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Aldolase

Mechanism + Description

There are a number of different classes of aldolase, some are metal dependant (Zn2+) in the active site and some work through a lysine in the active site. They couple aldehydes and ketones typically via conversion of one coupling partner from an electrophile to a nucleophilic imine/enamine followed by the normal aldol process to make the C-C bond. The enantioselectivity is controlled by the chiral environment of the active site. Scheme shows 2-Deoxy-d-ribose-5-phosphate aldolase (DERA), a class I aldolase, coupling acetaldehyde and chloroacetaldehyde.

Natural substrate of DERA aldolase

 

General comments

There are a range of aldolase enzymes used to make chiral carbon-carbon bonds. Some are very substrate specific and require phosphorylated molecules for binding in the active site, so are not that attractive as catalysts to make a wide range of unnatural substrates at scale. Of greatest interest at this time are DERA-type aldolases and threonine aldolases that will accept a reasonable range of non-phosphorylated substrates.

 

Key references

Schürmann, M.; Wolberg, M.; Panke, S.; Kierkels, H. The Development of Short, Efficient, Economic, and Sustainable Chemoenzymatic Processes for Statin Side Chains. In Green Chemistry in the Pharmaceutical Industry; Wiley-VCH Verlag GmbH & Co. KGaA, 2010; pp 127–144.

Gupta, P.; Mahajan, N.; Tanega, S. C. Recent advances in the stereoselective synthesis of 1,3-diols using biocatalysts. Catal. Sci. Technol. 2013, 3, 2462–2480.

Ranoux, A.; Hanefeld, U. Enzyme-Catalyzed Stereoselective C—C bond Formation Reactions in Total Syntheses. In Stereoselective Synthesis of Drugs and Natural Products; John Wiley & Sons, Inc., 2013.

Beaudoin, S. F.; Hanna, M. P.; Ghiviriga, I.; Stewart, J. D.; Progress in using threonine aldolases for preparative synthesis. Enzyme Microb. Technol. 2018, 119, 1–9.

Windle, C. L.; Berry, A.; Nelson, A.  Aldolase-catalysed stereoselective synthesis of fluorinated small molecules. Curr. Opin. Chem. Biol. 2017, 37, 33–38.

Greenberg, W. A.; Varvak, A.; Hanson, S. R.; Wong, K.; Huang, H.; Chen, P.; Burk, M. J. Development of an efficient, scalable, aldolase-catalyzed process for enantioselective synthesis of statin intermediates. PNAS, 2004, 10 (16), 5788–5793.

DeSantis, G.; Liu, J.; Clark, D. P.; Heine, A.; Wilson, I. A.; Wong, C.-H. Structure-Based mutagenesis approaches toward expanding the substrate specificity of d-2-Deoxyribose-5-phosphate aldolase. Bioorg. Med. Chem. 2003, 11, 43–52.

Ošlaj, M.; Cluzeau, J.; Orkić, D.; Kopitar, G.; Mrak, P. Časar, Z. A Highly Productive, Whole-Cell DERA Chemoenzymatic Process for of Key Lactonized Side-Chain Intermediates in Statin Synthesis. PLoS One 2013, 8 (5), e62250.

Müller, M. Chemoenzymatic Synthesis of Building Blocks for Statin Side Chains. Angew. Chem. Int. Ed. 2005, 44, 362–365.

Blesl, J.; Trobe, M.; Anderl, F.; Breinbauer, R.; Strohmeier, G. A.; Fesko, K. Application of Threonine Aldolases for the Asymmetric Synthesis of a-Quaternary alpha -Amino Acids. ChemCatChem 2018, 10, 3453–3458.

 

Relevant scale up examples

Biotechnol J. 2006, 1 (5), 537-548
100s kg scale

 

Org. Process Res. Dev. 2013, 17, 854-862
20 g scale

 

Org. Process Res. Dev. 2015, 19, 1317-1322
200 g scale

 

Green Review

  1. Atom efficiency (by-products, molecular weight)
    Catalytic technology—but enzyme loading should be optimized and steps using stoichiometric reagents like cofactor recycle should be optimized to avoid unnecessary excess of reagents.
  2. Safety Concerns
    Generally considered a safe technology to scale-up. Issues—enzymes/proteins can be sensitizers by inhalation, and some by mode of action can be skin irritants (proteases). If viable GMO cells are used, local regulations relating to use of GMOs need to be followed.
  3. Toxicity and environmental/aquatic impact
    No real concerns—enzymes are non-toxic and readily biodegradable. If viable GMO cells are used, these need to be passivated before discharge into the environment.
  4. Cost, availability & sustainable feedstocks
    Enzymes are produced from natural sustainable feedstocks.
  5. Sustainable implications
    Enzymes are made via fermentation; cloned and recombinant enzymes would be at no risk from depletion. Enzymes break down in the environment and the constituent amino acids are recycled in nature. The use of over expressed recombinant enzymes is generally much better than natural enzymes on a life cycle impact basis.