Development of a fluorogenic small substrate for dipeptidyl peptidase-4

Abstract

A series of aniline and m-phenylenediamine derivatives with electron-withdrawing 3,3,3-trifluoropropenyl substituents were synthesized as small and chemically stable fluorescent organic compounds. Their fluorescence performances were evaluated by converting 2,4-disubstituted aniline 1 to the non-fluorescent dipeptide analogue H-Gly-Pro-1 for the use as a fluorogenic substrate for dipeptidyl peptidase-4 (DPP-4). The progress of the enzymatic hydrolysis of H-Gly-Pro-1 with DPP-4 was monitored by fluorometric determination of 1 released into the reaction medium. The results suggest that 1 could be used as fluorophore in OFF–ON-type fluorogenic probes.

Synthesis of Small Fluorescent Molecules and Evaluation of Photophysical Properties

“…Aminopeptidase N was prepared from the Escherichia coli XL1-Blue containing pAN14 as described previously . The enzyme activity was assayed using H-Ala- 3f as a substrate following the procedure already described . The reaction mixture (total 100 μL) contained 0.1 M Tris-HCl (pH 7.0), 10–200 μM 3f , and 0.1–10 ng of the enzyme.…”

“…The title product was purified by column chromatography and was obtained in 68% yield (127 mg). A colorless solid; mp 48−49 °C (recrystallized from AcOEt and hexane); 1 H NMR (CDCl 3 ) δ: 3.81 (2H, br s), 6.13 (1H,dq,J = 16.0,6.4 Hz),6.72 (1H,dd,J = 8.0,0.8 Hz),m),m),7.25 (1H, dq, J = 16.0, 2.0 Hz), 7.29 (1H, dd, J = 7.6, 1.2 Hz); 13 C{ 1 H} NMR (CDCl 3 ) δ: 116.6 (q, J = 33.4 Hz), 116.8,119.2,119.4,123.6 (q,J = 267.4 Hz),127.9,130.9,133.3 (q, J = 6.7 Hz), 144.8; 19 F NMR (CDCl 3 ) δ: −63.03 (3F, dd, J = 6.4, 1.9 Hz); MS m/z 187 (M + ), 148, 118, 91; HRMS calcd for C 9 H 8 F 3 N 187.0609 (M + ), found 187.0603.…”

“…12 The enzyme activity was assayed using H-Ala-3f as a substrate following the procedure already described. 7 The reaction mixture (total 100 μL) contained 0.1 M Tris-HCl (pH 7.0), 10−200 μM 3f, and 0.1−10 ng of the enzyme. The reaction was initiated by the addition of the enzyme solution.…”

“…These fluorescence properties in aqueous solution enabled 2,4-TFPEaniline to be used as the fluorophore in a fluorogenic probe for dipeptidyl peptidase-4 (DPP-4). 7 In the course of our work on synthesizing small and high fluorescent compounds based on the 2-TFPE-aniline moiety, we found that 5-CN substituted for 2-TFPE-aniline (2-TFPE-5-CN-aniline) exhibited excellent fluorescent performance even in aqueous solution. In this paper, we describe the synthesis, fluorescent properties, and preliminary results of 2-TFPE-5-CN-aniline used as a fluorogenic probe for aminopeptidase N. It has been reported that benzene derivatives with a push− pull system have the propensity to become fluorescent compounds, despite the structure of a less extended π-system compared with polycyclic fluorophores.…”

“…Among them, 2,4-disubstituted aniline (2,4-TFPE-aniline) gave fluorescence quantum yields (Φ f ) of 0.27 at 461 nm even in an aqueous solution (water/DMSO = 9:1). These fluorescence properties in aqueous solution enabled 2,4-TFPE-aniline to be used as the fluorophore in a fluorogenic probe for dipeptidyl peptidase-4 (DPP-4) …”