Methods for generating analogs of coenzyme A
View Patent ↗Methods to generate analogs of coenzyme A in vivo are disclosed. The methods to generate analogs of coenzyme A in a cell comprise reacting pantetheine or a derivative thereof with a reporter to form labeled pantetheine or a derivative thereof, contacting the cell with the labeled pantetheine or derivative thereof such that the labeled pantetheine or derivative thereof enters the cell, phosphorylating the labeled pantetheine or derivative thereof to form phosphopantetheine or a derivative thereof, adenylating the labeled phosphopantetheine or derivative thereof to form a labeled dephosphoCoenzyme A or derivative thereof, and phosphorylating the 3′-hydroxyl of the labeled dephosphoCoenzyme A or derivative thereof to form a labeled coenzyme A analog or derivative thereof.
1. A method of generating analogs of coenzyme A in a cell, comprising:
labeling a pantetheine or a derivative thereof with a reporter to form labeled pantetheine or a derivative thereof;
contacting the cell with the labeled pantetheine or derivative thereof;
phosphorylating in vivo by a kinase the labeled pantetheine or derivative thereof to form labeled phosphopantetheine;
adenylating in vivo by an adenylyltransferase the labeled phosphopantetheine or derivative thereof to form a labeled-dephosphoCoenzyme A or a derivative thereof, and;
phosphorylating in vivo by a kinase the 3′-hydroxyl of the labeled-dephosphoCoenzyme A or derivative thereof to form a labeled-coenzyme A analog or a derivative thereof.
2. The method of claim 1 , wherein the pantetheine or derivative thereof comprises three modules.
3. The method of claim 2 , wherein the three modules are selected from a ω-functionalized amine, a sidechain, an α-aminoacid, a β-aminoacid, a linker, or a modulator.
4. The method of claim 3 , wherein the w-functionalized amine, the sidechain, the α-aminoacid, the β-aminoacid, the linker, or the modulator comprise a reporter (R).
5. The method of claim 3 , wherein the modulator is a dihydroxy acid, an aminohydroxy acid, a pantoic acid, or a homolog or derivative of pantoic acid.
6. The method of claim 1 , wherein the reporter is attached to the labeled coenzyme A analog by an azide-alkyne cycloaddition reaction.
7. The method of claim 1 , wherein the reporter is attached to the labeled coenzyme A analog by a ketone-hydroxylamine reaction.
8. The method of claim 1 , wherein the reporter is an affinity reporter, a colored reporter, a fluorescent reporter, a magnetic reporter, a radioisotopic reporter, a peptide reporter, a metal reporter, a nucleic acid reporter, a lipid reporter, a glycosylation reporter, a reactive a reporter, an enzyme inhibitor, or a biomolecular substrate.
9. The method of claim 1 , wherein the cell is a prokaryotic cell.
10. The method of claim 1 , wherein the cell is a eukaryotic cell.
11. The method of claim 1 , further comprising labeling a carrier protein domain with the labeled-coenzyme analog or derivative thereof to form a labeled protein.
12. The method of claim 11 , wherein the carrier protein domain comprises a fusion construct between a peptide or a carrier protein domain and a protein of interest.
13. The method of claim 11 , wherein the labeling is catalyzed by a phosphotransferase.
14. The method of claim 13 , wherein the phosphotransferase is 4′-phosphopantetheinyltransferase.
15. The method of claim 1 , wherein the labeled-phosphopantetheine analog can be used to identify proteins, characterize proteins, inhibit proteins, activate proteins, or examine the structure of proteins.
16. The method of claim 1 , wherein the labeled-dephosphocoenzyme A analog can be used to identify proteins, characterize proteins, inhibit proteins, activate proteins, or examine the structure of proteins.
17. The method of claim 1 , wherein the labeled-coenzyme A analog and related isomers of the labeled-coenzyme A analog can be used to identify proteins, characterize proteins, inhibit proteins, activate proteins, or examine the structure of proteins.