Proteases for biocatalysis. for smarter chemical synthesis

Transcription

1 Proteases for biocatalysis for smarter chemical synthesis

2 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 C 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 B D

3 Proteases Proteases (EC ) are enzymes which conduct proteolysis by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain forming the protein. Proteases belong to the class of enzymes known as hydrolases catalyzing the reaction of hydrolysis of various bonds with the participation of a water molecule. Proteases can be used in organic synthesis to resolve a pair of enantiomeric forms in racemic mixtures through kinetic resolution where one enantiomer in the mixture is more rapidly transformed than the other. Protease catalysts can resolve enantiomers through a variety of reactions such as: hydrolysis of esters or amides of carboxylic acid esterification or transesterification reactions amide/peptide bond formation Proteolysis of a peptide bond: Key applications of proteases Kinetic resolution of amino acids by hydrolysis of racemic amino esters which can be converted into dynamic kinetic resolution by addition of catalytic aldehyde 1 : Kinetic resolution of carboxylic acid by hydrolysis of carboxylic ester: Kinetic resolution of amino acid by hydrolysis of racemic amino ester: Other potential applications for proteases include: hydrolysis of selective amides2 formation of low molecular weight peptides3 transesterifications Serine proteases Serine proteases contain a serine group in their active site which is essential for substrate binding and cleavage. Serine proteases are characterized by their broad substrate specificity and their activity extends beyond purely peptidase to include esterase and amidase activities. The common reaction mechanism is in the form of a catalytic center containing serine as a nucleophile, aspartate as an electrophile and histidine as a base. The reaction mechanism involves the formation of covalently linked enzyme substrate intermediate through acylation resulting in loss of the corresponding amino acid or peptide fragment. Nucleophilic attack on the intermediate by water results in deacylation thereby completing hydrolysis of the peptide.

4 Subtilisin A Subtilisin A (E.C ) is an alkaline non-specific serine protease from Bacillus subtilis that initiates the nucleophilic attack on the peptide bond through a serine residue at the active site; it catalyzes the hydrolysis of proteins and peptide amides. Alcalase Alcalase acts as an esterase, enabling it to catalyze stereoselective hydrolysis of some esters. Alcalase also efficiently hydrolyzes amino esters which include heterocyclic amino esters. Savinase Savinase catalyzes stereoselective hydrolysis of some esters as well as strained amides under alkaline conditions. Esperase Esperase is an endo-peptidase with a broad specificity which performs well in alkaline conditions and at elevated temperatures as compared to other microbial serine proteases. Kinetic resolution of carboxylic acids by hydrolysis of carboxylic esters4: Kinetic resolution of strained amides2: Stability of proteases The graphs below represent alcalase ph and temperature stability. Effect of ph on alcalase activity Effect of temperature on alcalase activity Effect of ph on alcalase stability Relative activity (%) Relative activity (%) Relative activity (%) ph Temperature ( C) ph

5 Neutrase Neutrase (E.C ) is a neutral, zinc metallo endo-protease from Bacillus amyloliquefaciens that randomly hydrolyses internal peptide bonds and also facilitates enzymatic synthesis of oligopeptides by the reverse proteolysis reaction with zinc metal as co-catalyst. Neutrase belongs to the same protease family as thermolysin, a zinc dependent metallo endo-protease. Thermolysin in an immobilized form has been used successfully in industrial processes for synthesis of an Aspartame intermediate5. The reaction takes place in organic solvent and involves kinetic resolution of an amine methyl ester with high enatioselectivity and high regioselectivity in the amide bond formation of the a-carbonyl in Aspartic acid preferred over the b-carbonyl. rtrypsin rtrypsin (EC ) is an endopeptidase that preferentially hydrolyses ester bonds whose carboxyl groups are contributed by lysine (Lys) or arginine (Arg) except when either is followed by proline. The enzymatic mechanism of action is similar to other serine proteases. The aspartate residue located in the catalytic pocket of rtrypsin is responsible for attracting and stabilizing positively charged lysine and/or arginine, and is thus responsible for the specificity of the enzyme. 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 chemoselectivity less residual solvent carry over from reduced solvent use reduction of waste products produced and solvent usage higher energy savings Available from Novozymes Product name EC number Type Form Optimum usage conditions Activity Applications Alcalase 2.4 L FG C, ph AU-A/g Stereoselective hydrolysis of amino esters Alcalase 2.4 L, DX C, ph AU-A/g Stereoselective hydrolysis of amino esters Savinase 12 T Granulate C, ph KN-PU- S/g Stereoselective hydrolysis of amino esters Savinase 16 L C, ph KN-PU- S/g Stereoselective hydrolysis of amino esters Esperase 8.0 L C, ph KNPU-E/g Serine endoprotease that hydrolyzes internal peptide bonds Neutrase 0.8 L Metalloprotease C, ph AU-N/g Metallo endoprotease that hydrolyz-es internal peptide bonds rtrypsin Serin protease Granulate ph USP/mg Hydrolysis of amid end ester bonds of lysine and arginine at carboxyl terminal * K = Kilo, AU = Anson Unit, NPU = Novo Protease Unit, 1 AU = 1NPU, ASNU = Asparaginace Unit, USP = Trypsin activity unit using USP Crystallised Trypsin Reference Standard. The activity is determined relative to a protease A standard. The result is given in the same units as the standard. 1 ASNU is the amount of enzyme that produces 1 µmol Ammonia per minute under the standard reaction conditions.

6 References 1. Pietruszka, J., Simon, R.C., Kruska, F., and Braun, M. (2010) Euro.J.Org.Chem., Schonherr, H., Mollitor, J., and Schneider, C.(2010) Eur.J.Org.Chem., 20, Gedey, S., Lijebald, A., Lazar, L., Fulop, F., and Kanerva, L.T.,(2002), Can.J, Chem., 80, Ref. Carlos A. Martinez, Shanghui Hu, Yves Dumond, Junhua Tao, Patrick Kelleher, Liam Tully Organic Process Research & Development 2008, 12, Hari Krishna, S., Persson, M., and Bornscheuer, U.T. (2002) Tetrahedron Asymmetry, 13, Ozdermirhan, D., Sezer, S., and Sonmez, Y.(2008) Tetrahedron Asymmetry,19, 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 No 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 (DK) (US) (IN) (CN) Novozymes A/S Krogshoejvej 36 DK 2880 Bagsvaerd Denmark pharmaceuticals@novozymes.com novozymes.com