2-Azabicyclo[2.2.1]hept-5-en-3-one: Chemical Profile of a Versatile Synthetic Building Block and its Impact on the Development of Therapeutics

Singh, Rohit; Vince, Robert

doi:10.1021/cr2004822

Cited by 110 publications

(80 citation statements)

References 917 publications

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“…ABH has been used for the synthesis of carbocyclic nucleosides for more than 30 years, since Robert Vince recognised the potential of this intermediate to synthesise carbocyclic nucleosides with the required cis orientation of both hydroxymethyl and nucleobase groups. A recent review collected the different synthesis and numerous applications of ABH (Singh and Vince, 2012). Enzymatic resolution of ABH has been extensively studied, and there are several enzymes that provide excellent enantioselectivity and facilitate the preparation of enantiopure carbocyclic nucleosides.…”

Section: As Nucleoside Prodrugsmentioning

confidence: 99%

Biocatalytic approaches applied to the synthesis of nucleoside prodrugs

Iglesias

Lewkowicz

Médici

et al. 2015

Biotechnology Advances

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Section: As Nucleoside Prodrugsmentioning

confidence: 99%

Biocatalytic approaches applied to the synthesis of nucleoside prodrugs

Iglesias

Lewkowicz

Médici

et al. 2015

Biotechnology Advances

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“…Thus, much effort has been made to construct specific enzymes from promiscuous enzymes (Yoshikuni et al 2006). Chiral bicyclic γ-lactam, 2-azabicyclo[2.2.1]hept-5-en-3-one, is an important building block for the synthesis of a series of chemotherapeutic agents that are used as cardiac vasodilators and antiviral drugs (Singh and Vince 2012). Kinetic resolution of racemic γ-lactam by γ-lactamase is a powerful method for producing chiral γ-lactam (Wang et al 2014a).…”

Section: Introductionmentioning

confidence: 99%

Identification and regulation of the catalytic promiscuity of (−)-γ-lactamase from Microbacterium hydrocarbonoxydans

Sun

Zhao

Wang

et al. 2015

Appl Microbiol Biotechnol

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Mhg, a previously reported (-)-γ-lactamase from Microbacterium hydrocarbonoxydans, was identified to have perhydrolase activity by combining structure similarity search with activity assays. Kinetic studies illustrated that perhydrolysis was the native activity owing to lower K m and higher k cat/K m values. Experimental evidence showed that both hydrolysis and perhydrolysis reactions took place at the same active center. Engineering of the putative substrate-binding pocket revealed that Leu233 site played a vital role in the aspects of selective catalysis, soluble protein expression level and optimum temperature shift, etc. The mutants L233A, L233P, and L233T retained (-)-γ-lactamase activity but lost perhydrolase activity, while L233M only kept perhydrolase activity. Substitutions of Leu233 could dramatically influence the state of expressed protein. Computational analysis explicitly explained the relationships between mutations and γ-lactamase activity changes. Our investigations demonstrated that it was an efficient method to identify the enzyme catalytic promiscuity by combining 3D structure alignment with activity validations, and engineering of substrate-binding pocket could serve as a promising way to regulate activities of promiscuous enzymes.

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“…Mhg, a (Ϫ)-␥-lactamase with high stability and enantioselectivity, was first isolated and purified from Microbacterium hydrocarbonoxydans (13). Mhg can be utilized to prepare chiral ␥-lactam (14), which is an important chiral intermediate for the synthesis of a series of antiviral drugs, such as abacavir (targeting HIV) and peramivir (targeting hepatitis and pandemic influenza viruses). Recently, by performing structure similarity analysis combined with classical enzyme assays, we have found that Mhg has perhydrolase activity ( Fig.…”

mentioning

confidence: 99%

Facilitating the Evolution of Esterase Activity from a Promiscuous Enzyme (Mhg) with Catalytic Functions of Amide Hydrolysis and Carboxylic Acid Perhydrolysis by Engineering the Substrate Entrance Tunnel

Yan

Wang

Sun

et al. 2016

Appl Environ Microbiol

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Promiscuous enzymes are generally considered to be starting points in the evolution of offspring enzymes with more specific or even novel catalytic activities, which is the molecular basis of producing new biological functions. Mhg, a typical ␣/␤ fold hydrolase, was previously reported to have both ␥-lactamase and perhydrolase activities. However, despite having high structural similarity to and sharing an identical catalytic triad with an extensively studied esterase from Pseudomonas fluorescens, this enzyme did not show any esterase activity. Molecular docking and sequence analysis suggested a possible role for the entry of the binding pocket in blocking the entrance tunnel, preventing the ester compounds from entering into the pocket. By engineering the entrance tunnel with only one or two amino acid substitutions, we successfully obtained five esterase variants of Mhg. The variants exhibited a very broad substrate acceptance, hydrolyzing not only the classical p-nitrophenol esters but also various types of chiral esters, which are widely used as drug intermediates. Site 233 at the entrance tunnel of Mhg was found to play a pivotal role in modulating the three catalytic activities by adjusting the size and shape of the tunnel, with different amino acid substitutions at this site facilitating different activities. Remarkably, the variant with the L233G mutation was a very specific esterase without any ␥-lactamase and perhydrolase activities. Considering the amino acid conservation and differentiation, this site could be a key target for future protein engineering. In addition, we demonstrate that engineering the entrance tunnel is an efficient strategy to regulate enzyme catalytic capabilities. IMPORTANCEPromiscuous enzymes can act as starting points in the evolution of novel catalytic activities, thus providing a molecular basis for the production of new biological functions. In this study, we identified a critical amino acid residue (Leu233) at the entry of the substrate tunnel of a promiscuous enzyme, Mhg. We found that substitution of this residue with smaller amino acids such as Gly, Ala, Ser, or Pro endowed the enzyme with novel esterase activity. Different amino acids at this site can facilitate different catalytic activities. These findings exhibited universal significance in this subset of ␣/␤ fold hydrolases, including Mhg. Furthermore, we demonstrate that engineering the entrance tunnel is an efficient strategy to evolve new enzyme catalytic capabilities. Our study has important implications for the regulation of enzyme catalytic promiscuity and development of protein engineering methodologies. C atalytic promiscuity refers to the ability of an enzyme to catalyze different substrates and different types of chemical reactions in the same active center. Although high substrate specificity has been well known, increasing evidence indicates that enzyme catalytic promiscuity exists widely in nature (1, 2, 3, 4). Recently, enzyme promiscuity has captured much attention because it is related to the...

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2-Azabicyclo[2.2.1]hept-5-en-3-one: Chemical Profile of a Versatile Synthetic Building Block and its Impact on the Development of Therapeutics

Cited by 110 publications

References 917 publications

Biocatalytic approaches applied to the synthesis of nucleoside prodrugs

Biocatalytic approaches applied to the synthesis of nucleoside prodrugs

Identification and regulation of the catalytic promiscuity of (−)-γ-lactamase from Microbacterium hydrocarbonoxydans

Facilitating the Evolution of Esterase Activity from a Promiscuous Enzyme (Mhg) with Catalytic Functions of Amide Hydrolysis and Carboxylic Acid Perhydrolysis by Engineering the Substrate Entrance Tunnel

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