Cells treated with BI-BODIPY exhibited a solid mitotic arrest, seeing that exemplified by great and sustained cyclin B amounts (in comparison to DMSO control) (Fig

Cells treated with BI-BODIPY exhibited a solid mitotic arrest, seeing that exemplified by great and sustained cyclin B amounts (in comparison to DMSO control) (Fig. little molecule probes usually do not need hereditary manipulation of cells. Proteins kinases are in lots of ways ideal goals for the introduction of selective fluorescent little molecule probes. It is because proteins kinases get excited about many mobile adjustments and procedures within their localization, accessibility, and plethora are connected with adjustments in mobile state. Proteins kinases have already been utilized as biomarkers in cancers biology as the lack of endogenous kinase regulatory systems by stage mutations, gene deletions, gene amplifications, and chromosomal rearrangements continues to be well-established as an essential event in lots of cancers. Furthermore, medication chemical substance and breakthrough biology initiatives have got in latest years created many selective, cell-permeable little molecule ligands of particular mobile kinases. Right here we explain our initial tries to leverage existing, well-characterized kinase inhibitors to build up fluorescent little molecule probes for make use of as imaging equipment in cancers biology. Because of this work, we centered on Mps1-IN-1, a lately described inhibitor from the monopolar spindle 1 kinase (Mps1),1 and BI2536, a potent inhibitor of polo-like kinases (PLK1, PLK2, and PLK3).2, 3 These inhibitors focus on kinases that regulate cell routine progression which localize to distinct subcellular buildings during mitosis. Mps1 is normally a dual-specificity kinase whose activity is vital for the establishment and suffered activity of the spindle checkpoint.4C6 The polo-like kinases (PLKs) comprise a family group of conserved serine/threonine kinases that are highly conserved from fungus to human beings7 which are recognized to regulate cell routine development.2, 3, 8, 9 We reasoned which the synthesis and usage of Mps1- and PLK-selective little substances probes could facilitate the analysis of the kinases in various phases from the cell routine. In addition, such probes might be able to elucidate deregulated states and discover use as cancers diagnostics. Towards this final end, we endeavoured to convert Mps1-IN-1 and BI2536 to fluorescent probes of their particular kinase goals by conjugation to a cell-permeable fluorophore. 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)10 was chosen as the fluorescent dye since it includes a high quantum produce, it really is cell permeable, and its own derivatives have already been utilized to label proteins11C16 and DNA widely.17, 18 Synthesis of BODIPY-conjugated derivatives of Mps1-IN-1 and BI2536 (Mps1-IN-BODIPY, and BI-BODIPY, respectively) were adapted from published syntheses from the mother or father substances1, 19 and extra techniques to conjugate BODIPY are highlighted in Plans 1 and ?and2,2, respectively. Open in a separate window Plan 1 Synthesis of Mps1-IN-BODIPY. Reaction and conditions: (i) t-butyl 4-(1-(4-amino-3-methoxyphenyl)piperidin-4-yl)piperazine-1-carboxylate K2CO3, Pd2(dba)3, X-Phos, t-BuOH, 100 C; (ii) TFA, DCM, 0 C; LiOH?H2O, 25 C; (iii) 3-(2-carboxyethyl)-5,5-difluoro-7,9-dimethyl-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide (BODIPY acid), DCC, DMAP, THF, 0C25 C. Open in a separate window Plan 2 Synthesis of BI-BODIPY. Reaction and conditions: (i) t-butyl 4-((1R,4R)-4-aminocyclohexyl)piperazine-1-carboxylate, HBTU, DIEA, DMF, 25 C; (ii) TFA, DCM, 25 C; (iii) 3-(2-carboxyethyl)-5,5-difluoro-7,9-dimethyl-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide (BODIPY-acid), DCC, DMAP, THF, 0C25 C. For Mps1-IN-BODIPY, compound 1, which was prepared by following the reported process1, was reacted with tert-butyl 4-(1-(4-amino-3-methoxyphenyl)piperidin-4-yl)piperazine-1-carboxylate to give 2. Deprotection of 2 followed by amide coupling with the BODIPY acid produced the target Mps1-IN-BODIPY. For BI-BODIPY, compound 3, prepared according to previously reported procedures,19 was coupled with t-butyl 4-((1R,4R)-4-aminocyclohexyl)piperazine-1-carboxylate to afford 4.19 Acid mediated removal of the t-butoxycarbonyl group of 4 followed by amide coupling with BODIPY acid produced the target, BI-BODIPY. With Mps1-IN-BODIPY and BI-BODIPY in hand, we next performed dose-response experiments using a combination of biochemical and cellular assays to assess whether conjugation to BODIPY negatively affected the binding of the compounds to their kinase targets. Conjugation to BODIPY appeared to significantly reduce the activity of Mps-1-IN, as evidenced by a greater than ten-fold decrease in its binding affinity for recombinant Mps1 (Fig. 1A). Based on this result, we decided not to continue development of.4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)10 was determined as the fluorescent dye because it has a high quantum yield, it is cell Rabbit Polyclonal to MAP3K7 (phospho-Thr187) permeable, and its derivatives have been widely used to label proteins11C16 and DNA.17, 18 Synthesis of BODIPY-conjugated derivatives of Mps1-IN-1 and BI2536 (Mps1-IN-BODIPY, and BI-BODIPY, respectively) were adapted from published syntheses of the parent compounds1, 19 and additional actions to conjugate BODIPY are highlighted in Techniques 1 and ?and2,2, respectively. Open in a separate window Scheme 1 Synthesis of Mps1-IN-BODIPY. useful in live-cell and imaging experiments; moreover, unlike the expression of EGFP-fusion proteins, small molecule probes do not require genetic manipulation of cells. Protein kinases are in many ways ideal targets for the development of selective fluorescent small molecule probes. This is because protein kinases are involved in most cellular processes and changes in their localization, convenience, and large quantity are associated with changes in cellular state. Protein kinases have been used as biomarkers in malignancy biology because the loss of endogenous kinase regulatory mechanisms by point mutations, gene deletions, gene amplifications, and chromosomal rearrangements has been well-established as a crucial event in many cancers. In addition, drug discovery and chemical biology efforts have in recent decades produced many selective, cell-permeable small molecule ligands of specific cellular kinases. Here we describe our initial attempts to leverage existing, well-characterized kinase inhibitors to develop fluorescent small molecule probes for use as imaging tools in malignancy biology. For this effort, we focused on Mps1-IN-1, a recently described inhibitor of the monopolar spindle 1 kinase (Mps1),1 and BI2536, a potent inhibitor of polo-like kinases (PLK1, PLK2, and PLK3).2, 3 These inhibitors target kinases that regulate cell cycle progression and that localize to distinct subcellular structures during mitosis. Mps1 is usually a dual-specificity kinase whose activity is essential for the establishment and sustained activity of the spindle checkpoint.4C6 The polo-like kinases (PLKs) comprise a family of conserved serine/threonine kinases that are highly conserved from yeast to humans7 and that are known to regulate cell cycle progression.2, 3, 8, 9 We reasoned that this synthesis and use of Mps1- and PLK-selective small molecules probes could facilitate the study of these kinases in different phases of the cell cycle. In addition, such probes might be able to elucidate deregulated says and find use as malignancy diagnostics. Towards this end, we endeavoured to convert Mps1-IN-1 and BI2536 to fluorescent probes of their respective kinase targets by conjugation to a cell-permeable fluorophore. 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)10 was selected as the fluorescent dye because it has a high quantum yield, it is cell permeable, and its derivatives have been widely used to label protein11C16 and DNA.17, 18 Synthesis of BODIPY-conjugated derivatives of Mps1-IN-1 and BI2536 (Mps1-IN-BODIPY, and BI-BODIPY, respectively) were adapted from published syntheses from the mother or father substances1, 19 and extra measures to conjugate BODIPY are highlighted in Strategies 1 and ?and2,2, respectively. Open up in another window Structure 1 Synthesis of Mps1-IN-BODIPY. Response and circumstances: (i) t-butyl 4-(1-(4-amino-3-methoxyphenyl)piperidin-4-yl)piperazine-1-carboxylate K2CO3, Pd2(dba)3, X-Phos, t-BuOH, 100 C; (ii) TFA, DCM, 0 C; LiOH?H2O, 25 C; (iii) 3-(2-carboxyethyl)-5,5-difluoro-7,9-dimethyl-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide (BODIPY acidity), DCC, DMAP, THF, 0C25 C. Open up in another window Structure 2 Synthesis of BI-BODIPY. Response and circumstances: (i) t-butyl 4-((1R,4R)-4-aminocyclohexyl)piperazine-1-carboxylate, HBTU, DIEA, DMF, 25 C; (ii) TFA, DCM, 25 C; (iii) 3-(2-carboxyethyl)-5,5-difluoro-7,9-dimethyl-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide (BODIPY-acid), DCC, DMAP, THF, 0C25 C. For Mps1-IN-BODIPY, substance 1, that was prepared by following a reported treatment1, was reacted with tert-butyl 4-(1-(4-amino-3-methoxyphenyl)piperidin-4-yl)piperazine-1-carboxylate to provide 2. Deprotection of 2 accompanied by amide coupling using the BODIPY acidity created the prospective Mps1-IN-BODIPY. For BI-BODIPY, substance 3, prepared relating to previously reported methods,19 was in conjunction with t-butyl 4-((1R,4R)-4-aminocyclohexyl)piperazine-1-carboxylate to cover 4.19 Acid mediated removal of the t-butoxycarbonyl band of 4 accompanied by amide coupling with.1A). like a fusion having a fluorescent proteins circumvents the necessity for fixation but requires prior hereditary manipulation that’s not compatible with evaluation of clinical examples and major cells. Fluorescent little molecule probes that bind particularly to a proteins appealing may present some advantages of these regular strategies. Unlike antibodies, little molecule probes could be cell permeable and could be useful in live-cell and imaging tests therefore; furthermore, unlike the manifestation of EGFP-fusion protein, little molecule probes usually do not need hereditary manipulation of cells. Proteins kinases are in lots of ways ideal focuses on for the introduction of selective fluorescent little molecule probes. It is because proteins kinases get excited about most mobile processes and adjustments within their localization, availability, and great quantity are connected with adjustments in mobile state. Proteins kinases have already DEL-22379 been utilized as biomarkers in tumor biology as the lack of endogenous kinase regulatory systems by stage mutations, gene deletions, gene amplifications, and chromosomal rearrangements continues to be well-established as an essential event in lots of cancers. Furthermore, drug finding and chemical substance biology efforts possess in recent years created many selective, cell-permeable little molecule ligands of particular mobile kinases. Right here we explain our initial efforts to leverage existing, well-characterized kinase inhibitors to build up fluorescent little molecule probes for make use of as imaging equipment in tumor biology. Because of this work, we centered on Mps1-IN-1, a lately described inhibitor from the monopolar spindle 1 kinase (Mps1),1 and BI2536, a potent inhibitor of polo-like kinases (PLK1, PLK2, and PLK3).2, 3 These inhibitors focus on kinases that regulate cell routine progression which localize to distinct subcellular constructions during mitosis. Mps1 can be a dual-specificity kinase whose activity is vital for the establishment and suffered activity of the spindle checkpoint.4C6 The polo-like kinases (PLKs) comprise a family group of conserved serine/threonine kinases that are highly conserved from candida to human beings7 which are recognized to regulate cell routine development.2, 3, 8, 9 We reasoned how the synthesis and usage of Mps1- and PLK-selective little substances probes could facilitate the analysis of the kinases in various phases from the cell routine. Furthermore, such probes could probably elucidate deregulated claims and find use as malignancy diagnostics. Towards this end, we endeavoured to convert Mps1-IN-1 and BI2536 to fluorescent probes of their respective kinase focuses on by conjugation to a cell-permeable fluorophore. 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)10 was selected as the fluorescent dye because it has a high quantum yield, it is cell permeable, and its derivatives have been widely used to label proteins11C16 and DNA.17, 18 Synthesis of BODIPY-conjugated derivatives of Mps1-IN-1 and BI2536 (Mps1-IN-BODIPY, and BI-BODIPY, respectively) were adapted from published syntheses of the parent compounds1, 19 and additional methods to conjugate BODIPY are highlighted in Techniques 1 and ?and2,2, respectively. Open in a separate window Plan 1 Synthesis of Mps1-IN-BODIPY. Reaction and conditions: (i) t-butyl 4-(1-(4-amino-3-methoxyphenyl)piperidin-4-yl)piperazine-1-carboxylate K2CO3, Pd2(dba)3, X-Phos, t-BuOH, 100 C; (ii) TFA, DCM, 0 C; LiOH?H2O, 25 C; (iii) 3-(2-carboxyethyl)-5,5-difluoro-7,9-dimethyl-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide (BODIPY acid), DCC, DMAP, THF, 0C25 C. Open in a separate window Plan 2 Synthesis of BI-BODIPY. Reaction and conditions: (i) t-butyl 4-((1R,4R)-4-aminocyclohexyl)piperazine-1-carboxylate, HBTU, DIEA, DMF, 25 C; (ii) TFA, DCM, 25 C; (iii) 3-(2-carboxyethyl)-5,5-difluoro-7,9-dimethyl-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide (BODIPY-acid), DCC, DMAP, THF, 0C25 C. For Mps1-IN-BODIPY, compound 1, which was prepared by following a reported process1, was reacted with tert-butyl 4-(1-(4-amino-3-methoxyphenyl)piperidin-4-yl)piperazine-1-carboxylate to give 2. Deprotection of 2 followed by amide coupling with the BODIPY acid produced the prospective Mps1-IN-BODIPY. For BI-BODIPY, compound 3, prepared relating to previously reported methods,19 was coupled with t-butyl 4-((1R,4R)-4-aminocyclohexyl)piperazine-1-carboxylate to afford 4.19 Acid mediated removal of the t-butoxycarbonyl group of 4 followed by amide coupling with BODIPY acid produced the prospective, BI-BODIPY. With Mps1-IN-BODIPY and BI-BODIPY in hand, we next performed dose-response experiments using a combination of biochemical and cellular assays to assess whether conjugation to BODIPY negatively affected the binding of the compounds to their kinase focuses on. Conjugation to BODIPY appeared to significantly reduce the activity of Mps-1-IN, as evidenced by a greater than ten-fold decrease in its binding affinity for recombinant Mps1 (Fig. 1A). Based on this result, we decided not to continue development of this compound like a fluorescent probe of Mps1. In contrast, BI-BODIPY inhibited PLK1, PLK2, and PLK3 in biochemical kinase assays (Fig. 1B) with activities comparable to that of BI25363, demonstrating that linker-modification and conjugation to BODIPY did not significantly alter the biochemical activity of BI2536. To determine if BI-BODIPY elicits a cellular phenotype similar to that of the parent compound, we analysed its effects on cell cycle progression. PLK1 is definitely a critical regulator of cell cycle progression,.Hela S3 cells were arrested in the G1/S transition by 24-hour treatment with thymidine. proteins, small molecule probes do not require genetic manipulation of cells. Protein kinases are in many ways ideal focuses on for the development of selective fluorescent small molecule probes. This is because protein kinases are involved in most cellular processes and changes in their localization, convenience, and large quantity are associated with changes in cellular state. Protein kinases have been used as biomarkers in malignancy biology because the loss of endogenous kinase regulatory mechanisms by point mutations, gene deletions, gene amplifications, and chromosomal rearrangements has been well-established as a crucial event in many cancers. In addition, drug finding and chemical biology efforts possess in recent decades produced many selective, cell-permeable small molecule ligands of specific cellular kinases. Here we describe our initial efforts to leverage existing, well-characterized kinase inhibitors to develop fluorescent small molecule probes for use as imaging tools in malignancy biology. For this effort, we focused on Mps1-IN-1, a recently described inhibitor of the monopolar spindle 1 kinase (Mps1),1 and BI2536, a potent inhibitor of polo-like kinases (PLK1, PLK2, and PLK3).2, 3 These inhibitors target kinases that regulate cell cycle progression and that localize to distinct subcellular constructions during mitosis. Mps1 is definitely a dual-specificity kinase whose activity is essential for the establishment and sustained activity of the spindle checkpoint.4C6 The polo-like kinases (PLKs) comprise a family of conserved serine/threonine kinases that are highly conserved from candida to humans7 and that are known to regulate cell routine development.2, 3, 8, 9 We reasoned the fact that synthesis and usage of Mps1- and PLK-selective little substances probes could facilitate the analysis of the kinases in various phases from the cell routine. Furthermore, such probes could probably elucidate deregulated expresses and find make use of as cancers diagnostics. Towards this end, we endeavoured to convert Mps1-IN-1 and BI2536 to fluorescent probes of their particular kinase goals DEL-22379 by conjugation to a cell-permeable fluorophore. 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)10 was chosen as the fluorescent dye since it includes a high quantum produce, it really is cell permeable, and its own derivatives have already been trusted to label protein11C16 and DNA.17, 18 Synthesis of BODIPY-conjugated derivatives of Mps1-IN-1 and BI2536 (Mps1-IN-BODIPY, and BI-BODIPY, respectively) were adapted from published syntheses from the mother or father substances1, 19 and extra guidelines to conjugate BODIPY are highlighted in Plans 1 and ?and2,2, respectively. Open up in another window System 1 Synthesis of Mps1-IN-BODIPY. Response and circumstances: (i) t-butyl 4-(1-(4-amino-3-methoxyphenyl)piperidin-4-yl)piperazine-1-carboxylate K2CO3, Pd2(dba)3, X-Phos, t-BuOH, 100 C; (ii) TFA, DCM, 0 C; LiOH?H2O, 25 C; (iii) 3-(2-carboxyethyl)-5,5-difluoro-7,9-dimethyl-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide (BODIPY acidity), DCC, DMAP, THF, 0C25 C. Open up in another window System 2 Synthesis of BI-BODIPY. Response and circumstances: (i) t-butyl 4-((1R,4R)-4-aminocyclohexyl)piperazine-1-carboxylate, HBTU, DIEA, DMF, 25 C; (ii) TFA, DCM, 25 C; (iii) 3-(2-carboxyethyl)-5,5-difluoro-7,9-dimethyl-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide (BODIPY-acid), DCC, DMAP, THF, 0C25 C. For Mps1-IN-BODIPY, substance 1, that was prepared by following reported method1, was reacted with tert-butyl 4-(1-(4-amino-3-methoxyphenyl)piperidin-4-yl)piperazine-1-carboxylate to provide 2. Deprotection of 2 accompanied by amide coupling using the BODIPY acidity created the mark Mps1-IN-BODIPY. For BI-BODIPY, substance 3, prepared regarding to previously reported techniques,19 was in conjunction with t-butyl 4-((1R,4R)-4-aminocyclohexyl)piperazine-1-carboxylate to cover 4.19 Acid mediated removal of the t-butoxycarbonyl band of 4 accompanied by amide coupling with BODIPY acid created the mark, BI-BODIPY. With Mps1-IN-BODIPY and BI-BODIPY at hand, we following performed dose-response tests using a mix of biochemical and mobile assays to evaluate whether conjugation to BODIPY adversely affected the binding from the compounds with their kinase goals. Conjugation to BODIPY seemed to significantly decrease the activity of Mps-1-IN, as evidenced by a larger than ten-fold reduction in its binding affinity for recombinant Mps1 (Fig. 1A). Predicated on this result, we didn’t continue development of the compound being a fluorescent probe of Mps1. On the other hand, BI-BODIPY inhibited PLK1, PLK2, and PLK3 in biochemical kinase assays (Fig. 1B) with actions much like that of BI25363, demonstrating that linker-modification and conjugation to BODIPY didn’t considerably alter the biochemical activity of BI2536. To see whether BI-BODIPY elicits a mobile phenotype similar compared to that from the mother or father substance, we analysed its results on cell routine progression. PLK1 is certainly a crucial regulator of cell routine development, and both pharmacological inhibition of PLK1.Lowery DM, Lim D, Yaffe MB. little molecule probes could be cell permeable and could therefore end up being useful in live-cell and imaging tests; furthermore, unlike the appearance of EGFP-fusion protein, little molecule probes usually do not need hereditary manipulation of cells. Proteins kinases are in lots of ways ideal goals for the introduction of selective fluorescent little molecule probes. It is because proteins kinases get excited about most mobile processes and adjustments within their localization, ease of access, and plethora are connected with adjustments in mobile state. Proteins kinases have already been utilized as biomarkers in cancers biology as the lack of endogenous kinase regulatory systems by stage mutations, gene deletions, gene amplifications, and chromosomal rearrangements continues to be well-established as a crucial event in many cancers. In addition, drug discovery and chemical biology efforts have in recent decades produced many selective, cell-permeable small molecule ligands of specific cellular kinases. Here we describe our initial attempts to leverage existing, well-characterized kinase inhibitors to develop fluorescent small molecule probes for use as imaging tools in cancer biology. For this effort, we focused on Mps1-IN-1, a recently described inhibitor of the monopolar spindle 1 kinase (Mps1),1 and BI2536, a potent inhibitor of polo-like kinases (PLK1, PLK2, and PLK3).2, 3 These inhibitors target kinases that regulate cell cycle progression and that localize to distinct subcellular structures during mitosis. Mps1 is a dual-specificity kinase whose activity is essential for the establishment and sustained activity of the spindle checkpoint.4C6 The polo-like kinases (PLKs) comprise a family of conserved serine/threonine kinases that are highly conserved from yeast to humans7 and that are known to regulate cell cycle progression.2, 3, 8, 9 We reasoned that the synthesis and use of Mps1- and PLK-selective small molecules probes could facilitate the study of these kinases in different phases of the cell cycle. In addition, such probes might be able to elucidate deregulated states and find use as cancer diagnostics. Towards this end, we endeavoured to convert Mps1-IN-1 and BI2536 to DEL-22379 fluorescent probes of their respective kinase targets by conjugation to a cell-permeable fluorophore. 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)10 was selected as the fluorescent dye because it has a high quantum yield, it is cell permeable, and its derivatives have been widely used to label proteins11C16 and DNA.17, 18 Synthesis of BODIPY-conjugated derivatives of Mps1-IN-1 and BI2536 (Mps1-IN-BODIPY, and BI-BODIPY, respectively) were adapted from published syntheses of the parent compounds1, 19 and additional steps to conjugate BODIPY are highlighted in Schemes 1 and ?and2,2, respectively. Open in a separate window Scheme 1 Synthesis of Mps1-IN-BODIPY. Reaction and conditions: (i) t-butyl 4-(1-(4-amino-3-methoxyphenyl)piperidin-4-yl)piperazine-1-carboxylate K2CO3, Pd2(dba)3, X-Phos, t-BuOH, 100 C; (ii) TFA, DCM, 0 C; LiOH?H2O, 25 C; (iii) 3-(2-carboxyethyl)-5,5-difluoro-7,9-dimethyl-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide (BODIPY acid), DCC, DMAP, THF, 0C25 C. Open in a separate window Scheme 2 Synthesis of BI-BODIPY. Reaction and conditions: (i) t-butyl 4-((1R,4R)-4-aminocyclohexyl)piperazine-1-carboxylate, HBTU, DIEA, DMF, 25 C; (ii) TFA, DCM, 25 C; (iii) 3-(2-carboxyethyl)-5,5-difluoro-7,9-dimethyl-5H-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-4-ium-5-uide (BODIPY-acid), DCC, DMAP, THF, 0C25 C. For Mps1-IN-BODIPY, compound 1, which was prepared by following the reported procedure1, was reacted with tert-butyl 4-(1-(4-amino-3-methoxyphenyl)piperidin-4-yl)piperazine-1-carboxylate to give 2. Deprotection of 2 followed by amide coupling with the BODIPY acid produced the target Mps1-IN-BODIPY. For BI-BODIPY, compound 3, prepared according to previously reported procedures,19 was coupled with t-butyl 4-((1R,4R)-4-aminocyclohexyl)piperazine-1-carboxylate to afford 4.19 Acid mediated removal of the t-butoxycarbonyl group of 4 followed by amide coupling with BODIPY acid produced the target, BI-BODIPY. With Mps1-IN-BODIPY and BI-BODIPY in hand, we next performed dose-response experiments using a combination of biochemical and cellular assays to assess whether conjugation to BODIPY negatively affected the binding of the compounds to their kinase targets. Conjugation to BODIPY appeared to significantly reduce the activity of Mps-1-IN, as evidenced by a greater than ten-fold decrease in its binding affinity for recombinant Mps1 (Fig. 1A). Based on this result, we decided not to continue development of this compound as a fluorescent probe of.