Convergent Preparation and Photophysical Characterization of Dimaleimide Dansyl Fluorogens:  Elucidation of the Maleimide Fluorescence Quenching Mechanism

Abstract

Dimaleimide fluorogens are being developed for application to fluorescent protein labeling. In this method, fluorophores bearing two maleimide quenching groups do not fluoresce until both maleimide groups have undergone thiol addition reactions with the Cys residues of the target protein sequence [J. Am. Chem. Soc. 2005, 127, 559-566]. In this work, a new convergent synthetic route was developed that would allow any fluorophore to be attached via a linker to a dimaleimide moiety in a modular fashion. Series of dimaleimide and dansyl derivatives were thus prepared conveniently and used to elucidate the mechanism of maleimide quenching. Intersystem crossing was ruled out as a potential quenching pathway, based on the absence of a detectable triplet intermediate by laser flash photolysis. Stern-Volmer rate constants were measured with exogenous dimaleimide quenchers and found to be close to the diffusion-controlled limits, consistent with electron transfer being thermodynamically favorable. The thermodynamic feasibility of the photoinduced electron transfer (PET) quenching mechanism was verified by cyclic voltammetry. The redox potentials measured for dansyl and maleimide confirm that electron transfer from the dansyl excited state to a pendant maleimide group is exergonic and is responsible for fluorescence quenching of the fluorogens studied herein. Taking this PET quenching mechanism into account, future fluorogenic protein labeling agents will be designed with spacers of variable length and rigidity to probe the structure-property PET efficiency relationship.

Thermally Stable Fluorescent Maleimide/Isobutene Alternating Copolymers Containing Pyrenyl and Alkynylpyrenyl Moieties in the Side Chain

“…[5][6][7][8][9][10][11][12][13][14][15] N-(1-Pyrenyl)maleimide is popularly used as a biological probe for fluorescent protein labeling, as well as the other maleimide derivatives functionalized with a variety of fluorophores. [16][17][18][19][20] Recently, alkynylpyrenes have been developed as improved pyrene-based biomolecular probes with high fluorescence quantum yields and reduced quenching of the fluorescence. [21][22][23] Recently, it was reported that the alternating copolymerization of N-alkylmaleimides (RMI) with isobutene (IB) produced high-molecular-weight copolymers [poly(RMIalt-IB)s] with excellent thermal stabilities, as well as excellent mechanical and optical properties.…”

Dynamical perspective of protein-DNA interaction

“…In this regard, electron transfer dynamics from the dansyl to operator DNA has been studied. In principle, dansyl can also act as an electron donor or hole acceptor, retrieving the native state of guanine from its radical cation as the oxidation potential of N-ethyl dansylamide (0.94 V), a derivative of dansyl, is lower than the oxidation potential of guanine (58). The electron transfer dynamics of dansyl in the protein upon complexation with O R 1 and O R 2 DNA is evident from the faster fluorescence transients depicted in Figure 20 …”

Small photoactivatable molecules for controlled fluorescence activation in living cells

“…The reason for this is that the interaction with the analyte changes the redox potential of the quencher so that the photoinduced electron transfer is no longer possible and the fluorescence is restored. The two most prominent ways of annihilating a photoinduced electron transfer quenching system are either a chemical reaction (e.g., a Michael Type addition of a thiol on a maleimide analogue) [36][37][38] or the chelation of a cation (H + , Na + , K + , Ca 2+ , Zn 2+ , Cd 2+ , Hg 2+ , etc.). [5][6][7] Since these fluorescence activations are not photoinduced, we will not discuss them further.…”