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Back | Show only these items: | Structure of the tripeptide model Ac–Val–Tyr(Me)–NHMe and its cluster with water investigated by IR/UV double resonance spectroscopy

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

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj1

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.

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj1

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.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res1

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

Type: Conclusion | Advantage: None | Novelty: None | ConceptID: Con1

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.

Type: Result | Advantage: None | Novelty: None | ConceptID: Res2

Introduction

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

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot1

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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac1

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.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot2

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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac2

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.

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met1

This was first done by Levy and coworkers^1–3 who recorded R2PI spectra of the free amino acids phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp) as well as some di- and tripeptides.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac3

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.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot3

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

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met2

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

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met3

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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac4

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.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac4

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.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac5

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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac6

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.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac6

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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac7

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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac7

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

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met4

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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac8

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

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met5

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

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod1

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

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod1

In the case of a β-sheet related structure (β_L) 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.

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod1

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

Type: Model | Advantage: None | Novelty: None | ConceptID: Mod1

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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac9

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.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac10

In the case of Ac–Phe–OMe^11,12 only a β-sheet related (β_L) conformer has been observed as prominent isomer, whereas for Ac–Phe–NHMe¹⁴ four isomers are obtained with a β_L structure being the most stable one.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac10

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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac10

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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac11

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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac11

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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac11

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?

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot4

What is the preferred hydrogen bonding site?

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot4

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

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot4

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.

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot4

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.

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac12

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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac12

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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac13

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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac13

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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac13

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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac14

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

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met6

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.

Type: Method | Advantage: Yes | Novelty: Old | ConceptID: Met6

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

Type: Background | Advantage: None | Novelty: None | ConceptID: Bac15

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.

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj1

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

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj1

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

Type: Object | Advantage: Yes | Novelty: New | ConceptID: Obj1

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.

Type: Object | Advantage: Yes | Novelty: New | ConceptID: Obj1

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

Type: Object | Advantage: Yes | Novelty: New | ConceptID: Obj1

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

Type: Motivation | Advantage: None | Novelty: None | ConceptID: Mot5

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 (γ_L) structures similar to the one observed for isomers of Ac–Phe–NHMe,¹⁴ Ac–Trp–NH₂,¹⁶ and Ac–Trp–NHMe.¹⁶

Type: Object | Advantage: No | Novelty: New | ConceptID: Obj1

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.

Type: Object | Advantage: Yes | Novelty: New | ConceptID: Obj1

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

Type: Object | Advantage: None | Novelty: New | ConceptID: Obj1

Experimental

(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.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

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.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

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

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

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

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

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

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

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.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

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.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp1

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.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp2

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

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp2

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.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp2

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.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

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.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

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

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

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

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

The precipitating solid was filtered off and treated separately.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

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.

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

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

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

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).

Type: Experiment | Advantage: None | Novelty: None | ConceptID: Exp3

(b) Experimental set-up

The experimental set-up has been described elsewhere (cf^{refs. 11,12,36})..

Type: Method | Advantage: None | Novelty: Old | ConceptID: Met2

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).