A Bioorthogonally Synthesized and Disulfide-Containing Fluorescence Turn-On Chemical Probe for Measurements of Butyrylcholinesterase Activity and Inhibition in the Presence of Physiological Glutathione
Abstract
:1. Introduction
2. Results and Discussion
2.1. Synthesis of the FRET Chemical Probe 11
2.2. FRET and Fluorogenic Properties of the FRET Chemical Probe 11
2.3. Study of the Fluorogenic Reaction Mechanism of 11 with DTT
2.4. Deciphering the Ability of 11 to Discriminate Against Reactions with GSH
2.5. GSH not a Source of Interference in the 11-Based Fluorescence Turn-On Assay for Measuring BChE Activity
2.6. Direct and Accurate Determination of BChE Activity in Human Serum by the Fluorescence Turn-On Assay Based on 11
2.7. The 11-Based Fluorescence Assay for Kinetic Analysis of BChE Inhibition
3. Materials and Methods
3.1. Synthesis of the Key Bicyclononyne Derivatives (6)
3.1.1. (1. R,8S,9r,Z)-Ethyl Bicyclo [6.1.0]non-4-ene-9-Carboxylate (exo-2) and (1R,8S,9s,Z)-Ethyl Bicyclo[6.1.0]non-4-ene-9-Carboxylate (endo-2)
3.1.2. [(1. R,4Z,8S,9r)-Bicyclo[6.1.0]non-4-en-9-yl]Methanol (exo-3), [(1R,4Z,8S,9s)-Bicyclo[6.1.0]non-4-en-9-yl]Methanol (endo-3), (1R,8S,9r)-Bicyclo[6.1.0]non-4-yn-9-Ylmethanol (exo-4), (1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-Ylmethanol (endo-4), (1R,8S,9r)-Bicyclo[6.1.0]non-4-yn-9-Ylmethyl (4-nitrophenyl) Carbonate (exo-5), and (1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-Ylmethyl (4-Nitrophenyl) Carbonate (endo-5)
3.1.3. [(1. R,8S,9r)-Bicyclo[6.1.0]non-4-yn-9-yl]Methyl {2-[(2-Aminoethyl)Disulfanyl]ethyl}Carbamate (exo-6)
3.1.4. [(1. R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-yl]Methyl {2-[(2-Aminoethyl)Disulfanyl]ethyl}Carbamate (endo-6)
3.2. Synthesis of the Azido EDANS Derivative (10)
3.2.1. 3-Azidopropanoic acid (8) and 1-[(3-Azidopropanoyl)oxy]Pyrrolidine-2,5-Dione (9)
3.2.2. 5-{[2-(3-Azidopropanamido)Ethyl]Amino}Naphthalene-1-Sulfonate (10)
3.3. Synthesis of the EDANS–DABCYL-Paired FRET Chemical Probe (11)
3.4. Synthesis of the GSH Derivatives, 12 and 13
3.4.1. Methyl (2S)-2-Amino-5-({(2R)-1-[(2-Ethoxy-2-Oxoethyl)Amino]-1-oxo-3-Sulfanylpropan-2-yl}Amino)-5-Oxopentanoic Acid (12)
3.4.2. (2. S)-2-Amino-5-({(2R)-1-[(2-Methoxy-2-Oxoethyl)Amino]-1-oxo-3-Sulfanylpropan-2-yl}Amino)-5-Oxopentanoate (13)
3.5. Spectroscopic Measurements
3.6. Specificity and Mechanism of the FRET Chemical Probe 11 Reactions with Thiols
3.7. The 11-Based Assay for Fluorescence Turn-On Analysis of BChE Activity in Serum and in the Presence of a BChE Inhibitor
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
AChE | acetylcholinesterase |
BChE | butyrylcholinesterase |
BTCh | S-butyrylthiocholine iodide |
ChE | cholinesterase |
CHES | 2-(cyclohexylamino)ethanesulfonic acid |
DABCYL | 4-[4-(dimethylamino)phenylazo]benzoic acid |
DABCYL NHS ester | N-succinimidyl 4-[4-(dimethylamino)phenylazo]benzoate |
DCM | dichloromethane |
DIPEA | N,N-diisopropylethylamine |
DMF | dimethylformamide |
DTNB | 5′-dithiobis(2-nitrobenzoic acid) |
DTT | DL-dithiothreitol |
EDANS | 5-(2-aminoethylamino)-1-naphthalenesulfonic acid |
EPPS | 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid |
Et3N | trimethylamine |
Et2O | diethyl ether |
EtOH | ethanol |
FRET | fluorescence resonance energy transfer |
FT-MS | Fourier-transfer mass spectrometry |
GSH | glutathione |
LOD | limit of detection |
MeOH | methanol |
MES | 2-(N-morpholino)ethanesulfonic acid |
PB | phosphate buffer |
PBS | phosphate buffered saline |
PIPES | 1,4-piperazinediethanesulfonic acid |
SPAAC | strained-promoted azide-alkyne cycloaddition |
TCh | thiocholine |
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Gong, M.-M.; Dai, C.-Y.; Severance, S.; Hwang, C.-C.; Fang, B.-K.; Lin, H.-B.; Huang, C.-H.; Ong, C.-W.; Wang, J.-J.; Lee, P.-L.; et al. A Bioorthogonally Synthesized and Disulfide-Containing Fluorescence Turn-On Chemical Probe for Measurements of Butyrylcholinesterase Activity and Inhibition in the Presence of Physiological Glutathione. Catalysts 2020, 10, 1169. https://doi.org/10.3390/catal10101169
Gong M-M, Dai C-Y, Severance S, Hwang C-C, Fang B-K, Lin H-B, Huang C-H, Ong C-W, Wang J-J, Lee P-L, et al. A Bioorthogonally Synthesized and Disulfide-Containing Fluorescence Turn-On Chemical Probe for Measurements of Butyrylcholinesterase Activity and Inhibition in the Presence of Physiological Glutathione. Catalysts. 2020; 10(10):1169. https://doi.org/10.3390/catal10101169
Chicago/Turabian StyleGong, Ming-Mao, Chia-Yen Dai, Scott Severance, Chi-Ching Hwang, Bo-Kai Fang, Heng-Bo Lin, Chien-Hui Huang, Chi-Wi Ong, Jeh-Jeng Wang, Pei-Lun Lee, and et al. 2020. "A Bioorthogonally Synthesized and Disulfide-Containing Fluorescence Turn-On Chemical Probe for Measurements of Butyrylcholinesterase Activity and Inhibition in the Presence of Physiological Glutathione" Catalysts 10, no. 10: 1169. https://doi.org/10.3390/catal10101169