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

[D-Leu1]MC-LY (1) ([M + H]+ 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]+ m/z 1037.6041, Δ 1.0 ppm), [D-Leu1]MC-M(O)R (6) ([M + H]+ m/z 1071.5565, Δ 2.0 ppm) and [D-Leu1]MC-MR (7) ([M + H]+ 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]+ 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.


Introduction
Microcystins (MCs), such as MC-LR (2), are cyclic heptapeptide hepatotoxins ( Figure 1) produced primarily in cyanobacterial genera such as Microcystis, Dolichospermum (Anabaena), Nostoc and 2 dienoic acid (Adda) at position 5 ( Figure 1) [3]. Adda 5 and Glu 6 appear to be primarily responsible for the characteristic biological activity of MCs [2,3]. Protein phosphatase inhibition is directly related to the toxins' mechanism of action and animal studies have demonstrated that MCs are potent tumor promoters [1]. To date, the number of identified MCs continues to increase and more than 250 analogues have been characterized [4]. However, due to a lack of standards for these analogues, very few studies have adequately assessed their distribution in natural waters.
As part of feasibility studies for a cyanobacterial matrix reference material [5], a survey of cyanobacterial cultures from Canada was conducted using LC with UV and MS detection. Among the samples analyzed, two Microcystis aeruginosa cultures from Saskatchewan and Alberta, CPCC-464 and CPCC-299, showed the presence of a new microcystin tentatively identified as [D-Leu 1 ]MC-LY (1) [5] together with the previously reported [6,7] and well-characterized [8] [D-Leu 1 ]MC-LR (3). [D-Leu 1 ]MC-LY (1) was also tentatively identified recently by LC-HRMS/MS in a cyanobacterial bloom sample from southwestern Ontario, Canada [9], indicating that it may be a significant component of natural cyanobacterial blooms in this and other parts of the world. It is therefore necessary to verify the structure, and to evaluate its toxicity relative to other MCs because limited data are available on the toxicological consequences of varying the amino acid at position 1 and 2.

Results and Discussion
The analysis of M. aeruginosa culture CPCC-464 by LC-UV-MS/MS is shown in Figure 2. A very similar profile was observed with culture CPCC-299, with the only differences being in relative peak areas. The LC-UV chromatogram of CPCC-464 ( Figure 2a) showed two major peaks due to 1 and 3. As part of feasibility studies for a cyanobacterial matrix reference material [5], a survey of cyanobacterial cultures from Canada was conducted using LC with UV and MS detection. Among the samples analyzed, two Microcystis aeruginosa cultures from Saskatchewan and Alberta, CPCC-464 and CPCC-299, showed the presence of a new microcystin tentatively identified as [D-Leu 1 ]MC-LY (1) [5] together with the previously reported [6,7] and well-characterized [8] [D-Leu 1 ]MC-LR (3).
[D-Leu 1 ]MC-LY (1) was also tentatively identified recently by LC-HRMS/MS in a cyanobacterial bloom sample from southwestern Ontario, Canada [9], indicating that it may be a significant component of natural cyanobacterial blooms in this and other parts of the world. It is therefore necessary to verify the structure, and to evaluate its toxicity relative to other MCs because limited data are available on the toxicological consequences of varying the amino acid at position 1 and 2.

Results and Discussion
The analysis of M. aeruginosa culture CPCC-464 by LC-UV-MS/MS is shown in Figure 2. A very similar profile was observed with culture CPCC-299, with the only differences being in relative peak areas. The LC-UV chromatogram of CPCC-464 ( Figure 2a) showed two major peaks due to 1 and 3.
In the same experiment, the MS was operated with a precursor scan using the m/z 135 product ion for Adda, which is characteristic of most MCs [10]. Examination of all peaks in the total ion current chromatogram (Figure 2b In the same experiment, the MS was operated with a precursor scan using the m/z 135 product ion for Adda, which is characteristic of most MCs [10]. Examination of all peaks in the total ion current chromatogram (  Large scale culturing of CPCC-464 followed by centrifugation provided 188 g of biomass for purification of 1. This material was extracted with 70% MeOH-H2O, then taken through a preparative isolation procedure consisting of a hexane partitioning, C18 LC, LH-20 gel permeation, C18-flash chromatography, and semi-preparative HPLC. The total yield of 1 was 28.  Large scale culturing of CPCC-464 followed by centrifugation provided 188 g of biomass for purification of 1. This material was extracted with 70% MeOH-H 2 O, then taken through a preparative isolation procedure consisting of a hexane partitioning, C18 LC, LH-20 gel permeation, C18-flash chromatography, and semi-preparative HPLC. The total yield of 1 was 28.7 mg containing a small amount of [D-Leu 1 ,D-Glu(OMe) 6 ]MC-LY and a trace of what is believed to be [D-Leu 1 ,(6Z)-Adda 5 ]MC-LY (observed by LC-MS selected reaction monitoring modes).
The structure of 1 was elucidated from NMR spectra acquired in CD 3 OH in order to observe the exchangeable amide protons. The proton NMR spectrum had six resonances in the amide region with a profile similar to that of a peptide. Individual spin systems from each amide resonance were identified and assigned using 2D 1 H-1 H DIPSI-2 and 1 H-1 H COSY correlations (Table 1). Detailed spectra are provided in Figures S3-S10 and an overlay of chemical shifts on the proposed 2-dimensional chemical structure of 1 is shown in Figure S11. Carbon assignments were determined indirectly using 1 H-13 C HSQC and 1 H-13 C HMBC 2D NMR spectra. One carbon resonance was not assigned for Leu 1 (C1) due to spectral overlap. The Adda unit was assembled with the aid of the HMBC data, which determined the positions of the methyl groups. Trans-configuration of the 4,5-double bond is indicated by the large coupling constant between Adda-H4 and -H5 (15.5 Hz) and by the observation of a ROESY correlation between Adda-H4 and Adda-6-Me, and is consistent with the absence of a ROESY correlation between Adda-H4 to Adda-H5. The second double bond was also trans as a ROESY correlation was observed between Adda-H5 and Adda-H7 ( Figure 3). Carbon assignments were determined indirectly using 1 H-13 C HSQC and 1 H-13 C HMBC 2D NMR spectra. One carbon resonance was not assigned for Leu 1 (C1) due to spectral overlap. The Adda unit was assembled with the aid of the HMBC data, which determined the positions of the methyl groups. Trans-configuration of the 4,5-double bond is indicated by the large coupling constant between Adda-H4 and -H5 (15.5 Hz) and by the observation of a ROESY correlation between Adda-H4 and Adda-6-Me, and is consistent with the absence of a ROESY correlation between Adda-H4 to Adda-H5. The second double bond was also trans as a ROESY correlation was observed between Adda-H5 and Adda-H7 ( Figure 3). The relative stereochemistry of C2 and C3 of Adda was determined from the observation of a ROESY correlation between Adda-H3 and both Adda-H5 and Adda-2-Me, while Adda-H2 showed correlations to Adda-NH, Glu-NH, Adda-2-Me and Adda-H4, indicating that H2 and H3 are on the opposite faces of the Adda plane ( Figure 3). This is consistent with the ca. 9.7 Hz coupling constant between Adda-H2 and -H3. The glutamic acid unit (Glu), N-methyldehydroalanine (Mdha) and erthyo-β-methylaspartic acid (Masp) were identified in a similar manner, and their proton and carbon resonances were very similar to those previously published for 3 [8]. Two leucine units were identified by the similarities of their 1 H and 13 C resonances to those in the BMRB database (http://www.bmrb.wisc.edu/ref_info/; accessed September 2011) and those published for 3 [10]. The relative stereochemistry for H2 and H3 of the Masp unit was determined from the absence of Masp-H2 to Masp-3-Me correlation and the presence of a ROESY correlation between Masp-H2 and -H3, which places H2 and H3 on the same side of the plane. The tyrosine unit (Tyr) was assigned from the presence of the two doublets at 6.99 and 6.62 ppm, characteristic of a para-substituted phenyl ring. The aromatic protons, Tyr-H5 and -H9 correlated to a carbon at 36.3 ppm, characteristic of an aromatic amino acid. The accurate mass from LC-HRMS/MS indicated that the substituent on the phenyl ring was a hydroxyl group (Table 2), establishing its identity as Tyr.
The amino acid subunits assigned in the 1 H-1 H DIPSI-2, 1 H-1 H COSY and 1 H-13 C HMBC spectra were linked through correlations observed in the ROESY, NOESY ( Figure 3) and HMBC NMR spectra. In the HMBC spectra, a correlation between the N-methyl of Mdha and the carbonyl of Glu, and between the Masp-NH and the leucine carbonyl at 174.5 ppm, linked Glu 6 to Mdha 7 and Masp 3 to Leu 2 . Additionally, ROESY correlations were observed between Leu 2 -NH and both Leu 1 -NH and Masp 3 -NH. Furthermore, the Tyr-NH showed ROESY or NOESY correlations to Masp 3 -H3 and Adda 5 -NH, Adda 5 -NH showed correlations to Adda-H4 and Adda-H2, and Adda 5 -H2 showed correlations to Adda-H4, Adda-2-Me and Glu 6 -NH. These correlations show 1 to contain Leu-Leu-Masp-Tyr-Adda-Glu-Mdha, and the molecular formula established from LC-HRMS requires an amide linkage between Mdha 7 and Leu 1 moieties. That this linkage is present is demonstrated by the presence of numerous product ions in the HRMS/MS spectrum that are attributable to fragments containing both Leu 1 and Mdha 7 , such as those at m/z 169.1334, 197.1283 and 488.2745 ( Table 2). The ROESY correlations observed for 1 (Figure 3), especially those between the amide protons, were consistent with those expected based on the established 3-dimensional solution structure for MC-LR [12], which is reported to be very similar to that of [D-Leu 1 ]MC-LR (3) [8]. Thus, 1 has the same relative stereochemistry as 2 and 3. This is also supported by the close similarity of the 13 C NMR chemical shifts of 1 to those reported for [D-Leu 1 ]MC-LR (3) in the same solvent (Table S1). The fact both 1 and 3 are biosynthesized together by the MC synthetase of M. aeruginosa strain CPCC-464, and that 1 was subsequently found to have similar inhibitory potency to MC-LR (2) against protein phosphatase 2A (PP2A) (Figure 4), both indicate that 1 has the same absolute stereochemistry as 2 and 3 and that 1 is therefore [D-Leu 1 ]MC-LY ( Figure 1).  Calc  that was recently also reported by Foss et al. [11] in a cyanobacterial bloom sample, together with 6 and 7. Microcystins 6-8 from this sample and from M. aeruginosa CPCC-464 showed identical retention times and mass spectral characteristics. Methionine sulfoxide analogues of MCs appear to be formed by autoxidation [22], and it appears that the same process can also lead to formation of the corresponding sulfones. The stereochemistry of 6-8 cannot be verified by LC-MS methods. However, because 7 is presumably biosynthesized in the culture by the same synthetase that produces 1 and 3, and that 6 and 8 are autoxidation products of 7, 6-8 can therefore be assumed to have the same stereochemistry as 1 and 3 (Figure 1).
A careful non-targeted LC-MS analysis of a field sample by Foss et al. [11] recently reported more than 20 Leu 1 -containing MCs in a cyanobacterial bloom, with [D-Leu 1 ]MC-LR (3) as the major component, but no 1 was detected. Including the present study, 1 now appears to have been detected  Table 2). The negative ion MS/MS spectrum obtained from the FS/DIA (full scan/data independent acquisition) LC-HRMS of 1 showed a prominent product ion at m/z 128.0355, consistent with the presence of a MC containing Glu at position 6, and a neutral loss of m/z 112.0190, consistent with the presence of Masp at position 3 [17]. Careful comparison of the positive ion targeted LC-HRMS/MS spectrum of 1 with those of standards of 4 and 5 (Table 2) showed that product ions in 1 that contained amino acid-1 were consistently heavier by 42.047 Da (Leu vs. Ala) than the corresponding product ions from 5, and 42.047 (Leu 1 vs Ala 1 ), 92.026 (Tyr 4 vs Ala 4 ) or 134.073 Da (Leu 1 and Tyr 4 vs. Ala 1 and Ala 4 ) heavier than the corresponding product ions from 4 that contained amino acid-2, amino acid-4, or both amino acid-1 and -4, respectively ( Table 2, Figures S12-S18). Furthermore, the UV spectrum of 1 obtained during LC-UV analysis was identical to that of 5, and differed from that of 2 ( Figure S19), suggesting the presence of Tyr in 1 and 5 in addition to the UV-absorbing chromophores also present in 2 (i.e., Adda 5 and Mdha 7 ). The LC-MS/MS and LC-UV results are therefore entirely consistent with 1 being [D-Leu 1 ]MC-LY.A portion of the purified 1 was used to prepare a stock solution. This was quantitated using qNMR [18] and LC with chemiluminescence nitrogen detection (CLND) [19], then accurately diluted with 1:1 MeOH-H 2 O to prepare a reference material (RM) (~7.7 µM). LC-UV analysis of this RM showed the relative concentration of [D-Leu 1 ,D-Glu(OMe) 6 ]MC-LY to be 3.1%. The putative [D-Leu 1 ,(6Z)-Adda 5 ]MC-LY was below the limit of quantitation in LC-UV, but the relative concentration was estimated be to below 0.5% using HRMS/MS. Because MCs containing (6Z)-Adda 5 or D-Glu(OMe) 6 do not inhibit protein phosphatases [20], the RM of 1 was used for the PP2A inhibition assay without correcting for impurities. In the PP2A assay, the IC 50 for a certified RM (CRM) of MC-LR (2) was 0.62 nM (0.62 ng/mL), while that for the RM of 1 was 0.76 nM (0.80 ng/mL) (Figure 4). Matthiensen et al. [7] reported that MC-LR (2) and [D-Leu 1 ]MC-LR (3) had similar toxicities to mice when injected intraperitoneally, and that the IC 50 values of 2 and 3 in a PP1 assay were 3.1 and 4.4 nM, respectively. Similarly, Park et al. [6] independently found that 2 and 3 both had the same IC 50 value of 0.3 nM in their PP1 assay. Ikehara et al. [21] found that the IC 50 of MC-LF(9), which differs from MC-LY (5) only by the absence of a phenolic hydroxyl group on residue-2, was 3-fold higher than that of MC-LR (2) (0.096 vs. 0.032 nM) in their PP2A assay. Taken together with the data presented here, these results suggest that the replacement of D-Ala with D-Leu at position 1 in the MC structure has only a minor effect on the toxicity of MCs or on their inhibitory effects on PP1 and PP2A.
Authentic 3 in a cyanobacterial bloom extract from Poplar Island, MD, USA, whose structure has been verified as [D-Leu 1 ]MC-LR by purification and NMR analysis [11], had identical retention time and product ion spectra to the peak for 3 in CPCC-464 when analyzed by LC-HRMS/MS, thus verifying its identity as proposed by Hollingdale et al. [5].  ) were also detected in the culture extract, something that was recently also reported by Foss et al. [11] in a cyanobacterial bloom sample, together with 6 and 7. Microcystins 6-8 from this sample and from M. aeruginosa CPCC-464 showed identical retention times and mass spectral characteristics. Methionine sulfoxide analogues of MCs appear to be formed by autoxidation [22], and it appears that the same process can also lead to formation of the corresponding sulfones. The stereochemistry of 6-8 cannot be verified by LC-MS methods. However, because 7 is presumably biosynthesized in the culture by the same synthetase that produces 1 and 3, and that 6 and 8 are autoxidation products of 7, 6-8 can therefore be assumed to have the same stereochemistry as 1 and 3 (Figure 1).
A careful non-targeted LC-MS analysis of a field sample by Foss et al. [11] recently reported more than 20 Leu 1 -containing MCs in a cyanobacterial bloom, with [D-Leu 1 ]MC-LR (3) as the major component, but no 1 was detected. Including the present study, 1 now appears to have been detected in samples originating from three different locations in Canada [5,9], but so far, nowhere else in the world. Geographical differences in the distribution of microcystins are being reported [23,24]. Leu 1 -containing MCs have been implicated in bird deaths in both Canada and the USA [6,11] and have been reported in samples from cyanobacterial blooms in Brazil and Argentina [7,25,26] as well as in lichens from Argentina, USA, China, Japan, Norway, Sweden, and Finland [25,27]. Leu 1 variants may be more common and widespread than these studies indicate, as many analyses for MCs are conducted using highly targeted LC-MS/MS methods, and the Leu 1 -containing variants are heavier by 42 Da than the more common (and more commonly targeted) Ala 1 -containing MCs. Both types of variants would be readily detected if they were targeted in the LC-MS/MS method, or if untargeted LC-MS methods were used. Protein phosphatase inhibition assays, or immunoassays with appropriate cross-reactivities [28,29], can also be expected to detect both D-Ala 1 -and D-Leu 1 -containing MCs although they cannot indicate which type of variant is present.

Conclusions
[D-Leu 1 ]MC-LR (3) has been reported previously and its structure confirmed by NMR spectroscopy [8]. [5,9]. The results presented here firmly establish the identity of 1 and show that it has similar inhibitory potency towards PP2A as MC-LR (2). A calibration reference material has been prepared that can be used to identify and quantitate 1 in field samples and cultures. Microcystins containing D-Leu at position 1 may be fairly common in the Americas, and the data presented here and elsewhere suggest these to be only slightly less toxic than their more common D-Ala 1 -containing congeners. It is therefore important to consider the possible presence of a range of D-Leu 1 -containing MCs when analyzing bloom samples.

General Experimental Procedures
Purified 1 (250 µg) was dissolved in 30 µL of CD 3 OH for NMR spectroscopy. NMR spectra were acquired on a Bruker Avance III 600 MHz spectrometer (Bruker Biospin Ltd., Billerica, MA, USA) operating at a 1 H frequency of 600.28 MHz and 13 C frequency of 150.94 MHz using TOPSPIN 2.1 acquisition software with a 1.7 mm TXI gradient probe at 277 K. Standard Bruker pulse sequences were used for structure elucidation: one dimensional 1 H spectrum with composite pulse pre-saturation of water, double quantum filtered 1 H-1 H COSY, 1 H-1 H DIPSI-2 (mixing time 120 ms), 1 H- 13  The initial survey of cultures for the presence of MCs was performed by LC-UV-MS using an Agilent (Mississauga, ON, Canada) 1200 LC coupled with a SCIEX (Concord, ON, Canada) API 4000 Q-Trap mass spectrometer with UV monitoring at 238 nm and positive electrospray ionization MS, with full scans, m/z 135 precursor scans, product ion scans, and selected reaction monitoring. The LC column (50 × 2.1 mm; Agilent) was packed with 1.8 µm Zorbax SB-C18 and maintained at 40 • C. The flow rate was 0.3 mL/min, with a gradient of 10%-80% B over 30 min. Solvent A was water and B was 95% acetonitrile, each with 50 mM formic acid and 2 mM ammonium formate.
LC-HRMS was conducted with a Q Exactive-HF Orbitrap mass spectrometer equipped with a HESI-II heated electrospray ionization interface (ThermoFisher Scientific, Waltham, MA, USA) with an Agilent 1200 G1312B binary pump, G1367C autosampler, and G1316B column oven. Analyses were performed with a 3.5 µm Symmetry Shield C18 column (100 × 2.1 mm; Waters) held at 40 • C with mobile phases A and B of H 2 O and CH 3 CN, respectively, each of which contained formic acid (0.1% v/v). A linear gradient (0.3 mL min −1 ) was used from 20% to 90% B over 18 min, then to 100% B over 0.1 min, followed by a hold at 100% B (2.9 min), then returned to 20% B over 0.1 min with a hold at 20% B (3.9 min) to equilibrate the column. Injection volume was typically 1-5 µL. In positive ion mode the mass spectrometer was calibrated from m/z 74-1622, the spray voltage was 3.7 kV, the capillary temperature was 350 • C, and the sheath and auxiliary gas flow rates were 25 and 8 units, respectively, with MS data acquired from 2 to 20 min. Mass spectral data were collected using a combined FS/DIA method. FS data were collected from m/z 500-1400 using the 60000 resolution setting, an AGC target of 1 × 10 6 and a max IT of 100 ms. DIA data were collected using the 15000 resolution setting, an AGC target of 2 × 10 5 , max IT set to 'auto' and a stepped collision energy of 30, 60 and 80 V. Precursor isolation windows were 62 m/z wide and centered at m/z 530, 590, 650, 710, 770, 830, 890, 950, 1010, 1070, 1130, 1190, 1250, 1310, and 1370. DIA chromatograms were extracted for product ions at m/z 121.1011, 121.0647, 135. 0804, 135.1168, 375.1915, 389.2072, 361.1758, 213.0870, 426.2096, 440.2252, 454.2409, 412.1939, 393.2020, 379.1864, 585.3395, 599.3552, and 613.3709. Putative MCs detected using the above FS/DIA method were further probed in a targeted manner using the PRM scan mode with a 0.7 m/z precursor isolation window, typically using the 30,000 resolution setting, an AGC target of 5 ×

Toxins and Other Materials
Distilled H 2 O was further purified using a UV purification system (ThermoFisher Scientific) or a Milli-Q water purification system (Millipore Ltd., Oakville, ON, Canada). MeOH and CH 3 CN (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).

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 −2 s −1 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 −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].

Toxin Isolation from Culture Biomass
Wet cell biomass of CPCC-464 (104.8 g) was extracted four times with 70% MeOH-H 2 O (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-H 2 O (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-H 2 O extracts were partially evaporated, pre-adsorbed oñ 14 g of Waters 55-105µm prep C18 and packed on top of a vacuum liquid chromatography column After analyzing the fractions, those containing 1 were purified using a 3 µm Luna C18 (2) Figure S19).

Preparation of Reference Material
An aliquot containing [D-Leu 1 ]MC-LY (1) (4.3 mg) was evaporated under N 2 and dissolved in 3.0 mL 90% CD 3 OH-H 2 O. This stock solution was quantitated directly by 1 H NMR using high purity caffeine as the external calibrant as described previously [18]. A dilution of the stock solution was prepared with 50% MeOH-H 2 O 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-H 2 O (0.2% HCOOH) for 1. The external calibrant was also caffeine, with serial dilutions prepared gravimetrically in deionized H 2 O. Caffeine was eluted with 40% MeOH-H 2 O (0.2% HCOOH). The concentration of contaminating [D-Leu 1 ,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-H 2 O 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.

Protein Phosphatase Inhibition Assay
Ampoules of the RM of 1, along with the CRM of 2 (CRM-MCLR), were sent to Abraxis LLC (Warminster, PA, USA) for evaluation of toxicity. PP2A assays were performed using the microcystin-PP2A plate kit according to the kit's standard procedures [32].