Glycorandomization and the production of novel erythronolide and coumarin analogs

Abstract

The present invention provides combinatorial methods for rapidly generating a diverse library of glycorandomized structures, comprising incubating one or more aglycons and a pool of NDP-sugars in the presence of a glycosyltransferase. The glycosyltransferase may be one that is associated with or involved in production of natural secondary metabolites, or one which is putatively associated with or involved in production of natural secondary metabolites. The glycosyltransferase may show significant flexibility with respect to its NDP-sugar donors and/or its aglycons. NDP-sugar donors may be commercially available, or may be produced by utilizing mutant or wild type nucleotidyltransferases significant flexibility with respect to their substrates.

1 . A method of preparing a glycosylated compound comprising steps of: 
 (a) preparing a nucleotide sugar by combining a nucleotide triphosphate (NTP) and a sugar phosphate in the presence of an isolated nucleotidylyltransferase mutated at an amino acid position relative to an amino acid position selected from the group consisting of V173, G147, W224, N112, G175, D111, E162, T201, I200, E199, R195, L89, L109, Y146 and Y177 of  Salmonella enterica  LT2 rmlA-encoded alpha-D-glucopyranosyl phosphate thymidylyltransferase (Ep);    (b) combining the nucleotide sugar prepared in step (a) with a glycosyltransferase and a moiety capable of being glycosylated to produce a glycosylated compound; and    (c) recovering the glycosylated compound.    
     
     
         2 . The method according to  claim 1  wherein said isolated nucleotidylyltransferase is a glucose-1-phosphate nucleotidylyltransferase.  
     
     
         3 . The method according to  claim 1  wherein said isolated nucleotidylyltransferase is a thymidylyltransferase.  
     
     
         4 . The method according to  claim 1  wherein said isolated nucleotidylyltransferase is Ep.  
     
     
         5 . The method according to  claim 1  wherein said isolated nucleotidylyltransferase possesses a different substrate specificity for sugar phosphates relative to a corresponding non-mutant nucleotidylyltransferase.  
     
     
         6 . The method according to  claim 1  wherein the method is carried out in vitro.  
     
     
         7 . The method of  claim 1 , further wherein the nucleotide sugar prepared in step (a) is selected from the group consisting of Thymidine 5′-(α-D-glucopyranosyl diphosphate); Uridine 5′-(α-D-glucopyranosyl diphosphate); Thymidine 5′-(2-deoxy-α-D-glucopyranosyl diphosphate); Uridine 5′-(2-deoxy-α-D-glucopyranosyl diphosphate); Thymidine 5′-(3-deoxy-α-D-glucopyranosyl diphosphate); Uridine 5′-(3-deoxy-α-D-glucopyranosyl diphosphate); Thymidine 5′-(4-deoxy-α-D-glucopyranosyl diphosphate); Uridine 5′-(4-deoxy-α-D-glucopyranosyl diphosphate); Thymidine 5′-(6-deoxy-α-D-glucopyranosyl diphosphate); Uridine 5′-(6-deoxy-α-D-glucopyranosyl diphosphate); Thymidine 5′-(α-D-mannopyranosyl diphosphate); Uridine 5′-(α-D-mannopyranosyl diphosphate); Thymidine 5′-(α-D-galactopyranosyl diphosphate); Uridine 5′-(α-D-galactopyranosyl diphosphate); Thymidine 5′-(α-D-allopyranosyl diphosphate); Thymidine 5′-(α-D-altropyranosyl diphosphate); Uridine 5′-(α-D-allopyrano-syl diphosphate); Uridine 5′-(α-D-altropyranosyl diphosphate); Thymidine 5′-(α-D-gulopyranosyl diphosphate); Uridine 5′-(α-D-gulopyranosyl diphosphate); Thymidine 5′-(α-D-idopyranosyl diphosphate); Uridine 5′-(α-D-idopyranos-yl diphosphate); Thymidine 5′-(α-D-talopyranosyl diphosphate); Uridine 5′-(α-D-talopyranosyl diphosphate); Thymidine 5′-(6-amino-6-deoxy-α-D-glucopyranosyl diphosphate); Uridine 5′-(6-amino-6-deoxy-α-D-glucopyranosyl diphosphate); Thymidine 5′-(4-amino-4-deoxy-α-D-glucopyranosyl diphosphate); Uridine 5′-(4-amino-4-deoxy-α-D-glucopyranosyl diphosphate); Thymidine 5′-(3-amino-3-deoxy-α-D-glucopyranosyl diphosphate); Uridine 5′-(3-amino-3-deoxy-α-D-glucopyranosyl diphosphate); Thymidine 5′-(2-amino-2-deoxy-α-D-glucopyranosyl diphosphate); Uridine 5′-(2-amino-2-deoxy-α-D-glucopyranosyl diphosphate); Thymidine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyl diphosphate); Uridine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyl diphosphate); Thymidine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyl diphosphate); Uridine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyl diphosphate); Thymidine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyl diphosphate); Uridine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyl diphosphate); Thymidine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyl diphosphate); Uridine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyl diphosphate); Thymidine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyl diphosphate); Uridine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyl diphosphate); Thymidine 5′-(α-D-glucopyran-6-uronic acid diphosphate); Uridine 5′-(α-D-glucopyran-6-uronic acid diphosphate); Thymidine 5′-(α-D-arabinopyranosyl diphosphate); Uridine 5′-(α-D-arabinopyranosyl diphosphate);  
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         8 . The method according to  claim 1  wherein said method provides a diverse population of glycosylated compounds in a single reaction vessel.  
     
     
         9 . The method of  claim 1  further wherein the moiety capable of being glycosylated is selected from the group consisting of natural and synthetic metabolites, pyran rings, furan rings, enediynes, anthracyclines, angucyclines, aureolic acids, orthosomycins, macrolides, aminoglycosides, non-ribosomal peptides, polyenes, steroids, lipids, indolocarbazoles, bleomycins, amicetins, benzoisochromanequinones, coumarins, polyketides, pluramycins, aminoglycosides, oligosaccharides, peptides, and proteins.  
     
     
         10 . The method of  claim 1  further wherein the moiety capable of being glycosylated is selected from the group consisting of aglycons of bioactive anthracyclines, angucyclines, nonribosomal peptides, macrolides, enediynes, indolocarbazoles, pluramycins, aurelolic acids, orthosomycins, aminoglycosides, coumarins, bleomycins, amicetins, polyenes, benzoisochromanequinones, and angucyclines.  
     
     
         11 . The method of  claim 1  further wherein the glycosyltransferase is selected from the group consisting of CalB, CalE, CalN, CalU, Gra orf14, Gra orf5, LanGT1, LanGT2, LanGT3, LanGT4, MtmGI, MtmGII, MtmGTIII, MtmGTIV, NovM, RhlB, Rif orf 7, SnogD, SnogE, SnogZ, UrdGT1a, UrdGT1b, UrdGT1c, UrdGT2, AknK, AknS, DesVII, DnrS, OleG1, OleG2, TylCV, TylMII, TylN, DauH, DnrH, EryBV, EryCIII, Ngt, BgtA, BgtB, BgtC, GftA, GftB, GftC, GftD, GftE, Gp1-1, Gp1-2, RtfA, AveBI, BlmE, BlmF, MgtA, NysD1, OleD, OleI, SpcF, SpcG, StrH, Ugt51B1, Ugt51C1, UGT52, UgtA, UgtB, UgtC, UgtD and homologs thereof.  
     
     
         12 . The method of  claim 1  wherein the glycosylated compound prepared according to  claim 1  is subjected to repeated cycles of said method thereby yielding a multiply-glycosylated compound.  
     
     
         13 . A method of preparing a glycosylated compound comprising steps of: 
 (a) preparing a nucleotide sugar by combining a nucleotide triphosphate (NTP) and a sugar phosphate in the presence of an isolated nucleotidylyltransferase mutated at an active site amino acid position wherein said isolated nucleotidylyltransferase possesses a different substrate specificity for sugar phosphates relative to a corresponding non-mutant nucleotidylyltransferase;    (b) combining the nucleotide sugar prepared in step (a) with a glycosyltransferase and a moiety capable of being glycosylated to produce a glycosylated compound; and    (c) recovering the glycosylated compound.    
     
     
         14 . The method according to  claim 13  wherein said isolated nucleotidylyltransferase is mutated at an active site amino acid position relative to an amino acid position selected from the group consisting of V173, G147, W224, N112, G175, D1, E162, T201, 1200, E199, R195, L89, L109, Y146 and Y177 of  Salmonella enterica  LT2 rmlA-encoded alpha-D-glucopyranosyl phosphate thymidylyltransferase (Ep).  
     
     
         15 . The method according to  claim 13  wherein said isolated nucleotidylyltransferase is a glucose-1-phosphate nucleotidylyltransferase.  
     
     
         16 . The method according to  claim 13  wherein said isolated nucleotidylyltransferase is a thymidylyltransferase.  
     
     
         17 . The method according to  claim 13  wherein said isolated nucleotidylyltransferase is Ep.  
     
     
         18 . The method according to  claim 13  wherein the method is carried out in vitro.  
     
     
         19 . The method according to  claim 13  wherein said method provides a diverse population of glycosylated compounds in a single reaction vessel.  
     
     
         20 . The method according to  claim 13  wherein the glycosylated compound prepared according to  claim 13  is subjected to repeated cycles of said method thereby yielding a multiply-glycosylated compound.