WO2019207443A1

WO2019207443A1 - An enzymatic process for the preparation of (r)-sitagliptin

Info

Publication number: WO2019207443A1
Application number: PCT/IB2019/053257
Authority: WO
Inventors: Dhananjay Sathe; Sudeep Kumar; Mamata KATDARE; Ramesha N.
Original assignee: Unichem Laboratories Ltd
Priority date: 2018-04-24
Filing date: 2019-04-19
Publication date: 2019-10-31

Abstract

The present invention relates to an enzymatic process for the preparation of (R)- Sitagliptin. The process involves reductive amination of Pro-Sitagliptin to (R)- Sitagliptin using novel transaminase enzymes. The invention also relates to the Nucleotide sequences encoding novel transaminase enzymes and process to prepare transaminase enzymes.

Description

AN ENZYMATIC PROCESS FOR THE PREPARATION OF (R)-

SITAGLIPTIN

Technical Field of the Invention:

The present invention relates to a highly efficient enzymatic process to produce (R)-Sitagliptin baseusing a variant of transaminase enzyme derived from fungal source and expressed in soluble form in Escherichia coli ( E . coli).

Background of the Invention:

(R)-Sitagliptin [(R)-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] (Formula I) is a dipeptidyl-peptidase inhibitor (DPP-4 inhibitor) that is approved for the therapy of type 2 diabetes. It is marketed as the phosphate salt.

Formula I

(R)-Sitagliptin is prepared using two methods. Primarily, by the transamination of pro-sitagliptin [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]of Formula II, to obtain a racemic mixture, followed by separation of the enantiomer of interest using chiral acid. Alternatively, it is synthesized by the transamination of pro-sitagliptin using a specific chiral catalyst to yield enantiomer of interest in higher quantity.

Formula II In the first method, the two step process of transamination of pro-sitagliptin followed by separation of the particular enantiomer of Sitagliptin is tedious, time consuming, lacks reproducibility and results in low yield.

The second method of converting the ketone using a specific chiral catalyst is expensive, low yielding, less reproducible and commercially nonviable.

Owing to the limitations of the aforesaid methods of transamination, enzymatic transformation of ketone to amine is being studied. There are few methods described in the references mentioned herein disclosing biocatalytic/enzymatic process to prepare (R)-sitagliptin from pro-sitagliptin ketone of Formula II. These methods disclose transaminase enzyme derived from various sources used for preparation of (R)-sitagliptin base with better yield and purity as compared to the non-enzymatic methods.

Savile, C.K. et al (Science 329, 305-309, 2010) reports an efficient enzymatic process for the preparation of Sitagliptin. A variant from Ar/hrobac/erspKNK 168 was prepared to show broad applicability toward the synthesis of chiral amines. The variants were prepared using various mutation techniques. Under optimal conditions the best variant reported here converted 200 g/L of pro-sitagliptin to (R)-sitagliptin with >99.95% enantiomeric excess (e.e.) by using 6 g/L enzyme. The concentration of DMSO in the reaction mixture was 50% with a 92% assay yield. The enzyme reacted with a broad substrate range and had tolerance to high concentrations of isopropylamine and organic solvents that enhances their practical utility.

US patent 8293507 describes the preparation of variants of genes, encoding the enzyme transaminase derived from Arthrobactersp KNK168. Transaminase libraries were constructed and cloned into vector pCKl 10700 or pCKl 10900, transformed and expressed in E. coli W3110. Techniques used for the generation of these mutants were random mutagenesis, site saturation mutagenesis, etc. The enzyme variants demonstrate efficient conversion of pro-sitagliptin to (R)- sitagliptin with enantiomeric purity of at least 99%. The enzyme transaminase was used in the form of lyophilized powder and stored at -80°C until use. Polyethyleneimine was added to the lysate, followed by addition of sodium sulfate. The resulting suspension was clarified by centrifugation and the supernatant was concentrated by ultrafiltration. The concentrated solution was lyophilized and used for the bioconversion reactions. Further, a process for the isolation of sitagliptin has been described, which consist of using isopropyl acetate for the extraction of the product in alkaline condition.

EP2723763 and EP2961838 describe a process for the preparation of an immobilized transaminase enzyme, which is stable in organic solvents and can be used in non-aqueous conditions also. In this the conversion of pro-sitagliptin to (R)-sitagliptin is carried out using the resultant immobilized enzyme. The process requires use of ultra-filtration, lyophilization, -80°C deep freezer and immobilization of enzymes that adds to the cost of the process, thus making the process expensive and industrially nonfeasible.

It is the need of the hour to develop an economical and industrially feasible process to prepare (R)-sitagliptin. The present inventors have constructed variants of transaminase enzyme from various fungal sources, to bring about the conversion of pro-sitagliptin to (R)-sitagliptin. The present inventors have attended the unmet need and demonstrated an economical and feasible process for the conversion of pro-sitagliptin to (R)-sitagliptin, using the variants of transaminase enzyme.

Object of the Invention

The object of the invention is to prepare E. coli clones, which encode variants of transaminase enzymes derived from fungal sources. These variant enzymes convert pro-sitagliptin exclusively to (R)-Sitagliptin.

Another object of the invention is to use cell lysate as the source of the variant of transaminase enzyme.

Yet another object of the invention is to demonstrate a reproducible, economical and an industrially feasible enzymatic process to produce (R)-Sitagliptin from pro-sitagliptin ketone. Another object of the invention is to provide a process for the isolation and purification of (R)-Sitagliptin.

Summary of the Invention:

The present invention relates to variants of transaminase and nucleotide sequences encoding amino acid sequences of variants of transaminase. The invention further relates to the clones for the preparation of variants of transaminase. Each clone comprises nucleotide sequence encoding a variant of transaminase, a vector and a bacterial host cell. The said variants of transaminase are derived from fungal sources.

The present invention further relates to the preparation of clone encoding a variant of transaminase, wherein the process comprises:

a. preparing a nucleotide sequence encoding the variant of transaminase derived from a fungus;

b. constructing a vector pET28a containing the nucleotide sequence prepared in step‘a’;

c. transforming the vector constructed in step‘b’ into the bacterial host cell to prepare a clone comprising of nucleotide sequence encoding a variant of transaminase, a vector and a bacterial host cell.

The present invention relates to the process to produce cell lysate comprising a variant of transaminase, wherein the process comprises:

c. transforming the vector constructed in step‘b’ into the bacterial host cell to prepare a clone comprising nucleotide sequence encoding a variant of transaminase, a vector and a bacterial host cell; d. growing the clone obtained in step‘c’ by fed batch fermentation, harvesting the cells from fermentation broth and lysing the cells in suitable buffer to prepare cell lysate comprising a variant of transaminase;

e. separating cell lysate obtained in step‘d’ by centrifugation from the lysed cell in step‘d’.

The invention further relates to the process to produce (R)-Sitagliptin from pro- sitagliptin using cell lysate comprising a variant of transaminase, wherein the process comprises:

c. transforming the vector constructed in step‘b’ into the bacterial host cell to prepare a clone comprising nucleotide sequence encoding a variant of transaminase, a vector and a bacterial host cell;

d. growing the clone obtained in step‘c’ by fed batch fermentation, harvesting the cells from fermentation broth and lysing the cells in suitable buffer to prepare cell lysate comprising a variant of transaminase; e. separating cell lysate obtained in step‘d’ by centrifugation from the lysed cell in step‘d’. f. reacting pro-sitagliptin with the cell lysate comprising a variant of transaminase obtained in step‘e’, to give (R)-sitagliptin;

g. isolating and purifying (R)-sitagliptin base obtained in step‘f .

The invention further relates to the process for isolation and purification of (R)- Sitagliptin from the reaction mixture comprising (R)-Sitagliptin, wherein the said process comprises:

a. acidifying the reaction mixture comprising (R)-Sitagliptin to pH 1.0 to 4.0, at 30°C - 50°C for lh - 3h to obtain acidified reaction mix;

b. basifying the acidified reaction mixture obtained in step‘a’ to pH 10.0-11.0 to obtain basified reaction mix; c. adding celite to the basified reaction mixture obtained in step‘b’, stirring at 20°C - 30°C for 15 min - lh and filtering on hyflow bed to obtain aqueous solution containing (R)-Sitagliptin;

d. extracting (R)-Sitagliptin from the aqueous solution obtained in step‘c’ using ethyl acetate to obtain the extract comprising (R)-Sitagliptin;

e. washing the extract comprising (R)-Sitagliptin obtained in step‘d’, with water and subsequently with brine to obtain a clear extract containing (R)-Sitagliptin; f. concentrating the clear extract containing (R)-Sitagliptin obtained in step‘e’ to obtain (R)-Sitagliptin.

The invention also relates to (R)-Sitagliptin prepared using processes in the present invention. The invention further relates to (R)-Sitagliptin prepared using a variant of transaminase disclosed herein.

Brief Description of the Accompanying Sequences:

• SEQ ID NO. 1 : represents Nucleotide Sequence encoding modified amino acid sequence of the transaminase variant of SEQ ID NO: 2.

• SEQ ID NO. 2: represents modified amino acid sequence of the transaminase variant derived from Aspergillus fumigatus.

• SEQ ID NO. 3: represents Nucleotide Sequence encoding modified amino acid sequence of the transaminase variantof SEQ ID NO: 4.

• SEQ ID NO. 4: represents modified amino acid sequence of the transaminase variant derived from Nectriahaematococca.

• SEQ ID NO. 5: represents Nucleotide Sequence encoding modified amino acid sequence of the transaminase variantof SEQ ID NO: 6.

• SEQ ID NO. 6: represents modified amino acid sequence of the transaminase variant derived from Aspergillus terreus.

• SEQ ID NO. 7: represents Nucleotide Sequence encoding modified amino acid sequence of the transaminase variantof SEQ ID NO: 8.

• SEQ ID NO. 8: represents modified amino acid sequence of the transaminase variant derived from Aspergillus fischeri. • SEQ ID NO. 9: represents Nucleotide Sequence encoding modified amino acid sequence of the transaminase variantof SEQ ID NO: 10.

• SEQ ID NO. 10: represents modified amino acid sequence of the transaminase variant derived from Fusarium oxysporum.

Brief Description of the Figures:

Figure 1 : Annotated diagram of pET28a Vector Map with SEQ ID NO: 1- Nucleotide Sequence Encoding variant of transaminase derived from amino acid sequence of SEQ ID NO: 2.

Figure 2: Annotated diagram of pET28a Vector Map with SEQ ID NO: 3- Nucleotide Sequence Encoding variant of transaminase derived from amino acid sequence of SEQ ID NO: 4.

Figure 3: Annotated diagram of pET28a Vector Map with SEQ ID NO: 5- Nucleotide Sequence Encoding variant of transaminase derived from amino acid sequence of SEQ ID NO: 6.

Figure 4:Annotated diagram of pET28a Vector Map with SEQ ID NO: 7- Nucleotide Sequence Encoding variant of transaminase derived from amino acid sequence of SEQ ID NO: 8.

Figure 5: Annotated diagram of pET28a Vector Map with SEQ ID NO: 9- Nucleotide Sequence Encoding variant of transaminase derived from amino acid sequence of SEQ ID NO: 10.

Figure 6: SDS-PAGE analysis of recombinant variant of Transaminase enzyme of Clone with amino acid sequence SEQ ID NO. 2 encoded by nucleotide sequence of SEQ ID NO. 1 in cell lysate.

Figure 7: SDS-PAGE analysis of recombinant variant of Transaminase enzyme of Clone with amino acid sequence SEQ ID NO. 4 encoded by nucleotide sequence of SEQ ID NO. 3 in cell lysate

Figure 8: SDS-PAGE analysis of recombinant variant of Transaminase enzyme of Clone with amino acid sequence SEQ ID NO. 6 encoded by nucleotide sequence of SEQ ID NO. 5 in cell lysate Figure 9: SDS-PAGE analysis of recombinant variant of Transaminase enzyme of Clone with amino acid sequence SEQ ID NO. 8 encoded by nucleotide sequence of SEQ ID NO. 7 in cell lysate

Figure 10: SDS-PAGE analysis of recombinant variant of Transaminase enzyme of Clone with amino acid sequence SEQ ID NO. 10 encoded by nucleotide sequence of SEQ ID NO. 11 in cell lysate

Detailed description of the Invention:

Definitions:

The terms‘Transaminase’ and‘Transaminase enzyme’ refer to an enzyme that converts keto functional group to the chiral amine. In this invention the said enzyme is selected from amino acid sequence at least 85% identical to the amino acid sequence of Seq ID 2, 4, 6, 8 or 10, which are variants of transaminase enzyme obtained from fungal sources.

The terms‘nucleic acid’ or‘nucleotide’ or‘polynucleotide’ herein means at least two nucleotides covalently linked together and encodes at least one polypeptide or peptide with transaminase activity. The nucleic acid is DNA molecule in both genomic and cDNA form, wherein the nucleic acid contains any combination of deoxyribonucleotides, and any combination of bases, including adenine (A), thymine (T), cytosine (C), guanine (G), isocytosine, isoguanine, or their functional analogs etc. As used herein, the term‘nucleotide’ encompasses both nucleotides and nucleosides as well as nucleoside and nucleotide analogs, and modified nucleotides such as amino modified nucleotides.

The terms‘Protein’,‘polypeptide’ or‘peptide’ are used interchangeably herein to refer a polymer of at least two amino acids covalently linked by an amide bond and having transaminase activity, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristilation, ubiquitination, etc.). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids.

The term‘gene’ means the segment of DNA involved in encoding a polypeptide. The terms‘coding sequence’ refers to that portion of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.

The term‘recombinant’ when used with reference to, e.g., a cell, nucleic acid, or polypeptide, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature, or is identical thereto but produced or derived from synthetic materials and/or by manipulation using recombinant techniques. Non limiting examples include, among others, recombinant cells expressing genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise expressed at a different level.

The terms‘percentage of sequence identity’,‘percent identity’,‘% identity’ and ‘identical’ are used herein to refer to comparisons between polynucleotide sequences or polypeptide sequences, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

The term‘modified from’,‘derived from’ and‘variant’ are used interchangeably herein in the context of engineered enzymes identifies the originating enzyme, and/or the gene encoding such enzyme, upon which the engineering was based.

In this specification the words substrate, Pro-sitagliptin or Pro-sitagliptin ketone refer to the compound of Formula II.

The present invention relates to several variants of transaminase enzyme and nucleotide sequences encoding amino acid sequences of variants of transaminase. The invention further relates to the clones for the preparation of variants of transaminase. Each clone comprises nucleotide sequence encoding a variant of transaminase, a vector and a bacterial host cell. Amino acid sequence for the preparation of the said variants of transaminase are derived from fungal sources.

Particularly the present invention relates to a transaminase enzyme having amino acid sequence of at least 85% identical to the amino acid sequence of Seq ID 2 derived from Aspergillus fumigatus and the nucleotide sequence of Seq ID 1 encoding amino acid sequence at least 85% identical to the amino acid sequence of Seq ID 2. The invention further relates to the clones for the preparation of transaminase having amino acid sequence at least 85% identical to the amino acid sequence of Seq ID 2. Each clone comprises a nucleotide sequence encoding an amino acid sequence for variants of transaminase enzyme, having amino acid sequence at least 85% identical to the amino acid sequence of Seq ID 2, a vector and a bacterial host cell.

According to the aspect of the invention the amino acid sequence obtained from Aspergillus fumigatus, was used as a base for gene designing and modification, to obtain a nucleotide sequence of Seq ID 1, that encodes a transaminase enzyme having amino acid sequence of Seq ID 2, which is capable of converting pro- sitagliptin to (R)-sitagliptin with more than 99% e.e. The gene of interest encoding the enzyme transaminase was modified using molecular biology tools known to those skilled in the art.

The present invention also particularly relates to transaminase having amino acid sequence at least 85% identical to the amino acid sequence of Seq ID 4 derived from Nectriahaematococcaand the nucleotide sequence of Seq ID 3 encoding amino acid sequence at least 85% identical to the amino acid sequence of Seq ID 4. The invention further relates to the clones for the preparation of transaminase having amino acid sequence at least 85% identical to the amino acid sequence of Seq ID 4. Each clone comprises nucleotide sequence Seq ID 3 encoding transaminase having amino acid sequence at least 85% identical to the amino acid sequence of Seq ID 4, a vector and a bacterial host cell. According to this aspect of the invention the amino acid sequence obtained from Nectriahaematococca was used as a base for gene designing and modification, to obtain a nucleotide sequence of Seq ID 3, that encodes an efficient transaminase enzyme having amino acid sequence of Seq ID 4, which is capable of converting pro-sitagliptin to (R)-sitagliptin with more than 99% e.e. The gene of interest encoding the enzyme transaminase was modified using molecular biology tools known to those skilled in the art.

The present invention particularly relates to transaminase having amino acid sequence at least 85% identical to the amino acid sequence of Seq ID 6 derived from Aspergillus terreusand the nucleotide sequence of Seq ID 5 encoding amino acid sequence at least 85% identical to the amino acid sequence of Seq ID 6. The invention further relates to the clones for the preparation of transaminase having amino acid sequence at least 85% identical to the amino acid sequence of Seq ID 6. Each clone comprises nucleotide sequence Seq ID 5 encoding transaminase having amino acid sequence at least 85% identical to the amino acid sequence of Seq ID 6, a vector and a bacterial host cell.

According to this aspect the amino acid sequence obtained from Aspergillus terreus, was used as a base for gene designing and modification, to obtain a nucleotide sequence of Seq ID 5, which encodes an efficient transaminase enzyme having amino acid sequence of Seq ID 6, which is capable of converting pro- sitagliptin to (R)-sitagliptin with more than 99% e.e. The gene of interest encoding the enzyme transaminase was modified using molecular biology tools known to those skilled in the art.

In particularly invention relates to transaminase having amino acid sequence at least 85% identical to the amino acid sequence of Seq ID 8 derived from Aspergillus fischeriand the nucleotide sequence of Seq ID 7 encoding amino acid sequence at least 85% identical to the amino acid sequence of Seq ID 8. The invention further relates to the clones for the preparation of transaminase having amino acid sequence at least 85% identical to the amino acid sequence of Seq ID 8. Each clone comprises nucleotide sequence Seq ID 7 encoding transaminase having amino acid sequence at least 85% identical to the amino acid sequence of Seq ID 8, a vector and a bacterial host cell. According to this aspect of the invention the amino acid sequence obtained from Aspergillus fischeri, was used as a base for gene designing and modification, to obtain a nucleotide sequence of Seq ID 7, that encodes an efficient transaminase enzyme having amino acid sequence of Seq ID 8, which is capable of converting pro-sitagliptin to (R)-sitagliptin with more than 99% e.e. The gene of interest encoding the enzyme transaminase was modified using molecular biology tools known to those skilled in the art.

Invention also particularly relates to transaminase having amino acid sequence at least 85% identical to the amino acid sequence of Seq ID 10 derived from Fusarium oxysporum&nd the nucleotide sequence of Seq ID 9 encoding amino acid sequence at least 85% identical to the amino acid sequence of Seq ID 10. The invention further relates to the clones for the preparation of transaminase having amino acid sequence at least 85% identical to the amino acid sequence of Seq ID 10. Each clone comprises nucleotide sequence Seq ID 9 encoding transaminase having amino acid sequence at least 85% identical to the amino acid sequence of Seq ID 10, a vector and a bacterial host cell.

According to this aspect the amino acid sequence obtained from Fusarium oxysporum was used as a base for gene designing and modification, to obtain a nucleotide sequence of Seq ID 9, which encodes an efficient transaminase enzyme having amino acid sequence of Seq ID 10, which is capable of converting pro- sitagliptin to (R)-sitagliptin with more than 99% e.e. The gene of interest encoding the enzyme transaminase was modified using molecular biology tools known to those skilled in the art.

According to the aspect of the invention the best results are obtained using pET28a vector. The bacterial host cell is chosen such that it expresses the protein of interest at an optimal level. The host cell is selected from the strains of E. coir, such as E. coli BL21 DE3 Gold cells and E. coli Origami B (DE3) cells preferably, E. coli BL21 DE3 Gold cells.

Yet another embodiment of the present invention is a process for preparation of clone encoding a variant of transaminase, wherein the process comprises: a. preparing a nucleotide sequences encoding the variant of transaminase derived from a fungus;

b. constructing a vector pET28a containing the nucleotide sequencing prepared in step‘a’;

According to this aspect particular variant of transaminase is amino acid sequence which is at least 85% identical to the amino acid sequence of Seq ID 2, 4, 6, 8 or 10

Accordingly, nucleotide sequences 1, 3, 5, 7 and 9 encode transaminase having amino acid sequences of Seq ID 2, 4, 6, 8 and 10, respectively.

Thus amino acid sequence obtained from Aspergillus fumigatus was used as a base for gene designing and modification, to obtain a nucleotide sequence of Seq ID 1. Similarly the amino acid sequence obtained from Nectriahaematococca was used as a base for gene designing and modification, to obtain a nucleotide sequence of Seq ID 3. The amino acid sequence obtained from Aspergillus terreus , was used as a base for gene designing and modification, to obtain a nucleotide sequence of Seq ID 5. The amino acid sequence obtained from Aspergillus fischeri was used as a base for gene designing and modification, to obtain a nucleotide sequence of Seq ID 7 and the amino acid sequence obtained from Fusarium oxysporum was used as a base for gene designing and modification, to obtain a nucleotide sequence of Seq ID 9.

According to the aspect of the invention nucleotide sequence was modified using molecular biology tools known to those skilled in the art. The designed nucleotide sequence was compared using structural analytical tools and was subjected to bio- informatics analysis to confirm the sequence.

According to yet another aspect the vector is pET28a. The bacterial host cell is chosen such that it expresses the protein of interest at an optimal level. The host cell is selected from the strains of E. coli such as E. coli BL2l-Gold (DE3) cells, and E. coli B (DE3) Origami cells preferably, E. coli BL2l-Gold (DE3) cells.

A construct with each of the nucleotide sequence was prepared in vector, pET28a. This vector having the gene of interest was then transformed into the expression host, E. coli BL2l-Gold (DE3) cells to prepare a clone comprising nucleotide sequence of interest, a vector and a bacterial host cell. The resultant clone is grown at 30°C-40°C, preferably 35°C - 39°C, induced with 0.1 mM - 2mM IPTG to check the expression of transaminase enzyme.

d. growing the clone obtained in step‘c’ by fed batch fermentation, harvesting and lysing the cells in fermentation broth to prepare cell lysate comprising a variant of transaminase;

e. separating cell lysate obtained in step‘d’ by centrifugation from fermentation broth in step‘d’.

Thus amino acid sequence obtained from Aspergillus fumigatus was used as a base for gene designing and modification, to obtain a nucleotide sequence of Seq ID 1.

Similarly the amino acid sequence obtained from Nectriahaematococca was used as a base for gene designing and modification, to obtain a nucleotide sequence of Seq ID 3. The amino acid sequence obtained from Aspergillus terreus , was used as a base for gene designing and modification, to obtain a nucleotide sequence of Seq ID 5. The amino acid sequence obtained from Aspergillus fischeri was used as a base for gene designing and modification, to obtain a nucleotide sequence of Seq ID 7 and the amino acid sequence obtained from Fusarium oxysporum was used as a base for gene designing and modification, to obtain a nucleotide sequence of Seq ID 9.

According to yet another aspect the vector is pET28a. The bacterial host cell is chosen such that it expresses the protein of interest at an optimal level. The host cell is selected from the strains of E. coir, such as E. coli BL21 DE3 Gold cells, and E.coli BL21 DE3 Origami cells preferably, E. coli BL21 DE3 Gold cells.

A construct with each of the nucleotide sequence was prepared in vector, pET28a. This vector having the gene of interest was then transformed into the expression host, E. coli BL21 DE3 Gold cells to prepare a clone comprising nucleotide sequence of interest, a vector and a bacterial host cell. The resultant clone was cultured at 30°C-40°C preferably 35°C - 39°C in inoculum medium containing 10 gL ¹ - 30 gL ¹ of Luria Broth, 3 gL ¹ - 7 gL ¹ of dextrose, 6 gL ¹- 9 gL isodium hydrogen phosphate, 0.5 gL ¹ - 2.0 gL ¹ Magnesium Sulphate, 0.5 to 3.0 mLL ¹ Trace metal solution, 0.005 - 0.02 mgL ¹ Kanamycin sulphate. A300 ml of culture was used as inoculum for 3L fermenter. Fermentation was carried out at 30°C - 40°C preferably between 35°C-39°C for lOh -l4h under fed-batch mode using a glycerol-yeast extract based medium. The rate of feeding of glycerol-yeast extract in fed-batch stage was 2-6 gL^h ¹ from lh-5h, 4-10 gL^h ¹ for 5-8h, 2-6 gL^h ¹ for 8-l4h. The carbon to nitrogen (C: N) ratio was maintained in the range of 3: 1 to 5: 1. The culture was induced for the expression of the transaminase enzyme using O. lmM -2mM IPTG when the OD₆₀o_nm of the culture reached 120-150. After completion of the fermentation, the culture reached an OD₆oo_nm of not less than 190. The culture broth was centrifuged at 7,000-12,000 rpm for 20-40 min at l0°C -20°C. The culture supernatant was carefully decanted to separate the cell pellet. An output of more than 180 g of wet cell mass per liter of the culture broth was obtained. This was the cell lysate containing a variant of transaminase enzyme. Another aspect of this invention is isolation of cell lysate containing a variant of transaminase enzyme wherein the cell mass was uniformly suspended in 0.08- 0.12M triethanolamine buffer of pH 7.5-9.5, preferably 8.2-8.8, containing 0.05- 0.15 mM of pyridoxal-5’ -phosphate (PLP) in a ratio of 1 :6 to 1 : 15 (w/v). The mixture was stirred on overhead stirrer for 1-2 h to form a homogeneous suspension. The suspended cell mass was then lysed using a homogenizer at -15,000-20,000 psi pressure. The lysate was centrifuged at 7,000-12,000 rpm for 20-40 min at l0°C - 20°C, to obtain cell lysate containing a variant of transaminase enzyme.

e. separating cell lysate obtained in step‘d’ by centrifugation from fermentation broth in step‘d’;

f. reacting pro-sitagliptin with the cell lysate comprising a variant of transaminase obtained in step‘e’, to give (R)-sitagliptin;

g. isolating and purifying (R)-sitagliptin base obtained in step‘f .

Accordingly, nucleotide sequences 1, 3, 5, 7 and 9 encode transaminase having amino acid sequences of Seq ID 2, 4, 6, 8 and 10, respectively. Thus amino acid sequences obtained from Aspergillus fumigatus was used as a base for gene designing and modification, to obtain a nucleotide sequence of Seq ID 1.

Similarly the amino acid sequence obtained from Nectriahaematococca was used as a base for gene designing and modification, to obtain a nucleotide sequence of Seq ID 3. The amino acid sequence obtained from Aspergillus terreus was used as a base for gene designing and modification, to obtain a nucleotide sequence of Seq ID 5. The amino acid sequence obtained from Aspergillus fischeri was used as a base for gene designing and modification, to obtain a nucleotide sequence of Seq ID 7 and the amino acid sequence obtained from Fusarium oxysporum was used as a base for gene designing and modification, to obtain a nucleotide sequence of Seq ID 9.

A construct with each of the nucleotide sequence was prepared in vector, pET28a. This vector having the gene of interest was then transformed into the expression host, E. coli BL21 DE3 Gold cells to prepare a clone comprising nucleotide sequence of interest, a vector and a bacterial host cell. The resultant clone was cultured at 30°C-40°C preferably 35°C - 39°C in inoculum medium containing 10 gL ¹ - 30 gL ¹ of Luria Broth, 3 gL ¹ - 7 gL ¹ of dextrose, 6 gL ¹- 9 gL isodium hydrogen phosphate, 0.5 gL ¹ - 2.0 gL ¹ Magnesium Sulphate, 0.5 to 3.0 mLL ¹ Trace metal solution, 0.005 - 0.02 mgL ¹ Kanamycin sulphate. A300 ml of culture was used as inoculum for 3L fermenter. Fermentation was carried out at 30°C - 40°C preferably between 35°C-39°C for lOh -l4h under fed-batch mode using a glycerol-yeast extract based medium. The rate of feeding of glycerol-yeast extract in fed-batch stage was 2-6 gL^h ¹ from lh-5h, 4-10 gL^h ¹ for 5-8h, 2-6 gL^h ¹ for 8-l4h. The carbon to nitrogen (C: N) ratio was maintained in the range of 3: 1 to 5: 1. The culture was induced for the expression of the transaminase enzyme using O. lmM -2mM IPTG when the OD₆₀o_nm of the culture reached 120-150. After completion of the fermentation, the culture reached an OD₆oo_nm of not less than 190. The culture broth was centrifuged at 7,000-12,000 rpm for 20-40 min at l0°C -20°C. The culture supernatant was carefully decanted to separate the cell pellet. An output of more than 180 g of wet cell mass per liter of the culture broth was obtained. This was the cell lysate containing a variant of transaminase enzyme.

Another aspect of this invention is isolation of cell lysate containing a variant of transaminase enzyme wherein the cell mass was uniformly suspended in 0.08- 0.12M triethanolamine buffer of pH 7.5-9.5, preferably 8.2-8.8, containing 0.05- 0.15 mM of pyridoxal-5’-phosphate in a ratio of 1 :6 to 1: 15 (w/v). The mixture was stirred on overhead stirrer for 1-2 h to form a homogeneous suspension. The suspended cell mass was then lysed using a homogenizer at -15,000-20,000 psi pressure. The lysate was centrifuged at 7,000-12,000 rpm for 20-40 min at l0°C - 20°C. The centrifuged cell lysate is used as such for the conversion of pro- sitagliptin to (R)-sitagliptin.

According to yet another aspect pro-sitagliptin is reacted with cell lysate comprising a variant of transaminase enzyme, to obtain (R)-Sitagliptin in the presence of 0.5M - 1.2M isopropylamine and 0.5 mM to 3.0 mM pyridoxal-5’- phosphate while maintaining the pH between 8.0 - 9.0 at temperature of 40°C - 50°C. The completion of the reaction was achieved in lOh -24h.

Pro-sitagliptin was dissolved in DMSO and was added to the reaction mixture such that the total amount of DMSO per liter of reaction mixture is maintained in the range of 250 mL to 550 mL.

It is observed that up to 300 g/L of pro-sitagliptin was converted within 10 h - 24 h at 40°C-50°C.

a. acidifying the reaction mixture comprising (R)-Sitagliptin to pH 1.0 to 4.0, at 30°C - 50°C for lh - 3h to obtain acidified reaction mixture; b. basifying the acidified reaction mixture obtained in step‘a’ to pH 10.0-11.0 to obtain basified reaction mixture;

c. adding celite to the basified reaction mixture obtained in step‘b’, stirring at 20°C - 30°C for 15 min - lh and filtering on hyflow bed to obtain aqueous solution containing (R)-Sitagliptin;

According to this aspect of the invention, the reaction mixture was acidified to pH 1.0 to 4.0 using hydrochloric acid (HC1) and stirred well at 30°C - 50°C for lh - 3h. The pH of the reaction mixture was readjusted to 10.0-11.0 using 20% NaOH solution. Celite was added to the reaction mixture with pro-sitagliptin ketone to celite ratio of 1 :0.2 to 1 :2.0 (w/w) and stirred at 20°C-30°C for 15 min to 1 h. The reaction mixture was filtered through hyflow bed to obtain aqueous solution containing the product. 1 to 3 volumes of ethyl acetate was added to the aqueous filtrate and mixed vigorously. Aqueous and organic layers were separated and aqueous layer was re-extracted with ethyl acetate. The ethyl acetate extracts were pooled; water wash was given, twice, to the extracts to remove any unwanted inorganics and color impurities, followed by brine wash. The resultant extract was treated with 5-20% (w/v) activated charcoal, stirred for 15 min - lh at 20°C-30°C and filtered on hyflow bed. The filtrate was concentrated to obtain the product (R)-Sitagliptin.

The present invention thus provides an alternate process to produce (R)- Sitagliptin, from pro-sitagliptin by producing a variant of transaminase enzyme derived from a fungal source. Thus, the present inventors have designed a reproducible, economical and industrially feasible enzymatic process to produce (R)-sitagliptin base. Examples:

Following examples of the present invention demonstrate the best mode of carrying out the present invention. These examples do not limit the scope of invention in any manner and should be considered as purely illustrative.

DNA sequence encoding a polypeptide, derived from Aspergillus fumigatus, Nectriahaematococca , Aspergillus terreus , Aspergillus fischeri , and Fusarium oxysporum , was codon optimized, chemically synthesized by Genscript, ETSA and cloned into pET28a expression vector and vector had an N-terminal short amino acid residues. Vector carrying the desired gene sequence was then transformed into a propagation host, E. coli DH5a. Positive colonies were selected on the basis of colony PCR screening with pET forward and reverse primers and restriction digestion of the isolated plasmid was carried out from these colony PCR positive colonies. Plasmids were then transformed into expression host, E. coli BL21 DE3 Gold cells, to express the desired polypeptide when induced with 0.5-1 mM IPTG, at 35°C - 40°C preferably 37°C. Plasmids from these colonies were isolated and verified by DNA sequencing and a 100% match with original sequence was obtained.

Positive colonies were inoculated into a suitable inoculum media and transferred into Luria Broth containing the antibiotic Kanamycin. Incubation was carried out at 35-40°C preferably at 37 C until suitable growth is achieved followed by induction of the medium with 0.5 to 1 mM IPTG for 2-4 h preferably 4 h. Cells were harvested by centrifugation and lysed on a cell disruptor. The expression was analyzed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and it was found that 20-40% of the desired polypeptide was produced in soluble form.

Example 1: Construction of a Clone that expresses variant Transaminase Enzyme:

Five nucleotide sequences each encoding different amino acid sequences having SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8 and SEQ ID No. 10 were designed, codon optimized and each nucleotide sequence was cloned in an expression vector pET28a. The vector carrying the nucleotide sequence was then transformed into E. coli BL21 DE3 Gold expression host cells, to express the desired polypeptide when induced with lmM IPTG, at 37°C±l°C. The nucleotide sequences from these colonies were verified by DNA sequencing.

The expression of the enzyme was analyzed by SDS-PAGE and it was found that 40-50% of the enzyme was produced in the soluble form.

Example 2: Screening of clones for Transamination of Pro-sitagliptin to (R)- Sitagliptin:

The five clones obtained in example 1, were individually grown at 37°C ±1°C and induced with 1 mM IPTG. The cells were harvested by centrifugation and re- suspended in 0.1 M triethanolamine buffer, pH 8.5 (at 1 : 10 ratio w/v containing 0.1 mM of PLP) and lysed using a ultrasonic cell sonicator under chilled condition. The cell lysate was centrifuged and the supernatant was used as a source of enzyme transaminase.

The reactions mixture comprised of 1.5 mL cell lysate containing transaminase enzyme along with 0.1 mM PLP and 250 pL of 4M Isopropylamine (pH was adjusted to 8.0). A 5 mg of pro-sitagliptin ketone was solubilized in 200 pL of DMSO and added in to each of the reaction mixtures. Reactions were carried out at 30°C±l°C on a rotary shaker at 150 rpm for 24 h. pH of the reaction mixtures were adjusted to -8.4-8.6- using neat isopropylamine. After 24hr, conversion of the substrate pro-sitagliptin ketone was analyzed on TLC by HPLC and found that conversion is not less than 70.6%.

Also, 5 mL of the lysates from all the above five clones were used in a separate reaction. Each of the reaction mixtures comprised of 5mL cell lysate containing transaminase enzyme along with O.lmM PLP and 2mL of 4M Isopropylamine (pH 8.4). A 500 mg of pro-sitagliptin was solubilized in 3mL of DMSO and added to each of the reaction mixtures. The reaction was carried out at 45°C±l°C on a rotary shaker at l50rpm for 24 h. pH was adjusted to -8.4-8.6 at regular intervals using neat isopropylamine. After 24hr, the enantiomeric purity of the product (R)- Sitagliptin was analyzed by HPLC and was found to be 99.9% in each of the five clones.

Example 3: Production of Transaminase enzyme at 3L Fermenter scale:

Clone obtained from nucleotide of SEQ ID 3 was cultured at 37°C±l°C in inoculum medium containing Luria Broth 20 gL ¹, Glucose 5 gL ¹, di-Sodium hydrogen phosphate 7.5 gL ¹, Magnesium Sulphate 1.0 gL ¹, Trace metal solution 1.0 mLL ¹, Kanamycin Sulphate 0.010 mgL ¹ and 300 ml culture used as inoculum for 3L fermenter. Fermentation medium comprises Yeast Extract 10 gL ¹ , GlucoselO gL ¹, KH₂P0₄ 3 gL ¹, Na₂HP0₄ 7 gL ¹, (NH₄)₂S0₄ 2 gL ¹, NaCl 0.33 gL ¹, MgS0₄.7H₂0 1.0 gL ¹, Thiamine 0.01 gL ¹, Trace metal solution 1.0 mLL ¹ and Kanamycin 0.02 gL ¹. Fermentation was carried out at 37°C±l°C for l2h under fed batch mode using glycerol-yeast extract based feed. The pH was maintained around 6.8 throughout the fermentation using 10N NaOH. When the OD₆₀o_nm of the culture reached -135, the cells were induced with lmM IPTG. At the end of the fermentation, the final OD₆₀o_nm obtained was more than 190. The culture broth was harvested by centrifugation at 9000 rpm at l5°C±l°C for 15 min. An output of more than l80g of wet cell mass per litre of culture was obtained.

Example 4: Preparation of Cell Lysate as Source of Enzyme:

Cell mass obtained in Example 3 was processed to obtain the cell lysate. The cell mass was suspended in Triethanolamine buffer, (0.1 M pH 8.5) containing O. lmM PLP in a ratio of 1 : 10 (w/v) and stirred for at least for l-2h on an overhead stirrer to form a homogenous suspension. The process was carried out on ice throughout the work. The cell suspension was lysed on a homogenizer at -18000 psi. Two passes were carried out to achieve maximum lysis of the cells. The lysate was centrifuged at 9,000 rpm for 30 min at l5°C±l°C. The supernatant was used as the source of enzyme for the bioconversion reaction. Example 5: Production of (R)-Sitagliptin:

The reaction mixture comprised of 2.0L cell lysate containing transaminase enzyme along with l.7g of PLP and 1L of 4M Isopropylamine (pH 8.5). A 250g of pro-sitagliptin was solubilized in 2.2 L of DMSO and added to the reaction mixture. The final volume of the reaction mixture was ~5.0 L. Reaction mass was stirred on an overhead stirrer at 45°C±l°C for 12 h. pH was adjusted at regular intervals to -8.4-8.6 using neat Isopropylamine. The progress of the reaction was monitored during the course of the reaction to analyze conversion of the pro- sitagliptin to the product (R)-sitagliptin and determine the enantiomeric purity.

Example 6: Purification of (R)-Sitagliptin;

A 5.0 L reaction mixture was acidified with HC1 solution to pH 2.0-3.0 and was stirred at 45° C for 2 h. The pH was re-adjusted to 11.0 using 20% NaOH. A 250 gcelite was added to the alkalified reaction mixture and stirred at room temperature for 30 min. The reaction mixture was filtered to obtain aqueous solution of product. A 2.5 L of ethyl acetate was added to the reaction mixture and mixed well for 15-20 min. Aqueous and organic layers were separated and aqueous phase was re-extracted with 1.5 L of Ethyl Acetate. The extracts were pooled and washed twice with 1 L purified water followed with 1 L brine solution. A 25g of activated charcoal was added to the extract and stirred for 30min. The extract was filtered on hyflow bed and the resultant filtrate was concentrated through distillation under vacuum. Around 225g of (R)-sitagliptin was obtained having more than 99% e.e.

Claims

We Claim:

1. A recombinant nucleotide sequence of:

(i) SEQ ID 1;

(ii) SEQ ID 3;

(iii) SEQ ID 5;

(iv) SEQ ID 7;

(v) SEQ ID 9;

(vi) a nucleotide sequence encoding amino acid sequence of SEQ ID 2 or SEQ ID 4 or SEQ ID 6 or SEQ ID 8 or SEQ ID 10; or

(vii) a nucleotide sequence which is at least 85% identical to the nucleotide sequence of (i), (ii), (iii), (iv), (v) or (vi).

2. A vector comprising the recombinant nucleotide as claimed in claim 1.

3. A host cell transformed with the vector as claimed in claim 2.

4. The host cell as claimed in claim 3, wherein the preferred host cell is Escherichia coli cells.

5. A clone comprising a host cell transformed by a vector comprising a recombinant nucleotide sequence, wherein the recombinant nucleotide sequence is selected from:

(i) SEQ ID 1;

(ii) SEQ ID 3;

(iii) SEQ ID 5;

(iv) SEQ ID 7;

(v) SEQ ID 9;

6. The clone as claimed in claim 5, wherein the preferred vector is pET28a and the preferred host cell is Escherichia coli cells.

7. A process for preparation of clone as claimed in claim 5, wherein the process comprises: a. preparing a nucleotide sequence selected from:

(i) SEQ ID 1;

(ii) SEQ ID 3;

(iii) SEQ ID 5;

(iv) SEQ ID 7;

(v) SEQ ID 9;

(vii) a nucleotide sequence which is at least 85% identical to the nucleotide sequence of (i), (ii), (iii), (iv), (v) or (vi). b. constructing a vector pET28a containing the nucleotide sequencing prepared in step‘a’; and

c. transforming the vector constructed in step‘b’ into the bacterial host cell to prepare a clone comprising nucleotide sequence, a vector and a host cell.

8. The process as claimed in claim 7, wherein the preferred host cell is E. coli cells.

9. The process as claimed in claim 7, wherein the nucleotide sequence in step ‘a’ is prepared by codon optimization and chemical synthesis.

10. A process for soluble expression of amino acid sequence selected from SEQ ID 2 or SEQ ID 4 or SEQ ID 6 or SEQ ID 8 or SEQ ID 10, wherein the process comprises:

a. preparing a clone comprising a host, a vector containing a nucleotide sequence encoding targeted amino acid sequence;

b. fed-batch fermentation of clone obtained in step ‘a’ in fermentation broth; and

c. separation of soluble form of encoded amino acid from the fermentation broth in step‘b’.

11. The process as claimed in claim 10 wherein the nucleotide sequence in step ‘a’, is selected from:

(i) SEQ ID 1 for encoding the amino acid sequence of SEQ ID 2, (ii) SEQ ID 3 for encoding the amino acid sequence of SEQ ID 4,

(iii) SEQ ID 5 for encoding the amino acid sequence of SEQ ID 6,

(iv) SEQ ID 7 for encoding the amino acid sequence of SEQ ID 8, or;

(v) SEQ ID 9 for encoding the amino acid sequence of SEQ ID 10.

12. The process as claimed in claim 10 wherein the fermentation broth in step‘b’, comprises glycerol-yeast based feed medium.

13. The process as claimed in claim 12 wherein the rate of feeding of glycerol- yeast extract in fed-batch stage is 2-6 gL V from lh-5h, 4-10 gL V for 5- 8h, 2-6 gL ¹h ¹ for 8-l4h.

14. The process as claimed in claim 10 wherein the separation process in step‘c’, involves centrifugation at 7000-12000 rpm.

15. (R)-Sitagliptin prepared by transamination of pro-sitagliptin ketone using one or more enzymes encoded by recombinant nucleotide sequence selected from:

(i) SEQ ID 1;

(ii) SEQ ID 3;

(iii) SEQ ID 5;

(iv) SEQ ID 7;

(v) SEQ ID 9;

16. (R)-Sitagliptin prepared by transamination of pro-sitagliptin ketone using one or more variants of transaminase selected from amino acid sequence of SEQ ID 2 or SEQ ID 4 or SEQ ID 6 or SEQ ID 8 or SEQ ID 10.

17. A process to produce (R)-Sitagliptin comprising enzymatic transamination of pro-sitagliptin by cell lysate containing a variant of transaminase wherein the process comprises steps of:

a. reacting pro-sitagliptin with the cell lysate containing a variant of transaminase, to give (R)-Sitagliptin; b. isolating and purifying (R)-Sitagliptin obtained in step‘a’.

18. The process as claimed in claim 17, wherein cell lysate containing a variant of transaminase in step‘a’ is prepared by the process comprising the steps of: a. preparing a nucleotide sequence encoding the variant of transaminase; b. constructing the vector pET28a containing nucleotide sequence prepared in step‘a’;

d. growing the clone obtained in step ‘c’ by fed batch fermentation, harvesting the cells from fermentation broth and lysing the cells in suitable buffer to prepare cell lysate comprising a variant of transaminase; and

e. separating cell lysate containing a variant of transaminase obtained in step‘d’ by centrifugation from the lysed cell in step‘d’.

19. The process as claimed in claim 17, wherein isolating and purifying (R)-

Sitagliptin in step‘b’ comprises the steps of:

a. acidifying the reaction mixture comprising (R)-Sitagliptin to pH 1.0-4.0, at 30°C - 50°C for lh - 3h to obtain acidified reaction mix;

b. basifying the acidified reaction mixture obtained in step‘a’ to pH 10.0- 11.0 to obtain basified reaction mix;

e. washing the extract comprising (R)-Sitagliptin obtained in step‘d’, with water and subsequently with brine to obtain a clear extract containing (R)-Sitagliptin; and

f. concentrating the clear extract containing (R)-Sitagliptin obtained in step ‘e’ to obtain (R)-Sitagliptin.

20. (R)-Sitagliptin not less than 99% pure and having at least 99% enantiomeric excess, produced enzymatically, wherein the enzyme is in cell lysate.

21. A transaminase derived from fungus, wherein transaminase is selected from amino acid sequence of SEQ ID 2 or SEQ ID 4 or SEQ ID 6 or SEQ ID 8 or SEQ ID 10.

22. The transaminase as claimed in claim 21, wherein the fungus is selected from Aspergillus fiimigatus or Nectriahaematococca or Aspergillus terreus or Aspergillus fischeri or Fusarium oxysporum.