*4.2. Toxins and Other Materials*

Distilled H2O was further purified using a UV purification system (ThermoFisher Scientific) or a Milli-Q water purification system (Millipore Ltd., Oakville, ON, Canada). MeOH and CH3CN (Optima LC–MS grade) were from ThermoFisher Scientific. Hexanes was from Caledon. Formic acid and trifluoroacetic acid were from Sigma-Aldrich (Oakville, ON, Canada). A certified reference material for **2** (CRM-MCLR (Lot # 20070131)) and in-house reference materials for **4** and **5** were from the National Research Council Canada (Biotoxin Metrology, Halifax, NS, Canada).

#### *4.3. Biological Material*

*M. aeruginosa* cultures CPCC-464 and CPCC-299 were obtained from the University of Toronto Culture Collection (now the Canadian Phytoplankton Culture Collection housed at the University of Waterloo, ON, Canada). CPCC-464 was isolated from Trampling Lake, Saskatchewan, Canada, July 1998 and deposited by D. Parker as UWOCC#E7. CPCC-299 was isolated from Pretzlaff Pond, Alberta, Canada, August 1990 and deposited by E. Prepas and A. Lam as sample #45-2A. Bulk cultures of CPCC-464 were prepared in two aerated Brite-boxes (250 and 300 L), which are self-contained fiberglass boxes that optimize temperature and light to maximize biomass production. All cultures were grown on BG11 medium [30,31] made using filtered (1 μM) lake water that had pasteurized for 6 h at 85 ◦C. Light was provided by internally mounted cool white fluorescent tubes shaded with nylon mesh for an approximate intensity of 75–100 μmol m−<sup>2</sup> s−<sup>1</sup> on a 14:10 h light:dark cycle. Temperature was maintained at 20 ◦C and pH was monitored and remained constant at 8.6. When cultures reached late exponential stage, 188 g of wet biomass was harvested using a tangential flow centrifuge (IEC Centra MP-4R CEPA Z41 with an 804S rotor (GMI, Ramsey, MN, USA)) with a flow rate of 2–3 L min<sup>−</sup>1. The biomass was stored at −20 ◦C. An extract of lyophilized material from a cyanobacterial bloom at Poplar Island, MD, USA, which contained authentic 3 as well as the tentatively identified 6–8, was available from an earlier study [11].

#### *4.4. Toxin Isolation from Culture Biomass*

Wet cell biomass of CPCC-464 (104.8 g) was extracted four times with 70% MeOH–H2O (400 mL). After centrifugation, the supernatants were pooled (1.7 L) and partitioned with hexanes (700 mL). The hexane portion was back-extracted with 85% MeOH–H2O (300 mL) and combined with the first extract. The cleaned extract was adjusted to 85% MeOH and partitioned a second time with hexane (300 mL). The combined MeOH–H2O extracts were partially evaporated, pre-adsorbed on ~14 g of Waters 55-105μm prep C18 and packed on top of a vacuum liquid chromatography column (4 cm × 11 cm) containing Waters 55–105 μm Prep C18-silica. Fractions were eluted with increasing percentages of MeOH and analyzed by LC–MS. [D-Leu1]MC-LY (**1**) was present in fractions containing 40–80% MeOH–H2O and further purified on a Sephadex LH-20 column (1.6 cm × 68.0 cm), which was eluted isocratically with MeOH. Fractions containing **1** were combined and subjected to a flash chromatography column (Bakerbond 40 μm C18-silica, 1.5 cm × 22.0 cm) eluted with 55% MeOH–H2O. After analyzing the fractions, those containing **1** were purified using a 3 μm Luna C18(2) column (250 × 10 mm; Phenomenex, Torrance, CA, USA) eluted isocratically with 48% CH3CN–H2O containing 0.05% TFA at 2 mL/min, with UV monitoring at 238 nm. To remove TFA the collected fractions were diluted to 14% CH3CN–H2O and loaded on Waters Oasis HLB (6 cc, 500 mg) cartridges, washing the acid out with H2O and eluting **1** with MeOH.

Purity assessment of the final purified material by LC–MS was carried out on a SCIEX API 4000 mass spectrometer using full scan (*m*/*z* 700–1200) and selected reaction monitoring with positive electrospray ionization. Separations were performed by linear gradient elution on an Agilent 2.7 μm Poroshell 120 SB-C18 column (2.1 × 150 mm) held at 40 ◦C with mobile phase A: H2O, and B: 95% CH3CN, each containing 2 mM ammonium formate and 50 mM formic acid. The gradient was 25–75% B over 25 min, increased to 100% B over 2 min, and held for 11 min, at 0.2 mL/min. LC–UV analysis of the pure product was conducted on an Agilent 1290 Infinity LC System with diode array detector (DAD) with UV monitoring at 238 and 210 nm (same LC column and conditions as LC–MS monitoring except mobile phase A: H2O, and B: CH3CN, each containing 0.1% trifluoroacetic acid).

[D-Leu1]MC-LY (**1**): white solid; 1H and 13C NMR (Table 1); HRMS [M + H]<sup>+</sup> *m*/*z* 1044.5660 (calculated for C55H78O13N7 <sup>+</sup> 1044.5652 (<sup>Δ</sup> 0.8 ppm)), LC–HRMS/MS (Table 2); HRMS [M <sup>−</sup> H]<sup>−</sup> *m*/*z* 1042.5515(calculated for C55H76O13N7 − 1042.5507 (Δ 0.8 ppm)), LC–HRMS/MS (DIA) 1024.5418 (42%), 587.2759 (7), 325.2246 (28), 307.2141 (17), 128.0355 (100). λmax (LC–UV) 233, 280 nm (Figure S19).

#### *4.5. Preparation of Reference Material*

An aliquot containing [D-Leu1]MC-LY (1) (4.3 mg) was evaporated under N2 and dissolved in 3.0 mL 90% CD3OH–H2O. This stock solution was quantitated directly by 1H NMR using high purity caffeine as the external calibrant as described previously [18]. A dilution of the stock solution was prepared with 50% MeOH–H2O for analysis by LC–UV-CLND [19] using an Agilent 1100 HPLC system with a 1050 UV detector connected to a model 8060 CLND (Antek PAC, Houston, TX, USA). Separations were performed on an Agilent 3.5 μm Poroshell SB-C8 (2.1 × 150 mm) maintained at 40 ◦C. Isocratic elution was at 0.2 mL/min, using 65% MeOH–H2O (0.2% HCOOH) for 1. The external calibrant was also caffeine, with serial dilutions prepared gravimetrically in deionized H2O. Caffeine was eluted with 40% MeOH–H2O (0.2% HCOOH). The concentration of contaminating [D-Leu1,D-Glu(OMe)6]MC-LY was measured using the UV detector at 238 nm, with an accurate dilution of the RM of 1 as the calibrant.

After quantitation, the stock solution was quantitatively transferred using 50% high purity degassed MeOH–H2O to a calibrated volumetric flask, then diluted to the mark with the same. The solution was packaged under argon in flame sealed ampoules using an automatic ampouling machine (Cozzolli, Model FPS1-SS-428, NJ, USA), then stored at −80 ◦C.
