Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.A GC-MS method for the rapid and unambiguous identification of the amino acid labionin (Lab) occurring in type-III lantibiotics is presented. This method will constitute a valuable tool for the characterisation and structure elucidation of labyrinthopeptins and their differentiation from lanthionine-type lantibiotics

Henkel, Maik

Pesic, Alexander

Süßmuth, Roderich D.

Chemical Communications

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This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 7401–7403 7401Cite this: Chem. Commun., 2011, 47, 7401–7403Identiﬁcation of the amino acid labionin and its desulfurised derivative inthe type-III lantibiotic LabA2 by means of GC/MSwAlexander Pesic,z Maik Henkelz and Roderich D. Su¨ssmuth*Received 18th March 2011, Accepted 19th April 2011DOI: 10.1039/c1cc11573aA GC-MS method for the rapid and unambiguous identiﬁcationof the amino acid labionin (Lab) occurring in type-III lantibioticsis presented. This method will constitute a valuable tool for thecharacterisation and structure elucidation of labyrinthopeptinsand their diﬀerentiation from lanthionine-type lantibiotics.Lantibiotics are ribosomally synthesised peptide antibioticscontaining the thioether amino acids (2S,6R)-meso-lanthionine(Lan, 1) and/or (2S,3S,6R)-3-methyl-lanthionine (MeLan, 2).1As further structural modiﬁcations lantibiotics may containa,b-unsaturated amino acids such as 2,3-didehydroalanine(Dha, 3) and 2,3-didehydroaminobutyric acid (Dhb, 4).Lantibiotics are produced by a wide range of Gram-positivebacteria, e.g. Staphylococci and Lactococci, and mostly haveantimicrobial activity against other Gram-positive bacteria.Recently, we identiﬁed a new structural type of lantibiotics,named labyrinthopeptins, which are produced by the Actino-mycete Actinomadura namibiensis DSM6313.2 The structuremodel of Labyrinthopeptin A2 (LabA2, 6a) showed thepresence of an unusual a,a-disubstituted amino acid, namedlabionin (Lab, 5). From X-ray data it was deduced that Lab(5) has (2S,4S,8R)-conﬁguration. Lab (5) can be structurallyconsidered as a triamino triacid consisting of a basic lanthioninemotif, which is further extended at the quaternary C-a-atomby a methylene bridge and an a-amino acyl residue, thusenabling the formation of two ring systems within a peptidechain. From the sequence of labyrinthopeptin prepropeptidesit was deduced that a Ser/Ser/Cys motif is the precursor of Lab(5). Searches in databases containing genomic data showedthat gene clusters with similar architecture and high sequencehomology to the labyrinthopeptin gene cluster are widespreadamong Actinomycetes, e.g. Streptomyces coelicolor, S. avermitilis,and Saccharopolyspora erythraea. Based on those similaritieswe assume that they are producers of labyrinthopeptin-typelantibiotics. It is particularly interesting that their lantibioticprecursor peptides also contain the Ser/Ser/Cys motif, whichcould be a precursor of the Lab (5) motif (Fig. 1).However, structure elucidation of labyrinthopeptins as wellas of related type-III lantibiotics from abovementionedbacterial producers is challenging. The high cyclisation degreeof LabA2 (6a) poses signiﬁcant problems to mass spectro-metric sequencing. This is aggravated by two possiblealternative isobaric posttranslational modiﬁcations Dha+Lan(3+1) and Lab (5) in type-III lantibiotics. Furthermore,structure elucidation of labyrinthopeptins by NMR spectro-scopy is hampered by aggregation-prone properties.2 In thiscontext GC-MS appears as a very useful technique for thedetection, quantitation and structure elucidation of buildingblocks or modiﬁcations of peptidic natural products, e.g.amino acids, carbohydrates and lipids. Amino acids arecommonly derivatised as N-triﬂuoroacetyl or N-pentaﬂuoro-phenyl amino acid esters.3 The analytics of building blockswith higher molecular mass by GC-MS however is aggravatedby a decrease in volatility of the derivatives with increasingmolecular mass. Polar functional groups require additionalFig. 1 (A) Structures of the amino acids Lan (1), MeLan (2), Dha (3)and Dhb (4) characteristic of lantibiotics. Amino acid Lab (5) inLabA2 is characteristic for recently discovered type-III lantibiotics ofthe labyrinthopeptin-type. (B) Schemes of LabA2 (6a) and desulfo-LabA2 (6b). (C) Scheme of hydrolysis and microderivatisation forsubsequent GC/MS analysis of Lab (7) and a-methyl-2,4-diamino-glutaric acid (Dmg, 9): N-triﬂuoroacetyl/ethyl ester derivatisation to 8and 10 by (i) 6N HCl; 24 h; 110 1C, (ii) reductive desulfurisation withNiCl2/NaBH4; 6N HCl; 24 h; 110 1C, (iii, iv) AcCl; EtOH; TFAA.Technische Universita¨t Berlin, Institut fu¨r Chemie, Strasse des 17,Juni 124, 10623 Berlin, Germany.E-mail: suessmuth@chem.tu-berlin.de; Fax: +49 30 314 79651;Tel: +49 30 314 78774w Electronic supplementary information (ESI) available: MS data andsynthesis of amino acid standard. See DOI: 10.1039/c1cc11573az Both authors contributed equally to this work.ChemComm Dynamic Article Linkswww.rsc.org/chemcomm COMMUNICATIONPublished on 23 May 2011. Downloaded by TU Berlin - Universitaetsbibl on 31/03/2016 14:56:33. View Article Online / Journal Homepage / Table of Contents for this issue7402 Chem. Commun., 2011, 47, 7401–7403 This journal is c The Royal Society of Chemistry 2011derivatisations exhausting the limits of GC-MS as an analyticalmethod.The identiﬁcation of the amino acid Lan (1) and MeLan (2)in lantibiotics by means of GC/MS is known for several years,4in contrast, no analytical protocols were available for thedetection of recently discovered triamino triacid Lab (7).In this contribution for the ﬁrst time we report on theidentiﬁcation of the amino acid Lab (7) and its desulfurisedderivative acid a-methyl-2,4-diaminoglutaric acid (Dmg, 9)from lantibiotics of the labyrinthopeptin-type by means ofGC-PCI-MS (PCI = Positive Chemical Ionisation). Thisachievement provides rapid access to an unambiguousidentiﬁcation of Lab (7) and analogues and will ultimatelyassist in the assignment of unknown lantibiotics to distinctlantibiotic classes.We sought to develop robust protocols for a diﬀerentiationbetween Lan/MeLan (1/2) and Lab (5). Therefore, totalhydrolysis of the lantibiotic nisin as a model system wasperformed and the hydrolysate was derivatised as N-triﬂuoro-acetyl amino acid ethylesters. Detection of Lan/MeLan (1/2)by means of GC-PCI-MS in Nisin (ESIw, 25, Fig. S7 and S10)was established according to previous reports.4 In the samemanner we subsequently performed total hydrolysis of LabA2(6a) and derivatisation of the hydrolysate. As expected, thesignals of the corresponding N-triﬂuoroacetyl/ethyl esterscould be identiﬁed and assigned to Ala, Gly, Thr, Leu, Asp,Glu, Phe and Trp with the exception of (Cys)2 (Fig. 2A).Additionally the GC-MS chromatogram revealed a signal atRt = 36.3 min (Fig. 2B) assigned to derivatised Lab (8). Thiswas corroborated by indicative molecular ions [M + H]+ =668 Da and [M + C2H5]+ = 696 Da of derivatised Lab (8)under mild GC-PCI-MS conditions (Fig. 2C) and assignmentof characteristic ion fragments (Fig. S11, ESIw). These ionsshow characteristic fragmentations, which strongly depend onthe structure of analysedN-triﬂuoroacetyl amino acid ester.5 TheXIC for [M + H]+ = 668 Da (Fig. 2B) revealed also a peak atRt = 35.9 min with identical mass spectrum (data not shown) asforRt = 36.3 min (Fig. 2C), which suggests epimerization of Labas a likely explanation for this observation.The relative signal intensities in the GC-MS compared toother amino acid constituents were signiﬁcantly reduced andseveral other compounds eluting from the column could not beassigned (Fig. 2A).In order to give additional proof on the occurrence andthe assignment of Lab (7), degradation of LabA2 (6a) bydesulfurisation of thioethers was applied as previously reportedfor subtilosin A6 using NiCl2/NaBH4 treatment under retentionof the stereochemistry.7 Progress of desulfurisation wasmonitored by LC-ESI-MS and the reaction was completedwithin 24 hours. Fig. 3 shows the HR-ESI(+)-orbitrap massspectra of LabA2 (6a, in black) before [(M+ 2H)2+= 962 Da]and after (6b, in red) desulfurisation [(M + 2H)2+ = 931 Da]corresponding to a formal loss of two sulfur atoms and formaladdition of four hydrogen atoms. Under the reaction conditionsapplied, the integrity of cystine remained untouched.From the progress of the desulfurisation monitored byLC-MS transformation of Lab (7) into a-methyl-2,4-diamino-glutaric acid (Dmg, 9) and Ala was expected. Conversionof the hydrolysate of desulfo-LabA2 (6b) into theN-triﬂuoroacetyl/ethyl esters and subsequent GC-MS analyticsgave a new signal at Rt = 20.9 min (Fig. 4A and 5B) whereasthe previous signal from derivatised Lab (8) had disappeared.Trp was not detected after desulfurisation (Fig. 4A) ofLabA2 (6a) whereas several other compounds eluted fromthe column which could not be identiﬁed. Nevertheless,characteristic molecular ions for Dmg derivative (10) (Rt =20.9 min), [M +H]+ = 425 Da and [M+ C2H5]+ = 453 Dashown in Fig. 4B and characteristic mass fragments wereassigned (Fig. S12, ESIw).Our intention was to independently verify the identity ofDmg (9) by synthesis of the racemate according to Scheme 1.Synthetic access was obtained by Claisen-type rearrangementreaction of Cbz-L-alanine-allylester (13) to Cbz-a-allyl-alanineas the central step.8 Subsequent to this step a Sharplessdihydroxylation9 of the allyl moiety (15) and selective protectionof the primary alcohol with TBDMS (17a/b) was performed.By a Mitsunobu reaction an azide group was introducedand further steps comprised TBDMS deprotection, oxidationof the resulting alcohol, removal of protecting groups, hydro-genolysis and N-triﬂuoroacetyl/ethylester derivatisation whichFig. 2 (A) GC-MS chromatogram of the hydrolysate of LabA2 (6a)(6N HCl, 24 h) after N-triﬂuoroacetyl/ethylester derivatisation.(B) Enlarged section of (A) Lab (8) (TIC in black and normalisedXIC [M + H]+ = 668 Da in red). (C) PCI mass spectrum ofN-triﬂuoroacetyl/ethylester of Lab (8) at Rt = 36.3 min. Peaks markedwith asterisk (*) are unidentiﬁed byproducts.Fig. 3 HR-ESI(+)-orbitrap mass spectra of LabA2 before desulfuri-sation (black, 6a) [(M + 2H)2+ = 962 Da] and after desulfurisation(red, 6b) [(M + 2H)2+ = 931 Da].Published on 23 May 2011. Downloaded by TU Berlin - Universitaetsbibl on 31/03/2016 14:56:33. View Article OnlineThis journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 7401–7403 7403ﬁnally led to synthetic Dmg (23a/b). The stereochemistry ofsynthetic Dmg diastereomers (23a/b) was afterwardsdeduced by comparison of the retention time of Dmg (10).Fig. 5(A) shows the GC-MS analysis of synthetic 23a/b.The gradient allowed to separate the diastereomeric pairs23b at Rt = 20.6 min and 23a at Rt = 20.9 min,respectively. As expected both pairs showed identicalfragmentations patterns under PCI-MS conditions (23a/b,Fig. S8, ESIw) compared to the natural product derivedcompound Dmg (10). The identity in retention time (Fig. 5Aand B) and fragmentation patterns of synthetic referencecompounds 23a unequivocally proved the identity of thisamino acid as Dmg (10, Fig. 4B and 5B).The derivatisation protocol combined with GC-MS analysisdeveloped for Lab (7)/Dmg (9) was applied to the analysisof Labyrinthopeptin A1 (LabA1) and desulfurised LabA1(ESIw, 24a/b, Fig. S5 and S13), respectively. LabA1 isa member of the labyrinthopeptin family, for which thestructure has mainly been derived from genomic data of thebiosynthesis gene cluster.2 In accordance to the analytics ofLabA2 (6a/b) the GC-MS data of LabA1 (ESIw, 24a/b,Fig. S5, S9, and S13) also could unambiguously prove thepresence of Lab (7) and Dmg (9) for the ﬁrst time.In summary, we have established a protocol for thedetection of the unusual triamino triacid Lab (7) by GC/MSfrom LabA2 hydrolysates. Furthermore, the above strategycombined with NiCl2/NaBH4-mediated desulfurisation ofLab-containing type-III lantibiotics can be independentlyapplied for GC-MS analysis of derivative Dmg (9). Sub-sequently, this protocol was successfully applied to thedetection of Lab (7) and Dmg (9) from labyrinthopeptinLabA1 and desulfurised LabA1 (ESIw, 24a/b, Fig. S13),respectively. The detection of Lab (7) and Dmg (9) comple-ments the presently available methods for the analytics oflantibiotics. Our future attempts will extend on other type-IIIlantibiotics to prove the occurrence of Lab (7) (ESIw, 24a,Fig. S13) in these lantibiotics and we hypothesise that Lab (7)could be considered as a common structural motif characteristicfor many type-III lantibiotics.The work was supported by a research grant from theDeutsche Forschungsgemeinschaft (SU239/8-1).Notes and references1 J. M. Willey and W. A. van der Donk, Annu. Rev. Microbiol., 2007,61, 447.2 (a) K. Meindl, T. Schmiederer, K. Schneider, A. Reicke, D. Butz,S. Keller, G. Nicholson, H. Gu¨hring, L. Ve´rtesy, J. Wink,H. Hoﬀmann, M. Broenstrup, G. M. Sheldrick and R. D.Su¨ssmuth, Angew. Chem., Int. Ed., 2010, 49, 1151; (b) W. M.Mu¨ller, T. Schmiederer, P. Ensle and R. D. Su¨ssmuth, Angew.Chem., Int. Ed., 2010, 49, 2436.3 H. Frank, G. J. Nicholson and E. Bayer, J. Chromatogr., A, 1978,167, 187.4 (a) E. Ko¨sters, H. Allgaier, G. Jung and E. Bayer, Chromatographia,1984, 18, 287; (b) B. Li, D. Sher, L. Kely, Y. Shi, K. Huang,P. J. Knerr, I. Joewono, D. Rusch, S. W. Chisholm and W. A. vander Donk, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 10430.5 A. Prox and J. Schmid, Org. Mass Spectrom., 1969, 2, 105.6 E. K. Kawulka, T. Sprules, C. M. Diper, R. M. Whittal,T. R. McKay, P. Mercier, P. Zuber and J. C. Vederas, Biochemistry,2004, 43, 3385.7 T. G. Back, D. L. Baron and K. J. Yang, J. Org. Chem., 1993, 58,2407.8 U. Katzmaier and S. Maier, Tetrahedron, 1996, 52, 941.9 H. C. Kolb, M. S. van Nieuwenhze and K. B. Sharpless,Chem. Rev.,1994, 94, 2483.Fig. 4 (A) GC-MS chromatogram of the desulfo-LabA2 (6b)hydrolysate after N-triﬂuoroacetyl/ethylester derivatisation. (B) PCImass spectrum of the N-triﬂuoracetyl/ethylester of Dmg (10) at Rt =20.9 min.Fig. 5 GC-MS analytics. (A) Synthetic diastereomers of Dmg(23a/b) (TIC in black and normalised XIC [M + H]+ = 425 Dain red). (B) Dmg (10) from desulfurised hydrolysate of LabA2 (6b)(TIC in black and normalised XIC [M+H]+= 425 Da in red). Peaksmarked with asterisk (*) are undeﬁned byproducts.Scheme 1 Synthesis of Dmg (23a/b) as a standard for assignment ofLab derivatives after desulfurisation, hydrolysis and N-triﬂuoroacetyl/ethylester derivatisation. (i) LDA; ZnCl2, (ii) BF3*OEt; TBTA,(iii) AD-Mix-b, (iv) TBDMSCl; imidazol, (v) PPh3; DEAD; DPPA,(vi) TBAF, (vii) TEMPO; KBr; NaOCl, (viii) TFA; TES, (ix) Pd/C;H2, (x) AcCl; EtOH; TFAA.Published on 23 May 2011. Downloaded by TU Berlin - Universitaetsbibl on 31/03/2016 14:56:33. View Article Online

Identification of the amino acid labionin and its desulfurised derivative in the type-III lantibiotic LabA2 by means of GC/MS

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Identification of the amino acid labionin and its desulfurised derivative in the type-III lantibiotic LabA2 by means of GC/MS

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