4-(2-(5-(2-(tert-Butoxycarbonyl)hydrazinecarbonyl)-2-methylthiophen-3-yl)cyclopent-1-enyl)-5-methylthiophene-2-carboxylic Acid

Abstract

Diarylethene (DAE) molecular photoswitches draw attention as building units in the preparation of diverse photoactive molecules. An interesting class of these molecules are photoactive peptides. A way to build DAE moiety into peptides/peptidomimetics is via DAE amino acids, an example of which has been demonstrated in bioactive cyclic peptides, wherein the DAE Fmoc-amino acid was prepared and used. Herein, the preparation of DAE Boc-amino acid is presented using a modified method of synthesis. This contribution to the DAE amino acid collection could be useful in the further enhancement of diversity in designing different routes to photoactive peptides.

1. Introduction

Since peptide molecules are the protagonists of numerous bio-interactions, the idea of making peptides/peptidomimetics with light controllable molecular structures is an ongoing challenge that will hopefully lead to new smart drugs [1]. Further, photosensitive peptides are interesting as building units in light-sensitive supramolecular systems, which could have different applications as smart materials [2].

For photosensitive peptide synthesis, the most intuitive solution is to incorporate a photo-controllable amino acid into the peptide backbone, moving the research focus to the synthesis of amino acids with a photosensitive moiety.

Herein, the so-called photochromic moieties, defined by the possibility of light-induced switching to another isomer or chemical species [3], become interesting, as these structural changes are spectacular enough to impact the potential peptide conformation, thereby influencing its bioactivity and/or ability to self-assemble. One of the photochromic moieties drawing considerable attention is the diarylethene (DAE) family moiety, namely the diarylethene (DAE) molecular photoswitch [4,5,6,7]. Its applicability as a functional photoswitch (Scheme 1) lies on a quality line [5] (p. 12175): it is a P-type or thermally irreversible photochromic moiety, the “back and forth” switch process is fast and has a respectable iteration limit number, and finally, it also operates in the solid state.

It is shown that the DEA molecular photoswitch can impact the bioactivity of peptides or peptidomimetics. This was achieved by incorporating the DAE Fmoc-protected hydrazino-amino acid into a gramicidin S analogue, allowing the bioactivity to be regulated using UV/Vis light [8]; the bioactivity of a DAE bis-pyrene diamide targeting DNA/RNA and BSA was shown to be susceptible to light control [9].

Herein, the synthesis of DAE Boc-hydrazino-amino acid [10], Figure S16, is described (Scheme 2). The amino acid can be further used in the preparation of DAE photoactive peptides on a larger scale.

Scheme 1. A member of the DAE family with a 2-methylthiophen ring. Upon UV irradiation, the open structure (left) “switches” forward to the closed structure (right). The “back” process is triggered using Vis light. A more detailed presentation of DAE and DAE-_closed molecules’ stereochemistry is given [11].

Scheme 1. A member of the DAE family with a 2-methylthiophen ring. Upon UV irradiation, the open structure (left) “switches” forward to the closed structure (right). The “back” process is triggered using Vis light. A more detailed presentation of DAE and DAE-_closed molecules’ stereochemistry is given [11].

Scheme 2. Synthesis of Boc-amino acid (2) with an incorporated DAE moiety.

Scheme 2. Synthesis of Boc-amino acid (2) with an incorporated DAE moiety.

2. Results and Discussion

In this work, a type of DAE moiety depicted in Scheme 1 was used to synthesize a photoswitchable Boc-protected amino acid 2 (Scheme 2). Somewhat differently to the described procedure of analogous DAE Fmoc-amino acid synthesis [8], which was accomplished by coupling the starting DAE dicarboxylic acid 1 [11,12] with Fmoc-carbazate using reagent pair N,N-Diisopropylcarbodiimide (DIC)/N,N-Diisopropylethylamine (DIPEA) in dimethylformamide (DMF), in this case (Scheme 2), the starting compound DAE dicarboxylic acid 1 [Appendix B] was coupled to tert-Butyl-carbazate using coupling reagent pair chloro-4,6-dimethoxytriazine (CDMT)/N-methylmorpholine (NMM) in dry dichloromethane (CH₂Cl₂). Analogously to the described procedure [8], compound 2, along with the by-product diamide 3, was synthesized (Scheme 2).

In the described Fmoc-amino acid preparation [8], the target compound was further used (solid-phase synthesis) as a mixture of compounds, together with the by-product Fmoc-diamide, since, as fully protected, the diamide was not reactive. Herein, compound 2 was separated from Boc-diamide 3 by means of simple liquid–liquid extraction using organic solvent and alkaline water. This method resulted in a fairly pure target compound 2 according to ¹H NMR spectra (Figure S1). Using this method, compound 2 was isolated in a 36% yield, and raw diamide 3 was isolated in a 39% yield. The method herein described was performed on a scale of a few hundred mgs: 630 mg of product 2 was isolated.

Repeated trials of the method were performed. If the extraction steps were omitted, a mixture of 2 and 3 was obtained. The original procedure [8] describes the isolation of pure compounds by means of HPLC. Herein, the mixture of compounds 2:3 (10:4, 76 mg) was separated using preparative TLC (7% MeOH/CH₂Cl₂) to obtain fairly pure compound 2 (20 mg, Figures S3–S7).

The main features of the described DAE Fmoc- and Boc-amino acid synthesis presented herein are depicted in

Table 1. Target DAE Fmoc- and Boc-amino acid synthesis features.

	Reagents	Isol./mg	Yield/%	Method of Isolation
Fmoc	DIC/DIPEA/DMF	-	50 1	HPLC
Boc	CDMT/NMM/CH2Cl2	630	36	Extraction/TLC

¹ It is not depicted whether the data refers to the isolated target compound.

.

Synthesized DAE Boc-amino acid 2 and the by-product, diamide 3, were subjected to the photo-on and -off switch process depicted in Scheme 1. The compounds (c = 10⁻⁴ mol dm⁻³, CH₃CN) were “switched-on” using UV light (254 nm) and “switched-off” by exposure to room light (Figure 1, Figures S17 and S18).

The presumed closed forms, molecules 2c and 3c, were obtained in an amount sufficient for NMR analysis by irradiating (254 nm) higher concentrations of 2 and 3 (4.8 and 5.8 × 10⁻⁴ mol dm⁻³, respectively). The NMR spectra of open and presumed closed forms for each compound (2 and 3 vs. 2c and 3c) are evidently different (Figures S23–S33), indicating an occurrence of a predicted photoproduct. The occurrence of new ¹³C signals at 66.8 in 2c and 68.4 in 3c indicates the formation of a 4H-Indene ring. These signals suggest the presence of new quaternary 4 °C atoms. In both cases, there is a noticeable decrease of the thiophene ring H chemical shifts in the ¹H NMR spectra. Specifically, the chemical shifts change from 7.53 and 7.37 to 6.78 and 6.76 for 2 → 2c (CDCl₃) and from 7.29 to 6.74 for 3 → 3c (CDCl₃). These shifts are most likely due to the loss of aromaticity in the thiophene ring.

The method of synthesis herein presented gives a fairly pure photoswitchable DAE Boc-amino acid on a scale of a few hundred mgs. This allows, besides further Boc-solid-phase synthesis steps, further synthesis of photoswitchable peptides on a scale of a few hundred mgs in solution.

3. Materials and Methods

3.1. General

Prior to use in the coupling reaction, CH₂Cl₂ was refluxed and distilled over calcium hydride dust (CaH₂), and the collected distillate was deposited on type 4Å molecular sieves. Petroleter 40–70 °C was used. The reagents and solvents were used as supplied unless otherwise mentioned. EtOAc and CH₂Cl₂ were distilled and used as supplied. For the solid phase extraction of preparative TLC, CH₂Cl₂ (HPLC grade) was used. The preparative plate chromatography was conducted on homemade plates (Appendix B) (Kieselgel silica gel 60 F254, 0.063–0.200 mm, Merck, KGaA, Darmstadt, Germany). Analytical plates (Kieselgel silica gel 60 F254, Merck) were used, and the spots were visualized using UV light (254 nm; see Figure S12). For the establishment of inert atmosphere gas, argon (Ar) 5.0 was used. Given yields refer to isolated amounts.

¹H and ¹³C NMR spectra were obtained using NMR spectrometers: AV600 (magnet 14 T, Bruker BioSpin GmbH, Rheinstetten, Germany) or AV300 (magnet 7 T, Bruker BioSpin GmbH, Rheinstetten, Germany). The NMR spectra shift was calibrated according to standard TMS or solvent residual signal.

HRMS data were collected on a high-resolution mass spectrometer//HPLC system: 6546 LC/Q-TOF//1290 Infinity II (Agilent, Santa Clara, CA, USA).

IR spectra were obtained with Perkin Elmer FT-IR UATR (Shelton, CT, USA.)

Melting points were checked on an Original Kofler Mikroheitztisch apparatus (Reichert, Wien, Austria) and were uncorrected.

The UV-Vis spectra were obtained on Varian Cary 100 Bio UV-Visible Spectrophotometer (Varian Medical Systems, Paolo Alto, CA, USA).

The photoswitch process (analysis of 2 and 3) was induced on the compounds (2, 3) in a solution (c = 10⁻⁴ mol dm⁻³, CH₃CN (HPLC grade), quartz cuvette, 1 mL/1 cm) using light sources at different times: UV light/19 s (Luzchem photochemical reactor LZC-14, 254 nm, 8 × 8 W) and Vis light/1 h 47 min (room light, discharge lamps: PHILIPS master 2 × TL-D 36 W/840 and OSRAM 3 × L36 W/765). For photoswitch process (preparation of 2c and 3c) the differences in conditions are given in Appendix A.

Wherever possible, the procedures were performed, covering the glassware with Al foil.

For long periods, depositions of compounds are placed at +4 °C, and vials are wrapped in Al foil.

3.2. Experimental

3.2.1. Synthesis of 4-(2-(5-(2-(tert-Butoxycarbonyl)hydrazinecarbonyl)-2-methylthiophen-3-yl)cyclopent-1-enyl)-5-methylthiophene-2-carboxylic Acid (2)

In a two-neck glass vessel, under an atmosphere of Ar, compound 1 (1310 mg, 3.76 mmol) was suspended in dry CH₂Cl₂ (17 mL) at room temperature (20 °C). NMM (830 µL, 7.52 mmol) was added to the suspension, after which the suspension turned into a solution. The reaction solution was stirred at 500 rpm and cooled on ice. After cooling (10 min), CDMT (647 mg, 3.68 mmol) was added using a spatula in 3–4 portions for 15 min. The reaction mixture was stirred in Ar atmosphere, on ice, for 2 h. Then, another aliquot of NMM was added (830 µL, 7.52 mmol), followed by the addition of tert-Butyl-carbazate (487 mg, 3.68 mmol), wherein the reagent’s vial was washed with dry CH₂Cl₂ (3 mL). The reaction mixture was stirred on ice for another hour and then left to spontaneously reach room temperature overnight (the water bath reached 24 °C).

The reaction mixture was poured into a separation funnel with additional CH₂Cl₂ (2 × 35 mL) and extracted with KOHaq (0.5 mol dm⁻³, 66 mL), which produced a white suspension. To obtain good separation of the organic and water phases, diethyl ether (200 mL) and water (50 mL) were added. The layers were mixed and left to separate, which produced the organic phase O1 and the water phase.

The separated water phase was washed with CH₂Cl₂ (50 mL) and diethyl ether (100 mL). After the separation of layers, the bottom water layer was washed with additional diethyl ether (2 × 50 mL). The phases were separated, and the organic layers were combined into an organic phase (O2).

The obtained water phase, a dark brown solution, was identified as basic (pH 12–14). The water phase was placed on an ice bath and acidified to pH 1 with KHSO₄aq (8.5 g/10 mL), which produced a very slight whitish precipitate and the appearance of an isolated dark brown rubbery semi-solid. The water phase was decanted through sinter glass. Very small amounts of precipitate and the dark brown semi-solid were dissolved in EtOAc (100 mL). The EtOAc solution analysed on analytical TLC showed an indicative intense UV–visible spot (10% MeOH/CH₂Cl₂). The EtOAc solution was washed with 5% NaHCO₃ (50 mL), 0.5 mol dm⁻³ HCl (10 mL), and water (50 mL) to remove the possible residues of starting compound 1. The organic phase was dried over anhydrous Na₂SO₄ for 3 h, filtered off, and the solvent removed under reduced pressure to produce the final product 2 at first as colourless oil in fairly good purity (Figures S1 and S2) (630 mg, 1.36 mmol, 36%).

Compound 2 was also obtained from a mixture of 2 and the by-product diamide 3 (2:3 = 10:4, 76 mg, Figure S10) using two parallel preparative TLC plates eluting with 7% MeOH/CH₂Cl₂. The separated stripes on the TLC were visualized under a UV lamp (254 nm) (Figure S11). The removed two SiO₂ stripes were suspended in 7% MeOH/CH₂Cl₂ (20 and 40 mL), stirred for 25 min, and filtered through sinter glass-3 with the addition of the eluent (20 mL each portion). The solvent was removed under reduced pressure at r.t. to obtain compound 2 (Figures S3–S7 and S12). At +4 °C, the compound shows as a whitish solid (20 mg, 0.04 mmol, 43% in regard to the starting mixture). mp 162–172 °C; ¹H NMR (300 MHz, (CD₃)₂CO): δ 7.59 (s, 1H), 7.51 (s, 1H), 2.88–2.80. (m, 4H), 2.09–2.08 (m, 2H), 2.01 (s, 3H), 1.98 (s, 3H), 1.43 (s, 9H); ¹H NMR (600 MHz, CDCl₃): δ 8.13 (bs, 1H, NH), 7.53 (s, 1H), 7.37 (s, 1H), 6.81 (bs, 1H, NH), 2.81–2.74 (m, 4H), 2.06 (qui, J = 7.6 Hz, 2H), 1.94 (s, 3H), 1.90 (s, 3H), 1.48 (s, 9H); ¹³C{1H, APT} NMR (150 MHz, CDCl₃): * δ 165.8, 161.7, 156.1, 144.4, 141.5, 137.0, 136.6, 135.8 (CH), 135.0, 134.8, 130.9 (CH), 128.8, 82.5 (C_Boc), 38.7 (CH₂(b,c)), 28.3 ((CH₃)₃), 22.9 (CH₂(a)), 15.1 (CH₃), 14.8 (CH₃); IR (ATR): ν 3261, 2926, 2852, 1663, 1460, 1246, 1156, 735 cm^–1; HRMS (+ESI): m/z calcd. for [C₂₂H₂₆N₂O₅S₂]⁺: 463,1361 [M + H]⁺, found 463,1362; R_f (7% MeOH/CH₂Cl₂): 0.35.

* Atoms Cb,c,d,e are represented by two signals (Figures S4–S6 and S22).

3.2.2. Details on by-Product 3 (Scheme 2)

The method of isolation and characterisation and spectroscopic data are available in Appendix A and Supporting Information (Figures S8–S15, S17–S19, and S21).

3.2.3. Details on Closed Forms 2c and 3c

The method of preparation and NMR data are available in Appendix A.2 and Supporting Information (Figures S23–S35).

4. Conclusions

The synthesis of a diarylethene (DAE) Boc-protected hydrazino-amino acid has been presented. The described procedure, with all its issues and novelties, is a contribution to the “know how” knowledge of preparing DAE building units. The information provided herein might be of good use to researchers planning to synthesize photochromic peptides with a DAE photoswitch.