Isolation and Characterization of [D-Leu1]microcystin-LY from Microcystis aeruginosa CPCC-464

Abstract

[D-Leu¹]MC-LY (<b>1</b>) ([M + H]⁺<i>m</i>/<i>z</i> 1044.5673, Δ 2.0 ppm), a new microcystin, was isolated from <i>Microcystis aeruginosa</i> strain CPCC464. The compound was characterized by ¹H and ¹³C NMR spectroscopy, liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) and UV spectroscopy. A calibration reference material was produced after quantitation by ¹H NMR spectroscopy and LC with chemiluminescence nitrogen detection. The potency of <b>1</b> in a protein phosphatase 2A inhibition assay was essentially the same as for MCLR (<b>2</b>). Related microcystins, [D-Leu¹]MC-LR (<b>3</b>) ([M + H]⁺<i>m</i>/<i>z</i> 1037.6041, Δ 1.0 ppm), [D-Leu¹]MC-M(O)R (<b>6</b>) ([M + H]⁺<i>m</i>/<i>z</i> 1071.5565, Δ 2.0 ppm) and [D-Leu¹]MC-MR (<b>7</b>) ([M + H]⁺<i>m</i>/<i>z</i> 1055.5617, Δ 2.2 ppm), were also identified in culture extracts, along with traces of [D-Leu¹]MC-M(O₂)R (<b>8</b>) ([M + H]⁺<i>m</i>/<i>z</i> 1087.5510, Δ 1.6 ppm), by a combination of chemical derivatization and LC-HRMS/MS experiments. The relative abundances of <b>1</b>, <b>3</b>, <b>6</b>, <b>7</b> and <b>8</b> in a freshly extracted culture in the positive ionization mode LC-HRMS were ca. 84, 100, 3.0, 11 and 0.05, respectively. These and other results indicate that [D-Leu¹]-containing MCs may be more common in cyanobacterial blooms than is generally appreciated but are easily overlooked with standard targeted LC-MS/MS screening methods.