Structure of the tripeptide model Ac–Val–Tyr(Me)–NHMe and its cluster with water investigated by IR/UV double resonance spectroscopy

In this paper the structures of the isolated tripeptide model Ac–Val–Tyr(Me)–NHMe (Val = valine, Tyr = tyrosine) and its cluster with water are investigated by mass-, isomer- and state-selective IR/UV double resonance spectroscopy.

From the IR spectra both in the region of the NH and CO stretching vibrations and in combination with force field and ab initio calculations it can unambiguously be derived that the peptide contains a stretched, β-sheet related structure.

Thus the peptide serves as an ideal candidate for β-sheet model systems.

By adding one water molecule to the peptide the β-sheet related structure seems to be preserved with a water molecule being attached to the NHMe group.


The conformational preferences of isolated protein building blocks, amino acids and peptides, have been subject of great interest in recent years.

Due to their flexibility peptides can occur in many different conformers.

In order to learn something about the driving forces to form a conformer, investigations can start by applying molecular beam experiments on isolated amino acids and peptides.

The experimental discrimination between different conformers requires the use of mass-, isomer- and state-selective methods.

If the investigated species contains an aromatic chromophore (like the common amino acids phenylalanine, tyrosine or tryptophan) R2PI (resonant 2-photon ionization) spectroscopy can be used.

This was first done by Levy and coworkers1–3 who recorded R2PI spectra of the free amino acids phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp) as well as some di- and tripeptides.

In order to get information on the structure of the amino acids in the electronic grounds states (S0) vibrational frequencies of the important functional groups have to be recorded.

IR/UV double resonance spectroscopy (IR/R2PI; see e.gref. 4–13). turns out to be an excellent method to investigate the IR spectra of different neutral species in their S0 states.

In combination with force field, ab initio, and density functional theory (DFT) calculations structural assignments can be derived.

The first applications of the IR/R2PI technique to the amino acids phenylalanine12 and tryptophan 10 have been performed by Simons and coworkers.

The IR spectra have been recorded in the region of the NH and OH stretching vibrations and several isomers have been deduced from the IR spectra containing intramolecular hydrogen bonds between the COOH and NH2 group.

In a recent publication Bakker et al13. investigated the spectral fingerprint region of tryptophan from 330 to 1500 cm−1 by using a free electron laser as IR light source.

In our group the protected amino acids Ac–Phe–OMe (Ac = acetyl, Me = methyl)11,12 and Ac–Phe–NHMe14 have been investigated.

In these studies not only the NH stretch region but also the region of the CO stretching and NH bending vibrations (about 1500–1800 cm−1) has been investigated.

In most cases information on the CO stretching vibrations are necessary to yield an unambiguous assignment of a structural arrangement.

In order to open the new spectral window in the region of 1700 cm−1 for the IR/R2PI spectroscopy of CO stretching vibrations a new laser system has been constructed.12,15

Zwier and coworkers investigated the protected amino acids Ac–Trp–NH2 and Ac–Trp–NHMe in the region of the NH stretching vibrations by applying IR/laser induced fluorescence (LIF) spectroscopy.16

Three (two) isomers of Ac–Trp–NHMe (Ac–Trp–NH2) have been observed.

In order to discuss the conformational dynamics of Ac–Trp–NHMe after vibrational excitation the new hole-filling spectroscopy has been applied.17

The backbones of (protected) amino acids and peptides are characterized by a set of torsional angles ϕ and ψ for each amino acid (Ramachandran plot).18,19

Furthermore, the side-chain configurations are characterized by the dihedral angles χ1 and χ2, cf. Fig. 1.

In the case of a β-sheet related structure (βL) the angles ϕ and ψ fall into the region of about −180° < ϕ < −120° or 120° < ϕ < 180° and −180° < ψ < −120° or 120° < ψ < 180°, i.e. NH and CO groups of an amino acid are nearly parallel to each other.

Structures forming an intramolecular hydrogen bond between a NH and a CO group can form γL arrangements with angles ϕ and ψ of the backbone lying between: −120° < ϕ < 0°; 0° < ψ < 120°.

A detailed description of the nomenclature with respect to the angles ϕ, ψ, χ1 and χ2 is given in .refs. 11,19

In contrast to the unprotected amino acids the protected species (at N and C terminus) cannot form intramolecular hydrogen bonds between the polar acid (COOH) and basic (NH2) groups leading to a higher selectivity of the structures.

In the case of Ac–Phe–OMe11,12 only a β-sheet related (βL) conformer has been observed as prominent isomer, whereas for Ac–Phe–NHMe14 four isomers are obtained with a βL structure being the most stable one.

These protected amino acids serve both as most simple model systems to describe β-sheet related structures as well as β-sheet models.

β-sheet models contain peptides or protected amino acids that undergo intermolecular hydrogen bonds like two amino acids in a β-sheet secondary structure.11,12,14,20

The conformational properties of amino acids depend on many different influences.

The interaction with water plays an important role in directing an amino acid or peptide into a certain conformer.

In order to investigate the process of microsolvation the following questions have to be answered: What will happen if water is added to an isolated peptide?

What is the preferred hydrogen bonding site?

How many water molecules have to be attached to cause a conformational change?

Clusters of amides and amino acids with water have been investigated by different molecular beam experiments,21–28 but no hydrated peptides have been investigated up to now.

Snoek et al25. have been the first to examine the hydrated clusters of a free amino acid, tryptophan, with up to three water molecules by applying IR/R2PI spectroscopy and UV/UV hole-burning spectroscopy.

In the case of the investigated unprotected amino acid the water molecules are attached to the polar end groups.

There are some experimental studies on larger neutral peptides, like dipeptides or tripeptides.

The first IR/R2PI spectrum on a dipeptide has been published by our group choosing the Ac–Val–Phe–OMe (Val = valine) system.29

The IR spectra have been recorded both in the region of the CH and NH as well as the CO stretching modes.

In very recent publications other groups also extended their applications of IR/R2PI spectroscopy to peptides containing tryptophan and glycine30 or phenylalanine and proline.31

It should be mentioned that UV/UV hole burning spectroscopy32 has also been applied to some dipeptides.33,34

These method yields no direct information on the structure of the electronic ground state, but the UV/UV-hole burning spectroscopy is an ideal tool to distinguish between different isomers especially in the case of congested R2PI spectra.

An overview on newest developments in the field of biological molecules in the gas phase is given in .ref. 35

In this paper we extend our investigations on the model tripeptide Ac–Val–Tyr(Me)–NHMe (cf. Fig. 1) and its cluster with one water molecule.

Tyr(Me) represents the amino acid tyrosine with a methylated OH group in the side-chain.

This modification is important to avoid any hydrogen bonds via a polar side-chain.

Due to the use of a NHMe instead of an OMe protecting group, Ac–Val–Tyr(Me)–NHMe contains three amid groups and serves as model for a tripeptide.

Since Val and Tyr contain bulky side-chains both amino acids are ideal candidates to form β-sheet related structures.

The existence of β-sheet related structures is important to build up an extended β-sheet model.

On the other hand the molecule gets more flexible compared to Ac–Val–Phe–OMe, i.e. the additional NH group may lead to intramolecular hydrogen-bonded (γL) structures similar to the one observed for isomers of Ac–Phe–NHMe,14 Ac–Trp–NH2,16 and Ac–Trp–NHMe.16

In contrast to unprotected amino acids the polar NH and CO groups of Ac–Val–Tyr(Me)–NHMe are located in the backbone and not at the end of the molecule, i.e. water will be attached to the backbone of the peptide.

In this paper we will report on the influence of the first water molecule attached to the model tripeptide Ac–Val–Tyr(Me)–NHMe.


(a) Synthesis of Ac–Val–Tyr(Me)–NHMe

295 mg (1.00 mmol, 1.00 eq) N-tert-butyloxycarbonyl-O-methyl-(S)-tyrosine were dissolved in 5 ml of dry dichloromethane under argon and cooled to −10 °C.

To this solution 162 mg (1.00 mmol, 1.00 eq) of N,N′-carbonyldiimidazole were added and the reaction mixture was stirred for another 2 h at −10 °C.

Subsequently, 500 μl (1.10 mmol, 1.10 eq) of a 2 M solution of methylamine in tetrahydrofuran were added.

The mixture was warmed to 0 °C and stirred for 15 min.

The excess of methylamine was removed by passing an argon gas stream through the reaction vessel for 15 min.

The organic phase was then washed three times with 3% aqueous citric acid, three times with sodium carbonate/sodium bicarbonate buffer (pH 10) and once with water, and finally dried over magnesium sulfate.

After evaporation of the solvent (membrane pump) 250 mg (810 μmol, 81%) of a colorless solid (N-tert-butyloxycarbonyl-O-methyl-(S)-tyrosine methyl amide, cf. Fig. 2a) were obtained.

230 mg (746 μmol, 1.00 eq) N-tert-butyloxycarbonyl-O-methyl-(S)-tyrosine methyl amide (cf. Fig. 2a) were stirred in a mixture of 5 ml trifluoroacetic acid and 20 ml of dichloromethane for 2.5 h with ice cooling.

The solvent was removed in vacuum with toluene as azeotropic mixture.

After vacuum-drying the product O-methyl-(S)-tyrosine methyl amide trifluoracetate (cf. Fig. 2b) could be isolated in quantitative yield as a ductile colorless oil.

190 mg (1.20 mmol, 1.20 eq) of N-acetyl-(S)-valine, 494 mg (1.30 mmol, 1.30 eq) of the coupling reagent HATU, and 408 mg (3.00 mmol, 3.00 eq) of the coupling reagent HOAt, as well as 350 μl (3.00 mmol, 3.00 eq) of lutidine were dissolved under argon in a solvent mixture of dry dichloromethane/DMF (5:1) and stirred for 10 min at 0 °C.

Subsequently, a mixture of 322 mg (1.00 mmol, 1.00 eq) O-methyl-(S)-tyrosine methyl amide trifluoroacetate (cf. Fig. 2b) and 107 μl (1.00 mmol, 1.00 eq) of lutidine in little dichloromethane/DMF (5:1) were added.

The reaction mixture was initially stirred with ice-cooling for 2 h.

The organic layer was extracted three times with 1 M aqueous KHSO4.

The precipitating solid was filtered off and treated separately.

The organic layer was subsequently washed three times with saturated sodiumbicarbonate and once with water and with saturated sodium chloride solution, as well as the solid.

The organic layer was dried over sodium sulfate and the solvent was completely removed at the rotatory evaporator.

The resulting solid was combined with the previously filtered and thoroughly washed solid; the combined solids were vacuum dried, affording 158 mg (453 μmol, 45%) of the colorless solid Ac–Val–Tyr(Me)–NHMe (cf. Fig. 1).

(b) Experimental set-up

The experimental set-up has been described elsewhere (cfrefs. 11,12,36)..

Thus only a short description is given: The R2PI and IR/R2PI spectra were measured in a vacuum system consisting of a differentially pumped linear time-of-flight mass spectrometer and a pulsed valve (General Valve Iota One, 500 μm orifice) for skimmed jet expansion (X/D = 130).

A frequency-doubled dye laser (Lumonics HD 300), pumped by a Nd:YAG laser (Lumonics HY 400), was used for excitation to the S1 state and for ionization.

The IR light in the region of 2.63–3.08 μm (3250–3800 cm−1) was generated with a LiNbO3 crystal by difference frequency mixing of the fundamental (1064 nm) of a seeded Nd:YAG laser (Spectra-Physics PRO-230) and the output of a dye laser (Sirah, Precision Scan) pumped by the second harmonic (532 nm) of the same Nd:YAG laser.

The IR output is amplified by an optical parametric amplification (in a LiNbO3 crystal) of the output of the IR laser (2.63–3.08 μm) and the fundamental of the Nd:YAG laser.36

IR light in the region of 6 μm is generated by a third nonlinear process12,15 The signal (1.79–1.82 μm, 5500–5600 cm−1) and idler (2.56–2.63 μm, 3800–3900 cm−1) of the OPA process are the inputs to a difference frequency mixing process in an AgGaSe2 crystal leading to the IR light of 1600–1800 cm−1.

Since the time delay chosen for the two lasers is not longer than 100 ns, all lasers have been spatially overlapped.

In order to obtain IR/R2PI spectra the IR laser is fired 60 ns prior to the UV laser.

The substance (Ac–Val–Tyr(Me)–NHMe) and the valve are heated to 150 °C.

Helium was used as carrier gas (2000 mbar).

Results and discussion

(a) Structure of Ac–Val–Tyr(Me)–NHMe

The IR/R2PI spectrum of Ac–Val–Tyr(Me)–NHMe in the range of 1600–1800 and 3300–3600 cm−1 is depicted in Fig. 3.

The spectrum has been recorded via the electronic origin of the S1 ← S0 transition at 35 434 cm−1.

No further prominent transitions can be observed in the R2PI spectrum from 35 200 to 35 900 cm−1.

In the region of the NH stretching modes of the IR/R2PI spectrum three vibrational transitions are observed at 3411, 3440 and 3477 cm−1.

All vibrations are located above 3400 cm−1 which strongly indicates that a linear β-sheet related structure and no intramolecular hydrogen bonds are formed.

The frequencies of hydrogen-bonded NH stretching modes are typically located below 3400 cm−1.11,14,16

The NH stretching frequency at 3440 cm−1 is very close to the value of the NH stretching mode of Val at 3441 cm−1 in the spectrum of Ac–Val–Phe–OMe29 and can therefore be assigned to the NH stretching mode of the valine moiety.

The identity of the observed frequencies is not surprising, since the structural arrangements in the linear conformers of Ac–Val–Phe–OMe and Ac–Val–Tyr(Me)–NHMe should be nearly identical.

According to the results on Ac–Phe–NHMe14 and Ac–Trp–NHMe16 it is known that the NH stretching vibration of the NHMe protecting group is located around 3470 cm−1.

Therefore the transition at 3477 cm−1 is assigned to the N3–H3 (cf. Fig. 1) vibration of Ac–Val–Tyr(Me)–NHMe.

Thus the remaining transition at 3411 cm−1 has to be correlated with the N–H vibration of the tyrosine moiety.

Unfortunately the protected amino acid Ac–Tyr(Me)–NHMe has not been investigated up to now.

Thus a direct comparison of the NH stretching frequency of the tyrosine moiety in Ac–Val–Tyr(Me)–NHMe with the one in Ac–Tyr(Me)–NHMe is not possible but the observations obtained from other protected amino acids and peptides explain the relative low frequency of 3411 cm−1 (compared to the value of 3451 cm−1 of the Phe residue in Ac–Val–Phe–OMe29): By comparing the IR spectra of the protected amino acids Ac–Phe–OMe11 and Ac–Phe–NHMe14 as well as Ac–Trp–OMe37 and Ac–Trp–NHMe16 strong red-shifts (20 and 25 cm−1) of the NH stretching frequency of the Phe and Trp residues are observed resulting from the change of the OMe to NHMe protecting groups.

By going from the protected amino acid Ac–Phe–OMe to the protected dipeptide Ac–Val–Phe–OMe an additional small red shift (7 cm−1) of the (Phe) NH stretching mode is observed, i.e. both the choice of a NHMe protecting group and the extension of the backbone (from a protected amino acid to a tripeptide model) leads to a red-shift of the NH stretching frequency of the tyrosine residue.

The assignment to a linear β-sheet related structure is further supported by vibrational frequencies obtained for the CO groups of Ac–Val–Tyr(Me)–NHMe.

The IR/R2PI spectrum exhibits three transitions at 1694, 1700, and 1715 cm−1.

The transition at 1715 cm−1 is in good agreement with frequencies of the C1O1 (acetyl) group (cf. Fig. 1) observed for Ac–Val–Phe–OMe (1711 cm−1.29)

Furthermore, the value of 1694 cm−1 is nearly identical to the frequency observed for the corresponding valine residue of Ac–Val–Phe–OMe (1696 cm−1.29)

Thus the vibration at 1694 cm−1 can easily be assigned to the stretching vibration of the C2O2 group of Ac–Val–Tyr(Me)–NHMe.

Finally the remaining transition at 1700 cm−1 has to be correlated with the C3O3 stretching mode (cf. Fig. 1) of the Tyr residue.

In order to support the vibrational assignments force field, ab initio (Hartree–Fock, HF) and DFT (B3LYP functional) calculations have been performed using the Discover38 and Gaussian 9839 programs.

To generate starting geometries for ab initio calculations, a preselection via molecular dynamics using the CFF force field38,40–43 has been done.

Molecular dynamics have been performed at a temperature of 750 K and with a time step of 1 fs.

After 1000 steps the structure has been minimized.

This structure has been used to continue the dynamics in the same manner.

The following minimized structure has been compared to the first via its relative energy to discriminate between different minima.

With this technique 30,000 structures have been minimized and compared to each other.

The dynamics have been repeated several times to explore the PES.

The preselection using the CFF force field shows that β-sheet related structures turn out to be the most stable conformers.

This is in agreement with the analysis of the IR spectra indicating to β-sheet related structures, which can differ in their side-chain arrangements.

In a general overview on several peptides and clusters44 we will show that the class II force field CFF can be taken for the interpretation of “high resolution” IR spectra of isolated species.

Due to the size of the system and in order to compare the results with earlier studies, we have chosen the 3-21G(d) basis set for the subsequent HF calculations.

All structures with β-sheet related backbone arrangements have been fully optimized and the relative energies have been corrected by the zero point energies.

The calculated frequencies have been scaled with factors derived from our investigations on Ac–Phe–OMe (0.9076 for the NH vibrations and 0.9140 for the CO stretching modes.12)

The most stable isomer has a βV(g−)–βT(a) (superscripts: V = valine, T = tyrosine) arrangement (cf. Fig. 1).

The torsional angles characterizing the backbone and side-chain conformations have the following values: ϕV(C1–N1–CVα–C2) = −140°, ψV(N1–CVα–C2–N2) = 164°, χV1(N1–CVα–CVβ–CVγ) = −68°, ϕT(C2–N2–CTα–C3) = −166°, ψT(N2–CTα–C3–N3) = 173°, χT1(N2–CTα–CTβ–CTγ) = −147°.

It has to be mentioned that two structures of all βV–βT conformers have to be discussed which only differ by the orientation of the OMe group in the tyrosine side-chain.

If the angle χT2 (cf. Fig. 1) has a positive (negative) value the structure is characterized by a+(−) sign.

All ± structures of a given conformer have nearly identical energies.

The structure shown in Fig. 1 is a βV(g−)–βT(a)+ arrangement with a torsional angle of χT2 (CTα–CTβ–CTγ–CTδ) = 68°.

In the βV(g−)–βT(a) structure the OMe group would be rotated about the C–O axis by 180°.

The values ϕ and ψ of the βV(g−)–βT(a)± structures deviate not more than 7° from the values obtained for the minimum energy structure of Ac–Val–Phe–OMe.29

There are two different structural arrangements (βV(g+)–βT(a)±; βV(a)–βT(a)±) which are close in energy to the most stable structure of Ac–Val–Tyr(Me)–NHMe, i.e. the energy difference between the structures is about 100 cm−1 including zero point energy corrections.

The calculation of relative energies cannot be satisfactory at the HF/3-21G(d) level of theory and more sophisticated theoretical applications have to be performed in order to define the global minimum energy structure of Ac–Val–Tyr(Me)–NHMe.

In contrast to the relative energies the scaled frequencies obtained from calculations at the HF/3-21G(d) level of theory are often in good agreement with the experimental values which is a result of a cancellation of errors.

The vibrational frequencies obtained for the βV(g−)–βT(a)+ structure are listed in Table 1 yielding a reasonable agreement to the experimentally observed frequencies.

Additionally HF calculations using the 6-31G(d,p) basis and DFT calculations (B3LYP functional, 6-31+G(d) basis set) have been performed for the βV(g−)–βT(a)+ structure.

The following dihedral angles of the backbone structure are obtained from the HF/6-31G(d,p) (B3LYP/6-31+G(d)) calculations: ϕV = −129° (−130°), ψV = 160° (161°), χV1 = −63° (−62°), ϕT = −157° (−157°), ψT = 172° (160°), χT1 = −169° (−167°), χT2 = 74° (70°).

The resulting vibrational frequencies are also listed in Table 1.

The frequencies obtained from the DFT calculations strongly support our vibrational assignments.

(b) Structure of Ac–Val–Tyr(Me)–NHMe(H2O)1

The IR/R2PI spectrum of Ac–Val–Tyr(Me)–NHMe(H2O)1 in the spectral region from 3250 to 3800 cm−1 is shown in Fig. 4.

The spectrum has been recorded via the electronic origin of the S1 ← S0 transition at 35636 cm−1.

In order to get an interpretation of the spectrum the experimentally observed NH stretching frequencies are compared with the one of the Ac–Val–Tyr(Me)–NHMe monomer; in addition the OH stretching frequencies of the water moiety have to be discussed.

A water molecule can be attached as hydrogen acceptor (by forming an N–H⋯O bond) or as hydrogen donor (by forming an CO⋯H bond).

These arrangements lead to significantly different OH stretching frequencies.

In case of a CO⋯H hydrogen donor bond an OH donor stretching frequency in the region of about 3450–3600 cm−1 would be expected, e.g. in trans-N-phenylformamide(H2O)123 or trans-N-benzylformamide22 the vibrations are located at 3536 and 3525 cm−1, respectively.

Similar results are obtained in investigations on other hydrated clusters with aromatic chromophores45 or in our investigations on Ac–Phe–OMe(H2O)1, a hydrated cluster of a protected amino acid with water.46

In Ac–Phe–OMe(H2O)1 two isomers are discussed.

The first one contains a CO⋯H donor bond and in the second one a water molecule forms a bridge between a N–H (hydrogen bond acceptor) and a CO group (hydrogen bond donor).

Also for the second isomer containing a combined donor/acceptor binding motif the OH stretching frequency is significantly below 3600 cm−1 (at 3575 cm−1.46)

If water acts as proton donor the remaining free OH stretching mode would be expected around 3720 cm−1 (cf. e.grefs. 6,8,45,47)..

The experimental spectrum shows two transitions in the OH stretching region at 3635 and 3718 cm−1 (cf. Fig. 4).

The value observed for the free OH stretching mode is in very good agreement with the value expected for a donor bonded H2O molecule but no transition can be observed between 3450 and 3600 cm−1.

Thus no OH stretching donor vibration can be assigned.

On the other hand the transition at 3635 cm−1 is only shifted by −22 cm−1 with respect to the free symmetric vibration of isolated water48,49 strongly indicating that water cannot act as a donor.

If the water molecules acts as proton acceptor like in indole(H2O)1,50 phenol(H2O)16 or N-phenylformamide(H2O)123 a frequency of about 3650 cm−1 is obtained for the symmetric OH stretching mode.

In some cases this value can also shift to frequencies of about 3635 cm−1, e.g. in the π-bonded fluorene(H2O)1 cluster (3632 cm−1.51)

It is interesting to note that a π-interaction can also be discussed for the Ac–Val–Tyr(Me)–NHMe(H2O)1 cluster (see below).

Concerning the asymmetric OH stretching vibration its frequency is often located at about 3745 cm−1,6,8,50i.e. a few wavenumbers lower than the corresponding frequency of the free water molecule at 3756 cm−1.48,49

But there are also some acceptor bound species which show a much stronger shift down to about 3710 cm−1, cf. e.g. 2-naphthyl-1-ethanol(H2O)2 with an asymmetric OH stretching vibration of the acceptor water at 3708 cm−1.52

This water molecule also undergoes a π-interaction with the aromatic chromophore.

Additionally the π-bonded fluorene(H2O)151 is an example which shows a shift of the asymmetric OH stretching mode to 3719 cm−1.

Furthermore, it is interesting that the splitting of symmetric (ν1) and asymmetric (ν3) OH stretching mode in Ac–Val–Tyr(Me)–NHMe(H2O)1 is only 83 cm−1.

This value is lower than the corresponding difference of an isolated water molecule (99 cm−148,49) or other acceptor bound clusters6,8,45,50, but a similar low splitting has also been observed for the water dimer (about 85 cm−153) and for the fluorene(H2O)1 cluster (87 cm−1.51)

It should be noted that our ab initio calculations performed at the HF/6-31G(d,p) level of theory (see below) predict a splitting of 87 cm−1, if the difference is scaled to the corresponding value obtained for the free water molecule calculated at the same level of theory, i.e. the ν1/ν3 splitting is significantly reduced in the complex of Ac–Val–Tyr(Me)–NHMe with water.

This is in agreement with the experimental result.

According to the discussion of the OH stretching vibrations it can be assumed that the water molecule undergoes a hydrogen acceptor bond, i.e. water is attached to one of the three NH groups and forms a N–H⋯O hydrogen bond.

In order to find out which NH group is hydrogen-bonded the remaining transitions at 3393, 3418 and 3435 cm−1 (cf. Fig. 4) are compared with the NH stretching frequencies of the Ac–Val–Tyr(Me)–NHMe monomer.

It is obvious that the bands at 3435 and 3418 cm−1 show only small shifts (−5 and +7 cm−1) with respect to the corresponding transitions of the monomer (3440 and 3411 cm−1) and are therefore assigned to free NH stretching modes of the valine and tyrosine residues.

Since no transition is observed in the region from 3435 to 3635 cm−1, the free NH stretching vibration of the N3–H3 group (located at 3477 cm−1 in the monomer) can definitely not be observed.

This leads to the conclusion that the water molecule is hydrogen-bonded to the N3–H3 group of the NHMe protecting group (cf. Fig. 1), yielding a broader N–H stretching transition at 3393 cm−1.

The experimentally observed shift (84 cm−1) of the N3–H3 stretching vibration between monomer and cluster is in agreement with the value obtained for the N-phenylformamide cluster.23

It can be concluded that the IR/R2PI spectrum of Ac–Val–Tyr(Me)–NHMe(H2O)1 can be interpreted by predicting a structure which contains a water molecule undergoing a N3–H3⋯O acceptor bond.

As mentioned above the NH stretching frequencies of the Val and Tyr residues are very close to their corresponding values in the monomer.

Thus it is very likely to assume that the conformation of the backbone has not changed significantly.

Ab initio calculations at the HF level of theory are performed using the same basis sets (3-21G(d) and 6-31G(d,p)) as for the monomer.

According to the experimental results a β-sheet related structure with water attached to the N3–H3 group has been used as starting geometry.

Subsequently, structures including all possible side-chain configurations have been optimized at the HF/3-21G(d) level of theory.

As in the monomer a βV(g−)–βT(a) conformer turns out to be the most stable structure (cf. Fig. 5).

Thus this βV(g−)–βT(a) structure has been further optimized at the HF/6-31G(d,p) level yielding a complex with an almost linear intermolecular hydrogen bond, i.e. the angle between N3, H3 (cf. Fig. 1) and the oxygen atom of the water molecule is 174°, the N3–H3⋯OH2 bond length is 2.04 Å.

One hydrogen atom of the water molecule lies above the center of the aromatic ring (Tyr residue, cf. Fig. 5).

This π-interaction may lead to an additional stabilization of this structure.

The torsional angles of backbone and side-chain of the Val moiety have identical values with respect to the monomer.

Due to the influence of the attached water at the NHMe group, the torsional angles of the Tyr residue show a small shift (2° for ϕT, 16° for ψT and 0° for χT1), but the βL-arrangement is still conserved.

The frequencies calculated for the NH stretching vibrations of the N3–H3 group as well as the Tyr and Val moieties (3400, 3440 and 3441 cm−1) are in agreement with the experimental frequencies (3393, 3418 and 3435 cm−1).

The calculated frequencies have been scaled with the same factor as the one obtained for the monomer at the same level of theory.

Furthermore, a stabilization energy of 1288 cm−1 (15.4 kJ mol−1) is obtained for the cluster at the HF/6-31G(d,p) level including BSSE corrections54.


The tripeptide model Ac–Val–Tyr(Me)–NHMe is an extended molecule that is flexible enough to form several structural conformers, both stretched (β-sheet related) and intramolecular hydrogen-bonded arrangements.

From our spectroscopic investigations a β-sheet related arrangement can be derived.

This result indicates a high selectivity with respect to β-sheet related structures if appropriate amino acids are chosen.

According to these results Ac–Val–Tyr(Me)–NHMe is a good candidate for β-sheet model systems, i.e. intermolecular hydrogen-bonded cluster with other peptides can be formed leading to a structural configuration similar to the one observed between peptides in an extended β-sheet.

It is interesting to note that the monomer seems only slightly influenced by the addition of one water molecule.

Water forms an almost linear hydrogen bond to the NH group of NHMe which is the strongest donor of the molecule.

It will be a further question, whether the addition of more water molecules will change the backbone configuration of the peptide.