Single step biocatalytic amidation
A technology for biocatalyzing amides and amino acids, applied in biochemical equipment and methods, organic chemistry, enzymes, etc., can solve problems such as biocatalytic amidation of underivatized amino acids that have not yet been completed
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[0077] The following examples are set forth to aid in the understanding of the invention but are not intended and should not be construed as limiting the scope of the invention in any way. The examples do not include detailed descriptions of conventional methods. Such methods are well known to those of ordinary skill in the art.
example 1-L
[0078] Conversion of Example 1-L-proline
[0079] Small scale batch standard reaction with L-proline
[0080] For biotransformation under standard conditions, L-proline (1.9 mg, 33 mM) and immobilized CalB (8.3 mg), CalB435 from Novozymes or an immobilized CalB variant (unless otherwise stated) were used. Reactions were carried out in small glass vials sealed with caps and septa containing approximately 0.5M NH 3 The organic solvent (2-methyl-2-butanol, 1,4-dioxane or t-BuOH, 500 μL) was performed, and the Eppendorf Thermoshaker (Eppendorf Thermoshaker) was operated at 700 rpm and 70 °C. Comfort) for 16 hours with shaking.
[0081] After the reaction, methanol (500 μL) was added to completely dissolve the substrate and product. Samples were diluted with methanol to 1 mM substrate concentration (1:33). D,L-norvaline (0.5 mM) was added as an internal standard and samples were vortexed, centrifuged (13,000 rpm, 2 minutes), filtered through cotton wool and analyzed by HPLC-M...
example 3
[0096] Example 3 - Reaction with a new substrate
[0097] use CalB 435 and NH 3 test different acids
[0098] Different acids (33mM) and CalB 435 (8.3mg) in 0.5M NH 3 Incubate with 1,4-dioxane (500 μL). Reaction samples plus enzyme-free reference samples were prepared and incubated for each substrate. After a reaction time of 16 hours at 70°C, samples were diluted and analyzed. Product formation was confirmed by HPLC-MS (using the method described above for Cbz-proline) before analyzing the reaction by GC-MS or HPLC-UV. GC-MS: Dilute the sample and derivatize the unconverted substrate to the corresponding methyl ester. The derivatization procedure was performed by adding methanol (60 μL) to the sample (150 μL) followed by 2M trimethylsilyldiazomethane in hexane (10 μL). After incubation for 10 minutes at room temperature, samples were measured by GC-MS. Compare the ratio of substrate and product peak areas. An Agilent 7890A GC system equipped with an Agilent 5975C ...
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