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.

Graphical Abstract

INTRODUCTION

Peptides and proteins are cornerstones of the cellular machinery, essential for many biochemical processes. Appropriate fluorogenic labeling of peptides and proteins, which enables real-time imaging studies, is of key importance to enable analysis of their function at a molecular level, including their intermolecular interactions and cellular localization.^1-5 Many of these labels are attached to reactive side chains or to the N- or C-terminus, which has the potential to significantly impact the characteristics of the peptide or protein.^6,7 Unlike fluorescent fusion proteins⁸ and self-labeling tags⁹ which involve large structural modifications, small fluorescent amino acid analogues have emerged as alternative labeling tools for selective labeling of peptides and proteins.^10-12 Environmentally sensitive fluorescent amino acids may exhibit a change in fluorescence intensity or wavelength as the environment surrounding the amino acid changes from polar to apolar or vice versa, offering the advantage of a greater signal-to-noise ratio.^12-16 Over the past few years, several benzodiazole-based amino acids that possess solvatochromic fluorophores have been described and are of increasing interest (Figure 1i-iii).^15,16

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).

Benzo(heteroatom)diazole (BD, heteroatom X = O, S, Se) derivatives have been utilized in many biological studies due to their small size, charge neutrality, significant solvatochromism and environmental sensitivities.^17-20 These probes are valuable for live-cell imaging, where they can be used to visualize the localization and dynamics of specific targets within cells without the need for extensive washing procedures.^15,16 Here we focus on nitrobenzothiadiazoles (X = S) (NBTDs) as the core structure because these have been shown to exhibit far greater photostability than congeners where X = O, Se with minimal effects on other photophysical properties such as wavelength or quantum yield; they also exhibited greater chemical stability.^21-23

Another important issue is the strategy used to embed these probes within peptides and proteins, thus enabling control of dipole orientation.^24-28 Recently, we introduced conformationally constrained fluorescent azole-containing dipeptidomimetics into peptides and proteins by chemical synthesis or by the use of modified bacterial ribosomes; the derivatized products absorbed and emitted in the UV–visible spectral region (Figure 1iv). When an oxazole-based fluorescent dipeptidomimetic was successfully introduced at position 66 of green fluorescent protein (GFP) the modified GFP analogue emitted with ~10-fold greater fluorescence intensity compared to wild-type.²⁶ In general, short-wavelength UV spectral region fluorophores are not suitable for continuous observation using live-cell imaging due to their phototoxicity and exhibit excitation and emission wavelengths not detectable using lasers commonly associated with confocal microscopes. Thus, utilizing fluorophores that function at longer wavelengths is an attractive alternative. Benzothiadiazole derivatives are capable of achieving longer wavelength emission through reasonable structural modifications.²² Presently, the synthesis and photophysical properties of nitrobenzothiadiazole based dipeptidomimetics (e.g., NBTD-Gly) (Figure 1v) are described. This approach involves constructing the fluorescent units within the peptide backbone rather than as part of amino acid side chains.

The nitrobenzothiadiazole scaffold was functionalized with amino- and carboxy-termini, resulting in a conformationally restricted dipeptidomimetic scaffold (NBTD-Gly), that can be embedded within a peptide or protein of interest and used as a biophysical tool for studying protein structure and function. This represents a general strategy designed to take advantage of the ability to impose local conformational restraints to enhance fluorescent sensitivity and to expand the chemical diversity of the proteome beyond and in synergy with what can presently be accomplished by expanding the genetic code. The use of the prepared NBTD-Gly dipeptidomimetic for developing novel fluorescent probes with enhanced sensitivity for biological applications will be reported in due course.

RESULTS AND DISCUSSION

Compound Synthesis and Characterization.

The objective of this study was the synthesis and characterization of fluorescent properties of a new class of benzothiadiazole (BTD) based dipeptidomimetic scaffold (NBTD-Gly). Substitution of the BTD nucleus, primarily with C4 or C7 substituents, has been shown to be a facile method for tuning the dipole moment and absorption of the fluorophore, because these positions contribute directly to the push–pull dipole of the fluorophore. We introduced modifications at positions 4, 5, or 6 of the parent NBTD nucleus (1) (Figure 2) to investigate their impact on the photophysical properties of this nucleus.

Figure 2.

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

Initially, we synthesized NBTD analogues 1–6 (Figure 2). Compounds 1 and 2 were synthesized from commercially available 4-chloro-7-nitro-2,1,3-benzothiadiazole (12) in one step via nucleophilic aromatic substitution as depicted in Scheme 1. The synthesis of NBTD derivatives 3 and 4 began with commercially available 2,3-difluoro-1-methyl-4-nitroben-zene, as outlined in Scheme 2. An 89% yield of 2-fluoro-3-methyl-6-nitrobenzenamine (13) was realized by nucleophilic aromatic substitution of the activated fluoride ortho to the nitro group using ammonium hydroxide in THF. The nitro substituent in 13 was reduced over 5% palladium-on-carbon in methanol to give diamine 14 in 80% yield. Treatment with thionyl chloride and triethylamine in toluene then afforded 15 in 65% yield. Compound 15 was then nitrated with sodium nitrate in H₂SO₄ to introduce the critical NO₂ group, affording 4-fluoro-5-methyl-7-nitro-2,1,3-benzothiazole (16). This NBTD derivative was then treated with glycine tert-butyl ester or N-methylglycine tert-butyl ester to introduce amine-based electron donating groups that favored a push–pull dipole and afforded compounds 3 and 4, respectively.

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

For the preparation of compounds 5 and 6, commercially available 2-fluoro-4-methyl-6-nitroaniline underwent catalytic reduction with hydrogen over 5% palladium-on-carbon to give a diamine intermediate. The intermediate was converted directly to 4-fluoro-6-methyl-2,1,3-benzothiadiazole (17) via an oxidative ring closure with thionyl chloride (Scheme 3). Compound 17 was then nitrated with sodium nitrate in H₂SO₄ to afford compound 18 in 70% yield. Intermediate 18 was then treated with glycine tert-butyl ester or N-methylglycine tertbutyl ester to synthesize both NBTD derivatives 5 and 6, in 77 and 81% yields, respectively. As documented in the Supporting Information, NBTD derivatives 1–6 were all characterized fully, as were the synthetic intermediates leading to those compounds.

Scheme 3.

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

We next turned our attention to installation of the benzylic amino group essential for our dipeptidomimetic (NBTD-Gly) design. The key synthetic steps are outlined in Scheme 4, leading to the more stable N-phthalimide derivatives of the benzylic derivatives (22 and 23). The benzylic position in compound 15 was activated by radical bromination using N-bromosuccinimide (NBS) in the presence of benzoyl peroxide (BPO) to afford bis-halogenated 19 in 82% yield by controlling both the rate of addition of NBS and reaction time to avoid dibromination of the benzylic position. The Gabriel synthesis was then employed to introduce the benzylic amine using potassium phthalimide to afford protected amine 20 in 85% yield. Nitration of 20 with sodium nitrate in H₂SO₄ introduced the critical NO₂ group, providing nitrated intermediate 21. This intermediate was subjected to nucleophilic aromatic substitution with glycine tert-butyl ester or N-methylglycine tert-butyl ester to synthesize 22 and 23, respectively, in good yields. The phthaloyl group was deprotected using ethylenediamine, affording the free benzylic amine intermediates from their respective phthalimides (22 and 23), thus enabling further functionalization at this site. Subsequent acylation with 4-pentenoyloxy succinimide, yielded 4-pentenoyl protected 7 and 8. This protecting group has been found suitable for preparing a variety of misacylated tRNAs, and can subsequently be removed under mild conditions with dilute iodine.²⁹ This is typically done just prior use, making these compounds of potential utility for the preparation of activated tRNAs able to incorporate NBTD derivatives into proteins of interest. We have used this method in routine fashion to activate suppressor tRNAs with a wide range of unnatural amino acids, including cyclic dipeptides,^25,30 and azole-based dipeptidomimetic analogues.^25-28

Scheme 4.

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

Tripeptidomimetic analogues 9 and 10 were synthesized analogously by treatment of the benzylic amine intermediates resulting from deprotection of phthalimides 22 and 23 with Boc-glycine N-hydroxysuccinimide ester. NBTD analogue 11 was prepared by acylation of the benzylic free amine resulting from phthalimide deprotection of 22 with N-(9-fluorenylme-thoxycarbonyloxy) succinimide (Scheme 4). The Fmoc-protected dipeptidomimetic 11, represents a potential building block for solid phase peptide synthesis (SPPS). With desired compounds 1–11 in hand, we next explored their photophysical properties.

Photophysical Properties.

The photophysical properties of NBTD derivatives 1–11 were investigated by absorption and fluorescence spectroscopy, and a summary of data acquired in toluene is summarized in Table 1. Regioisomers can exhibit significantly different photophysical properties, as described previously for various modified fluorescent scaffolds.^31-34 Thus, exploring different substitution patterns can be a promising approach for tuning fluorophore emission or brightness. It seemed interesting to compare the photophysical parameters of such dyes with NBTD derivative 1 which lacks any methyl group on the NBTD benzenoid core or N⁴ atom. Commercially available NBTD and reported NBD-based noncanonical alpha-amino acids (Fmoc-l-Dap(NBTD)-OH (i), Fmoc-l-Dap(NBD)-OH (ii), and Fmoc-l-Dap(NBSeD)-OH (iii)) (Figure 1) also lack methyl or alkyl groups, reflecting a relative paucity of such derivatives. As described above, we have demonstrated a simple methylation strategy by introducing a methyl group(s) on the NBTD nucleus at positions 5, 6, and N⁴ before functionalizing the benzylic position to introduce the amino terminus of our new dipeptidomimetic NBTD derivatives 7–11. Interestingly, methylated regioisomer 3 exhibited a red shift in both absorption (Δλ = 20 nm) and emission (Δλ = 25 nm) spectra, and a 2.3-fold increase in quantum yield when compared to its unmethylated NBTD analogue 1 in toluene (Figure 3, Table 1). On the other hand, isomer 5 exhibited a blue shift (Δλ = 10 nm) in absorption and a red shift (Δλ = 25 nm) in emission spectra when compared to unmethylated NBTD analogue 1 (Figure 3). The quantum yield was also significantly affected by the position of the substituents when measured in toluene (Table 1). Methyl substitution at position 6 (regioisomer 5) was strongly detrimental to quantum yield (0.20 → 0.01), while the quantum yield for regioisomer 3, having a methyl group at position 5 was significantly enhanced (0.20 → 0.45) (Figure 3, Table 1). There seems to be substantial molecular conformation and steric constraint between the NBTD unit and the methyl groups at positions 5 and 6 that affect the overall transition dipole of the electronic excitation.

Table 1.

Spectral Properties of Fluorogenic Nitrobenzothiadiazole Based Probes (NBTDs 1–11) in Toluene Using Coumarin 153 as a Standard^a

NBTD	abs λabs (nm)	λem (nm)	ε (M−1 cm−1)	brightness	ΦF (toluene) ± % error
NBTD 1	435	500	16,800	3000	0.20 ± 3.1
NBTD 2	460	510	17,900	8100	0.46 ± 2.0
NBTD 3	455	525	9040	3800	0.45 ± 5.0
NBTD 4	460	530	7200	1200	0.18 ± 3.0
NBTD 5	425	525	9700	100	0.01 ± 0.4
NBTD 6	440	530	11,200	200	0.02 ± 0.4
NBTD 7	455	520	29,600	12,800	0.46 ± 4.8
NBTD 8	450	519	7000	200	0.04 ± 1.1
NBTD 9	455	526	9000	4500	0.52 ± 3.8
NBTD 10	450	515	6600	100	0.03 ± 0.4
NBTD 11	455	521	10,300	4700	0.47 ± 2.1

^aExcitation was carried out at the corresponding UV–vis maximum wavelength for each compound.

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).

We next turned our attention to the effect of N⁴-methylation on the photophysical properties of the NBTD derivatives. N⁴-methylated NBTD (2) exhibited red-shifted absorption and emission (Δλ = 25 and 10 nm in toluene, respectively) in relation to NBTD 1 (Table 1 and Figure 3). Similarly, the addition of an N⁴-methyl group to 3 (affording 4) and 5 (affording 6) effected the red shift of their absorption and emission spectra in toluene (Figure 3).

In general, for a given donor–acceptor type such as NBTD fluorophores, factors that suppress the intramolecular charge transfer (ICT) character upon irradiation with light in dipolar fluorophores lead to a reduction in the transition dipole moment, and hence a reduction in the fluorescence intensity.^35-37 The electron-donating ability of the donor group and electron-withdrawing ability of the acceptor group play crucial roles in determining the magnitude of the ICT. Introducing a β-carbonyl substituent (e.g., N-methylglycine ethyl ester) into the push–pull system of NBD fluorophore as a donor has been reported as a successful strategy to enhance fluorescence, due to the blockade of nonradiative decay pathways through inhibiting the dark twisted intramolecular charge transfer (TICT) configurations.³⁸ Thus, the introduction of N-methylglycine tert-butyl ester on NBTD fluorophores 2 and 6 as donors was similarly consistent with the β-carbonyl substituent strategy to attain a planar intramolecular charge transfer (PICT) state that emits strongly, rather than a TICT state that emits poorly.^34,38 In detail, N-methylation of 1 to afford 2 increased the quantum yield by 2.5-fold, while N-methylation of 5 doubled the quantum yield of 6 (Table 1).

In comparison, this was not the case for NBTD 4, as steric factors (notably 1,3-allylic strain) may also be of importance in the varying behaviors of dialkylamino compounds with similar donor capabilities.^35-37,39,40 The quantum yield of NBTD 3 dropped substantially in the presence an N⁴-methyl group (NBTD 4) in toluene (Table 1). A plausible explanation for the lower quantum efficiency of NBTD 4 is the formation of a TICT state, which is consistent with similar reported 1,3-allylic strain cases.^39,40 The steric effect exerted by the methyl group is responsible for the nonplanar arrangement of donor and acceptor, which inhibited intramolecular charge transfer. To evaluate the impact of N-methylated NBTD derivatives on TICT suppression, we selected NBTD 4 fluorophore as a model compound, using (time-dependent) density functional theory (DFT/TD-DFT) calculations. The pretwisting dihedral angle between the N⁴-methyl group and the NBTD unit for NBTD 4 (θ = 8.4°) was found to be much more significant than that in unmethylated N⁴ NBTD 3 (θ = 0°) (Figure 4). This twisting, originating due to the clash between methyl groups at C5 and N⁴, retards effective electronic interaction between the donor and the acceptor in the NBTD push–pull system. In keeping with published examples,^39,40 this suggests that fluorophore 4 absorbs a photon to give an excited state (4*), which is followed by electron transfer from the nitrogen atom to the NBTD ring system with concomitant twisting of the C4–N bond. TICT would be expected to be energetically more favorable in the NBTD 4 derivative due to an appreciable increase in the 1,3-allylic steric hindrance from the adjacent methyl group at C5. The amino donor group (N⁴) could lose its planarity and become a pretwisted conformation, which is highly susceptible to TICT rotations by lowering the rotation barrier and enhancing TICT formation (Figure 4).^35,39 The TICT form relaxes without emission of a photon, leading to rapid nonradiative decay of the excited state.

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.

Our investigation explored the TICT tendencies across the highlighted (colored orange) C4–N bond (Figure 4B). Specifically, we computed the rotational barriers (E_RB) and driving energies (E_DE) of NBTD 3 and NBTD 4 from the emissive ICT state to the dark TICT state (90° rotations). NBTD 4, displayed less rotation barrier transition to the dark TICT state on S₁ potential energy surface (E_RB = 0.25 eV; Figure 4C), with considerable driving energy (E_DE = −0.70 eV; Figure 4C) in vacuum, indicating its low quantum yield (Table 1). In contrast, compound 3, exhibited a higher energy barrier (E_RB = 0.61 eV; Figure 4C) and a reduced driving energy (E_DE = − 0.57 eV; Figure 4C), suggesting stronger resistance to TICT formation than N-methylated NBTD 4 and enhanced fluorescence quantum yield. This observation is consistent with the widely accepted TICT mechanism.^35-37,39,40

It is worth noting that the TICT state is typically nonemissive and reactive. Suppressing TICT formation in organic fluorophores is crucial for the development of high-performance fluorescent labels. Our quantum chemical calculations show that the unmethylated NBTD 3 has a transition dipole moment (1.94 D) larger than that of N-methylated derivative (NBTD 4) (1.41D), indicative of the importance of the allylic strain. These observations suggest that dual C5 and N⁴ methylation plays a significant role in the photophysical properties due to steric interaction. A similar trend in photophysical properties was observed for additional NBTD-Gly derivatives, notably for the methylation of NBTD 7 and 9, to afford their methylated derivatives 8 and 10, respectively (Table 1).

To understand the interaction of dyes with solvent in ground and excited states, solvatochromism was studied using different solvents in the presence of dipeptidomimetic derivative NBTD 9. The absorption and emission spectral variation observed due to solvent polarity is displayed in Figures 5 and 6. The fluorescent emission efficiency of NBTD 9 was diminished markedly with increasing solvent polarity (toluene→ dioxane → ethanol → PBS buffer) (Figure 5), or in mixtures of 1,4-dioxane and water (Figure 6). It was observed that NBTD 9 displayed solvent sensitive absorption and emission spectra. The absorption and emission maxima progressively increased by increasing solvent polarity (Figure 5). This clearly points to enhanced charge separation in the excited state and its stabilization by polar solvents. This solvatofluorochromism behavior is a common trait of BD-based chromophores, which have been shown to undergo rapid internal conversion with increasing solvent polarity.^21,22 In addition to evaluating their fluorescence properties, we assessed the chemical and photostability for selected NBTD derivatives 3, 4, and 9 (Table S1, Figures S1 and S2, Supporting Information). The good chemical and photostability of these dyes is notable, and consistent with their potential broader utility.

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).

CONCLUSIONS

We have described the synthesis and photophysical characterization of a series functionalized NBTD derivatives. The positional effect of methyl groups modulates the photophysical properties of NBTD derivatives, which is clear from the spectral differences between these regioisomeric dyes. We believe this study will benefit the strategy of molecular design for dipeptidomimetic NBTD-containing probes (NBTD-Gly). By analogy to NBTD 3, we have been able to synthesize NBTD 7 which exhibits a set of desirable photophysical properties that can be considered a suitable red-shifted fluorescent dipeptidomimetic building block for preparing misacylated tRNAs. Preparation of chemically misacylated semisynthetic nonsense suppressor tRNAs can be employed in biosynthetic incorporation of these novel dipeptidomimetic NBTD-containing probes into proteins of interest as we have shown before for our solvatochromic dipeptidomimetic based oxazole and thiazole derivatives.

Supplementary Material

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.joc.5c00700.

Details of the synthesis, computational analyses and photophysical characterization of new compounds. NMR spectra of all new compounds (PDF)

Data Availability Statement

The data underlying this study are available in the published article and its Supporting Information