10/17/11 1 Examples of green chemistry • Pressure treated wood – replacement of CCA (containing As and Cr) with ACQ • Polystyrene blowing agent – replacement of CFCs with CO 2 • Mushroom packaging – use of waste agricultural materials to grow packaging materials • Synthesis of sitaglipIn (Januvia) Synthesis of sitaglipIn • Trade name: Januvia • Used for treatment of Type 2 diabetes • Approved by FDA in October 2006 • Marketed beginning April 2007 N O N N N F F F F F F NH2 1 st generaIon synthesis (2005) • Hansen, K. B. et al. First GeneraIon Process for the PreparaIon of the DPP‐IV Inhibitor SitaglipIn. Org. Proc. Res. Dev. 2005, 9, 634. • Desai, A. A. SitaglipIn Manufacture: A Compelling Tale of Green Chemistry, Process IntensificaIon, and Industrial Asymmetric Catalysis. Angew. Chem. Int. Ed. 2011, 50, 1974. asymmetric hydrogenaIon EDC coupling/ Mitsunobu rxn. amide hydrolysis EDC coupling deprotecIon Overall yield: 52% Problems with 1 st generaIon • EDC coupling/Mitsunobu sequence had poor atom economy and produced a large amount of waste. • Scale‐up would be difficult. 2 nd generaIon synthesis (2009) • Hansen, K. B. et al. Highly Efficient Asymmetric Synthesis of SitaglipIn. J. Am. Chem. Soc. 2009, 131, 8798. • Desai, A. A. SitaglipIn Manufacture: A Compelling Tale of Green Chemistry, Process IntensificaIon, and Industrial Asymmetric Catalysis. Angew. Chem. Int. Ed. 2011, 50, 1974. asymmetric hydrogenaIon catalyIc coupling amidaIon enamine formaIon Overall yield: 65%
Transcript
10/17/11
1
Examples of green chemistry
• Pressure treated wood – replacement of CCA (containing As and Cr) with ACQ
• Polystyrene blowing agent – replacement of CFCs with CO2
• Mushroom packaging – use of waste agricultural materials to grow packaging materials
• Synthesis of sitaglipIn (Januvia)
Synthesis of sitaglipIn
• Trade name: Januvia • Used for treatment of Type 2 diabetes • Approved by FDA in October 2006 • Marketed beginning April 2007
N
O
N
N
N
F
F
F
F
F
F
NH2
1st generaIon synthesis (2005)
• Hansen, K. B. et al. First GeneraIon Process for the PreparaIon of the DPP‐IV Inhibitor SitaglipIn. Org. Proc. Res. Dev. 2005, 9, 634.
• Desai, A. A. SitaglipIn Manufacture: A Compelling Tale of Green Chemistry, Process IntensificaIon, and Industrial Asymmetric Catalysis. Angew. Chem. Int. Ed. 2011, 50, 1974.
complete stereocontrol directly from 11 (Scheme 1c). Eventhough the transformation 11!12 is embedded in the one-potsequence in the 2nd generation process, it still represents aunit operation for the process. This is eliminated in the directreductive amination of 11 into 13, which thus represents animprovement over the 2nd generation process (Scheme 1c).
Despite the overall brevity and efficiency of the 2ndgeneration process (Schemes 1b and c), its endgame left openroom for improvement because of the inherent drawbacks ofutilizing a transition metal mediated hydrogenation step. Thisnecessitated the use of specialized high-pressure equipmentand a process for the complete removal of the transition metalfrom the product stream, both of which were significant cost
drivers. Moreover, in the Rh-catalyzed process, the relativelylow stereocontrol in the asymmetric hydrogenation step(95% ee) necessitated the incorporation of an additionalcrystallization to obtain optically pure 1.
To circumvent these drawbacks, Merck and Codexisresearchers have recently engineered a highly evolved trans-aminase biocatalyst to transform 11 into 13 (Scheme 1d; 3rdgeneration process).[7,16] Starting from an R-selective trans-aminase enzyme for which 11 was not a natural substrate, acombination of computational modeling and iterative direct-ed evolution was utilized to engineer the optimal biocatalyst.This evolved enzyme contains 27 mutations, which are foundnot only in the active site, but also in the interface of the
Scheme 1. Three generations of process research and development towards the manufacture of sitagliptin phosphate 1. a) 1st generation process.b) 2nd generation process. c) Improvement upon the 2nd generation process. d) 3rd generation process. Bn=benzyl, binap=2,2’-bis(diphenyl-phosphino)-1,1’-binaphthyl, cod=1,5-cyclooctadiene, DIAD=diisopropyl azodicarboxylate, DMAP=4-dimethylaminopyridine, NMM=N-methyl-morpholine, segphos= (4,4’-bi-1,3-benzodioxole)-5,5’-diylbis(diphenylphosphine).
• EDC coupling/Mitsunobu sequence had poor atom economy and produced a large amount of waste.
• Scale‐up would be difficult.
complete stereocontrol directly from 11 (Scheme 1c). Eventhough the transformation 11!12 is embedded in the one-potsequence in the 2nd generation process, it still represents aunit operation for the process. This is eliminated in the directreductive amination of 11 into 13, which thus represents animprovement over the 2nd generation process (Scheme 1c).
Despite the overall brevity and efficiency of the 2ndgeneration process (Schemes 1b and c), its endgame left openroom for improvement because of the inherent drawbacks ofutilizing a transition metal mediated hydrogenation step. Thisnecessitated the use of specialized high-pressure equipmentand a process for the complete removal of the transition metalfrom the product stream, both of which were significant cost
drivers. Moreover, in the Rh-catalyzed process, the relativelylow stereocontrol in the asymmetric hydrogenation step(95% ee) necessitated the incorporation of an additionalcrystallization to obtain optically pure 1.
To circumvent these drawbacks, Merck and Codexisresearchers have recently engineered a highly evolved trans-aminase biocatalyst to transform 11 into 13 (Scheme 1d; 3rdgeneration process).[7,16] Starting from an R-selective trans-aminase enzyme for which 11 was not a natural substrate, acombination of computational modeling and iterative direct-ed evolution was utilized to engineer the optimal biocatalyst.This evolved enzyme contains 27 mutations, which are foundnot only in the active site, but also in the interface of the
Scheme 1. Three generations of process research and development towards the manufacture of sitagliptin phosphate 1. a) 1st generation process.b) 2nd generation process. c) Improvement upon the 2nd generation process. d) 3rd generation process. Bn=benzyl, binap=2,2’-bis(diphenyl-phosphino)-1,1’-binaphthyl, cod=1,5-cyclooctadiene, DIAD=diisopropyl azodicarboxylate, DMAP=4-dimethylaminopyridine, NMM=N-methyl-morpholine, segphos= (4,4’-bi-1,3-benzodioxole)-5,5’-diylbis(diphenylphosphine).
• Hansen, K. B. et al. Highly Efficient Asymmetric Synthesis of SitaglipIn. J. Am. Chem. Soc. 2009, 131, 8798.
• Desai, A. A. SitaglipIn Manufacture: A Compelling Tale of Green Chemistry, Process IntensificaIon, and Industrial Asymmetric Catalysis. Angew. Chem. Int. Ed. 2011, 50, 1974.
asymmetric hydrogenaIon
catalyIc coupling amidaIon
enamine formaIon
Overall yield: 65%
10/17/11
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2nd generaIon
• 2nd generaIon synthesis won a PresidenIal Green Chemistry Award in 2006
• ReducIon of waste : 50 kg rather than 250 kg per kg of product
• Total waste eliminaIon of 150 million kg over lifeIme of drug.
• Drawbacks: – use of Rh catalyst requires high pressure and a separaIon step.
– Only 95% e.e. necessitates a final crystallizaIon step.
Further improvements
• One step to sitaglipIn from intermediate
• Improved overall yield and efficiency
complete stereocontrol directly from 11 (Scheme 1c). Eventhough the transformation 11!12 is embedded in the one-potsequence in the 2nd generation process, it still represents aunit operation for the process. This is eliminated in the directreductive amination of 11 into 13, which thus represents animprovement over the 2nd generation process (Scheme 1c).
Despite the overall brevity and efficiency of the 2ndgeneration process (Schemes 1b and c), its endgame left openroom for improvement because of the inherent drawbacks ofutilizing a transition metal mediated hydrogenation step. Thisnecessitated the use of specialized high-pressure equipmentand a process for the complete removal of the transition metalfrom the product stream, both of which were significant cost
drivers. Moreover, in the Rh-catalyzed process, the relativelylow stereocontrol in the asymmetric hydrogenation step(95% ee) necessitated the incorporation of an additionalcrystallization to obtain optically pure 1.
To circumvent these drawbacks, Merck and Codexisresearchers have recently engineered a highly evolved trans-aminase biocatalyst to transform 11 into 13 (Scheme 1d; 3rdgeneration process).[7,16] Starting from an R-selective trans-aminase enzyme for which 11 was not a natural substrate, acombination of computational modeling and iterative direct-ed evolution was utilized to engineer the optimal biocatalyst.This evolved enzyme contains 27 mutations, which are foundnot only in the active site, but also in the interface of the
Scheme 1. Three generations of process research and development towards the manufacture of sitagliptin phosphate 1. a) 1st generation process.b) 2nd generation process. c) Improvement upon the 2nd generation process. d) 3rd generation process. Bn=benzyl, binap=2,2’-bis(diphenyl-phosphino)-1,1’-binaphthyl, cod=1,5-cyclooctadiene, DIAD=diisopropyl azodicarboxylate, DMAP=4-dimethylaminopyridine, NMM=N-methyl-morpholine, segphos= (4,4’-bi-1,3-benzodioxole)-5,5’-diylbis(diphenylphosphine).
• Hansen, K. B. et al. Highly Efficient Asymmetric Synthesis of SitaglipIn. J. Am. Chem. Soc. 2009, 131, 8798.
• Desai, A. A. SitaglipIn Manufacture: A Compelling Tale of Green Chemistry, Process IntensificaIon, and Industrial Asymmetric Catalysis. Angew. Chem. Int. Ed. 2011, 50, 1974.
(11, 12). To assess the feasibility of developing anenzyme for sitagliptin synthesis, we generated astructural homology model of ATA-117 (17) todevelop hypotheses for initial library designs.Docking studies using this model suggested thatthe enzyme would be unable to bind prositaglip-tin ketone (Fig. 2B) because of steric interferencein the small binding pocket and potentially un-desired interactions in the large binding pocket(Fig. 2C). By using a substrate walking approach(18) with a truncated substrate (Fig. 2D), we firstengineered the large binding pocket of the en-zyme and then evolved that enzyme for activitytoward prositagliptin ketone.
Consistentwith themodel, ATA-117was poorlyactive on the truncated methyl ketone analog(Fig. 2D), giving 4% conversion at 2 g/l substrateloading (table S2). Site saturation libraries of res-idues lining the large pocket of the active site pro-vided new variants with increased activity towardthe methyl ketone analog. The best variant con-tained a S223 ! P223 [S223P (19)] mutation andshowed an 11-fold activity improvement (Fig. 3and table S1). On the basis of this improved var-iant (ATA-117: S223P), we generated a smalllibrary of enzyme variants for potential activityon prositagliptin ketone. Analysis of the enzymemodel suggested four residues that could poten-tially interact with the trifluorophenyl group [V69,F122, T283, and A284 (19)]. Each of these po-sitions was individually subjected to saturationmutagenesis and also included in a combinatoriallibrary that evaluated several residues at each po-sition on the basis of structural considerations[V69!G69 andV69!A69 (V69GA),F122AVLIG,T283GAS, and A284GF; library size of 216 var-iants]. A variant containing four mutations, threein the small binding pocket and one in the largepocket, provided the first detectable transaminaseactivity on prositagliptin ketone (Fig. 3 and tableS3). No detectable activity was identified in anyof the variants from the single amino acid site sat-uration libraries. Initial activity was accomplishedvia an F122I, V, or L mutation in combinationwith V69G or A284G. Docking studies indicatedthat these mutations may relieve the steric inter-ference in the small binding pocket (Fig. 2E). En-zyme loading of 10 g/l provided 0.7% conversionof 2 g/l of ketone over 24 hours, corresponding toan estimated turnover of 0.1 per day. Screeningthe same combinatorial library in the ATA-117context without the S223P large binding pocketmutation did not provide any variant with detect-ible activity toward prositagliptin ketone. Havingattained activity through computer-aided catalystdesign, we started evolving an enzyme variant fora practical, large-scale process.
The variant with the highest activity towardprositagliptin ketone from round 1b was chosenas the parent for the second round of evolution,and all the beneficial mutations from both thesmall-pocket combinatorial library and the large-pocket saturation mutagenesis libraries werecombined into a new library. Screening of this li-brary resulted in a variant with 75-fold increased
Fig. 1. (A) The current synthesis of sitagliptin involves enamine formation followed by asymmetric hy-drogenation at high pressure (250 psi) using a rhodium-based chiral catalyst, providing sitagliptin in 97%e.e.,with trace amounts of rhodium. Recrystallization to upgrade e.e. followed by phosphate salt formationprovides sitagliptin phosphate. (B) Our biocatalytic route features direct amination of prositagliptin ketoneto provide enantiopure sitagliptin, followed by phosphate salt formation to provide sitagliptin phosphate.
Fig. 2. Previous substrate range studies suggested that the active site of transaminase consists of large (L)and small (S, typically limited to substituents about the size of a methyl group) binding pockets as mappedon the structure of acetophenone (A). Accordingly, the structure of prositagliptin ketone (B) can be mappedon these binding pockets and docked intothe active site of the homology model (C). A prositagliptin ketoneanalog (D) was designed to fit the large pocket for initial optimization of this part of the active site. Afterinitial engineering of the large pocket, an enzyme variant was generated with activity on the desiredsubstrate (E) by excavating the small pocket (gray/blue, transaminase homology model; orange, largebinding pocket; turquoise, small binding pocket; green, PLP and catalytic residues).
16 JULY 2010 VOL 329 SCIENCE www.sciencemag.org306
• 3rd generaIon won PresidenIal Green Chemistry Award in 2010.
• Savile, C. K. et al. BiocatalyIc Asymmetric Synthesis of Chiral Amines from Ketones Applied to SitaglipIn Manufacture. Science. 2010, 329, 305.
• Desai, A. A. SitaglipIn Manufacture: A Compelling Tale of Green Chemistry, Process IntensificaIon, and Industrial Asymmetric Catalysis. Angew. Chem. Int. Ed. 2011, 50, 1974.
Examples of green chemistry
• Pressure treated wood – replacement of CCA (containing As and Cr) with ACQ
• Polystyrene blowing agent – replacement of CFCs with CO2
• Mushroom packaging – use of waste agricultural materials to grow packaging materials
• Synthesis of sitaglipIn (Januvia) – mulIple generaIons of syntheses to improve yield and purity.
Homework
• Read your chosen arIcle from Real‐World Cases in Green Chemistry and prepare a short presentaIon (~10 min) – Describe the chemistry – Which principles does it illustrate? How?
• Wriien homework: Ch. 3, #7‐9 • OpIonal reading: I’ll post some arIcles about atom economy, which we’ll discuss next week.
