Lipases forbiocatalysisfor smarter chemical synthesis

Biocatalysis Biocatalysis involves the implementation of natural catalysts, such as enzymes, in place of chemical catalysts in synthetic processes. Compared to chemical catalysts, enzymes offer:

• higher reaction rates• milder reaction conditions• high reaction specificity with no side products

This change can enable new, more sustainable routes for the production of intermediates and active pharmaceutical ingredients (APIs). However, please note Novozymes products do not comply with manufacturing according to pharmaceutical standards and Novozymes products must not be used as active pharmaceutical ingredients (APIs) or excipients.

Biocatalysis has become an increasingly important tool for medicinal, process and polymer chemists, allowing the development of efficient and highly attractive synthetic processes on an industrial scale. Use of enzymes in catalysis is a well-established technology within the chemical industry. An advantage of enzymes in organic synthesis is their remarkable selective properties, which provide commercial benefits including:

• high selectivity in production of single stereoisomers• fewer side reactions• less reprocessing and purification steps• easier product separation• less pollution

The combination of all of these advantages leads to a reduction in costs.

Enzyme catalysts work by lowering the activation energy (Ea‡) for a reaction, thus dramatically increasing the rate of the reaction. As a result, products are formed faster and reactions reach their equilibrium state more rapidly. Most enzyme reaction rates are millions of times faster than those of comparable uncatalyzed reactions. As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions. However, enzymes do differ from most other catalysts in that they are highly specific for their substrates.

A

B

C

D

E

A. Various microorganisms can

be used to produce natural catalysts

such as enzymes

B. The enzymes are separated from the

microorganisms and subsequently

partially purified and formulated

C. An enzyme attracts specific

substrates to its active site

D. It catalyzes the chemical reaction by

which products are formed

E. It then allows the products

to separate from the enzyme surface

Lipozyme® CALBLipozyme® CALB is a non-specific lipase from Candida antarctica B. Lipozyme® CALB is stable over a broad pH range, especially in the alkaline pH range. This enzyme exhibits a high degree of substrate specificity, allowing large groups on the carboxylic acid and resulting in highly regio- and enantioselective conversions.

CALB has been used extensively in the resolution of racemic alcohols, amines, and acids, and in the preparation of optically active compounds from meso substrates. The resulting optically pure compounds are highly difficult to obtain by alternative routes and can be of great synthetic value. CALB has also been used as a regio-selective catalyst in selective acylation of different carbohydrates.

Dynamic kinetic resolution of racemic methyl ester1:

Desymmetrisation by enantioselective hydrolysis of diethylester2:

Transesterification of β-amino ester3:

Lipozyme® TL 100LLipozyme® TL 100L is a 1,3 specific lipase originating from Thermomyces lanuginosus. It is a very effective catalyst for transesterification, interesterification, ester hydrolysis and desymmetrisation of esters. Lipozyme® TL 100L exhibits a high degree of substrate selectivity allowing bulky side chains/large groups on the alcohol and acid part of the molecule.

One use of Lipozyme® TL 100L is in the commercial manufacture of Pregabalin. The key synthesis step is the enzymatic resolution of a racemic cyano diethylester (CNDE) to produce the desired monoacid enantiomer. In this application, Lipozyme® TL 100L performs well in activity and enantioselectivity.

Kinetic Resolution by desymmetrisation of cyano diethylester intermediate for Pregabalin4:

Novocor® AD LNovocor® AD L is a non-specific lipase originating from Candida antarctica A (CALA). CALA is stable over a relatively broad pH range especially at acidic pH. CALA is an extremely thermostable enzyme and has its temperature optimum above 90°C. In the esterification of sterically hindered substrates, CALA is the only lipase with the ability to catalyze the desired reactions. The enzyme can accept highly branched acyl groups as well as sterically hindered side chains at the alcohol.

Resolution of 2-phenylbut-3-yn-2-ol5:

Resolution of 1-methyl-1,2,3,4-tetrahydronapthalen-1-ol6:

Resinase® HTResinase® HT is a heat stable lipase originating from Aspergillus oryzae. It is a highly effective catalyst for ester hydrolysis of triglycerides.

Palatase® 20000 LPalatase®20000 L is a 1,3 specific lipase originating from Rhizomucor miehei. It is an effective catalyst for hydrolysis of small fatty acids on the carboxylic acid part.

Novozym® 51032Novozym® 51032 is a lipase originating from an Aspergillus micro-organism. It is an effective catalyst for hydrolysis of triglycerides as well as a variety of molecules containing ester linkages and has a specific hydrolytic activity on polyvinyl acetate.

Lipases Lipases (EC Number 3.1.1.3) are one of the most commonly used classes of enzymes in biocatalysis. Lipases catalyze the hydrolysis of triacylglycerols to diacylglycerol, monoacylglycerol, glycerol and free fatty acids. The reaction reverses under anhydrous conditions and the enzyme is able to synthesize new molecules by esterification, alcoholysis and transesterification. All reactions can be performed with high regio- and enantioselectivity under mild reaction conditions. Lipases have been used on a variety of substrates and show very broad substrate specificity due to the ubiquity in nature and the heterogeneity of lipases from different sources.

Regioselective hydrolysis of a triacylglycerol:

Key applications of lipasesLipase catalysts are used in industrial applications and the chemical transformations include:

• enantioselective resolution of esters and amides• acylation of alcohols and amines to form ester and amides• mild hydrolysis or acylation of sensitive substrates• kinetic resolution by transesterification of racemic alcohols• kinetic resolution by hydrolysis of racemic ester• desymmetrisation of diesters

* K = Kilo, LU = Lipase unit, PLU = Propyl Laurate Unit. 1LU is the amount of enzyme activity which liberates 1 µmol of tritratable butyric acid from the substrate

glycerol tributyrate per minute under defined standard conditions.

Benefits of enzymes as biocatalysts in organic transformations

Cost savings Improved productivity Improved quality of API/intermediate Environmental friendliness

• reduction in raw material input

• avoids use of costly chiral resolving agents or costly metal based catalysts

• lower equipment, labor and energy costs

• shortened synthesis routes

• more batches resulting in increased capacity

• avoids laborious protection and de-protection

• higher yields

• fewer or no by-products, leading to reduced impurities in the final products

• high stereo-, regio-, and chemo-selectivity

• less residual solvent carry over from reduced solvent use

• reduction of waste products produced and solvent usage

• higher energy savings

Novozymes offers a product portfolio of six lipases in liquid formulationProduct name Source Activity* pH optimum Temp optimum Substrate specificity

Lipozyme® CALB Candida Antartica B 5000 LU/g pH 5-9 30-60°C Esters and alcohols

Novocor® AD L Candida Antartica A 6000 LU/g pH 5-9 30-60°C Sterically hindered esters

Lipozyme® TL 100 L Thermomyces lanuginosa 100 KLU/g pH 7-10 20-50°C Esters and dicarboxylates

Resinase® HT Aspergillus oryzae 50 KLU/g pH 5-8 up to 90°C Esters and triglycerides

Palatase® 20000 L Rhizomucour miehei 20000 LU/g pH 7-10 30-50°C Esters

Novozym® 51032 Humicola insolens 15 KLU/g pH 7-10 35-70°C Esters

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References1. Pietruszka, J., Simon, R.C., Kruska, F., and Braun, M. (2010) Euro.J.Org.Chem., 6217-6224 2. Schonherr, H., Mollitor, J., and Schneider, C.(2010) Eur.J.Org.Chem., 20, 3908-3918 3. Gedey, S., Lijebald, A., Lazar, L., Fulop, F., and Kanerva, L.T.,(2002), Can.J, Chem., 80, 565-570 4. Ref. Carlos A. Martinez, Shanghui Hu, Yves Dumond, Junhua Tao, Patrick Kelleher, Liam Tully Organic Process Research & Development 2008, 12, 392-398 5. Hari Krishna, S., Persson, M., and Bornscheuer, U.T. (2002) Tetrahedron Asymmetry, 13, 2693-269 6. Ozdermirhan, D., Sezer, S., and Sonmez, Y.(2008) Tetrahedron Asymmetry ,19, 2717-2720.

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Novozymes does not promote or support the use of enzymes as Active Pharmaceutical Ingredients (APIs) or excipients.

Novozymes enzymes are industrial bulk products and are not produced according to pharmaceutical standards. If enzymes are

used as process aids, it is the responsibility of the API or excipient manufacturer to assure that the enzymes are suitable for the

intended use, including but not limited to evaluation of purity and absence of carry over in the final product. Laws, regulations,

and/or third party rights may prevent customers from importing, using, processing, and/or reselling the products described

herein in a given manner.

Without separate, written agreement between the customer and Novozymes to such effect, this document does not constitute

a representation or warranty of any kind and is subject to change without further notice.

About Novozymes Novozymes is the world leader in biological solutions. Together with customers, partners and the global community, we improve industrial performance while preserving the planet’s resources and helping to build better lives. As the world’s largest provider of enzyme and microbial technologies, our bioinnovation enables higher agricultural yields, low-temperature washing, energy-efficient production, renewable fuel and many other benefits that we rely on today and in the future. We call it Rethink Tomorrow.June 2016 - No. 2014-12478-04

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