Design And Synthesis Of Six-membered Spiro Rhodamine And Detection Of Copper And Mercury Ions

Heavy metal pollution is still one of the main environmental problems faced by human beings.It is necessary to develop simple and effective sensing methods to monitor and detect the level of heavy metals in the environment.Rhodamines are basic dyes based on xanthenes,with high absorption coefficient and broad fluorescence in the visible region of electromagnetic spectrum,high fluorescence quantum yield and photostability.Due to the special structure and corresponding fluorescence characteristics,rhodamine fluorescent dyes are often used to synthesize“off-on”fluorescent probes.The preparation of five-member spirolactam derivatives have met an enormous progress.Reaction of Rho B and Rho 6G with primary amines either by reflux in ethanol or by formation of acyl chloride with POCl₃ can at present be considered as a standard method for the preparation of metal chemosensors.Compared with rhodamine spirolactam,expanding five-member spiro to six-member spiro provides a new idea for the development of rhodamine fluorescent probes and an opportunity for selectivity,sensitivity and high molar extinction coefficient.In the first part,a universal one-pot three step synthetic process was developed.The inexpensive 2-carboxyl rhodamine used as material successively reacted with phosphorus oxychloride and sodium azide to generate 2-acyl azide rhodamine.The reaction mixtures were heated at 82℃to produce 2-isocyanate rhodamine.Then a novel six-membered spiro rhodamine derivative SRh-NH with urea structure was synthesized by 2-isocyanate rhodamine as a general platform molecule.The p Ka values of probe SRh-NH were 4.62±0.13（Abs）and 4.76±0.10（FI）,respectively.The p H response range of probe SRh-NH was 3-6.5.As a new six-membered spiro rhodamine,SRh-NH showed a dual-ion response mode to copper ions and mercury ions.SRh-NH showed good anti-interference ability to some common metal ions in the detection of copper ions.The detection limit of SRh-NH for Cu²⁺was 64 n M,and that for Hg²⁺was 2.7μM,which was calculated by the three-fold signal-to-noise ratio（S/N=3）formula.SRh-NH had a very fast response to Cu²⁺.The fluorescence intensity of SRh-NH changed instantaneously with addition of 0.02 m M（1 equiv.）Cu²⁺,and then reached the maximum within 7 seconds.However,when 1 m M（50equiv.）Hg²⁺was added,the fluorescence intensity reached the maximum after 2 min.According to Job’s curve,the stoichiometric ratio of SRh-NH to Hg²⁺was 1:1.In the second part of this paper,a six-membered spiro rhodamine SRh-OH containing hydroxyurea structure was synthesized by the reaction of 2-isocyanate rhodamine with hydrazine hydrate.Compared with five-membered rhodamine spirolactam（Rh-OH）,six-membered SRh-OH exhibited higher selectivity and sensitivity to Cu²⁺in aqueous solution.Upon addition of 10 equiv.Cu²⁺,the molar extinction coefficient of SRh-OH at 563 nm can be up to 4.73×10~4 L·mol^-1cm^-1.In the range of Cu²⁺from 0 to 28μM,there was an excellent linear relationship between the absorption or emission intensity and Cu²⁺concentration.The detection limit of SRh-OH was as low as 26 n M（S/N=3）.The reversible binding mode of SRh-OH with Cu²⁺was further confirmed by S^2-addition.Bio-imaging showed SRh-OH can map the distribution of Cu²⁺not only in mitochondria but also in lysosomes of Hela cells.