The overall goal of this procedure is to synthesize and purify Chiral Amino Alcohols, which are compounds with a wide range of applications from Chiral auxiliaries for organic synthesis to pharmaceutical therapy. This is an all yielding meter to gain access to chiral amino alcohol, derived from the natural amino acid, lysine in two or three enzymatic steps in a one part procedure. The main advantages of this procedure is the simplicity of the protocol and the efficiency of the purification steps.
Demonstrating the proceudre will be Aurelie Fossey-Jouenne, the technician from my laboratory. To synthesize the di-hydroxilated lysine derivative, add hepes buffer, l-lysine, alpaquita glutaric acid, sodium ascorbate, and more salt to a 250 ml erlenmyer flask. Then, add water to a final volume of 10 ml, taking into account the volume of the enzyme solution to be added.
Add the deoxygenase KD-01 to a final concentration of 0.075mg per ml. Shake the reaction mixture at room temperature at 300 rpm for the appropriate duration. After mixing, transfer 10mcl of the reaction mixture, to a 1.5ml ependorf tube.
Then, add aquias sodium bicarbonate solution, ethanol, and 2.5mg per ml, of DNFB solution in ethanol. Close the microtube, and shake the solution for 1 hour at 1000rpm, and 65 degrees Celsius. When finished, use a mini centrifuge to settle down the solution.
Quench the reaction mixture with 10mcl of 1M hydrochlroic acid. Vortex the solution, and then use a mini centrifuge to settle it down. Using a 1ml lure syringe, filter the mixture over a 4mm diameter non sterile syringe filter, with a 0.22mcm pour size hydrophilic polyvinylidene flouride membrane.
Following this, place the derivitized reaction mixture sample in a HPLC instrument. Inject 10mcl into the C-18 column Using a UV detection, at 400 nanometers to analyze the product. When the reaction monitoring indicates a completed reaction, add alphaquita glutaric acid, sodium ascorbate, and more salt to the flask.
Add the deoxygenase KD-02 to an apporoximate final concentration of 0.5mg per ml. Calculated using the initial reaction volume. Shake the reaction mixture at room temperature at 300rpm, for 18 hours.
When the reaction monitoring indicates a completed reaction, add 100mcl of 100 milimolar DTT to the reaction mixture. Plus 100mcl of 100 milimolar PLP. Add purified decarboxylase Dccpin at an approximate final concentration of 0.5mg per ml.
Calculated using the initial reaction volume. Then, shake the reaction mixture at room temperature at 300 rpm for 18 hours. Once the reaction is complete, cool down the mixture by placing the flask in an ice bath.
Carefully add 0.25ml of 6 molar hcl, and gently shake the cold reaction mixture during the addition. Now, transfer the acdic mixture into a 50ml conical bottom centrifuge tube, using a glass pasture pipette. Centrifuge the mixture at 1, 680 times gravity, and 4 degrees Celsius, for 15 minutes.
Following centrifugation, transfer the supernatant to a 250ml roundbottom flask. Next, add 10ml of Deionized water to the centrifuge tube containing the pellet. Vortex to re suspend the pellet.
After centrifuging the sample, transfer the supernatant to the round bottom flask, containing the first supernatant. Freeze the collected supernatants, by emerging of the flask into liquid nitrogen with constant hand swirling. Transfer the flask immediately to a bench-top manifold freeze dryer to prevent the material from thawing.
After the freeze-drying process is complete, remove the flask from the freeze dryer and carry out the purification steps. For the biocatalytic decarboxilation of mono-hydroxyl L-lysines the DC from S Rhumerentium, exhibited activity towards all the mono-hydroxy lysines. Where the best conversion observed for the three and five derivitives of the corresponding chiral hydroxy-diamines.
As expected, Dccpin turned out to be the most suitable for the decarboxylation of 4R-hydroxy-L-lysine 2. Under standard reaction conditions, the conversion of 3S-hydroxy-L-lysine 1 into its decarboxylated counterpart 5, with DCcpin was low. And no activity was observed towards 5R-hydroxy-L-lysine.
For the biocatalytic decarboxyilation of dehydroxy-L-lysines, only 3R, 4R, dihydroxy-L-lysine 3 was quantatatively converted into the corresponding dihydroxy-diamine 7 under the standard reaction conditions. The conversion of 45-dihydroxy-L-lysine 8 was moderate but was improved by increasing the enzyme loading. Neither PLP-DC was active towards 35-dihydroxy-L-Lysine 9.
The Enzymatic cascade reactions exhibiting quantatative conversion as determined by HPLC monitoring, were successfully scaled up. The amino alcohols were purified from complex enzymatic reaction mixtures, and excellent yeilds, and characterized by NMR. Once mastered this enzymatic cascade synthesis and subsequent purification can be done in 48 hours if its preformed properly.
While attempting this procedure, its important to remember, that good oxygenation of the reaction major is essential for the dioxygenated reactions. As well as a careful monitoring, to ensure that all of the lysine is consumed before adding the dicarboxidase. The main drawback of this protocol is limited subsidized ranges of the dioxygenase and decarboxylase enzyme.
Nevertheless, biochatalitic synthesis of various amino alcohol, from amino acids, can be explored by using a different set of enzymes. After watchin this video, you should have a good understanding of how to preform an enzymatic cascade reaction, and how to purify amino alcohols, from complex reaction mixtures. Don't forget that working with DNFB can be hazardous and precautions such as wearing personal protective equipment, and preforming the reaction in a fume hood, should otherwise be taken.
