Convergent synthesis and properties of photoactivable NADPH mimics targeting nitric oxide synthases

Abstract

A new series of photoactivable NADPH mimics bearing one or two O-carboxymethyl groups on the adenosine moiety have been readily synthesized using click chemistry. These compounds display interesting one- or two-photon absorption properties. Their fluorescence emission wavelength and quantum yields (Φ) are dependent on the solvent polarity, with a red-shift in a more polar environment (λ = 460-467 nm, Φ > 0.53 in DMSO, and λ = 475-491 nm, Φ < 0.17 in Tris). These compounds show good binding affinity towards the constitutive nNOS and eNOS, confirming for the first time that the carboxymethyl group can be used as a surrogate of phosphate. Two-photon fluorescence imaging of nanotriggers in living cells showed that the presence of one carboxymethyl group (especially on the 3' position of the ribose) strongly favors the addressing of nanotriggers to eNOS in the cell context.

Design of Light-Sensitive Triggers for Endothelial NO-Synthase Activation

“…The synthesis of the second generation of NTs (NT2_3, NT2_5, and NT2_7) and the third generation was previously reported [ [18], NH Nguyen, PhD Thesis, 2015 ENS Cachan]. Synthetic protocols will be published elsewhere [25].…”

“…NT1 is a photoactivable analogue of NADPH [15][16][17][18]23], designed to preserve the binding of the cofactor by the nucleotide moiety of the NADPH to its binding pocket within the NOS enzymes (red box in Figure 1). To introduce a photoactivable moiety in NT1, the flexible nicotinamide group of NADPH was replaced by a chromophore (purple box in Figure 1).…”

“…Compounds of these types would be intrinsically limited by diffusion of the cofactor or the active moiety to its binding site, measured in milliseconds, too slow a timescale for many catalytic events, including eNOS catalysis [13,14]. To overcome the diffusion limitation, we have designed photoactivable NADPH derivatives targeting the NADPH binding site in the NOS reductase domain [15][16][17][18]. The first derivative, called nanotrigger NT1 (Figure 1), kept the nucleotidic part of NADPH, which was further linked to a conjugated, photoactivable chromophore substituted with two donor groups ( Figure 1).…”

“…This 2' phosphate group was conserved in NT1, enabling the targeting of e/nNOS at their NADPH site in the reductase domain. In the second generation of NT, this phosphate group was substituted by either a 2' or 3' carboxylate group or both 2' and 3' carboxylate groups [18]. Interestingly only the 3' carboxylate derivative was able to generate NO upon two-photon excitation.…”

“…The R1 "hook" substituent of NT1 was an ethyl group. To further characterize how the charge carried by the ribose influenced binding to endothelial Nitric Oxide-Synthase (eNOS), the nucleotidic ribose was substituted by one or two carboxylate groups at either the 2' or 3' position or both, yielding NT2_7, NT2_5, and NT2_3, respectively [18,19]. In order to increase the affinity of the nanotrigger for e/nNOS, a charged R1 "hook1" was added, which targets additional charged residues of eNOS located within 7…”