A NADPH substitute for selective photo-initiation of reductive bioprocesses via two-photon induced electron transfer{ Anne-Claire Robin, a Said Gmouh, a Olivier Mongin, a Viatcheslav Jouikov, a Martinus H. V. Werts, a Cle ´ment Gautier, b Anny Slama-Schwok b and Mireille Blanchard-Desce* a Received (in Cambridge, UK) 27th October 2006, Accepted 21st December 2006 First published as an Advance Article on the web 18th January 2007 DOI: 10.1039/b615628b A NADPH substitute where the nicotinamide moiety is replaced by a chromophoric unit having much larger two- photon absorption cross-section and able to transfer electrons to flavins only upon excitation is described as an effective two- photon nanotrigger for selective photo-activation of electron transfer in bioreductive processes. NADPH is an important protein cofactor that plays an ubiquitous role in numerous bioprocesses. NADPH acts as the electron donor in a number of reductive biosyntheses such as fatty acid and nucleic acid synthesis. It is involved in the electron transfer chain of several proteins including cytochrome P450 reductase (CPR), 1 NO-synthase (NOS), 1–3 ferredoxin reductase where it acts as a primary electron donor. The electron transfer directed mostly towards flavin units (such as FAD in the case of flavocytochrome P450 BM3 4 or NO-synthase) is thus of fundamental importance as a crucial step in the enzymatic machinery. A molecular tool that would mimic NADPH in terms of protein affinity (i.e. binding to NADPH site) but would deliver electron transfer to adjacent electron acceptors such as flavins only upon appropriate activation would be of particular interest. Such a compound could be used to trigger the primary electron transfer in NADPH proteins in situ on demand. As such, it could be used as a ‘‘nanotrigger’’ that would allow molecular synchronization of an ensemble of proteins in solution providing an interesting alternative to single molecule addressing. Using laser pulses as the external command is particularly appealing for such a goal. This could be put into practice by designing a nanotrigger that undergoes photo-induced electron transfer to flavins upon photo- excitation. The selectivity and efficiency of photo-excitation are of course key parameters. With this aim in mind, our strategy has been to design a nanotrigger that allows efficient and selective photo-activation via two-photon excitation. Indeed two-photon excitation would provide a number of advantages for in situ excitation 5 which include (i) intrinsic 3D resolution, (ii) improved penetration depth in biological media (by using NIR excitation sources in the 700–1200 nm range thus reducing scattering and minimizing absorption) and (ii) higher selectivity and lower photo- damage (by decreasing significantly the excitation of endogenous chromophores such as tyrosine, tryptophane, flavins…). The design of such molecular tool is detailed in Fig. 1. The NADPH substitute is based on the combination of (i) a chromophoric unit that becomes strongly reductive upon excita- tion and has a significant two-photon absorption (TPA) cross- section in the red–NIR region (that allows better penetration for in situ experiments, typically in the 700–1000 nm range) and (ii) a molecular recognition unit that allows selective and efficient recognition of the NADPH binding site. The overall shape and length of NADPH is preserved. An adenosine unit is used as the recognition moiety and the chromophoric moiety is a bis-donor diphenyl-butadiene. A structurally-related symmetrical lipophilic chromophore 6 bearing two dialkylamino end-groups (i.e. NBu 2 ) was previously reported to have a suitable redox potential (i.e. 0.44 V/SCE in the ground state for the C 2+ /C couple and 21.04 V/SCE in the excited state for the C 2+ / C* couple) thus allowing photo- induced electron transfer to a FAD moiety (Eu = 20.06 V/NHE). Such a chromophore is also expected to have significantly larger TPA cross-section in the red–NIR region, 7 than NADPH 8,9 and adenine 10,11 thus allowing efficient two-photon excitation and triggering of electron transfer. The replacement of one of the dialkyamino group by a NH 2 unit although expected to lead to a slight reduction in maximum TPA cross section as compared to its lipophilic symmetrical analogue, was meant to enhance the water- solubility and reinforce the affinity for the NADPH binding site in proteins. a Synthe `se et Electrosynthe `se Organiques (CNRS, UMR 6510), Universite ´ de Rennes 1, Campus Scientifique de Beaulieu, Ba ˆt. 10A, F-35042, Rennes Cedex, France. E-mail: mireille.blanchard-desce@univ-rennes1.fr; Fax: (+33) 2 23 23 62 77; Tel: (+33) 2 23 23 62 77 b Laboratoire Optique et Biosciences (INSERM U696), Ecole Polytechnique, Palaiseau, France { Electronic supplementary information (ESI) available: Synthesis of nanotrigger NT and experimental details of spectroscopic measurements. See DOI: 10.1039/b615628b Fig. 1 Design of a NADPH substitute for selective two-photon triggering of bioreduction. COMMUNICATION www.rsc.org/chemcomm | ChemComm 1334 | Chem. Commun., 2007, 1334–1336 This journal is ß The Royal Society of Chemistry 2007