Ultrafast deactivation mechanisms of protonated aromatic amino acids following UV excitation

Deactivation pathways of electronically excited states have been investigated in three protonated aromatic amino acids: tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe).

The protonated amino acids were generated by electrospray and excited with a 266 nm femtosecond laser, the subsequent decay of the excited states being monitored through fragmentation of the ions induced and/or enhanced by another femtosecond pulse at 800 nm.

The excited state of TrpH+ decays in 380 fs and gives rise to two channels: hydrogen atom dissociation or internal conversion (IC).

In TyrH+, the decay is slowed down to 22.3 ps and the fragmentation efficiency of PheH+ is so low that the decay cannot be measured with the available laser.

The variation of the excited state lifetime between TrpH+ and TyrH+ can be ascribed to energy differences between the dissociative πσ* state and the initially excited ππ* state.


Among naturally occurring amino acids, three possess fluorescent aromatic chromophores: tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe).

Among them Trp has been the most studied owing to its strong fluorescence yield and the rich variety of information it provides according to the possible environments.1

By monitoring the Trp fluorescence in proteins, one can non-invasively diagnose malignancy in cells.2,3

To understand the mechanism of fluorescence spectroscopy for cancer detection and to further improve its efficiency, the understanding of the primary photo-processes occurring in the bare molecule is nevertheless required.

Interestingly, Trp fluorescence lifetime and intensity vary by orders of magnitude when the pH varies, and drops dramatically at low pH when Trp is fully protonated, emphasizing a predominant non-radiative process.1

In the case of neutral aromatic molecules such as phenol and indole (the chromophores of Tyr and Trp, respectively)4–9 and DNA bases,10,11 it has been recently shown that non-radiative decays play a key role in the photo-physical properties of these molecules.

An electronically excited state with a πσ* character, repulsive along the N–H or O–H coordinate of the aromatic cycle, governs the competition between the direct H-atom loss reaction and internal conversion occurring through a crossing back to the electronic ground state.4,12,13

For protonated aromatic amino acids, UV photo-induced dissociation (PID) mass spectrometry reveals some of the non-radiative deactivation channels that cannot be directly studied by means of fluorescence spectroscopy.

Among the non-radiative pathways, inter system crossing (ISC), chemical reaction, isomerisation, internal conversion IC and bond cleavage, the two latter processes, namely IC and H-atom loss, have been evidenced and distinguished in our previous study of protonated tryptamine.14

Through IC, the photon energy (4.66 eV at 266 nm) is converted into internal energy within the electronic ground state.

This internal energy is large enough to induce fragmentations.

For instance, less than 2 eV only are required for an ammonia loss.15

This excess energy is randomized and consequently leads to a statistical-type dissociation of the ion on the ground state potential energy surface, as also observed in collision-induced dissociation (CID) experiments.

On the other hand, during the H-atom loss reaction, most of the energy initially deposited in the excited state is imparted to the leaving hydrogen atom after the amino N–H bond cleavage.

The elimination of the hydrogen atom is a direct and fast process and results in the formation of a radical cation that exhibits a specific fragmentation pattern.

In this study, the excited state lifetimes of protonated aromatic amino acids were investigated by femtosecond pump–probe photo-induced dissociation (PID) mass spectrometry as previously done for protonated tryptamine.

The lowest energetic site of protonation for the three amino acids is predicted to be the terminal amino nitrogen.16,17

Very recently, the UV spectroscopy of TrpH+ has been reported18 and was found to be very similar to that of the neutral Trp molecule.19

Furthermore, even though several isomers could be formed using ESI technique, including several sites of interaction for the protonated amino group, theoretical calculations at the DFT/MRCI level predict a small effect of the position of the proton on the energy of the optically excited Lb state.18

These calculations also point out a diffuse electronic excited state in TrpH+ with an isomer-dependent energy.

In the meanwhile, we have emphasized, in our recent study of protonated tryptamine,14 the existence of a dissociative excited state along the N–H stretch coordinate of the amino group with a πσ* Rydberg-like character that could correspond to the above mentioned diffuse electronic state.

This excited state is thought to be responsible for the ultra-fast, non-ergodic, deactivation pathway observed in protonated tryptamine by monitoring the H-atom loss channel.


The experimental setup has been described in a previous publication.14,20

Briefly, a commercial electrospray ionization (ESI) source (Analytica of Bradford Inc.) generates the protonated amino acid ions from a 0.5 mM solution of amino acids (Aldrich) in water and methanol (50 : 50 by volume).

A hexapole ion guide is used as an ion trap, at the exit of which the ions are excited and dissociated by femtosecond laser pulses propagating orthogonal to the ESI axis.

The parent and fragment ions are focused between two grids of a home-made time-of-flight mass spectrometer (TOF MS), orthogonal to the ESI and to the laser axes, located 10 cm downstream from the hexapole trap.

The ions are mass-analyzed and detected by a pair of micro-channel plates.

The pump laser pulse (266 nm, 50–100 μJ) is the third harmonic of a Ti : sapphire laser (Spitfire, Spectra Physics) operating at 1 kHz, and the probe is its fundamental wavelength at 800 nm (150 μJ).

The relative time delay between the pump and the probe pulses is varied by a linear translator (Micro-Controle) by 50 fs steps.

The cross-correlation between the pump and the probe pulses was measured to be 250 fs by fitting the ionization signal of neutral toluene with a step-rise function.

The spectra are recorded by averaging 10 to 20 scans with 100 laser shots per scan step.


PID mass spectrometry

Fig. 1c shows the PID mass spectrum of TrpH+ obtained by subtracting a mass spectrum corresponding to the output of the ESI source (Fig. 1a) from a mass spectrum recorded with the UV laser (Fig. 1b).

The negative signal at m/z = 205 corresponds to a depletion of the parent ion mass peak while the fragment ions appear as positive mass peaks.

The insets show the structures of the protonated tryptophan parent ion and of the m/z = 130 Trp side-chain cation.

The fragmentation yield of this amino acid is rather high, reaching 10% at high UV intensity (100 μJ).

This yield is deduced from the ratio of the sum of fragment intensities observed with the dissociation laser versus the parent ion intensity (without the dissociation laser).

This is not an absolute measurement since the estimation of the temporal and spatial overlaps of the laser with the ion bunch is very difficult.

It is meaningful in the comparison between different species under the same experimental conditions.

Note that the power-dependency study reveals a linear behavior of the fragmentation yield as a function of the UV laser power.

The observed mass peak at m/z = 204 corresponds to the H-atom loss reaction and the m/z = 130 ion is a fingerprint of this atom loss reaction.

The H-atom loss from TrpH+ generates the radical cation Trp+ (m/z = 204), which in turn undergoes a ‘classical’ Cα–Cβ bond cleavage with the available internal energy leading to the Trp side chain cation at m/z = 130 as in electron-impact ionization/dissociation of neutral tryptophan21 or in collision-induced dissociation experiments of TrpH+.22

On the other hand, in collision-induced dissociation experiments on protonated Trp, the H-atom loss channel has never been reported and the production of the m/z = 130 fragment is weak, even at large collisional energy (center-of-mass energy).15

One should emphasize that it is quite difficult to observe the m/z = 204 fragment since this requires the full available UV power together with a high parent ion signal.

The other fragments detected are commonly seen in collision-dissociation experiment and can be divided into two groups according to their fragmentation mechanisms and following the attributions given by El Aribi et al.15

The mass peak at m/z = 188 results from ammonia loss and further fragments into m/z = 170 (–H2O) and m/z = 146 (–CH2CO).

The mass peak at m/z = 159 is the immonium ion of TrpH+, related to the concomitant loss of CO + H2O 23 and further dissociates into m/z = 132 (–HCN) and to a lower extent into m/z = 130 when the collision energy exceeds 4 eV (center-of-mass energy and assuming a complete inelastic collision).15

According to the relative intensity of each fragment in the PID mass spectrum, the branching ratio between H-atom loss reaction (m/z = 204 and 130) and the other fragmentation mechanisms is roughly 50%.

Fig. 2 presents the mass spectra obtained in the photo-induced dissociation of protonated tyrosine, Fig. 2c being the PID mass spectrum of TyrH+ obtained by subtracting a mass spectrum corresponding to the output of the ESI source (Fig. 2a) from a mass spectrum recorded with the UV laser (Fig. 2b).

The fragmentation yield is in the order of 6%, slightly lower than for TrpH+.

Even though the H-atom loss is not directly observed, we do detect the fingerprint of the H-atom loss reaction through the observation of the Tyr side chain cation at m/z = 107.

This cation is by far the most abundant dissociation fragment of the Tyr radical cation21,22 but is barely observed in CID experiments on TyrH+.15

Other fragments result from ammonia loss (m/z = 165) followed by H2O and CH2CO loss to give the mass peaks at m/z = 147 and 123, respectively and from the loss of CO + H2O at m/z = 136 that might further fragment into the Tyr side chain cation to a lower extent, as reported in collision induced experiments when the center-of-mass collision energy exceeds 4 eV.15

The mass peak at m/z = 119 corresponds to the concerted loss of CO + H2O and ammonia.

The H-atom loss reaction in Tyr can thus be monitored on the m/z = 107 mass peak and represents about 25% of the fragmentation yield, two times lower than for protonated Trp.

While we observed efficient PID of TrpH+ and TyrH+ with 266 nm excitation, PheH+ does not fragment easily under the same conditions.

The fragmentation yield is in the order of 0.2% at high UV intensity (100 μJ), 40 times lower than for TrpH+, excluding the possibility of any pump–probe experiment on this system.

The pump laser energy (37 500 ± 50 cm−1) is very close to the 0–0 transition of neutral (Phe) in the gas phase (37 537 cm−1)24 and the protonated ion may not absorb the 266 nm light.

Femtosecond transient spectroscopy

The femtosecond pump–probe PID scheme used in this series of experiments has been previously explained in details.14

As revealed by the PID mass spectra (Fig. 1 and Fig. 2), several fragmentation channels are already open by the UV pump excitation.

In order to observe a time-dependent signal on those fragments, the 800 nm probe photon must modify the branching ratio of the fragmentation reactions.

Absorbing a 800 nm probe-photon increases the internal energy of the system by 1.55 eV.

Internal conversion dynamics is quite difficult to study with our pump–probe technique since the excess energy imparted to the protonated molecule following UV excitation is already large and thus the fragmentation probability does not change much with the probe photon.14

Furthermore, the probe-photon can no longer be absorbed after IC is achieved because the molecule in its electronic ground state has no transition in the near IR.

In fact, most of the observed signal-to-noise ratios corresponding to fragments arising from internal conversion are usually poor.

On the other hand, during the H-atom loss reaction, most of the initial excess energy is released in the N–H bond cleavage that produces “colder” radical cations in a doublet spin-state that possess low lying electronic state in the near IR.

The absorbed probe-laser enhances further fragmentation of the nascent products and gives rise to a pump–probe signal on the fragment ions.

A femtosecond pump–probe transient PID signal of TrpH+ recorded on the m/z = 130 fragment for delays up to 17 µs is displayed in Fig. 3.

The experimental data are fitted by a model function which is a sum of a mono-exponential decay and a step function convoluted with the laser cross-correlation.

The fitting procedure provides a decay time constant of 380 ± 50 fs.

This ultra-fast decay is assigned to a very efficient coupling between the initially excited ππ* state and a dissociative πσ* state as observed in protonated tryptamine.

The second component of the model function, which is assumed to be a step function, represents the excitation of the Trp+ radical cations produced by H-atom loss with low internal energy and are thus stable on the experimental time-scale (up to 100 ps).

The excitation increases the energy in the radical cations which therefore undergo a more efficient fragmentation.14

While the pump–probe signal on the side chain TrpH+ fragment exhibits a complex decay signal with an ultra-fast component, the TyrH+ side chain fragment presents a single exponential decay with a slowed-down rate.

As shown in Fig. 4, the decay time-constant recorded for 50 ps on the m/z = 107 ion is 22.3 ± 1 ps, much slower recorded on the m/z = 130 fragment of TrpH+.

As explained above, no pump–probe transient PID signal could be obtained with PheH+ due to its extremely low fragmentation efficiency.


Time-dependent density functional calculations have shown that, in protonated tryptophan20 and tryptamine,14 there exists a charge-transfer (CT) state, with πσ* character, into which an electron is transferred from the π orbital of the indole ring to a σ* orbital on the protonated amino group.

This state becomes lower than the ππ* when the NH coordinate is stretched.

Upon this CT transition, the protonated amino group is neutralized and becomes a hypervalent R–NH3 group which is expected to be unstable.

The CT (πσ*) state crosses both the locally excited state and the electronic ground state and then acts as the driving force responsible for the photofragmentation reaction in this system.

It thus plays a very important role in the deactivation of UV excited amino acids.

This energy level diagram is the same as that describing the excited state dynamics of aromatic azine and enol molecules4 where the key role is played by an excited singlet state of πσ* character which has a repulsive potential-energy function with respect to the stretching of OH or NH bonds.

The 1πσ* potential-energy functions intersect not only the bound potential-energy functions of the 1ππ* excited states, but also that of the electronic ground state.

The lifetime of the 1ππ* states is controlled by the first crossing (barrier height) and the second conical intersection with the ground state triggers an ultrafast internal-conversion process, which is essential for the photo-stability of biomolecules.

In aromatic enol or azine molecules, the dissociation coordinate is the OH or NH in the plane of the aromatic ring whereas in the present case, the dissociation coordinate is the NH on the terminal amino group, and the absence of symmetry in the protonated species changes the conical intersections into avoided crossings.

The variation of excited state lifetimes in the different amino acids can then be understood in terms of variation of the energy of the CT (or πσ*) state relative to the optically active ππ* state.

A very simple model can provide some clues about the relative position of these states.

We will postulate that the potential energy function of the πσ* state along the N–H coordinate is the same for all amino acids at long N–H distance because it is mainly localized on one of the amino hydrogen atoms and it is largely unaffected by any substitution of the aromatic group.

We further assume that the diabatic (non-mixed potential energy curve) πσ* state also has the same shape for the three molecules even near the ground state equilibrium geometry.

These rough assumptions allow to estimate the position of the πσ* state in TyrH+ and PheH+ in comparison to TrpH+ using simple energetic considerations.

Fig. 5 shows schematic potential energy curves for protonated amino acids along one of the amino N–H coordinate at the diabatic limit, i.e. without mixing of ππ* and πσ* states.

The H-atom dissociation energy (De) of the protonated amino acid is calculated from asymptotic values of the proton affinity (PA), ionization potential (IP) of the amino acid and ionization potential of the hydrogen atom (IPH = 13.6 eV), according to the following equation:De = PA + IP − IPHAll the values are summarized in Table 1 along with the S0–S1 origin transition energy of neutral amino acids in the gas phase.

Since the first absorption band of protonated Trp 18 has been reported to be similar to the absorption of neutral Trp,19 the transition energies of the other protonated amino acids are assumed to be that of the neutrals.

The proton affinities being similar within 0.3 eV for the three amino acids, De largely depends on the ionization potentials which differ by more than 1 eV and is used to estimate the position of the πσ* state relative to the ππ* state.

In TrpH+, which has the lowest IP among the three amino acids, the ππ* and the πσ* states are probably very close at the ground state geometry.

This is substantiated by the very short lifetime of the excited state and the calculation of .ref. 20

In tyrosine, the transition energy is only slightly higher than in Trp.

The 4.66 eV pump-photon can excite the ππ–ππ* transition and the ππ* state is predissociated by the πσ* as revealed by the relatively high fragmentation efficiency.

But since the ionization potential is 0.8 eV higher than in Trp, the decay takes longer because the πσ* state is higher in energy, implying a higher barrier for the ππ*–πσ* crossing.

For PheH+, very likely the S1 state cannot be reached with our present pump energy.

However, even with sufficient pump energy, the probability for H-atom loss or IC to induce fragmentation should be very low since a dissociation energy De close to the pump energy implies that the πσ* state will lie high above the ππ* state.

No ultrafast non-radiative channel is expected but radiative decay should dominate the deactivation pathway of PheH+ in the near UV region.

Interestingly, protonated tyramine, an intermediate derived by decarboxylation of Tyr, does not seem to fragment under UV excitation, since we did not get any photodissociation products under our experimental conditions.

According to our simple energetic model, the IP of tyramine being 8.41 eV,21 close to that of Phe (8.5 eV), it seems that the πσ* state is too high in this species to trigger any non-radiative reaction.

This study sheds light on the fluorescence lifetime and intensity of aromatic amino acids in solution.

Protonated Trp and Tyr represent the form of these amino acids at low PH where the amino group is protonated and the carboxylic group is neutralized.

Titration curves of Trp, Tryptamine and Tyr show a dramatically decrease of the fluorescence quantum yield and shortening of the lifetime at low pH25 while tyramine does not show the same pH dependence as the others.


The excited state lifetimes of protonated tryptophan and tyrosine were investigated by femtosecond pump–probe photo-induced dissociation, while that of PheH+ could not be measured.

When excited by 266 nm UV radiation, TrpH+ decays in less than 400 fs while the TyrH+ lifetime is 22.3 ps.

This variation of the excited state lifetime is consistent with a model into which the lifetime is controlled by the crossing of the initially excited ππ* state with a dissociative πσ* state.

The relative energy of these two states is mainly determined by the IP of the corresponding neutral molecule and therefore a direct link can be assessed between this latter value and the excited state lifetime of the protonated ion.