A NADPH substitute for selective photo-initiation of reductive bioprocesses via two-photon induced electron transfer

Article abstract of 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 f

Full text of DOI:10.1039/b615628b

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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-Schwokb 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
minimizing absorption) and (ii) higher selectivity and lower photo-
damage (by decreasing significantly the excitation of endogenous
chromophores such as tyrosine, tryptophane, flavins…).
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.
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
chromophore6 bearing two dialkylamino end-groups (i.e. NBu2)
was previously reported to have a suitable redox potential (i.e. 0.44
V/SCE in the ground state for the C2+/C couple and 21.04 V/SCE
in the excited state for the C2+/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 NADPH8,9 and
adenine10,11 thus allowing efficient two-photon excitation and
triggering of electron transfer. The replacement of one of the
dialkyamino group by a NH2 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.
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 BM34 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
excitation5 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
aSynthe`se et Electrosynthe`se Organiques (CNRS, UMR 6510),
Universite´ de Rennes 1, Campus Scientifique de Beaulieu, Baˆt. 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
bLaboratoire 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.
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Scheme 1 i: a, 2, THF, NaH; b, AcOH, CH2Cl2 (92%); ii: 4, NaH, THF (63%); iii: HCl, CH2Cl2 (95%); iv: a, TsCl, pyridine; b, NaN3,
N,N-dimethylacetamide (69%); v: a, Ph3P, toluene; b, THF, H2O (100%); vi: NaOH, EtOH (75%); vii: 10, HBTU, DMF, Et3N (57%); viii: TFA, H2O,
THF (96%); ix: a, (iPr)2N–P(OBn)2, tetrazole; b, O2 (69%); x, Me3SiBr, CH2Cl2 (88%).
The synthesis of the NADPH substitute NT is described in
Scheme 1. The chromophoric moiety 9 was prepared in a six-step
sequence from aldehyde 1,12 which was first reacted with
tributyl[(1,3-dioxolan-2-yl)methyl]phosphonium bromide13 (2), to
afford, after hydrolysis under mild conditions, the homologous
a,b-unsaturated aldehyde 3. Horner-Emmons-Wadsworth con-
densation of 3 with phosphonate 414 gave stereospecifically the
E,E-diene 5. The tetrahydropyranyl protective group was then
removed under acidic conditions, and the resulting alcohol (6) was
then converted into amine (8), by reduction of the intermediate
azide 7. The chromophoric moiety 9 was obtained after cleavage of
the benzamide group under basic conditions. The coupling
reaction between 9 and the adenosine moiety 1015 in the presence
of HBTU afforded 11, the isopropylidene group of which was
removed with trifluoroacetic acid. The phosphate group was then
introduced on 12 with dibenzyldiisopropylphosphoramidite. The
resulting phosphoramidate was oxidized, and the benzyl protective
groups were finally removed using bromotrimethylsilane to afford
NT (Scheme 1).
Moreover, when excited in the NIR range (700–1000 nm), NT
generates fluorescence, demonstrating that NT can be effectively
photo-excited via a TPA process.
Two-photon excited fluorescence (TPEF) experiments solution
were then conducted to determine the TPA spectra of NT. The
experiments demonstrate that NT exhibits much larger TPA cross-
sections than both NADPH and flavins (Fig. 3) as well as other
NT is water-soluble and displays intense absorption in the near
UV region (Fig. 2). Its absorption maximum is slightly
hypochromically and hypsochromically shifted with respect to
the symmetrical lipophilic analogue of its chromophoric part
(whose absorption peaks at 398 nm in CH2Cl2),6 in agreement
with the lower electron-donating ability of the NH2 group as
compared to NBu2. Yet, due to the presence of this chromophoric
unit, the absorptivity of NT in the near UV is about one order of
magnitude larger than that of NADPH (Table 1).
Fig. 2 Absorption and emission spectra of nanotrigger NT in H2O.
Table 1 Photophysical characteristics of nanotrigger NT compared
to that of NADPH
labs/nm
e/M21 cm21
lem/nm
Wa
NT
NADPH
376
342
60 000
6 600
517
450
0.14
0.01916
a
Fluorescence quantum yield determined relative to fluorescein in
0.1 N NaOH.
NT emits slight fluorescence (Table 1) which shifts from the
visible blue in organic solvents to visible green in water (Fig. 2).
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Fig. 3 Two-photon absorption spectra of nanotrigger NT, NADPH8
and FAD8 in Tris buffer (pH 7.6).
Fig. 4 Quenching of the TPEF fluorescence (excited at 790 nm) of NT
(1 mM in Tris buffer) as a function of the concentration of FAD.
endogenous chromophores (including amino-acids).17 This vali-
dates the molecular design in terms of effective and selective
excitation of the NADPH substitute via two-photon excitation in
biological samples. At 790 nm, NT has a 1800 times larger TPA
cross-section than NADPH and 110 times larger than FAD.
Interestingly, we observe that NT shows a larger TPA cross-
section—in the 750–800 nm region (which corresponds to the one-
photon allowed transition)—than its symmetrical lipophilic
analogue.7 Hence although the NH2 group is a less effective
donating group than NR2, the breaking of centro-symmetry
induced by the replacement of one NR2 end-group by a NH2
group has a positive effect on the magnitude of TPA in the target
spectral window for biological applications because the one-
photon transition (corresponding to the lowest energy excited-
state) is no longer two-photon forbidden.
In conclusion we have designed and synthesized a NADPH
substitute that allows efficient two-photon excitation induced
electron transfer to flavins As such it represents a unique tool for
triggering the primary electron transfer in a variety of proteins
involved in bioreductive processes with low photo-damage and
excellent spatial resolution.
Pascal Saint-Val is acknowledged for technical assistance in the
synthesis.
Notes and references
{ Performed following the same protocol as described in the literature.6
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Cyclic voltammetry experiments conducted in acetonitrile/0.1 M
Bu4NPF6 show that the chromophoric moiety of NT (i.e.
chromophore 9) undergoes a two-electron oxidation at 0.61
V/SCE. The oxidation potential is shifted to a more positive value
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consequence of the lower electron-donating ability of the NH2
end-group as compared to the NBu2 moiety. Chemical oxidation
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undergoes oxidation to a dicationic species strongly absorbing in
the visible region (lmax = 573 nm) which is close to that of its
symmetrical lipophilic analogue (lmax = 583 nm).6 The redox
potential of the 92+/9* couple (#20.6 V/NHE estimated from the
oxidation potential and absorption and emission properties)
suggests that photo-induced electron transfer from NT to adjacent
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1336 | Chem. Commun., 2007, 1334–1336
This journal is ß The Royal Society of Chemistry 2007