Advanced biocatalysis for green chemical synthesis at commercial scale

The twelve principles of Green Chemistry as formulated by Anastas and Warner (Green Chemistry: Theory and Practice, Oxford University Press: New York, 1998, p.30) guide the development of less wasteful and overall environmentally friendlier chemical manufacturing processes. The first principle states that “it is better to prevent waste than to treat or clean up waste after it has been created”. The effectiveness of adherence to this principle is best illustrated in nature where living organism efficiently convert mixtures of organic chemicals through various intermediates into energy and new cellular materials. Very few by-products are made. Enzymes are nature’s catalysts for the efficient interconversion of chemicals and have evolved over millions of years to be highly regioselective, chemoselective, as well as stereoselective. Consequently, certain enzymes are used commercially for the manufacture of bulk products such as high fructose corn syrup (HFCS), acrylamide, and a few pharmaceutical intermediate, for instance (-)-lactam for abacavir. Unfortunately however, the application of natural enzymes has been limited to only a few processes as their performance is typically insufficient for chemical manufacturing processes where for economic reasons substrate and product concentrations need to be much higher than observed in nature and the process specifics may demand the need for use of non-natural organic solvents. To overcome limitations in natural enzymes, R&D has focused on enzyme stabilization and reuse via immobilization technology and reactor engineering. In parallel, the development of chemocatalyst using rare and expensive transition metals in attempts to imitate the selectivity of enzymes has seen a big surge. Despite intense research however, the excellent selectivity of enzymes has been unsurpassed by the many chemocatalysts that have been designed and evaluated over the past several decades.

In the mid 1990s directed evolution technologies entered the field of enzyme engineering and advanced biocatalysts have been generated for a range of manufacturing processes.  Through its manufacturing partners, Codexis is currently producing several pharmaceutical products at multi-tonnes scale.  Our technology applies the principles of natural evolution at the laboratory scale as we iteratively modify the genetic information that encode enzymes of interest and identify improved enzyme variants via high throughput screening.  By designing the best possible process and applying the corresponding reaction conditions to our screens, we develop catalysts for green-by-design manufacturing processes which deliver both environmental and economic advantages.

By way of illustration, Codexis developed a two-step, three enzyme process for the manufacture of ethyl (R)-4-cyano-3-hydroxybutyrate  or hydroxynitrile, the key chiral building block for atorvastatin (Lipitor®).  Key features of this new process are that it does not require metal catalysts or chemical derivatization steps, is carried out at room temperature and pressure at neutral pH in water and takes less time than the conventional process.  Because the enzymes impart nearly perfect stereocontrol in the reduction reaction as well as regiocontrol in the cyanide addition reaction, the product is manufactured in very high purity.  As a result, no complex and expensive work-up is required and the product is produced in high yield with an overall E-factor of 5.8 (kg waste per kg product).  Codexis has been manufacturing hydroxynitrile at a rate of ~10 mT per month.

Ketoreductases (KREDs) are enzymes that convert ketones to chiral alcohols to give product that typically is of >99.9% enantiomeric excess.  Because chiral alcohols are frequently seen as intermediates in pharmaceutical manufacture, Codexis developed a kit of advanced KRED biocatalysts that can be rapidly screened in the laboratory to provide catalysts for initial (and even commercial) make.  If such catalysts require further optimization, the screening data can be incorporated in the rapid development of better variants for the process of interest.   With this KRED kit we have developed enzymes for commercial scale manufacture of the chiral alcohol intermediates for montelukast (Singulair®), duloxetine (Cymbalta®), ezetimibe (Zetia®) and others (reviewed in Curr. Opin. Chem. Biol. 2010, 14:122-129).

Chiral alcohols are often used as intermediates for the manufacture of chiral amines.  The direct synthesis of chiral amines from prochiral ketones is a difficult but highly desirable reaction for process chemists.  In the 2007 paper on "Key green chemistry research areas" representatives from the pharmaceutical industry described the direct conversion of ketones and ammonia to chiral amines as an aspirational research objective (Green Chem. 2007, 9:411-420).  In nature transaminases are responsible for the synthesis of various amino acids from α-keto acids.  A wide variety of transaminases is known but their application in commercial processes has been hampered by their limited substrate range and susceptibility to chemical manufacturing conditions.  In collaboration with scientists from Merck, we embarked on a program to develop a transaminase catalyst for the direct manufacture of sitagliptin (Januvia® and Janumet®).  After first creating a catalysts that exhibited barely detectable activity on this bulky substrate we then improved it over 25,000-fold to function efficiently under process conditions including 40+% DMSO and reaction temperatures >50^oC (Science 2010, 329:305-309).  The paper describes that the new process may offer a 10-13 percent overall increase in yield of sitagliptin over the current process, a 53% increase in productivity, a 19% decrease in total waste, elimination of all heavy metals and a reduction in total manufacturing cost.  This work underscores the maturation of biocatalysis to enable efficient, economical and environmentally-benign processes for the manufacture of pharmaceuticals.  Based on the successful results of this collaboration, in 2010, Codexis and Merck received the US EPA Presidential Green Chemistry award. 

With these new enzyme engineering technologies all previous notions about the limitations of enzymes for large-scale use have been dispelled.   Advanced biocatalysts will continue to provide attractive options for the enablement of highly efficient green manufacturing processes that deliver safety advantages for plant operators, economic advantages to process adopters and environmental benefits for society.