic, since after several days of hydrogenoly-sis, we still observed the presence of multiple chlorine atoms byMALDI. Therefore, for the hexamers were hydrogenated underhigh pressure to achieve complete conversion in 48 h. The rela-tively low yield is caused by the inefficiency of the photocleavagefrom the resin together with the rather low reactivity of thebuilding blocks, which led to the presence of deletionsequences. Finally, ten different PGN glycan backbones of alter-nating MurNAc and GlcNAc moieties, equipped with an amino-linker were obtained: the two monomers 8 and 9, two disac-charides 10 and 11, two trimers 12 and 13, two tetramers 14and 15, and two hexamers 16 and 17.ConclusionsTen oligosaccharide fragments of the PGN backbone weresynthesized. The use of a new MurNAc BB enabled the fastsynthesis of a collection of oligosaccharides up to hexamers.These oligosaccharides can be used to create glycan micro-arrays for the characterization of peptidoglycan directedantibodies. Advancing the idea from recently publishedapproaches, where molecules on microarrays were modifieddirectly on the glass slide after in situ synthesis37 or print-ing,38 we will follow a similar strategy. We envision tocouple different stem peptide variants from solution,forming the amide bond directly with the arrayed oligosac-charides. These arrays will support the identification of epi-topes recognized by anti-PGN antibodies, and can poten-tially help to close current knowledge gaps in autoimmunedisorders.Conflicts of interestThere are no conflicts to declare.Scheme 3 Automated glycan assembly of PGN oligosaccharides. Reagents and conditions: (a) 1 × 5 equiv. BB1, TMSOTf, CH2Cl2, −30 °C (5 min) →−15 °C (35 min); (b) 1 cycle of a 10% acetic anhydride and 2% methanesulfonic acid in CH2Cl2, 25 °C; (c) 3 cycles of 20% piperidine in DMF, 25 °C(5 min) (Module B); (d) CH2Cl2, hν (365 nm); (e) LiOH, THF/1,4-dioxane/H2O, 2–24 h; (f ) H2, Pd/C, H2O/HOAc, 16–48 h. 8: 27%; 9: 42%; 10: 20%; 11:10%; 12: 11%; 13: 9%; 14: 3%; 15: 8%; 16: 3%; 17: 2%.Organic & Biomolecular Chemistry CommunicationThis journal is © The Royal Society of Chemistry 2021 Org. Biomol. Chem.Open Access Article. Published on 25 October 2021. Downloaded on 11/25/2021 10:00:31 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineAcknowledgementsThe authors thank the German Federal Ministry of Educationand Research (BMBF, grant no. 13XP5050A), the Max-Planck-Fraunhofer Collaboration Project (Glyco3Display), and the MaxPlanck Society for financial support. In addition, the authorswould like to thank Martina Delbianco for the fruitful discus-sions and the members of the Department of BiomolecularSystems for the technical support, especially Eva Settels andOlaf Niemeyer. Open Access funding provided by the MaxPlanck Society.References1 J. V. Höltje, Microbiol. Mol. Biol. Rev., 1998, 62, 181–203.2 W. Vollmer, D. Blanot and M. A. de Pedro, FEMS Microbiol.Rev., 2008, 32, 149–167.3 J. H. Rogers, H. R. Perkins and J. B. Ward, Microbial CellWalls and Membranes, Springer, Netherlands, 1980.4 P. Schumann, in Methods in Microbiology, 2011, vol. 38, pp.101–129.5 O. Irazoki, S. B. Hernandez and F. Cava, Front. Microbiol.,2019, 10, 500.6 A. K. Yadav, A. Espaillat and F. Cava, Front. Microbiol.,2018, 9, 2064.7 M. A. Kohanski, D. J. Dwyer and J. J. Collins, Nat. Rev.Microbiol., 2010, 8, 423–435.8 S. E. Girardin, I. G. Boneca, L. A. M. Carneiro, A. Antignac,M. Jéhanno, J. Viala, K. Tedin, M.-K. Taha, A. Labigne,U. Zähringer, A. J. Coyle, P. S. D. Stefano, J. Bertin,P. J. Sansonetti and D. J. Philpott, Science, 2003, 300, 1584–1587.9 S. E. Girardin, L. H. Travassos, M. Hervé, D. Blanot,I. G. Boneca, D. J. Philpott, P. J. Sansonetti and D. Mengin-Lecreulx, J. Biol. Chem., 2003, 278, 41702–41708.10 K. L. Woodhams, J. M. Chan, J. D. Lenz, K. T. Hackett andJ. P. Dillard, Infect. Immun., 2013, 81, 3490–3498.11 M. A. Boudreau, J. F. Fisher and S. Mobashery,Biochemistry, 2012, 51, 2974–2990.12 A. Gonzalez-Santana and R. D. Heijtz, Trends Mol. Med.,2020, 26, 729–743.13 Z. Huang, J. Wang, X. Xu, H. Wang, Y. Qiao, W. C. Chu,S. Xu, L. Chai, F. Cottier, N. Pavelka, M. Oosting,L. A. B. Joosten, M. Netea, C. Y. L. Ng, K. P. Leong,P. Kundu, K.-P. Lam, S. Pettersson and Y. Wang, Nat.Microbiol., 2019, 4, 766–773.14 P. J. Shaw, M. J. Barr, J. R. Lukens, M. A. McGargill, H. Chi,T. W. Mak and T.-D. Kanneganti, Immunity, 2011, 34, 75–84.15 B. L. Jutras, R. B. Lochhead, Z. A. Kloos, J. Biboy, K. Strle,C. J. Booth, S. K. Govers, J. Gray, P. Schumann, W. Vollmer,L. K. Bockenstedt, A. C. Steere and C. Jacobs-Wagner, Proc.Natl. Acad. Sci. U. S. A., 2019, 116, 13498–13507.16 L. Visser, H. J. d. Heer, L. A. Boven, D. van Riel, M. vanMeurs, M.-J. Melief, U. Zähringer, J. van Strijp,B. N. Lambrecht, E. E. Nieuwenhuis and J. D. Laman,J. Immunol., 2005, 174, 808–816.17 I. A. Schrijver, M. van Meurs, M.-J. Melief, C. W. Ang,D. Buljevac, R. Ravid, M. P. Hazenberg and J. D. Laman,Brain, 2001, 124, 1544–1554.18 W. G. Branton, J. Q. Lu, M. G. Surette, R. A. Holt, J. Lind,J. D. Laman and C. Power, Sci. Rep., 2016, 6, 37344.19 J. D. Kriesel, P. Bhetariya, Z.-M. Wang, D. Renner,C. Palmer and K. F. Fischer, Sci. Rep., 2019, 9, 1387.20 N. Wang, A. Hirata, K. Nokihara, K. Fukase andY. Fujimoto, Pept. Sci., 2016, 106, 422–429.21 S. Inamura, K. Fukase and S. Kusumoto, Tetrahedron Lett.,2001, 42, 7613–7616.22 S. Inamura, Y. Fujimoto, A. Kawasaki, Z. Shiokawa,E. Woelk, H. Heine, B. Lindner, N. Inohara, S. Kusumotoand K. Fukase, Org. Biomol. Chem., 2006, 4, 232–242.23 N. Wang, H. Hasegawa, C.-Y. Huang, K. Fukase andY. Fujimoto, Chem. – Asian J., 2017, 12, 27–30.24 M. Lee, D. Hesek, D. A. Dik, J. Fishovitz, E. Lastochkin,B. Boggess, J. F. Fisher and S. Mobashery, Angew. Chem.,Int. Ed., 2017, 56, 2735–2739.25 O. J. Plante, E. R. Palmacci and P. H. Seeberger, Science,2001, 291, 1523–1527.26 P. H. Seeberger, Acc. Chem. Res., 2015, 48, 1450–1463.27 M. Guberman and P. H. Seeberger, J. Am. Chem. Soc., 2019,141, 5581–5592.28 T. Tyrikos-Ergas, V. Bordoni, G. Fittolani, M. A. Chaube,A. Grafmüller, P. H. Seeberger and M. Delbianco, Chem. –Eur. J., 2021, 27, 2321–2325.29 J. Kandasamy, F. Schuhmacher, H. S. Hahm, J. C. Kleinand P. H. Seeberger, Chem. Commun., 2014, 50, 1875–1877.30 L. Kröck, D. Esposito, B. Castagner, C.-C. Wang,P. Bindschändler and P. H. Seeberger, Chem. Sci., 2012, 3,1617–1622.31 R. Enugala, M. J. D. Pires and M. M. B. Marques,Carbohydr. Res., 2014, 384, 112–118.32 M. P. DeNinno, J. B. Etienne and K. C. Duplantier,Tetrahedron Lett., 1995, 5, 669–672.33 W. Kinzy and R. R. Schmidt, Liebigs Ann. Chem., 1987, 407–415.34 M. Oikawa, W.-C. Liu, Y. Nakai, S. Koshida, K. Fukase andS. Kusumoto, Synlett, 1996, 1179–1180.35 S. Eller, M. Collot, J. Yin, H. S. Hahm and P. H. Seeberger,Angew. Chem., Int. Ed., 2013, 52, 5868–5861.36 Y. Yu, A. Kononov, M. Delbianco and P. H. Seeberger,Chem. – Eur. J., 2018, 24, 6075–6078.37 M. Mende, A. Tsouka, J. Heidepriem, G. Paris, D. S. Mattes,S. Eickelmann, V. Bordoni, R. Wawrzinek,F. F. Fuchsberger, P. H. Seeberger, C. Rademacher,M. Delbianco, A. Mallagaray and F. F. Loeffler, Chem. – Eur.J., 2020, 26, 9954–9963.38 C. Ruprecht, M. P. Bartetzko, D. Senf, A. Lakhina,P. J. Smith, M. J. Soto, H. Oh, J.-Y. Yang, D. Chapla,D. V. Silva, M. H. Clausen, M. G. Hahn, K. W. Moremen,B. R. Urbanowicz and F. Pfrengle, Angew. Chem., Int. Ed.,2020, 59, 12494–12498.Communication Organic & Biomolecular ChemistryOrg. Biomol. Chem. This journal is © The Royal Society of Chemistry 2021Open Access Article. Published on 25 October 2021. Downloaded on 11/25/2021 10:00:31 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online

Automated glycan assembly of peptidoglycan backbone fragments

NARCIS 

   University of GroningenAutomated glycan assembly of peptidoglycan backbone fragmentsDallabernardina, Pietro; Benazzi, Valentina; Laman, Jon D.; Seeberger, Peter H.; Loeffler,Felix F.Published in:Organic & Biomolecular ChemistryDOI:10.1039/d1ob01987bIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2021Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Dallabernardina, P., Benazzi, V., Laman, J. D., Seeberger, P. H., & Loeffler, F. F. (2021). Automatedglycan assembly of peptidoglycan backbone fragments. Organic & Biomolecular Chemistry, 45, 9829-9832.https://doi.org/10.1039/d1ob01987bCopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 21-06-2022Organic &Biomolecular ChemistryCOMMUNICATIONCite this: DOI: 10.1039/d1ob01987bReceived 10th October 2021,Accepted 22nd October 2021DOI: 10.1039/d1ob01987brsc.li/obcAutomated glycan assembly of peptidoglycanbackbone fragments†Pietro Dallabernardina,a Valentina Benazzi,a,b Jon D. Laman, cPeter H. Seebergera,d and Felix F. Loeffler *aWe report the automated glycan assembly (AGA) of different oligo-saccharide fragments of the bacterial peptidoglycan (PGN) back-bone. Iterative addition on a solid support of an acetyl glucos-amine and a new muramic acid building block is followed by clea-vage from the solid support and final deprotection providing 10oligosaccharides up to six units.Peptidoglycans (PGN) are an essential component of the cellwall of both Gram-positive and Gram-negative bacteria. PGNserve a structural function, counteracting osmotic pressureand maintaining cell shape, and protect against externalthreats.1,2 The main PGN structure is made of linear glycanstrands cross-linked by short peptide chains (Fig. 1b).3 Severalstructural modifications at both the glycan part, such as acetyl-ation, and the stem peptide are possible in order to give rise tohundreds of structural variants.4 Nevertheless, the structure ofthe glycan backbone is generally conserved and it is composedof two alternating amino sugars (Fig. 1a), N-acetylglucosamine(GlcNAc) and N-acetylmuramic acid (MurNAc), linked by aβ-1–4 bond. The D-lactoyl group of each MurNAc residue is con-nected through an amide bond to a stem peptide that can beup to five amino acid residues long.5 Many modifications canoccur at the peptide chain,2 in particular at the third aminoacid. The majority of Gram-positive bacteria have an L-Lys atposition three, while the majority of Gram-negative bacteriahave a non-proteinogenic amino acid A2pm (2,6-diaminopime-lic acid). The final dipeptide might present some modifi-cations due to acquired resistance to antibiotics.6 Stem pep-tides from different glycan strands may be cross-linked eitherdirectly or by an interpeptide bridge consisting of one to sevenamino acid residues.2In the past two decades, PGNs drew interest not only as apossible target for antibiotics,7 but also for their role as immu-nostimulatory molecules,8–11 as well as in mouse brain devel-opment and behavior.12 It was suggested that PGN fragmentshave many diverse roles as signalling molecules, includingcommunication between bacteria and bacteria with the host,as well as pathogenesis in animals and plants.5 PGNs havealso been correlated with some autoimmune and chronicinflammatory diseases.13–16 In particular, some studies14,17–19have demonstrated that peptidoglycan fragments are presentwithin phagocytes in the brain of multiple sclerosis patientsand that anti-PGN antibodies are present in cerebrospinalfluid during active disease, suggesting a possible link betweenFig. 1 (a) Symbolic representation and structures of the two aminosugars, N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid(MurNAc). (b) Schematic representation of the peptidoglycan structure.The different glycan strands are branched on the MurNAc residues witha sequence of amino acids (dark green circles) called stem peptide. Aninterpeptide bridge (light green box) may connect the two stempeptides.†Electronic supplementary information (ESI) available. See DOI: 10.1039/d1ob01987baDepartment of Biomolecular Systems, Max Planck Institute of Colloids andInterfaces, Am Muehlenberg 1, 14476 Potsdam, Germany.E-mail: felix.loeffler@mpikg.mpg.debUniversity of Pavia, Department of Organic Chemistry, V.le Torquato Taramelli, 10,27100 Pavia, ItalycDepartment of Pathology & Medical Biology, University Medical Center Groningen,Groningen, The NetherlandsdFreie Universität Berlin, Institute of Chemistry and Biochemistry, Arnimallee 22,14195 Berlin, GermanyThis journal is © The Royal Society of Chemistry 2021 Org. Biomol. Chem.Open Access Article. Published on 25 October 2021. Downloaded on 11/25/2021 10:00:31 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journalthese molecules and disease. Therefore, the level and identityof PGNs in the blood, urine, and tissues, might serve as apotential marker for progression of autoimmune and inflam-matory diseases, and, hence, as a tool in therapy monitoring.13Given the biological relevance of these molecules, insightinto PGN structure is essential to understand the role of PGNsin pathogenesis, discovering the mechanisms involved in theirrelease, and how they are detected by bacterial versus humanhost cells.5 Even though different proteins with PGN bindingactivity have been identified, the exact epitopes they recognizeremain poorly characterized.20 A major drawback in thecharacterization of the binding motif is the complexity in thepurification of the PGN fragments from biological sources.20Therefore, to obtain pure compounds it is necessary to resortto chemical synthesis. The chemical synthesis of such oligo-saccharide libraries is not trivial and requires an immense syn-thetic effort.21–24 Automated glycan assembly (AGA)25 hasalready proven its versatility in the generation of oligosacchar-ide collections.26,27 Iterative coupling of different buildingblocks (BB) on a solid support allows for the synthesis of well-defined oligo- and polysaccharide structures in a fast manner.Here, we report the AGA of ten fragments related to theglycan strands of PGNs using two different monosaccharideBBs. To prepare the compound collection, we designed BBs 1and 2 (Scheme 1) that can be synthesized starting from anunprotected glucosamine. The amine at C2 was equipped witha trichloroacetyl (TCA) as participating protecting group, toensure β selectivity during glycosylation. The TCA can be con-verted into the desired acetyl group upon reduction during thefinal deprotection.Fluorenylmethoxycarbonyl (Fmoc) was installed at C4 andused as a temporary protecting group for β1–4 chain elonga-tion. A lactic ester was installed in position three of the BB 2as precursor for the lactic acid moiety of the muramic acid. Allother positions were protected with benzyl (Bn) permanentprotecting groups. The phosphate leaving group was preferredto other leaving groups due to observed higher couplingefficiency in AGA for glucosamine BBs.28 The two desired BBs1 and 2 are accessible starting from the unprotected glucos-amine with a divergent synthetic pathway from glucosaminethioglycoside 3.29 Glycosyl phosphate building block 1 was pre-pared in nine steps, following published procedures.28–30BB 2 was synthesized starting from thiogycoside 329(Scheme 2) that was treated with NaH and (–)-ethyl (S)-2-trifluro-methanesulfonyl propionate, previously prepared in situ fromethyl L-(–)-lactate and triflic anhydride,31 to obtain the monosac-charide 4 with the ethyl propionate at C3. Different solventswere tested. Initially, DMF was used, but only starting materialwas recovered. In THF the reaction appeared to proceed to com-pletion, but the desired compound was obtained in an insepar-able mixture with different side products. Compound 3 provedinsoluble in dichloromethane. Therefore, we added some dropsof DMF, to completely dissolve the starting material. Thismixture gave the best result, affording 4 in 53% yield. The regio-selective opening of the benzylidene was attempted using theBroensted acid trifluoroacetic acid and triethylsilane32 ashydride donor. These conditions provided a low yield since aside product originated from the condensation of the freehydroxyl group on position 4 and the ethyl ester. This sideproduct was previously reported when Kinzy et al. used aBroensted acid to hydrolyze the benzylidene acetal.33Therefore, another strategy for the selective benzylideneopening, involving Me3N·BH3 and the Lewis acid BF3·OEt2 inacetonitrile was adopted.34 Under these conditions, the for-mation of the lactone side product was not observed. Finally,the hydroxyl group on C4 was protected with Fmoc to give 6and subsequently, the thioglycoside was transformed into thecorresponding α-glycosyl phosphate 2.With BB 1 and 2 in hand, we moved forward with the syn-thesis of our collection of oligosaccharides using AGA(Scheme 3). Merrifield resin functionalized with a photocleavablelinker was used as solid support.35 The BBs were coupled to thesolid support using TMSOTf-promoted glycosylation, employingone cycle and five equivalents of BB. Then, an acidic cappingsolution was used to acetylate the unreacted hydroxyl groupsand, thereby, minimizing deletion sequences.36Subsequently, the Fmoc carbonate was cleaved from thestructure, preparing the monosaccharide for the installation ofScheme 1 Retrosynthetic approach to obtain the glycan backbone ofPGN and the two BB required for the synthesis.Scheme 2 Synthesis of the MurNAc BB 2. Reagents and conditions: (a)(–)-ethyl (S)-2-trifluoromethylsulfonyloxy propionate, NaH, DMF/CH2Cl2, 0 °C → rt, 53%; (b) Me3N·BH3, BF3·OEt2, CH3CN, 0 °C → rt, 65%;(c) FmoCl, pyridine, CH2Cl2, rt, 93%; (d) (BuO)2P(O)OH, NIS, TfOH, 0 °C→ rt, 83%.Communication Organic & Biomolecular ChemistryOrg. Biomol. Chem. This journal is © The Royal Society of Chemistry 2021Open Access Article. Published on 25 October 2021. Downloaded on 11/25/2021 10:00:31 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinethe next unit. This cycle of coupling, capping, and deprotectionwas repeated until the desired sequence was achieved. OnceAGA was completed, the oligosaccharides were cleaved from theresin using UV light (365 nm) in a continuous flow photo-reactor.35 After purification of the desired oligosaccharides fromthe deletion sequences, the basic hydrolysis of the lactoyl estersto carboxylic acids was performed using LiOH in a solution ofTHF/dioxane/H2O.31 The remaining protecting groups werecleaved by hydrogenolysis with Pd/C and H2. Under these con-ditions, the TCA group was reduced to acetamide. This last stepwas more problematic, since after several days of hydrogenoly-sis, we still observed the presence of multiple chlorine atoms byMALDI. Therefore, for the hexamers were hydrogenated underhigh pressure to achieve complete conversion in 48 h. The rela-tively low yield is caused by the inefficiency of the photocleavagefrom the resin together with the rather low reactivity of thebuilding blocks, which led to the presence of deletionsequences. Finally, ten different PGN glycan backbones of alter-nating MurNAc and GlcNAc moieties, equipped with an amino-linker were obtained: the two monomers 8 and 9, two disac-charides 10 and 11, two trimers 12 and 13, two tetramers 14and 15, and two hexamers 16 and 17.ConclusionsTen oligosaccharide fragments of the PGN backbone weresynthesized. The use of a new MurNAc BB enabled the fastsynthesis of a collection of oligosaccharides up to hexamers.These oligosaccharides can be used to create glycan micro-arrays for the characterization of peptidoglycan directedantibodies. Advancing the idea from recently publishedapproaches, where molecules on microarrays were modifieddirectly on the glass slide after in situ synthesis37 or print-ing,38 we will follow a similar strategy. We envision tocouple different stem peptide variants from solution,forming the amide bond directly with the arrayed oligosac-charides. These arrays will support the identification of epi-topes recognized by anti-PGN antibodies, and can poten-tially help to close current knowledge gaps in autoimmunedisorders.Conflicts of interestThere are no conflicts to declare.Scheme 3 Automated glycan assembly of PGN oligosaccharides. Reagents and conditions: (a) 1 × 5 equiv. BB1, TMSOTf, CH2Cl2, −30 °C (5 min) →−15 °C (35 min); (b) 1 cycle of a 10% acetic anhydride and 2% methanesulfonic acid in CH2Cl2, 25 °C; (c) 3 cycles of 20% piperidine in DMF, 25 °C(5 min) (Module B); (d) CH2Cl2, hν (365 nm); (e) LiOH, THF/1,4-dioxane/H2O, 2–24 h; (f ) H2, Pd/C, H2O/HOAc, 16–48 h. 8: 27%; 9: 42%; 10: 20%; 11:10%; 12: 11%; 13: 9%; 14: 3%; 15: 8%; 16: 3%; 17: 2%.Organic & Biomolecular Chemistry CommunicationThis journal is © The Royal Society of Chemistry 2021 Org. Biomol. Chem.Open Access Article. Published on 25 October 2021. Downloaded on 11/25/2021 10:00:31 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineAcknowledgementsThe authors thank the German Federal Ministry of Educationand Research (BMBF, grant no. 13XP5050A), the Max-Planck-Fraunhofer Collaboration Project (Glyco3Display), and the MaxPlanck Society for financial support. In addition, the authorswould like to thank Martina Delbianco for the fruitful discus-sions and the members of the Department of BiomolecularSystems for the technical support, especially Eva Settels andOlaf Niemeyer. Open Access funding provided by the MaxPlanck Society.References1 J. V. Höltje, Microbiol. Mol. Biol. Rev., 1998, 62, 181–203.2 W. Vollmer, D. Blanot and M. A. de Pedro, FEMS Microbiol.Rev., 2008, 32, 149–167.3 J. H. Rogers, H. R. Perkins and J. B. Ward, Microbial CellWalls and Membranes, Springer, Netherlands, 1980.4 P. Schumann, in Methods in Microbiology, 2011, vol. 38, pp.101–129.5 O. Irazoki, S. B. Hernandez and F. Cava, Front. Microbiol.,2019, 10, 500.6 A. K. Yadav, A. Espaillat and F. Cava, Front. Microbiol.,2018, 9, 2064.7 M. A. Kohanski, D. J. Dwyer and J. J. Collins, Nat. Rev.Microbiol., 2010, 8, 423–435.8 S. E. Girardin, I. G. Boneca, L. A. M. Carneiro, A. Antignac,M. Jéhanno, J. Viala, K. Tedin, M.-K. Taha, A. Labigne,U. Zähringer, A. J. Coyle, P. S. D. Stefano, J. Bertin,P. J. Sansonetti and D. J. Philpott, Science, 2003, 300, 1584–1587.9 S. E. Girardin, L. H. Travassos, M. Hervé, D. Blanot,I. G. Boneca, D. J. Philpott, P. J. Sansonetti and D. Mengin-Lecreulx, J. Biol. Chem., 2003, 278, 41702–41708.10 K. L. Woodhams, J. M. Chan, J. D. Lenz, K. T. Hackett andJ. P. Dillard, Infect. Immun., 2013, 81, 3490–3498.11 M. A. Boudreau, J. F. Fisher and S. Mobashery,Biochemistry, 2012, 51, 2974–2990.12 A. Gonzalez-Santana and R. D. Heijtz, Trends Mol. Med.,2020, 26, 729–743.13 Z. Huang, J. Wang, X. Xu, H. Wang, Y. Qiao, W. C. Chu,S. Xu, L. Chai, F. Cottier, N. Pavelka, M. Oosting,L. A. B. Joosten, M. Netea, C. Y. L. Ng, K. P. Leong,P. Kundu, K.-P. Lam, S. Pettersson and Y. Wang, Nat.Microbiol., 2019, 4, 766–773.14 P. J. Shaw, M. J. Barr, J. R. Lukens, M. A. McGargill, H. Chi,T. W. Mak and T.-D. Kanneganti, Immunity, 2011, 34, 75–84.15 B. L. Jutras, R. B. Lochhead, Z. A. Kloos, J. Biboy, K. Strle,C. J. Booth, S. K. Govers, J. Gray, P. Schumann, W. Vollmer,L. K. Bockenstedt, A. C. Steere and C. Jacobs-Wagner, Proc.Natl. Acad. Sci. U. S. A., 2019, 116, 13498–13507.16 L. Visser, H. J. d. Heer, L. A. Boven, D. van Riel, M. vanMeurs, M.-J. Melief, U. Zähringer, J. van Strijp,B. N. Lambrecht, E. E. Nieuwenhuis and J. D. Laman,J. Immunol., 2005, 174, 808–816.17 I. A. Schrijver, M. van Meurs, M.-J. Melief, C. W. Ang,D. Buljevac, R. Ravid, M. P. Hazenberg and J. D. Laman,Brain, 2001, 124, 1544–1554.18 W. G. Branton, J. Q. Lu, M. G. Surette, R. A. Holt, J. Lind,J. D. Laman and C. Power, Sci. Rep., 2016, 6, 37344.19 J. D. Kriesel, P. Bhetariya, Z.-M. Wang, D. Renner,C. Palmer and K. F. Fischer, Sci. Rep., 2019, 9, 1387.20 N. Wang, A. Hirata, K. Nokihara, K. Fukase andY. Fujimoto, Pept. Sci., 2016, 106, 422–429.21 S. Inamura, K. Fukase and S. Kusumoto, Tetrahedron Lett.,2001, 42, 7613–7616.22 S. Inamura, Y. Fujimoto, A. Kawasaki, Z. Shiokawa,E. Woelk, H. Heine, B. Lindner, N. Inohara, S. Kusumotoand K. Fukase, Org. Biomol. Chem., 2006, 4, 232–242.23 N. Wang, H. Hasegawa, C.-Y. Huang, K. Fukase andY. Fujimoto, Chem. – Asian J., 2017, 12, 27–30.24 M. Lee, D. Hesek, D. A. Dik, J. Fishovitz, E. Lastochkin,B. Boggess, J. F. Fisher and S. Mobashery, Angew. Chem.,Int. Ed., 2017, 56, 2735–2739.25 O. J. Plante, E. R. Palmacci and P. H. Seeberger, Science,2001, 291, 1523–1527.26 P. H. Seeberger, Acc. Chem. Res., 2015, 48, 1450–1463.27 M. Guberman and P. H. Seeberger, J. Am. Chem. Soc., 2019,141, 5581–5592.28 T. Tyrikos-Ergas, V. Bordoni, G. Fittolani, M. A. Chaube,A. Grafmüller, P. H. Seeberger and M. Delbianco, Chem. –Eur. J., 2021, 27, 2321–2325.29 J. Kandasamy, F. Schuhmacher, H. S. Hahm, J. C. Kleinand P. H. Seeberger, Chem. Commun., 2014, 50, 1875–1877.30 L. Kröck, D. Esposito, B. Castagner, C.-C. Wang,P. Bindschändler and P. H. Seeberger, Chem. Sci., 2012, 3,1617–1622.31 R. Enugala, M. J. D. Pires and M. M. B. Marques,Carbohydr. Res., 2014, 384, 112–118.32 M. P. DeNinno, J. B. Etienne and K. C. Duplantier,Tetrahedron Lett., 1995, 5, 669–672.33 W. Kinzy and R. R. Schmidt, Liebigs Ann. Chem., 1987, 407–415.34 M. Oikawa, W.-C. Liu, Y. Nakai, S. Koshida, K. Fukase andS. Kusumoto, Synlett, 1996, 1179–1180.35 S. Eller, M. Collot, J. Yin, H. S. Hahm and P. H. Seeberger,Angew. Chem., Int. Ed., 2013, 52, 5868–5861.36 Y. Yu, A. Kononov, M. Delbianco and P. H. Seeberger,Chem. – Eur. J., 2018, 24, 6075–6078.37 M. Mende, A. Tsouka, J. Heidepriem, G. Paris, D. S. Mattes,S. Eickelmann, V. Bordoni, R. Wawrzinek,F. F. Fuchsberger, P. H. Seeberger, C. Rademacher,M. Delbianco, A. Mallagaray and F. F. Loeffler, Chem. – Eur.J., 2020, 26, 9954–9963.38 C. Ruprecht, M. P. Bartetzko, D. Senf, A. Lakhina,P. J. Smith, M. J. Soto, H. Oh, J.-Y. Yang, D. Chapla,D. V. Silva, M. H. Clausen, M. G. Hahn, K. W. Moremen,B. R. Urbanowicz and F. Pfrengle, Angew. Chem., Int. Ed.,2020, 59, 12494–12498.Communication Organic & Biomolecular ChemistryOrg. Biomol. Chem. This journal is © The Royal Society of Chemistry 2021Open Access Article. Published on 25 October 2021. Downloaded on 11/25/2021 10:00:31 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online

University of Groningen

   University of GroningenAutomated glycan assembly of peptidoglycan backbone fragmentsDallabernardina, Pietro; Benazzi, Valentina; Laman, Jon D.; Seeberger, Peter H.; Loeffler,Felix F.Published in:Organic & Biomolecular ChemistryDOI:10.1039/d1ob01987bIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2021Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Dallabernardina, P., Benazzi, V., Laman, J. D., Seeberger, P. H., & Loeffler, F. F. (2021). Automatedglycan assembly of peptidoglycan backbone fragments. Organic & Biomolecular Chemistry, 45, 9829-9832.https://doi.org/10.1039/d1ob01987bCopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 29-04-2023Organic &Biomolecular ChemistryCOMMUNICATIONCite this: DOI: 10.1039/d1ob01987bReceived 10th October 2021,Accepted 22nd October 2021DOI: 10.1039/d1ob01987brsc.li/obcAutomated glycan assembly of peptidoglycanbackbone fragments†Pietro Dallabernardina,a Valentina Benazzi,a,b Jon D. Laman, cPeter H. Seebergera,d and Felix F. Loeffler *aWe report the automated glycan assembly (AGA) of different oligo-saccharide fragments of the bacterial peptidoglycan (PGN) back-bone. Iterative addition on a solid support of an acetyl glucos-amine and a new muramic acid building block is followed by clea-vage from the solid support and final deprotection providing 10oligosaccharides up to six units.Peptidoglycans (PGN) are an essential component of the cellwall of both Gram-positive and Gram-negative bacteria. PGNserve a structural function, counteracting osmotic pressureand maintaining cell shape, and protect against externalthreats.1,2 The main PGN structure is made of linear glycanstrands cross-linked by short peptide chains (Fig. 1b).3 Severalstructural modifications at both the glycan part, such as acetyl-ation, and the stem peptide are possible in order to give rise tohundreds of structural variants.4 Nevertheless, the structure ofthe glycan backbone is generally conserved and it is composedof two alternating amino sugars (Fig. 1a), N-acetylglucosamine(GlcNAc) and N-acetylmuramic acid (MurNAc), linked by aβ-1–4 bond. The D-lactoyl group of each MurNAc residue is con-nected through an amide bond to a stem peptide that can beup to five amino acid residues long.5 Many modifications canoccur at the peptide chain,2 in particular at the third aminoacid. The majority of Gram-positive bacteria have an L-Lys atposition three, while the majority of Gram-negative bacteriahave a non-proteinogenic amino acid A2pm (2,6-diaminopime-lic acid). The final dipeptide might present some modifi-cations due to acquired resistance to antibiotics.6 Stem pep-tides from different glycan strands may be cross-linked eitherdirectly or by an interpeptide bridge consisting of one to sevenamino acid residues.2In the past two decades, PGNs drew interest not only as apossible target for antibiotics,7 but also for their role as immu-nostimulatory molecules,8–11 as well as in mouse brain devel-opment and behavior.12 It was suggested that PGN fragmentshave many diverse roles as signalling molecules, includingcommunication between bacteria and bacteria with the host,as well as pathogenesis in animals and plants.5 PGNs havealso been correlated with some autoimmune and chronicinflammatory diseases.13–16 In particular, some studies14,17–19have demonstrated that peptidoglycan fragments are presentwithin phagocytes in the brain of multiple sclerosis patientsand that anti-PGN antibodies are present in cerebrospinalfluid during active disease, suggesting a possible link betweenFig. 1 (a) Symbolic representation and structures of the two aminosugars, N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid(MurNAc). (b) Schematic representation of the peptidoglycan structure.The different glycan strands are branched on the MurNAc residues witha sequence of amino acids (dark green circles) called stem peptide. Aninterpeptide bridge (light green box) may connect the two stempeptides.†Electronic supplementary information (ESI) available. See DOI: 10.1039/d1ob01987baDepartment of Biomolecular Systems, Max Planck Institute of Colloids andInterfaces, Am Muehlenberg 1, 14476 Potsdam, Germany.E-mail: felix.loeffler@mpikg.mpg.debUniversity of Pavia, Department of Organic Chemistry, V.le Torquato Taramelli, 10,27100 Pavia, ItalycDepartment of Pathology & Medical Biology, University Medical Center Groningen,Groningen, The NetherlandsdFreie Universität Berlin, Institute of Chemistry and Biochemistry, Arnimallee 22,14195 Berlin, GermanyThis journal is © The Royal Society of Chemistry 2021 Org. Biomol. Chem.Open Access Article. Published on 25 October 2021. Downloaded on 11/25/2021 10:00:31 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journalthese molecules and disease. Therefore, the level and identityof PGNs in the blood, urine, and tissues, might serve as apotential marker for progression of autoimmune and inflam-matory diseases, and, hence, as a tool in therapy monitoring.13Given the biological relevance of these molecules, insightinto PGN structure is essential to understand the role of PGNsin pathogenesis, discovering the mechanisms involved in theirrelease, and how they are detected by bacterial versus humanhost cells.5 Even though different proteins with PGN bindingactivity have been identified, the exact epitopes they recognizeremain poorly characterized.20 A major drawback in thecharacterization of the binding motif is the complexity in thepurification of the PGN fragments from biological sources.20Therefore, to obtain pure compounds it is necessary to resortto chemical synthesis. The chemical synthesis of such oligo-saccharide libraries is not trivial and requires an immense syn-thetic effort.21–24 Automated glycan assembly (AGA)25 hasalready proven its versatility in the generation of oligosacchar-ide collections.26,27 Iterative coupling of different buildingblocks (BB) on a solid support allows for the synthesis of well-defined oligo- and polysaccharide structures in a fast manner.Here, we report the AGA of ten fragments related to theglycan strands of PGNs using two different monosaccharideBBs. To prepare the compound collection, we designed BBs 1and 2 (Scheme 1) that can be synthesized starting from anunprotected glucosamine. The amine at C2 was equipped witha trichloroacetyl (TCA) as participating protecting group, toensure β selectivity during glycosylation. The TCA can be con-verted into the desired acetyl group upon reduction during thefinal deprotection.Fluorenylmethoxycarbonyl (Fmoc) was installed at C4 andused as a temporary protecting group for β1–4 chain elonga-tion. A lactic ester was installed in position three of the BB 2as precursor for the lactic acid moiety of the muramic acid. Allother positions were protected with benzyl (Bn) permanentprotecting groups. The phosphate leaving group was preferredto other leaving groups due to observed higher couplingefficiency in AGA for glucosamine BBs.28 The two desired BBs1 and 2 are accessible starting from the unprotected glucos-amine with a divergent synthetic pathway from glucosaminethioglycoside 3.29 Glycosyl phosphate building block 1 was pre-pared in nine steps, following published procedures.28–30BB 2 was synthesized starting from thiogycoside 329(Scheme 2) that was treated with NaH and (–)-ethyl (S)-2-trifluro-methanesulfonyl propionate, previously prepared in situ fromethyl L-(–)-lactate and triflic anhydride,31 to obtain the monosac-charide 4 with the ethyl propionate at C3. Different solventswere tested. Initially, DMF was used, but only starting materialwas recovered. In THF the reaction appeared to proceed to com-pletion, but the desired compound was obtained in an insepar-able mixture with different side products. Compound 3 provedinsoluble in dichloromethane. Therefore, we added some dropsof DMF, to completely dissolve the starting material. Thismixture gave the best result, affording 4 in 53% yield. The regio-selective opening of the benzylidene was attempted using theBroensted acid trifluoroacetic acid and triethylsilane32 ashydride donor. These conditions provided a low yield since aside product originated from the condensation of the freehydroxyl group on position 4 and the ethyl ester. This sideproduct was previously reported when Kinzy et al. used aBroensted acid to hydrolyze the benzylidene acetal.33Therefore, another strategy for the selective benzylideneopening, involving Me3N·BH3 and the Lewis acid BF3·OEt2 inacetonitrile was adopted.34 Under these conditions, the for-mation of the lactone side product was not observed. Finally,the hydroxyl group on C4 was protected with Fmoc to give 6and subsequently, the thioglycoside was transformed into thecorresponding α-glycosyl phosphate 2.With BB 1 and 2 in hand, we moved forward with the syn-thesis of our collection of oligosaccharides using AGA(Scheme 3). Merrifield resin functionalized with a photocleavablelinker was used as solid support.35 The BBs were coupled to thesolid support using TMSOTf-promoted glycosylation, employingone cycle and five equivalents of BB. Then, an acidic cappingsolution was used to acetylate the unreacted hydroxyl groupsand, thereby, minimizing deletion sequences.36Subsequently, the Fmoc carbonate was cleaved from thestructure, preparing the monosaccharide for the installation ofScheme 1 Retrosynthetic approach to obtain the glycan backbone ofPGN and the two BB required for the synthesis.Scheme 2 Synthesis of the MurNAc BB 2. Reagents and conditions: (a)(–)-ethyl (S)-2-trifluoromethylsulfonyloxy propionate, NaH, DMF/CH2Cl2, 0 °C → rt, 53%; (b) Me3N·BH3, BF3·OEt2, CH3CN, 0 °C → rt, 65%;(c) FmoCl, pyridine, CH2Cl2, rt, 93%; (d) (BuO)2P(O)OH, NIS, TfOH, 0 °C→ rt, 83%.Communication Organic & Biomolecular ChemistryOrg. Biomol. Chem. This journal is © The Royal Society of Chemistry 2021Open Access Article. Published on 25 October 2021. Downloaded on 11/25/2021 10:00:31 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinethe next unit. This cycle of coupling, capping, and deprotectionwas repeated until the desired sequence was achieved. OnceAGA was completed, the oligosaccharides were cleaved from theresin using UV light (365 nm) in a continuous flow photo-reactor.35 After purification of the desired oligosaccharides fromthe deletion sequences, the basic hydrolysis of the lactoyl estersto carboxylic acids was performed using LiOH in a solution ofTHF/dioxane/H2O.31 The remaining protecting groups werecleaved by hydrogenolysis with Pd/C and H2. Under these con-ditions, the TCA group was reduced to acetamide. This last stepwas more problematic, since after several days of hydrogenoly-sis, we still observed the presence of multiple chlorine atoms byMALDI. Therefore, for the hexamers were hydrogenated underhigh pressure to achieve complete conversion in 48 h. The rela-tively low yield is caused by the inefficiency of the photocleavagefrom the resin together with the rather low reactivity of thebuilding blocks, which led to the presence of deletionsequences. Finally, ten different PGN glycan backbones of alter-nating MurNAc and GlcNAc moieties, equipped with an amino-linker were obtained: the two monomers 8 and 9, two disac-charides 10 and 11, two trimers 12 and 13, two tetramers 14and 15, and two hexamers 16 and 17.ConclusionsTen oligosaccharide fragments of the PGN backbone weresynthesized. The use of a new MurNAc BB enabled the fastsynthesis of a collection of oligosaccharides up to hexamers.These oligosaccharides can be used to create glycan micro-arrays for the characterization of peptidoglycan directedantibodies. Advancing the idea from recently publishedapproaches, where molecules on microarrays were modifieddirectly on the glass slide after in situ synthesis37 or print-ing,38 we will follow a similar strategy. We envision tocouple different stem peptide variants from solution,forming the amide bond directly with the arrayed oligosac-charides. These arrays will support the identification of epi-topes recognized by anti-PGN antibodies, and can poten-tially help to close current knowledge gaps in autoimmunedisorders.Conflicts of interestThere are no conflicts to declare.Scheme 3 Automated glycan assembly of PGN oligosaccharides. Reagents and conditions: (a) 1 × 5 equiv. BB1, TMSOTf, CH2Cl2, −30 °C (5 min) →−15 °C (35 min); (b) 1 cycle of a 10% acetic anhydride and 2% methanesulfonic acid in CH2Cl2, 25 °C; (c) 3 cycles of 20% piperidine in DMF, 25 °C(5 min) (Module B); (d) CH2Cl2, hν (365 nm); (e) LiOH, THF/1,4-dioxane/H2O, 2–24 h; (f ) H2, Pd/C, H2O/HOAc, 16–48 h. 8: 27%; 9: 42%; 10: 20%; 11:10%; 12: 11%; 13: 9%; 14: 3%; 15: 8%; 16: 3%; 17: 2%.Organic & Biomolecular Chemistry CommunicationThis journal is © The Royal Society of Chemistry 2021 Org. Biomol. Chem.Open Access Article. Published on 25 October 2021. Downloaded on 11/25/2021 10:00:31 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineAcknowledgementsThe authors thank the German Federal Ministry of Educationand Research (BMBF, grant no. 13XP5050A), the Max-Planck-Fraunhofer Collaboration Project (Glyco3Display), and the MaxPlanck Society for financial support. In addition, the authorswould like to thank Martina Delbianco for the fruitful discus-sions and the members of the Department of BiomolecularSystems for the technical support, especially Eva Settels andOlaf Niemeyer. Open Access funding provided by the MaxPlanck Society.References1 J. V. Höltje, Microbiol. Mol. Biol. Rev., 1998, 62, 181–203.2 W. Vollmer, D. Blanot and M. A. de Pedro, FEMS Microbiol.Rev., 2008, 32, 149–167.3 J. H. Rogers, H. R. Perkins and J. B. Ward, Microbial CellWalls and Membranes, Springer, Netherlands, 1980.4 P. Schumann, in Methods in Microbiology, 2011, vol. 38, pp.101–129.5 O. Irazoki, S. B. Hernandez and F. Cava, Front. Microbiol.,2019, 10, 500.6 A. K. Yadav, A. Espaillat and F. Cava, Front. Microbiol.,2018, 9, 2064.7 M. A. Kohanski, D. J. Dwyer and J. J. Collins, Nat. Rev.Microbiol., 2010, 8, 423–435.8 S. E. Girardin, I. G. Boneca, L. A. M. Carneiro, A. Antignac,M. Jéhanno, J. Viala, K. Tedin, M.-K. Taha, A. Labigne,U. Zähringer, A. J. Coyle, P. S. D. Stefano, J. Bertin,P. J. Sansonetti and D. J. Philpott, Science, 2003, 300, 1584–1587.9 S. E. Girardin, L. H. Travassos, M. Hervé, D. Blanot,I. G. Boneca, D. J. Philpott, P. J. Sansonetti and D. Mengin-Lecreulx, J. Biol. Chem., 2003, 278, 41702–41708.10 K. L. Woodhams, J. M. Chan, J. D. Lenz, K. T. Hackett andJ. P. Dillard, Infect. Immun., 2013, 81, 3490–3498.11 M. A. Boudreau, J. F. Fisher and S. Mobashery,Biochemistry, 2012, 51, 2974–2990.12 A. Gonzalez-Santana and R. D. Heijtz, Trends Mol. Med.,2020, 26, 729–743.13 Z. Huang, J. Wang, X. Xu, H. Wang, Y. Qiao, W. C. Chu,S. Xu, L. Chai, F. Cottier, N. Pavelka, M. Oosting,L. A. B. Joosten, M. Netea, C. Y. L. Ng, K. P. Leong,P. Kundu, K.-P. Lam, S. Pettersson and Y. Wang, Nat.Microbiol., 2019, 4, 766–773.14 P. J. Shaw, M. J. Barr, J. R. Lukens, M. A. McGargill, H. Chi,T. W. Mak and T.-D. Kanneganti, Immunity, 2011, 34, 75–84.15 B. L. Jutras, R. B. Lochhead, Z. A. Kloos, J. Biboy, K. Strle,C. J. Booth, S. K. Govers, J. Gray, P. Schumann, W. Vollmer,L. K. Bockenstedt, A. C. Steere and C. Jacobs-Wagner, Proc.Natl. Acad. Sci. U. S. A., 2019, 116, 13498–13507.16 L. Visser, H. J. d. Heer, L. A. Boven, D. van Riel, M. vanMeurs, M.-J. Melief, U. Zähringer, J. van Strijp,B. N. Lambrecht, E. E. Nieuwenhuis and J. D. Laman,J. Immunol., 2005, 174, 808–816.17 I. A. Schrijver, M. van Meurs, M.-J. Melief, C. W. Ang,D. Buljevac, R. Ravid, M. P. Hazenberg and J. D. Laman,Brain, 2001, 124, 1544–1554.18 W. G. Branton, J. Q. Lu, M. G. Surette, R. A. Holt, J. Lind,J. D. Laman and C. Power, Sci. Rep., 2016, 6, 37344.19 J. D. Kriesel, P. Bhetariya, Z.-M. Wang, D. Renner,C. Palmer and K. F. Fischer, Sci. Rep., 2019, 9, 1387.20 N. Wang, A. Hirata, K. Nokihara, K. Fukase andY. Fujimoto, Pept. Sci., 2016, 106, 422–429.21 S. Inamura, K. Fukase and S. Kusumoto, Tetrahedron Lett.,2001, 42, 7613–7616.22 S. Inamura, Y. Fujimoto, A. Kawasaki, Z. Shiokawa,E. Woelk, H. Heine, B. Lindner, N. Inohara, S. Kusumotoand K. Fukase, Org. Biomol. Chem., 2006, 4, 232–242.23 N. Wang, H. Hasegawa, C.-Y. Huang, K. Fukase andY. Fujimoto, Chem. – Asian J., 2017, 12, 27–30.24 M. Lee, D. Hesek, D. A. Dik, J. Fishovitz, E. Lastochkin,B. Boggess, J. F. Fisher and S. Mobashery, Angew. Chem.,Int. Ed., 2017, 56, 2735–2739.25 O. J. Plante, E. R. Palmacci and P. H. Seeberger, Science,2001, 291, 1523–1527.26 P. H. Seeberger, Acc. Chem. Res., 2015, 48, 1450–1463.27 M. Guberman and P. H. Seeberger, J. Am. Chem. Soc., 2019,141, 5581–5592.28 T. Tyrikos-Ergas, V. Bordoni, G. Fittolani, M. A. Chaube,A. Grafmüller, P. H. Seeberger and M. Delbianco, Chem. –Eur. J., 2021, 27, 2321–2325.29 J. Kandasamy, F. Schuhmacher, H. S. Hahm, J. C. Kleinand P. H. Seeberger, Chem. Commun., 2014, 50, 1875–1877.30 L. Kröck, D. Esposito, B. Castagner, C.-C. Wang,P. Bindschändler and P. H. Seeberger, Chem. Sci., 2012, 3,1617–1622.31 R. Enugala, M. J. D. Pires and M. M. B. Marques,Carbohydr. Res., 2014, 384, 112–118.32 M. P. DeNinno, J. B. Etienne and K. C. Duplantier,Tetrahedron Lett., 1995, 5, 669–672.33 W. Kinzy and R. R. Schmidt, Liebigs Ann. Chem., 1987, 407–415.34 M. Oikawa, W.-C. Liu, Y. Nakai, S. Koshida, K. Fukase andS. Kusumoto, Synlett, 1996, 1179–1180.35 S. Eller, M. Collot, J. Yin, H. S. Hahm and P. H. Seeberger,Angew. Chem., Int. Ed., 2013, 52, 5868–5861.36 Y. Yu, A. Kononov, M. Delbianco and P. H. Seeberger,Chem. – Eur. J., 2018, 24, 6075–6078.37 M. Mende, A. Tsouka, J. Heidepriem, G. Paris, D. S. Mattes,S. Eickelmann, V. Bordoni, R. Wawrzinek,F. F. Fuchsberger, P. H. Seeberger, C. Rademacher,M. Delbianco, A. Mallagaray and F. F. Loeffler, Chem. – Eur.J., 2020, 26, 9954–9963.38 C. Ruprecht, M. P. Bartetzko, D. Senf, A. Lakhina,P. J. Smith, M. J. Soto, H. Oh, J.-Y. Yang, D. Chapla,D. V. Silva, M. H. Clausen, M. G. Hahn, K. W. Moremen,B. R. Urbanowicz and F. Pfrengle, Angew. Chem., Int. Ed.,2020, 59, 12494–12498.Communication Organic & Biomolecular ChemistryOrg. Biomol. Chem. This journal is © The Royal Society of Chemistry 2021Open Access Article. Published on 25 October 2021. Downloaded on 11/25/2021 10:00:31 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online

Institutional Repository of the Freie Universität Berlin

   University of GroningenAutomated glycan assembly of peptidoglycan backbone fragmentsDallabernardina, Pietro; Benazzi, Valentina; Laman, Jon D.; Seeberger, Peter H.; Loeffler,Felix F.Published in:Organic & Biomolecular ChemistryDOI:10.1039/d1ob01987bIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2021Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Dallabernardina, P., Benazzi, V., Laman, J. D., Seeberger, P. H., & Loeffler, F. F. (2021). Automatedglycan assembly of peptidoglycan backbone fragments. Organic & Biomolecular Chemistry, 45, 9829-9832.https://doi.org/10.1039/d1ob01987bCopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 20-11-2022Organic &Biomolecular ChemistryCOMMUNICATIONCite this: DOI: 10.1039/d1ob01987bReceived 10th October 2021,Accepted 22nd October 2021DOI: 10.1039/d1ob01987brsc.li/obcAutomated glycan assembly of peptidoglycanbackbone fragments†Pietro Dallabernardina,a Valentina Benazzi,a,b Jon D. Laman, cPeter H. Seebergera,d and Felix F. Loeffler *aWe report the automated glycan assembly (AGA) of different oligo-saccharide fragments of the bacterial peptidoglycan (PGN) back-bone. Iterative addition on a solid support of an acetyl glucos-amine and a new muramic acid building block is followed by clea-vage from the solid support and final deprotection providing 10oligosaccharides up to six units.Peptidoglycans (PGN) are an essential component of the cellwall of both Gram-positive and Gram-negative bacteria. PGNserve a structural function, counteracting osmotic pressureand maintaining cell shape, and protect against externalthreats.1,2 The main PGN structure is made of linear glycanstrands cross-linked by short peptide chains (Fig. 1b).3 Severalstructural modifications at both the glycan part, such as acetyl-ation, and the stem peptide are possible in order to give rise tohundreds of structural variants.4 Nevertheless, the structure ofthe glycan backbone is generally conserved and it is composedof two alternating amino sugars (Fig. 1a), N-acetylglucosamine(GlcNAc) and N-acetylmuramic acid (MurNAc), linked by aβ-1–4 bond. The D-lactoyl group of each MurNAc residue is con-nected through an amide bond to a stem peptide that can beup to five amino acid residues long.5 Many modifications canoccur at the peptide chain,2 in particular at the third aminoacid. The majority of Gram-positive bacteria have an L-Lys atposition three, while the majority of Gram-negative bacteriahave a non-proteinogenic amino acid A2pm (2,6-diaminopime-lic acid). The final dipeptide might present some modifi-cations due to acquired resistance to antibiotics.6 Stem pep-tides from different glycan strands may be cross-linked eitherdirectly or by an interpeptide bridge consisting of one to sevenamino acid residues.2In the past two decades, PGNs drew interest not only as apossible target for antibiotics,7 but also for their role as immu-nostimulatory molecules,8–11 as well as in mouse brain devel-opment and behavior.12 It was suggested that PGN fragmentshave many diverse roles as signalling molecules, includingcommunication between bacteria and bacteria with the host,as well as pathogenesis in animals and plants.5 PGNs havealso been correlated with some autoimmune and chronicinflammatory diseases.13–16 In particular, some studies14,17–19have demonstrated that peptidoglycan fragments are presentwithin phagocytes in the brain of multiple sclerosis patientsand that anti-PGN antibodies are present in cerebrospinalfluid during active disease, suggesting a possible link betweenFig. 1 (a) Symbolic representation and structures of the two aminosugars, N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid(MurNAc). (b) Schematic representation of the peptidoglycan structure.The different glycan strands are branched on the MurNAc residues witha sequence of amino acids (dark green circles) called stem peptide. Aninterpeptide bridge (light green box) may connect the two stempeptides.†Electronic supplementary information (ESI) available. See DOI: 10.1039/d1ob01987baDepartment of Biomolecular Systems, Max Planck Institute of Colloids andInterfaces, Am Muehlenberg 1, 14476 Potsdam, Germany.E-mail: felix.loeffler@mpikg.mpg.debUniversity of Pavia, Department of Organic Chemistry, V.le Torquato Taramelli, 10,27100 Pavia, ItalycDepartment of Pathology & Medical Biology, University Medical Center Groningen,Groningen, The NetherlandsdFreie Universität Berlin, Institute of Chemistry and Biochemistry, Arnimallee 22,14195 Berlin, GermanyThis journal is © The Royal Society of Chemistry 2021 Org. Biomol. Chem.Open Access Article. Published on 25 October 2021. Downloaded on 11/25/2021 10:00:31 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journalthese molecules and disease. Therefore, the level and identityof PGNs in the blood, urine, and tissues, might serve as apotential marker for progression of autoimmune and inflam-matory diseases, and, hence, as a tool in therapy monitoring.13Given the biological relevance of these molecules, insightinto PGN structure is essential to understand the role of PGNsin pathogenesis, discovering the mechanisms involved in theirrelease, and how they are detected by bacterial versus humanhost cells.5 Even though different proteins with PGN bindingactivity have been identified, the exact epitopes they recognizeremain poorly characterized.20 A major drawback in thecharacterization of the binding motif is the complexity in thepurification of the PGN fragments from biological sources.20Therefore, to obtain pure compounds it is necessary to resortto chemical synthesis. The chemical synthesis of such oligo-saccharide libraries is not trivial and requires an immense syn-thetic effort.21–24 Automated glycan assembly (AGA)25 hasalready proven its versatility in the generation of oligosacchar-ide collections.26,27 Iterative coupling of different buildingblocks (BB) on a solid support allows for the synthesis of well-defined oligo- and polysaccharide structures in a fast manner.Here, we report the AGA of ten fragments related to theglycan strands of PGNs using two different monosaccharideBBs. To prepare the compound collection, we designed BBs 1and 2 (Scheme 1) that can be synthesized starting from anunprotected glucosamine. The amine at C2 was equipped witha trichloroacetyl (TCA) as participating protecting group, toensure β selectivity during glycosylation. The TCA can be con-verted into the desired acetyl group upon reduction during thefinal deprotection.Fluorenylmethoxycarbonyl (Fmoc) was installed at C4 andused as a temporary protecting group for β1–4 chain elonga-tion. A lactic ester was installed in position three of the BB 2as precursor for the lactic acid moiety of the muramic acid. Allother positions were protected with benzyl (Bn) permanentprotecting groups. The phosphate leaving group was preferredto other leaving groups due to observed higher couplingefficiency in AGA for glucosamine BBs.28 The two desired BBs1 and 2 are accessible starting from the unprotected glucos-amine with a divergent synthetic pathway from glucosaminethioglycoside 3.29 Glycosyl phosphate building block 1 was pre-pared in nine steps, following published procedures.28–30BB 2 was synthesized starting from thiogycoside 329(Scheme 2) that was treated with NaH and (–)-ethyl (S)-2-trifluro-methanesulfonyl propionate, previously prepared in situ fromethyl L-(–)-lactate and triflic anhydride,31 to obtain the monosac-charide 4 with the ethyl propionate at C3. Different solventswere tested. Initially, DMF was used, but only starting materialwas recovered. In THF the reaction appeared to proceed to com-pletion, but the desired compound was obtained in an insepar-able mixture with different side products. Compound 3 provedinsoluble in dichloromethane. Therefore, we added some dropsof DMF, to completely dissolve the starting material. Thismixture gave the best result, affording 4 in 53% yield. The regio-selective opening of the benzylidene was attempted using theBroensted acid trifluoroacetic acid and triethylsilane32 ashydride donor. These conditions provided a low yield since aside product originated from the condensation of the freehydroxyl group on position 4 and the ethyl ester. This sideproduct was previously reported when Kinzy et al. used aBroensted acid to hydrolyze the benzylidene acetal.33Therefore, another strategy for the selective benzylideneopening, involving Me3N·BH3 and the Lewis acid BF3·OEt2 inacetonitrile was adopted.34 Under these conditions, the for-mation of the lactone side product was not observed. Finally,the hydroxyl group on C4 was protected with Fmoc to give 6and subsequently, the thioglycoside was transformed into thecorresponding α-glycosyl phosphate 2.With BB 1 and 2 in hand, we moved forward with the syn-thesis of our collection of oligosaccharides using AGA(Scheme 3). Merrifield resin functionalized with a photocleavablelinker was used as solid support.35 The BBs were coupled to thesolid support using TMSOTf-promoted glycosylation, employingone cycle and five equivalents of BB. Then, an acidic cappingsolution was used to acetylate the unreacted hydroxyl groupsand, thereby, minimizing deletion sequences.36Subsequently, the Fmoc carbonate was cleaved from thestructure, preparing the monosaccharide for the installation ofScheme 1 Retrosynthetic approach to obtain the glycan backbone ofPGN and the two BB required for the synthesis.Scheme 2 Synthesis of the MurNAc BB 2. Reagents and conditions: (a)(–)-ethyl (S)-2-trifluoromethylsulfonyloxy propionate, NaH, DMF/CH2Cl2, 0 °C → rt, 53%; (b) Me3N·BH3, BF3·OEt2, CH3CN, 0 °C → rt, 65%;(c) FmoCl, pyridine, CH2Cl2, rt, 93%; (d) (BuO)2P(O)OH, NIS, TfOH, 0 °C→ rt, 83%.Communication Organic & Biomolecular ChemistryOrg. Biomol. Chem. This journal is © The Royal Society of Chemistry 2021Open Access Article. Published on 25 October 2021. Downloaded on 11/25/2021 10:00:31 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinethe next unit. This cycle of coupling, capping, and deprotectionwas repeated until the desired sequence was achieved. OnceAGA was completed, the oligosaccharides were cleaved from theresin using UV light (365 nm) in a continuous flow photo-reactor.35 After purification of the desired oligosaccharides fromthe deletion sequences, the basic hydrolysis of the lactoyl estersto carboxylic acids was performed using LiOH in a solution ofTHF/dioxane/H2O.31 The remaining protecting groups werecleaved by hydrogenolysis with Pd/C and H2. Under these con-ditions, the TCA group was reduced to acetamide. This last stepwas more problematic, since after several days of hydrogenoly-sis, we still observed the presence of multiple chlorine atoms byMALDI. Therefore, for the hexamers were hydrogenated underhigh pressure to achieve complete conversion in 48 h. The rela-tively low yield is caused by the inefficiency of the photocleavagefrom the resin together with the rather low reactivity of thebuilding blocks, which led to the presence of deletionsequences. Finally, ten different PGN glycan backbones of alter-nating MurNAc and GlcNAc moieties, equipped with an amino-linker were obtained: the two monomers 8 and 9, two disac-charides 10 and 11, two trimers 12 and 13, two tetramers 14and 15, and two hexamers 16 and 17.ConclusionsTen oligosaccharide fragments of the PGN backbone weresynthesized. The use of a new MurNAc BB enabled the fastsynthesis of a collection of oligosaccharides up to hexamers.These oligosaccharides can be used to create glycan micro-arrays for the characterization of peptidoglycan directedantibodies. Advancing the idea from recently publishedapproaches, where molecules on microarrays were modifieddirectly on the glass slide after in situ synthesis37 or print-ing,38 we will follow a similar strategy. We envision tocouple different stem peptide variants from solution,forming the amide bond directly with the arrayed oligosac-charides. These arrays will support the identification of epi-topes recognized by anti-PGN antibodies, and can poten-tially help to close current knowledge gaps in autoimmunedisorders.Conflicts of interestThere are no conflicts to declare.Scheme 3 Automated glycan assembly of PGN oligosaccharides. Reagents and conditions: (a) 1 × 5 equiv. BB1, TMSOTf, CH2Cl2, −30 °C (5 min) →−15 °C (35 min); (b) 1 cycle of a 10% acetic anhydride and 2% methanesulfonic acid in CH2Cl2, 25 °C; (c) 3 cycles of 20% piperidine in DMF, 25 °C(5 min) (Module B); (d) CH2Cl2, hν (365 nm); (e) LiOH, THF/1,4-dioxane/H2O, 2–24 h; (f ) H2, Pd/C, H2O/HOAc, 16–48 h. 8: 27%; 9: 42%; 10: 20%; 11:10%; 12: 11%; 13: 9%; 14: 3%; 15: 8%; 16: 3%; 17: 2%.Organic & Biomolecular Chemistry CommunicationThis journal is © The Royal Society of Chemistry 2021 Org. Biomol. Chem.Open Access Article. Published on 25 October 2021. Downloaded on 11/25/2021 10:00:31 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineAcknowledgementsThe authors thank the German Federal Ministry of Educationand Research (BMBF, grant no. 13XP5050A), the Max-Planck-Fraunhofer Collaboration Project (Glyco3Display), and the MaxPlanck Society for financial support. In addition, the authorswould like to thank Martina Delbianco for the fruitful discus-sions and the members of the Department of BiomolecularSystems for the technical support, especially Eva Settels andOlaf Niemeyer. Open Access funding provided by the MaxPlanck Society.References1 J. V. Höltje, Microbiol. Mol. Biol. Rev., 1998, 62, 181–203.2 W. Vollmer, D. Blanot and M. A. de Pedro, FEMS Microbiol.Rev., 2008, 32, 149–167.3 J. H. Rogers, H. R. Perkins and J. B. Ward, Microbial CellWalls and Membranes, Springer, Netherlands, 1980.4 P. Schumann, in Methods in Microbiology, 2011, vol. 38, pp.101–129.5 O. Irazoki, S. B. Hernandez and F. Cava, Front. Microbiol.,2019, 10, 500.6 A. K. Yadav, A. Espaillat and F. Cava, Front. Microbiol.,2018, 9, 2064.7 M. A. Kohanski, D. J. Dwyer and J. J. Collins, Nat. Rev.Microbiol., 2010, 8, 423–435.8 S. E. Girardin, I. G. Boneca, L. A. M. Carneiro, A. Antignac,M. Jéhanno, J. Viala, K. Tedin, M.-K. Taha, A. Labigne,U. Zähringer, A. J. Coyle, P. S. D. Stefano, J. Bertin,P. J. Sansonetti and D. J. Philpott, Science, 2003, 300, 1584–1587.9 S. E. Girardin, L. H. Travassos, M. Hervé, D. Blanot,I. G. Boneca, D. J. Philpott, P. J. Sansonetti and D. Mengin-Lecreulx, J. Biol. Chem., 2003, 278, 41702–41708.10 K. L. Woodhams, J. M. Chan, J. D. Lenz, K. T. Hackett andJ. P. Dillard, Infect. Immun., 2013, 81, 3490–3498.11 M. A. Boudreau, J. F. Fisher and S. Mobashery,Biochemistry, 2012, 51, 2974–2990.12 A. Gonzalez-Santana and R. D. Heijtz, Trends Mol. Med.,2020, 26, 729–743.13 Z. Huang, J. Wang, X. Xu, H. Wang, Y. Qiao, W. C. Chu,S. Xu, L. Chai, F. Cottier, N. Pavelka, M. Oosting,L. A. B. Joosten, M. Netea, C. Y. L. Ng, K. P. Leong,P. Kundu, K.-P. Lam, S. Pettersson and Y. Wang, Nat.Microbiol., 2019, 4, 766–773.14 P. J. Shaw, M. J. Barr, J. R. Lukens, M. A. McGargill, H. Chi,T. W. Mak and T.-D. Kanneganti, Immunity, 2011, 34, 75–84.15 B. L. Jutras, R. B. Lochhead, Z. A. Kloos, J. Biboy, K. Strle,C. J. Booth, S. K. Govers, J. Gray, P. Schumann, W. Vollmer,L. K. Bockenstedt, A. C. Steere and C. Jacobs-Wagner, Proc.Natl. Acad. Sci. U. S. A., 2019, 116, 13498–13507.16 L. Visser, H. J. d. Heer, L. A. Boven, D. van Riel, M. vanMeurs, M.-J. Melief, U. Zähringer, J. van Strijp,B. N. Lambrecht, E. E. Nieuwenhuis and J. D. Laman,J. Immunol., 2005, 174, 808–816.17 I. A. Schrijver, M. van Meurs, M.-J. Melief, C. W. Ang,D. Buljevac, R. Ravid, M. P. Hazenberg and J. D. Laman,Brain, 2001, 124, 1544–1554.18 W. G. Branton, J. Q. Lu, M. G. Surette, R. A. Holt, J. Lind,J. D. Laman and C. Power, Sci. Rep., 2016, 6, 37344.19 J. D. Kriesel, P. Bhetariya, Z.-M. Wang, D. Renner,C. Palmer and K. F. Fischer, Sci. Rep., 2019, 9, 1387.20 N. Wang, A. Hirata, K. Nokihara, K. Fukase andY. Fujimoto, Pept. Sci., 2016, 106, 422–429.21 S. Inamura, K. Fukase and S. Kusumoto, Tetrahedron Lett.,2001, 42, 7613–7616.22 S. Inamura, Y. Fujimoto, A. Kawasaki, Z. Shiokawa,E. Woelk, H. Heine, B. Lindner, N. Inohara, S. Kusumotoand K. Fukase, Org. Biomol. Chem., 2006, 4, 232–242.23 N. Wang, H. Hasegawa, C.-Y. Huang, K. Fukase andY. Fujimoto, Chem. – Asian J., 2017, 12, 27–30.24 M. Lee, D. Hesek, D. A. Dik, J. Fishovitz, E. Lastochkin,B. Boggess, J. F. Fisher and S. Mobashery, Angew. Chem.,Int. Ed., 2017, 56, 2735–2739.25 O. J. Plante, E. R. Palmacci and P. H. Seeberger, Science,2001, 291, 1523–1527.26 P. H. Seeberger, Acc. Chem. Res., 2015, 48, 1450–1463.27 M. Guberman and P. H. Seeberger, J. Am. Chem. Soc., 2019,141, 5581–5592.28 T. Tyrikos-Ergas, V. Bordoni, G. Fittolani, M. A. Chaube,A. Grafmüller, P. H. Seeberger and M. Delbianco, Chem. –Eur. J., 2021, 27, 2321–2325.29 J. Kandasamy, F. Schuhmacher, H. S. Hahm, J. C. Kleinand P. H. Seeberger, Chem. Commun., 2014, 50, 1875–1877.30 L. Kröck, D. Esposito, B. Castagner, C.-C. Wang,P. Bindschändler and P. H. Seeberger, Chem. Sci., 2012, 3,1617–1622.31 R. Enugala, M. J. D. Pires and M. M. B. Marques,Carbohydr. Res., 2014, 384, 112–118.32 M. P. DeNinno, J. B. Etienne and K. C. Duplantier,Tetrahedron Lett., 1995, 5, 669–672.33 W. Kinzy and R. R. Schmidt, Liebigs Ann. Chem., 1987, 407–415.34 M. Oikawa, W.-C. Liu, Y. Nakai, S. Koshida, K. Fukase andS. Kusumoto, Synlett, 1996, 1179–1180.35 S. Eller, M. Collot, J. Yin, H. S. Hahm and P. H. Seeberger,Angew. Chem., Int. Ed., 2013, 52, 5868–5861.36 Y. Yu, A. Kononov, M. Delbianco and P. H. Seeberger,Chem. – Eur. J., 2018, 24, 6075–6078.37 M. Mende, A. Tsouka, J. Heidepriem, G. Paris, D. S. Mattes,S. Eickelmann, V. Bordoni, R. Wawrzinek,F. F. Fuchsberger, P. H. Seeberger, C. Rademacher,M. Delbianco, A. Mallagaray and F. F. Loeffler, Chem. – Eur.J., 2020, 26, 9954–9963.38 C. Ruprecht, M. P. Bartetzko, D. Senf, A. Lakhina,P. J. Smith, M. J. Soto, H. Oh, J.-Y. Yang, D. Chapla,D. V. Silva, M. H. Clausen, M. G. Hahn, K. W. Moremen,B. R. Urbanowicz and F. Pfrengle, Angew. Chem., Int. Ed.,2020, 59, 12494–12498.Communication Organic & Biomolecular ChemistryOrg. Biomol. Chem. This journal is © The Royal Society of Chemistry 2021Open Access Article. Published on 25 October 2021. Downloaded on 11/25/2021 10:00:31 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online

https://refubium.fu-berlin.de/bitstream/fub188/33029/1/d1ob01987b.pdf

Automated glycan assembly of peptidoglycan backbone fragments

Abstract

Similar works

Full text

Available Versions

ARTS repository - University of Groningen

NARCIS

ARTS repository - University of Groningen

University of Groningen

Institutional Repository of the Freie Universität Berlin

University of Groningen