Synthesis of a macrocyclic rhodamine 110 enzyme substrate as an intracellular probe for caspase 3 activity Anthony P. Guzikowski,* John J. Naleway, Christina T. Shipp and Rod C. Schutte Marker Gene Technologies Inc., University of Oregon Riverfront Research Park, 1850 Millrace Dr., Eugene, OR 97403-1927, USA Received 16 November 1999; revised 24 April 2000; accepted 27 April 2000 Abstract The synthesis of the macrocyclic rhodamine 110 caspase 3 substrate 8 is described. The key step is a high dilution intramolecular cyclization reaction of an in situ generated primary amine with a 4-nitrophenyl ester. Substrate 8 reacts with recombinant caspase 3 to yield a ¯uorescent signal but virtually no signal is detected in the absence of caspase 3 or in the presence of the caspase 3 inhibitor Ac-DEVD-CHO. Notably, 8 selectively stains live cells that have been induced to undergo apoptosis with etoposide. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: cyclization; enzymes and enzyme reactions; ¯uorescence; macrocycle. Fluorogenic enzyme substrates oer a convenient and sensitive means to measure enzyme activity. Substrates incorporating the peptide sequence DEVD and a ¯uorophore such as 7-amino- 4-tri¯uoromethylcoumarin 1 or rhodamine 110 2 (1, R110) have been prepared to measure caspase 3 activity in studies of apoptosis. However, these substrates cannot measure caspase 3 activity in intact cells since they are impermeable to the cell membrane. Masking the charge of the free car- boxy groups of the glutamate and aspartate residues may improve substrate permeability of the cell membrane. We reasoned that the synthesis of a macrocyclic DEVD substrate may mask these charges since the carboxy groups should be more prone toward intramolecular hydrogen bonding compared to a non-cyclic analog. Therefore, we initiated studies into the synthesis of the macro- cyclic DEVD-R110 substrate 8. Initially, the mono-protected R110 analog 2 was prepared by the reaction of 1 with 1 equiv. of FMOC chloride and N,N-diisopropylethylamine (DIEA) in DMF (35±40%, Scheme 1). 3a Com- pound 2 was coupled to a suitably protected DEVD peptide. The a,a-dimethyl-3,5-dimethoxy- benzyloxycarbonyl (Ddz) group was chosen for protection of the terminal amine 4 while t-butyl esters were employed for the protection of the side chain carboxy groups. 5 The coupling of 2 and 0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0040-4039(00)00708-5 Tetrahedron Letters 41 (2000) 4733±4735 * Corresponding author. Tel: 541-342-3760; fax: 541-687-7963; e-mail: apguzik@oregon.uoregon.edu