Excited-state proton-transfer reactions of 7-azaindole with water,ammonia and mixed water–ammonia: microsolvated dynamics simulation

Dynamics simulations of excited-state multiple proton transfer (ESMPT) reactions in 7-azaindole (7AI) with ammonia, mixed water–ammonia, and water molecules were investigated by quantum dynamics simulations in the first-excited state using RI-ADC(2)/SVP-SV(P) in the gas phase. 7AI(WW), 7AI(WA), 7AI(AW) and 7AI(AA) clusters (W, water and A, ammonia) show very high probability of the excited-state triple proton transfer (ESTPT) occurrence in ranges from 20% for 7AI(WA) to 60% for 7AI(AW), respectively. Furthermore, 7AI(AW) clusters with ammonia placed near N–H of 7AI has the highest probability among other isomers. In 7AI with three molecules of bridged-planar of water, ammonia and mixed water–ammonia clusters, the excited-state quadruple proton transfer reactions occur ineffectively and rearrangement of hydrogen-bonded network on solvents also takes place prior to either ESTPT or excited-state double proton transfer. The role played by mixed-solvent is revealed with replacing H₂O with NH₃ in which the ESMPT is found to be more efficient corresponding to lower barrier in the excited state. The preferential number of solvent surrounding 7AI that facilitates the proton transfer process is two for methanol and water but this preferential number for ammonia is one.

Highlights: (i) replacing H₂O with NH₃ assists ESPT corresponding to lower barrier in the excited state; (ii) the ESMPT time of 7AI with mixed water–ammonia is in the sub-picosecond timescale; (iii) the PT tends to be concerted process with at least one ammonia, but synchronous without ammonia.     

Protic solvent effects on the photophysical properties of O=Ti(IV)TSPP: photoinduced electron transfer.

The photophysical properties of oxotitanium(IV)meso-tetra(4-sulfonatophenyl) porphyrin (O=Ti(IV)TSPP) have been investigated in water and methanol by laser spectroscopic techniques. The fluorescence emission spectrum of O=Ti(IV)TSPP in methanol exhibits two strong emission bands at 610 and 670 nm at room temperature with the decay time of ca. 310 +/- 10 ps and the rise time shorter than 30 ps, in contrast to the extremely weak emission with the decay time of ca. 27 +/- 4 ps in water, indicating that the fluorescence emissive states are different in the two solvents as supported by the solvent dependences of the excitation spectrum. The transient Raman spectra of O=Ti(IV)TSPP in water has been observed to exhibit a remarkable enhancement of phenyl-related mode at 1599 cm(-1), while in methanol, the Raman frequencies of the porphyrin skeletal modes (upsilon2 and upsilon4) are down-shifted without any apparent enhancement of the phenyl-related mode, indicating different interactions of the two solvents with the excited O=Ti(IV)TSPP. These Raman studies reveal that methanol molecule interacts with the photoexcited O=Ti(IV)TSPP more strongly than water, forming the exciplex, O=Ti(IV)TSPP(MeOH)*, suggesting that the two different emissive states are the singlet Franck-Condon state and the exciplex state in methanol and water, respectively. A broad triplet transient absorption of O=Ti(IV)TSPP has been also observed at 480 nm in water as well as in methanol, which is decreased upon addition of methyl viologen (MV2+) with appearance of a new absorption band at 620 nm. This indicates that the photoinduced electron transfer (PET) takes place from the porphyrin to MV2+ in both solvents. The kinetic analysis of the transient absorption band exhibits the PET rate constants of 4.76 x 10(5) s(-1) and 3,03 x 10(4) s(-1) in methanol and water, respectively. All these results infer that the PET takes place from the (d,pi) CT state and the triplet state of the excited porphyrin in methanol and water, respectively.     

Excited state intramolecular proton transfer in 3-hydroxy flavone and 5-hydroxy flavone: A DFT based comparative study

Potential energy (PE) curves for the intramolecular proton transfer in the ground (GSIPT) and excited (ESIPT) states of 3-hydroxy-flavone (3HF) and 5-hydroxy-flavone (5HF) were studied using DFT/B3LYP (6-31G (d,p)) and TD-DFT/B3LYP (6-31G (d,p)) level of theory respectively. Our calculations suggest the non-viability of ground state intramolecular proton transfer for both the compounds. Calculated PE curves of 3HF for the ground and excited singlet states proton transfer process explain its four state laser diagram. Excited states PE calculations support the ESIPT process to both 5HF and 3HF. The difference in ESIPT emission process of 3HF and 5HF have been explained in terms of HOMO and LUMO electron distribution of the enol and keto tautomer of these two compounds.     

One- and two-photon-induced fluorescence from recombinant green fluorescent protein

The fluorescence spectral properties of recombinant green fluorescent protein (rGFP) were examined with one- and two-photon excitations using femtosecond pulses from a Ti:sapphire laser. Intensity-dependent properties of the two-photon-induced fluorescence from rGFP excited by an 800-nm, 100-fs laser beam were reported, and the two-photon excitation cross section of rGFP was measured at 800 nm as about 160 x 10(-50) cm(4)s/photon. The possible excited-state proton transfer between two electronic states at about 400 nm in protonated (RH) species and 478 nm in deprotonated (R(-)) species in rGFP was confirmed by fluorescence and fluorescence excitation anisotropy spectra. A subelectronic state (or vibronic progression) at about 420 nm in RH species was identified, which was relatively stable and not involved in the excited state proton transfer in rGFP upon irradiation.     

Reduced formation of bipyrimidine photoproducts in DNA UV irradiated at high intensity

DNA was irradiated using an excimer laser (248 nm) at low intensity (3.15 x 10(7) watts/m2) or high intensity (1.25 x 10(11) watts/m2). Fluences up to 30 kJ/m2 were delivered at either intensity. Following irradiation, DNA damage products were measured, yielding the following findings: 1) the rate of formation of thymine-thymine and thymine-cytosine cyclobutane dimers and the bipyrimidine photoadduct 6-4'-[pyrimidine-2'-one]thymine were reduced at high intensity by about 2-fold and 2) extensive release of free thymine and thymine decomposition fragments occurred at high intensity, but not at low intensity. The effects of high intensity UV are due to promotion of low-lying excited state(s) by absorption of a second photon, producing higher excited state(s) with consequent ionization and base loss. Possible excited state intermediates in this process are the lowest triplet state of DNA bases and prolonged singlet states associated with excimer formation. The depletion of these excited states via promotion may be the cause of the diminished yield of bimolecular pyrimidine photoproducts, suggesting that these photoproducts are formed at low UV intensity in part from long-lived excited states. Long-lived excited states present at conventional UV intensities may contribute to formation of some photoproducts that occur rarely, but are of potential biologic importance, such as dimers between nonadjacent pyrimidines on the same strand and interstrand dimers forming DNA cross-links.     

Imino proton exchange in DNA catalyzed by ammonia and trimethylamine: evidence for a secondary long-lived open state of the base pair

The base-pair opening kinetics of the self-complementary oligomer d(CGCGAATTCGCG)(2) has been derived from NMR measurements of the imino proton exchange. In general, it has previously been found that imino proton exchange in duplex DNA is limited by the proton-transfer step from the open state and that the dependence of the exchange times on the inverse concentration of an added exchange catalyst is linear. In the present study, a curvature is observed for, in particular, the innermost AT base pair with both ammonia and trimethylamine (TMA) as exchange catalysts. The two catalysts act on the same open states, but the accessibility of TMA is reduced by a factor of 2-3 compared to ammonia. Assuming that ammonia accesses the imino proton equally in the open state of the base pair and in the mononucleoside, the curvature is consistent with 7-9% of the openings ending in open states with lifetimes of about 1 micros while the bulk of open-state lifetimes fall in the nanosecond range. A curvature is also found for the exchange times of the imino protons in the A-tract sequence CGCA(8)CGC/GCGT(8)GCG. This curvature becomes increasingly pronounced from the 5'-end toward the center of the tract and hereby seems to be correlated with the contraction of the minor groove. Thus, while the base-pair lifetimes deduced from the present study are in accordance with previous measurements, a substantial fraction of the open states formed by the central AT-base pairs in the two oligomers exhibits microsecond lifetimes in contrast to previous estimates in the nanosecond range. These findings may be of relevance for the way sequence specific recognition is accomplished by proteins and ligands.     

Exploring hydrogen bond in the excited state leading toward intramolecular proton transfer: detailed analysis of the structure and charge density topology along the reaction path using QTAIM

Excited state intramolecular proton transfer (ESIPT) reaction along the O-H[Symbol: see text][Symbol: see text][Symbol: see text][Symbol: see text]O hydrogen bond of o-hydroxy benzaldehyde (OHBA), methyl salicylate (MS) and salicylic acid (SA) was investigated by ab-initio quantum chemical calculation and theory of atoms and molecules (QTAIM) for the first time. Variation in several geometric as well as QTAIM parameters along the reaction coordinate was monitored in the fully relaxed excited state potential energy curve (PEC) obtained from intrinsic reaction coordinate (IRC) analysis. Although, the excited state barrier height for the forward reaction (?E (0) (#) ) reduces substantially in all the systems, MS and SA do not show any obvious asymmetry for proton transfer. For MS and SA, the crossover of the bond index as well as the lengths of the participating bonds at the saddle point is assigned due to this symmetry in accordance with bond energy - bond order (BEBO) model, which does not hold true in OHBA both in the ground and excited states. Bond ellipticity, covalent and metallic character were examined for different structures along the reaction path within the QTAIM framework. The QTAIM analysis was found to be able to uniquely distinguish between the ground and excited states of the OHBA molecule as well as both determining the effects on the bonding character of adding different substituent groups and differentiating between the ESIPT reactions in the SA and MS molecules.     

Dynamics of proton transfer and enzymatic activity

The rate-determining elementary reaction step, i.e. proton transfer from the chymotrypsin active centre to the scissile substrate bond has been studied in the present work. On the basis of our theoretical results a hypothesis was formulated to explain chymotrypsin enzymatic efficiency. After ES complex formation excited vibrational states are populated in the enzyme molecule. In the rate-determining elementary reaction step, the proton transfer takes place from the first excited vibrational state of the N-H bond in the imidazole group of His57. This proton transfer is realised by quantum mechanical tunneling mechanism.     

NMR characterization of the energy landscape of SUMO-1 in the native-state ensemble

Characterizing the low energy excited states in the energy landscape of a protein is one of the exciting and demanding problems in structural biology at the present time. These describe the adaptability of the protein structure to external perturbations. In this context, we used here non-linear dependence of amide proton chemical shifts on temperature to identify residues accessing alternative conformations in SUMO-1 in the native state as well as in the near-native states created by sub-denaturing concentrations of urea. The number of residues accessing alternative conformations increases and the profiles of curved temperature dependence also change with increasing urea concentration. In every case these alternative conformations lie within 2 kcal/mol from the ground state, and are separated from it by low energy barriers. The residues that access alternative conformations span the length of the protein chain but are located at particular regions on the protein structure. These include many of the loops, beta2 and beta5 strands, and some edges of the helices. We observed that some of the regions of the protein structure that exhibit such fluctuations coincide with the protein's binding surfaces with different substrate like GTPase effector domain (GED) of dynamin, SUMO binding motifs (SBM), E1 (activating enzyme, SAE1/SAE2) and E2 (conjugating enzyme, UBC9) enzymes of sumoylation machinery, reported earlier. We speculate that this would have significant implications for the binding of diversity of targets by SUMO-1 for the variety of functions it is involved in.     

Sleep-dependent changes in the coupling between heart period and blood pressure in human subjects

We investigated whether in human subjects, the pattern of coupling between the spontaneous fluctuations of heart period (HP) and those of systolic blood pressure (SBP) differs among wake-sleep states. Polysomnographic recordings and finger blood pressure measurements were performed for 48 h in 15 nonobese adults without sleep-disordered breathing. The cross-correlation function (CCF) between the fluctuations of HP and SBP at frequencies <0.15 Hz was computed during quiet wakefulness (QW), light (stages 1 and 2) and deep (stages 3 and 4) nonrapid-eye-movement sleep (NREMS), and rapid-eye-movement sleep (REMS). A positive correlation between HP and the previous SBP values, which is the expected result of baroreflex feedback control, was observed in the sleep states but not in QW. In deep NREMS, the maximum CCF value was significantly higher than in any other state, suggesting the greatest baroreflex contribution to the coupling between HP and SBP. A negative correlation between HP and the subsequent SBP values was also observed in each state, consistent with the mechanical feed-forward action of HP on SBP and with central autonomic commands. The contribution of these mechanisms to the coupling between HP and SBP, estimated from the minimum CCF value, was significantly lower in deep NREMS than either in light NREMS or QW. These results indicate that the pattern of coupling between HP and SBP at low frequencies differs among wake-sleep states in human subjects, with deep NREMS entailing the highest feedback contribution of the baroreflex and a low effectiveness of feed-forward mechanisms.     

A quantum chemical investigation of the electronic structure of thionine

We have examined the electronic and molecular structure of 3,7-diaminophenothiazin-5-ium dye (thionine) in the electronic ground state and in the lowest excited states. The electronic structure was calculated using a combination of density functional theory and multi-reference configuration interaction (DFT/MRCI). Equilibrium geometries were optimized employing (time-dependent) density functional theory (B3LYP functional) combined with the TZVP basis set. Solvent effects were estimated using the COSMO model and micro-hydration with up to five explicit water molecules. Our calculated electronic energies are in good agreement with experimental data. We find the lowest excited singlet and triplet states at the ground state geometry to be of π→π* (S(1), S(2), T(1), T(2)) and n→π* (S(3), T(3)) character. This order changes when the molecular structure in the electronically excited states is relaxed. Geometry relaxation has almost no effect on the energy of the S(1) and T(1) states (~0.02 eV). The relaxation effects on the energies of S(2) and T(2) are moderate (0.14-0.20 eV). The very small emission energy results in a very low fluorescence rate. While we were not able to locate the energetic minimum of the S(3) state, we found a non-planar minimum for the T(3) state with an energy which is very close to the energy of the S(1) minimum in the gas phase (0.04 eV above). When hydration effects are taken into account, the n→π* states S(3) and T(3) are strongly blueshifted (0.33 and 0.46 eV), while the π→π* states are only slightly affected (<0.06 eV).     

Excited state properties of lanthanide complexes: Beyond ff states

Generally, metal-centered ff states dominate the discussion of the excited state properties of lanthanide complexes. In particular, the luminescence properties of Eu(III) and Tb(III) compounds have been studied in great detail for many decades. However, other types of excited states such as MC fd, MLCT, LMCT, MMCT and IL are also of interest. In this context, we have recently examined the excited state behavior of selected Ce(III), Ce(IV), Eu(II) and Gd(III) complexes which are luminescent and/or photoreactive.     

Protonation of excited state pyrene-1-carboxylate by phosphate and organic acids in aqueous solution studied by fluorescence spectroscopy

Pyrene-1-carboxylic acid has a pK of 4.0 in the ground state and 8.1 in the singlet electronic excited state. In the pH range of physiological interest (pH approximately 5-8), the ground state compound is largely ionized as pyrene-1-carboxylate, but protonation of the excited state molecule occurs when a proton donor reacts with the carboxylate during the excited state lifetime of the fluorophore. Both forms of the pyrene derivatives are fluorescent, and in this work the protonation reaction was measured by monitoring steady-state and time-resolved fluorescence. The rate of protonation of pyrene-COO(-) by acetic, chloroacetic, lactic, and cacodylic acids is a function of DeltapK, as predicted by Marcus theory. The rate of proton transfer from these acids saturates at high concentration, as expected for the existence of an encounter complex. Trihydrogen-phosphate is a much better proton donor than dihydrogen- and monohydrogen-phosphate, as can be seen by the pH dependence. The proton-donating ability of phosphate does not saturate at high concentrations, but increases with increasing phosphate concentration. We suggest that enhanced rate of proton transfer at high phosphate concentrations may be due to the dual proton donating and accepting nature of phosphate, in analogy to the Grotthuss mechanism for proton transfer in water. It is suggested that in molecular structures containing multiple phosphates, such as membrane surfaces and DNA, proton transfer rates will be enhanced by this mechanism.     

Photophysical behavior and intramolecular energy transfer in Os(II) diimine complexes covalently linked to anthracene.

The photophysical behavior of two Os(II) complexes having a bipyridine ligand with anthracene attached directly to the bipyridine (4-(9-anthryl)-2,2'-bipyridine, bpy-AN) is reported. The two complexes [(bpy)2Os(bpy-AN)]2+ and [(bpy-AN)2Os(CO)Br]+ have (3)MLCT excited states that differ in energy by less than 800 cm(-1). Despite this fact, the observed photophysical behavior of the two complexes is entirely different. The complex with the higher energy 3MCLT state, [(bpy-AN)2Os(CO)Br]+, is nonemissive at room temperature, but has a long lived excited state that is localized on the 3(pi-pi*) state of the anthracene substituent. The other complex, [(bpy)2Os(bpy-AN)]2+, exhibits emission at room temperature and has a transient absorption spectrum that is consistent with a localized 3MLCT state. The excited state decay behavior of the two complexes can be fit well assuming a model in which noninteracting 3MLCT and 3(pi-pi*) states are in equilibrium.     

The interaction of ammonia with the photosynthetic oxygen-evolving system

The reaction of ammonia with the oxygen-evolving system was investigated using EPR. Two sites with distinct binding properties were found. One site, previously known to be responsible for the modification by ammonia of the multiline EPR signal from the S₂ state and believed to be accessible in this state only, was found to bind ammonia also in the S₁ state although weaker. The second binding site, identified by the effect of bound ammonia on the shape and position of the g = 4.1 EPR signal, was also found to be accessible in both the S₁ and S₂ states. The apparent dissociation constants for ammonia at the two sites in the S₁ and S₂ states were determined. In neither state did the binding the ammonia account for the observed inhibition of oxygen evolution, suggesting that binding to other S states plays an important role in the inhibition. Chloride, which is known to interfere with ammonia-induced inhibition of oxygen evolution, was found to compete with ammonia at the site associated with the modification of the g = 4.1 EPR signal. The broadening of the hyperfine lines of the multiline EPR signal, seen in the presence of ¹⁷O-labeled water, was still observed after the modification of the signal by ammonia. This indicates that ammonia has not completely displaced water bound to the catalytic site in the S₂ state. The results of the binding studies are interpreted in terms of a two state — two site model, where the two states are identified by their EPR signals, the multiline and the g = 4.1 signal, respectively, and the two sites identified by the effects of ammonia on these signals and where the equilibrium between the two states is regulated by the binding of ligands to the sites.     

Picosecond kinetics of excited-state charge separation in 4-(dimethylamino)pyridine.

4-(Dimethylamino)pyridine (DMAP) shows solvent-dependent dual fluorescence from the initially excited state B* and a highly polar TICT state A*. Room-temperature time-resolved picosecond fluorescence investigations prove the bimodal kinetics of the excited-state electron transfer reaction B*-->A* in polar aprotic media. In medium polarity solvents (such as ethyl acetate) two emitting states of DMAP are shown to reach equilibrium within 50 ps. Both emitting states originate from the same ground state. The rate of excited-state charge separation depends on polarity and proton donating ability of the surrounding medium. The effects of temperature on the quantum yields of both fluorescences of DMAP in polar aprotic media indicate the transition from the kinetic regime (at low temperatures) to the equilibrium regime (at high temperatures). The kinetic behaviour of the dual luminescence of DMAP in protic solvents is more complex than in aprotic ones. In alcohols an efficient nonradiative channel competes with excited-state charge separation.     

Chromophore protonation state controls photoswitching of the fluoroprotein asFP595

Fluorescent proteins have been widely used as genetically encodable fusion tags for biological imaging. Recently, a new class of fluorescent proteins was discovered that can be reversibly light-switched between a fluorescent and a non-fluorescent state. Such proteins can not only provide nanoscale resolution in far-field fluorescence optical microscopy much below the diffraction limit, but also hold promise for other nanotechnological applications, such as optical data storage. To systematically exploit the potential of such photoswitchable proteins and to enable rational improvements to their properties requires a detailed understanding of the molecular switching mechanism, which is currently unknown. Here, we have studied the photoswitching mechanism of the reversibly switchable fluoroprotein asFP595 at the atomic level by multiconfigurational ab initio (CASSCF) calculations and QM/MM excited state molecular dynamics simulations with explicit surface hopping. Our simulations explain measured quantum yields and excited state lifetimes, and also predict the structures of the hitherto unknown intermediates and of the irreversibly fluorescent state. Further, we find that the proton distribution in the active site of the asFP595 controls the photochemical conversion pathways of the chromophore in the protein matrix. Accordingly, changes in the protonation state of the chromophore and some proximal amino acids lead to different photochemical states, which all turn out to be essential for the photoswitching mechanism. These photochemical states are (i) a neutral chromophore, which can trans-cis photoisomerize, (ii) an anionic chromophore, which rapidly undergoes radiationless decay after excitation, and (iii) a putative fluorescent zwitterionic chromophore. The overall stability of the different protonation states is controlled by the isomeric state of the chromophore. We finally propose that radiation-induced decarboxylation of the glutamic acid Glu215 blocks the proton transfer pathways that enable the deactivation of the zwitterionic chromophore and thus leads to irreversible fluorescence. We have identified the tight coupling of trans-cis isomerization and proton transfers in photoswitchable proteins to be essential for their function and propose a detailed underlying mechanism, which provides a comprehensive picture that explains the available experimental data. The structural similarity between asFP595 and other fluoroproteins of interest for imaging suggests that this coupling is a quite general mechanism for photoswitchable proteins. These insights can guide the rational design and optimization of photoswitchable proteins.     

Spectroscopic characteristics of the micro-environmentally induced H-bond transformation in anil-type species: experimental and theoretical study

The results of combined experimental and theoretical investigations of the spectral behavior of anil-type systems are presented. Two species: N-triphenylmethylsalicylidene imine (MS1) and N-salicylidene methylamine (SmA) were studied. The electronic (absorption, emission and excitation) spectra of MS1 at room temperature were investigated in pure isooctane as well as in acetonitrile and methanol solutions by the steady-state experiments. A mechanism of molecular processes in the ground and excited states in different microenvironments is also proposed. It includes formation of intra- and intermolecular hydrogen bonds, their role in stabilization of molecular conformations and conformation equilibria. The "solvent assisted" proton transfer reaction and rearrangement were modeled using complexes obtained by attaching methanol molecules to the species studied. The OH-rotamer of SmA was also considered. Infrared and Raman spectra were predicted for MS1 and SmA and compared with the experimental data. An analysis of fundamental vibration frequencies was carried out. Quantum chemical ab initio calculations at the HF/6-31G** level were performed for the species studied and their complexes. Chemical formula of anil-type compound: N-salicylidene methylamine (SmA), N-salicylideneaniline (SA) and N-triphenylmethylsalicylidene imine (MS1). [Structure: see text].     

Crystal structure of a Ca2+-discharged photoprotein: implications for mechanisms of the calcium trigger and bioluminescence

Ca2+-regulated photoproteins are members of the EF-hand calcium-binding protein family. The addition of Ca2+ produces a blue bioluminescence by triggering a decarboxylation reaction of protein-bound hydroperoxycoelenterazine to form the product, coelenteramide, in an excited state. Based on the spatial structures of aequorin and several obelins, we have postulated mechanisms for the Ca2+ trigger and for generation of the different excited states that are the origin of the different colors of bioluminescence. Here we report the crystal structure of the Ca2+-discharged photoprotein obelin at 1.96-A resolution. The results lend support to the proposed mechanisms and provide new structural insight into details of these processes. Global conformational changes caused by Ca2+ association are typical of the class of calcium signal modulators within the EF-hand protein superfamily. Accommodation of the Ca2+ ions into the loops of the EF-hands is seen to propagate into the active site of the protein now occupied by the coelenteramide where there is a significant repositioning and flipping of the His-175 imidazole ring as crucially required in the trigger hypothesis. Also the H-bonding between His-22 and the coelenterazine found in the active photoprotein is preserved at the equivalent position of coelenteramide, confirming the proposed rapid excited state proton transfer that would lead to the excited state of the phenolate ion pair, which is responsible for the blue emission of bioluminescence.     

Mild, rapid and selective alcoholysis of terpyridine-appended amide substrates by Cu-catalysis: protonation state and reactivity of the complex

An amide bond of a terpyridine-appended substrate, 6-(L-phenylalanylamino)-2,2′:6′,2″-terpyridine (2a), was cleaved to yield phenylalanine methyl ester quantitatively in the presence of catalytic amounts of Cu²⁺ not only in methanol but also in aqueous methanol at 30 °C. The reaction proceeds via formation of an N₃O (three terpyridine nitrogens and one carbonyl oxygen) type 1:1 metal complex 2a-Cu²⁺. From spectral titration, the structure of the 2a-Cu²⁺ complex was confirmed to have three different protonation states, i.e., A (non-deprotonated amide with α-ammonium), D1 (deprotonated-amide with α-ammonium) and D2 (deprotonated-amide with α-amino) states. Among them, the complex in the D2 state was exclusively responsible for the observed mild, rapid and selective alcoholysis, showing the first-order rate constant of 6 × 10⁻³ s⁻¹ or half-decay time of 2 min in methanol at 30 °C. Acidity of the amide proton was found to be higher than that of the ammonium proton in the complex, allowing formation of the highly reactive amide-deprotonated D2 state in methanol even without addition of external bases. Factors contributing to the high reactivity of the complex were discussed.