Reversible natural product glycosyltransferase-catalyzed reactions, compounds and related methods
The present invention relates to methods of use of glycosyltransferases and related compounds. The invention exploits the reversibility of glycosyltransferases to generate new sugars, unnatural biomolecules and numerous one-pot reactions for generation of new biomolecules having varied backbones such as enediynes, vancomycins, bleomycins, anthracyclines, macrolides, pluramycins, aureolic acids, indolocarbazoles, aminglycosides, glycopeptides, polyenes, coumarins, benzoisochromanequinones, calicheamicins, erythromycin, avermectins, ivermectins, angucyclines, cardiac glycosides, steroids or flavinoids. In preferred embodiments, the invention specifically relates to biosynthesis of anticancer (the enediyne calicheamicin, CLM), anthelmintic agents (the macrolides avermectin, ivermectin and erythromycin) and antibiotic (the glycopeptide vancomycin, VCM) natural product-based drugs developed by reversible, bidirectional glycosyltransferase-catalyzed reactions.
1. A method of synthesizing a sugar moiety A-nucleotide diphosphate, in-situ, from a biomolecule having a sugar moiety A, the method comprising the steps of:
(a) incubating the biomolecule having the sugar moiety A with a nucleotide diphosphate in the presence of a glycosyltransferase capable of glycosylating the biomolecule that is selected from the group consisting of Micromonospora echinospora calicheamicin glycosyltransferase 1 (CalG1), Micromonospora echinospora calicheamicin glycosyltransferase 2 (CalG2), Micromonospora echinospora calicheamicin glycosyltransferase 3 (CalG3), Micromonospora echinospora calicheamicin glycosyltransferase 4 (CalG4), Amycolatopsis orientalis glycosyltransferase D (GtfD), Amycolatopsis orientalis glycosyltransferase E (GtfE), Saccharopolyspora erythraea erythromycin glycosyltransferase BV (EryBV), and Streptomyces avermitilis avermectin glycosyltransferase (AveBI), wherein the sugar moiety A in the biomolecule is excised from the biomolecule, thereby generating the sugar moiety A-nucleoside diphosphate and a biomolecule aglycon; and
(b) isolating the sugar moiety A-nucleotide diphosphate from step (a),
wherein the biomolecule is an enediyne, a vancomycin, a bleomycin, an anthracycline, a macrolide, a pluramycin, an aureolic acid, an indolocarbazole, an aminglycoside, a glycopeptide, a polyene, a coumarin, a benzoisochromanequinone, a calicheamicin, an erythromycin, an avermectin, an ivermectin, an angucycline, a cardiac glycoside, a steroid or a flavinoid.
2. The method of claim 1 , wherein the biomolecule having the sugar moiety A is an enediyne, a vancomycin, a calicheamicin, an erythromycin, an avermectin or an ivermectin.
3. The method of claim 1 , wherein the sugar moiety A-nucleotide diphosphate is a UDP sugar or a TDP sugar.
4. The method of claim 3 , wherein the TDP sugar is selected from TDP-α-D-glucose, TDP-β-L-rhamnose, TDP-O-methylrhamnose, TDP-6-azidoglucose, TDP-β-L-vancosamine, TDP-β-L-olendrose and TDP-β-L-mycarose.
5. The method of claim 1 , wherein said synthesis is reversible, and whereby incubating the sugar moiety A-nucleotide diphosphate and the biomolecule aglycon in the presence of the glycosyltransferase capable of glycosylating the biomolecule provides the biomolecule having the sugar moiety A.