Cell-free preparation of carbapenems

Abstract

Provided herein are cell-free systems for generating carbapenems, e.g., a compound of the Formula (I): or salts thereof; wherein , R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are defined herein. Also provided are pharmaceutical compositions comprising a compound generated by the inventive cell-free system, and use of these compounds and compositions for the treatment of bacterial infections.

1 - 32 . (canceled) 
     
     
         33 . One or more bacterial cells, wherein each cell expresses one or more enzymes selected from the group consisting of:
 a. ProA and ProB, or γ-glutamyl kinase-GP-reductase multi-enzyme complex;   b. Glk;   c. Pgi;   d. PfkA;   e. Fba;   f. TpiA;   g. GapA;   h. Pgk;   i. GpmA;   j. Eno;   k. PykA;   l. PykF;   m. AceE;   n. AceF;   o. acetyl-CoA acetyltransferase;   p. acetyl-CoA carboxylase and malonyl-CoA reductase, or beta-alanine transaminase;   q. S-hydratase and 3-hydroxyacyl-CoA dehydrogenase, or beta-oxidation multi-enzyme;   r. crotonyl-CoA reductase;   s. threonine aldolase;   t. carboxymethyl-Pro synthase;   u. carbapenam synthetase, or beta-lactam synthetase;   v. carbapenem synthase;   
       and isozymes thereof;
 wherein the one or more cells, in combination, express all enzymes (a)-(v), or isozyme(s) thereof; and 
 wherein a lysate of the one or more cells is capable, upon contact with glucose and glycine, of generating a carbapenem of Formula (I-a): 
 
       
         
           
           
               
               
           
         
       
       or a salt or tautomer thereof, 
       wherein:
 the dashed line   represents a double bond; 
 R 1  is hydrogen; 
 R 2  is —CH 2 CH 3 ; 
 R 3  is hydrogen; 
 R 4  is hydrogen; 
 R 5  is —OH; and 
 R 7a  is hydrogen. 
 
     
     
         34 . One or more bacterial cells, wherein each cell expresses one or more enzymes selected from the group consisting of:
 a. ProA and ProB, or γ-glutamyl kinase-GP-reductase multi-enzyme complex;   b. Glk;   c. Pgi;   d. PfkA;   e. Fba;   f. TpiA;   g. GapA;   h. Pgk;   i. GpmA;   j. Eno;   k. PykA;   l. PykF;   m. AceE;   n. AceF;   o. acetyl-CoA carboxylase and malonyl-CoA reductase, or beta-alanine transaminase;   p. threonine aldolase;   q. carboxymethyl-Pro synthase;   r. carbapenam synthetase, or beta-lactam synthetase; and   s. carbapenem synthase;
 and isozymes thereof; 
   wherein the one or more cells, in combination, express all enzymes (a)-(s), or isozyme(s) thereof; and   
       wherein a lysate of the one or more cells is capable, upon contact with glucose and glycine, of generating a carbapenem of Formula (I-a): 
       
         
           
           
               
               
           
         
       
       or a salt or tautomer thereof, 
       wherein:
 the dashed line   represents a double bond; 
 R 1  is hydrogen; 
 R 2  is hydrogen; 
 R 3  is hydrogen; 
 R 4  is hydrogen; 
 R 5  is —OH; and 
 R 7a  is hydrogen. 
 
     
     
         35 . One or more bacterial cells, wherein each cell expresses one or more enzymes selected from the group consisting of:
 a. ProA and ProB, or γ-glutamyl kinase-GP-reductase multi-enzyme complex;   b. Glk;   c. Pgi;   d. PfkA;   e. Fba;   f. TpiA;   g. GapA;   h. Pgk;   i. GpmA;   j. Eno;   k. PykA;   l. PykF;   m. AceE;   n. AceF;   o. acetyl-CoA acetyltransferase;   p. S-hydratase and 3-hydroxyacyl-CoA dehydrogenase, or beta-oxidation multi-enzyme;   q. crotonyl-CoA reductase;   r. carboxymethyl-Pro synthase;   s. carbapenam synthetase, or beta-lactam synthetase;   t. carbapenem synthase;   
       and isozymes thereof;
 wherein the one or more cells, in combination, express all enzymes (a)-(t), or isozyme(s) thereof; and 
 wherein a lysate of the one or more cells is capable, upon contact with glucose and an optionally substituted glutamate of the formula (i): 
 
       
         
           
           
               
               
           
         
       
       or salt thereof,
 of generating a carbapenem of Formula (I-a): 
 
       
         
           
           
               
               
           
         
       
       or a salt or tautomer thereof, 
       wherein:
 the dashed line   represents a double bond; 
 R 1  is hydrogen; 
 R 2  s —CH 2 CH 3 ; 
 each instance of R 3  and R 4  is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl; 
 R 5  is hydrogen or —OR 8 ; 
 each instance of R 8  is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —C(═O)R 8a , —C(═O)OR 8a , —C(═O)SR 8a , —C(═O)N(R 8b ) 2 , —C(═NR 8b )R 8a , —C(═NR 8b )OR 8a , —C(═NR 8b )SR 8a , —C(═NR 8b )N(R 8b ) 2 , —C(═S)R 8a , —C(═S)OR 8a , —C(═S)SR 8a , —C(═S)N(R 8b ) 2 , —C(═O)NR 8b SO 2 R 8a , —S(═O)R 8a , —SO 2 R 8a , —SO 2 N(R 8a ) 2 , —Si(R 8a ) 3 , —P(O)(R 8a ) 2 , —P(═O)(OR 8a ) 2 , —P(═O)(R 8a )(OR 8a ), —P(═O)(R 8a )(N(R 8b ) 2 ), —P(═O)(N(R 8b ) 2 ) 2 , —P(═O) 2 R 8a , —P(═O) 2OR   8a , —P(═O) 2 N(R 8b ) 2 , —B(R 8a ) 2 , —B(OR 8a ) 2 , and —BR 8a (OR 8a ); 
 each instance of R 8a  is independently selected from the group consisting of optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, if attached to an oxygen atom an oxygen protecting group, and if attached to a sulfur atom a sulfur protecting group; 
 each instance of R 8b  is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and a nitrogen protecting group, or two R 8b  are joined to form an optionally substituted heterocyclic ring; 
 or two R 8a  groups or an R 8a  and R 8b  group are joined to form an optionally substituted heterocyclic ring; and 
 R 7a  is hydrogen. 
 
     
     
         36 . One or more bacterial cells, wherein each cell expresses one or more enzymes selected from the group consisting of:
 a. ProA and ProB, or γ-glutamyl kinase-GP-reductase multi-enzyme complex;   b. Glk;   c. Pgi;   d. PfkA;   e. Fba;   f. TpiA;   g. GapA;   h. Pgk;   i. GpmA;   j. Eno;   k. PykA;   l. PykF;   m. AceE;   n. AceF;   o. acetyl-CoA carboxylase;   p. carboxymethyl-Pro synthase;   q. carbapenam synthetase, or beta-lactam synthetase;   r. carbapenem synthase;   
       and isozymes thereof;
 wherein the one or more cells, in combination, express all enzymes (a)-(r), or isozyme(s) thereof; and 
 wherein a lysate of the one or more cells is capable, upon contact with glucose and an optionally substituted glutamate of the formula (i): 
 
       
         
           
           
               
               
           
         
       
       or salt thereof, —of generating a carbapenem of Formula (I-a): 
       
         
           
           
               
               
           
         
       
       or a salt or tautomer thereof, 
       wherein:
 the dashed line   represents a double bond; 
 R 1  is hydrogen; 
 R 2  is hydrogen; 
 each instance of R 3  and R 4  is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl; 
 R 5  is hydrogen or —OR 8 ; 
 each instance of R 8  is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —C(═O)R 8a , —C(═O)OR 8a , —C(═O)SR 8a , —C(═O)N(R 8b ) 2 , —C(═NR 8b )R 8a , —C(═NR 8b )OR 8a , —C(═NR 8b )SR 8a , —C(═NR 8b )N(R 8b ) 2 , —C(═S)R 8a , —C(═S)OR 8a , —C(═S)SR 8a , —C(═S)N(R 8b ) 2 , —C(═O)NR 8b SO 2 R 8a , —S(═O)R 8a , —SO 2 R 8a , —SO 2 N(R 8a ) 2 , —Si(R 8a ) 3 , —P(═O)(R 8a ) 2 , —P(═O)(OR 8a ) 2 , —P(═O)(R 8a )(OR 8a ), —P(═O)(R 8a )(N(R 8b ) 2 ), —P(═O)(N(R 8b ) 2 ) 2 , —P(═O) 2 R 8a , —P(═O) 2OR   8a , —P(═O) 2 N(R 8b ) 2 , —B(R 8a ) 2 , —B(OR 8a ) 2 , and —BR 8a (OR 8a ); 
 each instance of R 8a  is independently selected from the group consisting of optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, if attached to an oxygen atom an oxygen protecting group, and if attached to a sulfur atom a sulfur protecting group; 
 each instance of R 8b  is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heteroalkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and a nitrogen protecting group, or two R 8b  are joined to form an optionally substituted heterocyclic ring; 
 or two R 8a  groups or an R 8a  and R 8b  group are joined to form an optionally substituted heterocyclic ring; and 
 R 7a  is hydrogen. 
 
     
     
         37 . The one or more bacterial cells of  claim 33 , wherein one or more enzymes selected from the group consisting of carboxymethyl-Pro synthase, carbapenam synthetase, beta-lactam synthetase, carbapenem synthase, and isozymes thereof, are sequestered in the periplasmic space of the one or more bacterial cells. 
     
     
         38 . The one or more bacterial cells of  claim 34 , wherein one or more enzymes selected from the group consisting of carboxymethyl-Pro synthase, carbapenam synthetase, beta-lactam synthetase, carbapenem synthase, and isozymes thereof, are sequestered in the periplasmic space of the one or more bacterial cells. 
     
     
         39 . The one or more bacterial cells of  claim 35 , wherein one or more enzymes selected from the group consisting of carboxymethyl-Pro synthase, carbapenam synthetase, beta-lactam synthetase, carbapenem synthase, and isozymes thereof, are sequestered in the periplasmic space of the one or more bacterial cells. 
     
     
         40 . The one or more bacterial cells of  claim 36 , wherein one or more enzymes selected from the group consisting of carboxymethyl-Pro synthase, carbapenam synthetase, beta-lactam synthetase, carbapenem synthase, and isozymes thereof, are sequestered in the periplasmic space of the one or more bacterial cells. 
     
     
         41 . The one or more bacterial cells of  claim 35 , wherein R 3  and R 4  are independently hydrogen or —CH 3 . 
     
     
         42 . The one or more bacterial cells of  claim 35 , wherein R 5  is hydrogen. 
     
     
         43 . The one or more bacterial cells of  claim 35 , wherein R 5  is —OH. 
     
     
         44 . The one or more bacterial cells of  claim 35 , wherein R 3  is —CH 3 , R 4  is hydrogen, and R 5  is —OH. 
     
     
         45 . The one or more bacterial cells of  claim 35 , wherein R 3  is —CH 3 , R 4  is hydrogen, and R 5  is hydrogen. 
     
     
         46 . The one or more bacterial cells of  claim 36 , wherein R 3  and R 4  are independently hydrogen or —CH 3 . 
     
     
         47 . The one or more bacterial cells of  claim 36 , wherein R 5  is hydrogen. 
     
     
         48 . The one or more bacterial cells of  claim 36 , wherein R 5  is —OH. 
     
     
         49 . The one or more bacterial cells of  claim 36 , wherein R 3  is —CH 3 , R 4  is hydrogen, and R 5  is —OH. 
     
     
         50 . The one or more bacterial cells of  claim 36 , wherein R 3  is —CH 3 , R 4  is hydrogen, and R 5  is hydrogen. 
     
     
         51 . The one or more bacterial cells of  claim 35 , wherein R 5  is —OR 8 , and R 8  is optionally substituted alkyl, optionally substituted heteroalkyl, or optionally substituted heterocyclyl. 
     
     
         52 . The one or more bacterial cells of  claim 36 , wherein R 5  is —OR 8 , and R 8  is optionally substituted alkyl, optionally substituted heteroalkyl, or optionally substituted heterocyclyl. 
     
     
         53 . A cell lysate of the one or more bacterial cells of  claim 33 . 
     
     
         54 . A cell lysate of the one or more bacterial cells of  claim 34 . 
     
     
         55 . A cell lysate of the one or more bacterial cells of  claim 35 . 
     
     
         56 . A cell lysate of the one or more bacterial cells of  claim 36 .