Photodynamic properties of lysine and arginine derivatives of bacteriopurpurinimide - Supplementary materials.docx

Reviewer comments 

 Figure S1. Absorption spectra of compounds 1-3 in 1% CrEL emulsion immediately after dissolution and after an indicated period of storage at 4 ºC. Solutions were stored at high concentration (compounds 1, 2a, 2b at 2 mM and compound 3 at 0.2 mM) in 5% CrEL emulsion. Before the measurements, solutions were diluted to 25 µM in 1% CrEL emulsion. 

Figure S2. Irradiation-induced changes in the absorption spectra of solutions of compound 1 mixed with RNO and His (a, b) or with RNO (c, d). Spectral changes in the 380-500 nm region (a) report on the photoinduced generation of singlet oxygen, which, reacting with histidine, forms trans-annular peroxide intermediate capable of specifically bleaching RNO.  Irradiation does not induce bleaching of compound 1 (b, d). 

Figure S3. Irradiation-induced changes in the absorption spectra of the solution of compound 2a mixed with RNO and His. Spectral changes in the 380-500 nm region (a) report on the photoinduced generation of singlet oxygen, which, reacting with histidine, forms trans-annular peroxide intermediate capable of specifically bleaching RNO.  Irradiation does not induce bleaching of compound 2a (b). 

Figure S4 - The study of photoinduced superoxide anion radical formation by compounds 1 (a), 2b (b) and 3 (c).  Solutions of 1, 2b and 3 were irradiated with the 543 nm laser light for 30 min in the presence of NBT, the chemical trap of superoxide anion radicals. Absorption spectra were recorded every 10 min. The inserts demonstrate more precisely an absence of photoinduced changes in absorption in the 530-550 nm spectral range, i.e. the absence of both photoinduced formation of superoxide anion radical and photoinduced degradation of the photosensitizers. 

The ability of 1, 2b and 3 to photoinduced formation of superoxide anion radicals was studied using nitroblue tetrazolium (NBT) as a chemical trap of these radicals: superoxide reduces NBT to a monoformazane that absorbs light in the region of 540 nm [1, 2]. Air-saturated solutions (phosphate buffered saline, pH 7.4) containing NBT (85 µM) and 1, 2b or 3 (10 µM, 1% CrEL) were irradiated with a He-Ne laser (532 nm, 1.2 mW/cm2) for 30 min at 34°C, and the absorption spectra was recorded every 10 min.  During irradiation of solutions 1, 2b and 3 in 1% CrEL in the presence of NBT there was no change in the optical absorption of solutions at a wavelength of 540 nm (Fig. S2), indicating the absence of superoxide anion radical formation. No photoinduced degradation of photosensitizers was observed during these experiments.  

Fig. S5. Typical intracellular fluorescence emission spectra of compounds 1 (a), 2a (b), 2b (c) and 3 (d)  (blue and red dotted lines) and corresponding fluorescence emission spectra of these compounds in 1% CrEL (black solid line). Typical intracellular fluorescence spectra of 2a and 2b in vesicular structures are shown by blue line; the spectra from the cytoplasmic regions with diffuse distribution of the compounds are shown by red line. Spectra are very similar in shape and maximum.

审稿意见

补充图S1：化合物1~3在1% CrEL乳液中溶解后即刻，以及于4℃储存指定时长后的吸收光谱。实验中，将化合物1、2a、2b以2 mM、化合物3以0.2 mM的高浓度配制于5% CrEL乳液中储存。测量前，将溶液以1% CrEL乳液稀释至25 µM。

补充图S2：化合物1分别与组氨酸（Histidine, His）和RNO混合、仅与RNO混合的溶液经辐照后的吸收光谱变化（a、b与c、d）。380~500 nm波段的光谱变化（a）反映了光诱导单线态氧的生成：单线态氧与组氨酸反应生成跨环过氧化物中间体，可特异性漂白RNO。辐照不会导致化合物1自身发生漂白（b、d）。

补充图S3：化合物2a与RNO和组氨酸混合的溶液经辐照后的吸收光谱变化。380~500 nm波段的光谱变化（a）反映了光诱导单线态氧的生成：单线态氧与组氨酸反应生成跨环过氧化物中间体，可特异性漂白RNO。辐照不会导致化合物2a自身发生漂白（b）。

补充图S4：化合物1（a）、2b（b）及3（c）光诱导超氧阴离子自由基生成的研究。将1、2b、3的溶液置于超氧阴离子自由基的化学捕获剂NBT存在下，用543 nm激光辐照30 min，每10 min记录一次吸收光谱。插图更清晰地展示了530~550 nm波段未出现光诱导吸收变化，即未检测到超氧阴离子自由基的光诱导生成，也未观察到光敏剂的光降解。

本研究采用硝基蓝四唑（nitroblue tetrazolium, NBT）作为超氧阴离子自由基的化学捕获剂，探究1、2b及3光诱导生成超氧阴离子自由基的能力：超氧阴离子可将NBT还原为在540 nm波段具有特征吸收的单甲臜[1,2]。将含有NBT（85 µM）与1、2b或3（10 µM，1% CrEL）的空气饱和磷酸盐缓冲生理盐水（pH 7.4）溶液，以氦氖激光器（532 nm，1.2 mW/cm²）于34℃辐照30 min，每10 min记录一次吸收光谱。在1% CrEL乳液中辐照1、2b及3的NBT共存溶液时，540 nm处的溶液光吸收未发生变化（对应图S2），表明未生成超氧阴离子自由基。本实验过程中未观察到光敏剂的光降解现象。

补充图S5：化合物1（a）、2a（b）、2b（c）及3（d）典型的细胞内荧光发射光谱（蓝色与红色虚线），以及这些化合物在1% CrEL乳液中的对应荧光发射光谱（黑色实线）。其中，2a和2b在囊泡结构中的典型细胞内荧光光谱以蓝色线展示，化合物弥散分布于细胞质区域的荧光光谱以红色线展示。两类光谱的形状与峰值均高度相似。