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Artificial Biocatalytic Linear Cascades to Access Artificial Biocatalytic Linear Cascades to Access  Hydroxy Acids, Lactones, and α- and β-Amino Acids Hydroxy Acids, Lactones, and α‐ and β‐Amino Acids  2 and Wolfgang Kroutil 1,2, ID 2 and Wolfgang Kroutil  1,2,*  * , Stefan Velikogne Joerg H. Schrittwieser 11, Stefan Velikogne  Joerg H. Schrittwieser 

ID

1

Institute of Chemistry, Organic and Bioorganic Chemistry, University of Graz, NAWI Graz, BioTechMed   Institute of Chemistry, Organic and Bioorganic Chemistry, University of Graz, NAWI Graz, BioTechMed  Graz, Heinrichstrasse 28, 8010 Graz, Austria; [email protected] Graz, Heinrichstrasse 28, 8010 Graz, Austria; joerg.schrittwieser@uni‐graz.at  2 ACIB GmbH, c/o Department of Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria; 2  ACIB GmbH, c/o Department of Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria;  [email protected] [email protected]‐graz.at  **  Correspondence: Wolfgang.Kroutil@uni‐graz.at; Tel.: +43‐316‐380‐5350  Correspondence: [email protected]; Tel.: +43-316-380-5350 1







Received: 25 April 2018; Accepted: 8 May 2018; Published: 14 May 2018  Received: 25 April 2018; Accepted: 8 May 2018; Published: 14 May 2018

Abstract: α‐, β‐, and ω‐Hydroxy acids, amino acids, and lactones represent common building blocks  Abstract: α-, β-, and ω-Hydroxy acids, amino acids, and lactones represent common building blocks and  intermediates  various  target  molecules.  This  review  summarizes  artificial published cascades  and intermediates forfor  various target molecules. This review summarizes artificial cascades published  during  the  last 10 years leading  to  these products. Renewables  as well as  compounds  during the last 10 years leading to these products. Renewables as well as compounds originating originating from fossil resources have been employed as starting material. The review provides an  from fossil resources have been employed as starting material. The review provides an inspiration inspiration  for  new  cascade  and basis may to be design the  basis  to  design  variations  of  these  cascades  for new cascade designs and designs  may be the variations of these cascades starting either starting either from alternative substrates or extending them to even more sophisticated products.  from alternative substrates or extending them to even more sophisticated products. Keywords: cascade; biocatalysis; biotransformations; hydroxy acids; lactones and α‐ and β‐amino acids  Keywords: cascade; biocatalysis; biotransformations; hydroxy acids; lactones and α- and β-amino acids  

1. Introduction  1. Introduction Artificial cascades involving biocatalysts have received increased attention recently [1–29], also  Artificial cascades involving biocatalysts have received increased attention recently [1–29], also in in combination of enzymes with metal catalysis [30–37]. In this context, a cascade is defined as the  combination of enzymes with metal catalysis [30–37]. In this context, a cascade is defined as the combination of a minimum of two chemical reaction steps in a single reaction vessel without isolation  combination of a minimum of two chemical reaction steps in a single reaction vessel without isolation of the intermediate(s) [38]. Linear cascade means that the product of one step in the cascade is the  of the intermediate(s) [38]. Linear cascade means that the product of one step in the cascade is the substrate of a second subsequent step (Scheme 1) [1ac].  substrate of a second subsequent step (Scheme 1) [1ac].

 

  Scheme 1. Linear cascade comprising n steps (n ≥ 2).  Scheme 1. Linear cascade comprising n steps (n ≥ 2).

In this review, the focus is on biocatalytic cascades of the last 10 years, whereby all steps are  In this review, the focus is on biocatalytic cascades of the last 10 years, whereby all steps are performed simultaneously, leading to hydroxy acids, lactones, and α‐ and β‐amino carboxylic acids  performed simultaneously, leading to hydroxy acids, lactones, and α- and β-amino carboxylic acids as as final products.  final products. 2. Hydroxy Acids  2. Hydroxy Acids Hydroxy acids play an important role in chemical industry: α‐Hydroxycarboxylic acids are part  Hydroxy acids play an important role in chemical industry: α-Hydroxycarboxylic acids are of  numerous  natural  products,  such  as  as depsipeptides  or oraeruginosins,  various  part of numerous natural products, such depsipeptides aeruginosins,as aswell  wellas  as of  of various Catalysts 2018, 8, x; doi: FOR PEER REVIEW    Catalysts 2018, 8, 205; doi:10.3390/catal8050205

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pharmaceuticals  [39–41].  Their  ω‐hydroxy  counterparts,  on other the  hand, other  are hand,  are  in  the  pharmaceuticals [39–41]. Their ω-hydroxy counterparts, on the used in used  the production production of a variety of chemical products and intermediates such as polyesters, resins, lubricants,  of a variety of chemical products and intermediates such as polyesters, resins, lubricants, plasticizers, plasticizers, and perfumes [42,43]. Hence, efficient synthetic entries to these compounds are highly  and perfumes [42,43]. Hence, efficient synthetic entries to these compounds are highly desired. desired. 

2.1. Preparation of ω-Hydroxycarboxylic Acids from Fatty Acids 2.1. Preparation of ω‐Hydroxycarboxylic Acids from Fatty Acids  The biocatalytic synthesis of ω-hydroxycarboxylic acids has recently been realised starting from The biocatalytic synthesis of ω‐hydroxycarboxylic acids has recently been realised starting from  ricinoleic acid or oleic acid in a three- or four-step cascade (Scheme 1) [44]. ricinoleic acid or oleic acid in a three‐ or four‐step cascade (Scheme 1) [44].  Biotransformations producing the desired hydroxy acids were performed with a conversion Biotransformations  producing  the  desired  hydroxy  acids  were  performed  with  a  conversion  ranging from 60% to 70% at 1 mM substrate concentration employing E. coli cells co-expressing ranging from 60% to 70% at 1 mM substrate concentration employing E. coli cells co‐expressing all  allthe necessary enzymes. Several other fatty acids, such as 5‐hydroxydecanoic acid, linoleic acid, and  the necessary enzymes. Several other fatty acids, such as 5-hydroxydecanoic acid, linoleic acid, and lesquerolic acid, were transformed by this cascade leading to a similar range of conversion. lesquerolic acid, were transformed by this cascade leading to a similar range of conversion. Treating  Treating 5 gram per litre olive oil with lipase generated a product mixture containing 11.3 mM oleic 5 gram per litre olive oil with lipase generated a product mixture containing 11.3 mM oleic acid and  acid and 1.2 mM palmitic acid. The subsequent biotransformation was performed as outlined in 1.2 mM palmitic acid. The subsequent biotransformation was performed as outlined in Scheme 2B,  Scheme 2B, yielding 9-hydroxynonanoic acid (10) with 60% conversion. yielding 9‐hydroxynonanoic acid (10) with 60% conversion. 

 

  Scheme 2. Cascades to ω‐hydroxycarboxylic acids. (A) Biotransformation starting from ricinoleic acid  Scheme 2. Cascades to ω-hydroxycarboxylic acids. (A) Biotransformation starting from ricinoleic (1) to yield n‐heptanoic acid (5) and 11‐hydroxyundec‐9‐enoic acid (4); (B) Four‐step cascade starting  acid (1) to yield n-heptanoic acid (5) and 11-hydroxyundec-9-enoic acid (4); (B) Four-step cascade from  oleic  producing  n‐nonanoic  acid  (11) acid and  9‐hydroxynonanoic  acid  (10).  Enzyme  starting fromacid  oleic(6) acid (6) producing n-nonanoic (11) and 9-hydroxynonanoic acid (10). abbreviations: ADH, alcohol dehydrogenase; BVMO, Baeyer–Villiger monooxygenase.   Enzyme abbreviations: ADH, alcohol dehydrogenase; BVMO, Baeyer–Villiger monooxygenase.

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In In  a a  follow-up conditionsand  andexpression  expressionprotocol  protocol were follow‐up study study [45], [45], the the biotransformation biotransformation  conditions  were  optimised for the preparation of 11-hydroxyundec-9-enoic acid (4), which may have potential optimised for the preparation of 11‐hydroxyundec‐9‐enoic acid (4), which may have potential as an as anantifungal  antifungalagent  agent[46].  [46].Employing  Employingincreased  increased concentrations ricinoleic acid mM) and the concentrations  of  of ricinoleic  acid  (1; (1; 10 10 mM)  and  the  biocatalyst (>10 g cell dry-weight per litre), a final concentration of 4.0 g/L 11-hydroxyundec-9-enoic biocatalyst (>10 g cell dry‐weight per litre), a final concentration of 4.0 g/L 11‐hydroxyundec‐9‐enoic  acid (4) was achieved. acid (4) was achieved.  2.2.2.2. Conversion of α‐Amino Acids into α‐Hydroxy Acids  Conversion of α-Amino Acids into α-Hydroxy Acids The synthesis of (R)acids has beenhas  described starting from the corresponding The  synthesis  of  and (R)‐ (S)-α-hydroxy and  (S)‐α‐hydroxy  acids  been  described  starting  from  the  L -amino acids, applying anacids,  L -amino acid deaminase (L-AAD) Proteus(Lmyxofaciens, followed corresponding  L‐amino  applying  an  L‐amino  acid  from deaminase  ‐AAD)  from  Proteus by themyxofaciens, followed by the asymmetric reduction mediated by either  asymmetric reduction mediated by either L- or D-hydroxyisocaproate dehydrogenases (HicDHs), L‐ or  D‐hydroxyisocaproate  dehydrogenases (HicDHs), derived from Lactobacillus confusus DSM 20196 and Lactobacillus paracasei  derived from Lactobacillus confusus DSM 20196 and Lactobacillus paracasei DSM 20008, respectively DSM 20008, respectively (Scheme 3) [47].  (Scheme 3) [47].

 

  Scheme  Transformation  L‐amino  acids  the  corresponding  α‐hydroxy  in  a  two‐step  Scheme 3. 3.  Transformation of Lof  -amino acids intointo  the corresponding α-hydroxy acidsacids  in a two-step cascade. cascade.  Enzyme  abbreviations:  L‐AAD,  ‐amino  acid HicDH, deaminase;  HicDH,  α‐hydroxyisocaproate  Enzyme abbreviations: L-AAD, L-amino acidLdeaminase; α-hydroxyisocaproate dehydrogenase. dehydrogenase. 

The transformation of all tested amino acids allowed conversions of 97–99% (73–85% isolated The transformation of all tested amino acids allowed conversions of 97–99% (73–85% isolated  yield) within 7 h, even with high substrate loadings (up to 200 mM; see Table 1), furnishing the yield) within 7 h, even with high substrate loadings (up to 200 mM; see Table 1), furnishing the α‐ α-hydroxy acids 14 in optically pure form (>99% ee). hydroxy acids 14 in optically pure form (>99% ee).  Table 1. Transformation of α-amino acids 12 into α-hydroxy acids 14 via a two-step cascade (Scheme 3) a . Table 1. Transformation of α‐amino acids 12 into α‐hydroxy acids 14 via a two‐step cascade (Scheme 3) a.  b (%)  b (%)ee (%)  ee (%) Substrate (mM)  Substrate Conc. (mM)  HicDH  Conv.  Substrate (mM) Substrate Conc. (mM) HicDH Conv. 12a  L‐Hic  >99 (81)  >99 (S)  100  12a 100 L -Hic >99 (81) >99 (S) 12a  D‐Hic  >99 (84)  >99 (R)  100  12a 100 D -Hic >99 (84) >99 (R) 12b  L‐Hic  >99 (83)  >99 (S)  50  12b 50 L -Hic >99 (83) >99 (S) 12b  D‐Hic  >99 (83)  >99 (R)  50  12b 50 D -Hic >99 (83) >99 (R) 12c  L‐Hic  >99 (81)  12c 100 100  L -Hic >99 (81) >99 (S) >99 (S) 12c  D‐Hic  >99 (85)  100  12c 100 D -Hic >99 (85) >99 (R) >99 (R) 12d  L ‐Hic  >99 (79)  100  12d 100 L -Hic >99 (79) >99 (S) >99 (S) 12d  D ‐Hic  >99 (82)  100  12d 100 D -Hic >99 (82) >99 (R) >99 (R) 12e  ‐Hic  >99 (77)  12e 200 200  LL-Hic >99 (77) >99 (S) >99 (S) 12e  ‐Hic  >99 (73)  12e 200 200  DD-Hic >99 (73) >99 (R) >99 (R) a Reaction conditions: Substrate L‐12a–e (50–200 mM), HCO2NH4 (3 eq.), NAD+ (1 mM), FDH (formate  a Reaction conditions: Substrate L-12a–e (50–200 mM), HCO2 NH4 (3 eq.), NAD+ (1 mM), FDH (formate dehydrogenase, 42 U/mmol), L-AAD (15–30 U/mmol), L-Hic (132 U/mmol) or D-Hic (96DU/mmol), 1 bar O2 , 21 ◦ C, dehydrogenase, 42 U/mmol),  L‐AAD (15–30 U/mmol),  L‐Hic (132 U/mmol) or  ‐Hic (96 U/mmol), 1  b 7 h.bar O Isolated yield in bparentheses. 2, 21 °C, 7 h.   Isolated yield in parentheses. 

L‐Tyrosine (12f) was also transformed at high substrate concentrations (200 mM) and on a 100  L-Tyrosine (12f) was also transformed at high substrate concentrations (200 mM) and on a 100 mg

mg toscale  to  afford  the  α‐hydroxy  acid  (S)‐14f  80%  isolated  yield  and ee >99%  ee  (Scheme  The  scale afford the α-hydroxy acid (S)-14f in 80%in  isolated yield and >99% (Scheme 4). The4).  product 14f  is  an  important  building  block  for  various active biologically  active such compounds,  such  as  14fproduct  is an important building block for various biologically compounds, as Saroglitazar (15), Saroglitazar (15), which is used as a treatment for diabetes Type II [48].  which is used as a treatment for diabetes Type II [48].

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   Scheme  4.  Scheme Multi-enzyme cascade  cascade synthesis  synthesis of  of (S)‐14f,  (S)-14f, a  a building  building block for the preparation of the Scheme 4. 4. Multi‐enzyme  Multi‐enzyme  cascade  synthesis  of  (S)‐14f,  a  building block  block for  for the  the preparation  preparation of  of the  the  L ‐AAD,  L ‐amino acid deaminase; HicDH,  antidiabetic drug Saroglitazar (15). Enzyme abbreviations:  antidiabetic drug Saroglitazar (15). Enzyme abbreviations: LL-AAD, -amino acid deaminase; HicDH, ‐AAD, LL‐amino acid deaminase; HicDH,  antidiabetic drug Saroglitazar (15). Enzyme abbreviations:  α‐hydroxyisocaproate dehydrogenase; FDH, formate dehydrogenase.  α-hydroxyisocaproate dehydrogenase; FDH, formate dehydrogenase. α‐hydroxyisocaproate dehydrogenase; FDH, formate dehydrogenase. 

In a subsequent study, an extended version of the cascade was published to access α‐hydroxy  In a subsequent study, an extended version of the cascade was published to access α-hydroxy In a subsequent study, an extended version of the cascade was published to access α‐hydroxy  acids starting from phenol derivatives and pyruvate (Scheme 5) [49].  acids starting from phenol derivatives and pyruvate (Scheme 5) [49]. acids starting from phenol derivatives and pyruvate (Scheme 5) [49]. 

  

   Scheme  Biocatalytic  cascade  for  the  of  acids  18  starting  from  phenol  Scheme 5.5.  5. Biocatalytic Biocatalytic  cascade  for synthesis the  synthesis  synthesis  of  α‐hydroxy  α‐hydroxy  18 from starting  from  phenol  Scheme cascade for the of α-hydroxy acids 18 acids  starting phenol derivatives L‐tyrosine phenol lyase;  L‐AAD,  L‐amino  derivatives 16 and pyruvate. Enzyme abbreviations: TPL,  L‐tyrosine phenol lyase;  ‐AAD,  L‐amino  derivatives 16 and pyruvate. Enzyme abbreviations: TPL,  16 and pyruvate. Enzyme abbreviations: TPL, L-tyrosine phenol lyase; L-AAD, L-aminoLacid deaminase; acid deaminase; HicDH, α‐hydroxyisocaproate dehydrogenase.  acid deaminase; HicDH, α‐hydroxyisocaproate dehydrogenase.  HicDH, α-hydroxyisocaproate dehydrogenase.

As an additional step in this new reaction setup, the amino acid is formed within the cascade  As an additional step in this new reaction setup, the amino acid is formed within the cascade  As an additional step in this new reaction setup, the amino acid is formed within the cascade employing a tyrosine phenol lyase (TPL) from Citrobacter freundii, which performs a C–C coupling  employing a tyrosine phenol lyase (TPL) from Citrobacter freundii, which performs a C–C coupling  employing a tyrosine phenol lyase (TPL) from Citrobacter freundii, which performs a C–C coupling between substituted phenols 16 and pyruvate. The oxidative deamination and subsequent reduction  between substituted phenols 16 and pyruvate. The oxidative deamination and subsequent reduction  between substituted phenols 16 and pyruvate. The oxidative deamination and subsequent reduction are done by the same enzymes as described before. However, by introducing the additional TPL into  are done by the same enzymes as described before. However, by introducing the additional TPL into  are done by the same enzymes as described before. However, by introducing the additional TPL into the cascade, it has to be performed in sequential mode since small amounts of product 18 cause an  the cascade, it has to be performed in sequential mode since small amounts of product 18 cause an  the cascade, it has to be performed in sequential mode since small amounts of product 18 cause an inhibition of step one.  inhibition of step one.  inhibition of step one. The  H)  range  mM  The model  model compound  compound phenol (16a, R =  phenol (16a, R =  H) was  was converted  converted in a  in a  range of 23–46  of 23–46  mM with  with high  high  The model compound phenol (16a, R = H) was converted in a range of 23–46 mM with high conversions (93–97%) and ee (>99%) for both enantiomers. Related phenol substrates were accepted  conversions (93–97%) and ee (>99%) for both enantiomers. Related phenol substrates were accepted  conversions (93–97%) and ee (>99%) for both enantiomers. Related phenol substrates were accepted at at concentrations up to 92 mM reaching conversions up to >99% and perfect ee (>97% by HPLC; Table  at concentrations up to 92 mM reaching conversions up to >99% and perfect ee (>97% by HPLC; Table  concentrations up to 92 mM reaching conversions up to >99% and perfect ee (>97% by HPLC; Table 2). 2). A preparative reaction transforming 56.6 mg of substrate 16a afforded the optically pure hydroxy  2). A preparative reaction transforming 56.6 mg of substrate 16a afforded the optically pure hydroxy  A preparative reaction transforming 56.6 mg of substrate 16a afforded the optically pure hydroxy acid acid (S)‐18a with 97% conversion in 77% isolated yield (ee > 97%).  acid (S)‐18a with 97% conversion in 77% isolated yield (ee > 97%).  (S)-18a with 97% conversion in 77% isolated yield (ee > 97%).   

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Table 2. Results of cascade transforming phenols to p‐hydroxyphenyl lactic acids 18 (Scheme 5) a.  b (%)  Substrate (mM)  Substrate Conc. (mM)  HicDH  Conv.  Table 2. Results of cascade transforming phenols to p-hydroxyphenyl lactic acids ee (%)  18 (Scheme 5) a . 16a  L‐HicDH  97 (77)  >97 (S)  46  b (%) >97 (R)  16a  D‐HicDH  97 (73)  46  (mM) Substrate (mM) Substrate Conc. HicDH ee (%) Conv. 16b  L‐HicDH  92 (60)  >97 (S)  46  16a 46 L -HicDH 97 (77) >97 (S) 16b  D‐HicDH  92 (58)  >97 (R)  46  16a 46 D -HicDH 97 (73) >97 (R) L‐HicDH  96 (77)  >97 (S)  16b 16c  46 69  L -HicDH 92 (60) >97 (S) ‐HicDH  96 (75)  >97 (R)  16b 16c  46 69  DD -HicDH 92 (58) >97 (R) ‐HicDH  >99 (80)  >97 (S)  16c 16d  69 92  LL -HicDH 96 (77) >97 (S) ‐HicDH  >99 (77)  >97 (R)  16c 16d  69 92  DD -HicDH 96 (75) >97 (R) 16d 16e  92 92  LL -HicDH >99 (80) >97 (S) ‐HicDH  93 (81)  >97 (S)  16d 16e  92 92  DD -HicDH >99 (77) >97 (R) ‐HicDH  95 (80)  >97 (R)  16e 16f  92 23  LL -HicDH 93 (81) >97 (S) ‐HicDH  83 (63)  >97 (S)  16e 16f  92 23  D -HicDH 95 (80) >97 D‐HicDH  85 (63)  96 (R)  (R) 16f 23 L -HicDH 83 (63) >97 (S) 16g  L‐HicDH  96 (80)  >97 (S)  46  16f 23 D -HicDH 85 (63) 96 (R) 16g  D‐HicDH  95 (85)  >97 (R)  46  16g 46 L -HicDH 96 (80) >97 (S) a  Reaction  conditions:  Substrate  16  (23–92  mM),  pyruvate  (2  eq.),  NH4Cl  (4  eq.),  NAD+  (1  mM),  16g 46 D -HicDH 95 (85) >97 (R) 4HCOO (3 eq.), TPL (41 U/mmol),  L‐AAD (40 U/mmol),  D‐HicDH (114 U/mmol) or  ‐HicDH (68  aNH Reaction conditions: Substrate 16 (23–92 mM), pyruvate (2 eq.), NH4 Cl (4 eq.), NAD+ (1 mM), NH4LHCOO (3 eq.), TPL (41 U/mmol), L -AAD (40 U/mmol), D -HicDH (114 U/mmol) or L-HicDH (68oxidative  U/mmol), deamination  FDH (76 U/mmol), U/mmol),  FDH  (76  U/mmol),  21  °C,  24  h  for  C–C  coupling,  3  h  for  and  ◦ C, 24 h for C–C coupling, 3 h for oxidative deamination and reduction. b Isolated yields in parentheses. 21 b Isolated yields in parentheses.  reduction. 

Starting from L‐phenylalanine, (S)‐mandelic acid was produced via a six‐step cascade involving  -phenylalanine, (S)-mandelic acid was produced via a six-step cascade involving Starting from  ammonia decarboxylation, epoxidation, epoxide hydrolysis, and oxidation of the primary ammonia elimination, elimination,  decarboxylation,  epoxidation,  epoxide  hydrolysis,  and  oxidation  of  the  alcohol to the carboxylic acid (Scheme 6) [50]. Performing the reaction with 120 mM of L-phenylalanine primary alcohol to the carboxylic acid (Scheme 6) [50]. Performing the reaction with 120 mM of  L‐ to (S)-mandelic acid with E. coli cells co-expressing all enzymes in a single strain afforded 83% phenylalanine  to  (S)‐mandelic  acid  with  E.  coli  cells  co‐expressing  all  enzymes  in  a  single  strain  conversion within 10 h and 92% conversion within 24 h and a perfect ee of 99%. afforded 83% conversion within 10 h and 92% conversion within 24 h and a perfect ee of 99%. 

 

  Scheme 6. Cascade transforming  Scheme 6. Cascade transforming LL‐phenylalanine to (S)‐mandelic acid. Enzyme abbreviations: PAL,  -phenylalanine to (S)-mandelic acid. Enzyme abbreviations: PAL, phenylalanine  ammonia  lyase;  PAD,  phenylacrylic  acid  decarboxylase;  SMO,  styrene  phenylalanine ammonia lyase; PAD, phenylacrylic acid decarboxylase; SMO, styrene monooxygenase; monooxygenase;  EH,  epoxide  hydrolase;  ADH,  FAD‐dependent  alcohol dehydrogenase  AlkJ  from  EH, epoxide hydrolase; ADH, FAD-dependent alcohol dehydrogenase AlkJ from Pseudomonas putida; Pseudomonas putida; AlDH, aldehyde dehydrogenase.  AlDH, aldehyde dehydrogenase.

2.3. Deracemization of α‐Hydroxy Acids  Following  previous  reports  on  deracemization  [1n‐t],  selected  α‐hydroxy  acids  were  deracemized in an enantioselective oxidation–stereoselective reduction sequence (Scheme 7, Table 3) 

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2.3. Deracemization of α-Hydroxy Acids Following previous reports on  deracemization [1n-t], selected α-hydroxy acids were deracemized Catalysts 2018, 8, x FOR PEER REVIEW  6 of 23  in an enantioselective oxidation–stereoselective reduction sequence (Scheme 7, Table 3) [51]. [51].  was Oxidation  was using achieved  using  a  flavin‐mononucleotide‐dependent  (S)‐2‐hydroxy  acid  Oxidation achieved a flavin-mononucleotide-dependent (S)-2-hydroxy acid dehydrogenase dehydrogenase  [(S)‐2‐HADH]  from  Pseudomonas  aeruginosa.  For  the  asymmetric  reduction  of  [(S)-2-HADH] from Pseudomonas aeruginosa. For the asymmetric reduction of the intermediatethe  α-keto intermediate  α‐keto  acid,  an  NADH‐dependent  ketoreductase  from  Leuconostoc  mesenteroides,  was  acid, an NADH-dependent ketoreductase from Leuconostoc mesenteroides, was employed, whereby the employed,  whereby  the  cofactor  was  regenerated  via  GDH  (glucose  dehydrogenase)  from  cofactor was regenerated via GDH (glucose dehydrogenase) from Exiguobacterium sibiricum. For the Exiguobacterium  sibiricum.  For  the  substrates  investigated  at  20  mM  concentration,  the  ee  values  substrates investigated at 20 mM concentration, the ee values obtained ranged between 60% and >99%, obtained ranged between 60% and >99%, whereby in most cases optically pure product was obtained  whereby in most cases optically pure product was obtained with up to 98% conversion. with up to 98% conversion.   

  Scheme  7.  Deracemization  of  α‐hydroxy  acids  by  enantioselective  oxidation  and  stereoselective  Scheme 7. Deracemization of α-hydroxy acids by enantioselective oxidation and stereoselective reduction. Enzyme abbreviations: (S)‐2‐HADH, (S)‐2‐hydroxy acid dehydrogenase; (R)‐2‐KAR, (R)‐ reduction. Enzyme abbreviations: (S)-2-HADH, (S)-2-hydroxy acid dehydrogenase; (R)-2-KAR, 2‐keto acid reductase. 

(R)-2-keto acid reductase.

Table  3.  Deracemization  of  α‐hydroxy  acids  by  enantioselective  oxidation  and  stereoselective 

Tablereduction (Scheme 7).  3. Deracemization of α-hydroxy acids by enantioselective oxidation and stereoselective reduction (Scheme 7).   Substrate rac‐26  Reaction Time (h)  Conv. (%) ee of (R)‐26 (%)  a  4  95  >99  Substrate rac-26 b  Reaction Time Conv. ee of (R)-26 (%) 6  (h) 95  (%) >99  c  6  93 95 >99  a 4 >99 d  4  95 95 >99  b 6 >99 e  4  96 93 >99  c 6 >99 f  4  97 95 >99  d 4 >99 g  4  98 96 >99  e 4 >99 h  2  95 97 >99  f 4 >99 i  4  96 98 >99  g 4 >99 j  2  95 95 >99  h 2 >99 k  4  96 96 >99  i 4 >99 l  2  96 95 >99  j 2 >99 m  4  95 96 >99  k 4 >99 n  82  6  36 96 l 2 >99 o  6  35 95 72  m 4 >99 p  6  60 36 >99  n 6 82 q  6  43 35 >99  o 6 72 r  6  77 60 63  p 6 >99 s  6  58 43 60  q 6 >99

r 2.4. Styrenes to α‐Hydroxy Acids  s

6 6

77 58

63 60

An alternative route to α‐hydroxycarboxylic acids has recently been developed as a modular  multi‐enzyme  system Acids for  the  functionalisation  of  styrene  derivatives  (Scheme  8)  [52].  In  this  2.4. Styrenes to α-Hydroxy approach, aryl‐substituted styrenes 28 were converted into vicinal diols (S)‐30 using the enzymes of  An alternative route to α-hydroxycarboxylic acids has recently been developed as a modular Module 1, styrene monooxygenase and epoxide hydrolase. Selective oxidation of the primary alcohol 

multi-enzyme system for the functionalisation of styrene derivatives (Scheme 8) [52]. In this

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approach, aryl-substituted styrenes 28 were converted into vicinal diols (S)-30 using the enzymes Catalysts 2018, 8, x FOR PEER REVIEW    7 of 23  of Module 1, styrene monooxygenase and epoxide hydrolase. Selective oxidation of the primary Catalysts 2018, 8, x FOR PEER REVIEW    7 of 23  alcohol moiety to the carboxylic acid was then realised by a second bi-enzymatic module, moiety to the carboxylic acid was then realised by a second bi‐enzymatic module, comprising the  comprising the membrane-associated alcohol dehydrogenase from Pseudomonas putida well membrane‐associated  alcohol  dehydrogenase  AlkJ  from  AlkJ Pseudomonas  putida  as  well as as  moiety to the carboxylic acid was then realised by a second bi‐enzymatic module, comprising the  as membrane‐associated  phenylacetaldehyde dehydrogenase from E. coli. Eleven different mandelic acid derivatives (S)-32 phenylacetaldehyde  dehydrogenase  from  E.  coli.  Eleven  different  mandelic  acid  derivatives  (S)‐32  alcohol  dehydrogenase  AlkJ  from  Pseudomonas  putida  as  well  as  were thus produced with 69–99% overall conversion and ee values mandelic  ranging from to >99%. were thus produced with 69–99% overall conversion and ee values ranging from 96% to >99%.  phenylacetaldehyde  dehydrogenase  from  E.  coli.  Eleven  different  acid 96% derivatives  (S)‐32  were thus produced with 69–99% overall conversion and ee values ranging from 96% to >99%.     

    Scheme  8.  Modularised  multi‐enzyme  system  for  the  production  of  1,2‐diols  (S)‐30  or  α‐hydroxy 

Scheme 8. Modularised multi-enzyme system for the production of 1,2-diols (S)-30 or α-hydroxy acids acids (S)‐32 from styrene derivatives 28. Abbreviations: SMO, styrene monooxygenase; EH, epoxide  Scheme  Modularised  multi‐enzyme  system  for  the styrene production  of  1,2‐diols  EH, (S)‐30  or  α‐hydroxy  (S)-32 from 8.  styrene derivatives 28. Abbreviations: SMO, monooxygenase; epoxide hydrolase; hydrolase;  ADH,  FAD‐dependent  alcohol  dehydrogenase  AlkJ  from  Pseudomonas  putida;  AlDH,  acids (S)‐32 from styrene derivatives 28. Abbreviations: SMO, styrene monooxygenase; EH, epoxide  ADH, FAD-dependent alcohol dehydrogenase AlkJ from Pseudomonas putida; AlDH, aldehyde aldehyde dehydrogenase; Q, ubiquinone.  hydrolase;  ADH,  FAD‐dependent  alcohol  dehydrogenase  AlkJ  from  Pseudomonas  putida;  AlDH,  dehydrogenase; Q, ubiquinone. aldehyde dehydrogenase; Q, ubiquinone. 

2.5. Access to α β‐Hydroxy Dicarboxylic Acid Derivative Ethyl (R)‐3‐Hydroxyglutarate  2.5. Access to α β-Hydroxy Dicarboxylic Acid Derivative Ethyl (R)-3-Hydroxyglutarate 2.5. Access to α β‐Hydroxy Dicarboxylic Acid Derivative Ethyl (R)‐3‐Hydroxyglutarate  The  biocatalytic  synthesis  of  ethyl  (R)‐3‐hydroxyglutarate  (36),  a  key  intermediate  in  the  The biocatalytic synthesis of ethyl (R)-3-hydroxyglutarate (36), a key intermediate in the preparation of the cholesterol‐lowering drug rosuvastatin, was achieved in a three‐step cascade with  The  biocatalytic  synthesis  of  ethyl  (R)‐3‐hydroxyglutarate  (36),  a  key  intermediate  in  the  preparation of the cholesterol-lowering drug rosuvastatin, inas  a starting  three-step cascade two  enzymes  [53].  Ethyl  (S)‐4‐chloro‐3‐hydroxybutyrate  (33) was was achieved employed  material,  preparation of the cholesterol‐lowering drug rosuvastatin, was achieved in a three‐step cascade with  with two enzymes [53]. Ethyl (S)-4-chloro-3-hydroxybutyrate (33) was employed as starting material, which  was  converted  to  the  corresponding  epoxide  by  a (33)  halohydrin  dehalogenase  (HHDH)  from  two  enzymes  [53].  Ethyl  (S)‐4‐chloro‐3‐hydroxybutyrate  was  employed  as  starting  material,  which was converted to the corresponding epoxide by a halohydrin dehalogenase (HHDH) from Agrobacterium radiobacter AD1, followed by a ring opening and subsequent hydrolysis to the product,  which  was  converted  to  the  corresponding  epoxide  by  a  halohydrin  dehalogenase  (HHDH)  from  Agrobacterium radiobacter AD1, followed by a ring opening and subsequent hydrolysis to the product, using a nitrilase from Arabidopsis thaliana (Scheme 9).  Agrobacterium radiobacter AD1, followed by a ring opening and subsequent hydrolysis to the product,  using a nitrilase from Arabidopsis thaliana (Scheme 9). using a nitrilase from Arabidopsis thaliana (Scheme 9).     

    Scheme  9.  Biocatalytic  transformation  of  ethyl  (S)‐4‐chloro‐3‐hydroxybutyrate  (33)  into  the  pharmacologically  important  ethyl  (R)‐3‐hydroxyglutarate  (36).  Enzyme  abbreviation:  HHDH,  Scheme  Biocatalytic  transformation  of  (33)  Scheme 9. 9.  Biocatalytic transformation of ethyl  ethyl (S)‐4‐chloro‐3‐hydroxybutyrate  (S)-4-chloro-3-hydroxybutyrate (33)into  intothe the halohydrin dehalogenase.  pharmacologically  important  ethyl  (R)‐3‐hydroxyglutarate  (36).  Enzyme  abbreviation:  HHDH,  pharmacologically important ethyl (R)-3-hydroxyglutarate (36). Enzyme abbreviation: HHDH, halohydrin dehalogenase.  halohydrin dehalogenase.

Using  E.  coli  cells  co‐expressing  the  two  required  enzymes,  up  to  300  mM  substrate  were  transformed within 1.5 h. However, at a substrate loading of 600 mM unfortunately only about 20%  Using  E.  coli  cells  co‐expressing  the  two  required  enzymes,  up  to  300  mM  substrate  were  Using E. coli cells co-expressing the two required enzymes, up to 300 mM substrate were of the target hydroxy acid was detected after 5 h reaction time while intermediate 35 accumulated.  transformed within 1.5 h. However, at a substrate loading of 600 mM unfortunately only about 20%  transformed within 1.5 h. However, at a substrate loading of 600 mM unfortunately only about Studying the inhibitory effect of the substrate 33 on the nitrilase, it was shown that above 300 mM a  of the target hydroxy acid was detected after 5 h reaction time while intermediate 35 accumulated.  significant decrease in activity was observed. In order to access high product titres, the cascade was  Studying the inhibitory effect of the substrate 33 on the nitrilase, it was shown that above 300 mM a  significant decrease in activity was observed. In order to access high product titres, the cascade was 

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20% of the target hydroxy acid was Catalysts 2018, 8, x FOR PEER REVIEW    detected after 5 h reaction time while intermediate 35 accumulated. 8 of 23  Studying the inhibitory effect of the substrate 33 on the nitrilase, it was shown that above 300 mM aperformed in a fed‐batch mode; thus, 300 mM of substrate was added twice over a period of 2 h. By  significant decrease in activity was observed. In order to access high product titres, the cascade using this approach, the reaction went to completion within 6 h. Adding 300 mM substrate for a third  was performed in a fed-batch mode; thus, 300 mM of substrate was added twice over a period of 2 h. time, 35% of intermediate 35 remained in the reaction mixture while 33 was completely consumed.  By using this approach, the reaction went to completion within 6 h. Adding 300 mM substrate for a Poorly balanced activity levels of the two co‐expressed enzymes were identified as the reason for this  third time, 35% of intermediate 35 remained in the reaction mixture while 33 was completely consumed. accumulation  of activity the  reaction  intermediate  as  the  nitrilase  was  expressed  in assignificantly  lower  Poorly balanced levels of the two co-expressed enzymes were identified the reason for this amounts  than  the  HHDH.  Consequently,  whole‐cell  preparations  expressing  the  enzymes  accumulation of the reaction intermediate as the nitrilase was expressed in significantly lower amounts individually  were  used  and  the  ratio  of  preparations the  amount  expressing of  different the cells  was  adjusted  to  obtain  than the HHDH. Consequently, whole-cell enzymes individually were comparable activity levels for the two enzymes. By employing this setup, 900 mM of compound 33  used and the ratio of the amount of different cells was adjusted to obtain comparable activity levels for were completely converted into the final product within 6 h, and (R)‐36 was isolated in 84% yield.  the two enzymes. By employing this setup, 900 mM of compound 33 were completely converted into the final product within 6 h, and (R)-36 was isolated in 84% yield. 3. α‐ and β‐Amino Acids  3. α- and β-Amino Acids Natural  and  non‐natural  amino  acids  and  their  derivatives  are  pivotal  building  blocks  for  Natural and non-natural amino drugs,  acids and their derivatives are pivotal building blocks agrochemicals,  bioactive  polypeptide  pharmaceuticals,  and  polymers  [54–57].  Hence,  the  for agrochemicals, bioactive polypeptide drugs, pharmaceuticals, and polymers [54–57]. Hence, efficient synthesis of these compounds has received tremendous interest  the efficient synthesis these compounds has received interest The  synthesis  of of the  non‐natural  α‐amino  acid  L‐tremendous phenylglycine  and  its  derivatives  has  been  The synthesis of the non-natural α-amino acid L-phenylglycine and its derivatives has been described using a three‐enzyme cascade, starting from racemic mandelic acid. While in an older study  described usingacid  a three-enzyme cascade, starting racemic mandelic acid. have  Whilebeen  in aninvestigated  older study only  mandelic  was  transformed  [58],  other from mandelic  acid  derivatives  only was [58], other mandelic acid derivatives been investigated more more mandelic recently acid [59].  In transformed this  redox‐neutral  cascade  that  proceeds  via have ‘hydrogen  borrowing’,  an  recently [59]. In this redox-neutral cascade that proceeds via ‘hydrogen borrowing’, an engineered engineered mandelate racemase (MRM) from Pseudomonas putida, a  D‐mandelate dehydrogenase ( D‐ mandelate racemase (MRM) from Pseudomonas putida, a D -mandelate dehydrogenase ( D -MDH) from MDH)  from  Lactobacillus  brevis,  and  an  L‐leucine  dehydrogenase  (LeuDH)  from  Exiguobacterium  Lactobacillus brevis, and an L-leucine dehydrogenase (LeuDH) from Exiguobacterium sibiricum were sibiricum were selected to produce  L‐phenyl glycine derivatives 38 in a one‐pot simultaneous fashion  selected to produce L-phenyl glycine derivatives 38 in a one-pot simultaneous fashion (Scheme 10). (Scheme 10). 

 

  Scheme 10. 10.  Three-step Three‐step  cascade cascade  for for  the the  conversion conversion  of of racemic racemic  phenyllactic phenyllactic  acid acid  derivatives derivatives  32 32 into into  Scheme D‐ optically active phenylglycine derivatives 38. Enzyme abbreviations: MRM, mandelate racemase;  optically active phenylglycine derivatives 38. Enzyme abbreviations: MRM, mandelate racemase; D‐mandelate dehydrogenase; LeuDH,  L‐leucine dehydrogenase.  MDH,  D -MDH, D -mandelate dehydrogenase; LeuDH, L -leucine dehydrogenase.

Under optimized conditions, 50 mM of substrate 32a (R = H) were transformed into the desired  Under optimized conditions, 50 mM of substrate 32a (R = H) were transformed into the desired product with 97% conversion and a perfect ee of >99% after 24 h. When the substrate loading was  product with 97% conversion and a perfect ee of >99% after 24 h. When the substrate loading was increased  to  up  to  200  mM,  good  conversions  of  more  than  95%  were  observed,  whereas  at  even  increased to up to 200 mM, good conversions of more than 95% were observed, whereas at even higher concentrations (500 mM) the conversion was reduced to 63%. To prove the applicability of this  higher concentrations (500 mM) the conversion was reduced to 63%. To prove the applicability of this cascade, 30.4 g (200 mM) of rac‐mandelic acid were transformed at a 1 L scale reaction, leading to  cascade, 30.4 g (200 mM) of rac-mandelic acid were transformed at a 1 L scale reaction, leading to >96% >96% conversion and an isolated product yield of 86.5%.    conversion and an isolated product yield of 86.5%. Further, mandelic acid derivatives were converted to the corresponding phenylglycine products  as well, showing moderate to good conversions (Table 4). Of special interest is product 38b (R = o‐ Cl), which is a building block for Clopidogrel, an antiplatelet drug.     

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Further, mandelic acid derivatives were converted to the corresponding phenylglycine products as well, showing moderate to good conversions (Table 4). Of special interest is product 38b (R = o-Cl), which is a building block for Clopidogrel, an antiplatelet drug. Catalysts 2018, 8, x FOR PEER REVIEW    9 of 23  Table 4. Conversion of mandelic acid derivatives 32 into phenyl glycine derivatives 38 (Scheme 10). Table 4. Conversion of mandelic acid derivatives 32 into phenyl glycine derivatives 38 (Scheme 10). 

Substrate  Time (h)  Substrate Time (h) Conv. (%)  Conv. (%) rac‐32a (R = H)  12  97  rac-32a (R = H) 12 97 rac‐32b (R = o‐Cl)  48  49  49 rac-32b (R = o-Cl) 48 rac‐32c (R = m‐Cl)  48  90  90 rac-32c (R = m-Cl) 48 rac‐32d (R = p‐Cl)  24  95  95 rac-32d (R = p-Cl) 24 rac-32e (R = p-OH) 48 rac‐32e (R = p‐OH)  48  66  66 + (0.1 mM), MRM (5 U/mL),  Reaction conditions: Substrate rac‐32 (50 mM), MgCl 2 (3 mM), NAD ‐MDH  Reaction conditions: Substrate rac-32 (50 mM), MgCl2 (3 mM), NAD+ (0.1 mM), MRM (5 U/mL), D-MDH (10DU/mL), ◦ C, 12–48 h. LeuDH (10 U/mL), ammonium chloride buffer (2.0 M, pH 9.5), 30 (10 U/mL), LeuDH (10 U/mL), ammonium chloride buffer (2.0 M, pH 9.5), 30 °C, 12–48 h. 

An analogous transformation of mandelic acid derivatives into phenylglycine derivatives has An analogous transformation of mandelic acid derivatives into phenylglycine derivatives has  been as an system for  for the been  developed developed  as  an  extension extension  of of  the the  above-mentioned above‐mentioned  modular modular  multi-enzyme multi‐enzyme  system  the  functionalisation of ring-substituted styrenes (Scheme 8) [52]. The (S)-mandelic acid derivatives 32, functionalisation of ring‐substituted styrenes (Scheme 8) [52]. The (S)‐mandelic acid derivatives 32,  which ultimately derived from styrenes 28, were oxidised to oxoacids 37 using hydroxymandelate which were were  ultimately  derived  from  styrenes  28,  were  oxidised  to  oxoacids  37  using  oxidase from Streptomyces coelicolor. Reductive amination of 37 by the branched-chain aminoacid hydroxymandelate oxidase from Streptomyces coelicolor. Reductive amination of 37 by the branched‐ transaminase fromtransaminase  E. coli using from  L -glutamate as amino donor and -glutamate dehydrogenase for L‐glutamate  as Lamino  donor  and  L‐glutamate  chain  aminoacid  E.  coli  using  regeneration of the latter afforded the L-phenylglycine derivatives 38 (cf. Schemes 8 and 10). The overall dehydrogenase for regeneration of the latter afforded the  L‐phenylglycine derivatives 38 (cf. Schemes  synthesis sequence starting from styrenes 28 involved eight enzymes (six in a linear sequence) arranged 8 and 10). The overall synthesis sequence starting from styrenes 28 involved eight enzymes (six in a  in three modules (each expressed in a separate E. coli host) and provided products L-38 in 16–86% linear sequence) arranged in three modules (each expressed in a separate E. coli host) and provided  overall yield and excellent optical purity (91% to >99% ee). products  L‐38 in 16–86% overall yield and excellent optical purity (91% to >99% ee).  -phenylglycine (38a) was also obtained in an eight-step cascade starting from L-phenylalanine L‐phenylglycine (38a) was also obtained in an eight‐step cascade starting from  ‐phenylalanine  (19), was transformed acid (25) as  (25) as shown 6, followed further (19),  which which was  transformed  to to  (S)-mandelic (S)‐mandelic  acid  shown  in in  Scheme Scheme 6,  followed  by by further  conversion into 38a by the sequence described in the previous paragraph [50]. Transformation of 40 mM conversion into 38a by the sequence described in the previous paragraph [50]. Transformation of 40  of (S)-of  L -phenylalanine with E.with  coli cells co-expressing all requiredall  enzymes afforded L -phenylglycine mM  (S)‐L‐phenylalanine  E.  coli  cells  co‐expressing  required  enzymes  afforded  L‐ with 85% conversion and 99% ee within 24 h. The challenging eight-step biocatalytic cascade was phenylglycine with 85% conversion and 99% ee within 24 h. The challenging eight‐step biocatalytic  successfully achieved with a single recombinant strain co-expressing 10 enzymes. cascade was successfully achieved with a single recombinant strain co‐expressing 10 enzymes.  To access To  access  LL-tyrosine ‐tyrosine  and and  its its derivatives derivatives  substituted substituted  on on  the the  aromatic aromatic  ring, ring,  aa two-step-one-pot two‐step‐one‐pot  cascade was designed consisting of the P450 monooxygenase BM3 from Bacillus megaterium and a cascade was designed consisting of the P450 monooxygenase BM3 from Bacillus megaterium and a  tyrosine phenyl lyase (TPL) from the microorganism Citrobacter freundii (Scheme 11) [60]. tyrosine phenyl lyase (TPL) from the microorganism Citrobacter freundii (Scheme 11) [60]. 

 

  Scheme  11.  Two‐step‐one‐pot  Scheme 11. Two-step-one-pot biosynthesis  biosynthesis of  of LL‐tyrosine  -tyrosine derivatives  derivatives 41  41 starting  starting from  from arenes  arenes 39.  39. Enzyme abbreviations: P450‐BM3, cytochrome P450 monooxygenase from Bacillus megaterium; TPL,  Enzyme abbreviations: P450-BM3, cytochrome P450 monooxygenase from Bacillus megaterium; TPL, L‐tyrosine phenol lyase.  -tyrosine phenol lyase.

Under non‐optimized conditions the model substrate 39c (R = OMe) yielded ~5% of the amino  Under non-optimized conditions the model substrate 39c (R = OMe) yielded ~5% of the amino acid acid  product  with  the  majority  of  intermediate  40c  remaining,  proving  steps  can  be  product with the majority of intermediate 40c remaining, proving that both that  stepsboth  can be performed performed simultaneously. An inhibition study of the cascade revealed that the amount of NADP+  and of the P450 have a substantial effect on the productivity of the TPL enzyme; however, the most  critical parameter was found to be the pH drop due to the release of gluconic acid during NADPH  regeneration.  Furthermore,  small  amounts  (97  10 >99 68  84  >97  28 10  53 >99  >97  21 28  65 53  >97 

>97 >97 >97 >97 >97 >97

a Reaction conditions: Substrate39f (R = Br)  21  >97 mM), NH4 Cl (180 mM), NADP+ 39 (5–20 mM), pyruvate (40 mM),65  D -glucose (100 (0.4 mM),a Reaction conditions: Substrate 39 (5–20 mM), pyruvate (40 mM),  PLP (40 µM), P450-BM3 (2 µM), TPL (3 mg/mL), GDH (12 U/mL), catalase (1200 U/mL),4Cl (180  Tris-HCl buffer D‐glucose (100 mM), NH (100 mM,mM), NADP pH 8.0), dimethylsulfoxide (DMSO) (1% v/v), room temperature, 6 h. b Selectivity is defined as the + (0.4 mM), PLP (40 μM), P450‐BM3 (2 μM), TPL (3 mg/mL), GDH (12 U/mL), catalase  percentage of L-41a–f in the final product mixture. (1200  U/mL),  Tris‐HCl  buffer  (100  mM,  pH  8.0),  dimethylsulfoxide  (DMSO)  (1%  v/v),  room 

temperature, 6 h. b Selectivity is defined as the percentage of L‐41a–f in the final product mixture. 

Besides α-amino acids, their β-counterparts have attracted attention since they might be applied in the synthesisBesides α‐amino acids, their β‐counterparts have attracted attention since they might be applied  of bioactive compounds as well as in the design of hybrid peptides [61–64]. A biosynthetic in  the  synthesis  bioactive  β-amino compounds  as  well  in  the  design  of  hybrid  peptides  [61–64].  A cascade route accessing variousof aromatic acids hasas  been reported using a two-step-one-pot biosynthetic route accessing various aromatic β‐amino acids has been reported using a two‐step‐one‐ starting from β-keto esters. The first step involves the hydrolysis of the ester by a lipase originating pot cascade starting from β‐keto esters. The first step involves the hydrolysis of the ester by a lipase  from Candida rugosa, followed by the amination of the keto acid by an ω-transaminase (ω-TA) from originating from Candida rugosa, followed by the amination of the keto acid by an ω‐transaminase (ω‐ Polaromonas sp. JS666 (Scheme 12) [65]. TA) from Polaromonas sp. JS666 (Scheme 12) [65].   

  Scheme 12. Biocatalytic synthesis of β‐amino acids 44 starting from β‐keto esters 42 using a lipase and 

Scheme 12. Biocatalytic synthesis of β-amino acids 44 starting from β-keto esters 42 using a lipase and a transaminase (TA).  a transaminase (TA). The  cascade  was  optimized  with  respect  to  the  use  of  co‐solvent,  amine  donor,  and  lipase 

Theconcentration; substrate 42a was converted under improved conditions with a conversion of 89% and  cascade was optimized with respect to the use of co-solvent, amine donor, and lipase a perfect ee of >99% for the (R)‐enantiomer. Several further β‐keto esters were transformed into their  concentration; substrate 42a was converted under improved conditions with a conversion of 89% and corresponding amino acids with conversions ranging between 26 and 99% with excellent ee values in  a perfect ee of >99% for the (R)-enantiomer. Several further β-keto esters were transformed into their all cases (Table 6).  corresponding amino acids with conversions ranging between 26 and 99% with excellent ee values in   all cases (Table 6).

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a Table 6. Transformation of β‐keto esters 42 into β‐amino acids 44 (Scheme 12)  a.  44 (Scheme 12) a ..  Table 6. Transformation of β-keto esters 42 into β-amino acids Table 6. Transformation of β‐keto esters 42 into β‐amino acids 44 (Scheme 12) 

Table 6. Transformation of β‐keto esters 42 into β‐amino acids 44 (Scheme 12)  Substrate  Conv. (%)  ee (%)  Substrate  Substrate Conv. (%)  ee (%)  Conv. (%) ee (%) 42a  >99%  42a  89  >99%  89  Substrate  Conv. (%)  ee (%)  42a 42b  89 >99%  >99% 42b  96  >99%  96  42a  89  >99%  42b 42c  96 >99%  >99% 42c  75  >99%  75  42b  96  >99%  42c 42d  75 73  >99%  >99% 42d  73  >99%  73 75  >99%  42d >99% 42c  42e  >99%  99  >99%  42e  99  73  >99%  42e 99 >99% 42d  61  >99%  42f  61  42f  >99%  61 99  >99%  42f >99% 42e  47  >99%  >99% 42g  47  42g  >99%  47 42g 42f  61  >99%  41  >99%  >99% 42h 42h  41  42h  >99%  41 42g  47  >99%  26  >99%  >99% 42i 42i  26  42i  >99%  26 42h  41  >99%  aa Reaction conditions: Substrate 42 (50 mM), (S)‐1‐phenylethylamine (150 mM), PLP (1 mM), lipase  a Reaction conditions: Substrate 42 (50 mM), (S)‐1‐phenylethylamine (150 mM), PLP (1 mM), lipase  Reaction conditions: Substrate 42 (50 mM), (S)-1-phenylethylamine (150 mM), PLP (1 mM), lipase 42i  26 (30 mg/mL), >99%  TA (20 mg/mL), Tris-HCl buffer (100 mM,a pH 8.0), DMSO (15% v/v), 37 ◦ C, 24 h. (30 mg/mL), TA (20 mg/mL), Tris‐HCl buffer (100 mM, pH 8.0), DMSO (15% v/v), 37 °C, 24 h.  (30 mg/mL), TA (20 mg/mL), Tris‐HCl buffer (100 mM, pH 8.0), DMSO (15% v/v), 37 °C, 24 h.   Reaction conditions: Substrate 42 (50 mM), (S)‐1‐phenylethylamine (150 mM), PLP (1 m

(30 mg/mL), TA (20 mg/mL), Tris‐HCl buffer (100 mM, pH 8.0), DMSO (15% v/v), 37 °C, 2

Phenylalanine and derivatives thereof were deracemized by a combination of E. coli whole cells Phenylalanine and derivatives thereof were deracemized by a combination of E. coli whole cells  Phenylalanine and derivatives thereof were deracemized by a combination of E. coli whole cells  expressing an LL‐amino acid deaminase and an engineered  -amino acid deaminase andDan engineered D -amino acid dehydrogenase to prepare expressing an  D‐amino acid dehydrogenase to prepare  expressing an  L‐amino acid deaminase and an engineered  ‐amino acid dehydrogenase to prepare  Phenylalanine and derivatives thereof were deracemized by a combination of E. optically pure D -phenylalanines (Scheme 13) [66]. Alternatively, of starting from the from  racemate, optically  pure  D ‐phenylalanines  (Scheme  13)  [66].  Alternatively,  instead  of the  starting  the  optically  pure  D‐phenylalanines  (Scheme  13)  [66].  Alternatively,  instead  of instead starting  from  expressing an  L‐amino acid deaminase and an engineered  D‐amino acid dehydrogen optically pure L -enantiomers were used as substrate leading to inversion of the absolute configuration. racemate, optically pure  L‐enantiomers were used as substrate leading to inversion of the absolute  racemate, optically pure  L‐enantiomers were used as substrate leading to inversion of the absolute  optically  pure  D‐phenylalanines  (Scheme  13)  [66].  Alternatively,  instead  of  star Various substituted derivatives of theD‐amino acids were isolated with good yields  D -amino acids D were isolated with good yields (69–83%) and configuration. Various substituted derivatives of the  ‐amino acids were isolated with good yields  configuration. Various substituted derivatives of the  racemate, optically pure  L‐enantiomers were used as substrate leading to inversion  excellent optical purities (95% to >99% ee). (69–83%) and excellent optical purities (95% to >99% ee).  (69–83%) and excellent optical purities (95% to >99% ee).  configuration. Various substituted derivatives of the  D‐amino acids were isolated w (69–83%) and excellent optical purities (95% to >99% ee). 

 

 

 

 

 

Scheme 13. Deracemization cascade for the preparation of  Scheme 13. Deracemization cascade for the preparation of D D‐amino acids 45. Enzyme abbreviations:  -amino acids 45. Enzyme abbreviations: Scheme 13. Deracemization cascade for the preparation of  D‐amino acids 45. Enzyme abbreviations:  L L‐AAD,  -AAD, LL‐amino acid deaminase; MDPD, meso‐diaminopimelate dehydrogenase.  -amino acid deaminase; MDPD, meso-diaminopimelate dehydrogenase. L‐AAD, L‐amino acid deaminase; MDPD, meso‐diaminopimelate dehydrogenase.  Scheme 13. Deracemization cascade for the preparation of  D‐amino acids 45. Enzyme ab

4. Lactones 

4. Lactones  4. Lactones

L‐AAD, L‐amino acid deaminase; MDPD, meso‐diaminopimelate dehydrogenase. 

4. Lactones  Lactones—and particularly γ‐ and δ‐lactones—are important flavour and fragrance compounds  Lactones—and particularly γ‐ and δ‐lactones—are important flavour and fragrance compounds  Lactones—and particularly γ- and δ-lactones—are important flavour and fragrance compounds are  found  in  juices,  various  fruits,  juices,  wines,  and  spirits  [67]. substituted  Furthermore,  substituted  γ‐ that  are  found that  in  various  fruits,  wines,  spirits  [67].  Furthermore,  γ‐γ-butyrolactones Lactones—and particularly γ‐ and δ‐lactones—are important flavour and fragran that are found in various fruits, juices,and  wines, and spirits [67]. Furthermore, substituted butyrolactones occur as a central motif in many natural products, for instance nephrosteranic acid or  butyrolactones occur as a central motif in many natural products, for instance nephrosteranic acid or  that  are  found  in  juices,  wines,  spirits [68]. [67].  Furthermore,  occur as a central motif in many natural products, forvarious  instance fruits,  nephrosteranic acid orand  arctigenin arctigenin  [68].  ɛ‐Caprolactone,  on  the  other  hand,  is  a  valuable  chemical  for  the  preparation  of  arctigenin  [68].  ɛ‐Caprolactone,  on  the  other  hand,  a  valuable  chemical  the  preparation  of  butyrolactones occur as a central motif in many natural products, for instance nephro -Caprolactone, on the other hand, is a is  valuable chemical for thefor  preparation of biodegradable polymers, biodegradable polymers, such as polycaprolactone [69] and a precursor for ɛ‐caprolactam.  biodegradable polymers, such as polycaprolactone [69] and a precursor for ɛ‐caprolactam.  [68].  such as polycaprolactone [69] andarctigenin  a precursor for ɛ‐Caprolactone,  -caprolactam. on  the  other  hand,  is  a  valuable  chemical  for  the  The chemical synthesis of these compounds usually involves a large number of steps, low overall  The chemical synthesis of these compounds usually involves a large number of steps, low overall  biodegradable polymers, such as polycaprolactone [69] and a precursor for ɛ‐caprolac The chemical synthesis of these compounds usually involves a large number of steps, low overall yields, toxic or otherwise hazardous reagents, and impractical reaction conditions, which makes them  yields, toxic or otherwise hazardous reagents, and impractical reaction conditions, which makes them  yields, toxic or otherwise hazardousThe chemical synthesis of these compounds usually involves a large number of ste reagents, and impractical reaction conditions, which makes neither  “green”  nor  nor atom‐economic  [70–73].  Hence,  biocatalytic  cascade  have  neither  “green” them nor  neither atom‐economic  [70–73].  Hence,  biocatalytic  cascade  transformations  have  yields, toxic or otherwise hazardous reagents, and impractical reaction conditions, wh “green” atom-economic [70–73]. Hence, biocatalytic cascadetransformations  transformations have become a useful tool in the preparation of lactones, as they avoid the necessity of time‐consuming  become a useful tool in the preparation of lactones, as they avoid the necessity of time‐consuming  neither  atom‐economic  [70–73].  Hence,  biocatalytic  become a useful tool in the preparation of “green”  lactones, nor  as they avoid the necessity of time-consuming and cascade  transfo and  yield‐reducing  isolation  and  purification  of  intermediates  and  reduce  waste  generation  to  a  and  yield‐reducing  isolation  and  purification  of become a useful tool in the preparation of lactones, as they avoid the necessity of ti intermediates  and  reduce  to  a  to a minimum. yield-reducing isolation and purification of intermediates and waste  reducegeneration  waste generation minimum.  minimum.  and  yield‐reducing  isolation  and in purification  of through intermediates  and  reduce  waste  g In most of these multi-enzyme sequences, the lactone is formed the final step oxidation In  most  of  these  multi‐enzyme  sequences,  the  lactone  is  formed  in  the  final  step  through  In  most  of of these  multi‐enzyme  the  lactone  is  formed (BVMO). in  the  final  step  through  minimum.  a cyclic ketone by asequences,  Baeyer–Villiger monooxygenase However, systems that derive the oxidation of a cyclic ketone by a Baeyer–Villiger monooxygenase (BVMO). However, systems that  oxidation of a cyclic ketone by a Baeyer–Villiger monooxygenase (BVMO). However, systems that  most  of reported. these  multi‐enzyme  sequences,  the  lactone  is  formed  in  the  fina lactone from an acyclic precursor haveIn  also been derive the lactone from an acyclic precursor have also been reported.  derive the lactone from an acyclic precursor have also been reported.  oxidation of a cyclic ketone by a Baeyer–Villiger monooxygenase (BVMO). Howeve derive the lactone from an acyclic precursor have also been reported. 

(30 mg/mL), TA (20 mg/mL), Tris‐HCl buffer (100 mM, pH 8.0), DMSO (15% v/v), 37 °C,  42h  41  42d  73  >99%  42i  26  42e  99  >99%  Phenylalanine and derivatives thereof were deracemized by a combination of E a Reaction conditions: Substrate 42 (50 mM), (S)‐1‐phenylet 42f  61  >99%  Catalysts 2018, 8, x FOR PEER REVIEW    expressing an  L‐amino acid deaminase and an engineered  D‐amino acid dehydroge (30 mg/mL), TA (20 mg/mL), Tris‐HCl buffer (100 mM, pH 8 42g  47  >99%  optically  pure  D‐phenylalanines  (Scheme  13)  42h  [66].  Alternatively,  instead  of  sta 41  >99%  Catalysts 2018, 8, 205 12 of 24 Table 6. Transformation of β‐keto esters 42 into β‐amino acids 44 (Sch racemate, optically pure  L‐enantiomers were used as substrate leading to inversion Phenylalanine and derivatives thereof were deracemiz 42i  26  >99%  Catalysts 2018, 8, x FOR PEER REVIEW    12 of 23  configuration. Various substituted derivatives of the  ‐amino acids were isolated w a Reaction conditions: Substrate 42 (50 mM), (S)‐1‐phenylethylamine (150 m expressing an  L‐amino acid deaminase and an engineered Substrate  DConv. (%)  ee (%)  (69–83%) and excellent optical purities (95% to >99% ee).  42a  89 (Scheme  >99%  pure  D‐phenylalanines  13)  [66].  Alte (30 mg/mL), TA (20 mg/mL), Tris‐HCl buffer (100 mM, pH 8.0), DMSO (15%  4.1. Formation of Lactones by Baeyer–Villiger Monooxygenases inoptically  Cascade Systems 4.1. Formation of Lactones by Baeyer–Villiger Monooxygenases in Cascade Systems  42b  96  >99%  racemate, optically pure  L‐enantiomers were used as subs Scheme 13. Deracemization cascade for the preparation of  D‐amino acids 45. Enzyme abbrev The combination of alcohol oxidation by an alcohol dehydrogenase (ADH) 42c  and Baeyer–Villiger The combination of alcohol oxidation by an alcohol dehydrogenase (ADH) and Baeyer–Villiger  75  >99%    configuration. Various substituted derivatives of the  D‐am Phenylalanine and derivatives thereof were deracemized by a combin L‐AAD, L‐amino acid deaminase; MDPD, meso‐diaminopimelate dehydrogenase.  oxidation of the resulting ketone by a BVMO represents perhaps the simplest biocatalytic cascade 42d  73  >99%  oxidation of the resulting ketone by a BVMO represents perhaps the simplest biocatalytic cascade  expressing an  L(69–83%) and excellent optical purities (95% to >99% ee).  ‐amino acid deaminase and an engineered  D‐amino acid d 99  >99%  system for lactone production. Although this bi-enzymatic reaction represents 42e  a double oxidation, system for lactone production. Although this bi‐enzymatic reaction represents a double oxidation, it  13)  [66].  Alternatively,  inste 4. Lactones  optically  pure  D‐phenylalanines  (Scheme  42f  61  >99%  it redox-neutralwith  withrespect  respectto tothe  therequired  required nicotinamide  nicotinamide cofactor, since  since the  the Baeyer–Villiger is  isredox‐neutral  Baeyer–Villiger  Catalysts 2018, 8, x FOR PEER REVIEW      cofactor,  racemate, optically pure  L‐enantiomers were used as substrate leading to 42g  47  Lactones—and particularly γ‐ and δ‐lactones—are important flavour and fragrance c monooxygenase requires NAD(P)H for reducing one oxygen atom of O22 to water while the other one  to water while the other one >99%  monooxygenase requires NAD(P)H for reducing one oxygen atom of O configuration. Various substituted derivatives of the  D‐amino acids were 42h  41 [67].  >99%  that  Hence, are  found  in  various  juices,  wines,  and co-substrate spirits  sub Table 6. Transformation of β‐keto esters 42 is incorporated into the substrate. molecular oxygenfruits,  is the only stoichiometric inFurthermore,  is incorporated into the substrate. Hence, molecular oxygen is the only stoichiometric co‐substrate in  (69–83%) and excellent optical purities (95% to >99% ee).  42i  26  >99%  butyrolactones occur as a central motif in many natural products, for instance nephroster the overall process. the overall process.  a Reaction conditions: Substrate 42 (50 mM), (S)‐1‐phenylethylamine (150 mM) Substrate  Conv.  arctigenin  on using the  other  hand,  is  a  valuable  chemical  for  the  pre The conversion of cyclohexanol (47)[68].  into ɛ‐Caprolactone,  -caprolactone (49) this cascade concept has been The conversion of cyclohexanol (47) into ɛ‐caprolactone (49) using this cascade concept has been   (30 mg/mL), TA (20 mg/mL), Tris‐HCl buffer (100 mM, pH 8.0), DMSO (15% v/v 42a  89  biodegradable polymers, such as polycaprolactone [69] and a precursor for ɛ‐caprolactam independently reported by two research groups in 2013 (Scheme 14) [74,75]. independently reported by two research groups in 2013 (Scheme 14) [74,75].  42b  96  The chemical synthesis of these compounds usually involves a large number of steps, 42c  75  Phenylalanine and derivatives thereof were deracemized by a combinat yields, toxic or otherwise hazardous reagents, and impractical reaction conditions, which m   42d  73  expressing an  L‐amino acid deaminase and an engineered  D‐amino acid deh 42e  transforma 99  neither  “green”  nor  atom‐economic  [70–73].  Hence,  biocatalytic  cascade  optically  pure  D‐phenylalanines  (Scheme  13)  [66].  Alternatively,  instead 42f  61  become a useful tool in the preparation of lactones, as they avoid the necessity of time‐ racemate, optically pure  L‐enantiomers were used as substrate leading to in 42g  Scheme 13. Deracemization cascade for the preparation of  D ‐amino acids 45. Enzyme ab 47  and  yield‐reducing  isolation  and  purification  of  intermediates  and  reduce  waste  gene configuration. Various substituted derivatives of the  D‐amino acids were iso 42h  41  L ‐AAD,  L ‐amino acid deaminase; MDPD, meso‐diaminopimelate dehydrogenase.  minimum.  (69–83%) and excellent optical purities (95% to >99% ee).  42i  26 

In  most  of  these  multi‐enzyme  sequences,  the  lactone  is  formed  in  the  final  st a Reaction conditions: Substrate 42 (50 mM), (S)‐1‐ph 4. Lactones  Scheme 13. Deracemization cascade for the preparation of  D oxidation of a cyclic ketone by a Baeyer–Villiger monooxygenase (BVMO). However, s   (30 mg/mL), TA (20 mg/mL), Tris‐HCl buffer (100 mM L‐AAD, L‐amino acid deaminase; MDPD, meso‐diaminopim derive the lactone from an acyclic precursor have also been reported.  Lactones—and particularly γ‐ and δ‐lactones—are important flavour and fragra that  are  found  in  various  fruits,  juices,  wines,  and  spirits  [67].  Furthermore, Phenylalanine and derivatives thereof were dera   4. Lactones  butyrolactones occur as a central motif in many natural products, for instance nephro expressing an  L‐amino acid deaminase and an engin Scheme 14. 14.  Transformation  of  cyclohexanol  (47)  to  ɛ‐caprolactone  (49)  using  an is alcohol  D‐amino acids 45. arctigenin  [68].  ɛ‐Caprolactone,  on  the  other  hand,  a  valuable  chemical  for  the Scheme Transformation of cyclohexanol (47) toScheme 13. Deracemization cascade for the preparation of  -caprolactone (49) using an alcohol dehydrogenase Lactones—and particularly γ‐ and δ‐lactones—are imp optically  pure  D‐phenylalanines  (Scheme  13)  [66]. dehydrogenase (ADH) and a Baeyer–Villiger monooxygenase (BVMO).  L ‐AAD,  L ‐amino acid deaminase; MDPD, meso‐diaminopimelate dehydrogen (ADH) and a Baeyer–Villiger monooxygenase (BVMO). biodegradable polymers, such as polycaprolactone [69] and a precursor for ɛ‐caprola that  racemate, optically pure  are  found  in  various  fruits,  juices,  wines,  and  s L‐enantiomers were used as The chemical synthesis of these compounds usually involves a large number of st butyrolactones occur as a central motif in many natural pro configuration. Various substituted derivatives of the Both studies show that an efficient transformation of cyclohexanol into ɛ‐caprolactone is feasible  4. Lactones  yields, toxic or otherwise hazardous reagents, and impractical reaction conditions, wh arctigenin  [68].  the  other  hand,  is  a  v Both studies show that an efficient transformation of cyclohexanol into ɛ‐Caprolactone,  -caprolactone is on  feasible (69–83%) and excellent optical purities (95% to >99% e (94% conversion of 60 mM 47, 80% conversion of 10 mM 47); however, both also observed reduced  neither  “green”  nor  atom‐economic  [70–73].  biocatalytic  biodegradable polymers, such as polycaprolactone [69] and (94% conversion of 60 mM 47, 80% conversion ofLactones—and particularly γ‐ and δ‐lactones—are important flavour  10 mM 47); however, both also Hence,  observed reduced cascade  transfo conversions at elevated substrate concentrations which was attributed to inhibition and deactivation  become a useful tool in the preparation of lactones, as they avoid the necessity of t that  which are  found  in  The chemical synthesis of these compounds usually inv various  fruits,  juices,  wines,  and  spirits  [67].  Fur conversions at elevated substrate concentrations was attributed and deactivation   to inhibition of the Baeyer–Villiger monooxygenase. This issue was addressed in subsequent studies carried out  and  yield‐reducing  and  purification  of  intermediates  and  reduce  waste  yields, toxic or otherwise hazardous reagents, and impracti of the Baeyer–Villiger monooxygenase. This butyrolactones occur as a central motif in many natural products, for insta issue was isolation  addressed in subsequent studies carried out as as a joint effort of the two involved research groups: [76,77] The ɛ‐caprolactone formed underwent in  minimum.  neither  “green”  nor  atom‐economic  [70–73].  Hence,  bio arctigenin  [68].  ɛ‐Caprolactone,  on  the  other  hand,  is  chemi a joint effort of the two involved research groups: [76,77] The -caprolactone formed underwent in situa  valuable  situ  ring‐opening  oligomerisation  enabled  by of  lipase  A multi‐enzyme  from  Candida sequences,  antarctica  (CAL‐A),  which  In  most  these  the  lactone  is  formed  in  the  fin become a useful tool in the preparation of lactones, as the biodegradable polymers, such as polycaprolactone [69] and a precursor fo ring-opening oligomerisation enabled by lipase A from Candida antarctica (CAL-A), which alleviated alleviated  product  inhibition  and  produced  oligo‐ ‐caprolactone  displaying  an  average  molecular  Scheme 13. Deracemization cascade for the preparation of  and  yield‐reducing  isolation  and  purification  of  intermed The chemical synthesis of these compounds usually involves a large nu product inhibition and producedoxidation of a cyclic ketone by a Baeyer–Villiger monooxygenase (BVMO). Howev oligo--caprolactone displaying an average molecular weight ofD‐amino acids 45. En weight of 375 g/mol. It is worth noting that no hydrolysis of 49 to the hydroxycarboxylic acid was  L‐AAD, L‐amino acid deaminase; MDPD, meso‐diaminopimelate dehydrogenase derive the lactone from an acyclic precursor have also been reported.  minimum.  yields, toxic or otherwise hazardous reagents, and impractical reaction cond 375 g/mol. It is worth noting that no hydrolysis of 49 to the hydroxycarboxylic acid was observed. observed.  In  an  additional  optimisation  approach,  the  authors  chose  to  use  a  stabilised  variant  of  most  of variant these  multi‐enzyme  sequences,  the casca lacto neither chose “green”  nor  atom‐economic  [70–73].  Hence,  biocatalytic  In an additional optimisation approach, the authors to use aIn stabilised of cyclohexanone cyclohexanone monooxygenase (CHMO variant C376L/M400I) [78] and to employ the ADH and the  4. Lactones  oxidation of a cyclic ketone by a Baeyer–Villiger monooxy become a useful tool in the preparation of lactones, as they avoid the ne monooxygenase (CHMO variant C376L/M400I) [78] and to employ the ADH and the monooxygenase monooxygenase as separate E. coli whole‐cell preparations. Moreover, despite the theoretical redox‐ derive the lactone from an acyclic precursor have also been and  yield‐reducing  and  purification nature of  intermediates  and  redu Lactones—and particularly γ‐ and δ‐lactones—are important flavour an as separate E. coli whole-cell preparations. Moreover, despite theisolation  theoretical redox-neutral of neutral nature of the sequence, acetone and  D‐glucose (1 equivalent each) were added to improve  minimum.  that  are each) found  in added various  fruits,  juices,  wines,  and  spirits  [67].  Furth the sequence, acetone and D-glucose (1 equivalent were to improve cofactor regeneration. cofactor regeneration. These reaction conditions enabled the conversion of 200 mM cyclohexanol (47)  In  of most  these  multi‐enzyme  sequences,  lactone  is  formed  butyrolactones occur as a central motif in many natural products, for instanc These reaction conditions enabled the conversion 200 of  mM cyclohexanol (47) within 48 h the  going within 48 h going to completion thereby affording a mixture of 75% oligo‐ɛ‐caprolactone and 25% of  oxidation of a cyclic ketone by a Baeyer–Villiger monooxygenase (BVMO arctigenin  [68].  ɛ‐Caprolactone,  on  25% the  other  hand,  is  a  to completion thereby affording a mixture of 75% oligo-caprolactone and of monomeric 49.valuable  chemical monomeric  49.  Recently,  the  co‐expression  of  CHMO  and  ADH  in  E.  coli  using  a  Duet™  vector  derive the lactone from an acyclic precursor have also been reported.  biodegradable polymers, such as polycaprolactone [69] and a precursor for ɛ Recently, the co-expression of CHMO and ADH in E. coli using a Duet™ vector allowed higher allowed  higher  conversion  values  of  the  substrate  47  in  whole‐cell  biocatalysis  compared  to  an  Scheme 13. Deracemization cascade for the preparatio The chemical synthesis of these compounds usually involves a large num conversion values of the substrate 47 in whole-cell biocatalysis compared to an expression of both expression  of  both  enzymes  from  two  separate  plasmids  [79].  Alternatively,  fusion  of  the  alcohol  L‐AAD, L‐amino acid deaminase; MDPD, meso‐diamin yields, toxic or otherwise hazardous reagents, and impractical reaction condit enzymes from two separate plasmids [79]. Alternatively, fusion of the alcohol dehydrogenase and dehydrogenase and the BMVO also allowed to engineer the reaction [80]. The same cascade could  neither [80]. “green”  nor  cascade atom‐economic  [70–73].  Hence,  biocatalytic  cascade the BMVO also allowed to engineer the reaction The same could also be achieved using also be achieved using whole cells of Geotrichum candidum CCT 1205 [81].    4. Lactones  become a useful tool in the preparation of lactones, as they avoid the neces whole cells of Geotrichum candidum CCT 1205 [81]. The cascade mentioned above has also been extended by a rhodium‐catalyzed hydrogenation  and been yield‐reducing  and  purification  of  intermediates  and  reduce Lactones—and particularly γ‐ and δ‐lactones—ar The cascade mentioned above has also extended byisolation  a rhodium-catalyzed hydrogenation step that converts phenol into cyclohexanol (47), which is then employed in the biocatalytic cascade  minimum.  that  are  found  in  various  fruits,  juices,  wines,  a step that converts phenol into cyclohexanol (47), which is then employed in the biocatalytic cascade without purification [82].  In  most  of  these  butyrolactones occur as a central motif in many natur multi‐enzyme  sequences,  the  lactone  is  formed  in  without purification [82]. The formation and in situ oligomerisation of a chiral lactone, (S)‐4‐methyl‐ɛ‐caprolactone (51),  oxidation of a cyclic ketone by a Baeyer–Villiger monooxygenase (BVMO).  arctigenin  [68].  ɛ‐Caprolactone,  on  the  other  hand,  The formation and in situ oligomerisation of a chiral lactone, (S)-4-methyl-caprolactone (51), from  4‐methylcyclohexanol  (used  as  a  mixture  of  cis  and  trans  isomers)  via  the  same  biocatalytic  derive the lactone from an acyclic precursor have also been reported.  from 4-methylcyclohexanol (used as a mixture of cis and transbiodegradable polymers, such as polycaprolactone [6 isomers) via the same biocatalytic cascade was also demonstrated (Scheme 15) [83].  The chemical synthesis of these compounds usua cascade was also demonstrated (Scheme 15) [83]. yields, toxic or otherwise hazardous reagents, and imp neither  “green”  nor  atom‐economic  [70–73].  Henc become a useful tool in the preparation of lactones,  and  yield‐reducing  isolation  and  purification  of  int minimum.  In  most  of  these  multi‐enzyme  sequences,  the

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Catalysts 2018, 8, x FOR PEER REVIEW   

13 of 23  Scheme 13. Deracemization cascade for the preparation of  D‐a

Catalysts 2018, 8, x FOR PEER REVIEW     

L‐AAD, L‐amino acid deaminase; MDPD, meso‐diaminopimela 13 of 23 

 

4. Lactones 

Scheme 13. Deracemization cascade for the preparatio Lactones—and particularly γ‐ and δ‐lactones—are impo L‐AAD, L‐amino acid deaminase; MDPD, meso‐diamino that  are  found  in  various  fruits,  juices,  wines,  and  spir   butyrolactones occur as a central motif in many natural produ 4. Lactones  Scheme  15.  Conversion  of  4‐methylcyclohexanol  (50)  into  oligo‐(S)‐4‐methyl‐ɛ‐caprolactone  (52)  arctigenin  [68].  ɛ‐Caprolactone,  Scheme 15. Conversion of 4-methylcyclohexanol (50) into oligo-(S)-4-methyl-caprolactone (52)on  using   the  other  hand,  is  a  val using an alcohol dehydrogenase (ADH), a Baeyer–Villiger monooxygenase (BVMO), and a lipase.  an alcohol dehydrogenase (ADH), a Baeyer–Villiger monooxygenase (BVMO), and a lipase. biodegradable polymers, such as polycaprolactone [69] and a Lactones—and particularly γ‐ and δ‐lactones—ar Scheme  15.  Conversion  of  4‐methylcyclohexanol  (50)  into  oligo‐(S)‐4‐methyl‐ɛ‐caprolactone  (52)  The chemical synthesis of these compounds usually invo that  are  found  in  various  fruits,  juices,  wines,  an using an alcohol dehydrogenase (ADH), a Baeyer–Villiger monooxygenase (BVMO), and a lipase.  When  the  alcohol‐to‐lactone  cascade  system  is  extended  upstream  by  a  hydroxylation  step,  yields, toxic or otherwise hazardous reagents, and impractical butyrolactones occur as a central motif in many natura When alcohol-to-lactone cascade system is extended upstream by a hydroxylation lactones the can  be  obtained  directly  from  cycloalkanes.  The  formation  of  ɛ‐caprolactone  (49),  ζ‐ step, When  the obtained alcohol‐to‐lactone  system  is  extended  upstream  by nor  a of hydroxylation  step,  [70–73].  neither  “green”  atom‐economic  Hence,  bioca arctigenin  [68].  ɛ‐Caprolactone,  on  the  other  hand,  i enantholactone (55a, n = 2) and η‐caprylolactone (55b, n = 3) from cyclohexane, cycloheptane, and  lactones can be directly cascade  from cycloalkanes. The formation -caprolactone (49), lactones  can  be  obtained  directly  from  cycloalkanes.  The  formation  of  ɛ‐caprolactone  (49),  ζ‐ cyclooctane, respectively, according to this concept has been demonstrated using a variant of the P450  become a useful tool in the preparation of lactones, as they  biodegradable polymers, such as polycaprolactone [69 ζ-enantholactone (55a, n = 2) and η-caprylolactone (55b, n = 3) from cyclohexane, cycloheptane, enantholactone (55a, n = 2) and η‐caprylolactone (55b, n = 3) from cyclohexane, cycloheptane, and  monooxygenase BM3 from Bacillus megaterium for introducing the alcohol functionality in the first  and  yield‐reducing  isolation  and  purification  of  intermedia The chemical synthesis of these compounds usual and cyclooctane, respectively, according to this concept has been demonstrated using a variant of the cyclooctane, respectively, according to this concept has been demonstrated using a variant of the P450  step (Scheme 16) [84]. The subsequent oxidation to the ketone was performed by an ADH from either  minimum.  yields, toxic or otherwise hazardous reagents, and imp P450 monooxygenase BM3 from Bacillus megaterium for introducing the alcohol functionality in the monooxygenase BM3 from Bacillus megaterium for introducing the alcohol functionality in the first  a  Thermus  sp.  or  from  Thermoanaerobacter  ethanolicus,  whereby  the  latter  gave  the  better  results  in  In neither  most  these  multi‐enzyme  sequences,  the  lactone “green”  nor  [70–73].  Hence first step (Scheme 16) [84]. The subsequent oxidation to the ketone wasof  performed by atom‐economic  an ADH from step (Scheme 16) [84]. The subsequent oxidation to the ketone was performed by an ADH from either  terms of conversion. The final oxidation to the desired lactone was achieved using a stabilised variant  oxidation of a cyclic ketone by a Baeyer–Villiger monooxyg become a useful tool in the preparation of lactones, a eithera aThermus  Thermussp.  sp.or orfrom  fromThermoanaerobacter  Thermoanaerobacter ethanolicus, whereby the latter gave the better results in ethanolicus,  whereby  the  latter  gave  the  better  results  in  of cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871.  derive the lactone from an acyclic precursor have also been re and  yield‐reducing  isolation variant and  purification  of  inte termsterms of conversion. The final oxidation to the desired lactone was achieved using a stabilised variant  of conversion. The final oxidation to the desired lactone was achieved using a stabilised of cyclohexanone 9871. of cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871.    monooxygenase from Acinetobacter sp. NCIMBminimum.  In  most  of  these  multi‐enzyme  sequences,  the    oxidation of a cyclic ketone by a Baeyer–Villiger mon derive the lactone from an acyclic precursor have also

  Scheme 16. Biocatalytic synthesis of lactones from cycloalkanes using a P450 monooxygenase (P450),    an alcohol dehydrogenase (ADH), and a Baeyer–Villiger monooxygenase (BVMO).  Scheme 16. Biocatalytic synthesis of lactones from cycloalkanes using a P450 monooxygenase (P450),  Scheme 16. Biocatalytic synthesis of lactones from cycloalkanes using a P450 monooxygenase (P450), an alcohol dehydrogenase (ADH), and a Baeyer–Villiger monooxygenase (BVMO).  Although the use of a “designer cell” co‐expressing all three enzymes did allow production of 

an alcohol dehydrogenase (ADH), and a Baeyer–Villiger monooxygenase (BVMO). the  desired  lactones,  yields  were  generally  low  (less  than  1  mM  from  149–185  mM  cycloalkane).  Although the use of a “designer cell” co‐expressing all three enzymes did allow production of  Therefore, a cascade was tested instead in which the biocatalysts were added as separate cell‐free  Although the of yields  acofactor  “designer cell” co-expressing all by  three enzymes didcoli  allow production the  desired  lactones,  were  generally  low  than  1  mM  from  149–185  mM  cycloalkane).  extracts  and  in use which  regeneration  was (less  realised  endogenous  E.  enzymes  and  the of the desired lactones, yields were generally low (less than 1 mM from 149–185 mM cycloalkane). Therefore, Therefore, a cascade was tested instead in which the biocatalysts were added as separate cell‐free  simple  addition  of  D‐glucose  and  glycerol.  Using  this  reaction  setup,  3.2  mM  of  ζ‐enantholactone  extracts  and  in  which  cofactor  regeneration  was  realised  by  endogenous  E.  coli  enzymes  and  the  a cascade instead in which thewithin  biocatalysts were addedof  asan  separate cell-free and in (55b) was were tested formed  from  cycloheptane  20  h.  The  addition  external  cofactor extracts recycling  simple  addition  of  D ‐glucose  and  glycerol.  Using  this  reaction  setup,  3.2  mM  of  ζ‐enantholactone  system,  consisting  of  sodium  formate  and  a  formate  dehydrogenase  (FDH),  the  final  of which cofactor regeneration was realised by endogenous E. coli enzymes and increased  the simple addition (55b) and were glycerol. formed  from  h. mM The  of addition  of  an  external  cofactor  concentration of 55b to 10.6 mM, and raising the biocatalyst amounts by a factor of 3.75 resulted in  D-glucose Usingcycloheptane  this reactionwithin  setup,20  3.2 ζ-enantholactone (55b) wererecycling  formed from system,  consisting  of  sodium  formate  and  a  formate  dehydrogenase  (FDH),  increased  the  final  the formation of 23.3 mM 55b. Table 7 summarizes the results obtained with all three investigated  cycloheptane within 20 h. The addition of an external cofactor recycling system, consisting of sodium concentration of 55b to 10.6 mM, and raising the biocatalyst amounts by a factor of 3.75 resulted in  substrates.  formate and a formate dehydrogenase (FDH), increased the final concentration of 55b to 10.6 mM, and the formation of 23.3 mM 55b. Table 7 summarizes the results obtained with all three investigated  raising the biocatalyst amounts by a factor of 3.75 resulted in the formation of 23.3 mM 55b. Table 7 substrates.  Table 7. Results of the biocatalytic synthesis of lactones from cycloalkanes (Scheme 16) a. 

summarizes the results obtained with all three investigated substrates.

Product Concentrations (mM)  a.  Table 7. Results of the biocatalytic synthesis of lactones from cycloalkanes (Scheme 16)  Substrate  TTN b  Alcohol  Ketone  Lactone  Table 7. Results of the biocatalytic synthesis of lactones from cycloalkanes (Scheme 16) a . Product Concentrations (mM)  Cyclohexane  822  0  0  5.18  Substrate  TTN b  Alcohol  Ketone  Lactone  Cycloheptane  4185  0.48  2.63  23.25  Product Concentrations (mM) Cyclooctane  1017  0.27  2.40  3.74  Cyclohexane  0  0  5.18  b Substrate TTN 822  Ketone23.25  Lactone a Reaction conditions: Substrate 53 (149–185 mM), P450 (6.3 μM), ADH (cell‐free extract of 150 mg  Cycloheptane  4185  Alcohol 0.48  2.63  Cyclooctane  8221017  0.27  2.40  0 3.74  wet‐cell weight per mL), CHMO (cell‐free extract of 75 mg wet‐cell weight per mL), FDH (cell‐free  Cyclohexane 0 5.18

a Reaction conditions: Substrate 53 (149–185 mM), P450 (6.3 μM), ADH (cell‐free extract of 150 mg  Cycloheptane 4185 D‐glucose (100 mM), glycerol (100 mM), sodium formate  0.48 2.63 23.25 extract of 75 mg wet‐cell weight per mL),  + (100 μM), NADP+ (100 μM), Tris‐HCl buffer (200 mM, pH 8.0), 30 °C, 24 h.  b Total  wet‐cell weight per mL), CHMO (cell‐free extract of 75 mg wet‐cell weight per mL), FDH (cell‐free  Cyclooctane 1017 0.27 2.40 3.74 (100 mM), NAD a Reaction ‐glucose (100 mM), glycerol (100 mM), sodium formate  extract of 75 mg wet‐cell weight per mL),  turnover number: molar amount of products formed per molar amount of P450.  conditions: Substrate 53 (149–185 mM),DP450 (6.3 µM), ADH (cell-free extract of 150 mg wet-cell weight + (100 μM), NADP+ (100 μM), Tris‐HCl buffer (200 mM, pH 8.0), 30 °C, 24 h.  b Total  (100 mM), NAD per mL), CHMO (cell-free extract of 75 mg wet-cell weight per mL), FDH (cell-free extract of 75 mg wet-cell weight per mL), D -glucose (100 mM), glycerol (100 mM), sodium formate (100 mM), NAD+ (100 µM), NADP+ (100 µM), turnover number: molar amount of products formed per molar amount of P450.  Tris-HCl buffer (200 mM, pH 8.0), 30 ◦ C, 24 h. b Total turnover number: molar amount of products formed per molar amount of P450.

optically  pure (30 mg/mL), TA (20 mg/mL), Tris‐HCl buffer (100 mM, pH 8.0), DMSO (15% D‐phenylalanines  (Scheme  13)    [66].  Alternatively,  instead  of  racemate, optically pure  L‐enantiomers were used as substrate leading to inversi Scheme 13. Deracemization cascade for the preparation of D‐amino acids 45. Enzyme abbreviations:  configuration. Various substituted derivatives of the  D‐amino acids were isolated Phenylalanine and derivatives thereof were deracemized by a comb L‐AAD, L‐amino acid deaminase; MDPD, meso‐diaminopimelate dehydrogenase.  (69–83%) and excellent optical purities (95% to >99% ee).  expressing an  L‐amino acid deaminase and an engineered  D‐amino acid Catalysts 2018, 8, x FOR PEER REVIEW    14 of 23  optically  pure  D ‐phenylalanines  (Scheme  13)  [66].  Alternatively,  ins Catalysts 2018, 8, 205 14 of 24 4. Lactones  Scheme 13. Deracemization cascade for the preparat Catalysts 2018, 8, x FOR PEER REVIEW    14 of 23    racemate, optically pure LL‐AAD,  ‐enantiomers were used as substrate leading  Interestingly, alcohol dehydrogenases and BVMOs have been combined for lactone synthesis  L‐amino acid deaminase; MDPD, meso‐diami Lactones—and particularly γ‐ and δ‐lactones—are important flavour and fragrance compounds  also  in a  second,  entirely different  way:  The configuration. Various substituted derivatives of the  Baeyer–Villiger  oxidation  of  cyclohexanone (48)  to  ɛ‐D‐amino acids wer that  are  found  in  various  fruits, alcohol juices, dehydrogenases wines,  and  spirits  [67].  Furthermore,  substituted  Interestingly, and BVMOs have been combined forγ‐lactone synthesis Interestingly, alcohol dehydrogenases and BVMOs have been combined for lactone synthesis  (69–83%) and excellent optical purities (95% to >99% ee).  caprolactone  (49)  catalysed  by  cyclohexanone  monooxygenase  has  been  coupled  with  the  double  4. Lactones  butyrolactones occur as a central motif in many natural products, for instance nephrosteranic acid or  also in a second, entirely different way: The Baeyer–Villiger oxidation of cyclohexanone (48) also  in a  second,  entirely different  way:  The  Baeyer–Villiger  oxidation  of  cyclohexanone (48)  to to ɛ‐ oxidation of hexane‐1,6‐diol (56) to 49 (via the intermediate lactol 57) catalysed by the ADH from  arctigenin  [68].  caprolactone  ɛ‐Caprolactone,  on  the  other  hand,  is  a  valuable  chemical  for  the  preparation  of  Lactones—and particularly γ‐ and δ‐lactones—a -caprolactone(49)  (49) catalysed by cyclohexanone monooxygenase has been coupled with the double catalysed  by  cyclohexanone  monooxygenase  has  been  coupled  with  the  double    convergent  cascade  system  is  redox‐neutral  with  Thermoanaerobacter  ethanolicus  [85].  The  resulting  biodegradable polymers, such as polycaprolactone [69] and a precursor for ɛ‐caprolactam.  that  found  by in the various  oxidation of hexane-1,6-diol (56) to 49 (via the intermediate lactol 57)are  catalysed ADH fruits,  from juices,  wines,  oxidation of hexane‐1,6‐diol (56) to 49 (via the intermediate lactol 57) catalysed by the ADH from  respect to the nicotinamide cofactor and theoretically produces three molecules of ɛ‐caprolactone (49)  The chemical synthesis of these compounds usually involves a large number of steps, low overall  butyrolactones occur as a central motif in many natu Thermoanaerobacter ethanolicus [85]. The resulting convergent cascade system is redox-neutral with Thermoanaerobacter  ethanolicus  [85].  The  resulting  convergent  cascade  system  is  redox‐neutral  with  from two molecules of 48 and one molecule of 56 (Scheme 17).  yields, toxic or otherwise hazardous reagents, and impractical reaction conditions, which makes them  arctigenin  [68].  on  the  other  hand, respect to the nicotinamide cofactor and theoretically produces three molecules of ɛ‐Caprolactone,  -caprolactone (49) respect to the nicotinamide cofactor and theoretically produces three molecules of ɛ‐caprolactone (49)  neither  “green” from two molecules of 48 and one molecule of 56 (Scheme 17).  nor  two atom‐economic  Hence,  biocatalytic  cascade  have  biodegradable polymers, such as polycaprolactone [6 from molecules of [70–73].  48 and one molecule of 56 (Scheme 17).transformations    become a useful tool in the preparation of lactones, as they avoid the necessity of time‐consuming  The chemical synthesis of these compounds usua and  yield‐reducing  generation  to  a  yields, toxic or otherwise hazardous reagents, and im   isolation  and  purification  of  intermediates  and  reduce  waste  minimum.  neither  “green”  nor  atom‐economic  [70–73].  Hen In  most  of  these  multi‐enzyme  sequences,  the  lactone  is  formed  in  the  final  step  through  become a useful tool in the preparation of lactones, Scheme 13. Deracemization cascade for the preparation of  ‐amino acids 45. Enzyme oxidation of a cyclic ketone by a Baeyer–Villiger monooxygenase (BVMO). However, systems that  and  yield‐reducing  isolation Dand  purification  of  in L‐AAD, L‐amino acid deaminase; MDPD, meso‐diaminopimelate dehydrogenase.  derive the lactone from an acyclic precursor have also been reported.  minimum.  In  most  of  these  multi‐enzyme  sequences,  th 4. Lactones  oxidation of a cyclic ketone by a Baeyer–Villiger mo Scheme 13. Deracemization cascade for the preparation of  D‐amino acids 4 derive the lactone from an acyclic precursor have als Lactones—and particularly γ‐ and δ‐lactones—are important flavour and frag  

L‐AAD,  L‐amino acid deaminase; MDPD, meso‐diaminopimelate dehydroge that  are  found  in  various  fruits,  juices,  wines,  and  spirits  [67].  Furthermo Scheme  17.  Convergent  cascade  synthesis  of  ɛ‐caprolactone  (49)  from  1,6‐hexanediol  (56)  and    butyrolactones occur as a central motif in many natural products, for instance nep cyclohexanone  (48).  Enzyme  abbreviations:  ADH,  alcohol  dehydrogenase;  BVMO,  Baeyer–Villiger  4. Lactones  Scheme  17.  cascade  synthesis  of  ɛ‐caprolactone  (49) (49) from  1,6‐hexanediol  (56)  arctigenin  [68].  on from the  other  hand,  is  and  a  valuable  chemical  for  t Scheme 17. Convergent  Convergent cascade synthesis of ɛ‐Caprolactone,  -caprolactone 1,6-hexanediol (56) monooxygenase .  cyclohexanone  (48).  Enzyme  abbreviations:  ADH, Lactones—and particularly γ‐ and δ‐lactones—are important flavou alcohol  dehydrogenase;  BVMO,  Baeyer–Villiger  and cyclohexanone (48). Enzyme abbreviations: ADH, alcohol dehydrogenase; BVMO, Baeyer biodegradable polymers, such as polycaprolactone [69] and a precursor for ɛ‐capr –Villiger monooxygenase. monooxygenase .  The chemical synthesis of these compounds usually involves a large number o are  found  in  of  various  fruits,  juices,  wines,  and  spirits  [67].  Fu However,  in  practice  the  formation  of that  three  equivalents  ɛ‐caprolactone  (49)  was  never 

yields, toxic or otherwise hazardous reagents, and impractical reaction conditions,  observed  despite  complete  depletion  of  the butyrolactones occur as a central motif in many natural products, for inst starting  materials  due  to  undesired  hydrolysis  and  However,  in  practice  the  formation  of  three  equivalents  of  ɛ‐caprolactone  (49)  was  never  neither  “green”  nor  atom‐economic  [70–73].  Hence,  biocatalytic  cascade chem tran arctigenin  [68].  on was the  other  hand,  is  a  valuable  However, in practice the formation of three equivalents of ɛ‐Caprolactone,  -caprolactone (49) never observed oligomerisation of 49 under the reaction conditions. Nevertheless, up to 18.3 mM of 49 were formed  observed  despite  complete  depletion  of  the  starting  materials  due  to  undesired  hydrolysis  and  become a useful tool in the preparation of lactones, as they avoid the necessity o biodegradable polymers, such as polycaprolactone [69] and a precursor f despite complete depletion of the starting materials due to undesired hydrolysis and oligomerisation from 20 mM of 48 and 10 mM of 56 within a reaction time of 72 h, corresponding to an analytical  oligomerisation of 49 under the reaction conditions. Nevertheless, up to 18.3 mM of 49 were formed  and  yield‐reducing  isolation  purification  of  intermediates  The chemical synthesis of these compounds usually involves a large  of 49 under the reaction conditions. Nevertheless, up to 18.3 mM ofand  49 were formed from 20 mM of 48 and  reduce  was yield of 61%.  from 20 mM of 48 and 10 mM of 56 within a reaction time of 72 h, corresponding to an analytical  minimum.  yields, toxic or otherwise hazardous reagents, and impractical reaction co and 10 mM of 56asymmetric  within a reaction time ofof  72prochiral  h, corresponding to an analytical yield of 61%. by  ene‐ When  the  reduction  α,β‐unsaturated  ketones  catalysed  yield of 61%.  In  most  of  these  multi‐enzyme  sequences,  the  lactone  is  formed  in  the  neither  “green”  nor  atom‐economic  [70–73].  Hence,  biocatalytic  casf When the asymmetric reduction of prochiral α,β-unsaturated ketones catalysed by ene-reductases reductases  is  combined  with  the  oxidation  by  a  Baeyer–Villiger  monooxygenase  catalysed  by  When  the  asymmetric  reduction  of  prochiral  α,β‐unsaturated  ketones  catalysed  by  ene‐ oxidation of a cyclic ketone by a Baeyer–Villiger monooxygenase (BVMO). How become a useful tool in the preparation of lactones, as they avoid the n is combined with the oxidation by a Baeyer–Villiger monooxygenase catalysed by BVMOs, BVMOs, chiral lactones are obtained. This concept has first been put into practice in a biocatalytic  reductases  is  combined  with  the  oxidation  by  been a  Baeyer–Villiger  monooxygenase  catalysed  by  derive the lactone from an acyclic precursor have also been reported.  and  yield‐reducing  isolation  purification  of  intermediates  and  red chiral lactones are obtained. This concept has first put into18),  practice inare  aand  biocatalytic synthesis of synthesis  of  5‐alkyl‐substituted  δ‐valerolactones  60  (Scheme  which  flavour  and  fragrance  BVMOs, chiral lactones are obtained. This concept has first been put into practice in a biocatalytic  minimum.  5-alkyl-substituted compounds [86].  δ-valerolactones 60 (Scheme 18), which are flavour and fragrance compounds [86]. synthesis  of  5‐alkyl‐substituted  δ‐valerolactones  60  which  are  flavour  and  fragrance  In (Scheme  most  of  18),  these  multi‐enzyme  sequences,  the  lactone  is  formed compounds [86].  oxidation of a cyclic ketone by a Baeyer–Villiger monooxygenase (BVM   derive the lactone from an acyclic precursor have also been reported. 

 

  Scheme  18. 18.  Preparation  2‐alkylidenecyclopentanones  58.  Scheme Preparation of  of (R)‐δ‐valerolactones  (R)-δ-valerolactones 60  60 starting  starting from  from 2-alkylidenecyclopentanones 58.   Enzyme  abbreviations:  Baeyer–Villiger  monooxygenase;  GDH,  D‐ Enzyme abbreviations: ERED,  ERED,ene‐reductase;  ene-reductase;BVMO,  BVMO, Baeyer–Villiger monooxygenase; GDH, Scheme  18.  Preparation  of  (R)‐δ‐valerolactones  60  starting  from  2‐alkylidenecyclopentanones  58.  glucose dehydrogenase.  D -glucose dehydrogenase. Enzyme  abbreviations:  ERED,  ene‐reductase;  BVMO,  Baeyer–Villiger  monooxygenase;  GDH,  D‐ glucose dehydrogenase.  In this study, Acinetobacter sp. RS1 was shown to be the best organism for the C=C‐reduction 

In this study, Acinetobacter sp. RS1 was shown to be the best organism for the C=C-reduction step regarding activity, growth and enantioselectivity by screening 200 alkane‐, toluene‐ or benzene‐  step In this study, Acinetobacter sp. RS1 was shown to be the best organism for the C=C‐reduction  regarding activity, growth and enantioselectivity by screening 200 alkane-, toluene- or benzenedegrading microbial strains. Already after 2 h, the reaction reached >99% conversion, producing (R)‐ degrading microbial strains. Already after 2 h, the reaction reached >99% conversion, producing step regarding activity, growth and enantioselectivity by screening 200 alkane‐, toluene‐ or benzene‐  59a  and  (R)‐59b  with  87%  and  92%  ee,  respectively.  For  the  following  Baeyer–Villiger  oxidation,  (R)-59a and (R)-59b with 87% and 92% ee, respectively. For the following Baeyer–Villiger oxidation, degrading microbial strains. Already after 2 h, the reaction reached >99% conversion, producing (R)‐ resting cells of Escherichia coli containing a cyclohexanone monooxygenase (CHMO) and a glucose  resting of Escherichia containing a cyclohexanone monooxygenase (CHMO) andoxidation,  a glucose 59a  and cells (R)‐59b  with  87% coli and  92%  ee,  respectively.  For  the  following  Baeyer–Villiger  dehydrogenase  (GDH)  were  employed.  In  initial  experiments,  an  undesired  oxidation  activity  on  dehydrogenase (GDH) were employed. In initial experiments, an undesired oxidation activity on resting cells of Escherichia coli containing a cyclohexanone monooxygenase (CHMO) and a glucose  substrates  58  was  observed;  consequently,  the  one‐pot  cascade  was  carried  out  in  a  sequential  substrates 58 was(GDH)  observed; consequently, theinitial  one-pot cascade was carried out inoxidation  a sequential fashion, dehydrogenase  were  employed.  In  experiments,  an  undesired  activity  on  fashion,  producing  (R)‐60a  and  (R)‐60b  with  83%  and  66%  conversion  and  98%  and  97%  ee,  producing (R)-60a and (R)-60b with 83% and 66% conversion and 98% and 97% ee, respectively. substrates  58  was  observed;  consequently,  the  one‐pot  cascade  was  carried  out  in  a  sequential  respectively. The improved enantiomeric excess of the products 60 compared to the intermediates 59  fashion,  producing  (R)‐60a  and  (R)‐60b  with  83%  and  66%  conversion  and  98%  and  97%  ee,  respectively. The improved enantiomeric excess of the products 60 compared to the intermediates 59 

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The improved enantiomeric excess of the products 60 compared to the intermediates 59 was shown was shown to be a consequence of the hydrolysis of the lactone catalysed by the Acinetobacter cells.  was shown to be a consequence of the hydrolysis of the lactone catalysed by the Acinetobacter cells.  to be a consequence of the hydrolysis of the catalysed by the Acinetobacter cells. Thereby was shown to be a consequence of the hydrolysis of the lactone catalysed by the Acinetobacter cells.  Thereby  the  (S)‐enantiomers  of  lactones  60 lactone were  preferentially  converted  (E  =  8–11).  Hence,  the the  Thereby  the  (S)‐enantiomers  of  lactones  60  were  preferentially  converted  (E  =  8–11).  Hence,  the of (S)-enantiomers of lactones were preferentially converted (E = 8–11). Hence, the optical Thereby  the  (S)‐enantiomers  of 60 lactones  60  were  preferentially  converted  (E  =  8–11).  Hence, purity the  optical purity of the desired products increased over time due to removal of the minor enantiomer.  the desired over time due to removal of the minor enantiomer. As a(40  final proof of optical purity of the desired products increased over time due to removal of the minor enantiomer.  optical purity of the desired products increased over time due to removal of the minor enantiomer.  As  a  final  products proof  of  increased concept,  preparative‐scale  transformations  with  58a  and  58b  mg)  were  As As  a  final  proof  of  concept,  preparative‐scale  transformations  with  58a  and  58b  (40  mg)  were  concept, preparative-scale transformations with 58a and 58b (40 mg) were performed and the lactones a  final  proof  of  concept,  preparative‐scale  transformations  with  58a  and  58b  (40  mg)  were  performed  and  the  lactones  60a  and  60b  were  isolated  in  56%  and  41%  yield  (98%  and  97%  ee),  performed  and  the  lactones  and  60b  were  isolated  in  56%  and  41%  yield  (98%  and  97%  ee),  60a andand  60b were isolated in60a  56% and 41% yield (98% in  and 97% ee), respectively. performed  the  lactones  60a  and  60b  were  isolated  56%  and  41%  yield  (98%  and  97%  ee),  respectively.  A similar combination of ene-reductases and Baeyer–Villiger monooxygenases, extended by an respectively.  respectively.  A similar combination of ene‐reductases and Baeyer–Villiger monooxygenases, extended by an  A similar combination of ene‐reductases and Baeyer–Villiger monooxygenases, extended by an  A similar combination of ene‐reductases and Baeyer–Villiger monooxygenases, extended by an  ADH-catalysed step, has been investigated for the preparation of ε-caprolactone derivatives 64 from ADH‐catalysed step, has been investigated for the preparation of ε‐caprolactone derivatives 64 from  ADH‐catalysed step, has been investigated for the preparation of ε‐caprolactone derivatives 64 from  cyclohexenol derivatives 61 (Scheme This particular  particular cascade  cascade was  was termed  termed an  an “artificial ADH‐catalysed step, has been investigated for the preparation of ε‐caprolactone derivatives 64 from  cyclohexenol  derivatives  61  (Scheme  19) 19)  [87,88]. [87,88].  This  “artificial  cyclohexenol  derivatives  61  (Scheme  19)  [87,88].  This  particular  cascade  was  termed  an  “artificial  cyclohexenol  derivatives  61  (Scheme  19)  [87,88].  This  particular  cascade  was  termed  an  “artificial in metabolic mini pathway” by the authors, meaning that all the required enzymes were co-expressed metabolic mini pathway” by the authors, meaning that all the required enzymes were co‐expressed  metabolic mini pathway” by the authors, meaning that all the required enzymes were co‐expressed  E. coli, while in nature they are not connected in a single organism in a metabolic context. metabolic mini pathway” by the authors, meaning that all the required enzymes were co‐expressed  in E. coli, while in nature they are not connected in a single organism in a metabolic context.  in E. coli, while in nature they are not connected in a single organism in a metabolic context.  in E. coli, while in nature they are not connected in a single organism in a metabolic context. 

   

 

    Scheme 19. “Artificial metabolic mini pathway” for the synthesis of ε‐caprolactone derivatives 64. 

 

Scheme 19. “Artificial metabolic mini pathway” for the synthesis of ε-caprolactone derivatives Scheme 19. “Artificial metabolic mini pathway” for the synthesis of ε‐caprolactone derivatives 64.  Scheme 19. “Artificial metabolic mini pathway” for the synthesis of ε‐caprolactone derivatives 64.  Enzyme abbreviations: ADH, alcohol dehydrogenase; ERED, ene‐reductase; BVMO, Baeyer–Villiger  64. Enzyme abbreviations: ADH, alcohol dehydrogenase; ERED, ene-reductase; BVMO, Enzyme abbreviations: ADH, alcohol dehydrogenase; ERED, ene‐reductase; BVMO, Baeyer–Villiger  Enzyme abbreviations: ADH, alcohol dehydrogenase; ERED, ene‐reductase; BVMO, Baeyer–Villiger  Enzyme abbreviations: ADH, alcohol dehydrogenase; ERED, ene‐reductase; BVMO, Baeyer–Villiger  monooxygenase.  Baeyer–Villiger monooxygenase. monooxygenase.  monooxygenase.  monooxygenase. 

Suitable enzyme candidates for each individual step of the cascade were identified whereby the  Suitable enzyme candidates for each individual step of the cascade were identified whereby Suitable enzyme candidates for each individual step of the cascade were identified whereby the  Suitable enzyme candidates for each individual step of the cascade were identified whereby the  ADH  from  Lactobacillus  kefir,  the  ene‐reductase  XenB  from  Pseudomonas  sp.,  and  the  well‐known  the ADH from Lactobacillus kefir, the ene-reductase XenB from Pseudomonas sp., and thewell‐known  well-known ADH  from  Lactobacillus  kefir,  the  ene‐reductase  XenB  from  Pseudomonas  sp.,  and  the  ADH  from  Lactobacillus  kefir,  the  ene‐reductase  XenB  from  Pseudomonas  sp.,  and  the  well‐known  cyclohexanone  monooxygenase  from  Acinetobacter  sp.  performed  best  for  most  of  tested  cyclohexanone monooxygenase from Acinetobacter sp. performed best for most of the tested the  substrates. cyclohexanone  monooxygenase  from  Acinetobacter  sp.  performed  best  for  most  of  the  tested  cyclohexanone  monooxygenase  from  Acinetobacter  sp.  performed  best  for  most  of  the  tested  substrates.  Cofactor  regeneration  was via performed  via  the ofmetabolism  of  the  E.  coli  host.  Table  8  Cofactor regeneration was performed the metabolism the E. coli host. Table 8 summarises the substrates.  Cofactor  regeneration  was  performed  via  the  metabolism  the  coli  host.  Table  substrates.  Cofactor  regeneration  was  performed  via  the  metabolism  of of  the  E. E.  coli  host.  Table  8  8  summarises the results of the substrates tested.    results of the substrates tested. summarises the results of the substrates tested.  summarises the results of the substrates tested.      Table  8. 8.  Overview  for for the the synthesis  of  of ε‐ Table Overviewof  ofresults  resultsof ofthe  the“artificial  “artificialmetabolic  metabolicmini  minipathway”  pathway” synthesis Table  8.  Overview  of  results  of  the  “artificial  metabolic  mini  pathway”  for  the  synthesis  of  ε‐ a Table  8.  Overview  of  results  of  the  “artificial  metabolic  mini  pathway”  for  the  synthesis  of  ε‐ a Table  8.  Overview  of  results  of  the  “artificial  metabolic  mini  pathway”  for  the  synthesis  of  ε‐ caprolactone derivatives 64 (Scheme 19)  ε-caprolactone derivatives 64 (Scheme 19).  . a.  caprolactone derivatives 64 (Scheme 19)  aa.  a.  caprolactone derivatives 64 (Scheme 19)  caprolactone derivatives 64 (Scheme 19)  Substrate  Product  Time (h)  Conv. (%)  ee/de (%)  Substrate Product Time (h) Conv.ee/de (%)  (%) ee/de (%) Substrate  Product  Time (h)  Time (h)  Conv. (%)  ee/de (%)  Substrate  Product  Conv. (%)  Substrate  Product  Time (h)  Conv. (%)  ee/de (%) 

 

>99  n.a. b  b bb  n.a. b >99 >99  n.a.  >99  n.a.   

20  20 20 20 20 

>99  >99  >99  >99 >99  >99  >99 >99  >99  >99 

20  20 20 20 20 

64  >99  64  64 >99  >99  >99 >99  64 64 

b

 

    

   

      

     

      

21  21 21 21

      

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Table 8. Cont. Catalysts 2018, 8, x FOR PEER REVIEW  Catalysts 2018, 8, x FOR PEER REVIEW        Catalysts 2018, 8, x FOR PEER REVIEW  Catalysts 2018, 8, x FOR PEER REVIEW  Catalysts 2018, 8, x FOR PEER REVIEW    Product Catalysts 2018, 8, x FOR PEER REVIEW  Catalysts 2018, 8, x FOR PEER REVIEW  Substrate       Catalysts 2018, 8, x FOR PEER REVIEW  Catalysts 2018, 8, x FOR PEER REVIEW 

Time (h)

Conv. (%)

ee/de (%)

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Table 6. Transformation of β‐keto esters 42 into β‐amino acids 44 (Scheme 12) a. 

         

Substrate  Conv. (%)  ee (%)  86  86 20 20 20  86  42a  89  >99%  86  20 20  86  20  86  20  86  42b  96  >99%  42c  75  >99%      42d  73  >99%        42e  99  >99%  42f  61  >99%  42g  47  >99%  63  63 20 20  63  42h  41  >99%  20 63  20 20  63  20  63  20  63  42i  26  >99% 

>99  >99  >99  >99  >99  >99 

>99

>99  >99  >99  >99  >99  >99 

>99

 Reaction conditions: Substrate 42 (50 mM), (S)‐1‐phenylethylamine (150 mM), PLP (1 mM), lipase  (30 mg/mL), TA (20 mg/mL), Tris‐HCl buffer (100 mM, pH 8.0), DMSO (15% v/v), 37 °C, 24 h.                      a

Phenylalanine and derivatives thereof were deracemized by a combination of E. coli whole cells  expressing an  L‐amino acid deaminase and an engineered  D‐amino acid dehydrogenase to prepare  optically  pure  D‐phenylalanines  (Scheme  13) 20  [66].  instead  of  starting  from  the  20  Alternatively,  >99  93 93  >99  20  93  >99  20 93 >99 20  93  >99  20  93  >99  20  93  >99  racemate, optically pure  L‐enantiomers were used as substrate leading to inversion of the absolute  configuration. Various substituted derivatives of the  D‐amino acids were isolated with good yields  (69–83%) and excellent optical purities (95% to >99% ee).           

 

         

20 20  20 20  20 20 20 

>99  >99  >99  >99  >99  >99  >99  >99  >99  >99 >99  >99  >99 

>99

                    a Reaction conditions: Substrate (2.5–4.0 mM), resting E. coli cells (concentration not reported), 24 °C,  a  Reaction conditions: Substrate (2.5–4.0 mM), resting E. coli cells (concentration not reported), 24 °C,  aa Reaction conditions: Substrate (2.5–4.0 mM), resting E. coli cells (concentration not reported), 24 °C,   Reaction conditions: Substrate (2.5–4.0 mM), resting E. coli cells (concentration not reported), 24 °C,  a Reaction conditions: Substrate (2.5–4.0 mM), resting E. coli cells (concentration not reported), 24 ◦ C, 48 h. b n.a.:  Reaction conditions: Substrate (2.5–4.0 mM), resting E. coli cells (concentration not reported), 24 °C,   Reaction conditions: Substrate (2.5–4.0 mM), resting E. coli cells (concentration not reported), 24 °C,  b b b b n.a.: not applicable.  48 h.   n.a.: not applicable.  48 h.  b b n.a.: not applicable.  bapplicable. b  n.a.: not applicable.  48 h.  48 h.  not  n.a.: not applicable.  48 h.   n.a.: not applicable.  48 h. 

aa aa

Interestingly,  substrate  rac‐61d  was  oxidized  by  ADH  from  Lactobacillus  kefir  with  100%  Interestingly,  substrate  rac‐61d  was  oxidized  by  ADH  from  Lactobacillus  kefir  with  100%  Interestingly,  substrate  rac‐61d  was  oxidized  by  ADH  from  Lactobacillus  kefir  with  100%  Interestingly, substrate rac-61d was oxidized by ADH from Lactobacillus kefir with 100% conversion, Interestingly,  substrate  rac‐61d  was  oxidized  by  ADH  from  Lactobacillus  kefir  with  100%  Interestingly,  substrate  rac‐61d  was  oxidized  by  ADH  from  Lactobacillus  kefir  with  100%  conversion, but the ene‐reductase from Pseudomonas sp. did not accept intermediate 62d, although it  conversion, but the ene‐reductase from Pseudomonas sp. did not accept intermediate 62d, although it  conversion, but the ene‐reductase from Pseudomonas sp. did not accept intermediate 62d, although it  but the ene-reductase from Pseudomonas sp. did not accept intermediate 62d, although it was active on conversion, but the ene‐reductase from Pseudomonas sp. did not accept intermediate 62d, although it  conversion, but the ene‐reductase from Pseudomonas sp. did not accept intermediate 62d, although it    was active  on on  the  regioisomeric  62c.  Therefore,  an additional  ERED  was  investigated,  namely  old  was active  the  regioisomeric  62c.  Therefore,  an additional  ERED  was  investigated,  namely  old  was active  the  regioisomeric  62c.  Therefore,  an additional  ERED  was  investigated,  namely  old  1 the regioisomeric 62c. Therefore, an additional ERED was investigated, namely old yellow enzyme was active  on on  the  regioisomeric  62c.  Therefore,  an additional  ERED  was  investigated,  namely  old  was active  on  the  regioisomeric  62c.  Therefore,  an additional  ERED  was  investigated,  namely  old  yellow  enzyme  1  (OYE1)  from  Saccharomyces  carlsbergensis,  which  yielded  optically  pure  (S)‐63d,  Scheme 13. Deracemization cascade for the preparation of  D‐amino acids 45. Enzyme abbreviations:  yellow  enzyme  1  from  Saccharomyces  carlsbergensis,  which  yielded  optically  pure  (S)‐63d,  yellow  enzyme  1  (OYE1)  (OYE1)  from  Saccharomyces  carlsbergensis,  which  yielded  optically  pure  (S)‐63d,  (OYE1) from1  carlsbergensis, which yielded optically pure (S)-63d, whichpure  was then further yellow  enzyme  1 Saccharomyces (OYE1)  from  Saccharomyces  carlsbergensis,  which  yielded  optically  pure  (S)‐63d,  yellow  enzyme  (OYE1)  from  Saccharomyces  carlsbergensis,  which  yielded  optically  (S)‐63d,  which was then further oxidized to lactone (S)‐64d.    which was then further oxidized to lactone (S)‐64d.    L‐AAD, L‐amino acid deaminase; MDPD, meso‐diaminopimelate dehydrogenase.  which was then further oxidized to lactone (S)‐64d.  oxidized to lactone (S)-64d. which was then further oxidized to lactone (S)‐64d.  which was then further oxidized to lactone (S)‐64d.       Preparative‐scale biotransformations with substrates rac‐61d and (1S,5S)‐61e (100 mg) afforded  Preparative‐scale biotransformations with substrates rac‐61d and (1S,5S)‐61e (100 mg) afforded  Preparative‐scale biotransformations with substrates rac‐61d and (1S,5S)‐61e (100 mg) afforded  Preparative-scale biotransformations with substrates rac-61d and (1S,5S)-61e (100 mg) afforded Preparative‐scale biotransformations with substrates rac‐61d and (1S,5S)‐61e (100 mg) afforded  Preparative‐scale biotransformations with substrates rac‐61d and (1S,5S)‐61e (100 mg) afforded  4. Lactones  the products (S)‐64d and (3R,6R)‐64e in 55% and 60% isolated yield, respectively, and in >99% ee/de.  the products (S)‐64d and (3R,6R)‐64e in 55% and 60% isolated yield, respectively, and in >99% ee/de.  the products (S)‐64d and (3R,6R)‐64e in 55% and 60% isolated yield, respectively, and in >99% ee/de.  the products (S)-64d and (3R,6R)-64e in 55% and 60% isolated yield, respectively, and in >99% ee/de. the products (S)‐64d and (3R,6R)‐64e in 55% and 60% isolated yield, respectively, and in >99% ee/de.  the products (S)‐64d and (3R,6R)‐64e in 55% and 60% isolated yield, respectively, and in >99% ee/de.  In a subsequent study, three ene‐reductases from Pseudomonas putida were used in a very similar  In a subsequent study, three ene‐reductases from Pseudomonas putida were used in a very similar  In a subsequent study, three ene‐reductases from Pseudomonas putida were used in a very similar  InLactones—and particularly γ‐ and δ‐lactones—are important flavour and fragrance compounds  a subsequent study, three ene-reductases from Pseudomonas putida were used in a very similar In a subsequent study, three ene‐reductases from Pseudomonas putida were used in a very similar  In a subsequent study, three ene‐reductases from Pseudomonas putida were used in a very similar  cascade,  in in  this  case  implemented  using  isolated  enzymes  (purified  enzymes  and  crude  cell‐free  cascade,  case  implemented  using  isolated  enzymes  (purified  enzymes  and  crude  cell‐free  that  are  found  in  various  fruits,  juices,  wines,  and  spirits  [67].  Furthermore,  substituted  cascade,  in  this  case  implemented  using  isolated  enzymes  (purified  enzymes  and  crude  cell‐free  cascade, inthis  this case implemented using isolated enzymes (purified enzymes and crude cell-freeγ‐ cascade,  in  this  case  implemented  using  isolated  enzymes  (purified  enzymes  and  crude  cell‐free  cascade,  in  this  case  implemented  using  isolated  enzymes  (purified  enzymes  and  crude  cell‐free  extracts)  [89].  Cyclohexenol,  cyclohexanone,  and  cyclopentenone  were  investigated,  leading  to  extracts)  [89].  Cyclohexenol,  cyclohexanone,  and  cyclopentenone  were  investigated,  leading  to  δ‐ δ‐ butyrolactones occur as a central motif in many natural products, for instance nephrosteranic acid or  extracts)  [89].  Cyclohexenol,  cyclohexanone,  and  cyclopentenone  were  investigated,  leading  extracts) [89]. Cyclohexenol, cyclohexanone, and cyclopentenone were investigated, leading extracts)  [89].  Cyclohexenol,  cyclohexanone,  and  cyclopentenone  were  investigated,  leading  to to  δ‐ δ‐to extracts)  [89].  Cyclohexenol,  cyclohexanone,  and  cyclopentenone  were  investigated,  leading  to  δ‐ valerolactone or ɛ‐caprolactone as products. Conversions between 19% and 99% were obtained at a  valerolactone or ɛ‐caprolactone as products. Conversions between 19% and 99% were obtained at a  arctigenin  [68].  the  other  hand,  is  a between valuable  chemical  for  the  obtained preparation  valerolactone or ɛ‐caprolactone as products. Conversions between 19% and 99% were obtained at a  δ-valerolactone or ɛ‐Caprolactone,  -caprolactone ason  products. Conversions 19% and 99% were at a of  valerolactone or ɛ‐caprolactone as products. Conversions between 19% and 99% were obtained at a  valerolactone or ɛ‐caprolactone as products. Conversions between 19% and 99% were obtained at a  substrate concentration of 3 mM within 1 h.  substrate concentration of 3 mM within 1 h.  biodegradable polymers, such as polycaprolactone [69] and a precursor for ɛ‐caprolactam.  substrate concentration of 3 mM within 1 h.  substrate concentration of 3 mM within 1 h. substrate concentration of 3 mM within 1 h.  substrate concentration of 3 mM within 1 h.  The chemical synthesis of these compounds usually involves a large number of steps, low overall  4.2. Formation of Lactones from Acyclic Precursors  4.2. Formation of Lactones from Acyclic Precursors  4.2. Formation of Lactones from Acyclic Precursors 4.2. Formation of Lactones from Acyclic Precursors  4.2. Formation of Lactones from Acyclic Precursors  yields, toxic or otherwise hazardous reagents, and impractical reaction conditions, which makes them  4.2. Formation of Lactones from Acyclic Precursors  The  formation  form  acyclic  precursors  a  acommon  strategy  The  formation  of of  lactones  form  acyclic  precursors  is  is  not  only  a  common  strategy  in in  organic  neither  “green”  nor  atom‐economic  [70–73].  Hence,  biocatalytic  cascade  transformations  have  The formation oflactones  lactones form acyclic precursors isnot  notonly  only common strategy inorganic  organic The  formation  lactones  form  acyclic  precursors  not  only  a  common  strategy  organic  The  formation  of of  lactones  form  acyclic  precursors  is  is  not  only  a  common  common  strategy  in in  organic  The  formation  of  lactones  form  acyclic  precursors  is  not  only  a  strategy  in  organic  synthesis, but also takes place in the biosynthesis of many naturally occurring lactones, for instance  become a useful tool in the preparation of lactones, as they avoid the necessity of time‐consuming  synthesis, but also takes place in the biosynthesis of many naturally occurring lactones, for instance  synthesis, but also takes place in the biosynthesis of many naturally occurring lactones, for instance synthesis, but also takes place in the biosynthesis of many naturally occurring lactones, for instance  synthesis, but also takes place in the biosynthesis of many naturally occurring lactones, for instance  synthesis, but also takes place in the biosynthesis of many naturally occurring lactones, for instance  macrolides.  These  molecules  are  produced  by by  polyketide  synthases  (PKSs)—large  multi‐enzyme  and  yield‐reducing  isolation  and  purification  of  intermediates  and  reduce  waste  generation  to  a  macrolides.  These  molecules  are  produced  synthases  (PKSs)—large  multi‐enzyme  macrolides. These molecules are produced bypolyketide  polyketide synthases (PKSs)—large multi-enzyme macrolides.  These  molecules  are  produced  polyketide  synthases  (PKSs)—large  multi‐enzyme  macrolides.  These  molecules  are  produced  by by  polyketide  synthases  (PKSs)—large  multi‐enzyme  macrolides.  These  molecules  are  produced  by  polyketide  synthases  (PKSs)—large  multi‐enzyme  complexes—from acyclic precursors that are assembled by consecutive Claisen condensations while  complexes—from acyclic precursors that are assembled by consecutive Claisen condensations while  minimum.  complexes—from acyclic precursors that are assembled by consecutive Claisen condensations while  complexes—from acyclic precursors that are assembled by consecutive Claisen condensations while  complexes—from acyclic precursors that are assembled by consecutive Claisen condensations while  bound to an acyl‐carrier protein and are finally cyclised by a thioesterase domain of the PKS [90,91].  In  most  of  these  multi‐enzyme  sequences,  the  lactone  is  formed  in  the  final  step  through  bound to an acyl‐carrier protein and are finally cyclised by a thioesterase domain of the PKS [90,91].  bound to an acyl‐carrier protein and are finally cyclised by a thioesterase domain of the PKS [90,91].  bound to an acyl‐carrier protein and are finally cyclised by a thioesterase domain of the PKS [90,91].  bound to an acyl‐carrier protein and are finally cyclised by a thioesterase domain of the PKS [90,91].  The  exploitation  of of  this  biocatalytic  machinery  for for  polyketide  synthesis  in in  vitro  has  proven  oxidation of a cyclic ketone by a Baeyer–Villiger monooxygenase (BVMO). However, systems that  The  exploitation  this  biocatalytic  machinery  polyketide  synthesis  vitro  has  proven  The  exploitation  this  biocatalytic  machinery  polyketide  synthesis  vitro  has  proven  The  exploitation  of of  this  biocatalytic  machinery  for for  polyketide  synthesis  in in  vitro  has  proven  The  exploitation  of  this  biocatalytic  machinery  for  polyketide  synthesis  in  vitro  has  proven  challenging,  but  recently  researchers  have  succeeded  in  producing  triketide  lactones  via  multi‐ challenging,  but  recently  researchers  have  succeeded  in  producing  triketide  lactones  via  multi‐ derive the lactone from an acyclic precursor have also been reported.  challenging,  but  recently  researchers  have  succeeded  producing  triketide  lactones  via  multi‐ challenging,  but  recently  researchers  have  succeeded  in in  producing  triketide  lactones  via  multi‐ challenging,  but  recently  researchers  have  succeeded  in  producing  triketide  lactones  via  multi‐ enzyme  reactions  that  combine  functional  domains  from  several  polyketide  synthases  [92].  The  enzyme  reactions  that  combine  functional  domains  from  several  polyketide  synthases  [92].  The  enzyme  reactions  that  combine  functional  domains  from  several  polyketide  synthases  [92].  The  enzyme  reactions  that  combine  functional  domains  from  several  polyketide  synthases  [92].  The  enzyme  reactions  that  combine  functional  domains  from  several  polyketide  synthases  [92].  The 

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complexes—from acyclic precursors that are assembled by consecutive Claisen condensations while bound to an acyl-carrier protein and are finally cyclised by a thioesterase domain of the PKS [90,91]. The exploitation of this biocatalytic machinery for polyketide synthesis in vitro has proven Catalysts 2018, 8, x FOR PEER REVIEW    17 of 23  challenging, but recently researchers have succeeded in producing triketide lactones via multi-enzyme reactions that combine functional domains from several polyketide synthases [92]. The authors authors used the malonyl‐CoA ligase from Streptomyces coelicolor for producing the extender unit 67  used the malonyl-CoA ligase from Streptomyces coelicolor for producing the extender unit 67 from from methylmalonic acid (66) and N‐acetylcysteamine (65) at the expense of stoichiometric amounts  methylmalonic acid20).  (66)In  andparallel,  N-acetylcysteamine (65) at the expense of stoichiometric amounts of ATP of  ATP  (Scheme  an  asymmetric  reduction  of  β‐keto‐thioesters  68  by  isolated  (Scheme 20). Indomains  parallel, of  anbacterial  asymmetric reduction of β-keto-thioesters 68 starter  by isolated ketoreductase ketoreductase  PKSs  was  performed  to  furnish  the  units  for  the  PKS‐ domains of bacterial PKSs was performed to furnish the starter units for the PKS-catalysed Claisen catalysed  Claisen  condensation.  The  crude  reaction  mixtures  obtained  from  the  two  condensation. The crude reaction mixtures obtained from the two biotransformations were then biotransformations  were  then  combined  and  supplemented  with  the  terminal  PKS  module  combined and supplemented with the terminal PKS module (consisting of acyl carrier protein, acyl (consisting of acyl carrier protein, acyl transferase, ketoreductase, and ketosynthase domains) fused  transferase, ketoreductase, and ketosynthase fused to the thioesterase of the erythromycin to  the  thioesterase  of  the  erythromycin  PKS domains) from  Saccharopolyspora  erythraea.  This  “minimal  PKS”  PKS from Saccharopolyspora erythraea. This “minimal PKS” catalysed the Claisen condensation of 67 catalysed the Claisen condensation of 67 and 69 as well as the reduction of the resulting ketone and  and 69 as well as the reduction of the resulting ketone and the final cyclization to the product lactone the final cyclization to the product lactone 70. Although the isolated yields of the overall sequence  70. Although the13%,  isolated of the overall sequence did not 13%,amounts  this method allowed the did  not  exceed  this yields method  allowed  the  preparation  of  exceed substantial  of  the  triketide  preparation of substantial of the triketideThe  lactones 70 (3.4–773‐ketolactones  mg) as single stereoisomers. lactones  70  (3.4–77  mg)  as amounts single  stereoisomers.  corresponding  could  also  be  The corresponding 3-ketolactones could also be accessed by isolating intermediate 69 and performing accessed by isolating intermediate 69 and performing the final biotransformation in the absence of  the final biotransformation in the absence of an NADPH regeneration system. an NADPH regeneration system.   

  Scheme 20. Synthesis of triketide lactones 70 via coupling of enzymatically prepared precursors 67  Scheme 20. Synthesis of triketide lactones 70 via coupling of enzymatically prepared precursors 67 and 69 by a polyketide synthase (PKS).  and 69 by a polyketide synthase (PKS).

Another  study  aimed  at  the  synthesis  of  chiral  lactones  from  acyclic  precursors  relied  on  Another study aimed at the synthesis of chiral lactones from acyclic precursors relied spontaneous  ring  closure  and  used  ene‐reductase  YqjM  from  Bacillus  subtilis  and  alcohol  on spontaneous ring closure and used ene-reductase YqjM from Bacillus subtilis and alcohol dehydrogenases  from  different  microbial  sources  for  controlling  the  formation  of  up  to  three  dehydrogenases from different microbial sources for controlling the formation of up to three contiguous contiguous stereogenic centres (Scheme 21) [93].  stereogenic centres (Scheme 21) [93].   In the first step of the synthetic sequence, (E)- or (Z)-oxo-esters 71 were reduced by YqjM—a reaction that proceeded with varying stereochemical outcomes: While both double bond isomers of substrate 71a (R1 = H, R2 = Me, R3 = Et) afforded the same product (R)-72a and only the (E)-isomer of substrate 71c (R1 , R2 = Me, R3 = Et) was converted, substrate 71b (R1 = Me, R2 = H, R3 = Et) displayed an E/Z-dependent switch in selectivity. The (E)-isomer was reduced to (S)-72b, while the (Z)-isomer gave the (R)-enantiomer, and in both cases optically pure products (ee > 99%) were obtained. The subsequent reduction of intermediates 72 catalysed by alcohol dehydrogenases from Lactobacillus kefir (Lk-ADH), Lactobacillus brevis (Lb-ADH), Thermoanaerobacter sp. (ADH-T), or from

  Scheme  21.  Two‐step  cascade  leading  to  substituted  γ‐butyrolactones  (74)  using  an  ene‐reductase 

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Catalysts 2018, 8, 205

Scheme 20. Synthesis of triketide lactones 70 via coupling of enzymatically prepared precursors 67  and 69 by a polyketide synthase (PKS). 

a commercial supplier (evocatal, evo-1.1.030) was more predictable, proceeding with the expected selectivity in all cases. The at  ethyl 73a–c spontaneous cyclisation to therelied  desired Another  study  aimed  the esters synthesis  of underwent chiral  lactones  from  acyclic  precursors  on  lactones 74a–c,ring  whileclosure  the reaction a tert-butyl ester the hydroxyacid stage (73d). spontaneous  and employing used  ene‐reductase  YqjM stopped from  at Bacillus  subtilis  and  alcohol  All products werefrom  easilydifferent  isolated by extraction and purified by columnthe  chromatography. The to  isolated dehydrogenases  microbial  sources  for  controlling  formation  of  up  three  yields and optical purities are summarised in Table 9. contiguous stereogenic centres (Scheme 21) [93]. 

 

Catalysts 2018, 8, x FOR PEER REVIEW    Catalysts 2018, 8, x FOR PEER REVIEW  Catalysts 2018, 8, x FOR PEER REVIEW  Catalysts 2018, 8, x FOR PEER REVIEW       Catalysts 2018, 8, x FOR PEER REVIEW  Catalysts 2018, 8, x FOR PEER REVIEW 

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In the first step of the synthetic sequence, (E)‐ or (Z)‐oxo‐esters 71 were reduced by YqjM—a  In the first step of the synthetic sequence, (E)‐ or (Z)‐oxo‐esters 71 were reduced by YqjM—a  In the first step of the synthetic sequence, (E)‐ or (Z)‐oxo‐esters 71 were reduced by YqjM—a  In the first step of the synthetic sequence, (E)‐ or (Z)‐oxo‐esters 71 were reduced by YqjM—a  In the first step of the synthetic sequence, (E)‐ or (Z)‐oxo‐esters 71 were reduced by YqjM—a  In the first step of the synthetic sequence, (E)‐ or (Z)‐oxo‐esters 71 were reduced by YqjM—a  reaction that proceeded with varying stereochemical outcomes: While both double bond isomers of  reaction that proceeded with varying stereochemical outcomes: While both double bond isomers of  reaction that proceeded with varying stereochemical outcomes: While both double bond isomers of  reaction that proceeded with varying stereochemical outcomes: While both double bond isomers of  reaction that proceeded with varying stereochemical outcomes: While both double bond isomers of  reaction that proceeded with varying stereochemical outcomes: While both double bond isomers of  11111111 = H, R 22222222 = Me, R 33333333 = Et) afforded the same product (R)‐72a and only the (E)‐isomer of  substrate 71a (R  = H, R  = Me, R  = Et) afforded the same product (R)‐72a and only the (E)‐isomer of  substrate 71a (R  = H, R  = Me, R  = Et) afforded the same product (R)‐72a and only the (E)‐isomer of  substrate 71a (R 1 = H, R 2 = Me, R 2 = Me, R 3 = Et) afforded the same product (R)‐72a and only the (E)‐isomer of  3 = Et) afforded the same product (R)‐72a and only the (E)‐isomer of  substrate 71a (R1 = H, R  = H, R  = Me, R  = Et) afforded the same product (R)‐72a and only the (E)‐isomer of  substrate 71a (R substrate 71a (R 11111, R 22222 = Me, R 33333 = Et) was converted, substrate 71b (R 1 2 3 11111111 = Me, R 22222222 = H, R 33333333 = Et) displayed  substrate 71c (R , R  = Me, R  = Et) was converted, substrate 71b (R  = Me, R  = H, R  = Et) displayed  substrate 71c (R  = Me, R  = H, R  = Et) displayed  111, R 222 = Me, R 333 = Et) was converted, substrate 71b (R substrate 71c (R 1 = Me, R 2 = H, R 2 = H, R 3 = Et) displayed  3 = Et) displayed  substrate 71c (R1, R , R2 = Me, R  = Me, R3 = Et) was converted, substrate 71b (R  = Et) was converted, substrate 71b (R1 = Me, R  = Me, R  = H, R  = Et) displayed  substrate 71c (R , R  = Me, R  = Et) was converted, substrate 71b (R substrate 71c (R an E/Z‐dependent switch in selectivity. The (E)‐isomer was reduced to (S)‐72b, while the (Z)‐isomer  an E/Z‐dependent switch in selectivity. The (E)‐isomer was reduced to (S)‐72b, while the (Z)‐isomer  an E/Z‐dependent switch in selectivity. The (E)‐isomer was reduced to (S)‐72b, while the (Z)‐isomer  an E/Z‐dependent switch in selectivity. The (E)‐isomer was reduced to (S)‐72b, while the (Z)‐isomer  an E/Z‐dependent switch in selectivity. The (E)‐isomer was reduced to (S)‐72b, while the (Z)‐isomer  an E/Z‐dependent switch in selectivity. The (E)‐isomer was reduced to (S)‐72b, while the (Z)‐isomer  gave the (R)‐enantiomer, and  the (R)‐enantiomer, and in  in both  both cases  cases optically  optically pure  pure products  products (ee > 99%) were  (ee > 99%) were obtained.  obtained. The  The  gave  the (R)‐enantiomer, and  in  both  cases  optically  pure  products  (ee > 99%) were  obtained.  The  gave  gave  the (R)‐enantiomer, and  in  both  cases  optically  pure  products  (ee > 99%) were  obtained.  The  gave  the (R)‐enantiomer, and  in  both  cases  optically  pure  products  (ee > 99%) were  obtained.  The  gave  the (R)‐enantiomer, and  in  both  cases  optically  pure  products  (ee > 99%) were  obtained.  The  subsequent reduction of intermediates 72 catalysed by alcohol dehydrogenases from Lactobacillus kefir  subsequent reduction of intermediates 72 catalysed by alcohol dehydrogenases from Lactobacillus kefir  subsequent reduction of intermediates 72 catalysed by alcohol dehydrogenases from Lactobacillus kefir  subsequent reduction of intermediates 72 catalysed by alcohol dehydrogenases from Lactobacillus kefir  subsequent reduction of intermediates 72 catalysed by alcohol dehydrogenases from Lactobacillus kefir  subsequent reduction of intermediates 72 catalysed by alcohol dehydrogenases from Lactobacillus kefir  (Lk‐ADH), Lactobacillus  Lactobacillus brevis  brevis (Lb‐ADH),  (Lb‐ADH), Thermoanaerobacter  Thermoanaerobacter sp.  sp. (ADH‐T),  (ADH‐T), or  or from  from a  a commercial  commercial  (Lk‐ADH),  Lactobacillus  brevis  (Lb‐ADH),  Thermoanaerobacter  sp.  (ADH‐T),  or  from  a  commercial  (Lk‐ADH),  (Lk‐ADH),  Lactobacillus  brevis  (Lb‐ADH),  Thermoanaerobacter  sp.  (ADH‐T),  or  from  a  commercial  (Lk‐ADH),  Lactobacillus  brevis  (Lb‐ADH),  Thermoanaerobacter  sp.  (ADH‐T),  or  from  commercial  (Lk‐ADH),  Lactobacillus  brevis  (Lb‐ADH),  Thermoanaerobacter  sp.  (ADH‐T),  or  from  a a  commercial  supplier (evocatal, evo‐1.1.030) was more predictable, proceeding with the expected selectivity in all  supplier (evocatal, evo‐1.1.030) was more predictable, proceeding with the expected selectivity in all  supplier (evocatal, evo‐1.1.030) was more predictable, proceeding with the expected selectivity in all  supplier (evocatal, evo‐1.1.030) was more predictable, proceeding with the expected selectivity in all  supplier (evocatal, evo‐1.1.030) was more predictable, proceeding with the expected selectivity in all  supplier (evocatal, evo‐1.1.030) was more predictable, proceeding with the expected selectivity in all  cases. The ethyl esters 73a–c underwent spontaneous cyclisation to the desired lactones 74a–c, while    cases. The ethyl esters 73a–c underwent spontaneous cyclisation to the desired lactones 74a–c, while  cases. The ethyl esters 73a–c underwent spontaneous cyclisation to the desired lactones 74a–c, while  cases. The ethyl esters 73a–c underwent spontaneous cyclisation to the desired lactones 74a–c, while  cases. The ethyl esters 73a–c underwent spontaneous cyclisation to the desired lactones 74a–c, while  cases. The ethyl esters 73a–c underwent spontaneous cyclisation to the desired lactones 74a–c, while  the reaction employing a tert‐butyl ester stopped at the hydroxyacid stage (73d). All products were  the reaction employing a tert‐butyl ester stopped at the hydroxyacid stage (73d). All products were  the reaction employing a tert‐butyl ester stopped at the hydroxyacid stage (73d). All products were  the reaction employing a tert‐butyl ester stopped at the hydroxyacid stage (73d). All products were  Scheme 21. 21.  Two‐step  cascade  leading leading  to to substituted substituted γ-butyrolactones γ‐butyrolactones  (74) (74)  using using an an ene-reductase ene‐reductase  the reaction employing a tert‐butyl ester stopped at the hydroxyacid stage (73d). All products were  the reaction employing a tert‐butyl ester stopped at the hydroxyacid stage (73d). All products were  Scheme Two-step cascade easily isolated by extraction and purified by column chromatography. The isolated yields and optical  easily isolated by extraction and purified by column chromatography. The isolated yields and optical  easily isolated by extraction and purified by column chromatography. The isolated yields and optical  (ERED) and an alcohol dehydrogenase (ADH) for controlling up to three adjacent stereogenic centres.  easily isolated by extraction and purified by column chromatography. The isolated yields and optical  easily isolated by extraction and purified by column chromatography. The isolated yields and optical  (ERED) and an alcohol dehydrogenase (ADH) for controlling up to three adjacent stereogenic centres. easily isolated by extraction and purified by column chromatography. The isolated yields and optical  purities are summarised in Table 9.  purities are summarised in Table 9.  purities are summarised in Table 9.  purities are summarised in Table 9.  purities are summarised in Table 9.  purities are summarised in Table 9.  Table 9. Results of the two-step biocatalytic synthesis of substituted γ-butyrolactones (74; Scheme 21) a . aaaaaaaa.  Table 9. Results of the two‐step biocatalytic synthesis of substituted γ‐butyrolactones (74; Scheme 21)  Table 9. Results of the two‐step biocatalytic synthesis of substituted γ‐butyrolactones (74; Scheme 21)  . . .  Table 9. Results of the two‐step biocatalytic synthesis of substituted γ‐butyrolactones (74; Scheme 21)  a.  a.  Table 9. Results of the two‐step biocatalytic synthesis of substituted γ‐butyrolactones (74; Scheme 21)  Table 9. Results of the two‐step biocatalytic synthesis of substituted γ‐butyrolactones (74; Scheme 21)  Table 9. Results of the two‐step biocatalytic synthesis of substituted γ‐butyrolactones (74; Scheme 21)  c bb b  Substrate Product Yield ee (%) de (%)  de (%) ADH b  b  c (%)  Substrate  ADH b  Product  Yield ccccccc(%)  (%)  ee (%)  ee (%)  de (%)  Substrate  ADH  Product  Yield  ee (%)  b  b  b  Substrate  ADH  Product  Yield   (%)  de (%) 

Substrate  Substrate  Substrate 

ADH b b  ADH  ADH 

Product  Product  Product 

c (%)  Yield c (%)   (%)  ee (%)  ee (%)  de (%)  de (%)  Yield  ee (%)  de (%)  Yield 

ADH‐T ADH‐T ADH-T          ADH‐T ADH‐T ADH‐T ADH‐T   

90  90  90 90  90  90  90 

>99  >99  >99 >99  >99  >99  >99 

>99  >99 >99  >99  >99  >99  >99 

Lk‐ADH Lk‐ADH Lk-ADH          Lk‐ADH Lk‐ADH Lk‐ADH Lk‐ADH   

80  80  80 80  80  80  80 

>99  >99  >99 >99  >99  >99  >99 

>99  >99 >99  >99  >99  >99  >99 

evo‐1.1.030  evo‐1.1.030  evo‐1.1.030  evo-1.1.030 evo‐1.1.030  evo‐1.1.030  evo‐1.1.030 

82  82  82  82 82  82  82 

>99  >99  >99  >99 >99  >99  >99 

>99  >99 >99  >99  >99  >99  >99 

90  90  90  90  90  90 90 

>99  >99  >99  >99  >99  >99 >99 

>99  >99  >99  >99  >99  >99 >99 

63  63  63  63  63  63  63

98  98  98  98  98  98  98

>99  >99  >99  >99  >99  >99  >99

50  50  50  50  50  50 

98  98  98  98  98  98 

>99  >99  >99  >99  >99  >99 

        

        

        

        

         Lk‐ADH  Lk‐ADH  Lk‐ADH  Lk‐ADH  Lk‐ADH  Lk-ADH Lk‐ADH 

        

         evo‐1.1.030  evo‐1.1.030  evo‐1.1.030  evo‐1.1.030  evo‐1.1.030  evo‐1.1.030  evo-1.1.030

        

         Lk‐ADH  Lk‐ADH  Lk‐ADH  Lk‐ADH  Lk‐ADH  Lk‐ADH 

        

        

Lk‐ADH  Lk‐ADH  Lk‐ADH  Lk‐ADH  Catalysts 2018, 8, 205

     

>99  >99  >99  >99 

>99  >99  >99  >99 

     

evo‐1.1.030  evo‐1.1.030  evo‐1.1.030  evo‐1.1.030  Substrate

90  90  90  90 

ADH b

     

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Table 9. Cont. Product

63  63  63  63  Yield c (%)

     

Lk‐ADH  Lk‐ADH  Lk-ADH Lk‐ADH  Lk‐ADH       

98  98  98  98  ee (%)

>99  >99  >99  >99  de (%)

50  50  50 50  50 

98  98  98 98  98 

>99  >99  >99  >99 >99 

70  70  70  70  70

>99  >99  >99  >99  >99

>99  >99  >99  >99 >99 

     

ADH‐T  ADH‐T  ADH‐T  ADH‐T  ADH-T Catalysts 2018, 8, x FOR PEER REVIEW  Catalysts 2018, 8, x FOR PEER REVIEW          Catalysts 2018, 8, x FOR PEER REVIEW  Catalysts 2018, 8, x FOR PEER REVIEW  Catalysts 2018, 8, x FOR PEER REVIEW  Catalysts 2018, 8, x FOR PEER REVIEW 

     

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Lb‐ADH  Lb‐ADH  Lb-ADH Lb‐ADH  Lb‐ADH  Lb‐ADH        

62  62  62 62  62  62 

>99  >99  >99 >99  >99  >99 

98  98  98 98 98  98 

75  75  75  75  75 75 

>99  >99  >99  >99  >99 >99 

>99  >99  >99  >99  >99 >99 

85  85  85  85  85  85

>99  >99  >99  >99  >99  >99

>99  >99  >99  >99  >99  >99

       evo‐1.1.030  evo‐1.1.030  evo‐1.1.030  evo‐1.1.030  evo-1.1.030 evo‐1.1.030 

      

       Lk‐ADH  Lk‐ADH  Lk‐ADH  Lk‐ADH  Lk‐ADH  Lk-ADH

      

      

+++   (0.67  +  (0.67  Reaction  conditions:  Substrate  71  (67  mM),  D‐glucose  ‐glucose  (250  mM),  NADP mM),  GDH  (0.067     Reaction  conditions:  Substrate  71  (67  mM),  D (250  mM),  NADP mM),  GDH  (0.067  +(0.67  +  (0.67  Reaction  conditions:  Substrate  71  (67  mM),  D‐glucose  ‐glucose  (250  mM),  NADP mM),  GDH  (0.067  Reaction  conditions:  Substrate  71  (67  mM),  D (250  mM),  NADP mM),  GDH  (0.067  + NADP   Reaction  Substrate  mM),  D (250  mM),    (0.67  mM),  (0.067  D‐glucose  Reactionconditions:  conditions: Substrate 71 71  (67 (67  mM), D -glucose (250 mM), NADP (0.67 mM), GDH (0.067GDH  U/mL), ERED U/mL),  ERED  (0.167  U/mL;  purified  enzyme),  ADH  (0.73–1.67  U/mL;  cell  free  extract),  phosphate  U/mL),  ERED  (0.167  U/mL;  purified  enzyme),  ADH  (0.73–1.67  U/mL;  cell  free  extract),  phosphate  U/mL),  ERED  (0.167  U/mL;  purified  enzyme),  ADH  (0.73–1.67  U/mL;  cell  free  extract),  phosphate  U/mL),  (0.167  U/mL;  purified  ADH  (0.73–1.67  U/mL;  cell  free  extract),  phosphate  (0.167ERED  U/mL; purified enzyme), ADHenzyme),  (0.73–1.67 U/mL; cell free extract), phosphate buffer (100 mM, pH 7.0), U/mL),  ERED  (0.167  U/mL;  purified  enzyme),  ADH  (0.73–1.67  U/mL;  cell  free  extract),  phosphate  bbb ADH‐T, alcohol dehydrogenase from Thermoanaerobacter sp.;  buffer (100 mM, pH 7.0), 30 °C, 24 h.   ADH‐T, alcohol dehydrogenase from Thermoanaerobacter sp.;  30 ◦ C, 24 h. b ADH-T, alcohol dehydrogenase from Thermoanaerobacter sp.; Lb-ADH, alcohol dehydrogenase buffer (100 mM, pH 7.0), 30 °C, 24 h.  bb ADH‐T, alcohol dehydrogenase from Thermoanaerobacter sp.;  b ADH‐T, alcohol dehydrogenase from Thermoanaerobacter sp.;  buffer (100 mM, pH 7.0), 30 °C, 24 h.   ADH‐T, alcohol dehydrogenase from Thermoanaerobacter sp.;  buffer (100 mM, pH 7.0), 30 °C, 24 h.  buffer (100 mM, pH 7.0), 30 °C, 24 h.  from Lactobacillus brevis.; Lk-ADH, alcohol dehydrogenase from Lactobacillus kefir; evo-1.1.030, commercial alcohol Lb‐ADH,  alcohol  dehydrogenase  from  Lactobacillus  brevis.;  Lk‐ADH,  alcohol  dehydrogenase  from  Lb‐ADH,  alcohol  dehydrogenase  from  Lactobacillus  brevis.;  Lk‐ADH,  alcohol  dehydrogenase  from  Lb‐ADH,  alcohol  dehydrogenase  from  Lactobacillus  brevis.;  Lk‐ADH,  alcohol  dehydrogenase  from  Lb‐ADH,  alcohol  dehydrogenase  from  Lactobacillus  brevis.;  Lk‐ADH,  alcohol  dehydrogenase  from  Lb‐ADH,  alcohol  dehydrogenase  from  Lactobacillus  brevis.;  Lk‐ADH,  alcohol  dehydrogenase  from  dehydrogenase from evocatal. c Isolated yields after column chromatography. c c cc Isolated yields after   Isolated yields after  Lactobacillus kefir; evo‐1.1.030, commercial alcohol dehydrogenase from evocatal.  Lactobacillus kefir; evo‐1.1.030, commercial alcohol dehydrogenase from evocatal.  cc Isolated yields after   Isolated yields after  Lactobacillus kefir; evo‐1.1.030, commercial alcohol dehydrogenase from evocatal.   Isolated yields after  Lactobacillus kefir; evo‐1.1.030, commercial alcohol dehydrogenase from evocatal.  Lactobacillus kefir; evo‐1.1.030, commercial alcohol dehydrogenase from evocatal.  column chromatography.  column chromatography.  column chromatography.  column chromatography.  column chromatography.  aaa a a a a

In a later study, the cascade was extended, starting from allylic cyclic alcohols using fusion proteins; however, conversions did not exceed 34% [94].from  In  a a  later  study,  the  cascade  was  extended,  starting  from  allylic  cyclic  alcohols  using  fusion  In  a  later  study,  the  cascade  was  extended,  starting  allylic  cyclic  alcohols  using  fusion  In  a  later  study,  the  cascade  was  extended,  starting  from  allylic  cyclic  alcohols  using  fusion  In  a  later  study,  the  cascade  was  extended,  starting  from  allylic  cyclic  alcohols  using  fusion  In  later  study,  the  cascade  was  extended,  starting  from  allylic  cyclic  alcohols  using  fusion  proteins; however, conversions did not exceed 34% [94].  proteins; however, conversions did not exceed 34% [94].  proteins; however, conversions did not exceed 34% [94].  proteins; however, conversions did not exceed 34% [94].  proteins; however, conversions did not exceed 34% [94].  5. Conclusions

5. Conclusions  5. Conclusions  Cascade reactions possess the advantage of circumventing the need of isolation of reaction 5. Conclusions  5. Conclusions  5. Conclusions  intermediates, which saves resources, reagents, and time. They may also minimize the risk of undesired Cascade  reactions  possess  the  advantage  of  circumventing  the  need  of  isolation  of  reaction  Cascade  reactions  possess  the  advantage  of  circumventing  the  need  of  isolation  of  reaction  Cascade  reactions  possess  the  advantage  of  circumventing  the  need  of  isolation  of  reaction  Cascade  reactions  possess  the  advantage  of  circumventing  the  need  of  isolation  of  reaction  reactions  possess  the  advantage  of  circumventing  the  need  of  isolation  of  reaction  sideCascade  reactions of unstable intermediates. Furthermore, the cascade approach is expected to lead to intermediates,  which  saves  resources,  reagents,  and  time.  They  may  also  minimize  the  risk  of of  intermediates,  which  saves  resources,  reagents,  and  time.  They  may  also  minimize  the  risk  of  intermediates,  which  saves  resources,  reagents,  and  time.  They  may  also  minimize  the  risk  intermediates,  which  saves  resources,  reagents,  and  time.  They  may  also  minimize  the  risk  of  intermediates,  which  saves  reagents,  and  time.  They  may  also  minimize  the  risk  higher yields compared to a resources,  classical sequence of single-step transformations and at the same time of  can undesired side reactions of unstable intermediates. Furthermore, the cascade approach is expected to  undesired side reactions of unstable intermediates. Furthermore, the cascade approach is expected to  undesired side reactions of unstable intermediates. Furthermore, the cascade approach is expected to  undesired side reactions of unstable intermediates. Furthermore, the cascade approach is expected to  undesired side reactions of unstable intermediates. Furthermore, the cascade approach is expected to  increase the synthetic efficiency by reducing operational work-up steps and resources. Consequently, lead to higher yields compared to a classical sequence of single‐step transformations and at the same  lead to higher yields compared to a classical sequence of single‐step transformations and at the same  lead to higher yields compared to a classical sequence of single‐step transformations and at the same  lead to higher yields compared to a classical sequence of single‐step transformations and at the same  lead to higher yields compared to a classical sequence of single‐step transformations and at the same  cascades enable to reduce the amount of waste, due to minimization of the amount of chemicals used time  can  increase  the  synthetic  efficiency  by  reducing  operational  work‐up  steps  and  resources.  time  can  increase  the  synthetic  efficiency  by  reducing  operational  work‐up  steps  and  resources.  time  can  increase  the  synthetic  efficiency  by  reducing  operational  work‐up  steps  and  resources.  time  can  increase  the  synthetic  efficiency  by  reducing  operational  work‐up  steps  and  resources.  time  the  synthetic  efficiency  by  reducing  operational  work‐up  steps  and  resources.  (e.g.,can  by increase  circumventing work-up). Consequently, cascades enable to reduce the amount of waste, due to minimization of the amount of  Consequently, cascades enable to reduce the amount of waste, due to minimization of the amount of  Consequently, cascades enable to reduce the amount of waste, due to minimization of the amount of  Consequently, cascades enable to reduce the amount of waste, due to minimization of the amount of  Consequently, cascades enable to reduce the amount of waste, due to minimization of the amount of  The cascades discussed in this review represent a fraction of possible artificial biocatalytic cascades chemicals used (e.g., by circumventing work‐up).  chemicals used (e.g., by circumventing work‐up).         chemicals used (e.g., by circumventing work‐up).  chemicals used (e.g., by circumventing work‐up).  chemicals used (e.g., by circumventing work‐up).  [1ac], of which the number will significantly increase during the next years. This will be a result of The  cascades  discussed  in  this  review  represent  a a  fraction  of  possible  artificial  biocatalytic  The  cascades  discussed  in  this  review  represent  a  fraction  of  possible  artificial  biocatalytic  The  cascades  discussed  in  this  review  represent  a  fraction  of  possible  artificial  biocatalytic  The  cascades  discussed  in  this  review  represent  a  fraction  of  possible  artificial  biocatalytic  The  cascades  discussed  in  this  review  represent  fraction  of  possible  artificial  (novel) enzymes becoming more easily available, improved expression tools, as well biocatalytic  as improved cascades [1ac], of which the number will significantly increase during the next years. This will be a  cascades [1ac], of which the number will significantly increase during the next years. This will be a  cascades [1ac], of which the number will significantly increase during the next years. This will be a  cascades [1ac], of which the number will significantly increase during the next years. This will be a  cascades [1ac], of which the number will significantly increase during the next years. This will be a  enzyme engineering methods. result  of  (novel)  enzymes  becoming  more  easily  available,  improved  expression  tools,  as  well  as  result  of  (novel)  enzymes  becoming  more  easily  available,  improved  expression  tools,  as  well  as  result  of  (novel)  enzymes  becoming  more  easily  available,  improved  expression  tools,  as  well  as  result  of  (novel)  enzymes  becoming  more  easily  available,  improved  expression  tools,  as  well  as  result  of  (novel)  enzymes  becoming  more  easily  available,  improved  expression  tools,  as  well  as  This review shows that artificial cascades may can use different feedstocks as substrates: either improved enzyme engineering methods.  improved enzyme engineering methods.  improved enzyme engineering methods.  improved enzyme engineering methods.  improved enzyme engineering methods.  from renewables such as amino acids but also from fossil resources, such as phenol or styrenes, etc. This review shows that artificial cascades may can use different feedstocks as substrates: either  This review shows that artificial cascades may can use different feedstocks as substrates: either  This review shows that artificial cascades may can use different feedstocks as substrates: either  This review shows that artificial cascades may can use different feedstocks as substrates: either  This review shows that artificial cascades may can use different feedstocks as substrates: either  from renewables such as amino acids but also from fossil resources, such as phenol or styrenes, etc.  from renewables such as amino acids but also from fossil resources, such as phenol or styrenes, etc.  from renewables such as amino acids but also from fossil resources, such as phenol or styrenes, etc.  from renewables such as amino acids but also from fossil resources, such as phenol or styrenes, etc.  from renewables such as amino acids but also from fossil resources, such as phenol or styrenes, etc.  The  target  compounds  discussed  in  the  review,  namely  hydroxy  acids,  amino  acids,  and  lactones  The  target  compounds  discussed  in  the  review,  namely  hydroxy  acids,  amino  acids,  and  lactones  The  target  compounds  discussed  in  the  review,  namely  hydroxy  acids,  amino  acids,  and  lactones  The  target  compounds  discussed  in  the  review,  namely  hydroxy  acids,  amino  acids,  and  lactones  The  target  compounds  discussed  in  the  review,  namely  hydroxy  acids,  amino  acids,  and  lactones  represent important building blocks and intermediates. The cascades shown enabled the successful  represent important building blocks and intermediates. The cascades shown enabled the successful  represent important building blocks and intermediates. The cascades shown enabled the successful  represent important building blocks and intermediates. The cascades shown enabled the successful  represent important building blocks and intermediates. The cascades shown enabled the successful  preparation of these compounds within 2–10 artificially combined steps.  preparation of these compounds within 2–10 artificially combined steps.  preparation of these compounds within 2–10 artificially combined steps.  preparation of these compounds within 2–10 artificially combined steps.  preparation of these compounds within 2–10 artificially combined steps.  A A  number  of  cascades  have  already  been  described  to  be  performed  at  elevated  substrate  A  number  of  cascades  have  already  been  described  to  be  performed  at  elevated  substrate  A  number  of  cascades  have  already  been  described  to  be  performed  at  elevated  substrate  A  number  of  cascades  have  already  been  described  to  be  performed  at  elevated  substrate  number  of  cascades  have  already  been  described  to  be  performed  at  elevated  substrate 

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The target compounds discussed in the review, namely hydroxy acids, amino acids, and lactones represent important building blocks and intermediates. The cascades shown enabled the successful preparation of these compounds within 2–10 artificially combined steps. A number of cascades have already been described to be performed at elevated substrate concentration, e.g., reaching in the best case e.g., 600 mM as reported in this review and are therefore ideally suited for preparative transformations and applications; however, in other cases the substrate concentration is as low as 1 mM, which is due to the limitation of the most sensitive enzyme in the cascade. Thus, a cascade can only be as good in terms of efficiency (space time yield) as is given by the “weakest” enzyme. Limiting reactions are still hydroxylation transformations of C–H bonds but also cross-inhibition by products/substrates of various steps may force to keep concentrations low. Therefore, there is still a need for further improved enzymes. Another point which would be desirable is that co-expression constructs for various cascades may be deposited and made broadly available for subsequent studies. This may allow to generate the possibility to design rather fast novel complex networks including the now designed and optimized cascades. Acknowledgments: We gratefully acknowledge the Federal Ministry for Digital and Economic Affairs (bmwd), the Federal Ministry for Transport, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol, Government of Lower Austria and ZIT—Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG. Conflicts of Interest: The authors declare no conflict of interest.

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42a  89  >99%  Table 6. Transformation of β‐keto esters 42 into β‐amino acids 44 (Scheme 12) a.  42b  96  >99%  42c  75  >99%  Substrate  Conv. (%)  ee (%)  42d  73  >99%  42a  89  >99%  42e  99  >99%  42b  96  >99%  42f  61  >99% 23 of 24 Catalysts 2018, 8, 205 42c  75  >99%  42g  47  >99%  42d  73  >99%  42h  41  >99%  42e  99  >99%  42i  of L-tyrosine 26  derivatives >99%  from 60. Dennig, A.; Busto, E.; Kroutil, K. Biocatalytic 42f W.; Faber,61  >99%  one-pot synthesis a Reaction conditions: Substrate 42 (50 mM), (S)‐1‐phenylethylamine (150 mM), PLP (1 mM monosubstituted benzenes, 42g  pyruvate, and ACS Catal. 2015, 5, 7503–7506. [CrossRef] 47 ammonia. >99%  D.J. 61. Seebach, D.; Beck, A.K.; Bierbaum, 42h  (30 mg/mL), TA (20 mg/mL), Tris‐HCl buffer (100 mM, pH 8.0), DMSO (15% v/v), 37 °C, 24 h 41 The world >99% of β-and γ-peptides comprised of homologated 42i other components. 26  >99%  Biodivers. 2004, 1, 1111–1239. [CrossRef] [PubMed] proteinogenic amino acids and Chem. a Reaction conditions: Substrate 42 (50 mM), (S)‐1‐phenylethylamine (150 mM), PLP (1 mM), lipase  62. Steer, D.L.; Lew, R.A.; Perlmutter,Phenylalanine and derivatives thereof were deracemized by a combination of E. col P.; Smith, I.A.; Aguilar, M.-I. β-Amino acids: Versatile peptidomimetics. Curr. Med. Chem. 2002, 9, 811–822. [CrossRef]L‐amino acid deaminase and an engineered  [PubMed] expressing an  D‐amino acid dehydrogenas (30 mg/mL), TA (20 mg/mL), Tris‐HCl buffer (100 mM, pH 8.0), DMSO (15% v/v), 37 °C, 24 h.  63. Kuhl, A.; Hahn, G.M.; Dumi´coptically  , M.; Mittendorf, J. Alicyclic β-amino acids in medicinal chemistry. Amino Acidsinstead  of  startin pure  D‐phenylalanines  (Scheme  13)  [66].  Alternatively,  Phenylalanine and derivatives thereof were deracemized by a combination of E. coli whole cells  2005, 29, 89–100. [CrossRef] racemate, optically pure  [PubMed] L‐enantiomers were used as substrate leading to inversion of  64. Fülöp, F.; Martinek, T.A.; configuration. Various substituted derivatives of the  Tóth, G.K. Application of alicyclic β-amino acids in peptide chemistry. expressing an  L‐amino acid deaminase and an engineered  D‐amino acid dehydrogenase to prepare  D‐amino acids were isolated with Chem. Soc. Rev. 2006, 35,(Scheme  323–334. [CrossRef] [PubMed] optically  pure  D‐phenylalanines  13)  [66].  Alternatively,  instead  of  starting  from  the  (69–83%) and excellent optical purities (95% to >99% ee).  65. Mathew, S.; L Jeong, S.-S.; Chung, T.; Lee, S.-H.; Yun, H. Asymmetric synthesis of aromatic β-amino acids racemate, optically pure  ‐enantiomers were used as substrate leading to inversion of the absolute  using ω-transaminase: Optimizing the lipase concentration to obtain thermodynamically unstable β-keto configuration. Various substituted derivatives of the  D‐amino acids were isolated with good yields    acids. Biotechnol. J. 2016, 11, 185–190. [CrossRef] [PubMed] (69–83%) and excellent optical purities (95% to >99% ee).  66. Parmeggiani, F.; Ahmed, S.T.; Thompson, M.P.; Weise, N.J.; Galman, J.L.; Gahloth, D.; Dunstan, M.S.; Leys, D.; Turner, N.J. Single-biocatalyst synthesis of enantiopure D-arylalanines exploiting an engineered D-amino   acid dehydrogenase. Adv. Synth. Catal. 2016, 358, 3298–3306. [CrossRef] 67. Marchelli, R. The potential of enantioselective analysis as a quality control tool. Trends Food Sci. Technol. 1996, 7, 113–119. [CrossRef] 68. Hoffmann, H.M.R.; Rabe, J. Synthese und biologische Aktivität von α-Methylen-γ-butyrolactonen. Angew. Chem. 1985, 97, 96–112. [CrossRef] 69. Labet, M.; Thielemans, W. Synthesis of polycaprolactone: A review. Chem. Soc. Rev. 2009, 38, 3484–3504. [CrossRef] [PubMed] 70. Mattes, H.; Hamada, K.; Benezra, C. Stereospecificity in allergic contact dermatitis to simple substituted methylene lactones derivatives. J. Med. Chem. 1987, 30, 1948–1951. [CrossRef] [PubMed] 71. Moslandl, A.; Günther, C. Stereoisomeric flavor compounds. 20. Structure and properties of γ-lactone Scheme 13. Deracemization cascade for the preparation of D‐amino acids 45. Enzyme abbre enantiomers. J. Agric. Food Chem. 1989, 37, 413–418. [CrossRef] L‐AAD, L‐amino acid deaminase; MDPD, meso‐diaminopimelate dehydrogenase.  72. Carreno, M.C.; Sanz-Cuesta, M.J. Conjugate additions of Me2 CuLi to enantiopure γ-hydroxy-δ-sulfinyl and   sulfonyl pentenoates. J. Org.4. Lactones  Chem. 2005, 70, 10036–10045. [CrossRef] [PubMed] Scheme 13. Deracemization cascade for the preparation of D‐amino acids 45. Enzyme abbreviations:  73. Brown, E.; Deroye, C.; Touet, J. Synthesis of versatile chiral intermediates by enantioselective conjugate L‐AAD, L‐amino acid deaminase; MDPD, meso‐diaminopimelate dehydrogenase.  Lactones—and particularly γ‐ and δ‐lactones—are important flavour and fragrance addition of alkenyl Grignard reagents to enamides deriving from (R)-(−)- or (S)-(+)-2-aminobutan-1-ol. that  are  found  in  various  fruits,  juices,  wines,  and  spirits  [67].  Furthermore,  su Tetrahedron Asymmetry 1998, 9, 1605–1614. [CrossRef] 4. Lactones  74. Staudt, S.; Bornscheuer, U.T.;butyrolactones occur as a central motif in many natural products, for instance nephroste Menyes, U.; Hummel, W.; Gröger, H. Direct biocatalytic one-pot-transformation Lactones—and particularly γ‐ and δ‐lactones—are important flavour and fragrance compounds  arctigenin  on  the  other Technol. hand,  is  a  valuable  chemical  for  the  pr of cyclohexanol with molecular oxygen[68].  into ɛ‐Caprolactone,  -caprolactone. Enzyme Microb. 2013, 53, 288–292. that  are  found  in  various  fruits,  juices,  wines,  and  spirits  [67].  Furthermore,  substituted  γ‐ [CrossRef] [PubMed] biodegradable polymers, such as polycaprolactone [69] and a precursor for ɛ‐caprolactam butyrolactones occur as a central motif in many natural products, for instance nephrosteranic acid or  75. Mallin, H.; Wulf, H.; Bornscheuer,The chemical synthesis of these compounds usually involves a large number of steps U.T. A self-sufficient Baeyer–Villiger biocatalysis system for the synthesis arctigenin  [68].  on  the yields, toxic or otherwise hazardous reagents, and impractical reaction conditions, which other  hand,  a  valuable  for  the  preparation  of  of ɛ‐Caprolactone,  -caprolactone from cyclohexanol. Enzymeis Microb. Technol.chemical  2013, 53, 283–287. [CrossRef] [PubMed] 76. Schmidt, S.; Scherkus, C.; Muschiol, J.; Menyes, U.; Winkler, T.; Hummel, W.; Gröger, H.; Liese, A.; Herz, H.G.; biodegradable polymers, such as polycaprolactone [69] and a precursor for ɛ‐caprolactam.  neither  “green”  nor  atom‐economic  [70–73].  Hence,  biocatalytic  cascade  transform Bornscheuer, U.T. An enzyme cascade synthesis of ε-caprolactone and its oligomers. Angew. Chem. Int. Ed. The chemical synthesis of these compounds usually involves a large number of steps, low overall  become a useful tool in the preparation of lactones, as they avoid the necessity of time 2015, 54, 2784–2787. [CrossRef] [PubMed] yields, toxic or otherwise hazardous reagents, and impractical reaction conditions, which makes them  and  yield‐reducing  isolation  and  purification  of  intermediates  and  reduce  waste  gen 77. Scherkus, C.; Schmidt, S.; Bornscheuer, U.T.; Gröger, H.; Kara, S.; Liese, A.transformations  Kinetic insights intohave  -caprolactone neither  “green”  nor  atom‐economic  [70–73].  Hence,  biocatalytic  cascade  minimum.  synthesis: Improvement of an enzymatic cascade reaction. Biotechnol. Bioeng. 2017, 114, 1215–1221. become a useful tool in the preparation of lactones, as they avoid the necessity of time‐consuming  In  most  of  these  multi‐enzyme  sequences,  the  lactone  is  formed  in  the  final  s [CrossRef] [PubMed] and  yield‐reducing  isolation  and  purification  of  intermediates  and  reduce  waste  generation  to  a  oxidation of a cyclic ketone by a Baeyer–Villiger monooxygenase (BVMO). However, s 78. Opperman, D.J.; Reetz, M.T. Towards practical Baeyer–Villiger-monooxygenases: Design of cyclohexanone minimum.  derive the lactone from an acyclic precursor have also been reported.  monooxygenase mutants with enhancedthe  oxidative stability. ChemBioChem 2010, 11, 2589–2596. In  most  of  these  multi‐enzyme  sequences,  lactone  is  formed  in  the  final  step  through [CrossRef] [PubMed] oxidation of a cyclic ketone by a Baeyer–Villiger monooxygenase (BVMO). However, systems that  79. Kohl, A.; Srinivasamurthy, V.; Böttcher, D.; Kabisch, J.; Bornscheuer, U.T. Co-expression of an alcohol derive the lactone from an acyclic precursor have also been reported.  dehydrogenase and a cyclohexanone monooxygenase for cascade reactions facilitates the regeneration of the NADPH cofactor. Enzyme Microb. Technol. 2018, 108, 53–58. [CrossRef] [PubMed] 80. Aalbers, F.S.; Fraaije, M.W. Coupled reactions by coupled enzymes: Alcohol to lactone cascade with alcohol dehydrogenase–cyclohexanone monooxygenase fusions. Appl. Microbiol. Biotechnol. 2017, 101, 7557–7565. [CrossRef] [PubMed]

Catalysts 2018, 8, 205

81.

82.

83. 84. 85.

86. 87.

88.

89.

90. 91. 92. 93. 94.

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