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Ubrogepant IM
Wild-type enzyme :
• Stability : 70 % activity loss in 50 % DMSO
• Selectivity : Dr < 0.5
• Space-time-yield : < 2 g L- 1 d- 1
�
,%.
V
leer / on
• Stability : Tolerates 50 % DMSO
• Selectivity : Dr > 120
• Space-time-yield : > 30 g L- 1 d- 1
4 rounds of evolution
|
Figure 2 - 100 mL reaction results for final transaminase variant ( a ) & comparison of initial and evolved enzyme ( b ) acid - that are coupled by simple amide bond formation in the subsequent step .
The lactam contains three chiral centres and the original synthetic route using a chiral serine derivative results in a mixture of racemic products , making chiral supercritical fluid chromatography separation necessary for purification . 3 In this article , we describe the development of an evolved transaminase for asymmetric synthesis of the lactam via dynamic kinetic transamination ( Figure 1 ).
In total , four rounds of evolution were conducted for improving the activity and selectivity . An already evolved enzyme from a previous study displaying improved stability served as the starting point .
During the investigation with the starting enzyme , methanol as a solvent was found to be more beneficial regarding activity and selectivity , although DMSO had been previously reported in literature as optimal . 4 Consequently , methanol was selected as co-solvent in the beginning . The initial backbone could tolerate 35 % methanol and 20 % DMSO producing predominantly the undesired products 11 and 12 .
After the first round of evolution the resulting enzyme showed similar tolerance towards methanol , while it tolerated at least 50 % DMSO . Under these conditions , the activity and selectivity were improved by more than 60-fold . In the second round of evolution , the substrate loading was now targeted and could successfully be increased from 5 to 30 g / L .
Furthermore , the temperature was raised from 45 ° C to 55 ° C in the screening which improved the selectivity of the reaction twofold , while the activity remained at the same level . In the third round of evolution , the substrate was found to be hydrolysed when methanol was used as a cosolvent and the hydrolysis could not be prevented by changing the reaction conditions .
At that stage , the co-solvent was switched back from methanol to DMSO to avoid hydrolysis . During this round of evolution , the selectivity was improved 1.5-fold and the stability , in particular with regard to isopropylamine
( IPM ) - the amine donor used at high concentrations to drive the reaction towards the product - was improved . In the last round of evolution , the best evolved enzyme for this process was showing high selectivity , but slightly decreased activity and stability compared to its direct predecessor while still meeting the process cost targets . It is worth mentioning that under reaction conditions ( 55 ° C , 50 % DMSO , pH 10.5 , 1 m IPM ), the racemisation of the phenyl group ( 3 and 4 to 1 and 2 ) proceeded in a fast manner and also the lactam formation in the final step occurred spontaneously .
In the end the evolved transaminase was able to convert 95 % of 50 g / L of substrate within 72 hours . The diastereomeric ratio of the desired products 9 and 10 to 11 and 12 could be maintained at above 120 ( Figure 2 ).
Pregabalin IM synthesis via engineered d-hydantoinase
( S ) -3-isobutyl-γ-aminobutyric acid ( Pregabalin ) is a generic drug associated with endogenous inhibitory neurotransmitters and has
Figure 3 - Chemo-enzymatic synthesis of Pregabalin
Hydantoinase
Hofmann rearrangement
13 14
OH
35