Bis-Rhodamines Bridged with a Diazoketone Linker: Synthesis, Structure, and Photolysis

Abstract

Two fluorophores bound with a short photoreactive bridge are fascinating structures and remained unexplored. To investigate the synthesis and photolysis of such dyes, we linked two rhodamine dyes via a diazoketone bridge (-COCN₂-) attached to position 5' or 6' of the pendant phenyl rings. For that, the mixture of 5'- or 6'-bromo derivatives of the parent dye was prepared, transformed into 1,2-diarylacetylenes, hydrated to 1,2-diarylethanones, and converted to diazoketones Ar¹COCN₂Ar². The high performance liquid chromatography (HPLC) separation gave four individual regioisomers of Ar¹COCN₂Ar². Photolysis of the model compound─C₆H₅COCN₂C₆H₅─in aqueous acetonitrile at pH 7.3 and under irradiation with 365 nm light provided diphenylacetic acid amide (Wolff rearrangement). However, under the same conditions, Ar¹COCN₂Ar² gave mainly α-diketones Ar¹COCOAr². The migration ability of the very bulky dye residues was low, and the Wolff rearrangement did not occur. We observed only moderate fluorescence increase, which may be explained by the insufficient quenching ability of diazoketone bridge (-COCN₂-) and its transformation into another (weaker) quencher, 1,2-diarylethane-1,2-dione.

Scheme 1. Combination of Two Caged Fluorophores Bound with a Linker (A) and an Alternative Based on a Single Photoreactive Caging Group Incorporated into a Linker (B)

Figure 1

Diazoketone linkers −COCN₂– connect two N,N′-bis(2,2,2-trifluorethyl)rhodamine residues via positions 5′ and 6′ of the pendant phenyl rings: four possible regioisomers 1a–d and their designations Iso4, Iso3, Iso1, and Iso2 (for isomers 1–4, respectively) according to high performance liquid chromatography (HPLC) retention times.

Scheme 2. Synthesis of Regioisomeric Bromorhodamines 6a and 6b Containing N,N′-Bis(2,2,2-trifluoroethyl) Groups

Conditions: (i) pyridine, CH₂Cl₂, rt, overnight; (iii) 48% aq. HBr, AcOH, reflux, 6 h; (iv) method A: 160 °C, 3 h; addition of 5 (2nd equiv), 85% aq. H₃PO₄, 160 °C, 3 h (47%); method B: 1,2-dichlorobenzene, 160 °C, 3 h, addition of 5 (2nd equiv), 160 °C, 3 h (31%).

Scheme 3. Bromides 6a,b and 1,2-Bis(tributystannyl)acetylene in the Synthesis of Bis(rhodamine) Acetylenes 7a–c as a Mixture of 5,5-, 5,6-, and 6,6-Regioisomers

Scheme 4. Hydration of 7a, 7b, and 7c Mixture in the Presence of Triflic F₃CSO₃H (Reagent) and Propionic (Solvent) Acids Leads to the Mixture of Ketones 8a [5(CH₂),5(CO)], 8b [6(CH₂),5(CO)], 8c [5(CH₂),6(CO)], and 8d [6(CH₂),6(CO)]

The Regitz diazotransfer with tosyl azide affords the target diazoketones 1a [5(N₂),5(CO)], 1b [6(N₂),5(CO)], 1c [5(N₂),6(CO)], and 1d [6(N₂),6(CO)]. For full structures of 1a–d, see Figure 1.

Scheme 5. Synthesis and Photolysis of Azibenzil (11)

Conditions: (i) aq. AcOH, Nafion NR50, 100 °C, 24 h, 70%; (ii) aq. Ga(F₃CSO₃)₃, 100 °C, 24 h, 59%; (iii) aq. F₃CCH₂OH, F₃CSO₃H, MW, 90 °C, 1 h, 90%; (iv) TsN₃, DBU, MeCN, 0 °C, 1 h, rt, 3–18 h, 53%; (v) MeCN, aq. HEPES buffer, pH 6.5, air; (vi) MeCN, aq. HCOONH₄ buffer, pH 7.3–7.4, air.

Figure 2

Irradiation of azibenzil (PhCOCN₂Ph) dissolved in aqueous acetonitrile (80% acetonitrile, 20% water, v/v) with HCOONH₄ buffer (pH 7.3) results in full conversion to a new substance (amide 16, see Figure S2) with the same retention time but without absorption maximum at 319 nm. (A) Absorption changes upon irradiation; inset: transient at 319 nm. (B) Chromatograms (2D maps) of the sample before (left) and after (right) irradiation. (C) Chromatogram at 260 nm (a shift was introduced for clarity); inset: absorption spectra of the main peaks.

Figure 3

Relative fluorescence of isomers 1–4 (Figure 1) in MeCN (80% v/v) and 10 mM HCOONH₄ buffer, pH = 7.4 (20% v/v). Yellow bars: starting materials. Green bars: after complete photolysis of the starting diazoketones. The numbers on top of the bars show the fluorescence quantum yields for the starting compounds and their increase upon photolysis to mixtures containing α-diketones as the main products (see Figure S3).

Scheme 6. Photolysis of the Bis(rhodamine) Diazoketones 1a, 1b, 1c, and 1d

The main product is shown. Solvent: acetonitrile/water 80/20 (v/v), aqueous HCOONH₄ buffer (pH 7.3–7.4).