Peptide Cations as Molecular Wires

7/31/2019 Peptide Cations as Molecular Wires

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Highly Efficient Charge Transfer in Peptide Cations in the Gas Phase: Threshold Effects

and Mechanism

R. Weinkauf,* P. Schanen, A. Metsala, and E. W. Schlag

Institut fur Physikalische und Theoretische Chemie, TU-Munchen, 85747 Garching,Lichtenbergstrasse 4, Germany

M. Bu1rgle and H. Kessler

Institut fur Organische Chemie und Biochemie, TU-Munchen, 85747 Garching,Lichtenbergstrasse 4, Germany

ReceiVed: March 27, 1996; In Final Form: August 26, 1996X

We present new experimental data demonstrating specific, photoactivated positive charge migration in isolatedpeptide radical cations. The effect exhibits a threshold behavior, which we can directly correlate with energeticsof local electronic states. A new very efficient mechanism for charge transfer in cations is proposed thatinvolves an extended coulomb state (EC) of shakeup character. Our investigations are performed on laser-desorbed, cooled, neutral peptides in the gas phase. Charge localization in the peptide is achieved by resonantUV two-photon ionization at an aromatic chromophore. Charge flow in the cations can be activated byabsorption of a first visible (VIS) photon. Presence of charge in the aromatic chromophore is probed by

resonant absorption of a second VIS photon and monitored by dissociation. While this charge detection isfound to work in isolated, positively charged chromophores or amino acids, it is efficiently quenched insome peptides. We explain this by photoactivated charge transfer and charge storage in nonaromatic groupsof the peptides. At threshold this process is found to be strongly dependent on amino acid substitution evenfar away from the site of photoactivation. For analysis we first set up a local molecular orbital model forpeptide cations and subsequently obtain a landscape of local electronic cation states formed by local hole andlow-lying extended coulomb states. Charge transfer is found to be a through-bond mechanism involvingenergetically accessible electronic states along the path of charge flow. Charge transfer between hole statesis mediated with very high efficiency through saturated carbon bridges by extended coulomb states. Thisnew mechanism seems to be generally applicable to large extended molecular radical cations. Only barriersof the size of a full length of a certain defined amino acid are found to block charge transfer. The modelqualitatively accounts for the order of the rates of the processes involved.

I. Introduction

The dynamics of proteins in its many facets is patentlyimportant and its nature is without doubt peculiar to theproperties of the constituent amino acids. One of the mostessential aspects of this dynamics involves the extremely rapidtransfer of charges and excitation energy often serving as anenergy pump for biological systems. These processes can besuccessfully seen and studied already in medium-sized polypep-tides. We introduce a new model for the understanding ofcharge-transfer processes in cationic peptides and demonstrateits applicability to a series of tailor-made polypeptides.

Charge transfer is a fundamental process in bioenergetics such

as in photosynthesis.

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In native charge-transfer systems anelectron is transported between donor and acceptor through largepeptides and proteins.3-6 Here through-bond and through-space mechanisms can contribute to charge transfer. Becauseof the complexity of the peptides, the importance of individualamino acids in controlling electron transport is not yet under-stood in detail. We will demonstrate here that it can be highlyspecific and strongly dependent from amino acid composition.

In typical charge-transfer model systems special donor-acceptor constituents are used to enable local photoexcitation

which triggers the charge-transfer process. We are investigatingcharge transfer and charge migration in native peptide cationswithout additional donor and acceptor units attached, in orderto understand the detailed contribution of the individual aminoacids to the charge migration process not mitigated by bridge-head structures at both ends.

Bridges spacing donor and acceptor complexes are usuallyconsisting of rigid saturated carbon chains, a molecular systemfar away in its properties from peptide chains. Only fewexperiments on model charge-transfer systems with donor andacceptor spaced by peptide oligomer bridges have been carriedout.7-9

Oligopeptides, due to their chainlike valence structure andtheir functional groups, spaced by short saturated carbon bridges(Figure 1a), provide a special one-dimensional model systems.It is this linear, repetitive structure which is causing specialeffects in transport of positive charge through peptides as wewill demonstrate. The positive charge conductivity of peptidescan be regarded as being analogous to a molecular wire, similarto concepts presented by Ratner and co-workers.10 We hereshow that a very efficient charge-transfer mechanism is activein radical cations of peptides.

For treating charge transfer in the gas phase, the relevantpotentials of course do not include solvent effects (see ref 11)but rather refer to normal coordinates of the molecule. In thiscase charge transfer becomes a pure intramolecular relaxation

* To whom correspondence should be addressed. Present address: Institute of Chemistry, Akadeemia tee 15, EE 0026

Tallinn, Estonia.X Abstract published in AdVance ACS Abstracts, November 1, 1996.

18567J. Phys. Chem. 1996, 100, 18567-18585

S0022-3654(96)00926-4 CCC: $12.00 1996 American Chemical Society


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process12-15 similar to internal conversion14,15 taking placebetween two electronic states located at different sites of themolecule. The theoretical description leads to an energy gaplaw for the charge-transfer rate15 similar to that found for internalconversion.16,17

For neutral molecules in solvents intramolecular energy isnot conserved, and the charge-separated state is stronglystabilized by solvent shielding. In the gas-phase molecular18-20

and cluster charge-transfer systems21-23 mostly implode and

form exciplexes. This is due to the large coulomb forceresulting from charge separation. Bixon, Jortner, and others12,13

have analyzed charge transfer in neutral isolated supermoleculesand suggested that charge separation should have a limited rangeof 7 . So far there is only one case where evidence for long-range electron transfer in the gas phase has been observed.20

There is no distance restriction expected for anions and cations.12

As this indicates the situation in cations for charge transfer isquite different than that for neutrals:

(i) By local ionization of peptides a positive hole is createdin the HOMO orbital of an aromatic chromophore. Due to thissurplus charge, transfer becomes a charge shift process.

(ii) The motion of the charge does not substantially affectthe global geometry because of the nonbonding character of

the electronic states involved and the lack of electron-holecoulomb attraction. This is clearly in contrast to neutral charge-transfer systems where due to the large coulombic forces (gasphase) or the strong shielding of the solvent (liquid) thegeometry of the charge-transfer state is typically stronglydistorted.

(iii) The electronic states involved are essentially localized.They correspond either to localization of the positive charge atdifferent functional sites in the peptide cation or to shakeupstates.

(iv) In cations quartet states are situated at high energies.Relaxation processes of excited states therefore are exclusivelyintra- or interchromophore processes of internal conversiontype.

(v) In linear extended cations shakeup states can be extremelylow in energy due to coulomb forces (extended coulomb states).They can efficiently mediate charge transfer through shortsaturated carbon bridges. The low energy of such states appearsas a general characteristic of linear extended radical cations.We believe that these states are the essential element facilitatingcharge migration in most large molecular radical cations.

(vi) Because of the small energetic spacings of electronicstates an energetic order of local electronic states can be tailoredin a staircase fashion: Charge transfer to the other side of the

peptide then becomes a multistep process probably without anybarrier. To our knowledge such a barrierless through-bondmultistep mechanism has so far not been investigated.

The arguments given in i-vi show that investigation of themechanism of charge flow in peptide cations in the gas phaserepresents a new approach to the study of charge transfer inpure proteins. New insights in the mechanism of chargemigration in radical cations are provided. Although theexperimental investigation of peptides in the gas phase is insome ways much more complicated, the above-mentionedadvantages greatly simplify the interpretation of data for thesemolecular systems and permit us to understand the mechanismby a molecular description in local molecular orbitals togetherwith their couplings. It should be noted that gas-phaseinvestigations of ionization, charge shift and dissociationprocesses have essential advantages:

(i) Ionization has large cross section because no chargerecombination after ionization as in solvents can occur.

(ii) No reorganization of the solvent has to be considered.(iii) The detectability of isolated ions and electrons in the

gas phase is high.Investigations of electron transfer in peptide cations without

cofactors are known only for few solution phase experiments.24-28

In those experiments, however, protonation and deprotonationwas involved. Little has been done so far on peptide radicalcations under isolated conditions.29-31

Special tailor-made peptides have been synthesized for these

investigations which can be photoexcited locally. Subsequentcharge migration is steered by choosing a suitable amino acidsequence. Thus control of the position of the charge will bepossible. Usually we have a single aromatic chromophore inthe peptide to ensure resonant photoexcitation and hence localionization of the chromophore site. As an example of a typicalsample the peptide alanyl-alanyl-alanyl-tyrosine (Ala-Ala-Ala-Tyr) is shown with its structure in Figure 1a. As discussedpreviously29 resonant (1+1)-UV photon absorption at thearomatic side chain of tryptophan (Trp), tyrosine (Tyr), orphenylalanine (Phe) is site selective. Local ionization is easilyachieved due to electronic selection rules and energetic condi-tions (see Figure 1b). This initial charge localization is theinherent precondition for the observation of charge flow in the

Figure 1. (a) Structure of the peptide Ala-Ala-Ala-Tyr. We typicallyuse peptides with a single aromatic chromophore in the C-terminalposition and nonaromatic amino acids in the chain and at theN-terminus. (b) In a local picture of the peptide by resonant two-photon excitation a local ionization at the aromatic side chain is possible(see text). Without further photon activation the positive charge staysthere because of the low ionization potential of the chromophore. (c)

Laser mass spectrum of Ala-Ala-Ala-Tyr: UV photon excitation ofthe radical cation results in an internal energy of 4-5 eV. The observedfragment ion of mass 44 Da can be identified as a positive chargedN-terminal fragment (see a). Its occurrence can be exclusivelyexplained by charge migration.

18568 J. Phys. Chem., Vol. 100, No. 47, 1996 Weinkauf et al.


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cation. In our previous paper29 and in Figure 1 we show thatafter UV excitation of peptide cations charge migration canoccur. This is derived by the type of fragment ions found inthe mass spectra. For example in the UV laser dissociationmass spectrum of the Ala-Ala-Ala-Tyr cation (Figure 1c) afragment ion of mass 44 Da is observed, which is undoubtedlydue to the positively charged immonium fragment ion shownon the left hand in Figure 1a. Despite that the positive chargewas initially located at the hydroxyphenyl chromophore ((1+1)-ionization) the charge is finally found at the N-terminal fragment

ion. Such charge transfer at high internal energies has beenobserved for Leu-Leu-Tyr,29 Leu-Leu-Leu-Tyr, Ala-Ala-Tyr,Ala-Ala-Tyr-Ala-Ala, and other peptides.32

The process of charge migration exists in peptide cations andmust have a threshold. Our goal here is to investigate thisthreshold and to study how charge transfer can be correlated tothe properties of the peptides and its amino acid subunits.Charge transfer directly after ionization has been shown to occurby Levy and co-workers for some bichromophoric molecules33

and by us for N,N-dimethylphenylethylamine.34 For peptidescontaining only one aromatic amino acid, it is obvious thatelectron transfer directly after ionization is impossible forenergetic reasons (see local ionization energies in Figure 1band Table 2). Charge migration in these peptide cations requires

photoactivation and takes place in excited electronic states. Theabsorption shift to the green, which can be found for aromaticmolecules upon electron removal and the fact that chargednonaromatic amino acids as glycine and leucine do not absorbin this wavelength range allows site- and charge-sensitivephotoexcitation (see section V.II). Detection of a doublyresonant two-photon excitation of the peptide cations is thenrealized via dissociation. Note that the kind of fragment ion insuch an experiment is not of interest but only the total fragmention current. If charge is stored outside the aromatic chro-mophore throughout the duration of the laser pulse, nofragmentation will be detected.

In this work we describe our pump-probe technique in detailand present new results for peptides differing in size, in

chromophores, and in nonaromatic constituents. For somepeptides we observe two-photon absorption in the cation, forsome not. Investigations show that the effect of quenching ofthe two-photon excitation is not simply correlated to themolecular size or to absorption changes. Several alternativeexplanations are discussed in detail, but charge transfer is arguedto be the only reasonable mechanism.

Furthermore, we present a model of charge transfer in peptidecations based on a local molecular orbital picture and alandscape of local electronic states. This picture allows us toestimate the energetics of the process. We find that energeticallyaccessible electronic states of functional groups are efficientlycoupled by low energetic extended coulomb states. In case ofcharge transfer we find a staircase-like situation without any

barrier for charge flow. Time scales of rates are qualitativelyin agreement with this model.

II. Experimental Setup

Large biological molecules are thermally unstable. Thus, forobservation in the gas phase, they have to be evaporated byspecial laser desorption techniques.35 Ionization of neutrals isperformed by multiphoton ionization by UV laser pulses, atechnique that has been proven to be a suitable tool for massspectrometrical analysis.30,31

The experimental setup used here is similar to that of ourprevious experiments.29 In the first vacuum chamber thenonvolatile molecules and peptides were laser desorbed by Nd:

YAG laser pulses (1064 nm, 1 mJ/pulse, 200 ns pulse duration).The sample holder was a stainless steel rod homogeneouslycovered by the peptide sample by means of electrospray. Thesample rod was turned by a stepping motor to ensure that foreach cycle a new surface was desorbed. The desorption processtook place in a closed channel (diameter 1 mm, 6 mm long)which is filled by Ar gas through a pulsed nozzle (3 barbackpressure, nozzle diameter 400 m). In the Ar beam themolecules are cooled and transported through a skimmer intothe ionization chamber of a reflectron time-of-flight instrument.

The pressure there was typically 10-6

mbar. Only neutralmolecules can reach the ion source due to the permanentlyapplied voltages of+1600 V (Repeller) and +1000 V (secondion source electrode).

The local ionization of the intact and cold neutral moleculesis performed by resonant two-photon UV ionization with thefrequency doubled output of an excimer laser pumped dye laser(UV pulse energy 100 J, focus diameter 500 m, pulse width5-7 ns, wavelength range 260-290 nm). By carefully adjustingthe UV laser intensity, it is possible to achieve softionization.29-31 Thus, neither neutral fragmentation nor sub-sequent ionization of fragments contribute to our mass spectra,nor is a further UV photon absorbed by the cation. The latterstatement can be derived from the low dissociation threshold

for formation of the immonium ion as discussed previously. 29Photodissociation is exclusively performed by a second dyelaser in the visible (VIS) wavelength region, which was pumpedby the same excimer laser. We employed a time delay of 0and 10 ns between ionization laser (laser 1, UV) and photo-fragmentation laser (laser 2, VIS) and found that the results havebeen independent of this delay and fixed it to 10 ns. Typicalpulse energies of the visible laser radiation have been 1.5 mJat a focus diameter of 1 mm. The pulse width was 7-10 ns atwavelengths of 480-550 nm. To ensure complete overlapbetween the UV and VIS lasers, resonant multiphoton dissocia-tion of benzene was monitored for the adjustment before andafter each peptide experiment. Mass separation was performedin a reflectron time-of-flight mass spectrometer. The ion signal

was digitized by a transient recorder and stored in a computerat a repetition rate of 6 Hz. Mass spectra have been averagedover 100 shots.

Calculation of electronic state energies and molecular struc-tures have been performed on a silicon graphics work stationand a CONVAX by MOPAC at a PM336 level taking intoaccount configuration interaction. Due to the time-consumingcalculations for peptides, we confined our investigations at thislevel of theory to the tripeptide Gly-Gly-Tyr. Structures of otherpeptides have been calculated at a MNDO level. The energeticpositions of extended coulomb states in peptides have beenestimated by calculation of such states for small molecules at aPM3 level including configuration interaction. We are awarethat due to the three singly occupied orbitals these energetic

values only qualitatively describe the situation (see sectionV.II.I).

III. Concept of the Site-Sensitive Charge Probe

In the tailor-made peptides we employ here, charge transfercan occur only in electronic excited states of the cation (seesection V.II). For site-selective activation and site-selectivecharge probing we make use of the fact that the absorption ofaromatic chromophores changes strongly upon electron removal.This dependence on the charge state of the chromophoreproduces a switching effect since the VIS absorption is switchedon or off. For the understanding of the relevant processes inpeptides we first need a short review of the photoinducedprocesses in isolated aromatic chromophores.

Charge Transfer in Peptide Cations J. Phys. Chem., Vol. 100, No. 47, 1996 18569


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III.I. Photoexcitation in Isolated Aromatic Cations. Theabsorption of aromatic molecules strongly changes upon electronremoval. Whereas neutral aromatic molecules absorb UV (firstband at 240-290 nm), their corresponding radical cations absorbresonantly both UV and visible light. This is well-known fromfluorescence spectroscopy,37 photodissociation spectroscopy,33,38-43

HeI photoelectron spectra,44,45 and absorption spectra of aminoacid ions in glasses.46

The reason for the absorption change upon electron removal

is easily understood by the molecular orbital scheme (Figure2) of the aromatic chromophores, here benzene. Each electronicstate (Figure 2a) represents an electron configuration in themolecular orbital scheme (Figure 2b). Positions of the molecularorbitals can be taken from band positions in HeI photoelectronspectra as described in section V.II.

Resonant (1+1) UV excitation via a * orbital results inionization (UV1, UV2 in Figure 2a,b). By ionization a hole inthe HOMO orbital is created. Note that now in all photoexci-tation processes the positive hole is moved downward to lowermolecular chromophore orbitals (see Figure 2b VIS1,2 andUV3). Absorption of UV photons in aromatic cations isresonant as proven in various experiments29-31 (UV3 in Figure2b). In small peptide cations such UV photon absorption can

be detected by dissociation. At low UV laser intensitiesphotoabsorption in the cation can be avoided and exclusivelyparent cations are produced. In charged aromatic chromophoresalso resonant photoexcitation processes can be performed bytransitions in the manifold of bonding orbitals (VIS1,2 in Figure2a,b). Due to their small spacing the resulting phototransitionsare in the infrared (X-A transition) and green wavelength range(X-B and B-E transition, in Figure 2a). In peptides we usethe B-X transition for activation of charge transfer.

In the B-state VIS photon excitation is also resonant with

highly excited electronic states (E state in Figure 2a). Theexistence of such resonant electronic states can be taken fromHeI PE spectra44,45 and the fact that UV photons (hUV ) 2hVIS)are resonantly absorbed (see above). Thus, in aromatic cationstypically a multiphoton excitation with wavelengths of 480-530 nm is multiresonant.

Lifetimes of Excited Electronic States. Excited electronicstates in aromatic cations typically have short lifetimes.37,47,48

This lifetime shortening is attributed to fast and ultrafast47,48

internal conversion (IC). In cations quartet states are higher inenergy and internal conversion is the only relaxation mechanismof the first excited states.49 In the MO picture, after photoex-citation of the cation to the B states, the internal conversionprocess fills the deeper hole with a HOMO electron (see Figure

2b). A large IC rate in aromatic cations can be understood bythe relatively small energy gap of 1.5-2 eV between electronicstates and the energy gap law.17 Ultrafast processes are due topotential intersections in one or several reaction coordinates andmostly appear at higher energies (for benzene for example atthe C state level47,48). In our chromophores the internalconversion rate acts like an internal clock which is measuringother competing processes in comparison to its own period.

Therefore in the B state, which we excite in the VISphotoactivation step, further photoabsorption has to competewith fast internal conversion. In nanosecond laser pulseexcitation usually optical pumping rates are small (1/100 ps).Therefore they cannot compete with internal conversion pro-cesses, and further photon absorption occurs indirectly.

After fast internal conversion to the electronic cation groundstate, the B state can be reexcited by a second VIS photon butfrom a higher vibrational level. Explicitly for C2H2+ we couldshow that resonant vibrational states with lifetimes of 2 ps canbe detected by dissociation after a multiphoton excitation.39 Thetwo-photon resonance has been proven as well to hold for thefluorobenzene cation38 where excitation to the excited state (firstgreen photon) and photodissociation (several green photons) wasseparated in time by some microseconds. This agrees well withFranck-Condon considerations which show that such a transi-tion would be favored by small differences in vibrationalquantum numbers. Hence we generalize this observation to allour aromatic chromophore cations and rule out that internal

conversion processes are blocking the second VIS photoab-sorption.Either direct or via internal conversion (see Figure 2a)

multiresonant multiphoton absorption is a signature of chargedaromatic chromophores and hence inherently a result of thepresence of the positive charge. Annihilation of the positivecharge would turn back the absorption to the UV wavelengthrange and interrupt photoabsorption of VIS photons. Dissocia-tion occurs after a resonant two-photon or multiphoton excita-tion. Thus, the yield of dissociation is an appropriate signalfor detection of the efficiency of the resonant VIS two-photonabsorption processes in the cation. This concept was previouslyapplied by us and others for spectroscopy of short livedelectronic excited states in cations.39-43,50,51 In analogy to

Figure 2. Photoabsorption of aromatic chromophores turns fromultraviolet to green by electron removal. (a) UV-VIS ionization andion excitation scheme in aromatic molecules. Multiresonant multipho-ton excitation either direct or after internal conversion can be detectedby dissociation. (b) Molecular orbital scheme and photoexcitationsteps: Excitation of the cation is explained as electron hole shiftingdownward in the manifold of bonding electronic states. Due to thesmall spacing of the bound states excitation by several green photonsis always multiresonant. (c) VIS photon dissociation spectrum of the

benzene cation, X-B transition: resonant absorption of several VISphotons (500-550 nm) is detected by dissociation. The dissociationcan take place at the three photon energy level. In peptides becauseof the high density of vibronic transitions broad absorption bands canbe expected.



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multiphoton ionization, by tuning laser wavelength and detectionof all fragment ions, spectroscopy of the resonant intermediatestates of cations is possible. This technique can be applied tothe cations of the amino acids tryptophan, tyrosine,29 andphenylalanine.

As an example for the multiresonant VIS multiphoton

absorption in aromatic cations, the spectrum of the X-

Btransition of the benzene cation is shown in Figure 2c. Thespectrum was obtained by monitoring the signal of the C6H5+

fragment ion as a function of the VIS laser wavelength. Thefirst fragmentation channel produces C6H5+ and is reached bya three-VIS-photon excitation. The observed structure corre-sponds to vibrational features of the B state. These structurescover the wavelength range 500-550 nm and continue below500 nm (C state). The assignment of the vibrational featuresis described elsewhere.40 We know that the peak width of thespectrum is mostly determined by saturation and power broad-ening because of the high intensity needed for the three-photonstep to achieve dissociation.38 A lifetime of some picosecondsor shorter can be assumed for the B state.38

The multiple resonant VIS multiphoton excitation is a generalfeature of all aromatic chromophores. It is worth noting thatthe photodissociation spectrum agrees well with the firstelectronic band in the HeI PE spectrum.44 Differences invibrational structures are due to different selection rules causedby the high symmetry in benzene. Such symmetry selectionrules are weakened in tyrosine and tryptophan, and thusabsorption bands of aromatic cations can be predicted as energydifferences of bands in HeI PE spectra.44,45 In section V.II wewill make use of this concept for amino acids.

III.II. Site-Selective Activation and Site-Selective Charge

Probe in Peptide Cations. For site-selective activation andsite-selective charge probing in the peptide cations we makeuse of the optical properties of the charged aromatic chro-

mophores described above. As for photoabsorption of aromaticchromophores embedded in amino acids, it is known from HeIphotoelectron spectra52-55 that the energies of the local electronicchromophore states are not or only slightly shifted. In our tailor-made peptides the chromophore is always substituted at theC-terminus and hence spaced from the side chains of the nextamino acid by two saturated carbon atoms and the peptide bridge(five bonds). Therefore, we assume that their electronicenergies and absorption cross sections stay the same in peptides.The optical spectra in peptide cations are, however, expectedto be broad and diffuse due to the large density of vibrationalstates and a continuous tuning of the excitation wavelength isnot required.

In Figure 3a the scheme of the charge-sensitive detection inpeptide cations is shown for Leu-Leu-Trp. The excitationscheme of the chromophore is the same as in Figure 2a. Aftersite-selective ionization the first resonant VIS absorption (VIS1in Figure 3a) photoactivates the peptide cation. This processis resonant for all the tailor-made peptides we investigate. Asmentioned above photoactivation performs a transport of the

positive hole to a lower molecular orbital of the chromophore.In contrast to the case of the isolated chromophore at thisenergy level, charge transfer from the nearest chain unit canoccur and annihilate the positive hole in the aromatic chro-mophore (see Figure 3a: CT). This charge transfer process isin direct competition to the intrachromophore internal conversionrate, and we only observe can two limiting cases in which eithercharge transfer is very fast or slow in comparison with theintrachromophore internal conversion rate, which, in thisfunction, acts as a clock. The charge annihilation in thechromophore returns back to its neutral ground-state absorbingUV. No VIS photoabsorption is observed for the chargeds-bonded amino acid cations such as Gly, Ala, and Leu, eitheras N-terminal or chain amino acid in the peptide in the

Figure 3. (a) Detection scheme for charge transfer in peptide cations, example Leu-Leu-Tyr: UV1,2: Site selective ionization at Tyr. VIS1:photoactivation. CT: charge transfer to the nonaromatic chain. VIS2: detection of the positive charge in the chromophore. IC: internal conversion

and subsequent VIS2 excitation. Diss: dissociation after two VIS photoabsorption. (b-d) Two-color UV-vis mass spectra. Note the largedifferences in the two-photon absorption behavior despite the same chromophore. We here clearly observe an effect in which the whole peptideis involved. Selective local and fragment free two-photon UV ionization at 287 nm is not shown. VIS1 and VIS2 photon excitation of the cationsis performed in the wavelength range 490-550 nm. (b) In Gly-Gly-Trp two photon VIS absorption is detected by dissociation. (c) In Leu-Leu-Trpresonant two-photon VIS absorption is efficiently quenched (no fragmentation). We take this as indication of charge flow out of the aromaticchromophore at the one photon VIS energy. (d) In Leu-Gly-Leu-Trp fragmentation by absorption of two VIS photons is observed. We interpretthis by glycyl acting as barrier (see text).



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wavelength range 480-530 nm (see Figure 3a and section V.II).Therefore as long as the positive charge is stored in thenonaromatic part of the peptide during the laser pulse no secondphoton absorption is observed.

No dissociation occurs in peptides at the one-VIS-photonenergy level due to energetic reasons (section V.II). Photoab-sorption of two-VIS-photons leads to fragmentation. At 4.4-5eV internal energy dissociation takes place. Neither the kindof fragment ions nor charge transfer at the two-photon level ispart of our immediate interest: Simply the yield of fragment

ions of any kind is used for detection of the presence of thecharge in the chromophore site after photoactivation. In short,dissociation of our tailor-made peptides with VIS light meansno charge transfer after activation; no dissociation means thatafter photoactivation charge transfer and storage of the chargeoutside the chromophore occures. In our first paper on thissubject we could show that the VIS two-photon excitation inthe cation works for the amino acids tyrosine and tryptophaneand for the tripeptide Gly-Gly-Trp but interestingly not for Leu-Leu-Tyr. We explained this by charge transfer in the Leu-Leu-Tyr peptide cation at the one-photon energy level.29

IV. Results

In this part we present new results of UV-VIS two-colorexperiments at tailor-made small peptides and give an overviewof all peptides investigated up to now. Initial charge localizationin peptides is performed by resonant (1+1) UV two-photonionization of aromatic chromophores. The UV wavelengths forionization have been chosen in accord with the neutral reso-nances in the amino acids.29,56,57 For each peptide it waspossible to obtain a completely fragment-free UV ionizationmass spectrum, which indicates that peptide absorption stopsafter resonant two-photon UV ionization (see section V). Inour first publication on this subject we always displayed theUV ionization mass spectra which have the parent cation as asingle peak. In this work, for simplicity, we display only thetwo laser UV-VIS mass spectra.

Photoexcitation of the peptide cations was performed 10 nsafter ionization by a second laser. Photoactivation and chargeprobe is performed by the same visible laser (pulse width 5ns). Charge probing in the aromatic chromophore has beendetected by cation fragmentation in a reflectron time-of-flightmass spectrometer.

We showed previously that two VIS photons can be absorbedin the wavelength range 480-530 nm in isolated amino acidstryptophan and tyrosine. In the following we show howphotoabsorption in these chromophores depends on compositionof the peptide.

Tryptophan-Containing Peptides. In Figure 3b-d theUV-VIS two-color mass spectra of some tryptophan-containingpeptides are shown. All peptides possess the aromatic amino

acid tryptophan as single aromatic chromophore and only thenonaromatic parts are exchanged. Because the VIS photoab-sorption takes place in the identical chromophore the propertiesof the three peptides should be very similar.

As Figure 3b-d shows, the differences in the two-color UV-VIS mass spectra are substantial: Fragmentation is observedfor Gly-Gly-Tyr and Leu-Gly-Leu-Tyr, whereas no fragmenta-tion is observed for Leu-Leu-Trp. The comparison of thepeptide sequences and the corresponding UV-VIS mass spectrain Figure 3b-d clearly shows that the effect of quenching ofthe VIS two-photon absorption is neither a simple internalchromophore effect nor a simple effect caused by one of thenonaromatic amino acids. If only one amino acid is added (asin Figure 3c-d) the two-photon absorption behavior of the

peptide is entirely changed. For example because the substitu-tion of glycyl versus leucyl has been performed at the non-neighboring amino acid of tryptophan (see Figure 3d) a simplechange of the absorption cross section of the charged aromaticchromophore or a shift of its absorption wavelength can be ruledout as very unlikely. According to our charge detection schemedescribed in section III, this bifurcational behavior is directlycorrelated with an absorption of a second VIS photon (chargein the chromophore, see Gly-Gly-Trp) or quenching of the VISabsorption by charge transfer (charge moves to the chain, see

Leu-Leu-Trp).We exclude experimental reasons for the differences in ourUV-VIS mass spectra of Figure 3b-d. To ensure that allparameters were optimized, we had to perform several tests ineach experiment:

(i) Laser overlap between the UV and the VIS laser was testedbefore and after each peptide mass spectrum, by switching to abenzene resonance.

(ii) In addition the VIS laser wavelength was tuned stepwisefor each peptide between 480 and 530 nm.

(iii) The laser pulse energy of the VIS laser was variedbetween 50 and 2 mJ without substantially changing thebifurcational character of the spectra.

In the latter experiment (iii) the differences of the two-photoncross section can be estimated: Whereas at laser pulse energiesof 2 mJ VIS light (5 ns pulse width) more than 90% of isolatedtryptophan and the tripeptide Gly-Gly-Trp can be dissociated,vanishing amount of dissociation is found for the tripeptide Leu-Leu-Trp. Hence we do not find effects of a few percent butmore of several orders of magnitude. To explain our result forLeu-Leu-Trp, the lifetime of the excited isolated chromophorestate B has to be shortened severely by charge annihilation. Byour rate model a charge transfer rate faster than 1/(50 ps) wasestimated. The complex behavior of the peptides in Figure3b-d shows that some matching of electronic states in the aminoacids is necessary for charge transfer at such a low energy levelto understand the mechanism. The results have to be correlatedto the energetic positions of molecular orbitals of tryptophan,leucyl and glycyl as will be discussed in section V.II.

A Barrier for Charge Transfer. The motivation for theinvestigation of the peptides Leu-Gly-Leu-Trp (see Figure 3d)and Leu-Gly-Gly-Leu-Trp (not shown here) was the search fora barrier for charge flow. For both molecules two-photon VISchromophore excitation and fragmentation is observed incontrast to Leu-Leu-Gly. Obviously glycyl in combination withtryptophan blocks charge migration and acts as a barrier. Thismust have energetic reasons as we show below. The results ofLeu-Gly-Leu-Trp and Leu-Gly-Gly-Leu-Trp are especiallyinteresting because they demonstrate several effects:

(i) An absorption shift of the excited state in the indolechromophore is not responsible for the quenching of the two-

photon absorption. For example, in Leu-Gly-Leu-Trp incomparison to the case Leu-Leu-Tyr the neighboring amino acidof tryptophan was not changed: Absorption shifts due to varioussubstitutions performed seven bonds away from the chro-mophore can be neglected.

(ii) Charge transfer proceeds through the peptide chain bonds.Otherwise a head-to-tail (i.e., through space) transfer wouldhave been able to occur even in the presence of the glycyl barrier(see section V.II).

(iii) We assume that charge is transferred to the first leucyl(see above) but then cannot overcome the barrier of glycyl andcharge is then transferred back into the aromatic chromophorewithin our laser pulse duration of 5 ns. Backflow of the chargeinto the chromophore leads to the ground state X (probably via



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the A state). As explained previously the second VIS photonexcitation then is again resonant with the X-B chromophoretransition, but in a higher vibrational manifold.

Charge Transfer in Biaromatic Peptides. To demonstratethat we really can move charges in peptide cations, weinvestigated a biaromatic peptide linked by nonaromatic aminoacids: The concept is detection of charge arrival at the otherside of the peptide. We gain this information by comparisonof two UV-VIS mass spectra of Leu-Leu-Leu-Tyr and Phe-Leu-Leu-Tyr.

A precondition for this experiment is the initial chargelocalization in Tyr. As is well-known by liquid- and gas-phasespectra of the amino acids Phe and Tyr,56,57 the UV absorptionof Tyr is red-shifted against those of Phe. We first test theionization to be localized at tyrosine by ionizing a mixture ofthe dipeptides Phe-Leu and Leu-Tyr at different wavelengths.In Figure 4a the mass spectrum of the mixture is shown at anionization wavelength of 266.25 nm (resonant for Phe and Tyr).Both peptides appear in the mass spectrum. At a wavelengthof 281.75 nm (resonant for Tyr only) the dipeptide Leu-Tyr isionized selectively (see Figure 4b). Thus, at a wavelength of281.75 nm in the peptide Phe-Leu-Leu-Tyr composed of bothparts we locally ionize in the aromatic side chain of tyrosine.

Charge transfer directly after ionization is impossible due to

energetic reasons. The ionization potential of tyrosine is by0,4 eV lower than that of phenylalanine (see Table 2). Thislarge difference in ionization potentials and the energetic ordermakes through-space resonant charge exchange directly afterionization impossible.

In both peptides Leu-Leu-Leu-Tyr and Phe-Leu-Leu-Tyrcation photoactivation and charge probing by visible light(shown in Figure 4c,d) has been performed under absolutelyequal conditions. The VIS photon excitation does not lead todissociation for Leu-Leu-Leu-Tyr but does for Phe-Leu-Leu-

Tyr. Apparently the second aromatic chromophore plays anactive role in the photoabsorption of the cation.In accordance with our results given above the lack of

fragmentation for Leu-Leu-Leu-Tyr indicates charge transfer tothe peptide chain. This implies that in Phe-Leu-Leu-Tyr afterVIS photoactivation the charge transfer at least to the leucylcan occur. The ionization potential at the N-terminus is 8.5eV. If the charge would be once in the chain, in Phe-Leu-Leu-Tyr charge can be transferred to the second aromatic chro-mophore Phe because of its lower ionization potential of 8.4eV. The side chain of phenylalanine then could act as a efficienttrap for the positive charge. The second VIS photon then canbe absorbed in the charged aromatic benzyl ring of theN-terminal Phe leading to fragmentation. Such a process would

Figure 4. Charge transfer to the other side of the peptide: (a, b) MPI mass spectra of a sample mixture of Phe-Leu (a) and Leu-Tyr (b) at theionization wavelengths of 266.25 and 281.75 nm. (b) Selective ionization of tyrosine can be achieved at 281.75 nm. We use this wavelength forsite-selective ionization of the peptide composed of both parts. (c, d) UV-VIS mass spectra of Leu-Leu-Leu-Tyr (c) and Phe-Leu-Leu-Tyr (d). (c)No dissociation indicates that after photoactivation charge is transferred out of the chromophore and stored in nonaromatic sites of the peptide.(d) After local ionization and VIS photon activation in Tyr we explain dissociation now by absorption of a second VIS photon in the chargedaromatic ring of Phe: for this, charge has been transferred to the other side of the peptide.



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explain the occurence of a second VIS photoabsorption inaccordance with the observation of dissociation.

Overview over the Peptides Investigated. We investigateda variety of peptides and found either quenching or absorptionof VIS light. An overview is given in Table 1. In most of thepeptides charge transfer is observed. Two-photon VIS absorp-tion is found only in cases where the amino acids glycyl andtryptophan were combined or in some biaromatic peptides.Obviously an energetic threshold for charge transfer in peptidecations exists. For peptides containing the chromophore tyrosineno two-photon absorption is observed despite the fact that theisolated amino acid tyrosine absorbs two photons of VIS lightefficiently.29 All molecules listed in Table 1 have been

investigated with VIS wavelengths in the range 480-550 nm.No substantial change in the yield of fragment ion productionas a function of wavelength was observed. Especially forpeptides where no quenching of the two-photon excitation wasobserved we tried without success to provoke charge transferat higher photon energies. In Table 1 we include the energeticsof the charge-transfer process along the chain: We only observecharge transfer in cases where the process is exoenergetic (Epositive) along the path to the N-terminus. For a detailedexplanation see section V.II.

The collection of different peptides investigated delivers heremore information than each individual result. In the following,by referring to Table 1, we explain some of these conclusions.For example, experiments 1 and 6 show that photodissociation

is not a pure side-chain effect. Experiments 1, 2, and 4 showthat photoabsorption is not a pure chromophore effect. Hencethe full molecule is participating at the process taking place.Experiments 3 and 4 as well as 8 and 9 show that two-photonabsorption is not due to a simple level shift of the electronicstate in the chromophore. Through-bond charge transfer canbe ruled out by experiments 2, 3, 4, and 5. Complete chargetransfer to the other side of the peptide has to be assumed bycomparing experiments 8 and 9. No length and size dependenceof the effect is observed for peptides larger than two aminoacids which indicates that two-photon dissociation still worksand is not suppressed by a slow statistical decay (see 2, 3, and7, 8). Back charge transfer even after the one helical can beruled out by experiments 3 and 8. Permanent charge storing

outside the aromatic chromophore is essential for our 5 nsdetection scheme. We therefore do not consider of dipeptides.Short pulse excitation is required to obtain results here. Ourexperiments for various peptides show (see Table 2) that theprocess is inherently coupled with properties of the constituentindividual amino acids. For the occurence of charge transferin polypeptides apparently a certain energy balancing betweenlocal electronic states of the amino acids has to be fulfilled.

V. Discussion

We observe large differences in two-photon absorption yieldsin peptide cations which we explain by photoactivated chargetransfer out of the aromatic chromophore and storage in anonaromatic part of the molecule. As we will show below, wecan readily explain all our observations for peptide cations witha unified model.

Charge transfer has been demonstrated to occur in peptidecations at an internal energy larger than 4 eV. (section I29).This points to the fact that charge transfer exists in peptidecations and must have a threshold. In this work we concentrateon threshold effects at various tailor-made peptides in order tofind the direct correlation of our results with peptide propertiesand properties of individual amino acids. We believe that inpeptide cations the threshold for charge transfer lies in the energy

range 2-2.5 eV. This corresponds to photoactivation by a onephoton process in the wavelength range 480-530 nm.V.I. Alternative Possible Mechanisms for the Quenching

of the Second VIS Photon Excitation Step in Peptide Cations.

For completeness we discuss various alternate possibilities toexplain our findings. We feel that we have strong evidencefor a threshold behavior of the charge-transfer process occurringin peptides at an energy of 2-2.5 eV. Local ionization at thearomatic chromophore is performed by resonant (1+1) UVIonization for all peptides investigated here. We here discussonly processes taking place in cations. Therefore, as mentionedabove our experiment consists of three steps: activation (firstVIS photon), charge probe (second VIS photon), and monitoringof two-photon excitation by dissociation. Therefore, to inves-

TABLE 1: UV-Vis Two-Color Results of Several SmallPeptides: Fragmentation, Charge-Transfer (CT) Behavior(+, -), and Energy (E) Balance for the Charge-TransferSteps (+: Exoenergetic)

peptidesfragmen-

tation CTEbalance for CT

steps 1, 2, 3, ... in eV

1 Gly-Gly-Trp + - +0.2, -0.1, +0.42 Leu-Leu-Trp - + +0.2, +0.2, +0.73 Leu-(Leu)3-Trp - + +0.2, +0.2, +0.2, +0.2, +0.74 Leu-Gly-Leu-Trp + CT and +0.2, +0.2, -0.1, +0.7

back CT5 Leu-(Gly)2-Leu-Trp + CT and +0.2, +0.2, -0.1, -0.1, +0.7

back CT6 Gly-Gly-Tyr - + +0.2 to nO C-term.

+1.6 to nN (Tyr)+1.3 to nN in chain+1.8 to nN N-term.

7 Leu-Leu-Tyr - + +0.2 to nO C-term.+1.6 to nN (Tyr)+1.6 nN in chain+2.1 to N-term.

8 Leu-(Leu)2-Tyr - + see above9 Phe-Leu-Leu-Tyr + + see above and text

+2.2 to highest (Phe)10 Ala-Ala-Tyr - + +0.2 to n0 C-term.

+1.6 to nN (Tyr)+1.4 nN in chain+2.0 to N-term.

TABLE 2: Energetic Positions of the Lowest ElectronicStates in Some Amino Acids As Taken from HeIPhotoelectron Spectraa

molecule EI band pos energy ref

leucine N-terminal nN 8.5 eV estimated, 55, 71, 72leucyl nN in chain 9.0 eV estimated, 55, 71, 72alanine N-terminal nN 8.6 eV 53, 55alanyl nN in chain 9.2 eV 53, 55, 67glycine N-terminal nN 8.8 eV 53, 55glycyl nN in chain 9.3 eV 53, 55, 70glycine C-terminal nO 10.5 eV 53, 55b

NH-CH2-CH2-NH CH2 like 11.3 eV 44CC 11.8 eV 44tyrosine band 8.0 eV 53, 58

band 8.7 eV 58nN in chain 9.0 eV estimated, 53nO C-terminal 10.4 eV 52, 53bband 10.6 eV 58

tryptophan band 7,4 eV 52, 54, 58band 8.0 eV 52, 54, 58nN in chain 9.0 eV estimated, 52, 67band 9.2 eV 52, 54, 58band 10.5 eV 52nO C-terminal 10.5 eV 58b

phenylalanine band 8.4 eV 53, 58N-terminal nN 8.5 eV 58band 8.8 eV 44, 58, estimatedband 10.9 eV 58

a All values are taken from the onset of HeI photoelectron spectra.b Same value taken as for glycine.



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tigate alternative mechanisms for the quenching of the secondVIS photon excitation, photoabsorption and dissociation at theone- and the two-VIS-photon level and intrachromophorerelaxation processes have to be investigated in detail.

V.I.I. Effects of Variation in VIS Photoabsorption. Forpeptides containing the same aromatic chromophore but differingin length and amino acid composition, differences in opticalabsorption could occur due to either (i) different initial internalenergies (ii) or shifts of electronic states at the one or two VISphoton level.

(i) As for differences in internal energies: The laser desorbedmolecules are cooled in a supersonic beam and ionized byresonant (1+1) UV ionization. Both processes do not contributesubstantial internal energy to the peptides and in addition therelevant parameters are very similar for all peptides. Neverthe-less, even an excess energy of 0,5 eV would result intemperatures far below room temperature because of the largenumbers of internal degrees of freedom. Large internal energiesin cations could be generated by further UV photon absorptionin the cation previous to VIS excitation. Such UV absorptionwould be resonant as explained in section III.I. The fact thatwe can ionize without any fragmentation, however, demon-strates, that peptide ions are formed in their electronic groundstate.

(ii) As for possible shifting of electronic states in energy asa result of the peptide environment: Large shifts of electronicstates either at the one or the two photon energy level can beexcluded. The aromatic amino acids were always attached atthe C-terminal side of the peptide and hence in all peptides, ifthe neighboring amino acid was changed, the site of substitutionis spaced by five bonds. Small or vanishing shifts are alsoconfirmed by the comparison of HeI photoelectron spectra ofamino acids52-55 and analogous molecules58 to the HeI PEspectra of the isolated chromophores.44,45

The most convincing argument, however, is that the quench-ing of fragmentation or even the occurrence of fragmentationis correlated with substitution of nonneighboring amino acids

(see Figure 3c,d) where the distance between site of substitutionand chromophore is enormous. In addition the absorption bandcan be assumed to be broad in peptides due to the high densityof vibrational states, thus making small shifts irrelevant for ourexperiment. Despite this, for resonant VIS excitation, wave-length have been varied between 480 and 530 nm. As forresonant excitation at the two-photon VIS level of the chro-mophore, we could show that for all peptides UV (hUV )2hVIS) absorption of the chromophore is possible.

V.I.II. Effects of Rate Variation of Intrachromophore

Relaxation Processes. Similar argumentation as above holdsfor influences of the environment on intrachromophore relax-ation processes as intrachromophore internal conversion andisomerization. As discussed in section III.I, relaxation of the

excited chromophore state by internal conversion is the pre-dominant pathway and would not stop resonant VIS multiphotonexcitation. Isomerization processes can be ruled out due to thelow internal energy at the one photon VIS level. This isespecially valid for all nonaromatic amino acids. In aromaticside chains the expected isomerization is the formation of a ringstructure consisting of seven carbon atoms similar to thecycloheptatriene cation: One carbon atom of the alkyl chainwould enter the aromatic ring forming a positively charged seven(Tyr and Phe) or six ring (Trp). For the phenylethyl cation itis known that its isomer, the methylcycloheptatriene, is 0.4 eVhigher in energy and that the barrier between both structures is1.1 eV.59 Other isomers are higher in energy or directlycorrelate to dissociation as for example the formation of the

tropylium cation. We are well aware that after one-photon VISexcitation we could be above the isomerization threshold.However, even if at the one-photon VIS level this isomerizationchannel would be accessible on the time scale of 5 ns (whichis improbable due to the high barrier), we expect that most ofthe molecules keep the six-ring structure because of the higherdensity of vibrational states there. Hence this would notcompletely switch on and off the photoabsorption process. Inaddition, this process should not depend on substitution far awayfrom the chromophore as observed.

V.I.III. Time Scale of Dissociation in Small Peptides. Apossible explanation for not seeing the same VIS photodisso-ciation for all peptides could perhaps be sought in molecularsize effects and the resulting various dissociation thresholds:The peptides investigated differ in number of atoms andmolecular composition. Both parameters can influence theenergetics and dynamics of dissociation, our detection step here.This can be excluded in as discussed below.

For dissociation at the one-photon VIS level, it is well-knownthat dissociation of large molecules place on a long time scaleeven for high internal energies.60,61 We know the localdissociation thresholds either N-terminal (IPN + 0.5 eV29) orC-terminal (IPC + 1.5 eV29). As shown previously29 the kindof dissociation depends from the relative position of the absolute

dissociation energies (which depends from the local IP) andthe internal energy. However for our peptides we find nocorrelation between the yield of fragment ions and our data.For example in Leu-Gly-Leu-Trp, due to the low ionizationenergy of Trp, at the one photon VIS energy level (2,5 eV) wewould be only 1.0 eV above dissociation threshold. In such alarge molecule no dissociation would be expected at such lowexcess energies.61 In contrast to this we observe fragmentation.This suggests a two-photon process. Other examples can befound which show that dissociation at the one-photon VISenergy does not occur.

After absorption of two VIS photons, molecules are excitedto an energy which is up to several electron volts (3.5-4.0 eV)above dissociation threshold. Due to the low dissociationthreshold at this high internal energy fast dissociation is expectedfor all peptides of size up to at least four amino acids. 61 Inaddition our results show that neither peptide length nor thenumber of atoms shows correlation with the dissociationobserved.

V.I.IV. Photo-Charge Transfer. For completeness we haveto discuss the possibility that the first charge-transfer step inpeptide cations could also be due to a direct photoexcitation.This would be an optical transition which excites an electronfrom the neighboring chain state into the HOMO orbital of thechromophore. Such a process would be possible in peptidescontaining tyrosine as a chromophore. We here observeabsorption of visible light below the estimated chromophore

absorption. No data, however, are available concerning directoptical excitation of charge-transfer states in large molecules.Optical excitation of charge transfer states has been observedto have a large transition moment in the weakly bound clustercation N2O+Ar.62 We cannot completely rule out a similarmechanism in our peptides.

We have good arguments to explain a fast charge transferand do not favor such a direct phototransition as the relevantmechanism. Especially the results of Gly-Gly-Trp and Leu-Gly-Leu-Trp sustain this concept: Optical transition to thecharge-transfer state by VIS light would be energetically possiblebut is not observed. Direct optical charge transfer would haveto compete with chromophore excitation. Transition momentsin aromatic chromophores, especially if they are charged, are



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large. To explain our results of complete highly efficientquenching of the two-photon VIS excitation, the optical transi-tion moment for the charge-transfer excitation has to be at leasta factor of 100 higher than that of the chromophore excitation,a value that is highly improbable. We therefore propose that adirect photo-charge transfer is not the main effect we observehere.

Another argument against a photo-charge-transfer process isthe exclusively observed local chromophore dissociation in 500fs laser pulse excitation of the polypeptides gramicidin D and

gramicidin S.63 Direct photo-charge transfer would havedistributed charge and photoexcitation over the molecule evenin femtosecond laser excitation, an effect that would lead tostatistical dissociation in contrast to our observation. Wetherefore assume photo-charge transfer to play an minor rolein the charge transfer we observe.

Our two-color experiments show that photoabsorption of twophotons of visible light is switched on or off in several peptidescontaining the same chromophore. The observed behavior ismultifarious but as such is highly informative: It is differentfor different chromophoric amino acids, different nonaromaticchain amino acids, different peptide length, barriers, andbiaromatic molecules. However, we can explain all these effectsby a charge-transfer mechanism and hence feel that this is the

most plausible explanation. As our results indicate, somematching between electronic states of the chromophores andpeptide chain amino acids has to be fulfilled in order to allowcharge transfer to occur. To verify this, we set up a zero-orderlocal molecular orbital model to obtain a landscape of localionization potentials for peptides. We discuss charge transferas a hole transfer involving charge positions in local orbitals offunctional groups. Extended coulomb states are found tomediate efficient charge transfer in peptide cations.

V.II. Energetics of Charge Transfer in Peptide Cations.

Our main goal here is the determination of the energetics ofthe charge-transfer process in peptide cations and the elucidationof the contribution of individual electronic states. So far thereis no experimental data of electronic states available for peptides.It is, however, well-known that theoretical calculations at aMNDO or PM3 level can well describe the conformation ofpeptides. Calculations of the ground state, excited electronicstates, shakeup states, and extended coulomb states of peptideions at a high level of accuracy is very time consuming. Thisis the reason we look for support mostly from experimental data.For this we have to develop a concept for determining energeticpositions of relevant electronic states of the peptides. Here wehave to distinguish two kinds of electronic states:

(i) Local hole states which correspond to electron removalfrom well-localized orbitals either lone pair orbitals or orbitalsof the aromatic chromophores (one molecular orbital is halfoccupied).

(ii) Electronic states which correspond to electron removaland simultaneous electron excitation into antibonding molecularorbitals. These states are called shakeup states64 and correspondto an electron configuration of three half-occupied orbitals. Inlinear chainlike molecules they can be extended and modifiedby coulomb forces and are called extended coulomb states.

Whereas local hole states are observed in HeI photoelectronspectra of amino acids and related molecules, valence shakeupstates are either not at all accessible by photoelectron spectro-scopy (two electron excitation)49 or in some cases appear assmall satellite structures.49 We there calculated their energeticposition by theory.

V.II.I. Electronic States That Correspond to Electron

Removal from Well-Localized Orbitals (Hole States). In

contrast to neutrals, in radical cations because of smallreorganization energies and the lack of large coulomb energyterms the energetic positions of the positive charge in varioussites of the peptide can be determined with high accuracy byHeI photoelectron spectra of amino acids and analogousmolecules. This is a zero-order description of localizedelectronic states in peptides, but to our knowledge this is thefirst attempt to determine energetic positions of electronic statesin peptides by experimental data. We therefore explain ourconcept in detail.

A special property of peptides is the localization of lone-pair and chromophore electronic states and their spacing by shortsaturated carbon bridges. As Figure 1a shows, the peptides,for our concern, can be analyzed in terms of several, well-spaced, functional parts: The amine group at the N-terminuson the left hand, the acid group at the C-terminus on the righthand, the nitrogen-oxygen site in the peptide chain skeleton,and the aromatic chromophore. In the following we derive theproperties of the functional groups in peptides from theirproperties in isolated well understood small molecular sub-systems. To simplify this task, we have to make someassumptions as follows:

(i) For determination of energetic positions of electronic statesin peptides, electronic states which are either lone pair or

aromatic side chain states and which are spaced by saturatedcarbon links can be treated as independent.

(ii) Couplings and energetic degeneracy of neighboringelectronic states have to be treated separately.

(iii) The energetic positions of the positive hole at variouslocations in the peptide chains are given by electronic bandpositions (ionization potentials) in HeI photoelectron spectraof amino acids and small analogous model molecules.

(iv) By using Koopmans theorem,65 electronic band positionsin HeI photoelectron spectra apart from the energetic positionsof electron holes describe the energetic positions of molecularorbitals.

The concept that electronic states which are spaced by

saturated carbon links can be treated as independent is amolecules in a molecule concept, similar to the compositemolecule approach.66 Due to the saturated carbon spacers wehere assume no or weak direct electronic coupling. In generalthe assumptions i-iv have been found to be useful for aminoacids52-55 and related molecules.58,67 For lone-pair orbitals innitrogen and oxygen a local approximation is widely used inHeI photoelectron spectroscopy and referred to as lone-pairspectroscopy.58,66-72 By substitution of methyl groups it hasbeen shown for amino acids and related molecules that lone-pair orbitals only slightly shift their energetic positions if thesite of substitution is spaced by two saturated carbon atoms.Similar considerations are valid also for interaction of lone-pair orbitals with aromatic ring systems isolated by two saturated

carbon bridges.58

Shifts and intensities of the electronic bandscan be predicted by substituent additivity effects66 with anaccuracy of (0.1 eV, in a similar way as the classicalWoodward rules73,74 are used for the absorption shifts in neutralmolecules.

Electronic States in Amino Acids and Related Molecules.

HeI photoelectron spectra of amino acids52-55 and relatedmolecules58,67,72 have been measured previously by others. Wemake use of these results in order to determine the energeticposition of the energetic position of molecular orbitals and thepositive hole at various locations in peptides.

Figure 5 shows reproductions of HeI photoelectron spectraof glycine (a),53 N-methylacetamide (b),67 and the aromaticamino acid tyrosine (c).53 The HeI photoelectron spectrum of



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the simplest amino acid glycine (see Figure 5a53) contains twowell-localized electronic states: The nN and the nO state whichare due to a removal of a lone-pair electron of either nitrogen(nN) or of the oxygen in the CdO group (nO). These twoorbitals are assumed to be independent and completely local-ized.53 The position of the electronic state corresponding tothe ionization in the amine group is situated at 8.8 eV (onset).Its smooth onset can be understood by ab initio calculations:75,76 The pyramidal amine group of the neutral amino acidbecomes planar in the cation. We therefore take the verybeginning of the onset as the position of the electronic origin.

The shift of this state on a substitution is about 0.2 eV for CH3(alanine) and 0.3 eV for CH2-CH2-(CH3)2 substitution (esti-mated from increments given for R, , and substitution inrefs 66 and 68-72). We take this amine lone-pair state and itsenergy value as characteristic of the highest occupied molecularorbital at the N-terminus. Strong localization of the nN orbitalhas been confirmed by electron momentum spectroscopy77 aswell by own MP336 and own ab initio calculations.75,76

Photoelectron spectra of the isolated amine group as for examplein ethylamine show a large gap between the ground and thefirst excited electronic state of the cation.44 Hence visible (480-530 nm) photon excitation is nonresonant.

In glycine the electronic state which is due to ionization ofthe C-terminal nO state is situated at an energy of 10.5 eV

(onset). The OO state ionization potential is found at 11.6 eV(onset53,55). Both energies are out of the range of the relevantprocesses for tryptophan-containing peptides, which makes themin our opinions into spectator orbitals. As for tyrosine contain-ing peptides, however, the C-terminal nO state plays an importantrole for charge transfer.

The electronic states of amino acids in the peptide chain(neither N nor C terminal) are well typified by the molecule

N-methylacetamide. The HeI PE spectrum and its structure areshown in Figure 5b. The peptide bond is simulated in this

molecule, resulting in a somewhat different spectrum incomparison to Figure 5a. The onset of the nN lone-pair state inthe amide group is shifted to higher energies (9.3 eV) incomparison to the amine group (8.8 eV). Despite the nomen-clature of others (ON53 and 267), we prefer the nomenclaturenN because of the close energetic position to the lone-pair nNband in the PE spectrum of the amine group.

In N-methylacetamide the oxygen lone-pair nO state is situatedmuch lower in energy in comparison to glycine resulting in anoverlapping with the nN state. This near-degenerancy of bothstates can be shown by substitution experiments as done in ref67. Due to the small energetic gap we are aware that both thenN orbital and the nO orbital are strongly coupled. Both states

together as a unit can be seen as localized around the amidegroup in the peptide chain. In the following, for simplification,we mostly refer to this states as a single chain state. Themost interesting result from the HeI PE spectrum of Figure 5bis the large gap to the higher electronic bands which are due toionization of the saturated carbon bridge. This energy gap isbroad enough to explain the lack of absorption of photons ofan energy below 2.5 eV in cations of non-aromatic amino acids(see arrow in Figure 5b). The same lack of VIS light absorption(2-2.5 eV) is valid for the amino acids alanine and leucine.

In Figure 5c the HeI photoelectron spectrum of tyrosinemeasured by Cannington et al.53 is shown. By comparison tothe HeI photoelectron spectrum of phenol44 it becomes evidentthat the observed bands in Figure 5c are due to the aromatic

chromophore. For tyrosine subsequent photoexcitation twophotons of energies between 2.2 and 2.4 eV is 2-fold resonantas shown by the two arrows in Figure 5c. This holds also fortryptophan and phenylalanine. In Table 2 the energetic positionsof the relevant electronic states of the amino acids leucine,alanine, glycine, tyrosine, tryptophan, and phenylalanine eitheras isolated amino acids or as chain are given. Errors are mostlydue to the broad structures of the electronic bands in the HeIphotoelectron spectra which are due to the high temperatures(500 K) at which the photoelectron spectra have been taken.To identify the excited-state bands in HeI photoelectron spectravalues have been taken from molecules analogous to aminoacids.58,67 The energetic position of the C-terminal nO band inTyr, Trp, and Phe has been estimated from Gly, because noidentification of this band was possible in the correspondingHeI spectra. Similarly the in-chain nN band of Tyr and Trpwas estimated to be the same as in Leu.

Assembling a Local Molecular Orbital Model for Peptides.

We assemble a local molecular orbital model for peptides bymaking use of the energetic positions of the highest molecularorbitals of the functional peptide sites as determined byphotoelectron spectra (see Table 2). The energies of theseorbitals as shown in Table 2 are taken to correspond to thepositive hole in this localized orbital where the rest of themolecule is neutral. Reorganization effects due to chargeremoval are completely included. It is this energetic informationof hole positions which is of direct interest to us.

Figure 5. Determination of energetic hole positions in peptides byaid of lone-pair HeI photoelectron spectroscopy of amino acids andrelated molecules: spectra taken from refs 53 (a, c) and 67 (b). (a)Glycine: Local orbitals exists in the cation due to electron removalfrom lone pair orbitals of either nitrogen (nN) or oxygen (nO). (b)N-methylacetamide contains the peptide bond: the nN orbital is shiftedto higher energies in comparison to (a). Note the wide energy gapbetween the first two and higher bands, which results in low crosssection for VIS laser excitation. (c) Tyrosine: the HeI photoelectronspectrum consists mostly of structures due to the aromatic chromophore.

Note the low ionization potential and the energetic position of excitedelectronic states which allow 2-fold resonance for VIS photoexcitation.



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We first constitute a molecular orbital picture for our peptides.We assume additivity of the local states as a zero-order model.

This is a reasonable starting point as explained above and issupported by our own theoretical calculations level, takingconfiguration interaction into account. As an example, in Table3 for the tripeptide Gly-Gly-Tyr experimental ionization po-tentials derived by our above-described technique are comparedto theoretical values. The energies of the two electronic statesof the peptide where the charge is on the chromophore or wherethe charge is in the amino group have been calculated by PM336

taking configuration interaction into account. The energeticpositions of the amino group to the chain states are reproduced.Note especially the high charge localization in either thechromophore site (state prepared by ionization) or the aminesite (positive charge site after charge transfer). The chro-mophore-amine distances are discussed in section V.IV.

Calculations have been quite time consuming, and therefore weconfined our investigations at the highest level (PM3, config-uration interaction included) on Gly-Gly-Tyr. For other peptidesMNDO calculations have been performed which also agree withour model. In Figure 6a-c a molecular orbital schemes forthe peptides Gly-Gly-Trp (a), Leu-Leu-Trp (b), and Tyr-Leu-Leu-Trp (c) are displayed. To show the local character of themolecular orbitals, we order them along the peptide chain. TheCH2 pseudo-orbitals at 11.3 eV and the C-C carbon orbitalsof the CH2-CH2 bridge at 11.8 eV are low in energy and notincluded. By ionization the positive hole is created in theHOMO orbital of the aromatic chromophore. Charge transferneeds further photoactivation. Excitation with VIS light (480-530 nm) transports an electron from a lower -orbital to the

HOMO orbital (VIS1, Figure 6). By this the positive hole istransferred to a lower orbital. Charge transfer is a through-bond transfer involving HOMO orbitals.

In Figure 6a the energetic situation of molecular orbital statesis shown for the peptide Gly-Gly-Trp for which experimentallyno quenching of two-photon absorption in the cation wasobserved. The position of the excited state hole of the indolechromophore is determined to be below the first chain orbitalnn, which still belongs to the tryptophan amino acid. Accordingto the energetic order of the bridge levels the first glycyl peptidebridge states nN and nO, however, are lower in energy than thehole position after chromophore excitation. Due to energetics,electron flow through this state is endoenergetic. We believethat transfer of the charge is energetically blocked at the glycyl

in agreement with our observation that charge probe in thearomatic chromophore is positive. The relaxation pathway givenin Figure 6a is the most natural if we consider the energy gaplaw for internal conversion16,17 to hold here.

Exchanging glycyl by leucyl shifts up the corresponding nNand nO chain states by more than 0.3 eV (see Figure 6b): Chargetransfer is now exoenergetic and the chain orbitals form adownward leading staircase (Table 1). This energetic situationagrees well with the fact that for the peptide Leu-Leu-Leu-Trpour two-color laser excitation spectra indicate charge transfer.Due to a strong coupling of the isoenergetic chain states thischarge transfer can be assumed to be mediated to the N-terminalend (see section V.III). Because the N-terminal nitrogen lone-pair state is somewhat lower than the chain nN orbital the hole

could be trapped there (for further explanation see section V.IV).This charge trapping in a nonaromatic site of the peptide is anessential process for our observation of charge transfer for ourexcitation scheme with ns laser pulses.

As our scheme shows charge transfer in Leu-Leu-Trp is aprocess involving energetically accessible intermediate electroniclevels in the chain spaced by short saturated carbon bridges.This is a rather untypical situation for charge transfer systemsup to now. The role of the CH2-CH2 bridge is discussed indetail below.

By MO considerations in biaromatic peptides as for Phe-Leu-Leu-Tyr (see Figure 6c) it can be expected that after activation

TABLE 3: Calculated and Experimental Data of theTripeptide Gly-Gly-Tyr

electronic statesof the cat ion IP(PM3+CI) IP(exp)

pos chargelocalization

R(N-C)()

ground state+ at chromophore

8.52 eV 8.0 eV(onset)

95% + atchromophore

5.56

excited state+ at NH2 group

9.07 eV 8.8 eV(onset)

80% + atNH2 group

5.87

Figure 6. Zero-order molecular orbital scheme for peptides: (a-c)Gly-Gly-Trp (a), Leu-Leu-Trp (b), and Phe-Leu-Leu-Tyr (c). Forenergetic positions see text and Table 1. Note that the orbitals of theCH2-CH2 and CH2-C bridges are low in energy and not shown here.Ion photoactivation by a VIS photon results in transport of the holeinto a lower chromophore orbital. Whereas electron transfer from thenitrogen chain state (nN) into the chromophore is energetically unfavor-able in (a) it can take place in (b) and (c). The charge can be transferredto the other side of the molecule and stored there in the N-terminalamine group as shown in (b). This is in agreement with ourexperimental results in Figure 3b,c. Complete charge transfer can bedetected in Phe-Leu-Leu-Tyr (see (c)) in agreement with experiments

shown in Figure 4d.



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by VIS light in tyrosine, charge now would flow to the benzylring of the N-terminal phenylalanine. Note that the HOMOorbital of Phe is situated above the N-terminal nN orbital.Therefore, after VIS photoactivation (VIS1) in Tyr chargetransfer can be completed by ending up in the Phe side chain.In the now charged aromatic benzyl ring a second VIS photoncould be absorbed. This energetic scheme is in completeagreement with our experimental finding (Figure 4d), and hencewe believe we have a self-consistent and plausible mechanism.

The local molecular orbital picture, just looking at energetics,

nicely explains the effects observed for this and other peptidesincluding barrier effects. So far, however, strong couplingsbetween local states in the peptides have been neglected, andonly states corresponding to localized hole positions have beenconsidered.

V.II.II. Extended Coulomb States and Couplings between

Localized Electronic States. Electronic coupling betweenelectronic states have to be classified into weak and strongcouplings. Strong electronic coupling causes large measurableenergetic shifts and energetic splittings. Most of our localpicture above is based on the observation that shifts andsplittings of localized electronic states in peptides are small andtherefore not observed in HeI photoelectron spectra. Fordetermination of the energetic positions of the relevant states,

in zero-order approximation we assume electronic states to beweakly coupled. A weak electronic coupling, however, can alsoresult in fast and ultrafast radiationless relaxation rates. Thiscan be due to the high isoenergetic density of vibrational statesor/and due to strong vibronic coupling in one or several reactioncoordinates. In this case despite the weak electronic couplingone would talk about a strongly coupled case in theory ofrelaxation dynamics.

Couplings between Electronic States in Neutrals. Inneutral molecules coupling between degenerate electronic stateseven when spaced by two saturated carbon atoms can berelatively large as calculations of Campbell et al. show.78 Thisis induced by through-bond coupling of the local electronicstates via orbitals of the -CH2-CH2- bridge.79 In the caseof our localized nitrogen lone-pair states in the peptides thismodel, however, is strongly overestimating the electroniccoupling: The coupling between lone-pair orbitals and bridgeorbitals we have is much smaller than for a orbital directlylinking with the bridge. We therefore assume the directelectronic coupling of Huckel type between chain states andchromophore states to be weak.

Through-space coupling via direct orbital overlap is an othermechanism which has to be discussed. It is clearly dependenton relative molecular orbital orientation and therefore stronglyvaries with bridge geometry.80 We believe that such couplingdoes not apply for the kind of small localized lone-pair orbitalsin peptides.

For radical cations couplings between localized states as thelone pair and the chromophore states are expected to be largerthan in neutrals. Beside couplings of type as discussed forneutrals, effects characteristic for radical cations such as chargeresonance and shakeup states have to be taken into account.

Charge Resonance in Radical Cations. Charge resonanceeffects have been discussed for degenerate chromophores inmolecular anions81 and cluster cations.82,83 The couplingsbetween degenerate local states does not influence stronglyenergetic positions of electronic states as observed in photo-electron spectra of ethylenediamine.44 Also in peptides somedelocalization of isoenergetic orbitals of different peptide bondsites can be qualitatively seen by population analysis in our PM3calculations.

Coupling by Extended Coulomb States (EC States). Webelieve, however, that besides the Huckel bridge coupling andcharge resonance there is a special mechanism active in peptidecations which is strongly influencing the dynamics of the charge-transfer process. This mechanism is involving electronic statesnot considered up to now. These electronic states correspondto a molecular orbital electron population with three openorbitals. The formation of such states from neutral moleculeswould require emission of an electron and a simultaneousexcitation of a second electron. Therefore these states are

termed shakeup states64

or non-Koopmans states49

and areusually not found in HeI photoelectron spectra. Only in fewmolecules such states have been observed as small satellites.49

In principle, however, they can be excited by photoabsorptionof the radical cation and can participate efficiently in all kindof relaxation processes. As HeI photoelectron spectra ofethylenediamine44 and substituted phenylethylamines58 show,the electronic coupling of EC states with local hole states seemsto be weak in a sense that the hole states keep their normalenergetic positions. This fact strongly supports our local picture.

The existence of low energetic EC states seems to be acharacteristic for extended linear radical cations: the displace-ment of charges, e.g., the extension is necessary for the strongenergetic lowering of some of these states by coulomb effects.

We therefore terme these states extended coulomb states (ECstates).

One of such shakeup states corresponds to electron transferbetween two localized chromophores through a saturated-CH2-CH2- carbon bridge. The corresponding molecularorbital population for such a process is shown in Figure 7a-cfor N-methylethyldiamine. Suppose that the positive chargeinitially can be located at the nonmethylated amine (see Figure7a). This positive charge then be transferred by electron transferthrough * orbitals of the -CH2-CH2- bridge (Figure 7b).The final state corresponds to an electron population where thecharge is situated in the methylated amine group (Figure 7c).

The shakeup state as shown in Figure 7b is characterized by

two positive charges on both sides and the surplus electron ina * orbital of the bridge. Normally one would excludecontributions of such a shakeup state to charge transfer incations, because the * orbitals of N-C and C-C bonds arehigh in energy. However, the electron in the CH2-CH2 bridgeis attracted by two positive charges on both sides. This situationcauses a strong reorganization of the nearby valence electronsand as a result lowers the energy of the shakeup state. Wetherefore term this state the extended coulomb state (EC state).The electronic state energies have been determined by PM3calculations including CI.36 The energetic position of the ECstate is calculated to be between the initial excited state (IP )8.7 eV, Figure 7a) and the final state (ground state, IP ) 7.8eV, Figure 7c). This holds as well for other EC states situated

in the N-C bond. It must be considered that such calculationsare quite difficult and can have considerable errors, but webelieve that the qualitative conclusion is sound that the EC stateis energetically situated between both hole states where thecharge is either at the left or right amine group. The intermedi-ate energetic position for the EC states in N-methylethyldiamineclearly shows that charge transfer through short saturated carbonbridges probably is nearly or completely activationless andtherefore is presumed to be extremely fast. This makes aconductive molecular wire out of certain peptide cations. Notethat during the charge-transfer process at no time is the hole inthe saturated carbon bridge.

The strong shifting of antibonding bridge states in EC statesseems to be a general feature of short saturated carbon bridges



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in extended linear cations. For this modified situation thequestion of strong coupling between initial, EC, and final stateneeds further investigation. It is however plausible and shown

by our own calculations, that the local C-C stretch reactioncoordinate is strongly changed in the EC state. Thereforevibronic couplings and conical intersections47,48 are interestingeffects that have to be expected.

The energetic position of the EC states clearly becomesstrongly dependent on the length of the saturated carbon bridge.Mediating charge transfer through long nonconducting bridgestherefore should become inefficient. Due to the special structureof peptides consisting of short saturated carbon bridges andrepetitive localized electronic states of functional groups, webelieve that the interplay of energetically accessible chain states(accessible hole positions) and EC states plays the predominantrole in charge transfer in peptide cations. The molecular orbitalscheme is no longer appropriate to describe this situation because

EC states must be taken into account. Therefore in the followingwe use a picture of localized ionic states. These consist oflocalized ionization potentials and localized EC states.

V.II.III. Landscape of Localized Electronic States Con-

sisting of Local Ionization Potentials and Localized Extended

Coulomb States. It is common usage that a molecule has asingle ionization energy. For large chainlike molecules, how-ever, we have a situation of states localized at various sites ofthe peptide which are characterized mostly by the localenvironment. Their ionization leads to energies which weattribute to local sites in peptides and therefore define as localionization potentials. This local ionization potential agrees withthe energetic positions of the positive charge in various sites ofthe peptide. The ensemble of all hole positions defines a

landscape of localized ionization potentials showing valleysand barriers. In Figure 8a the landscape of local ionizationpotentials of the peptide Leu-Leu-Tyr is shown. Excitedelectronic states and EC states (dotted lines) are included. Notethat in this picture each electronic state corresponds to anelectron configuration where open molecular orbitals and hencecharges are localized. The rest of the molecule is always neutral.The coordinate in Figure 8 is an electronic coordinate corre-sponding to the displacement of the hole along the peptide chain.The position of the EC states is energetically and spatiallybetween the local hole states and hence acts to efficientlymediate charge transfer in peptides. The staircase-like arrange-ment of the electronic states along the peptide chain to theN-terminus becomes evident in Figure 8a. Note as well the

high density of electronic states at energies larger than 1 eVabove the chromophore ground state, which resembles a bandstructure. The electronic states corresponding to the hole to bein orbitals of the saturated carbon bridge are high in energyand therefore should not contribute substantially to the charge-

Figure 7. Role of extended coulomb states in charge transfer demonstrated in the MO scheme for N-methylethyldiamine. The electronic stateenergies have been calculated by PM3 taking configuration interaction into account (see text). (a) Suppose the positive charge can be initiallylocalized in the nonmethylated anime group. (b) Transfer of the positive charge can be mediated by electron transfer via nonbonding * orbitalsof the C-C and C-N bonds. Due to the special configuration in radical cations the electron in the bridge is attracted by two positive charges,resulting in a strong lowering of the total state energy. (c) In the energetically optimized state the positive charge is situated at the methylatedamine group.

Figure 8. (a) Landscape of local electronic states in peptide cations:local ionization potentials, local excited states and extended coulombstates fo the peptide Leu-Leu-Trp. (b) Discussion of charge-transferrates. After photoexcitation, a staircaselike situation mediates chargetransfer to the N-terminus. Charge back transfer to the chromophoreis an improbable tunneling process along the entire peptide chain.



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transport mechanism. In the following we discuss the mech-anism and the rates of charge transfer in peptides in thelandscape of local electronic states.

V.III. Order of Time Scales. In our nanosecond experimentwe intrinsically cannot detect fast molecular processes directly.However, optical pumping rates, rates of intrachromophorerelaxation processes as well as the storage of the positive chargefor more than 5 ns in the nonaromatic part of the peptides givesus an order of magnitude of the time required for the processesinvolved. In the following we discuss these processes, their

mechanisms, and their theoretical description.Several processes can compete in photoactivated peptide

cations. The relevant rates are shown in Figure 8b again forthe tr

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Peptide Cations as Molecular Wires