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J. Phys. Chem. B 1999, 103, 8172-8179
Photophysical and Structural Features of Covalently Bound Peptide-Protoporphyrin-Peptide Compounds Carrying Naphthalene Chromophores Basilio Pispisa,*,† Mariano Venanzi,† Lorenzo Stella,† Antonio Palleschi,‡ and Giancarlo Zanotti§ Dipartimento di Scienze e Tecnologie Chimiche, UniVersita` di Roma “Tor Vergata”, 00133 Roma, Italy, Dipartimento di Chimica, UniVersita` di Roma “La Sapienza”, 00185 Roma, Italy, and Centro di Chimica del Farmaco, c/o Dipartimento di Studi Farmaceutici, UniVersita` di Roma “La Sapienza”, 00185 Roma, Italy ReceiVed: March 30, 1999; In Final Form: July 21, 1999
The photophysics of a series of covalently bound peptide-protoporphyrin-peptide compounds carrying naphthalene (N) were investigated in methanol solution by steady-state and time-resolved fluorescence experiments, and by transient absorption spectra as well. The general formula of the series is P(xN)2, where P refers to protoporphyrin IX, and x to the number of amino acids in the sequence Boc-Leu-Leu-Lys-(Ala)nLeu-Leu-Lys-OtBu of each backbone chain (n ) 0-3). Quenching of excited naphthalene takes place by electronic energy transfer from excited naphthalene to ground-state porphyrin and proceeds on a time scale of about 2 ns and 20-30 ns. IR spectra in methanol indicate that intramolecularly H-bonded conformations form, and CD data in both methanol and water-methanol mixtures suggest the presence of R-helix structure. According to fluorescence decay data coupled with molecular mechanics calculations, two conformers for each dimeric peptide are the major contributors to the observed phenomena. These conformers are characterized by a globular, protein-like structure, where the protoporphyrin resides in a central pocket, making it amenable to metalation for mimicking hemoprotein, while the two N groups are externally located. Of the four N linkages in the two conformers, three of them attain a very similar steric arrangement around the central P molecule, in terms of both center-to-center distance and mutual orientation, while the fourth experiences a different steric disposition as compared to the others. Where the theoretical structures and photophysical properties were correlated within the Fo¨rster-type mechanism, the kinetics of the energy transfer were well reproduced for all compounds investigated only when the mutual orientation of the chromophores was also taken into account. This implies that interconversion among conformational substates of probe linkages is slow on the time scale of the transfer process.
Introduction Model hemoproteins composed by peptide-heme-peptide or heme-peptide adducts are currently investigated and tested in a number of processes,1,2 such as electron transfer, oxidation catalysis, etc. In all cases, the peptide chains attain an R-helical conformation because of the presence of suitable amino acids3 or the use of proper solvent media.1a,4 We have already reported a hemoprotein model, formed by a polypeptide-tetrapyridineiron(III)-polypeptide adduct,5 and its activity in stereoselective electron-transfer reactions,5-7 but a more realistic model seems now appropriate. This prompted us to prepare a peptide-heme-peptide system, having the two peptide chains covalently linked to the central heme group. This because a covalently bound composite structure allows us to test a wider range of experimental conditions than an adduct, while maintaining the basic features of hemoproteins. The first step of this investigation was that of studying the physical-chemical and structural properties in methanol of a series of peptide-protoporphyrin-peptide compounds, as shown below. The second step, which shall be described later, refers to the metalation of the central porphyryl moiety and the use † Dipartimento di Scienze e Tecnologie Chimiche, Universita ` di Roma “Tor Vergata”. ‡ Dipartimento di Chimica, Universita ` di Roma “La Sapienza”. § Centro di Chimica del Farmaco, Universita ` di Roma “La Sapienza”.
of the so-formed covalently bound peptide-heme-peptide systems in electron-transfer reactions. The dimeric peptides under investigation consist of two chains of sequential oligopeptides carrying 1-naphthylacetic acid, covalently bound to the -amino group of a lysine residue, linked together through a protoporphyrin IX (P) molecule, which, in turn, is bound to the side chain of other lysine residues, as shown in Chart 1. The two oligopeptides were synthesized from two Leu-Leu-Lys triads (Leu ) L-leucine; Lys ) L-lysine), linked together through a spacer of n ) 0-3 L-alanine (Ala). The dimeric peptides are denoted as P(xN)2, where N is naphthalene and x refers to the number of amino acids in the sequence BocLeu-Leu-Lys-(Ala)n-Leu-Leu-Lys-OtBu of each chain, namely P(6N)2, P(7N)2, P(8N)2, and P(9N)2. The corresponding reference samples are instead denoted as xN and P(x)2, depending on the bound chromophore. The two peptide chains in the dimeric compounds are expected to attain an ordered, intramolecularly H-bonded structure, owing to the presence of both the good R-helixforming Leu and Ala residues3,8 and the bulky chromophores that play a concerted role in stabilizing the R-helical structure,9 as already observed in studying the monomeric species, i.e., PxN peptides.10 In principle, several geometric arrangements of the peptide chains around the central porphyrin could be envisaged in the
10.1021/jp9911043 CCC: $18.00 © 1999 American Chemical Society Published on Web 09/03/1999
Peptide-Protoporphyrin-Peptide Compounds CHART 1
dimeric compounds, and within each arrangement, a number of conformations can be also populated, owing to the internal degrees of freedom of the lysine side chains carrying the probes. As a result, one may expect a relatively large distribution of conformers. In fact, this is not the case, according to both fluorescence decay experiments and computational data, the main reason being the rigidity of the overall structure, because optimization of the interactions between the R-helical chains and porphyrin ring is achieved by bringing the protoporphyryl moiety to reside in a central pocket, sandwiched between the helical chains. This leads, on one hand, to a highly compact structure and, on the other, to a steric arrangement where the porphyrin is amenable to metalation for mimicking hemoproteins.11 By combining the results on the excited-state behavior of the chromophores bound in the dimeric peptides with IR and CD spectral results, and with those obtained by molecular mechanics calculations as well, we were able to build up the molecular model of the most probable structures of the compounds examined in methanol. Experimental Section Materials. The identity and purity of all peptides were checked by amino acid analysis and by 1H NMR, high-resolution mass spectrometry (FAB), and thin-layer chromatography, using silica gel Merck plates. The following abbreviations are used: Boc ) tert-butyloxycarbonyl, Z ) benzyloxycarbonyl, Ddz ) (R,R-dimethyl-3,5dimethoxybenzyl)oxycarbonyl, OtBu ) tert-butyl ester, Ac ) acetyl, iBCCl ) isobutyloxycarbonyl chloride, NAc ) naphthylacetyl (in Chart 1 simply denoted as N), NMM ) Nmethylmorpholine, DMF ) dimethylformamide, TFA ) trifluoroacetic acid, and THF ) tetrahydrofuran. Ddz and Boc groups were used to protect the amino terminus, and OtBu was used to protect the carbonyl terminus. The removal of Ddz and Boc groups was accomplished by 3% TFA in CH2Cl2 and absolute TFA, respectively. The Z group was used to protect the -amino group of Lys and removed with H2 over a Pd/C catalyst. The synthesis of the oligopeptides was performed by the conventional mixed anhydride (MA) method. Amino acid precursors were purchased form NOVA Biochem or Fluka, and
J. Phys. Chem. B, Vol. 103, No. 38, 1999 8173 naphthylacetic acid and protoporphyrin IX from Aldrich. Analytical-grade reagents and solvents were always used. Boc-Leu-Leu-Lys-(Ala)n-Leu-Leu-Lys(NAc)-OtBu. General Procedure. A 1 mmol amount of Boc-Leu-Leu-Lys(Z)(Ala)n-OH, as obtained by the method already reported,10b was dissolved in 30 mL of CH2Cl2-DMF (1:1) and treated at -10 °C with 136 mg of iBCCl (1 mmol) and 136 mg of NMM (1.34 mmol) under stirring. After 10 min, the solution of the amino component, prepared as follows, was added to this solution: 818 mg of Ddz-Leu-Leu-Lys(NAc)-OtBu (1 mmol) in 30 mL of CH2Cl2 was treated with 0.76 mL of TFA (10 mmol) for 30 min at room temperature to cleave the Ddz group and then was neutralized by 1.1 mL of NMM (10 mmol). After 3 h of stirring at room temperature (rt), the reaction mixture was washed with saturated aqueous NaHCO3 solution, 0.5 M KHSO4, and water, dried with Na2SO4, and evaporated to give a residue that was chromatographed on a Sephadex LH 20 column (2.5 × 250 cm) in CH3OH as eluant to afford the pure title compounds. Rf (CHCl3/CH3OH, 95/5) ) 0.30 (n ) 0), 0.30 (1), 0.30 (2), 0.35 (3), and 0.25 (4); yields ) 60, 36, 50, 50, and 15%, respectively. The Z group was removed by hydrogenolysis in methanol solution over 10% Pd on activated carbon for 24 h at rt. Boc-Leu-Leu-Lys-(Ala)2-Leu-Leu-Lys(Ac)-OtBu. The title compound was prepared as P(8)2 reference precursor. It was obtained from Boc-Leu-Leu-Lys(Z)-(Ala)2-OH and Ddz-LeuLeu-Lys(Ac)-OtBu following the same procedure previously described.4a Rf (CHCl3/CH3OH, 95/5) ) 0.30; yield ) 30%. Protoporphyryl Peptides. General Procedure. A 281 mg sample of protoporphyrin IX (0.5 mmol) was dissolved in 5 mL of DMF and treated at -10 °C with 78 mg of iBCCl (0.5 mmol) and 101 mg of NMM (1 mmol). After 10 min a precooled solution of 0.5 mmol of the above-described amino peptide component was added. The reaction mixture was stirred at room temperature for 4 h and then evaporated in vacuo. The residue, treated with 10 mL of CH3OH, was filtered from the insoluble material, and the filtrate was evaporated and chromatographed on a Sephadex LH 20 column (2.5 × 250 cm) using methanol as eluant. The fractions containing the protoporphyryl peptides were collected and evaporated, and the residue was further chromatographed on a silica gel column (2 × 50 cm) in CHCl3/ CH3OH (9/1) as eluant, to separate the monomeric from the dimeric peptides. Dimeric compounds: Rf (CHCl3/CH3OH, 9/1) ) 0.30 (n ) 0), 0.35 (1), 0.40 (2), and 0.45 (3); yields ) 5, 10, 4, and 2%, respectively, those of the blank (having acetyl instead of naphthylacetyl; see above) being 0.55 and 2%, respectively. HRMS (FAB: trinitrobenzyl alcohol matrix), m/e: calcd for C148H218O22N20, C154H228O24N22, C160H238O26N24, C166H248O28N26 (n ) 0, 1, 2, 3, respectively; see Chart 1), 2629.4, 2771.6, 2913.7, and 3055.9; found 2632.6, 2771.1, 2914.0, and 3059.4, respectively. Methods. Steady-state fluorescence spectra were recorded on a SPEX Fluoromax spectrofluorometer, operating in SPC mode (λex ) 280, λem ) 340 nm). Quantum yields were obtained by using naphthalene in cyclohexane as reference: Φ0 ) 0.23 ( 0.02. Nanosecond decays were measured by a CD900, SPC lifetime apparatus from Edinburgh Instruments. Excitation in the UV region was achieved by a flashlamp filled with ultrapure hydrogen (300 mmHg; fwhm ) 1.2 ns, 30 kHz repetition rate). The decay curves were fitted by a nonlinear least-squares analysis to exponential functions by an iterative deconvolution method. The time distribution analysis was performed by using the standard software supplied by Edinburgh Instruments. Fluorescence anisotropy measurements were carried out on the SPEX apparatus, equipped with Glan-Thomson polarizing
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prisms. The steady-state anisotropy coefficient is defined as r ) (I|| - I⊥)/(I|| + 2I⊥), where I|| and I⊥ are the fluorescence intensities measured with the emission polarizer set parallel or perpendicular to the excitation polarization direction, respectively. The average rotational correlation time τ′′ is derived from the following expression:
r ) r0/[1 + (τ/τ′′)] where τ is the fluorescence intensity decay time and r0 is the anisotropy in the absence of any motion (r0 ) 0.11 for protoporphyrin12). By approximating the peptide to a rigid sphere, one can also estimate τ′′ by the following expression:
τ′′ ) ηV/kT where η is the solvent viscosity, V is the molecular volume, k is the Boltzmann constant, and T is the absolute temperature. All fluorescence experiments were carried out in quartz cells, using solutions previously bubbled for 20 min with ultrapure nitrogen. Transient absorption measurements were carried out by a flash-photolysis setup, the pulsed excitation (308 nm) being achieved by a Xe/HCl excimer laser (Lamda Physik EMG 50E). The pulse width was about 15 ns, the laser energy less than 10 mJ/pulse, and the delay time
