Structural characterization of new microcystins containing tryptophan and oxidized tryptophan residues

Abstract

Microcystins are cyclic peptides produced by cyanobacteria, which can be harmful to humans and animals when ingested. Eight of the (more than) 90 microcystin variants presently characterized, contain the amino acid tryptophan. The well-researched oxidation products of tryptophan; kynurenine, oxindolylalanine, and N-formylkynurenine, have been previously identified in intact polypeptides but microcystin congeners containing oxidized tryptophan moieties have not been reported. Liquid chromatography-tandem mass spectrometric analysis of an extract of Microcystis CAWBG11 led to the tentative identification of two new tryptophan-containing microcystins (MC‑WAba and MC-WL), as well as eight other microcystin analogs containing kynurenine, oxindolylalanine and N‑formylkynurenine (Nfk). Investigation of one of these congeners (MC‑NfkA) by nuclear magnetic resonance spectroscopy was used to verify the presence of Nfk in the microcystin. Liquid chromatography-mass spectrometry analysis of a tryptophan oxidation experiment demonstrated that tryptophan-containing microcystins could be converted into oxidized tryptophan analogs and that low levels of oxidized tryptophan congeners were present intracellularly in CAWBG11. MC-NfkR and MC-LNfk were detected in standards of MC-WR and MC-LW, indicating that care during storage of tryptophan-containing microcystins is required.

Scheme 1

Products from the oxidation of tryptophan in proteins according to Taylor et al. [8].

Figure 1

Structures of the tryptophan-containing microcystin congeners found in Microcystis CAWBG11, with amino acid numbering (1–7) indicated (Configuration for 3 and 4 is assumed).

Figure 2

Negative ion liquid chromatography-mass spectra of MC-WAba (a) prior to β-mercaptoethanol (BME) derivatization and (b) after two hours of incubation at 30°C.

Figure 3

Tandem mass spectrometry fragment ions indicating the amino acid sequence in MC-WAba.

Figure 4

Structures of MC-WR and its oxidized tryptophan congeners, with amino acid numbering (1–7) indicated (Configuration for 5‑7 assumed).

Figure 5

Structures of MC-WA and its oxidized tryptophan congeners, with amino acid numbering (1–7) indicated (Configuration for 8‑10 assumed).

Figure 6

Structures of MC-WR and its oxidized tryptophan congeners, with amino acid numbering (1–7) indicated (Configuration assumed).

Figure 7

Structure of MC-NfkA (10) with atom numbering used for the nuclear magnetic resonance spectroscopy assignment.

Figure 8

Negative ion liquid chromatography-mass spectra of an extract of CAWBG11 performed under mild (extraction) conditions. Spectra focusing on (a) the -XR congeners, (b) the -XA congeners and (c) the -XAba congeners.