Bioluminescent Properties of Semi-Synthetic Obelin and Aequorin Activated by Coelenterazine Analogues with Modifications of C-2, C-6, and C-8 Substituents

Abstract

Ca2+-regulated photoproteins responsible for bioluminescence of a variety of marine organisms are single-chain globular proteins within the inner cavity of which the oxygenated coelenterazine, 2-hydroperoxycoelenterazine, is tightly bound. Alongside with native coelenterazine, photoproteins can also use its synthetic analogues as substrates to produce flash-type bioluminescence. However, information on the effect of modifications of various groups of coelenterazine and amino acid environment of the protein active site on the bioluminescent properties of the corresponding semi-synthetic photoproteins is fragmentary and often controversial. In this paper, we investigated the specific bioluminescence activity, light emission spectra, stopped-flow kinetics and sensitivity to calcium of the semi-synthetic aequorins and obelins activated by novel coelenterazine analogues and the recently reported coelenterazine derivatives. Several semi-synthetic photoproteins activated by the studied coelenterazine analogues displayed sufficient bioluminescence activities accompanied by various changes in the spectral and kinetic properties as well as in calcium sensitivity. The poor activity of certain semi-synthetic photoproteins might be attributed to instability of some coelenterazine analogues in solution and low efficiency of 2-hydroperoxy adduct formation. In most cases, semi-synthetic obelins and aequorins displayed different properties upon being activated by the same coelenterazine analogue. The results indicated that the OH-group at the C-6 phenyl ring of coelenterazine is important for the photoprotein bioluminescence and that the hydrogen-bond network around the substituent in position 6 of the imidazopyrazinone core could be the reason of different bioluminescence activities of aequorin and obelin with certain coelenterazine analogues.

Internal Light Sources‐Mediated Photodynamic Therapy Nanoplatforms: Hope for the Resolution of the Traditional Penetration Problem

“…Bioluminescence is a widespread luminescence phenomenon in natural organisms based on the principle that substrates containing chromophores produce autofluorescence under the catalytic effect of enzymes in organisms. [55,56] Similar to chemiluminescence, bioluminescence as an endogenous light source can activate PSs for anticancer therapy through RET. Due to the merits of bioluminescence, like high quantum yield, non-invasive damage, and high security, researchers have applied various bioluminescence systems, for instance, the firefly luciferase (FLuc)luciferin, renilla luciferase (RLuc)-coelenterazine, and NanoLuc (NLuc)-furimazine systems to PDT.…”

Crystal structure of semi-synthetic obelin-v after calcium induced bioluminescence implies coelenteramine as the main reaction product

“…Brought to life by the prospects of various analytical applications of CTZ-dependent bioluminescence proteins, the approach of chemical modification of CTZ to acquire luciferins which would alter properties of these proteins became very popular. In order to change bioluminescence intensity, kinetics, calcium sensitivity, or generate different emission color, a large number of CTZ analogues with various modifications of imidazopyrazinone core or its C2, C6 and C8 substituents have been synthesized and tested in many applications with different photoproteins or luciferases showing promising results [6][7][8][9][10] .…”