The pH-responsive profile of the traditional pH probes (the HH-type pH probes) is governed by the Henderson-Hasselbalch equation. Despite their widespread use, pH probes with further enhanced pH sensitivity, i.e., Hill-type pH probes, are sought after. As a result of positive cooperativity toward protonation, they exhibit a smaller acid-base transition width than that of a HH-type pH probe, which is 2 pH unit. As a result, the Hill-type pH probes have an improved capability to differentiate minor biorelevant pH changes. We previously devised a class of small-molecule Hill-type pH probes (PHX) with pKa in the range of 6.2-6.8. Subcellular organelles with a physiological pH lower than 6.2 are abundant and consequently Hill-type probes for use in these organelles are expected to exhibit a pKa lower than 6.2 as well. Through a rational and systematic structure-property relationship study, we showcased that the pKa values of the PHN type probes could be lowered by increasing the steric bulkiness or the electron-withdrawing capability of the dialkylamino groups on the bottom phenol moiety. The pKa values of the PHN-type probes now cover the wide biorelevant acidic range from 3.5 to 6.8. The potentials of these probes for biological imaging are exemplified and they are expected to be adopted in biological studies and medical diagnosis.
Keywords: Hill-type; acidic pH range; cell imaging; fluorescent pH probes; pKa modulating.