Peptaibols are oligomers, containing non-proteogenic amino acids. Some of them are considered as antifungal and antibiotic agents, therefore the elucidation of their structure is important. There have been a lot of efforts determining their amino acid sequences via mass spectrometry, but isobaric components cannot be determined using this technique. In this work, a HPLC method development was carried out for the determination of leucine, isoleucine, valine and isovaline in one chromatographic run. Furthermore, a chiral separation of the optical isomers of these compounds has been also solved

Endre Gábor

Kredics László

Marik Tamás

Papp Dóra

Szekeres András

Varga Mónika

Vágvölgyi Csaba

University of Szeged

 24th International Symposium on Analytical and Environmental Problems 283 DETERMINATION OF VALINE AND LEUCINE ISOMERS IN PEPTAIBOLS  Gábor Endre1,2, Tamás Marik1, László Kredics1 Mónika Varga1, Csaba Vágvölgyi1, András Szekeresa  1University of Szeged, Faculty of Science and Informatics, Departement of Microbiology, Közép fasor 52. Szeged H-6726 2Doctoral School in Biology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary e-mail: egabcy@gmail.com  Abstract Peptaibols are oligomers, containing non-proteogenic amino acids. Some of them are considered as antifungal and antibiotic agents, therefore the elucidation of their structure is important. There have been a lot of efforts determining their amino acid sequences via mass spectrometry, but isobaric components cannot be determined using this technique. In this work, a HPLC method development was carried out for the determination of leucine, isoleucine, valine and isovaline in one chromatographic run. Furthermore, a chiral separation of the optical isomers of these compounds has been also solved.  Introduction Peptaibols are peptid-like oligomers produced as extracellular secondary metabolites by Trichoderma species. Some peptaibols function as antibiotic and antifungal agents [1]. They are amphiphilic, which allows them to form voltage-dependent ion channels in cell membranes, making them leak and leading to the cell death. These compounds contain non-coded, non-proteinogeic amino acids, like α-aminoisobutyric acid, ethylnorvaline, isovaline and hydroxyproline. Their N-terminus is acetylated, and the C-terminal amino acid is hydroxylated into an acid alcohol [2]. Due to their diverse nature, the structure elucidations of newly discovered peptaibols are important. There have been a lot of efforts to determine their amino acid sequence by mass spectrometry [3-5], however, isobaric amino acids could be specified, and no configuration of the compounds could be observed. To apply liquid chromatography, problems could be solved-, e.g. isobaric amino acids could be separated and detected, as well as the D-, and L-isomers could be distinguished due to the proper chiral derivatisation. For the chiral determination of the desired amino acids, Marfey’s reagent can be used. The reaction was carried out by adding the chiral reagent into the amino acid sample forming two products, depending on the configuration of the connecting amino acid. When the reagent is reacting with an L-amino acid, the product could form an intramolecular H-bond that results in a ring-like system. In the case of D-amino acids reaction partners, another product occurs, that cannot form any H-bonds intramolecularly. This slight difference between these two products leaded to their baseline separation on the applied chromatographic column.  Experimental Trichoderma pleuroti (SZMC 12454) was cultivated on solid medium. Peptaibols were extracted using CHCl3/MeOH = 3/1 and the crude extract was separated on a normal phase column in a CHCl3/MeOH gradient. One selected fraction was evaporated and hydrolysed in cc. HCl overnight, to release the amino acids. After setting the pH to 7, the achiral and chiral derivatisations were applied. Derivatising agents, amino acid standards and other chemicals were purchased from Sigma Aldrich Inc. (Saint Louis, MO, USA). The achiral derivatisation of amino acids was done by  24th International Symposium on Analytical and Environmental Problems 284 adding 125 µl of borate buffer (pH = 10.2) to 50 µl of amino acid solution. After mixing vigorously, 25 µl of OPA solution and 20 µl of FMOC solution were added. The solution was diluted with 780 µl HPLC water and mixed. The resulted solution was injected into a HPLC-UV system. Chiral derivatisation of amino acids was done by Marfey’s reagent (Thermo Fischer Sicentific, Waltham, MA, USA). To 50 µl of amino acid solution, 100 µl of Marfey’s reagent’s solution (1 w% in acetone) and 20 µl solution of NaHCO3 (1 M) were added. After mixing, the reaction mixture was thermostated at 35°C for 60 minutes, which was followed by neutralisation by 10 µl of HCl solution (2 M). The mixture was evaporated to dryness and was dissolved in 500 µl HPLC grade DMSO. The resulted solution was diluted ten times and injected into a HPLC-UV system. HPLC method development and measurements were done using a Dionex Ultimate 3000 UHPLC instrument (Thermo Fischer Sicentific, Waltham, MA, USA). The instrument was equipped with a quaternary pump, a degasser, a well plate sampler, a column thermostat and a UV-VIS detector. Separation of achirally derivatised amino acids was performed using a Phenomenex Ez:faast AAA-MS 250 x 2 mm, 4 µm column (Phenomenex Inc., CA, USA). The mobile phase was the following: A: H2O + 41 mM acetate buffer (pH = 4), B: MeOH + 41 mM acetate buffer (pH = 4). The elution was isocratic with the ratio of A/B = 45/55 for 33 minutes, while the flow rate was 0.35 ml/min at 45°C. The injection volume was 1 µl and peaks were detected at λ = 338 nm and λ = 262 nm. Separation of chirally derivatised amino acids was performed using same column as above and the mobile phase was the following: A: H2O + 25 mM HCOONH4, B: MeOH + 25 mM HCOONH4. During the gradient elution the B % increased from 35% to 70 % for 30 minutes. The flow rate was 0.25 ml/min at 35°C during analyses and the injection volume was 5 µl and peaks were detected at λ = 340 nm and λ = 254 nm.  Results and discussion In the first stage of our work, standard amino acid solutions were derivatised with o-phthaldialdehyde (OPA) and 9-fluorenylmethyloxycarbonyl chloride (Fmoc). A non-chiral HPLC-UV method was developed for the separation of leucin, isoleucin, valine and isovaline. The components were separated in an isocratic elution within 33 minutes. The chirally derivatised L-, and D-amino acids were also separated, but in this case, a gradient elution from 35 % B to 70% B was used. After the separation method optimization, the Trichoderma pleuroti (SZMC 12454) strain was cultivated. At the end of the fermentation the mycelia were extracted and the crude peptaibol mixture was purified by flash chromatography using CHCl3/MeOH gradient. The peptaibol containing fractions eluted from 40% to 60% of MeOH and a fraction was selected from the collected ones contained the most amount of peptaibols. The oligomers were hydrolysed and the liberated amino acids derivatised achirally. The achiral amino acid separation resulted, that the peptaibol mixture contains valine and isovaline in 1 to 1 ratio and leucine and isoleucine in 1 to 1 ratio also as well. This peptaibol fraction was also chirally derivatised and analysed. Altogether L-isoleucine, D-isovaline and L-leucine were found, none of the two valine isomers could be detected.   Conclusion Two successful HPLC-UV methods were developed to analyse isobaric amino acids and to define their configuration. Group of peptaibols were successfully purified, hydrolysed and analysed. After analysing the real samples, it could be concluded that further purification of the peptaibols is needed to have the pure compounds hydrolysed and analysed.    24th International Symposium on Analytical and Environmental Problems 285 Acknowledgements This work was supported by the Hungarian Scientific Research Fund by grants NKFI-128659 and this work was connected to the project GINOP-2.3.2-15-2016-00012.   References [1] N. Stoppacher, B. Reithner, M. Omann, Rapid. Commun. Mass Sp. 21 (2007) 3963. [2] J. K. Chugh, B. A. Wallace, Biochem. Soc. T. 29 (2001) 565. [3] T. Marik, P. Urbán, C. Tyagi, Chem. Biochem. 14 (2017) e1700033. [4] J. Kirschbaum, C. Krause, R. K. Winzheimer, J. Peptide Sci. 9 (2003) 799. [5] T. Marik, Cs. Várszegi. L. Kredics, Acta Biol. Szegediensis 57 (2013) 109.    

Determination of valine and leucine isomers in peptaibols

 Book of Abstracts | 16th Wellmann International Scientific Conference| ISBN 978-963-306-589-1  115  DETERMINATION OF VALINE AND LEUCINE ISOMERS IN PEPTAIBOLS  GÁBOR ENDRE1,2, DÓRA PAPP1, TAMÁS MARIK1, LÁSZLÓ KREDICS1, MÓNIKA VARGA1, CSABA VÁGVÖLGYI1, ANDRÁS SZEKERES1  1University of Szeged, Faculty of Science and Informatics, Department of Microbiology, Közép fasor 52. Szeged H-6726 2Doctoral School in Biology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary egabcy@gmail.com (G.E.), andras.j.szekeres@gmail.com (A.Sz.)   Peptaibols are peptid-like oligomers produced as extracellular secondary metabolites by Trichoderma species. Some peptaibols known as effective antibacterial and antifungal agents. They are amphiphilic, which allows them to form voltage-dependent ion channels in cell membranes causing leaks, which finally lead to the death of the cells. These compounds contain non-ribosomally synthetized, non-proteinogenic amino acids, like α-aminoisobutyric acid, ethyl-norvaline, isovaline, and hydroxyproline. Regarding these molecules, the N-terminus is acetylated, and the C-terminal is an amino-alcohol. Due to their diverse nature and bioactivities, the comprehensive elucidation of the structure of newly discovered peptaibols is important. A lot of efforts have been made to analyse the amino acid sequence of peptaibols using mass spectrometry, however, the isobaric amino acids cannot be specified, thus the configuration of the compounds could not be determined. The hydrolysation of peptaibols followed by liquid chromatographic separation both problems could be solved-, e.g. isobaric amino acids could be separated, and due to chiral derivatisation D-, and L-isomers can be distinguished. In this work, we focused on the achiral and chiral separation of the isobaric amino acids, valine, isovaline, leucine and isoleucine by HPLC-UV analysis. For these purposes, two HPLC methods were developed. For achiral separation the standard amino acid mixture was firstly derivatised with o-phtaldialdehyde (OPA) and then 9-fluorenylmethyloxycarbonyl chloride (Fmoc). For chiral derivatisation, Marfey’s reagent was used to separate the D-, and L-amino acids. Finally, the developed methods were applied for hydrolysed pure peptaibol fractions.  Acknowledgements This work was supported by the Hungarian Government and the European Union within the frames of the Széchenyi 2020 Programme through grant GINOP-2.3.2-15-2016-00012. AS was supported through the New National  Book of Abstracts | 16th Wellmann International Scientific Conference| ISBN 978-963-306-589-1  116  Excellence Program of the Ministry of Human Capacities (ÚNKP-16-4). The infrastructural background was established with the support of GINOP-2.3.3-15-2016-00006 grant (Széchenyi 2020 Programme). 

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Determination of valine and leucine isomers in peptaibols

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