The mechanism of the water-catalyzed excited-state proton-transfer (ESPT) reaction for 7-azaindole (7AI) has long been investigated, but there are some controversial viewpoints. Recently, owing to the superiority of sensing biowaters in proteins by a 7AI analogue, 2,7-diazatryptophan, it is timely to reinvestigate water-catalyzed ESPT in 7AI and its analogues in an attempt to unify the mechanism. Herein, a series of 7AI analogues and their methylated derivatives were synthesized to carry out a systematic study on pKa, pKa*, and the associated fluorescence spectroscopy and dynamics. The results conclude that all 7AI derivatives undergo water-catalyzed ESPT in neutral water. However, for those derivatives with -H (7AI) and a electron-donating substituent at C(3), they follow water-catalyzed ESPT to form an excited N(7)-H proton-transfer tautomer, T*. T* is rapidly protonated to generate an excited cationic (TC*) species. TC* then undergoes a fast deactivation to the N(1)-H normal species in the ground state. Conversely, protonation in T* is prohibited for those derivatives with an electron-withdrawing group at the C(2) or C(3) or with the C(2) atom replaced by an electron-withdrawing nitrogen atom (N(2) in, e.g., 2,7-diazatryptophan), giving a prominent green T* emission. Additional support is given by the synthesis of the corresponding N(7)-CH3 tautomer species, for which pKa* of the cationic form, that is, the N(7)-CH3N(1)-H(+) species, is measured to be much greater than 7.0 for those with electron-donating C(3) substituents, whereas it is lower than 7.0 upon anchoring electron-withdrawing groups. For 7AI, the previously missing T* emission is clearly resolved with a peak wavelength at 530 nm in the pH interval of 13.0-14.3 (H- 14.2).

In this study, we used the tryptophan analogue, (2,7-aza)Trp, which exhibits water catalyzed proton transfer isomerization among N(1)-H, N(7)-H, and N(2)-H isomers, to probe the water environment of tryptophan-59 (Trp59) near the connecting loop region of ribonuclease Tl (RNase T1) by replacing the tryptophan with (2,7-aza)Trp. The resulting (2,7-aza)Trp59 triple emission bands and their associated relaxation dynamics, together with relevant data of 7-azatryptophan and molecular dynamics (MD) simulation, lead us to propose two Trp59 containing conformers in RNase T1, namely, the loop-close and loop-open forms. Water is rich in the loop-open form around the proximity of (2,7-aza)Trp59, which catalyzes (2,7-aza)Trp59 proton transfer in the excited state, giving both N(1)-H and N(7)-H isomer emissions. The existence of N(2)-H isomer in the loop-open form, supported by the MD simulation, is mainly due to the specific hydrogen bonding between N(2)-H proton and water molecule that bridges N(2)-H and the amide oxygen of Pro60, forming a strong network. The loop-close form is relatively tight in space, which squeezes water molecules out of the interface of α-helix and β2 strand, joined by the connecting loop region; accordingly, the water-scant environment leads to the sole existence of the N(1)-H isomer emission. MD simulation also points out that the Trp-water pairs appear to preferentially participate in a hydrogen bond network incorporating polar amino acid moieties on the protein surface and bulk waters, providing the structural dynamic features of the connecting loop region in RNase T1.