Functionalized Nitrobenzothiadiazoles as Embedded Fluorescent Probes

Abstract

Described herein is the synthesis and photophysical characterization of novel dipeptidomimetic fluorogenic probes. The new dipeptidomimetic cassette is based on the environment-sensitive fluorophore nitrobenzothiadiazole (NBTD) and is designed to be embedded within a polypeptide backbone, not attached as part of a conformationally unrestrained amino acid side chain. Compounds 1-6 were prepared to investigate the effect of introducing a methyl group at positions 5 and 6 of the nitrobenzothiadiazole scaffold on their photophysical properties. Additionally, we investigated the effect of N-methylation of the 4-amino group on their chemical and photophysical properties. The nitrobenzothiadiazole scaffold was functionalized with amino- and carboxy-termini, resulting in a conformationally restricted dipeptidomimetic scaffold (NBTD-Gly) that can potentially be embedded within peptides or proteins of interest and used as a biophysical tool for studying protein structure and function. Thus, we investigated the synthesis and photophysical properties of functionalized nitrobenzothiadiazoles (1-11). The amino-terminus of the dipeptidomimetic scaffold (NBTD-Gly) was introduced at the benzylic position of the benzothiadiazole core using Gabriel synthesis, followed by nitration. The carboxy terminus was introduced via nucleophilic aromatic substitution as part of the glycine moiety. Subsequent steps involved the removal of a phthaloyl protecting group and a condensation reaction to form protected dipeptidomimetic analogues 7-11.

Figure 1.

Chemical structures of representative examples of reported noncanonical solvatochromic fluorescent benzo(heteroatom)diazole amino acids, including Fmoc-l-Dap(NBTD)-OH (i), Fmoc-l-Dap(NBD)-OH (ii), and Fmoc-l-Dap(NBSeD)-OH (iii). Chemical structure of dipeptidomimetic oxazole amino acid (iv). Chemical structure of proposed nitrobenzothiadiazole glycine dipeptidomimetic scaffold (NBTD-Gly) (v).

Figure 2.

Chemical structures of nitrobenzothiadiazoles synthesized and studied (NBTDs 1–11).

Figure 3.

(A) Normalized UV–vis spectra and (B) fluorescence emission spectra of NBTDs 1–6 in toluene (all at 0.1 abs, excitation was carried out at individual NBTD UV–vis maximum wavelengths).

Figure 4.

(A) The distribution of torsional angle (ϕ) around the C4NBTD–N bond in NBTD 3 (solid blue line) and NBTD 4 (dotted red line) in the ground state from molecular dynamics (MD) simulations. The torsional angle values for the optimized conformation at the ground state for NBTD 3 and NBTD 4 were found to be ϕ = 00 and ϕ = 8.40, respectively. ϕ is denoted as the torsional angle between the NBTD unit and the glycine tert-butyl ester or its N-methylated glycine tert-butyl ester planes when viewed along the C4–N bond. (B) Illustration of NBTD 4 and its ICT excited states, showing the process of twisted internal charge transfer (TICT). Unmethylated NBTD derivative (3) shows a flat conformation (ϕ = 00) plausibly due to the reduced allylic strain, whereas the calculated ground state conformation of NBTD 4 shows a small torsional angle (ϕ = 8.40). Upon excitation, compound 3 thus seems to attain a planar internal charge transfer (PICT) state readily; whereas compound 4 may initially generate a partially twisted ICT state which is further twisted to form a fully twisted ICT state. (C) The comparison of S1 potential energy surface (PESs) as a function of θ (from the LE to the TICT states) of NBTD 3 (blue) and NBTD 4 (red). The rotation barrier (ERB) and driving energy (EDE) for TICT are labeled in the inset.

Figure 5.

(A) Normalized UV–vis spectra and (B) fluorescence emission spectra of dipeptidomimetic nitrobenzothiazole (NBTD-Gly, 9) in various solvents (toluene, dioxane, ethanol, PBS buffer (containing 20% DMSO)) (all at 0.1 abs, excitation was carried out at their corresponding UV–vis maximum wavelength).

Figure 6.

(A) Normalized UV–vis spectra and (B) fluorescence emission spectra of environment-sensitive dipeptidomimetic nitrobenzothiazole (NBTD 9) in mixtures of 1,4-dioxane and water (containing 20% DMSO) at 25 °C (excitation at 460 nm).

Scheme 1.

Synthetic Routes Employed for the Preparation of NBTD Derivatives 1 and 2

Scheme 2.

Synthetic Routes Employed for the Preparation of NBTD Derivatives 3 and 4

Scheme 3.

Synthetic Routes Employed for the Preparation of NBTD Derivatives 5 and 6

Scheme 4.

Synthetic Routes Employed for the Preparation of NBTD Derivatives 7, 8, 9, 10, and 11