Design and Synthesis of AX7574: A Microcystin-Derived ...

794 Bioconjugate Chem., Vol. 15, No. 4, 2004 Shreder et al.Scheme 1.Synthesis of the Microcystin-TAMRA Probe AX7574Downloaded by WUHAN UNIV on October 25, 2009 | http://pubs.acs.orgPublication Date (Web): June 18, 2004 | doi: 10.1021/bc04995804,6-dioxoheptanoic acid to form a carboxylic acid bearingderivative. However, this acid was not further elaboratedupon. In the end, they chose a synthetic route in which4,6-dioxoheptanoic acid was carbodiimide coupled tocephalexin and the resulting dione conjugated to pyoverdinin 20% yield. Because microcystin modification with4,6-dioxoheptanoic acid would yield a tricarboxylic acid,any resulting amidation with an amine bearing fluorophorederivative would be difficult to accomplish selectively.Consequently, we started our approach, analogousto the case of cephalexin, with the synthesis of a fluorophore-dionederivatization reagent. In particular, wechose to use tetramethylrhodamine (TAMRA) as thefluorophore, because of its previous success as a sensitiveand quantitative tag for ABPs.The requisite dione was prepared from the mixedsuccinimidyl esters of 5-(and 6)-carboxytetramethylrhodamine(TAMRA-OSu). Reaction of TAMRA-OSu with(Boc-4-aminomethyl)piperidine followed by treatment ofthe resulting product with 1:1 TFA/DCM produced thesecondary amine 1 (Scheme 1). When this amine wastreated with an excess of N,N′-carbonyldiimidazoleactivated4,6-dioxoheptanoic acid, compound 1 was quantitativelyconverted into the desired dione 2. Afterpurification via C 18 reverse phase HPLC, the TAMRAdione2 was isolated in 66% yield. As expected, 1 H NMRanalysis showed the presence of resonances for both theenol and dione form of compound 2. Furthermore, doublingof the enol, dione, and aromatic resonances wasobserved from the use of the mixed TAMRA isomerstarting material.The probe AX7574 was produced from the condensationof compound 2 with microcystin-LR under basic conditions.Microcystin-LR (580 µg) was heated with a 10-foldexcess of dione in a mixture of 20% dioxane/1 M NaHCO 3(pH 9.0) for 2 days at 65 °C. LC/MS analysis indicatedthe consumption of microcystin-LR and the presence ofa new TAMRA-containing peak (as evidenced by anabsorption at 550 nm) with the desired product mass(MALDI-FTMS (DHB): m/z 1625.8402 - C 87 H 112 N 14 O 17+ H requires 1625.8464). AX7574 was isolated from otherimpurities using C 18 reverse phase chromatography in4% yield.AX7574 was characterized structurally using tandemmass spectrometry. Fragmentation of the parent ion (MS/MS) resulted in two major ions, 1099.5 and 527.4. Theseions were assigned to the fragments that result from theN-CO cleavage of the tertiary amide bond found in thelinker. Further isolation and fragmentation of these ions(MS/MS/MS) were consistent with the expected structureof AX7574 (see Figure 2). For the former ion, fragmentationscorresponding to cleavage of the cyclic peptidebackbone, decarbonylation, and loss of the pyrimidinemoiety were seen. For the latter ion, fragments correspondingto cleavage about the amide bond of the linker-TAMRA moiety were observed.Once the structure of AX7574 was secured, attentionwas turned to demonstrating its ability to label PP-1.PP-1 (25 nM) was incubated with AX7574 (0.5 µM) in 50mM HEPES, 5% DMSO, 0.01% Triton x-100, pH 7.4, atambient temperature. At various timepoints, samplealiquots were removed, quenched with DTT-containingsample buffer, and then heat-denatured. The resultingsamples were subjected to denaturing SDS-PAGE, andlabeled enzyme was visualized using a flatbed laserinducedfluorescence scanner. Importantly, the TAMRAtag provided a sensitive fluorescent handle by which thedegree of labeling could be quantified (see ExperimentalProcedures). Consistent with the results of Holmes et al.(vide supra), the kinetics of PP-1 labeling was on theorder of hours (see Figure 3) with 65% of the labelingoccurring at ∼16 h. When the time course data were fitto a single exponential, maximal labeling was determinedto approach a ratio of 1:1 PP-1 to AX7574.Several additional experiments were run to determineif the observed labeling was specific for the active site of