Synthesis of a macrocyclic rhodamine 110 enzyme substrate as an intracellular probe for caspase 3 activity

Tetrahedron Letters

Volumn 41, Issue 24, 2000, Pages 4733-4735

  Guzikowski, Anthony P ^a   Naleway, John J ^a   Shipp, Christina T ^a   Schutte, Rod C ^a  

^a UNIVERSITY OF OREGON   (United States)

Author keywords

Cyclization; Enzymes and enzyme reactions; Fluorescence; Macrocycle

Indexed keywords

CASPASE 3; CASPASE INHIBITOR; RHODAMINE;

ARTICLE; CYCLIZATION; ENZYME ACTIVITY; ENZYME SUBSTRATE COMPLEX; MOLECULAR INTERACTION; SIGNAL DETECTION; SYNTHESIS;

EID: 0034659894     PISSN: 00404039     EISSN: None     Source Type: Journal    
DOI: 10.1016/S0040-4039(00)00708-5     Document Type: Article

References (13)

1. Gurtu, V.; Kain, S. R.; Zhang, G. Anal. Biochem. 1997, 251, 98-102.
2. Molecular Probes, Inc., Eugene, OR, USA.
3. (a) Greene, T. W. Protective Groups in Organic Synthesis; John Wiley & Sons: New York, 1981; pp. 225-226.
4. (b) ibid pp. 273-274.
5. Schmitt, W.; Zanotti, G.; Wieland, T.; Kessler, H. J. Am. Chem. Soc. 1996, 118, 4380-4387.
6. Peptide 3 was purchased from BioMol Research Laboratories, Plymouth Meeting, PA, USA.
7. N-Tr-6-Aminohexanoic acid was prepared by the reaction of methyl 6-aminohexanoate hydrochloride with Tr-chloride (DMF, DIEA) followed by saponification. The corresponding mixed anhydride was prepared by treating the protected amino acid with IBCF in the presence of NMM in THF at -10°C.
8. The preparation of a cyclic peptide employing a 4-nitrophenyl ester has been reported. Studer, R. O.; Lergier, W. Helv. Chim. Acta 1965, 460-470.
9. The mass spectral analysis was performed by Mass Consortium Corp., San Diego, CA, USA.
10. 2O) δ 0.97-1.80 (m, 6H), 1.21 (t, J=7 Hz, 27H), 1.36-2.78 (m, 28H), 2.97 (q, J=7 Hz, 18H), 4.06 (t, J=3 Hz, 1H), 4.38 (bs, 1H), 4.68-4.80 (m, 1H), 6.64 (d, J=9 Hz, 1H), 6.65 (d, J=9 Hz, 1H), 7.11 (d, J=9 Hz, 1H), 7.23 (d, J=8 Hz, 1H), 7.50-7.83 (m, 4H), 7.88-7.93 (m, 1H), 8.02 (d, J=8 Hz, 1H).
11. Assay conditions: [8]=10 μM; Recombinant caspase 3 was prepared in bacterial cell cultures and crude preparations were diluted to give a measurable fluorescent signal. An 1:50000 dilution gave about 1300 counts/h over 3 h. An 1:1000 dilution gave about 5000 counts/h over 12 h. Virtually no fluorescence was detected in the absence of recombinant caspase 3.
12. Garcia-Calvo, M.; Peterson, E. P.; Leiting, B.; Ruel, R.; Nicholson, D. W.; Thornberry, N. A. J. Biol. Chem. 1998, 273, 32608-32613. Experiments performed as described in Ref 10 but with the addition of Ac-DEVD-CHO (10 μM).
13. Assay conditions: NIH3T3 (mouse endothelial) and D5 (mouse melanoma) cell lines (ATCC, Rockville, MD), induced for apoptosis by treatment with etoposide (50 μg/mL in RPMI1640 media+10% CS for 18 h), with [8]=50 μM (for 1 h) showed significant and cell-specific labeling. These same cell lines were also stained with the fluorescent caspase 3 cell permeable inhibitor FITC-VAD-FMK (Promega Corp., Madison, WI) to verify apoptosis induction. Non-induced cell lines showed little or no detectable labeling under identical conditions.