Photoinduced Electron Transfer from Tryptophan to Ru^IITAP Complexes: The Primary Process for Photo-Cross-Linking with Oligopeptides

Abstract

The photoreaction mechanism of [Ru(TAP)(2)(phen)](2+) and [Ru(TAP)(3)](2+) (TAP = 1,4,5,8-tetraazaphenanthrene) with tryptophan (Trp), N-acetyl-Trp, and Lys-Trp-Lys is examined. The existence of a photoelectron-transfer process from the amino acid unit is demonstrated by laser flash photolysis experiments. The back electron transfer (BET) from the reduced complex to the oxidized amino acid, occurring at the microsecond time scale, corresponds approximately to an equimolecular-bimolecular process; however, it is disturbed by another reaction, originating from the oxidized Trp. Moreover, in competition with the BET, the reduced and oxidized intermediates give rise to an adduct. The latter is clearly detected by gel electrophoresis experiments in denaturing conditions, with a system composed of an oligonucleotide derivatized at the 3' end by the Ru(II)TAP complex and hybridized with the complementary sequence functionalized at the 5' end by the tripeptide Lys-Trp-Lys. Thus, upon illumination, a cross-linking between the two strands is observed, which originates from the presence of a Trp residue.

Photocrosslinking between Peptide–Peptide or Peptide–Oligonucleotide by RuII–TAP Complexes

“…The luminescence quenching [18,19] (by electron transfer) of their excited states by Lys-Trp-Lys and Ala-Trp-Ala was found to be close to the diffusion limit (see the Supporting Information). The photoreactions, under steady-state illumination of 1 and 2 in the presence of these two peptides under argon, were monitored by UV/Vis absorption spectroscopy as a function of the irradiation time ( Figure 2 and the Supporting Information).…”

“…Similar changes were observed with the mononucleotide GMP (guanosine-5'-monophosphate) and Trp; they have been attributed to formation of adducts upon illumination. [17,18] In order to characterize the adducts in this work the crude illuminated solutions were analyzed by MALDI mass spectrometry (see the Experimental Section). A spectrum is presented in Figure 3 …”

“…Based on our previous report, a covalent mono-adduct between the complex and the peptide has a structure such as that in Figure 1. [17][18][19] On the basis of this structure, the mass should correspond to the sum of the mass of the Ru complex (A) plus that of the peptide (B), minus that of two hydrogen atoms [A + BÀ2 H] due to the formation of one covalent bond. This is indeed observed in Figure 3 at m/z 1104.…”

“…[17,18] The resulting ion pairs, in competition with the back electron transfer (BET in the reaction scheme, Equation (3)) gives rise, after several steps, to formation of an adduct of the Trp unit on the complex [Eq. (4)], [17,19] the structure of which is shown in Figure 1 …”

“…The observation of such decomposition products would indicate that, in the mixture of singly charged cations, there are at least species in which the two peptides are covalently bound to the same TAP ligand since an addition on the phen ligand has never been detected during the numerous experiments performed in the past with GMP [16] and more recently with Trp. [17][18][19] In addition to these m/z 1379.5/ 1380.5 fragment ions, intense fragment ions were detected at m/z 921.2/922.2 and formally result from the direct loss of a TAP ligand covalently bound to a peptide ( Figure 5). Interestingly, these fragment ions were already observed in the MALDI mass spectrum presented in Figure 3.…”