Fluorogenic rhodamine B derivatives that become brighter at neutral pH.

Area of Science:

• Organic Chemistry
• Analytical Chemistry
• Biochemistry

Background:

• Rhodamine dyes are valuable fluorescent probes for activity-based sensing.
• Controlling the open/closed equilibrium of rhodamine amides is key for sensing applications.
• Rhodamine B amides are typically non-fluorescent at neutral pH due to their closed form.

Purpose of the Study:

• To develop a rhodamine B amide derivative that exhibits fluorescence at neutral pH.
• To create a novel strategy for shifting the open-close equilibrium of rhodamine amides.
• To demonstrate a new method for activity-based sensing at physiological pH.

Main Methods:

• Synthesized Rhodamine B derivatives with an N-(2'-carboxy)-phenyl substitution (RhoB-AA).
• Compared the fluorescence properties of RhoB-AA with unsubstituted N-phenyl derivatives (RhoB-Ph) at neutral pH (7.2).
• Investigated the effect of esterase activity on RhoB-AA derivatives.

Main Results:

• RhoB-AA derivatives showed significantly enhanced absorbance and emission at pH 7.2 (up to ~1500x) compared to RhoB-Ph.
• The fluorescence of RhoB-AA at neutral pH is dependent on the free carboxylic acid group.
• Esters of RhoB-AA were non-fluorescent but could be converted to fluorescent RhoB-AA by esterases.

Conclusions:

• A simple ortho anthranilic acid substitution effectively shifts the rhodamine B amide equilibrium to favor the open, fluorescent form at neutral pH.
• This strategy provides a powerful new tool for activity-based sensing at neutral pH.
• RhoB-AA derivatives offer a promising platform for developing novel fluorescent probes for biological and chemical sensing.