FIELD OF THE INVENTION:

Enzyme transaminase is used for the preparation of compound of Formula I comprising reuse of at least once used transaminase enzyme for stereoselective conversion of keto group of 4- Oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[l,2,4]triazolo[4,3-a ]pyrazine-7(8H)-yl]-l-(2,4,5- trifluorophenyl)butan-2-one (also referred herein Ketoamide) of Formula II. Generally, as disclosed in prior art the said transaminase enzyme is used once only and discarded after the reaction is completed. Alternately, prior art discloses the reuse of the once used enzyme after immobilization process. The inventors of the present invention disclose herein that the enzyme once used can be reused as such without any processing like immobilization for the future batches for the same chemical reaction step without any adverse impact on the yield and purity of the product. The recycling, also referred herein as reuse makes the process for preparing compound of Formula-I, which is a drug molecule Sitagliptin thereby making the said improved inventive process economical and green at industrial scale.

The rapid separation and efficient recovering of used enzymes after an enzymatic reaction is considered an important requirement along with the high catalytic performances of the used enzyme for the same chemical process step for future batch/es. In view of this, inventors of the present invention have developed a novel and innovative process for the preparation of compound of formula I and intermediates thereof that allows a good separation of enzyme from the reaction mass wherein fresh or used enzyme is used and reuse of said at least once used enzyme isolated from the reaction mass. The separation process is economical and technically feasible with a minimal topping with a fresh enzyme, if any.

BACKGROUND OF THE INVENTION:

Sitagliptin is chemically known as 7-[(3R)-3-amino-l-oxo-4-(2,4,5-trifluorophenyl) butyl]- 5,6,7,8-tetrahydro-3-(trifluoromethyl)-l,2,4-triazolo[4,3-a] pyrazine represented by structural Formula-I:

Sitagliptin is a dipeptidyl peptidase 4 (DPP-4) inhibitor and used to improve glycemic control in patients with type-2 diabetes mellitus. Sitagliptin is sold under the trade name JANUVIA as monotherapy and JANUMET in combination with Metformin hydrochloride.

US6699871B2(hereinafter referred as ‘871) discloses Sitagliptin and other related compounds which are inhibitors of the dipeptidyl peptidase-IV enzyme ('DP-IV inhibitors') and which are useful in the treatment or prevention of diseases in which the dipeptidyl peptidase-IV enzyme is involved, such as diabetes and particularly type 2 diabetes.

‘871 discloses a process for preparation of chiral beta amino acid derivative using chiral pyrazine derivative to introduce chiral amine, which after few subsequent steps give Sitagliptin and related compounds. The process involves many steps making process commercially inviable. The process also uses pyrophoric reagent butyl lithium, explosive reagent diazomethane, expansive reagents silver benzoate and EDC.

W02004085661A2 discloses a process for the preparation of enantiomerically enriched beta amino amide including Sitagliptin by treating ketoamide compound with a chiral auxiliary S- phenyl glycine, followed by diastereoselective reduction of the enamine by hydrogenation using Pt2O and finally debenzylation by palladium catalyzed hydrogenolytic removal of benzyl group. The process suffers from low diastereo- and enantioselectivity and lower yield. Also, the process uses expansive metal catalyst like Pt2O.

US7495123B2 discloses a process for the preparation of chiral beta amino amide by rhodium catalysed asymmetric hydrogenation of prochiral enamine in the presence of a chiral mono- or bisphosphine ligand. Drawback of this process lies on high cost of catalyst and low enantiomeric purity.

W02006081151A1 discloses a process for preparation of chiral beta amino amide that comprises an enantioselective hydrogenation of a prochiral beta-amino acrylic acid derivative substrate in the presence of an ammonium salt and a transition metal precursor complexed with a chiral ferrocenyl bisphosphine ligand.

Rhodium-catalized asymmetric enamine hydrogenation originally used for the large-scale manufacture of the antidiabetic compound Sitagliptin. In the recent era, Rhodium is replaced with a transaminase enzyme which ultimately has led to an enzymatic process that reduces waste, improves specificity, yield, safety and eliminates the need for a metal catalyst. Exploration for suitable catalysts has been expanded significantly from the last few decades because of the increasing demand of environmental friendly production in the industry. In this quest, biocatalysts have much to offer because of their ease of production, substrate specificity, and green chemistry. Progress in biotechnology has paved the way for the widespread application of biocatalysis in industrial organic synthesis.

A process for the preparation of Sitagliptin is reported in Science, 2010, 329, 305-309 using transaminase enzymes to reduce beta keto amide with high enantioselectivity. The biotransformation is performed in a mixture of triethanol amine buffer and DMSO. The process is capable for the synthesis of Sitagliptin with high enantiomeric purity. One of the main drawback of the process is high cost of enzyme. Another drawback of the process is use of water miscible DMSO as co-solvent, which makes work up process difficult. Further, difficulty in recovery of DMSO makes this process expensive and environmentally unsafe.

US8293507 discloses the use of transaminase for the biocatalytic conversion of 4-oxo-4-[3- (trifluoromethyl)-5,6-dihydro[l,2,4]triazolo[4,3-a]pyrazin-7 (8H)-yl]-l-(2,4,5- trifluorophenyl)butan-2-one (the “ketoamide substrate”) to (2R)-4-oxo-4-[3-(trifluoromethyl)- 5,6-dihydro[l,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-l-(2,4,5- trifluorophenyl)butan-2-amine (the “product) in presence of an amino group donor. However, due to substrate poorly water- soluble, DMSO is used in varying amount from 10-50% thereby making DMSO solvent recovery difficult, and cost is higher.

US9587229 and US9523107discloses the use of immobilized transaminases comprising a recombinant transaminase physically attached to a resin by ether hydrophobic interactions or covalent bonds, capable of converting 4-oxo-4-[3-(trifluoromethyl)-5,6- dihydro[l,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-l-(2,4,5-trif luorophenyl)butan-2-one to (2R)- 4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[l,2,4]triazolo[4,3- a]pyrazin-7(8H)-yl]-l-(2,4,5- trifluorophenyl)butan-2-amine in the presence of an amino group donor to levels measurable by an analysis technique. Immobilized transaminases are not changed during the reactions; these enzymes can be used as catalysts for a desired chemical transformation.

CN105018440 discloses transaminase from mycobacterium (Mycobacteriumvanbaalenii) PYR-lhas the function of transaminase, is a kind of new transaminase. The prochirality carbonyl compound is preferably 3- carbonyls -4- (2,4,5- trifluorophenyl) -butanoic acid esters. CN103014081 discloses the use of transaminase by 3- carbonyls -4- (2,4,5- trifluoros Phenylmethyl butyrate into R-3- amino -4- (2,4,5- trifluorophenyl)-methyl butyrate.

CN104805069B discloses immobilization of transaminase and its reuse. Restructuring transaminase is fixed on sodium alginate by the immobilization process. It discloses catalysis of prochiral carbonyl compounds using immobilised to obtain chiral amine.

Transaminases are a specific class of enzymes that catalyse the direct amination of ketones to chiral amines. Enantiomerically pure chiral amines are key intermediates in a number of pharmaceutical compounds that possess a wide range of biological activities. Besides, enzyme based catalysis is performed with much higher fidelity, under mild ambient environmental reaction conditions like temperature, pH, and pressure are highly efficient in terms of number of steps, giving them an edge over their chemical counter parts. The unique characteristic of enzyme makes them highly applicable for a number of chemical transformation reactions in pharmaceutical industries.

Although enzymes represent a key component in a broad range of reactions but their cost becomes a limiting factor in industries and academics. Further, despite great potential of enzymes, their industrial applications have been restricted because of the recovery of enzymes and difficulty in reusability limits their application in the industry. To overcome these limitations, enzymes have been immobilized on various solid supports. Most of the prior art cited herein above comprises the use of immobilization process which allows the enzymes to be recycled and reused, reducing cost in comparison with use of a fresh enzyme. The said immobilisation process as described herein requires a separate facility having requisite infrastructure and extra material viz solid support and the like but still cost efficient compared to process wherein a fresh enzyme is used.

Enzyme immobilization is defined as a process of confining the enzyme molecules to a solid support over which a substrate is passed and converted into product. It is done for protection from degradation and deactivation of enzyme with the intension of re-use of enzymes for reaction cycles, lowering the total production cost of enzyme mediated reactions. Enzyme immobilization is one of the most promising approaches for exploiting enzyme based processes in Biotransformation. Immobilized enzymes though a little cheaper than fresh unused enzyme but still are expensive as the process of immobilization process need an exclusive dedicated infrastructure which is expensive needing extra space, material and utilities. In addition, it requires specific material and additional process step which is time consuming. For immobilisation process sophisticated process equipment is needed. In the production process other problems arise, such as increased risk of contamination, need for extra control of temperature and pH, and substrate purity. Diffusion resistance may cause increased levels of by-products and may also reduce the apparent activity. It is evident that the process which comprises reuse of immobilised enzyme which requires extra step of immobilization is expensive and uneconomical compared to a process where the reuse of at least once used enzyme is used as such without any extra process step like immobilization.

As evident from foregoing the reuse of immobilized enzymes is also not economical thus not viable at industrial scale. In view of above inventors of the present invention disclose herein a novel and inventive process for recovering the used biocatalyst particularly transaminase from the reaction mass and reusing the same recovered biocatalyst as such without immobilization for the future production batch for the preparation of Sitagliptin and intermediates thereof thereby making the process economical and green, without having any additional cost of immobilization like processes.

There is no disclosure or even indication or teaching in the prior art about the safe and simple recovery of used enzyme from the reaction mass and reuses of said recovered enzyme as such without immobilization for subsequent batch for the same process.

TECHNICAL PROBLEMS ASSOCIATED WITH THE PRIOR ART:

• Immobilization of enzyme

• High cost and additional process step of immobilization

• Use of water soluble and costly solvent like DMSO does not provide effective recovery of the solvent thereby making the process commercially unviable at industrial scale.

• Organic solvent used, DMSO being water miscible makes the single phase solvent system and both the product and enzyme remain in the same solvent system so difficult to recover enzyme from the reaction mass.

• Generation of effluent as DMSO is not recovered from the single phase two solvents system comprising water and DMSO.

• Only option associated with process disclosed in the prior art is the use of immobilized transaminase enzyme with the intension of their stability, recycle and reuse for the preparation of Sitagliptin of formula I and key intermediates thereof. • Immobilization of transaminase enzyme has got the disadvantages as discussed herein before.

In view of above, inventors of the present invention realized that there is a dire need for the development of an enzymatic process for the preparation of Sitagliptin and intermediates thereof which addresses the issues disclosed herein above wherein enzyme to be used for the desired conversions is not immobilized enzymes.

There is no indication or teaching or motivation wherein a person skilled in the art can as such plan or think to recover and reuse of enzyme without immobilization using a biphasic solvent system, for said biochemical reaction and reuse of same recovered enzyme as such without immobilization for the same biotransformation reaction comprising stereospecific amination of keto group of ketoamide of Formula II to obtain chiral amino ester of Formula I or intermediate thereof.

Inventors of the present invention successfully based on exhaustive R&D efforts have evolved an efficient cost effective, green novel and inventive process comprising: developing an efficient biphasic solvent system for said biochemical reaction and the said biphasic solvent comprises water and water immiscible organic solvent; recovery of used enzyme from the biphasic solvent system; reuse of said recovered enzyme as such without immobilization; recovery of the organic solvent from the biphasic reaction mass containing compound of Formula I. no generation of effluent as both the solvents of the reaction mass are recovered ans reused.

The improved process disclosed herein for the preparation of compound of Formula I addresses all the issues therein in the biochemical process disclosed in the prior art and making the said improved process for industrial production of Sitagliptin and intermediates thereof green, economical and commercially viable at industrial scale.

Beside high enzymatic performance, a rapid separation and efficient recovery (also referred as recycling or reuse herein before and after) of the biocatalyst after enzymatic reaction are important requirements to reduce the production costs as well as to minimize waste generation, especially in industrial applications. The recovery and reuse of a catalyst is important from both the economic and the environmental point of view. This disclosure discloses herein a process comprising using biphasic solvent system, separation of used transaminase enzyme from the biphasic reaction mass and the said recovered used transaminase enzyme is reused as such without immobilization, making the process cost effective without affecting quality and quantity parameters. The said novel and inventive process also ensures minimum waste effluent.

Disclosed herein is a novel and innovative process particularly for the preparation of Sitagliptin of Formula I with high optical purity comprising reuse of at least once used transaminase enzyme as such without having any kind of immobilization or the like. Used enzyme is collected from the reaction mass of the biochemical enzymatic process for the preparation of Sitagliptin, wherein the said used transaminase enzyme is obtained just by separation of aqueous phase of the biphasic solvent system which contains said enzyme which is reused as such for the same biocatalytic amination of ketoamide of formula II into Sitagliptin of formula I using buffer and a water immiscible organic solvent selected from the group comprising isopropyl acetate, n-butanol, isobutanol, Methyl tert-butyl ether, 2-methyl tetrahydrofuran, cyclohexanol and the like or mixture thereof.

The process is also characterized by the fact that use of water immiscible solvent ensures good conversion due to non-precipitation of the ketoamide substrate of Formula II as in the single phase solvent system, substrate of Formula II being insoluble in water precipitates from a single phase solvent system and substantial quantity remains unreacted.

OBJECTS OF THE INVENTION:

One of the objective of the present invention is to make the process green avoiding generation of waste effluent and commercially viable at industrial scale for the production of Sitagliptin of Formula I comprising recycle and reuse of at least once used transaminase enzyme as such without immobilization or any other additional processing act and the said used enzyme is isolated directly from the reaction mass of amination used for the preparation of Sitagliptin. The process for recovery or isolation of at least once used enzyme from the reaction mass comprises separation of organic solvent layer from the reaction mass and separated aqueous layer contains used transaminase enzyme and the separated aqueous layer containing said used enzyme is reused as such without immobilization by adding substrate in a water immiscible organic solvent into the said separated aqueous layer containing used enzyme separated from the early production batch and proceeding for the biocatalytic reaction to obtain substantially pure Sitagliptin of Formula I in high yield.

Use of biphasic solvent system comprising water and water immiscible organic solvent enables the separation of enzyme from the reaction mass and the said separated enzyme is reused for the same biochemical reaction without having any other extra efforts like immobilization. Moreover, the novel process also enables the recovery of organic solvent. There is no teaching or motivation to a person skilled in the art to use biphasic solvent system for the biochemical reaction and to recover and reuse the enzyme as such without immobilization thus making the process green and economical at industrial scale.

One of the objectives of the present invention is to reuse of at least once used transaminase enzyme without having any kind of processing such as immobilization and the like.

One of the objectives of the present invention is to reduce the overall cost of the process for the preparation of Sitagliptin.

One of the objectives of the present invention is to use water immiscible solvent to simplify the workup procedure characterized by the feature that enzyme used remains in aqueous layer and easily isolated/recovered for reuse in the same water solvent for next batch for the preparation of same product.

One of the objectives of the present invention is to provide the robust process comprising using water immiscible solvent which otherwise may lead to incomplete conversion in water miscible solvent due to precipitation of the substrate.

One of the objectives of the present invention is to avoid the use of immobilized enzymes, thereby avoiding the expenses required for the process of immobilization.

Another objective is to recover the organic solvent which can be reused avoiding the generation of effluent making the process economical and green at industrial scale. Disclosed herein is a novel and innovative process to prepare Sitagliptin of Formula I with high optical purity comprising reuse of at least once used transaminase enzyme as such without any kind of processing like immobilization and the said used enzyme is recovered/isolated from the reaction mass of early batch. The process of recovery/isolation of used enzyme comprises separating aqueous layer of biphasic solvent system of reaction mass containing used enzyme and the same aqueous mother liquor containing used enzyme is used as such again and substrate of Formula II in a water immiscible organic solvent is added to the said aqueous mother liquor containing enzyme. Recycled and reused transaminase under the consideration of the present invention is used for the amination of ketoamide compound of formula II into Sitagliptin of Formula I. Also the separated organic solvent layer containing product (Sitagliptin of Formula I) is also separated and product isolated and solvent recovered which too can be used again avoiding the generation of effluent making the process economical and green at industrial scale.

ADVANTAGE OF THE INVENTION:

Major advantage of the present invention is the reuse of at least once used transaminase enzyme as such without involving extra efforts like immobilization and the like for the commercially economical production of Sitagliptin at of Formula I at industrial level with minimization of waste.

Use of biphasic solvent system makes work up and enzyme and solvent recovery simpler and cost effective. The process with water immiscible solvent makes the process robust which otherwise may lead to incomplete conversion in water miscible solvent as the ketoamide substrate of Formula II being insoluble in water precipitate out from the reaction mass. Separation of water layer containing enzyme and the organic layer containing desired products is separated at great ease and the used enzyme in water layer is reused as such for the next batch for the preparation of desired compounds. The product is isolated from the organic solvent layer and the solvent is recovered avoiding generation of effluent making the process economical and green.

DETAILED DESCREPTION OF THE PRESENT INVENTION:

Transaminases (also referred herein as TAs) are one of the most promising biocatalysts in organic synthesis for the preparation of chiral amino compounds. The concise reaction, excellent enantioselectivity, environmental friendliness and compatibility with other enzymatic or chemical systems have brought transaminase to the attention of scientists working in the area of biocatalysis. However, to utilize transaminase in a more efficient and economical way, attempts are made to optimize their performance using recycled and reuse of transaminase enzymes as such without involving any extra enzyme activation process like immobilization and the like.

The greenness of these outstanding enzymes (biocatalysts), including aspects such as shorter routes without functional group protecting steps, lower cost of equipment and energy, less environmental waste, easier procedures for work-up and even lower cost of catalysts considering the recycling of enzymes as disclosed herein, has made them one of the preferred options for industrial organic preparation.

Much attention and effort has been applied in recent years towards the reuse of immobilized enzymes, but no measures are taken for reusing non-immobilised enzyme as such and nor there is any such disclosure in the prior art. Furthermore, the process for preparation of Sitagliptin of Formula I disclosed therein in prior art discloses use of a single phase solvent system and the product and used enzyme both are present in the same single phase of the reaction mass, therefore, very difficult to isolate the enzyme and also recovery of the water miscible solvent. However, with great difficulty the enzyme is recovered and has never been used as such and instead is processed by the expensive and an extra process step of immobilization and the said immobilized enzyme only is reused for the same reaction step of stereoselective amination of a ketoamide of Formula II to prepare Sitagliptin of Formula I. Enzyme being expensive is the one of the major cost factor among the chemical materials used for the production of Sitagliptin of Formula I.

The present disclosure without any teaching, motivation from the prior art disclose herein use of biphasic solvent system comprising water and water immiscible organic solvent for the stereoselective amination of selectively a ketoamide of Formula II comprising reuse of at least once used enzyme as such without immobilisation. The advantages are as below:

1. The substrate of Formula II being soluble in organic solvent phase doesn’t precipitate whilst reaction is in progress, therefore maximum substrate of Formula II gets converted into corresponding compound of Formula I.

2. After the reaction is complete, the enzyme remains in the water phase and product of Formula I remain in water immiscible organic solvent phase. 3. Organic solvent is separated and the solvent is recovered and substantially pure compound of Formula I is obtained in good yield. Recovery of solvent ensures minimization of waste and making the process economical and green.

4. Water phase containing used enzyme, amine donor and buffer is separated and is reused as such for the next production batch for the production of compound of compound of Formula I comprising addition of the substrate ketoamide of Formula II in a water immiscible organic solvent into the said water solvent phase containing used enzyme from earlier batch.

Prior art discloses the said biocatalytic process for the preparation of Sitagliptin of Formula I wherein the fresh enzyme is used once and no provision of isolating the used enzyme from a single phase reaction mass. As disclosed in the prior art the used enzyme can be reused only after immobilization for the preparation of Sitagliptin. The immobilization process is expensive and time consuming as it comprises additional process steps. Inventors of the present invention disclose herein the reuse of recovered enzyme as such without any additional process step like immobilization. The used enzyme therein in the aqueous mother liquor separated from the biphasic solvent system is reused as such by the inventors of the present invention for the same purpose viz. amination of a ketoamide of compound of Formula II into amino group to prepare Sitagliptin of Formula I at industrial scale making the process economical and green.

Inventors of the present invention have been able to achieve this and as a result; disclose herein an efficient process for the preparation of Sitagliptin of Formula I.

As disclosed herein above this has been achieved by the inventors of present invention by using biphasic solvent system comprising water and water immiscible solvent which has following advantages.

1. In water immiscible organic solvent, good conversion of compound of Formula II into compound of Formula I is achieved. A water miscible solvent forms single phase with water and the substrate of Formula II being insoluble in water gets precipitated which leaves some of the substrate unreacted.

2. The amino product of Formula I obtained remains in the organic solvent and the enzyme, cofactor and buffer remains in the aqueous layer. The organic layer is separated and product isolated and solvent is recovered making the process economical and green as due to solvent recovery there is no generation of effluent.

3. The aqueous layer containing used enzyme, cofactor and buffer is reused as such as an aqueous part of the biphasic solvent system containing enzyme, cofactor and buffer to which substrate of Formula II in water immiscible organic solvent is added for same biocatalytic process.

4. As the organic solvent is recovered and water layer also is used for the next batch, there is no solvent waste making the process green.

The preferred embodiments described herein details for illustrative purposes only and are by no means limiting and can be further enhanced by many variations. It is understood that various omissions and substitutions of equivalents are contemplated as circumstances may suggest or render expedient but are intended to cover the application or implementation without departing from the spirit or scope of the present invention.

With detailed reference to certain embodiments of the present invention, the example of these embodiments illustrates with structure and the formula enclosed. Although the present invention will be illustrated in conjunction with row illustrated embodiments, but it is to be understood that they are not intended to limit the invention to those embodiments. On the contrary, it is contemplated that contain all alternative forms, modification and the equivalents that may be included in the scope of the invention as defined in the claims. The invention is not restricted to method described herein and material, but include any method similar or equivalent with method described herein and material that can be used for putting into practice the present invention and material. One or more in the list of references being incorporated to, patent or similar data is different from the application (including but not limited to defined term, term usage, described technology etc. or runs counter to the application, it is as the criteria with the application

In a general embodiment disclosed herein is a process for the preparation of compound of Formula I comprising: a) an aqueous layer is separated from the biphasic solvent system of the previous production lot of production of compound of Formula I and the said aqueous layer contains cofactor, buffer, water, alkyl amine and at least once used transaminase enzyme; b) pH of the said aqueous solvent phase is adjusted to about 7 to about 10 preferably about 8.5 using an acid; c) a solution containing ketoamide of Formula II in a water immiscible organic solvent is added slowly to the aqueous mixture of step b) and the temperature of the reaction mass is maintained at about 35°C to 70°C preferably about 45-50°C;adjusting pH between about 8.2 to about 9.5. Byproduct like acetone generated during the reaction is removed under vacuum or bubbling or both from the reaction mass to enable the reaction to proceed further and the stirring of the said reaction mass is continued until achievement of maximum conversion; d) the reaction mass of step (c) is cooled to room temperature; e) organic solvent layer is separated from the aqueous layer of the biphasic solvent reaction mass and the said aqueous layer is further extracted with same water immiscible organic used in step (c); f) combined organic solvent is concentrated by removing solvent by standard process like vacuum distillation and the like to obtain substantially pure desired amino product of Formula I and recovering the organic solvent; g) aqueous layer as separated in step e) from the water immiscible organic solvent layer and the said aqueous layer contains the residual used enzyme, buffer, alkyl amine and cofactor used for the biocatalytic amination to obtain compound of Formula I and the said aqueous layer containing used enzyme is used as a water solvent phase of the biphasic solvent system as such for the next batch/cycle for the same process as of the instant embodiment and the used enzyme in the said aqueous layer is reused for the same purpose.

Schematic representation:

SCHEME-I

Used enzyme is isolated after its first use for its further reaction as disclosed in the present invention simply by the separation of reaction mass into water immiscible organic solvent containing product of Formula I and aqueous layer containing used enzyme.

Separated aqueous layer herein above containing used enzyme, buffer, alkyl amine and cofactor is reused in subsequent cycle/batch for the preparation of compound of Formula I and likewise the aqueous layer containing reused enzyme is again reused for subsequent cycle/batch for the same purpose of converting compound of Formula II into compound of Formula I.

The process of the present invention is further characterized by the feature that use of biphasic solvent medium comprising water and water immiscible organic solvent makes work up, isolation of product simple and effective. Furthermore, said biphasic solvent medium also enables the recovery of enzyme and organic solvent from the reaction mass just by separation of each phase of the said biphasic solvent medium also refereed as system. The process with water immiscible solvent make the process robust which otherwise may lead to incomplete conversion in water miscible solvent due to precipitation of the substrate of molecule of Formula II.

Cofactor in step a) is Pyridoxal phosphate.

The alkyl amine is used as an amine donor moiety. The said alkyl amine is also acts as a base for pH adjustment. The alkyl amine used in herein above is selected from the group comprising isopropyl amine, isobutyl amine, isopropyl amine, 2-methyl benzyl amine, diphenyl methyl amine or any C-substitutes isopropyl amine and the mixture thereof and the likes.

The water immiscible solvent used herein above step c) is selected from alkyl ester, ether, water immiscible alcohols and 2-methyl tetrahydrofuran or mixture thereof.

Water immiscible alkyl ester is selected from the group comprising ethyl acetate, isopropyl acetate, isobutyl acetate, ter. Butyl acetate or mixture thereof and the likes. Water immiscible ether is selected from the group comprising diisopropyl ether, methyl, methyl isopropyl ether, methyl ter-butyl ether or mixture thereof and the likes.

Water immiscible alcohol is selected from the group comprising n-butanol, ter.-butanol, cyclohexanol, or mixture thereof and the likes.

The aq. Buffer used herein is selected from the group comprising triethanol amine buffer, phosphate buffer, Tris buffer, or any other buffer in which the said enzyme used herein is stable or mixture thereof.

In an another embodiment an aqueous phase is prepared comprising addition of cofactor Pyridoxal phosphate, buffer triethanolamine and isopropyl amine as an amine donor and transaminase enzyme, pH of said aq. Mixture is adjusted to about 8.5 using hydrochloric acid. The said buffer aq. mixture is stirred at room temperature to get the clear solution. Temperature of the said aq. Mixture is raised to about 45°C. A water immiscible organic solvent isopropyl acetate solution containing ketoamide of Formula II is added slowly to the said aq. mixture resulting into a biphasic solvent system also referred herein as reaction mass. Temperature of said reaction mass is maintained at about 45-50°C and pH is adjusted between about 8.2-9.5 using isopropyl amine. Acetone generated as a by-product therein is removed under the reduced pressure to enable the reaction to proceed to completion. Stirring is continued until achievement of maximum conversion. The reaction mass is cooled to room temperature. Organic solvent Isopropyl acetate layer is separated from aqueous layer of the biphasic solvent reaction mass and aqueous layer is further extracted with isopropyl acetate. Combined isopropyl acetate is distilled under vacuum to afford substantially pure Sitagliptin of Formula I as a product as oily mass which turns to solid on standing. Aqueous layer which contains used transaminase enzyme is reused as such for next cycle/batch for amination of a ketoamide of Formula II for the next production batch of Sitagliptin of Formula I as described herein before in earlier embodiment.

In still another embodiment aqueous layer obtained herein before from a reaction mass where reused enzyme is used and said reused enzyme is reused again for the preparation of Sitagliptin of Formula I. The said aqueous layer contains reused enzyme, cofactor pyridoxal phosphate, buffer triethanolamine and amine donor isopropyl amine and the said aqueous layer is heated to about 45 °C and a solution ketoamide of Formula II in isopropyl acetate is added slowly. The biphasic reaction mass is stirred at about 45-50°C by adjusting pH between about 8.2-9.5 using isopropyl amine. Acetone generated as by-product is removed under the reduced pressure to enable the reaction to proceed further to completion. Stirring of the said reaction mass is continued to achieve maximum conversion. The biphasic reaction mass is cooled to room temperature. Organic isopropyl acetate layer is separated and said aqueous layer is extracted further with isopropyl acetate to extract any product of Formula I, if present in said aqueous phase. Combined organic layer is distilled under vacuum to afford Sitagliptin of Formula I product as oily mass which turns to solid on standing. The water immiscible organic solvent is recovered.

In a specific embodiment disclosed herein is an economically efficient and green enzymatic process for preparation of Sitagliptin of formula I as presented in SCHEME - 1, comprising at least once used transaminase enzyme is reused for the enzymatic conversion of compound of formula II in a biphasic solvent medium comprising aqueous buffer and water immiscible organic solvent as described herein in earlier embodiments.

The embodiments of this invention are best understood and further illustrated by the following, non-limiting Examples:

Example 1: Preparation of 7-[(3R)-3-amino-l-oxo-4-(2,4,5-trifluorophenyl) butyl]-5, 6,7,8- tetrahydro-3-(trifluoromethyl)-l,2,4-triazolo[4,3-a]pyrazine (or Sitagliptin)

Triethanol amine (2.75g), water (50 mL) & Isopropyl amine were stirred together-and adjusted pH to 8.5 using cone. HC1. Pyridoxal phosphate (56 mg) and transaminase enzyme (1g) was added to the above solution. Temperature was raised to 45°C. A solution of ketoamide of formula I (25g) in isopropyl acetate was added to the above solution. The reaction mass was stirred at 45-50°C by adjusting pH between 8.2-9.5 using isopropyl amine while removing acetone formed in the reaction. The reaction mass was further cooled to room temperature. Isopropyl acetate layer was separated and aqueous layer was extracted with isopropyl acetate. Combined isopropyl acetate layer was distilled to afford Sitagliptin (90% yield).

Aqueous layer from the above process which contains enzyme was used for second cycle of biotransformation.

Example 2: pH of the aqueous layer from Example 1 was adjusted to 8.2-9.5 using con. HC1 and added pyridoxal phosphate (56 mg). Temperature was raised to 45°C. A solution of ketoamide (25g) in isopropyl acetate was added to the above aqueous solution. The reaction mass was stirred at 45-50°C by adjusting pH between 8.2-9.5 using isopropyl amine while removing acetone formed in the reaction. The reaction mass was further cooled to room temperature. Isopropyl acetate layer was separated and aqueous layer was extracted with isopropyl acetate. Combined isopropyl acetate layer was distilled to afford Sitagliptin (90% yield).

Example 3: Preparation of 7-[(3R)-3-amino-l-oxo-4-(2,4,5-trifluorophenyl) butyl]-5, 6,7,8- tetrahydro-3-(trifluoromethyl)-l,2,4-triazolo[4,3-a]pyrazine (or Sitagliptin)

Triethanol amine (1.1g), water (20 mL) & isopropyl amine were stirred together-and adjusted pH to 8.5 using cone. HC1. Pyridoxal phosphate (59 mg) and transaminase enzyme (170 mg) was added to the above solution. Temperature was raised to 45°C. A solution of ketoamide of formula I (10g) in isopropyl acetate was added to the above solution. The reaction mass was stirred at 45-50°C by adjusting pH between 8.8-9.5 using isopropyl amine while removing acetone formed in the reaction. The reaction mass was further cooled to room temperature. Isopropyl acetate layer was separated and aqueous layer was extracted with isopropyl acetate. Combined isopropyl acetate layer was distilled to afford Sitagliptin (90% yield).

Example 4: pH of the aqueous layer from Example 3 was adjusted to 8.2-9.5 using cone. HC1 and temperature was raised to 45°C. A solution of ketoamide (10g) in isopropyl acetate was added to the above aqueous solution. The reaction mass was stirred at 45-50°C by adjusting pH between 8.2-9.5 using isopropyl amine while removing acetone formed in the reaction. The reaction mass was further cooled to room temperature. Isopropyl acetate layer was separated and aqueous layer was extracted with isopropyl acetate. Combined isopropyl acetate layer was distilled to afford Sitagliptin (90% yield).

Example 5: Preparation of 7-[(3R)-3-amino-l-oxo-4-(2,4,5-trifluorophenyl) butyl]-5, 6,7,8- tetrahydro-3-(trifluoromethyl)-l,2,4-triazolo[4,3-a]pyrazine (or Sitagliptin)

Triethanol amine (11g), water (200 mL) & isopropyl amine were stirred together-and adjusted pH to 8.5 using cone. HC1. Pyridoxal phosphate (600 mg) and transaminase enzyme (2.5 g) was added to the above solution. Temperature was raised to 45°C. A solution of ketoamide of formula I (100g) in isopropyl acetate was added to the above solution. The reaction mass was stirred at 45-50°C by adjusting pH between 8.8-9.5 using isopropyl amine while removing acetone formed in the reaction. The reaction mass was further cooled to room temperature. Isopropyl acetate layer was separated and aqueous layer was extracted with isopropyl acetate. Combined isopropyl acetate layer was distilled to afford Sitagliptin (91% yield).

Example 6: pH of the aqueous layer (510 mL) from Example 5 was adjusted to 8.5-9.5 using cone. HC1. Added pyridoxal phosphate (608 mg) and transaminase enzyme (500 mg). Temperature was raised to 45°C. A solution of ketoamide (100g) in isopropyl acetate was added to the above aqueous solution. The reaction mass was stirred at 45-50°C by adjusting pH between 8.2-9.5 using isopropyl amine while removing acetone formed in the reaction. Isopropyl acetate layer was separated and aqueous layer was extracted with isopropyl acetate. Combined isopropyl acetate layer was distilled to afford Sitagliptin (89 % yield).

Example 7: pH of the aqueous layer from Example 7 was adjusted to 8.5-9.5. Added pyridoxal phosphate (608 mg) and transaminase enzyme (500 mg). Temperature was raised to 45 °C. A solution of ketoamide (100g) in isopropyl acetate was added to the above aqueous solution. The reaction mass was stirred at 45-50°C by adjusting pH between 8.2-9.5 using isopropyl amine while removing acetone formed in the reaction. Isopropyl acetate layer was separated and aqueous layer was extracted with isopropyl acetate. Combined isopropyl acetate layer was distilled to afford Sitagliptin (89 % yield).

Example 8: Preparation of 7-[(3R)-3-amino-l-oxo-4-(2,4,5-trifluorophenyl) butyl]-5, 6,7,8- tetrahydro-3-(trifluoromethyl)-l,2,4-triazolo[4,3-a]pyrazine phosphate.

A1000 mL round bottomed flask was charged isopropyl acetate (370mL), water (74 mL) and Sitagliptin free base (37g) and stirred at room temperature to get clear solution. Added orthophosphoric acid (10.43g) to form thick white precipitate. The mass is heated to 75°C to get clear solution. The solution is then cooled slowly to 25°C. During cooling white precipitate was formed. The precipitate was filtered and washed with isopropanol. The product was dried under reduced pressure at 45 °C to afford Sitagliptin phosphate (93% yield) as white powder with ee 99.9 %