A Nitroxide Derivative as a Probe for Conformational Studies of Short Linear Peptides in Solution. Spectroscopic and Molecular Mechanics Investigation Basilio Pispisa,* ,‡ Antonio Palleschi, § Lorenzo Stella, ‡ Mariano Venanzi, ‡ and Claudio Toniolo # 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 Studio sui Biopolimeri, C.N.R., Dipartimento di Chimica Organica, UniVersita ` di PadoVa, 35131 PadoVa, Italy ReceiVed: March 9, 1998; In Final Form: June 16, 1998 The photophysics of linear Aib-based hexapeptides (Aib )R-aminoisobutyric acid) of general formula Ac-Toac-(Aib) n -Trp-(Aib) r -OtBu (TnTrp), where n + r ) 4, and Toac and Trp are a nitroxide spin labeled C R,R -disubstituted glycine and tryptophan, respectively, were investigated in methanol and dioxane solutions by steady-state and time-resolved fluorescence measurements. Another hexapeptide, i.e., Boc-(S)Bin-Ala- Aib-Toac-(Ala) 2 -OtBu (T-Bin), carrying the same nitroxide derivative and a binaphthyl as the fluorophore was also studied by the same techniques. Quenching of the excited tryptophan or binaphthyl chromophore proceeds on a time scale from subnanoseconds to a few nanoseconds, depending on the conformers distribution in solution. CD and IR spectral patterns in methanol or CDCl 3 suggest that the backbone of the peptides examined is in the 3 10 -helical conformation, thus preserving the structural features of the crystal state, as earlier determined by X-ray diffraction measurements. The fluorescence results were satisfactorily described by a dipole-dipole interaction mechanism, in which electronic energy transfer takes place from the excited tryptophan or binaphthyl to Toac, provided the mutual orientation between the fluorophore and Toac is taken into account. This implies that interconversion among conformational substates is slow on the time scale of the transfer process. Molecular mechanics calculations coupled with time decay data allowed us to build up the most probable structures of these peptides in methanol solution. Introduction Extensive work has been carried out for understanding the mechanism through which excited states are quenched by paramagnetic species, such as nitroxyl radicals, but there is not yet a univocal interpretation of the phenomenon. 1-3 Apart from the intrinsic interest, the importance of these studies currently stems from the capability of the doublet quencher to probe the structural and dynamic features of membranes, 4,5 micelles, 6,7 and proteins surface. 1 In addition, unlike many free radical species, • NO displays selective reactivity, the major biological targets including heme-iron and iron-sulfur proteins. 8 Very recently, the rate constant for the reaction of nitroxide with tryptophan and tyrosine radicals in peptides and proteins was measured, suggesting that • Trp and • Tyr are likely and important targets for • NO generated in vivo. 9 Among the nitroxide derivatives currently used, Toac is a nitroxide spin-labeled C R,R -disubstituted glycine (Chart 1) that, when incorporated into a peptide chain, provides a means of restricting the range of backbone conformations. 10,11 Indeed, besides favoring stereochemical rigidity, Toac is able to probe 3 10 /R-helix conformations in peptides doubly labeled at ap- propriate relative positions. 12 In the research program aimed at elucidating the dynamics and conformational features of polypeptides 13 and linear oli- gopeptides 14 in solution by studying the photophysical behavior of these materials carrying suitable fluorophores, we have recently investigated short linear peptides containing tryptophan (Trp) and Toac (T), and (S)-binaphthyl (Bin) and Toac (Chart 1). The general formula for the former series of peptides is Ac-Toac-(Aib) n -Trp-(Aib) r -OtBu, where Ac is acetyl, OtBu tert- butoxy, Aib R-aminoisobutyric acid, and n + r ) 4, hereafter denoted as TnTrp, namely T0Trp, T1Trp, T2Trp, and T3Trp, to emphasize the length of the spacer between the chromophores in the hexapeptides. In the latter case, the formula is Boc-(S)- Bin-Ala-Aib-Toac-(Ala) 2 -OtBu, denoted as T-Bin, where Boc is tert-butyloxycarbonyl, and Ala L-alanine. According to X-ray diffraction results, 15,16 in the crystal state the backbone of the peptides examined is in the 3 10 -helical conformation, with right-handedness (r.h.) for TnTrp, and left- handedness (l.h.) for T-Bin. This latter structure is unusual for a peptide containing L-alanine residues, and is most probably due to the presence of the chiral (S)-binaphthyl moiety. Chart 2 illustrates the crystal state structures of T-Bin and T3Trp peptides, the former comprising two conformers, here denoted as a and b, differing from each other by the different puckering of the Toac moiety and its spatial orientation with respect to the binaphthyl group. ‡ Universita ` di Roma Tor Vergata. § Universita ` di Roma La Sapienza. # Universita ` di Padova. CHART 1 7890 J. Phys. Chem. B 1998, 102, 7890-7898 S1089-5647(98)01415-1 CCC: $15.00 © 1998 American Chemical Society Published on Web 09/15/1998