Ultrafast excited state dynamics in protonated GWG and GYG tripeptides

Abstract

The excited state dynamics of two protonated tripeptides GWG and GYG has been investigated by pump/probe femtosecond measurements on photofragments, to explore the behavior of peptides where the terminal protonated amino group is not directly linked to the aromatic residue. The dynamics observed are short and surprisingly similar to the dynamics observed on the free protonated tryptophan and tyrosine aromatic amino acids. Specific photofragments observed for protonated GWG are related to the formation of a radical species WG°⁺ after cleavage of the C_α–N bond near the tryptophan residue.

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In the recent years we have demonstrated the major role of excited states, dissociative along NH or OH coordinate, in the relaxation of neutral biological aromatic molecules.¹ We have also shown that the excited state lifetime of protonated tryptophan is very short and that photo-excitation induces fragmentation, one of the channels corresponding to a H atom loss.^2–5 This excited state dynamics of protonated tryptophan has been traced back to a coupling between the initially excited ππ* state and a dissociative πσ* state on the basis of both time-dependent density-functional theory (TD-DFT) calculations and coupled clusters calculations (RI-CC2).² The process can be viewed as driven by an electron transfer from the indole ring towards the NH₃⁺ group. The localization of the electron on the amino group produces a hypervalent radical, C–NH₃, which is electronically similar to NH₄. This radical moiety is unstable and undergoes fast dissociation by rupture of either the N–H or the C_α–N bond. In the course of the reaction, the excited-state potential energy (PE) surface of the πσ* state crosses the ground state PE surface. This crossing governs the branching ratio between H-atom or NH₃ loss reactions and recombination on the ground-state energy surface, i.e.internal conversion . In the case of protonated tryptamine⁴ or tryptophan,⁵ this mechanism is well characterized experimentally, the branching ratio between the H loss channel and internal conversion being about 50%.

This model has been able to explain the difference of lifetime between tryptophan and tyrosine. Briefly the lifetime of the excited state is linked to the energy gap between the ππ* and the πσ* excited states, which is larger in tyrosine than in tryptophan.³

In all our previous experiments^3,5,6 on protonated systems, the N terminal site was on the aromatic residue so that, by construction, the NH₃⁺ group was very close to the aromatic chromophore. The coupling between ππ* and πσ* state is then expected to be quite efficient and will lead to fast dissociation.

In a recent publication,⁷ the role of the distance between the aromatic chromophore and the NH₃ terminal group has been used to explain the drastic difference in the photo fragmentation efficiency between the singly protonated and the doubly protonated pentapeptide (AGWLK). In the doubly charged species the distance between the aromatic ring and the NH₃⁺ amino group is much larger than in the singly charged one, the coupling between the aromatic ring and the protonated amino group is much less efficient leading to a smaller fragmentation yield.

In the present work, we address the following question: what happens when the terminal amino group is not “directly” attached to the aromatic amino acid, i.e. when a residue is inserted between the aromatic amino acid residue and the NH₃⁺ terminal group. In other words, is the lifetime of the excited state of the chromophore drastically changed as compared to the one of the free protonated amino acid (tyrosine and tryptophan).

This question has recently been addressed in the case of LW by Schultz and coworkers⁸ and indeed the lifetime of this protonated species was found comparable to the free tryptophan lifetime but nothing is known on tyrosine containing systems.

We have shown in a previous paper on protonated tryptamine⁴ that, when the excited state dynamics is probed by an increase in fragmentation of the molecule, pump/probe measurements require that the pump laser (266 nm) alone produces more than one fragment. Indeed the pump/probe signal intensity is due to the variation of the branching ratio between different fragmentation channels resulting from pump only or pump + probe. If there is only one fragment, no pump/probe signal can be recorded. In most tyrosine containing dipeptides, only one fragment is observed after absorption of the pump UV laser and the experiment is not feasible. This is not the case in tripeptides.

The setup has been described in previous publications.^4,6 Briefly it consists of an electrospray source linked to a pulsed time of flight mass spectrometer . The ions are trapped in the hexapole trap of the electrospray and extracted a few microsecond before the laser pulse. The femtosecond 266 nm/800 nm pump/probe lasers intersect the ion bunch just after the exit of a focusing Einzel lens. Both parent and photofragment ions fly in a field free zone for about 10 µs before entering the first two plates of the TOF mass spectrometer located a few cm downstream where they are extracted/accelerated and mass-analyzed by pulsed high voltage towards the MCP detector. The mass spectra presented are obtained by subtracting the laser off signal (ion signal issued from the electrospray source) from the laser on ion signal.

Collision induced dissociation experiments have been performed in a commercial ion trap (Esquire 3000+ from Bruker Daltonics) equipped with an electrospray ion source. The ions of interest are mass-selected before collision with He buffer gas and the resulting fragments are then mass-analysed to extract full scan mass spectra.

The fragmentation mass spectra obtained with laser induced dissociation and collision induced dissociation for both protonated GWG at m/z 319 and GYG at m/z 296 are presented in Fig. 1 and summarized in Table 1. At first it seems that both excitation processes (collision and photon) lead to similar products. However, the CID spectra are dominated by b₂ ions (m/z 244 for GWGH⁺ and m/z 221 for GYGH⁺) while the LID spectra show a predominance of a₂ ions (m/z 216 for GWGH⁺ and m/z 193 for GYGH⁺). MS/MS collision induced dissociation of b₂ ions mostly gives a₂ ions. It thus seems that under UV excitation, secondary fragmentation from b₂ ions is enhanced as compared to CID condition.

Fig. 1 Fragmentation mass spectra of protonated GWG (left) and GYG (right). (a) GWG collision induced dissociation; (b) GWG laser induced dissociation; (c) GYG collision induced dissociation; (d) GYG laser induced dissociation. The laser wavelength is 266 nm.

Table 1 Fragmentation of GWGH⁺ and GYGH⁺ tripeptides: collision induced fragments are in normal characters, laser induced fragments in italic, in both cases the major fragments are in bold, and the immonium ion that corresponds to a double bond cleavage of the peptide chain is bold–italic

GWGH^+ fragments	CID % of total fragment ion signal	LID % of total fragment ion signal	LID % of total fragment ion signal	CID % of total fragment ion signal	GYGH^+ fragments
301 ammonia loss	2%	Absent	Absent	3‰	278 ammonia loss
262 ≡ y2	<0.1%	12%	24%	<1‰	239 ≡ y2
245 ≡ z2 (y2-NH3)	—	10%	3%	—	222 ≡ z2 (y2-NH3)
244 ≡ b2 or z2-H	82%	7%	3%	64%	221 ≡ b2 or z2-H
227 ≡ (b2-17)	<1%	∼1%
226 ≡ (b2-18)	<1%	∼1%
216 ≡ a2	8%	32%	59%	31%	193 ≡ a2
199		3%
187		7%
159 W immonium ion	<0.1%	4%	12%	4%	136 Y immonium ion
132	2.4%	18%
130 W side chain ion	<0.2%	4%	Absent	Absent	107 Y side chain ion
Ratio 130/132	0.08	0.20

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In both GWGH⁺ and GYGH⁺ tripeptides, y₂ ions (m/z 262 and 239) as well as z₂ ions (m/z 245 and 222) resulting from y₂ by ammonia loss are clearly observed under UV laser induced dissociation while missing under CID condition. Note also that the tryptophan immonium ion m/z 159 and the m/z 130 (C_α–C_β bond break) are barely seen in the GWGH⁺ CID mass spectrum in contrast to the LID mass spectrum.

In Fig. 2 is presented the femtosecond pump/probe signal recorded for selected photofragments issued from protonated GWG. Firstly, the effect of the probe laser on the photofragment yield depends on the recorded fragment. While the fragment yield of m/z 244, 159 and 130 is enhanced, all the others fragmentation channels including m/z 262, 216, 187 and 132 are depleted. Secondly, as already observed for protonated W⁵ or WL,⁶ the lifetime measured is short, 550 ± 80 fs. It should be noticed that for W and WL, a second 10 ps decay has been recorded, which is not observed here on any of the fragments. Similarly, the plateau observed in the pump/probe signal recorded on the m/z 130 fragment—corresponding to a C_α–C_β bond rupture of the W residue—in previous experiments on protonated tryptamine, W and WL is not observed: within the experimental uncertainties, the excited state of the indole chromophore in GWGH⁺ presents only one very fast decay.

Fig. 2 Pump (266 nm)/probe (800 nm) signal reported for selected fragments of protonated GWG. All fragments exhibit similar fast femtosecond dynamics, with a 550 ± 80 fs time constant.

The pump/probe signal observed on the GYGH⁺ photofragments is presented in Fig. 3. As for protonated tyrosine,³ the signal to noise ratio is not as good as that obtained for tryptophan containing species and the pump/probe signal can only be observed on two fragments m/z 136 and 221. Nevertheless, it can be clearly seen that the lifetime is ten times longer than for GWG, i.e. about 5 ps.

Fig. 3 Pump (266 nm)/probe (800 nm) signal recorded for selected fragments of protonated GYG. The picosecond decay is similar for the 2 fragments, with a 4.7 ± 0.2 ps time constant.

The present results can be understood in the framework of the model that we have developed in the last years, starting from simple systems (protonated tryptamine) going to more complex ones: protonated W and Y and recently WL. As already stated in the introduction, the basis of the model is the existence of a coupling between the locally excited ππ* state and a πσ* state located on the amino group, dissociative along the N–H or C–NH₃ coordinate. This coupling causes the very short lifetime of the locally excited ππ* state and is strongly dependent on the energy gap between these two states.

As a result of the light mass of the H atom and the repulsive character of the πσ* state, the dynamics of the H atom loss on the repulsive πσ* surface should be much faster than the 250 and 400 fs recorded in the photofragmentation of protonated tryptamine and tryptophan. These short decays are indeed the combination of the locally excited ππ* state lifetime and the time spent on the repulsive πσ* state until the conical intersection with the electronic ground state is reached.

The excited state dynamics is followed through absorption of the probe 800 nm photon to higher S_n excited states, either from the locally excited ππ* state or from the πσ* state. As the system evolves on the πσ* potential energy surface towards the conical intersection with the ground state , there is a branching between H-atom loss and internal conversion , and the effect of the probe laser strongly depends on the process:

(i) After internal conversion to S₀, the protonated tripeptide has a closed shell electronic structure with its first absorption band in the UV region, and cannot be excited with the 800 nm probe laser. Therefore, the subsequent fragmentations of hot-ground state ions are not influenced by the probe laser.

(ii) When the H-atom loss reaction occurs, a radical cation , i.e. a doublet state , is produced that has low lying electronic excited states in the near IR. Such a species can absorb the probe photon at 800 nm and a time evolution can be observed with our pump/probe technique. This latter process is responsible for the observation of a plateau in the pump/probe signal on the m/z 130 fragment (C_α–C_β bond rupture) issued from protonated tryptamine, tryptophan and tryptophan–leucine dipeptide.

The short excited state lifetimes recorded here for the protonated tripeptides seems to indicate that the coupling between the ππ* and πσ* is still efficient even if the NH₃⁺ protonated amino group is not directly linked to the aromatic residue. We observe an increase in the lifetime from GWG to GYG, as in the free aromatic amino acids where the gap between the ππ* and the πσ*excited states is larger in tyrosine than in tryptophan, so that similarly a larger ππ*/πσ* energy gap can be expected in GYG than in GWG.

The main difference between the present results and those obtained on smaller systems is the absence of a longer time component in the pump/probe signal assigned to the formation of a radical cation associated with the H-atom loss. It may be that efficient fragmentation occurs for structures where the NH₃⁺ group lies in close interaction with the aromatic ring, in a conformation such that the H atom loss would be suppressed by a kind of cage effect , the NH₃⁺ group being squeezed between the aromatic ring and the carbonyl group. A way to probe the structure of protonated peptides would be infrared multiphoton photodissociation (IRMPD) experiments that should give access to the different isomers populated at 300 °K.⁹ Besides, in a very recent experiment developed in the group of T. Rizzo,¹⁰ well-resolved UV photodissociation spectroscopy of cold protonated amino acids has been obtained revealing the contribution of several conformers. The combination of experiments in both frequency and time domains would certainly be able to tackle the issue of conformation dependence on the fragmentation efficiency.

It is in fact surprising that all the decays can be fitted with a single exponential in contrast to what has been observed before for W or WL that were exhibiting bi-exponential decays ascribed to the presence of many conformers populated at room temperature. We had expected a similar result for tripeptides. Either all the conformers share the same lifetime or most likely this can be due to the detection method. One observes a pump/probe signal mostly on conformers for which specific dissociation channels (different from those observed in CID) are open in the excited state .⁴

Since there is no evidence for a H-atom loss reaction in the tripeptides, photo-fragmentation is thought to occur through internal conversion and there should be little difference between laser induced and collision induced dissociation. There are however some differences: in both GWGH⁺ and GYGH⁺ tripeptides, y₂ ions (m/z 262 and 239) as well as z₂ ions (m/z 245 and 222) resulting from y₂ by ammonia loss are clearly observed under UV laser induced dissociation while missing under CID condition. Besides, for GWG, a main difference is the observation of the tryptophan immonium ion (m/z 159) and m/z 130 tryptophan side chain ion with laser induced dissociation. These latter fragments may be related to the formation of a WG°⁺ (m/z 261) radical cation , that further dissociates to m/z 244, 159 and in a lower extent to m/z 130 ions.¹¹

Both WG°⁺ and protonated GWG fragment to a m/z 244 ion, but with a different chemical structure: the m/z 244 ion corresponds to a simple b₂ ion in the GWGH⁺ fragmentation, while the radical WG°⁺ ion leads to a [z₂-H]°⁺ ion after NH₃ loss, through the cleavage of the C_α–N bond next to the tryptophan residue associated with hydrogen atom transfer to the N-terminal.¹¹ Note that the ion yield for these three specific photo-fragments increases with the probe laser, while the major LID fragment channels, at m/z 216 and 132, which are also important CID fragments, decrease with the probe laser. This result cannot be explained by complete energy redistribution after internal conversion , i.e. a statistical fragmentation type, because in such a case the internal energy due to the absorption of the probe photon should lead to a depletion of the larger masses into the lighter ones through secondary fragmentation. Besides, we have performed the MS/MS CID of m/z 244 b₂ ion, which gives m/z 216 and 132 as secondary fragments without any m/z 159 immonium ion. We therefore concluded that one of the UV deactivation pathways of protonated GWG ion leads to the formation of WG°⁺ radical species that further dissociates into m/z 244, 159 and 130 ions. The m/z 159 immonium ion has also been observed in the photoinduced dissociation at 266 nm of long protonated synthetic polypeptides (10 residues) containing one tryptophanyl residue¹² and was assigned to an incomplete vibrational redistribution of the energy in the system, which enhances fragmentation near the chromophore.

Notice that the tyrosine immonium ion (m/z 136) is also enhanced in photo-fragmentation as compared to CID.

In summary, CID leads exclusively to cleavage of the peptide bond on the C terminal side (b₂, a₂ fragment) while LID gives rupture of the peptide bond on both sides of the chromophore (a₂, y₂, z₂ and z₂-H).

The excited lifetimes of the protonated tripeptides GWG and GYG have been measured through pump/probe femtosecond scheme. The lifetimes are short (550 fs and 5 ps) and similar to those observed in the corresponding protonated amino acids, which indicates a strong interaction between the terminal amino group and the aromatic residue, even though they are separated by another amino acid residue. The absence of long time component seems to imply that there is no hydrogen loss reaction in such system. However the UV de-excitation pathways of protonated GWG ion leads to the formation of a WG°⁺ radical, as well as to fragmentations of the peptidic chain next to the absorbing chromophore, consistent with an incomplete energy redistribution.

Acknowledgements

The authors thank Dr J. Lemaire from the Laboratoire de Chimie Physique, Université Paris-Sud XI, for the loan of the esquire 3000+ ion trap (Bruker Daltonics). The authors are grateful to F. Gobert for the maintenance of the femtosecond laser. This work has been performed at the Centre de Cinétique Rapide ELYSE (Bat. 349, Université Paris-Sud, 91405 Orsay, France) and has been supported by the Conseil Général de l’Essone.

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