Combinatorial polyketide libraries produced using a modular PKS gene cluster as scaffold

Abstract

Combinatorial libraries of polyketides can be obtained by suitable manipulation of a host modular polyketide synthase gene cluster such as that which encodes the PKS for erythromycin. The combinatorial library is useful as a source of pharmaceutically active compounds. In addition, novel polyketides and antibiotics are prepared using this method.

What is claimed is:  
     
         1 . A process for preparing a compound of formula 1:  
       
         
           
           
               
               
           
         
       
       and to pharmaceutically acceptable salts thereof, wherein: 
 R 1  is an alpha-branched C 3 -C 8  alkyl, alkenyl, alkynyl, alkoxyalkyl or alkylthioalkyl group any of which may optionally be substituted by one or more hydroxyl groups; a C 5 -C 8  cycloalkylalkyl group wherein the alkyl group is an alpha-branched C 2 -C 5  alkyl group; a C 3 -C 8  cycloalkyl or C 5 -C 8  cycloalkenyl group, either of which may optionally be substituted by methyl or one or more hydroxyl or one or more C 1 -C 4  alkyl groups or halo atoms; or a 3 to 6 membered oxygen or sulphur containing heterocyclic ring which may be saturated, or fully or partially unsaturated and which may optionally be substituted by one or more C 1 -C 4  alkyl groups or halo atoms;  
 or R 1  is phenyl which may be optionally substituted with at least one substituent selected from C 1 -C 4  alkyl, C 1 -C 4  alkoxy and C 1 -C 4  alkylthio groups, halogen atoms, hydroxyl groups, trifluoromethyl, and cyano; or R 1  may be with formula (a) as shown below:  
                     
 wherein X is, S or —CH 2 —, a, b, c, and d are each independently 0-2 and a+b+c+d≦5;  
 R 2  is H or OH; R 3 -R 5  are each independently H, CH 3 , or CH 2 CH 3 ; R 6  is H or OH; and R 7  is H, CH 3 , or CH 3 CH 2 ; R 8  is H or desosamine; R 9  is H, CH 3 , or CH 2 CH 3 ; R 10  is OH, mycarose (R 13  is H), or cladinose (R 13  is CH 3 ), R 11  is H; or R 10 =R 11 =0; and R 12  is H, CH 3 , or CH 2 CH 3 ; or any of the above defined compounds modified by replacing one or more —CHOH or —CHOR groups by a keto group, which comprises fermenting an organism capable of producing erythromycin in the presence of a carboxylic acid of the formula R 1  CO 2 H wherein R 1  is as defined hereinabove, or a salt, ester or amide thereof or oxidative precursor thereof, and isolating the compound of formula 1.  
 
     
     
         2 . A process of  claim 1 , wherein the organism is  Saccharopolyspora erythraea  and which may optionally contain an effectively integrated plasmid capable of directing the biosynthesis of compounds of formula 1; said plasmid may optionally contain a Type II PKS promoter/activator gene.  
     
     
         3 . A process of  claim 2 , wherein the organism  Saccharopolyspora erythraea  is selected from strains NRRL 2338, 18643 or 21484 which may optionally contain an effectively integrated plasmid capable of directing the biosynthesis of compounds of formula 1; said plasmid may optionally contain the act promoter and its cognate activator gene act II -orf 4 .  
     
     
         4 . A process of  claim 3 , wherein the optionally effectively integrated plasmids is pAVLD, pIG1, pND30, pCJR26, pCJR49, pC-AT12, pc-ATX or other similar constructs.  
     
     
         5 . A process of  claim 3 , wherein the organism is  S. erythraea  ERMD1 , S. erythraea  NRRL 2338/pIG1 , S. erythraea  NRRL 2338/pND30, or other similar tranformants.  
     
     
         6 . A process for preparing a compound of formula 2:  
       
         
           
           
               
               
           
         
       
       and to pharmaceutically acceptable salts thereof, wherein: 
 R 1  is H, C 1 -C 8  alkyl, C 2 -C 8  alkenyl, C 2 -C 8  alkynyl, alkoxyalkyl or alkylthioalkyl containing from 1 to 6 carbon atoms in each alkyl or alkoxy group wherein any of said alkyl, alkoxy, alkenyl or alkynyl groups may be substituted by one or more hydroxyl groups or by one or more halo atoms; or a C 3 -C 8  cycloalkyl or C 5 -C 8  cycloalkenyl either of which may be optionally substituted by methyl or one or more C 1 -C 4  alkyl groups or halo atoms; or a 3 to 6 membered oxygen or sulphur containing heterocyclic ring which may be saturated or fully or partially unsaturated and which may optionally be substituted by one or more C 1 -C 4  alkyl groups or halo atoms; or a group of the formula SR 14  wherein R 14  is C 1 -C 8  alkyl, C 2 -C 8  alkenyl, C 2 -C 8  alkynyl, C 3 -C 8  cycloalkyl, C 5 -C 8  cycloalkenyl, phenyl or substituted phenyl wherein the substituent is C 1 -C 4  alkyl, C 1 -C 4  alkoxy or halo, or a 3-6 membered oxygen or sulphurcontaining heterocyclic ring which may be saturated, or fully or partially unsaturated and which may optionally be substituted by one or more C 1 -C 4  alkyl groups or halo atoms;  
 R 2  is H or OH; R 3 -R 5  are each independently H, CH 3 , or CH 2 CH 3 ; R 6  is H or OH; and R 7  is H, CH 3 , or CH 3 CH 2 ; R 8  is H or desosamine; R 9  is H, CH 3 , or CH 2 CH 3 ; R 10  is OH, mycarose (R 13  is H), or cladinose (R 13  is CH 3 ), R 11 , is H; or R 10 =R 11 =O; and R 12  is H, CH 3 , or CH 2 CH 3 , with the proviso that when R 3 -R 5  are CH 3 , R 7  is CH 3 , R 9  is CH 3  and R 12  is CH 3 , then R 1  is not H or C 1  alkyl; or any of the above defined compounds modified by replacing one or more —CHOH or —CHOR groups by a keto group, which comprises fermenting an organism capable of producing erythromycin in the presence of a carboxylic acid of the formula R 1  CO 2 H wherein R 1  is as defined hereinabove, or a salt, ester or amide thereof or oxidative precursor thereof, and isolating the compound of formula 2.  
 
     
     
         7 . A process of  claim 6 , wherein the organism is  Saccharopolyspora erythraea  and which may optionally contain an effectively integrated plasmid capable of directing the biosynthesis of compounds of formula 2; said plasmid may optionally contain a Type II PKS promoter/activator gene.  
     
     
         8 . A process of  claim 7 , wherein the organism  Saccharopolyspora erythraea  is selected from strains NRRL 2338, 18643 or 21484 which may optionally contain an effectively integrated plasmid capable of directing the biosynthesis of compounds of formula 2 said plasmid may optionally contain the act 1  promoter and its cognate activator gene act II -orf 4 .  
     
     
         9 . A process of  claim 8 , wherein the optionally effectively integrated plasmids is pAVLD, pIG1, pND30, pCJR26, pCJR49, pC-ATI2, pc-ATX or other similar constructs.  
     
     
         10 . A process of  claim 8 , wherein the organism is  S. erythraea  ERMD1 , S. erythraea  NRRL 2338/pIG1 , S. erythraea  NRRL 2338/pND30, or other similar tranformants.