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Hydrophobic Interactions in Donor-Disulphide-Acceptor (DSSA) Probes Looking Beyond Fluorescence Resonance Energy Transfer Theory

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Abstract

Donor –linker –acceptor (DSSA) is a concept in fluorescence chemistry with acceptor being a fluorescent compound (FRET) or quencher. The DSSA probes used to measure thiol levels in vitro and in vivo. The reduction potential of these dyes are in the range of −0.60 V, much lower than the best thiol reductant reported in literature, the DTT (−0.33 V). DSSA disulphide having an unusually low reduction potential compared to the typical thiol reductants is a puzzle. Secondly, DSSA probes have a cyclized rhodamine ring as acceptor which does not have any spectral overlap with fluorescein, but quenches its absorbance and fluorescence. To understand the structural features of DSSA probes, we have synthesized DSSANa and DSSAOr. The calculated reduction potential of these dyes suggest that DSSA probes have an alternate mechanism from the FRET based quenching, namely hydrophobic interaction or dye to dye quenching. The standard reduction potential change with increasing complexity and steric hindrance of the molecule is small, suggesting that ultra- low Eo’ has no contribution from the disulphide linker and is based on structural interactions between fluorescein and cyclized rhodamine. Our results help to understand the DSSA probe quenching mechanism and provide ways to design fluorescent probes.

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Abbreviations

DSSA:

Donor–Disulfide linker–acceptor

DSSAAl –DSSA:

Probe with cystamine as a linker, DSSAAr –DSSA probe probe with diaminophenyl disulfide as a linker

DSSANa – DSSA:

Probe with 2,2’-dithiodi (1-naphthylamine) as a linker

DSSAOr – DSSA:

Probe with o-diamino diphenyl disulphide as a linker

FRET:

Forster (Fluorescence) resonance energy transfer, chemiluminescence resonance energy transfer (CRET)

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Authors and Affiliations

  1. School of Bio Sciences and Technology, VIT University, Vellore, Tamil Nadu, 632014, India

    Shilpa Kammaradi Sanjeeva, Chandrasekhar B. Nair, P. V. Subba Rao, Phani Kumar Pullela & Ramamoorthy Siva

  2. Bigtec Labs, Bigtec Pvt. Ltd., 2nd Floor, 59th ‘C’ cross, Rajajinagar, Banglore, 560 010, India

    Shilpa Kammaradi Sanjeeva, Swathi Korrapati, Chandrasekhar B. Nair, P. V. Subba Rao & Phani Kumar Pullela

  3. School of Advanced Sciences, VIT University, Vellore, Tamil Nadu, 632014, India

    Swathi Korrapati, Phani Kumar Pullela & U. Vijayalakshmi

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  1. Shilpa Kammaradi Sanjeeva
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Sanjeeva, S.K., Korrapati, S., Nair, C.B. et al. Hydrophobic Interactions in Donor-Disulphide-Acceptor (DSSA) Probes Looking Beyond Fluorescence Resonance Energy Transfer Theory. J Fluoresc 24, 1297–1306 (2014). https://doi.org/10.1007/s10895-014-1414-z

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