Glycorandomization and Production of Novel Vancomycin 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 comprising incubating at least one moiety capable of being glycosylated and at least one thymidine or uridine nucleotide diphosphosugar comprising a sugar structure selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       in the presence of at least one first glycosyltransferase wherein at least one glycosylated compound is produced. 
     
     
         2 . A method according to  claim 1 , wherein the incubation is carried out in vitro. 
     
     
         3 . A method according to  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, proteins, hybrids consisting of one or more these components, analogs and bioactive aglycons thereof 
     
     
         4 . A method of  claim 1 , further wherein the moiety capable of being glycosylated is selected from the group consisting of vancomycin, teicoplannin, analogs, hybrids, and active aglycons thereof. 
     
     
         5 . A method of  claim 1 , further wherein at least one of the at least one first glycosyltransferase is selected from the group consisting of CalB, CalE, CalN, CalU, Gra orfl4, 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, TyIN, DauH, DnrH, EryBV, EryCIII, Ngt, BgtA, BgtB, BgtC, GftA, GftB, GftC, GftD, GftE, Gpl-1, Gpl-2, RtfA, AveBI, BlmE, BlmF, MgtA, NysD1, OleD, OleI, SpcF, SpcG, StrH, Ugt51B1, Ugt51C1, UGT52, UgtA, UgtB, UgtC, UgtD and homologs thereof. 
     
     
         6 . A method according to  claim 1 , further comprising incubating the at least one glycosylated compound with at least one second nucleotide diphosphosugar in the presence of at least one second glycosyltransferase to produce at least one twice-glycosylated compound having at least a first and a second glycosyl attachment. 
     
     
         7 . A method comprising incubating at least one moiety capable of being glycosylated and at least one thymidine or uridine nucleotide diphosphosugar comprising a sugar structure selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       in the presence of at least one first glycosyltransferase, wherein at least one glycosylated compound is produced. 
     
     
         8 . A method according to  claim 7 , 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, proteins, hybrids consisting of one or more these components, analogs and bioactive aglycons thereof. 
     
     
         9 . A method of  claim 7 , further wherein the moiety capable of being glycosylated is selected from the group consisting of vancomycin, teicoplannin, analogs, hybrids, and active aglycons thereof. 
     
     
         10 . A method of  claim 7 , further wherein at least one of the at least one first glycosyltransferase is selected from the group consisting of CalB, CalE, CalN, CalU, Gra orfl4, 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, Gpl-1, Gpl-2, RtfA, AveBI, BlmE, BlmF, MgtA, NysD1, OleD, Olel, SpcF, SpcG, StrH, Ugt51B1, Ugt51C1, UGT52, UgtA, UgtB, UgtC, UgtD and homologs thereof. 
     
     
         11 . A method according to  claim 7 , further comprising incubating the at least one glycosylated compound with at least one second nucleotide diphosphosugar in the presence of at least one second glycosyltransferase to produce at least one twice-glycosylated compound having at least a first and a second glycosyl attachment. 
     
     
         12 . A method comprising subjecting at least one glycosylated compound produced according to the method of  claim 8  to repeated cycles of incubation with at least one nucleotide diphosphosugar in the presence of at least one glycosyltransferase until a population of multiply-glycosylated compounds of the desired type and number of compounds is achieved. 
     
     
         13 . A method comprising incubating at least one chemoselectively ligatable moiety comprising a structure selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       and at least one glycosylated compound wherein at least one chemoselectively ligated compound is produced. 
     
     
         14 . A method according to  claim 13 , further, wherein the glycosylated compound is initially produced by incubating at least one moiety capable of being glycosylated and at least one thymidine or uridine nucleotide diphosphosugar comprising a sugar structure selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       in the presence of at least one first glycosyltransferase wherein the at least glycosylated compound is produced. 
     
     
         15 . A method comprising incubating at least one glycosylated compound produced by the method of  claim 14  that is capable of being glycosylated with and at least one second nucleotide diphosphosugar in the presence of at least one second glycosyltransferase to produce at least one twice-glycosylated compound having at least a first and a second glycosyl attachment. 
     
     
         16 . A method according to  claim 14 , 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, proteins, hybrids consisting of one or more these components, analogs and bioactive aglycons thereof. 
     
     
         17 . A method of  claim 14 , further wherein the moiety capable of being glycosylated is selected from the group consisting of vancomycin, teicoplannin, analogs, hybrids, and active aglycons thereof. 
     
     
         18 . A method according to  claim 14 , further comprising subjecting at least one glycosylated compound to repeated cycles of incubation with at least one nucleotide diphosphosugar in the presence of at least one glycosyltransferase until a population of multiply-glycosylated compounds of the desired type and number of compounds is achieved. 
     
     
         19 . A vancomycin derivative designated by the formula: 
       
         
           
           
               
               
           
         
       
       wherein R is 
       
         
           
           
               
               
           
         
       
     
     
         20 . A vancomycin derivative designated by the formula 
       
         
           
           
               
               
           
         
       
       wherein R is