Myxoprincomide, a secondary metabolite of the myxobacterium
Myxococcus xanthus DK 1622, is synthesised for the first time. The
central, unusual α-ketoamide is generated at the end of the synthesis to avoid side reactions during the synthesis of this rather
reactive subunit. Nevertheless, the synthetic natural product is
obtained as an isomeric mixture. Detailed analytical investigations
show that the identical isomeric mixture is found in the isolated
natural product

Dastbaz, Jan

Kazmaier, Uli

Kohr, Michael

Müller, Rolf

Walt, Christine

Organic & Biomolecular Chemistry

English

Acronym

Organic &Biomolecular ChemistryCOMMUNICATIONCite this: Org. Biomol. Chem., 2022,20, 9609Received 4th November 2022,Accepted 18th November 2022DOI: 10.1039/d2ob02021arsc.li/obcTotal synthesis of Myxoprincomide, a secondarymetabolite from Myxococcus xanthus†Michael Kohr,a Christine Walt,b Jan Dastbaz,b Rolf Müller b and Uli Kazmaier *a,bMyxoprincomide, a secondary metabolite of the myxobacteriumMyxococcus xanthus DK 1622, is synthesised for the first time. Thecentral, unusual α-ketoamide is generated at the end of the syn-thesis to avoid side reactions during the synthesis of this ratherreactive subunit. Nevertheless, the synthetic natural product isobtained as an isomeric mixture. Detailed analytical investigationsshow that the identical isomeric mixture is found in the isolatednatural product.Myxobacteria, Gram-negative δ-proteobacteria, widespread innature, are highly efficient producers of natural products.1Myxococcus xanthus DK 1622, the microbe of the year 2020,was the first myxobacterial strain whose complete genomesequence was published in 2006.2 With 9.8 million base pairs,this extremely large bacterial genome contains at least 18 bio-synthetic gene clusters encoding for polyketides, nonriboso-mal peptides or hybrids of both.3 Still, interestingly, only fivecompound classes can be isolated and characterised. Thisindicates that there are yet unassigned pathways, where nonatural products could be related with so far.4 Moderngenome mining techniques, combining targeted mutagenesis,liquid chromatography coupled with HRMS, and statisticaldata analysis identified Myxoprincomide as a new secondarymetabolite from Myxococcus xanthus DK 1622.5The linear nonapeptide contains some unusual buildingblocks (red in Fig. 1), such as β-hydroxylated valine, β-lysineand an α-keto-β-amino acid derived from valine. The tiny, iso-lated amounts allow for the structure elucidation of a newnatural product, but only a few investigations of the biologicalactivities of this compound have been conducted.6Because of our interest in the total synthesis of natural pro-ducts, we began working on the synthesis of Myxoprincomideto verify the proposed structure and obtain enough material inhand for further biological studies. The most remarkablebuilding block is the central α-keto-β-amino acid, whose bio-synthesis has yet to be explained. Similar building blocks arefound in a series of mainly cyclic peptides such as jamaicens-amide A,7 orbiculamide A 8 and the keramamides.9Furthermore, the α-ketoamino acid is presumably the mostcritical subunit in the molecule. In addition, the adjacentβ-stereogenic centre should be somewhat acidic and sensitiveto epimerisation. Similarly, the α-keto functionality should berelatively reactive, undergoing additions such as hydrate for-Fig. 1 Myxoprincomide.Scheme 1 Synthesis of α-hydroxy-β-amino acid 5.†Electronic supplementary information (ESI) available: Experimental details,compound characterization, copies of 1H and 13C NMR spectra, HPLC chromato-grams and MS2 analysis. See DOI: https://doi.org/10.1039/d2ob02021aaOrganic Chemistry, Saarland University, D-66123 Saarbrücken, Germany.E-mail: u.kazmaier@mx.uni-saarland.debDepartment Microbial Natural Products, Helmholtz Institute for PharmaceuticalResearch Saarland (HIPS), Helmholtz Centre for Infection Research (HZI), andDepartment of Pharmacy, Saarland University, D-66123 Saarbrücken, GermanyThis journal is © The Royal Society of Chemistry 2022 Org. Biomol. Chem., 2022, 20, 9609–9612 | 9609Open Access Article. Published on 18 November 2022. Downloaded on 6/12/2023 8:19:29 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journal  | View Issuemation.10 Therefore, we decided to generate the α-keto-func-tionality near the end of the synthesis by oxidation of thecorresponding α-hydroxy acid.In our work, we obtained the desired N-Boc-protectedderivative from Boc-valinal 1 by adding NaCN to provide cyan-hydrine 2 as a 1 : 1 diastereomeric mixture (Scheme 1). Theα-hydroxy nitrile 2 was converted into the α-hydroxy ester 3using acetylchloride in MeOH. Under these conditions, theBoc-protecting group was removed but reintroduced in thenext step. Finally, the saponification of the methyl ester pro-vided the free carboxylic acid 5, which could be used in thepeptide synthesis. The other unusual amino acids were pre-pared according to procedures in the literature or purchased.11The synthesis of the linear peptide chain, as shown inScheme 2, started with a prolongation of L-ornithine 6 12 via anArndt–Eistert homologation to generate the required β-lysine.Scheme 2 Synthesis of myxoprincomide.Communication Organic & Biomolecular Chemistry9610 | Org. Biomol. Chem., 2022, 20, 9609–9612 This journal is © The Royal Society of Chemistry 2022Open Access Article. Published on 18 November 2022. Downloaded on 6/12/2023 8:19:29 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineThe activation of 6 as a mixed anhydride and reaction with dia-zomethane provided diazoketone 7, with only traces ofornithine methyl ester as a side product. This issue was notconcerning since the diazoketone could be easily purified byflash chromatography. The diazoketone 7 could directly bereacted with alanine benzyl ester in the presence of silver ben-zoate to the desired dipeptide 8 in overall high yield. Next, thecoupling with the following two protected amino acids pro-ceeded under standard conditions. Tyrosine was used with anallyl-protecting group on the phenolic OH group to avoid sidereactions in the final oxidation step. The allyl group couldeasily be removed later using CpRu(MeCN)3PF6 as a catalyst.13The subsequent coupling with α-hydroxy acid 5 proceededwithout problems, and no O-protecting group was required.The final peptide couplings were performed using TBTU,which provided the best yields.The oxidation of the α-hydroxy acid subunit in 15 was per-formed using Dess Martin periodinane. The reaction had to becarried out in DMSO because of the insolubility of 15 in di-chloromethane, which is generally used in Dess Martin oxi-dations.14 Next, the O-allyl protecting group was removed fromthe tyrosine using the above-mentioned ruthenium complex.Finally, the benzyl-protecting groups were removed by catalytichydrogenation. Of note, we had to carry out the reaction in amixture of chloroform, ethyl acetate and methanol. Withoutchloroform, only uncomplete conversion was observed, as wellas a partial reduction of the α-keto group. Under the optimisedconditions, myxoprincomide could be obtained in high yield,sufficient for biological studies (Scheme 2).The characterisation and identification of the naturalproduct were not as trivial as expected. The NMR spectrashowed several sets of signals which could be partly explainedby the existence of rotamers since some sets disappeared at ahigher temperature. Nevertheless, the synthesised productcontained two ‘side products’, also confirmed by UPLC. Thepurification of myxoprincomide by preparative HPLC and rea-nalysis indicated that the three products are evidently in equi-librium and converting into each other. A detailed analysis ofthe NMR spectra of the isolated natural product clearly showedthat the same side products could also be found there.The minor signals can be likely explained by an epimerisa-tion of the α-keto amide or the formation of a hydrate at theketo functionality, but a clear identification was not possible.Therefore, we also performed MS measurements, which gaveidentical fragmentation patterns for the isolated and syn-thesised natural product (see ESI†).Although most 1H NMR signals fit almost perfectly, somesignals showed differences, especially for protons close to theamino group of the β-lysine incorporated. This differencemight be caused by a different pH or the water content of theNMR solvent. This phenomenon is common with such ahighly polar natural product containing basic side chains.With the synthetic myxoprincomide in hand, we carried outmany bioactivity assays, investigating the IC50 values againstseveral bacteria, fungi and cancer cell lines. Unfortunately, nosignificant activity was observed in any of these assays.Therefore, the ecological function of this secondary metaboliteof Myxococcus xanthus remains elusive.ConclusionsWe developed the first total synthesis of the myxobacterialnatural product myxoprincomide with the longest linearsequence of 19 steps and in 16% overall yield. The unusualα-ketoamide as the central part of the molecule was generatedat the end of the peptide synthesis to avoid side reactions atthis subunit. The natural product itself was obtained as amixture of isomers which equilibrate. Detailed investigationsindicate that the same isomers are also found in the isolatednatural product. Unfortunately, no significant biologicalactivity of myxoprincomide could be observed.Conflicts of interestThere are no conflicts to declare.AcknowledgementsFinancial support from Saarland University and the DFG(Bruker Neo 500 – 447298507) is gratefully acknowledged. Wealso thank Dr Susanne Kirsch-Dahmen for her biologicalstudies.References1 (a) H. Reichenbach, J. Ind. Microbiol. Biotechnol., 2001, 27,149–156; (b) R. O. Garcia, D. Krug and R. Müller, MethodsEnzymol., 2009, 458, 59–91; (c) K. J. Weissman andR. Müller, Nat. Prod. Rep., 2010, 27, 1276–1295.2 B. S. Goldman, W. C. Nierman, D. Kaiser, S. C. Slater,A. S. Durkin, J. Eisen, C. M. Ronning, W. B. Barbazuk,M. Blanchard, C. Field, C. Halling, G. Hinkle, et al., Proc.Natl. Acad. Sci. U. S. A., 2006, 103, 15200–15205.3 D. E. Whitworth, Myxobacteria: Multicellularity andDifferentiation, ASM Press, Chicago, 2007.4 (a) H. B. Bode and R. Müller, Angew. Chem., 2005, 117,6988–7007, (Angew. Chem., Int. Ed., 2005, 44, 6828–6846);(b) H. B. Bode, P. Meiser, T. Klefisch, N. S. Cortina,D. Krug, A. Göhring, G. Schwär, T. Mahmud, Y. A. Elnakadyand R. Müller, ChemBioChem, 2007, 8, 2139–2144;(c) S. C. Wenzel, P. Meiser, T. Binz, T. Mahmud andR. Müller, Angew. Chem., 2006, 118, 2354–2360, (Angew.Chem., Int. Ed., 2006, 45, 2296–2301); (d) V. Simunovic,J. Zapp, S. Rachid, D. Krug, P. Meiser and R. Müller,ChemBioChem, 2006, 7, 1206–1220; (e) P. Meiser,K. J. Weissman, H. B. Bode, D. Krug, J. S. Dickschat,A. Sandmann and R. Müller, Chem. Biol., 2008, 15, 771–781.Organic & Biomolecular Chemistry CommunicationThis journal is © The Royal Society of Chemistry 2022 Org. Biomol. Chem., 2022, 20, 9609–9612 | 9611Open Access Article. Published on 18 November 2022. Downloaded on 6/12/2023 8:19:29 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online5 N. S. Cortina, D. Krug, A. Plaza, E. Revermann andR. Müller, Angew. Chem., 2012, 124, 836–841, (Angew.Chem., Int. 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Evindar, D. Fishlock and G. A. Lajoie,Tetrahedron Lett., 2001, 42, 3807–3809.12 G. Pattenden and T. Thompson, Chem. Commun., 2001,717–718.13 S. Tanaka, H. Saburi, Y. Ishibashi and M. Kitamura, Org.Lett., 2004, 6, 1873–1875.14 D. B. Dess and J. C. Martin, J. Org. Chem., 1983, 48, 4155–4156.Communication Organic & Biomolecular Chemistry9612 | Org. Biomol. Chem., 2022, 20, 9609–9612 This journal is © The Royal Society of Chemistry 2022Open Access Article. Published on 18 November 2022. Downloaded on 6/12/2023 8:19:29 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online

Total synthesis of Myxoprincomide, a secondary metabolite from Myxococcus xanthus

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Total synthesis of Myxoprincomide, a secondary metabolite from Myxococcus xanthus

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