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

**Patricia LeBlanc 1, Nadine Merkley 1, Krista Thomas 1, Nancy I. Lewis 1, Khalida Békri 1,**†**, Susan LeBlanc Renaud 2, Frances R. Pick 2, Pearse McCarron 1, Christopher O. Miles <sup>1</sup> and Michael A. Quilliam 1,\***


Received: 16 December 2019; Accepted: 20 January 2020; Published: 23 January 2020

**Abstract:** [D-Leu1]MC-LY (**1**) ([M + H]<sup>+</sup> *m*/*z* 1044.5673, Δ 2.0 ppm), a new microcystin, was isolated from *Microcystis aeruginosa* strain CPCC-464. 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 **1** in a protein phosphatase 2A inhibition assay was essentially the same as for MC-LR (**2**). Related microcystins, [D-Leu1]MC-LR (**3**) ([M + H]<sup>+</sup> *m*/*z* 1037.6041, Δ 1.0 ppm), [D-Leu1]MC-M(O)R (**6**) ([M + H]<sup>+</sup> *m*/*z* 1071.5565, Δ 2.0 ppm) and [D-Leu1]MC-MR (**7**) ([M + H]<sup>+</sup> *m*/*z* 1055.5617, Δ 2.2 ppm), were also identified in culture extracts, along with traces of [D-Leu1]MC-M(O2)R (**8**) ([M + H]<sup>+</sup> *m*/*z* 1087.5510, Δ 1.6 ppm), by a combination of chemical derivatization and LC–HRMS/MS experiments. The relative abundances of **1**, **3**, **6**, **7** and **8** 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.

**Keywords:** microcystin; cyanotoxin; structure; PP2A inhibition; liquid chromatography; mass spectrometry; cyanobacteria

**Key Contribution:** Structure determination and toxicity evaluation of a new microcystin, [D-Leu1]MC-LY, and detection of related toxins in a culture of *Microcystis aeruginosa* (CPCC-464). This study and others indicate that [D-Leu1]-containing MCs are more common in cyanobacterial blooms than is generally appreciated but are easily overlooked with standard targeted LC–MS/MS screening methods.
