Controlled ultrafast ππ*-πσ* dynamics in tryptophan-based peptides with tailored micro-environment

The study investigates how the micro-environment at the atomic scale controls ultrafast photoinduced dynamics in biomolecular ions. In aromatic biomolecules, non-adiabatic couplings between ππ* and πσ* states drive ultrafast charge, energy transfer, and bond-specific dynamics. While many ultrafast gas-phase studies focus on neutral molecules, biologically relevant species are often ionic, and their charge state and adducts can crucially alter their potential energy landscapes. Using controlled adduction in gas-phase peptides containing tryptophan, the authors test the hypothesis that the relative energy gap between ππ* and πσ* states—and thus the ππ*-πσ* population transfer timescale—can be tuned by the identity and positioning of a single charged adduct. The work aims to establish a mechanistic framework for such control and quantify the resulting timescale changes using femtosecond pump–probe spectroscopy and ab initio calculations.

Prior ultrafast experiments on complex molecules in the gas phase have elucidated mechanisms of charge dynamics, structural rearrangements, and energy dissipation, often mediated by conical intersections and non-adiabatic couplings. In aromatic biomolecular systems, ππ*-πσ* couplings are known to enable ultrafast hydrogen detachment/transfer and bond fission, typically on sub-ps to few-ps timescales. Electrospray ionization has enabled studies of ionic biomolecules, where protonation, deprotonation, or metal adduction significantly affect photophysics and photochemistry. Control strategies in photochemistry include coherent control, vibrational (IR) preparation, and chemical substitution to tune state energies and couplings. The present study extends these concepts by tuning the micro-environment via a single adduct atom to control ππ*-πσ* dynamics in tryptophan-based ions, building on observations in substituted heteroaromatics and microsolvated ions where energy gaps govern ultrafast pathways.

Experimental: An on-the-fly femtosecond action spectroscopy setup couples an electrospray ionization (ESI) source to a triple-quadrupole mass spectrometer. Ions are mass-selected (MS1), intersected once with collinear UV pump and IR probe pulses, and fragments are mass-analyzed (MS2). A Ti:sapphire laser (800 nm, 25 fs, 2 mJ, 5 kHz) generates 267 nm pump pulses by third-harmonic generation (BBO crystals 200 µm and 50 µm). The residual 800 nm serves as the probe. UV and IR are recombined after a reflective delay line and focused (f=1 m) collinearly onto the ion beam; the IR focus is offset by 10 cm to overlap with the UV spot and suppress ionization. Typical energies: UV 5 µJ; IR 800 µJ; IR intensity 1–2×10^12 W cm−2 (no standalone IR-induced fragmentation). At each pump–probe delay, a mass spectrum is averaged over 40 s. Dynamics are monitored via delay-dependent changes in photofragment yields: for TrpNa+ (m/z 227), the UV-specific photofragment m/z 130 (Cα–Cβ cleavage) reports on excited-state population; for AlaTrpNa+, dynamics are tracked in fragments such as m/z 168. The UV-only signal is subtracted from the UV+IR signal at each delay. Data are fitted with a single-exponential decay plus step model; for TrpH+ the fit includes convolution with the instrument cross-correlation due to finite pulse durations. Solutions were prepared at 200 µM in 50:50 MeOH:H2O, with 0.1% acetic acid for TrpH+ production and 0.1 mM NaCl for TrpNa+ and AlaTrpNa+. Computational: Ground-state geometries for TrpH+, TrpNa+, and AlaTrpNa+ were optimized by DFT (B3LYP/6-311+G(d,p)). Vertical excited states and potential energy surfaces (PESs) were computed using RICC2/aug-cc-pVDZ for the most stable conformers, with checks on higher-energy conformers and different basis sets showing consistent energetic ordering. PES scans were generated by stretching N–H and N–Na bonds without further geometry optimization to locate ππ*–πσ* crossings. State characters were assigned via dominant orbital analysis, identifying ππ* states localized on the indole chromophore and πσ* states localized on the NH2X moiety.

• Time-resolved UV–IR pump–probe measurements reveal single-exponential ππ* population decays with markedly different timescales depending on the micro-environment: • TrpH+: 390 ± 100 fs (consistent with literature ≈380 ± 50 fs). • TrpNa+: 13 ± 3 ps. • AlaTrpNa+: 35 ± 8 ps. Overall, the decay slows by factors of ~30 (TrpH+ → TrpNa+) and ~100 (TrpH+ → AlaTrpNa+), demonstrating control over two orders of magnitude by changing the adduct and peptide environment.
• UV photofragmentation of TrpNa+ (m/z 227) produces a dominant m/z 130 fragment (Cα–Cβ cleavage) not favored in statistical CID, indicating specific excited-state dynamics.
• Ab initio calculations identify ππ* states (localized on indole) and nearby πσ* states (localized on the NH2X terminus). PESs show a non-adiabatic crossing: the πσ* state is bound along N–H but dissociative along N–Na, rationalizing observed fragmentation patterns.
• The πσ* vertical energy increases across the series (e.g., from about 4.77 to 5.16 eV), while ππ* energies change little; thus, the ππ*–πσ* gap Δε increases, reducing non-adiabatic transfer rates and lengthening lifetimes.
• A simple Global-Local Stabilization Separation (GLOSS) model quantifies the energy-gap change δΔε when switching adducts as the difference between (i) global stabilization via adduct affinity to the chromophore-bearing ion (ΔXA) and (ii) local Coulomb stabilization of the πσ* state at the NH2–X site (ΔECoul ∝ 1/RNX). For TrpH+ → TrpNa+, GLOSS predicts δΔε ≈ 395 meV, close to ab initio ≈310 meV. For AlaTrpNa+ → TrpNa+, GLOSS gives ≈326 meV vs calculations ≈230 meV.
• The ππ*→πσ* transfer can be viewed as a charge-transfer (CT) process moving positive charge from the NH3+ terminus toward the indole, and the study demonstrates this CT can be tuned via the micro-environment (adduct and peptide flexibility).

The experiments directly test the hypothesis that the micro-environment controls ππ*–πσ* dynamics by altering the relative state energies. The measured lifetimes increase dramatically with sodium adduction and further with peptide extension (AlaTrpNa+), aligning with calculations that show an increased ππ*–πσ* energy gap. The observed non-adiabatic crossing along N–Na provides a mechanistic route for ππ*→πσ* population transfer and accounts for specific UV-induced fragments. The GLOSS model disentangles contributions: global complexation stabilizes the ground and ππ* states similarly (as both are indole-localized), while local electrostatics at NH2–X selectively stabilizes πσ*. Increasing adduct affinity (global effect) widens the gap and slows transfer; shortening the NH2–X distance (local effect) narrows the gap and accelerates it. Quantitative agreement between GLOSS estimates and ab initio trends supports the model’s validity. Framing the dynamics as CT emphasizes broader relevance to controlling charge flow in biomolecular ions. Overall, the results demonstrate a practical route to manipulate femtosecond–picosecond non-adiabatic dynamics using angström-scale micro-environmental design.

This work shows that ultrafast ππ*–πσ* dynamics in tryptophan-based ionic peptides can be controlled over orders of magnitude by tailoring a single charged adduct and the immediate molecular environment. Time-resolved action spectroscopy and ab initio calculations establish that changes in the ππ*–πσ* energy gap, driven by global (complexation) and local (NH2–X electrostatics) stabilization, govern the non-adiabatic transfer timescale. The GLOSS model provides a transparent, semi-quantitative framework to predict and design such effects. These insights open avenues to engineer charge and energy flow in biomolecular ions via micro-environment design at the Å scale. Future research could extend to more complex peptides and proteins, explore other adducts and microsolvation, examine structural isomerization pathways, and assess spin-state (triplet) involvement to fully map controllable photodynamics.

• The study examines three specific systems; generality across broader biomolecular classes remains to be tested.
• Vertical excitation energies at ground-state geometries are used as common references; adiabatic energies and full-dimensional dynamics may refine quantitative comparisons.
• PES scans are 1D along selected bonds without full geometry relaxation; multidimensional effects and conical intersection topologies could influence dynamics.
• Signals are modeled with single-exponential decays; potential multi-component dynamics or coherent effects might be unresolved.
• Gas-phase, isolated ions may differ from condensed-phase environments; translation to biological conditions requires caution.
• UV-induced dynamics in these species are intrinsically complex; other pathways (e.g., isomerization, triplet involvement) are acknowledged but not explored here.