The authors thank V&#233;ronique de Berardinis for fruitful discussion and Alain Perret, Christine Pell&#233;, and Peggy Sirvain for technical support.

1. Enzyme Preparation
<ol>
	<li>Express and purify proteins as previously described26. 
	NOTE: Recombinant proteins were obtained with the following final concentrations: &#945;KAO from Catenulispora acidiphila, UniProtKB ID: C7QJ42 (KDO1), 1.35 mg/mL; &#945;KAO from C. pinensis, UniProtKB ID: C7PLM6 (KDO2), 2.29 mg/mL; PLP-DCs from S. rumirantium, UniProtKB ID: O50657 (LDCSrum), cell free extract with total enzyme at 12.44 mg/mL; PLP-DC from C. pinensis, UniProtKB ID: C7PLM7 (DCCpin), 2.62 mg/mL.</li>
</ol>
2. Preparation of Solutions
NOTE: All the solutions below are prepared in deionized water.
<ol>
	<li>Prepare 1 M of HEPES buffer, pH 7.5; adjust with 5 M of NaOH. 
	NOTE: During the pH adjustment step, wear protective gloves, protective clothing, eye protection, and face protection (P280). In case of contact with eyes (P305, P351, P338), rinse cautiously with water for several min. Remove contact lenses if possible. Continue rinsing and immediately call a poison center or doctor (P310). The P code refers to the precautionary statement (hazard class and category; see, e.g., pubchem.ncbi.nlm.nih.gov/ghs/).</li>
	<li>Prepare 100 mM L-lysine, 100 mM (5S)-hydroxy-L-lysine, 150 mM &#945;-ketoglutaric acid, 100 mM sodium ascorbate, 10 mM ammonium iron(II) sulfate hexahydrate (Mohr salt), 100 mM pyridoxal 5-phosphate (PLP), and 100 mM dithiothreitol (DTT).</li>
	<li>Prepare 1 M, 2M, and 6M of hydrochloric acid (HCl) from a 37% HCl solution (approximatively 12 M solution), following the same safety instructions as those described in 2.1. 
	NOTE: The solutions of &#945;-ketoglutaric acid, sodium ascorbate, and ammonium iron(II) sulfate hexahydrate must be made fresh on the day of use. The lysine solution can be stored for several months at room temperature. The PLP solution can be stored for one month at 4 &#176;C. The DTT solution can be stored for one month at -20 &#176;C.</li>
</ol>
3. Analytical-scale Reactions
<ol>
	<li>Method development for the decarboxylation reaction of (5R)-hydroxy-L-lysine
	<ol>
		<li>Add 11 &#181;L of 1 M solution of HEPES buffer pH 7.5 (final concentration 50 mM) to a 2 mL microtube. Then, add 22 &#181;L of 100 mM (5S)-hydroxy-L-lysine (final concentration 10 mM), 2.2 &#181;L of 100 mM PLP (final concentration 1 mM), and 2.2 &#181;L of 100 mM DTT (final concentration 1 mM). 
		NOTE: In the case of reaction with LDCSrum, the addition of DTT is not necessary.</li>
		<li>Add the purified PLP-DC (final concentration 0.1 mg/mL). Complete with H2O for a final volume of 220 &#181;L.</li>
		<li>Shake the reaction mixture at room temperature, with an open lid in open air, at 300 rpm for 3 h. 
		NOTE: Shaking the reaction mixture with an open lid in open air ensures a good oxygenation of the reaction media. It is not therefore necessary to bubble oxygen in the reaction media.</li>
		<li>Collect 10 &#181;L of the reaction mixture to be analyzed according to the reaction monitoring procedure described in step 6. The samples are preferably derivatized and analyzed directly after the sampling.</li>
	</ol>
	</li>
	<li>Method development for the enzymatic cascade reaction combining one hydroxylation step catalyzed by &#945;KAO with decarboxylation catalyzed by PLP-DC
	<ol>
		<li>Add 11 &#181;L of 1 M of HEPES buffer pH 7.5 (final concentration 50 mM) to a 2 mL microtube. Then, add 22 &#181;L of 150 mM of &#945;-ketoglutaric acid (final concentration 15 mM), 5.5 &#181;L of 100 mM of sodium ascorbate (final concentration 2.5 mM), 22 &#181;L of 10 mM of Mohr&#39;s salt (final concentration 1 mM), and 22 &#181;L of 100 mM of L-lysine (final concentration 10 mM).</li>
		<li>Complete with H2O for a final volume of 220 &#181;L, taking into account the volume of the enzyme solution to be added in step 3.2.3.</li>
		<li>Add the required purified &#945;KAO enzyme according to the targeted regioselectivity: KDO1 for hydroxylation in position C-3 or KDO2 for position C-4 of L-lysine. The final concentration of the purified enzyme (0.05-0.5 mg/mL) was determined to enable a complete conversion in approximatively 3 h.</li>
		<li>Shake the reaction mixture at room temperature, with an open lid in open air, at 300 rpm for 3 h.</li>
		<li>Add 2.2 &#181;L of 100 mM of PLP (final concentration ~ 1 mM) and 2.2 &#181;L of 100 mM of DTT (final concentration ~ 1 mM). 
		NOTE: In the case of reaction with LDCSrum, the addition of DTT is not necessary.</li>
		<li>Add the purified PLP-DC at a concentration enabling complete conversion in 18 h: LDCSrum at an approximate final concentration of 0.1 mg/mL for the cascade reaction with KDO1, and DCCpin at an approximate final concentration of 0.5 mg/mL for the cascade reaction with KDO2.</li>
		<li>Shake the reaction mixture at room temperature, with an open lid in open air at 300 rpm for 18 h.</li>
		<li>Run a monitoring procedure as in step 3.1.4.</li>
	</ol>
	</li>
	<li>Method development for the enzymatic cascade reaction combining two hydroxylation steps catalyzed by &#945;KAOs with decarboxylation catalyzed by PLP-DC
	<ol>
		<li>Run steps 3.2.1-3.2.2.</li>
		<li>Add KDO1 at a final concentration of 0.05 mg/mL. Shake the reaction mixture at room temperature, with an open lid in open air, at 300 rpm for 3h.</li>
		<li>Add KDO2 at an approximate final concentration of 0.5 mg/mL, calculated using the initial reaction volume. Shake the reaction mixture at room temperature, with an open lid in open air, at 300 rpm for 18 h.</li>
		<li>Run step 3.2.5.</li>
		<li>Add the purified PLP-DC DCCpin at an approximate final concentration of 0.5 mg/mL, calculated using the initial reaction volume. Shake the reaction mixture at room temperature, with an open lid in open air, at 300 rpm for 18 h.</li>
		<li>Run the monitoring procedure as in step 3.1.4.</li>
	</ol>
	</li>
</ol>
4. Semi-preparative One-pot Biocatalytic Reaction
NOTE: Enzymatic reactions are carried out on 0.1 mmol of L-lysine in an open-air 250 mL glass Erlenmeyer flask for a total volume of 10 mL.
<ol>
	<li>Synthesis of the monohydroxylated derivatives
	<ol>
		<li>Add 0.5 mL of 1 M HEPES buffer, pH 7.5 (final concentration 50 mM) to the flask. Then, add 1 mL of 100 mM L-lysine (final concentration 10 mM), 1 mL of 150 mM &#945;-ketoglutaric acid (final concentration 15 mM), 0.25 mL of 100 mM sodium ascorbate (final concentration 2.5 mM), and 1 mL of 10 mM Mohr&#39;s salt (final concentration 1 mM).</li>
		<li>Complete with H2O for a final volume of 10 mL, taking into account the volume of the enzyme solution to be added in step 4.1.3.</li>
		<li>Add the required purified &#945;KAO enzyme according to the targeted regioselectivity: KDO1 at a final concentration of 0.075 mg/mL for hydroxylation in position C-3 or KDO2 at a final concentration of 0.5 mg/mL for position C-4 of L-lysine. The final concentration of the purified enzyme was determined to enable a complete conversion in approximatively 3 h.</li>
		<li>Shake the reaction mixture at room temperature at 300 rpm for the appropriate duration. When the reaction monitoring indicates a completed hydroxylation reaction (run all the steps detailed in step 6 for the reaction monitoring protocol), proceed to step 4.1.5. A typical reaction time is 3 h.</li>
		<li>Add 100 &#181;L of 100 mM PLP to the &#945;KAO reaction mixture (final concentration ~ 1 mM). Then, add 100 &#181;L of 100 mM DTT (final concentration ~ 1 mM), except when using LDCSrum.</li>
		<li>Add purified PLP-DC: LDCSrum at an approximate final concentration of 0.05 mg/mL for the cascade reaction with KDO1 or DCCpin at an approximate final concentration of 0.5 mg/mL for the cascade reaction with KDO2.</li>
		<li>Shake the reaction mixture at room temperature, 300 rpm, for 18 h and continue directly to the steps detailed in step 4.3.</li>
	</ol>
	</li>
	<li>Synthesis of the dihydroxylated derivative
	<ol>
		<li>Add 0.5 mL of 1 M HEPES buffer, pH 7.5 (final concentration 50 mM), 1 mL of 100 mM L-lysine (final concentration 10 mM), 1 mL of 150 mM &#945;-ketoglutaric acid (final concentration 15 mM), 0.25 mL of 100 mM sodium ascorbate (final concentration 2.5 mM), and 1 mL of 10 mM Mohr&#39;s salt (final concentration 1 mM). Complete with H2O for a final volume of 10 mL, taking into account the volume of the enzyme solution to be added in step 4.2.2.</li>
		<li>Add KDO1 to a final concentration of 0.075 mg/mL. Shake the reaction mixture at room temperature, at 300 rpm for the appropriate duration.</li>
		<li>When the reaction monitoring indicates a completed hydroxylation reaction (see step 6 for the reaction monitoring protocol), proceed to step 4.2.5. A typical reaction time is 3 h.</li>
		<li>Add 1 mL of 150 mM &#945;-ketoglutaric acid (final concentration 15 mM), 0.25 mL of 100 mM sodium ascorbate (final concentration 2.5 mM), and 1 mL of 10 mM Mohr&#39;s salt (final concentration 1 mM).</li>
		<li>Add KDO2 to an approximate final concentration of 0.5 mg/mL, calculated using the initial reaction volume. Shake the reaction mixture at room temperature at 300 rpm for 18 h.</li>
		<li>When the reaction monitoring indicates a completed dihydroxylation reaction (see step 6 for the reaction monitoring protocol), proceed to step 4.2.7. A typical reaction time is 18 h.</li>
		<li>Add 100 &#181;L of 100 mM of DTT (final concentration ~ 1 mM), except when using the LDCSrum.</li>
		<li>Add the purified DCCpin at an approximate final concentration of 0.5 mg/mL, calculated using the initial reaction volume.</li>
		<li>Shake the reaction mixture at room temperature at 300 rpm for 18 h.</li>
	</ol>
	</li>
	<li>Quenching
	<ol>
		<li>Cool down the reaction mixture by placing the 250 mL glass Erlenmeyer flask in an ice bath.</li>
		<li>Add carefully, over approximatively 1 min, 0.25 mL of 6 M HCl by manually gently shaking the cooled reaction mixture. Follow the same safety instructions as those described in step 2.1.</li>
		<li>Transfer the acidic mixture into a 50 mL conical-bottom centrifuge tube using a glass Pasteur pipette equipped with a bulb, then centrifuge at 1,680 x g and 4 &#176;C for 15 min.</li>
		<li>Withdraw the supernatant and keep aside in a 250 mL round-bottom flask.</li>
		<li>Add 10 mL of deionized water to the conical-bottom centrifuge tube containing the pellet. Vortex to re-suspend the pellet.</li>
		<li>Centrifuge at 1,680 x g, 4 &#176;C, for 15 min.</li>
		<li>Withdraw the supernatant and add it to the 250 mL round-bottom flask containing the first supernatant (step 4.3.4).</li>
		<li>Freeze the collected supernatants by immersion of the flask into liquid nitrogen with constant hand swirling, and transfer the flask immediately to a benchtop manifold freeze-dryer to prevent the material from thawing.</li>
		<li>After the freeze-drying process is complete (overnight), remove the flask from the freeze-dryer.</li>
	</ol>
	</li>
</ol>
5. Purification of Amino Alcohols from Crude Enzymatic Reaction Mixture
<ol>
	<li>Ion exchange resin
	<ol>
		<li>Dissolve the freeze-dried product in the minimum amount of aqueous 0.1 M of HCl (approximatively 4 mL) until solid particles are no longer visible to the naked-eye.</li>
		<li>Condition a sulfonic acid functional group cation exchange resin, 200-400 mesh, in a glass column (20 x 250 mm) by eluting at atmospheric pressure 1M HCl (4 column volumes) followed by 0.1 M HCl (4 column volumes; column volume = 10 mL).</li>
		<li>Load the sample carefully at the top of the resin on the walls of the glass column using a 1,000 &#181;L micropipette. Then, rinse the conical-bottom centrifuge tube that contained the crude product three times with 1 mL 0.1 M of HCl aqueous solution per wash, and load the resulting rinse volumes onto the column in the same way.</li>
		<li>Elute with a non-linear gradient: 4 column volumes of 0.1 M HCl, 4 column volumes of water, 4 column volumes of 5% NH4OH, 4 column volumes of 10% NH4OH, 4 column volumes of 15% NH4OH, 4 column volumes of 20% NH4OH, 4 column volumes of 25% NH4OH and lastly 4 column volumes of 28% NH4OH.</li>
		<li>Monitor the purification by HPLC (see step 6).</li>
		<li>Pool the fractions containing the compound (NH4OH 20-28%) and freeze-dry as described in steps 4.3.8-4.3.9. 
		NOTE: The ion exchange column can be reused after elution with deionized water to neutralization of elution water (approximatively 50 mL) followed by reconditioning by eluting with 50 mL of 1 M of HCl.</li>
	</ol>
	</li>
	<li>SPE
	<ol>
		<li>Solubilize the freeze-dried compound in approximatively 2 mL of H3PO4/water (20 &#181;L/mL).</li>
		<li>Place a mixed mode cation-exchange SPE 6 mL-cartridge on an extraction manifold connected to a water pump. Condition the cartridge by drawing through 4 mL of methanol under reduced pressure provided by the manifold. Equilibrate the cartridge by drawing through 4 mL of H3PO4/water (20 &#181;L/mL).</li>
		<li>Load the sample onto the cartridge using a 1,000 &#181;L micropipette at the top of the resin on the walls of the glass column, and rinse the vial containing the product three times with 0.75 mL of H3PO4/water (20 &#181;L/mL) per wash. Load the resulting rinse volume onto the column in the same way.</li>
		<li>Wash the cartridge by eluting under reduced pressure, provided by the manifold, 4 mL of 2% HCOOH in water, then with 4 mL of water. Elute with a gradient of NH4OH in water (4 mL for each elution, starting from 4% NH4OH: 10%, 15%, 15%, 20%, and 28%).</li>
		<li>Monitor the purification by HPLC by analyzing each fraction according to the reaction monitoring protocol described in step 6. Keep all the fractions containing pure desired derivative according to ultraviolet (UV) chromatogram and proceed to step 5.2.6.</li>
		<li>Pool the fractions containing the desired compound into a 100 mL round bottom flask, rinse the tubes containing the fractions with water, and add the rinse volume to the 100 mL round bottom flask.</li>
		<li>Remove the solvent under reduced pressure using a rotary evaporator equipped with a thermostat water bath kettle at 40 &#176;C and connected to a vacuum pump set at 10 mbar.</li>
		<li>Solubilize the solid in a minimum volume of water and transfer the solution into a weighed 25 mL round bottom flask. Rinse the flask with a minimum volume of water, add the rinse volume to the 25 mL flask, and proceed to step 5.2.9.</li>
		<li>Weigh the purified product and calculate the yield.</li>
		<li>Dissolve the purified product in the minimum volume of 2M of HCl until solid particles are no longer visible to the naked-eye and freeze-dry as described in steps 4.3.8-4.3.9. 
		NOTE: The amino alcohols are sensitive compounds and are kept as their hydrochloride salts.</li>
	</ol>
	</li>
</ol>
6. Reaction Monitoring and Product Analysis
<ol>
	<li>Derivatization of substrates and products prior to HPLC analysis 
	NOTE: The substrates and products need to be derivatized as aromatic derivatives to increase their UV chromatographic detectivity.We used 1-fluoro-2,4-dinitrobenzene (DNFB). Each sample was injected into the HPLC immediately after derivatization.
	<ol>
		<li>Take 10 &#181;L of the enzymatic reaction and transfer to a 1.5 mL microtube. Add 10 &#181;L of 0.25 M of NaHCO3 aqueous solution, 100 &#181;L of ethanol, and 30 &#181;L of 2.5 mg/mL of DNFB solution in ethanol.</li>
		<li>Close the microtube and shake the resulting solution for 1 h at 65 &#176;C, at 1,000 rpm. Quench with 10 &#181;L 1 M of HCl.</li>
		<li>Filter over a 4 mm diameter non-sterile syringe filter with a 0.22 &#181;m pore size hydrophilic polyvinylidene fluoride (PVDF) membrane using a 1 mL Luer syringe.</li>
	</ol>
	</li>
	<li>Perform monitoring of the dioxygenase reaction using any system capable of separating derivatized amino acids. This protocol involves UV detection of amino acids derivatized by the dinitrobenzene (DNB) chromophore group27.
	<ol>
		<li>Fit the HPLC with C18 with a trimethyl silyl endcapping reversed phase column (5 &#181;m, 3 x 150 mm).</li>
		<li>Equilibrate the C18 column with (30:70) eluent B (MeCN + 0.1% TFA) to eluent A (H2O + 0.1% TFA) at a flow rate of 1 mL/min and 35 &#176;C for 15 min (corresponding to approximatively 20 column volumes).</li>
		<li>Inject 10 &#181;L of the derivatized reaction mixture (see step 6.1) onto the HPLC C18 column. Use a mixture of MeCN, 0.1% TFA/H2O and 0.1% TFA as eluent with a linear gradient (ratio 30:70 to 70:30 in 9 min, temperature 35 &#176;C, flow 1 mL/min).</li>
		<li>Use UV detection set at 400 nm to analyze the products eluted on the chromatographic column. DNB-lysine typically elutes around 6.5 min, hydroxylated DNB-lysine around 5.5 min, dihydroxylated DNB-lysine around 4.2 min, and DNB-aminodiol around 5.3 min.</li>
	</ol>
	</li>
</ol>
7. NMR Analysis of Purified Amino Alcohols
<ol>
	<li>Dissolve purified amino alcohols in D2O (700 &#181;L) and transfer the solution to a Nuclear Magnetic Resonance (NMR) tube.</li>
	<li>Acquire 1H and 13C NMR spectra according to appropriate NMR facility protocols28.</li>
</ol>

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Microbiol. Biotechnol. 91 (5), 1287-1296 (2011).</li><li>Schneider, J., Wendisch, V. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotechnological+production+of+polyamines+by+bacteria:+recent+achievements+and+future+perspectives.">Biotechnological production of polyamines by bacteria: recent achievements and future perspectives.</a> Appl. Microbiol. Biotechnol. 91 (1), 17-30 (2011).</li><li>Qian, Z. -. G., Xia, X. -. X., Lee, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+engineering+of+Escherichia+coli+for+the+production+of+cadaverine:+A+five+carbon+diamine.">Metabolic engineering of Escherichia coli for the production of cadaverine: A five carbon diamine.</a> Biotechnol. Bioeng. 108 (1), 93-103 (2011).</li><li>Shin, J. H., Lee, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+engineering+of+microorganisms+for+the+production+of+L-arginine+and+its+derivatives.">Metabolic engineering of microorganisms for the production of L-arginine and its derivatives.</a> Microb. Cell. Fact. 13, (2014).</li><li>Nguyen, A., Schneider, J., Reddy, G., Wendisch, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fermentative+Production+of+the+Diamine+Putrescine:+System+Metabolic+Engineering+of+Corynebacterium+Glutamicum.">Fermentative Production of the Diamine Putrescine: System Metabolic Engineering of Corynebacterium Glutamicum.</a> Metabolites. 5 (2), 211 (2015).</li><li>Kidron, H., Repo, S., Johnson, M. S., Salminen, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Classification+of+Amino+Acid+Decarboxylases+from+the+Alanine+Racemase+Structural+Family+by+Phylogenetic+Studies.">Functional Classification of Amino Acid Decarboxylases from the Alanine Racemase Structural Family by Phylogenetic Studies.</a> Mol. Biol. Evol. 24 (1), 79-89 (2007).</li><li>Takatsuka, Y., Onoda, M., Sugiyama, T., Muramoto, K., Tomita, T., Kamio, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Characteristics+of+Selenomonas+ruminantium+Lysine+Decarboxylase+Capable+of+Decarboxylating+Both+L-Lysine+and+L-Ornithine.">Novel Characteristics of Selenomonas ruminantium Lysine Decarboxylase Capable of Decarboxylating Both L-Lysine and L-Ornithine.</a> Biosci. Biotechnol. Biochem. 63 (6), 1063-1069 (1999).</li><li>Takatsuka, Y., Tomita, T., Kamio, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+Amino+Acid+Residues+Conferring+Substrate+Specificity+upon+Selenomonas+ruminantium+Lysine+Decarboxylase.">Identification of the Amino Acid Residues Conferring Substrate Specificity upon Selenomonas ruminantium Lysine Decarboxylase.</a> Biosci. Biotechnol. Biochem. 63 (10), 1843-1846 (1999).</li><li>Baud, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocatalytic+Approaches+towards+the+Synthesis+of+Chiral+Amino+Alcohols+from+Lysine:+Cascade+Reactions+Combining+alpha-Keto+Acid+Oxygenase+Hydroxylation+with+Pyridoxal+Phosphate-+Dependent+Decarboxylation.">Biocatalytic Approaches towards the Synthesis of Chiral Amino Alcohols from Lysine: Cascade Reactions Combining alpha-Keto Acid Oxygenase Hydroxylation with Pyridoxal Phosphate- Dependent Decarboxylation.</a> Adv. Syn. Catal. 359 (9), 1563-1569 (2017).</li><li>Ilisz, I., Berkecz, R., Peter, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+chiral+derivatizing+agents+in+the+high-performance+liquid+chromatographic+separation+of+amino+acid+enantiomers:+a+review.">Application of chiral derivatizing agents in the high-performance liquid chromatographic separation of amino acid enantiomers: a review.</a> J. Pharm. Biomed. Anal. 47 (1), 1-15 (2008).</li><li>. Organic Chemistry. Nuclear Magnetic Resonance (NMR) Spectroscopy Available from: <a target="_blank" href="https://www-jove-com.remotexs.ntu.edu.sg/science-education/5680/nuclear-magnetic-resonance-nmr-spectroscopy">https://www-jove-com.remotexs.ntu.edu.sg/science-education/5680/nuclear-magnetic-resonance-nmr-spectroscopy</a> (2017)</li><li>Hibi, M., Ogawa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+and+biotechnology+applications+of+aliphatic+amino+acid+hydroxylases+belonging+to+the+Fe(II)/alpha-ketoglutarate-dependent+dioxygenase+superfamily.">Characteristics and biotechnology applications of aliphatic amino acid hydroxylases belonging to the Fe(II)/alpha-ketoglutarate-dependent dioxygenase superfamily.</a> Appl. Microbiol. Biotechnol. 98 (9), 3869-3876 (2014).</li><li>H&#252;ttel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocatalytic+Production+of+Chemical+Building+Blocks+in+Technical+Scale+with+&#945;-Ketoglutarate-Dependent+Dioxygenases.">Biocatalytic Production of Chemical Building Blocks in Technical Scale with &#945;-Ketoglutarate-Dependent Dioxygenases.</a> Chem. Ing. Tec. 85 (6), 809-817 (2013).</li><li>Kourist, R., Guterl, J. -. K., Miyamoto, K., Sieber, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+Decarboxylation-An+Emerging+Reaction+for+Chemicals+Production+from+Renewable+Resources.">Enzymatic Decarboxylation-An Emerging Reaction for Chemicals Production from Renewable Resources.</a> ChemCatChem. 6 (3), 689-701 (2014).</li><li>Lee, J., Michael, A. J., Martynowski, D., Goldsmith, E. J., Phillips, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+diversity+and+the+structural+basis+of+substrate+specificity+in+the+beta/alpha-barrel+fold+basic+amino+acid+decarboxylases.">Phylogenetic diversity and the structural basis of substrate specificity in the beta/alpha-barrel fold basic amino acid decarboxylases.</a> J. Biol. Chem. 282 (37), 27115-27125 (2007).</li><li>Porter, J. L., Rusli, R. A., Ollis, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+Evolution+of+Enzymes+for+Industrial+Biocatalysis.">Directed Evolution of Enzymes for Industrial Biocatalysis.</a> ChemBiochem. 17 (3), 197-203 (2016).</li></ol>

The authors have nothing to disclose.

Chiral amino alcohols and derivatives have a wide range of applications, from chiral auxiliaries for organic synthesis to pharmaceutical therapy. Multistep synthesis for producing amino alcohols by conventional organic synthesis are numerous, but may not always be efficient because of tedious protection/deprotection steps together with a sensitive control of the stereochemistry16. A biocatalytic approach that dispenses with the protection/deprotection steps and is usually highly stereoselective represents a good alternative.
Previous work reported the synthesis of various &#946;-amino alcohols with a 2-phenylethan-1-amine backbone by dynamic kinetic asymmetric transformation (DYKAT). In this route, the hydroxy-substituted stereogenic center was introduced by aldolisation on benzaldehyde and derivatives, catalyzed by a threonine aldolase. The low diastereoselectivity and the moderate yield of this first step were overcome by the subsequent stereospecific decarboxylation catalyzed by L-tyrosine DC, leading to enantioenriched aromatic &#946;-amino alcohols11,12.
We reported herein a straightforward protocol for the synthesis of various amino alcohols starting from the readily available L-lysine. Although very efficient, this protocol suffers from drawbacks due to limited substrate ranges of the &#945;KAO and PLP-DC enzymes. Nevertheless, biocatalytic synthesis of various amino alcohols can be considered by using different combinations of other &#945;KAO and amino acid DCs29,30,31,32. It is worth noting that the two-step order is critical in the cascade as the PLP-DCs are also active towards the L-lysine. Care must be taken to ensure that all the L-lysine is consumed in the first oxidative step before running the decarboxylation reaction catalyzed by the PLP-DC. This is ensured through a careful monitoring of the reaction by HPLC after derivatization of the reaction media by a chromophore agent. The low tolerance of the enzyme to high substrate concentration is a limitation for further development. To address this issue, enzyme evolution strategies might be used for optimizing the enzyme properties, such as substrate concentration tolerance33. Also, enzyme immobilization can be considered to ensure reuse of the enzyme.
This protocol is easy to carry out, and one of its most attractive features is the efficiency of the purification steps. In our preceding work, the direct extraction of polar molecules from the complex enzymatic reaction mixture was an issue, and derivatization of the compounds by hydrophobic groups was necessary16. The purification of the amino alcohols directly from the reaction without derivatization required extensive work. In this protocol, the two-step purification procedure combining ion exchange resin and solid phase extraction removes glycerol (contained in the enzyme solution) and HEPES buffer, two polar molecules with physical-chemical properties close to the ones of the targeted amino alcohols and therefore difficult to separate from these compounds. This purification protocol can be adapted for the extraction of various polar molecules from complex reaction mixtures such as those from enzyme reactions.

Despite the benefits offered by biocatalysis, the integration of biocatalytic steps in synthetic pathways or total biocatalytic routes remains mostly limited to enzymatic kinetic resolutions. These routes have been widely used as a first step in asymmetric chemo-enzymatic synthesis, but biocatalysis offers many more possibilities in functional group interconversions with high stereoselectivity1,2,3. Moreover, as biocatalytic reactions are conducted in similar conditions, it is therefore feasible to perform cascade reactions in a one-pot fashion4,5.
Chiral amino alcohols are versatile molecules for use as auxiliaries or scaffolds in organic synthesis6. The amino alcohol moiety is frequently found in secondary metabolites and in active pharmaceutical ingredients (API). Primary &#946;-amino alcohols are readily available from the corresponding &#945;-amino acids by conventional chemical synthesis, but access to chiral &#947;-amino alcohols or secondary amino alcohols often requires tedious synthetic pathways together with sensitive control of the stereochemistry7,8,9,10. Due to its high stereoselectivity, biocatalysis may provide a superior synthetic route to these chiral building blocks11,12,13,14.
We previously reported the synthesis of mono- and di-hydroxy-L-lysines by diastereoselective enzymatic hydroxylation catalyzed by dioxygenases of the iron(II)/&#945;-ketoacid-dependent oxygenase family (&#945;KAO) (Figure 1)15. In particular, starting from L-lysine, the KDO1 dioxygenase catalyzes the formation of the (3S)-hydroxy derivative (1), while the (4R)-derivative (2) is formed by the reaction with KDO2 dioxygenase. Successive regiodivergent hydroxylations by KDO1 and KDO2 lead to the formation of the (3R,4R)-dihydroxy-L-lysine (3) in optically pure form. However, the limited substrate range of these enzymes impedes their large utilization in chemical synthesis, especially in the hydroxylation of simple amines, as a carboxylic acid moiety in the &#945;-position of the amino group is essential for activity16.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56926/56926fig1.jpg" /> 
Figure 1: Biocatalytic conversions of L-lysine. Conversion into (3S)-hydroxy-L-lysine (1) catalyzed by KDO1 dioxygenase; (4R)-hydroxy-L-lysine (2) catalyzed by KDO2 dioxygenase; and (3R,4R)-dihydroxy-L-lysine (3) by cascade reaction catalyzed successively by KDO1 and KDO2 dioxygenases. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56926/56926fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Decarboxylation is a common reaction in metabolism17. In particular, amino acid DCs (EC 4.1.1) are cofactor-free (pyruvoyl-dependent) or PLP-dependent enzymes, and catalyze the decarboxylation of amino acids into the corresponding polyamines in bacteria and higher organisms18,19,20,21,22. The mono- and dihydroxy compounds (Figure 3) 4-7, 10-11 correspond to hydroxylated cadaverine, the diamine obtained by decarboxylation of L-lysine. Cadaverine is a key building block for the chemical industry, specifically it is a component of polyamide and polyurethane polymers. Therefore, bio-based production of this diamine from renewable resources has attracted attention as an alternative to the petroleum-based route, and various microorganisms have been engineered for this purpose. In these metabolic pathways, lysine DC (LDC) is the key enzyme. LDC is a PLP-dependent enzyme belonging to the alanine racemase (AR) structural family23. The PLP-dependent DCs (PLP-DCs) are known to be highly substrate-specific. However, a few enzymes own the capability of slight promiscuity, being active towards both L-ornithine and L-lysine amino acids, as for example the LDC from Selenomonas rumirantium (LDCSrum), which has similar kinetic constants for lysine and ornithine decarboxylation24,25. This extended substrate specificity makes this enzyme a good candidate for the decarboxylation of mono- and di-hydroxy-L-lysine. In addition, to find DCs active towards the hydroxyl derivatives of lysine, we examined the genomic context of the genes encoding the &#945;KAO enzymes. Indeed, in prokaryotic genomes the genes encoding enzymes involved in the same biosynthetic pathway are generally co-localized in gene clusters. The KDO2 (from Chitinophaga pinensis) gene was found co-localized with a gene encoding putative PLP-DC (Figure 2). In contrast, no gene encoding for DC has been found when analyzing the genomic context of the KDO1 dioxygenase. The PLP-DC protein from C. pinensis (DCCpin) was therefore selected as a promising candidate to catalyze the decarboxylation step of the cascade reaction.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56926/56926fig2.jpg" /> 
Figure 2: Genomic context of KDO2 gene in C. pinensis. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56926/56926fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Consequently, we designed enzymatic cascade reactions involving dioxygenases and DCs to achieve the synthesis of aliphatic chiral &#946;- and &#947;-amino alcohols from amino acids (Figure 3). As previously reported, the C-H oxidation catalyzed by the &#945;KAO introduces the hydroxy-substituted stereogenic center with total diastereoselectivity; the C&#946;/&#947; chirality will be preserved in the decarboxylative step, which only affects the C&#945; carbon of the amino acid moiety16.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56926/56926fig3.jpg" /> 
Figure 3: Retrosynthetic analysis. (A) Retrosynthesis of &#946;- and &#947;-amino alcohols (R)-1,5- diaminopentan-2-ol (4) from (5R)-hydroxy-L-Lysine, and (S)-1,5-diaminopentan-2-ol (5) and 1,5-diaminopentan-3-ol (6) from L-lysine. (B) Retrosynthesis of &#946;,&#947;- and &#946;,&#948;-amino diols (2S,3S)-1,5-diaminopentane-2,3-diol (10) and (2R,4S)-1,5-diaminopentane-2,4-diol (11) starting from (5R)-hydroxy-L-lysine, and (2R,3R)-1,5-diaminopentane-2,3-diol (7) starting from L-lysine. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56926/56926fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Starting from L-lysine and its (5R)-hydroxy derivative, we herein report a two/three step, one pot, enzymatic procedure combining dioxygenases and PLP-DCs to obtain the target amino alcohols. Prior the synthesis at the laboratory scale of the target molecules, the method was developed at the analytical scale to adjust the reaction conditions, e.g., the enzyme concentrations, required to allow full conversion of the starting materials; we present this procedure as well.

<table>
	<tbody>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>L-lysine hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>L5626</td>
			<td></td>
		</tr>
		<tr>
			<td>(5S)-hydroxy-L-lysine</td>
			<td>Sigma Aldrich</td>
			<td>GPS NONH</td>
			<td>Out sourcing</td>
		</tr>
		<tr>
			<td>&#945;-ketoglutaric acid</td>
			<td>Sigma Aldrich</td>
			<td>75892</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium ascorbate</td>
			<td>Sigma Aldrich</td>
			<td>A7631</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Iron(II) sulfate hexahydrate</td>
			<td>Acros</td>
			<td>201370250</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridoxal phosphate (PLP)</td>
			<td>Sigma Aldrich</td>
			<td>82870</td>
			<td></td>
		</tr>
		<tr>
			<td>3,4-dimercaptobutane-1,2-diol (DTT)</td>
			<td>Sigma Aldrich</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr>
			<td>1-fluoro-2,4-dinitrobenzene (DNFB)</td>
			<td>Sigma Aldrich</td>
			<td>D1529</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>20825.290</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate</td>
			<td>Sigma Aldrich</td>
			<td>71631</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl 37%</td>
			<td>Sigma Aldrich</td>
			<td>435570</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl 0.1M</td>
			<td>Fluka</td>
			<td>35335</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile HiPerSolv CHROMANORM for LC-MS</td>
			<td>VWR</td>
			<td>83640.320</td>
			<td></td>
		</tr>
		<tr>
			<td>2,2,2-trifluoroacetic acid</td>
			<td>VWR</td>
			<td>153112E</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonia 28%</td>
			<td>VWR</td>
			<td>21182.294</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol HiPerSolv CHROMANORM for LC-MS</td>
			<td>VWR</td>
			<td>83638.32</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Acros</td>
			<td>270480010</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoric acid 85%</td>
			<td>Acros</td>
			<td>201145000</td>
			<td></td>
		</tr>
		<tr>
			<td>Deuterium oxide</td>
			<td>Acros</td>
			<td>320,710,075</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma Aldrich</td>
			<td>S5881</td>
			<td></td>
		</tr>
		<tr>
			<td>C18 HPLC column</td>
			<td>Phenomenex</td>
			<td>00F-4601-Y0</td>
			<td></td>
		</tr>
		<tr>
			<td>Accela UHPLC System</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accela PDA detector</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4mm syringe filters - 0,22&#181;m - PVDF</td>
			<td>Merck</td>
			<td>SLGVR04NL</td>
			<td></td>
		</tr>
		<tr>
			<td>Single-use tuberculin syringe with ml graduation, Luer tip</td>
			<td>VWR</td>
			<td>HSWA5010.200V0</td>
			<td></td>
		</tr>
		<tr>
			<td>Cation exchange resin 100-200&#160;mesh</td>
			<td>Sigma Aldrich</td>
			<td>217506</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixed mode cation-exchange solid-phase extraction cartridge 6 mL</td>
			<td>Waters</td>
			<td>186000776</td>
			<td></td>
		</tr>
		<tr>
			<td>Extraction manifold</td>
			<td>Waters</td>
			<td>WAT200609</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary evaporator</td>
			<td>B&#252;chi</td>
			<td>531-0103</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer alpha 1-2 LDplus</td>
			<td>Christ</td>
			<td>L083302</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette 20 &#181;L</td>
			<td>Eppendorf</td>
			<td>3121000031</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette 100 &#181;L</td>
			<td>Eppendorf</td>
			<td>3121000074</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette 500 &#181;L</td>
			<td>Eppendorf</td>
			<td>3121000112</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette 1000 &#181;L</td>
			<td>Eppendorf</td>
			<td>3121000120</td>
			<td></td>
		</tr>
		<tr>
			<td>300 MHz spectrometer</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL microtube</td>
			<td>CLEARLine</td>
			<td>CL20.002.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical-bottom centrifuge tube</td>
			<td>Fischer Scientific</td>
			<td>05-539-8</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL round-bottom flask 14/23</td>
			<td>Fischer Scientific</td>
			<td>10353331</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL round-bottom flask 29/32</td>
			<td>Fischer Scientific</td>
			<td>11786183</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL round-bottom flask 29/32</td>
			<td>Fischer Scientific</td>
			<td>11786183</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL erlenmeyer flask</td>
			<td>Fischerbrand</td>
			<td>15496143</td>
			<td></td>
		</tr>
	</tbody>
</table>

enzymatic cascade reactions for the synthesis of chiral amino alcohols from l-lysine

Amino alcohols are versatile compounds with a wide range of applications. For instance, they have been used as chiral scaffolds in organic synthesis. Their synthesis by conventional organic chemistry often requires tedious multi-step synthesis processes, with difficult control of the stereochemical outcome. We present a protocol to enzymatically synthetize amino alcohols starting from the readily available L-lysine in 48 h. This protocol combines two chemical reactions that are very difficult to conduct by conventional organic synthesis. In the first step, the regio- and diastereoselective oxidation of an unactivated C-H bond of the lysine side-chain is catalyzed by a dioxygenase; a second regio- and diastereoselective oxidation catalyzed by a regiodivergent dioxygenase can lead to the formation of the 1,2-diols. In the last step, the carboxylic group of the alpha amino acid is cleaved by a pyridoxal-phosphate (PLP) decarboxylase (DC). This decarboxylative step only affects the alpha carbon of the amino acid, retaining the hydroxy-substituted stereogenic center in a beta/gamma position. The resulting amino alcohols are therefore optically enriched. The protocol was successfully applied to the semipreparative-scale synthesis of four amino alcohols. Monitoring of the reactions was conducted by high performance liquid chromatography (HPLC) after derivatization by 1-fluoro-2,4-dinitrobenzene. Straightforward purification by solid-phase extraction (SPE) afforded the amino alcohols with excellent yields (93% to &#62;95%).

We have previously reported the synthesis of mono- and di-hydroxy-L-lysines by diastereoselective enzymatic hydroxylation catalyzed by dioxygenases of the iron(II)/&#945;KAO family (Figure 1)16. To optimize the protocol of the entire cascades presented here, which combine one or two hydroxylation steps catalyzed by an &#945;KAO followed by a decarboxylation step catalyzed by a PLP-DC, the reaction conditions were adjusted to satisfy the requirements of both enzymatic reactions. We started by investigating the activities of the two DCs, LDCSrum and DCCpin, towards the commercially available (5R)-hydroxy-L-lysine. Then we assayed the DC activities towards the mono derivatives, 3-hydroxy-L-lysine (1) and 4-hydroxy-L-lysine (2), in cascade with the oxidation step catalyzed by the appropriate &#945;KAO. Table 1 presents the results of the biocatalytic decarboxylations of the mono-hydroxy-L-lysines. Conversions were measured by HPLC after derivatization of the reaction mixture with DNFB to give the corresponding DNB derivatized substrates and products.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Entry</td>
			<td>Substrate</td>
			<td>PLP-DC</td>
			<td>Product</td>
			<td>Conversion</td>
		</tr>
		<tr>
			<td>1</td>
			<td>(5R)-hydroxy-L-lysine</td>
			<td>LDCSrumb)</td>
			<td>4</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>2</td>
			<td>(5R)-hydroxy-L-lysine</td>
			<td>DCCpinb)</td>
			<td>4</td>
			<td>n.d.</td>
		</tr>
		<tr>
			<td>3</td>
			<td>1a)</td>
			<td>LDCSrumb)</td>
			<td>5</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>4</td>
			<td>1a)</td>
			<td>DCCpinb)</td>
			<td>5</td>
			<td>29%</td>
		</tr>
		<tr>
			<td>5</td>
			<td>2a)</td>
			<td>LDCSrumc)</td>
			<td>6</td>
			<td>60%</td>
		</tr>
		<tr>
			<td>6</td>
			<td>2a)</td>
			<td>DCCpinc)</td>
			<td>6</td>
			<td>100%</td>
		</tr>
	</tbody>
</table>
Table 1: Biocatalytic decarboxylation of monohydroxy-L-lysines. Reaction conditions: substrate (10 mM), PLP-DC (0.1 to 0.15 mg mL-1), HEPES buffer (50 mM, pH 7.5), PLP (1 mM), DTT (1 mM; not used with LDCSrum), overnight, RT, 300 rpm. (a) Synthesized enzymatically in situ. (b) 0.1 mg/mL. (c) 0.15 mg/mL; n.d.: not detected
The DC from S. rumirantium (LDCSrum) exhibited activity towards all the mono hydroxy lysines with best conversion of 3- and 5- derivatives to the corresponding chiral hydroxy diamines (entries 1, 3, and 5). As expected, DCCpin, the DC of the &#945;KAO genomic context, turned out to be the most suitable for the decarboxylation of (4R)-hydroxy-L-lysine (2) (entry 6). Under standard reaction conditions the conversion of (3S)-hydroxy-L-lysine (1) into its decarboxylated counterpart (5) with DCCpin was low (entry 4), and no activity was observed towards (5R)-hydroxy-L-lysine (entry 2).
Lastly, we examined the activities of the two DCs towards the di-hydroxy-L-lysine derivatives 3, 8, and 9, synthesized in situ by one or two hydroxylation steps catalyzed by KDO1, KDO2, or a combination of the two (Figure 2). The results of the biocatalytic decarboxylation of the di-hydroxy-L-lysines are summarized in Table 2.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Entry</td>
			<td>Substrate</td>
			<td>PLP-DC</td>
			<td>Product</td>
			<td>Conversion</td>
		</tr>
		<tr>
			<td>1</td>
			<td>3</td>
			<td>LDCSrumb)</td>
			<td>7</td>
			<td>n.d.</td>
		</tr>
		<tr>
			<td>2</td>
			<td>3</td>
			<td>DCCpinb)</td>
			<td>7</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>3</td>
			<td>8a)</td>
			<td>LDCSrumb)</td>
			<td>10</td>
			<td>19%</td>
		</tr>
		<tr>
			<td>4</td>
			<td>8a)</td>
			<td>DCCpinb)</td>
			<td>10</td>
			<td>12%</td>
		</tr>
		<tr>
			<td>5</td>
			<td>9a)</td>
			<td>LDCSrumc)</td>
			<td>11</td>
			<td>n.d.</td>
		</tr>
		<tr>
			<td>6</td>
			<td>9a)</td>
			<td>DCCpinc)</td>
			<td>11</td>
			<td>n.d.</td>
		</tr>
	</tbody>
</table>
Table 2: Biocatalytic decarboxylation of dihydroxy-L-lysines. Reaction conditions: substrate (10 mM), PLP-DC (0.1 to 0.15 mg/mL), HEPES buffer (50 mM, pH 7.5), PLP (1 mM), DTT (1 mM; not used with LDCSrum), overnight, RT, 300 rpm. (a) Synthesized enzymatically in situ. (b) 0.1 mg/mL. (c) 0.15 mg/mL; n.d.: not detected.
Regarding the decarboxylation of the dihydroxy-L-lysines 3, 8, and 9, the results were not fully satisfactory. In the standard reaction conditions, only (3R,4R)-dihydroxy-L-lysine (3) was quantitatively converted into the corresponding dihydroxy diamine 7 (entries 1-2). The conversion of 4,5-dihydroxy-L-lysine (8) was moderate (entries 3-4) but it is worth noting that it could be improved by greatly increasing the enzyme loading. None of the two tested PLP-DCs were found active towards 3,5-dihydroxy-L-lysine (9) (entries 5-6).
The enzymatic cascade reactions exhibiting quantitative conversion as determined by HPLC monitoring were successfully scaled up (Figure 4).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56926/56926fig4v2.jpg" /> 
Figure 4: Representative analytical data for the three-step enzymatic cascade. (A) HPLC monitoring of biocatalytic conversion of L-lysine into (2R,3R)-1,5-diaminopentan-2,3-diol (7). RT = retention time. (B) 1H NMR and (C) 13C NMR of amino alcohol (7) after purification. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56926/56926fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The purification protocol presented herein allows efficient extraction of the amino alcohols from the complex enzymatic reaction mixture. The amino alcohols 4, 5, 6, and 7 were obtained in excellent yields (Figure 5).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56926/56926fig5.jpg" /> 
Figure 5: Enzymatic cascades for the synthesis of amino alcohols. Synthesis of (R)-1,5- diaminopentan-2-ol (4), (S)-1,5-diaminopentan-2-ol (5), 1,5-diaminopentan-3-ol (6), and (2R,3R)-1,5-diaminopentane-2,3-diol (7). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56926/56926fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine at JoVE.com

Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine

Chiral amino alcohols are versatile molecules for use as scaffolds in organic synthesis. Starting from L-lysine, we synthesize amino alcohols by an enzymatic cascade reaction combining diastereoselective C-H oxidation catalyzed by dioxygenase followed by cleavage of the carboxylic acid moiety of the corresponding hydroxyl amino acid by a decarboxylase.

Amino alcohols are versatile compounds with a wide range of applications. For instance, they have been used as chiral scaffolds in organic synthesis. ...

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12.0K Views.  CEA, CNRS, Univ Evry, Univ Paris-Saclay. Chiral amino alcohols are versatile molecules for use as scaffolds in organic synthesis. Starting from L-lysine, we synthesize amino alcohols by an enzymatic cascade reaction combining diastereoselective C-H oxidation catalyzed by dioxygenase followed by cleavage of the carboxylic acid moiety of the corresponding hydroxyl amino acid by a decarboxylase.

Video: Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine

Microwave-assisted Intramolecular Dehydrogenative Diels-Alder Reactions for the Synthesis of Functionalized Naphthalenes/Solvatochromic Dyes

Functionalized naphthalenes have applications in a variety of research fields ranging from the synthesis of natural or biologically active molecules to the preparation of new organic dyes. Although numerous strategies have been reported to access naphthalene scaffolds, many procedures still present limitations in terms of incorporating functionality, which in turn narrows the range of available substrates. The development of versatile methods for direct access to substituted naphthalenes is therefore highly desirable.
The Diels-Alder (DA) cycloaddition reaction is a powerful and attractive method for the formation of saturated and unsaturated ring systems from readily available starting materials. A new microwave-assisted intramolecular dehydrogenative DA reaction of styrenyl derivatives described herein generates a variety of functionalized cyclopenta[b]naphthalenes that could not be prepared using existing synthetic methods. When compared to conventional heating, microwave irradiation accelerates reaction rates, enhances yields, and limits the formation of undesired byproducts.
The utility of this protocol is further demonstrated by the conversion of a DA cycloadduct into a novel solvatochromic fluorescent dye via a Buchwald-Hartwig palladium-catalyzed cross-coupling reaction. Fluorescence spectroscopy, as an informative and sensitive analytical technique, plays a key role in research fields including environmental science, medicine, pharmacology, and cellular biology. Access to a variety of new organic fluorophores provided by the microwave-assisted dehydrogenative DA reaction allows for further advancement in these fields.

Microwave-assisted intramolecular dehydrogenative Diels-Alder (DA) reactions provide concise access to functionalized cyclopenta[b]naphthalene building blocks. The utility of this methodology is demonstrated by one-step conversion of the dehydrogenative DA cycloadducts into novel solvatochromic fluorescent dyes via Buchwald-Hartwig palladium-catalyzed cross-coupling reactions.

Functionalized naphthalenes have applications in a variety of research fields ranging from the synthesis of natural or biologically active molecules to ...

Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions

Isothermal titration calorimetry (ITC) is a well-described technique that measures the heat released or absorbed during a chemical reaction, using it as an intrinsic probe to characterize virtually every chemical process. Nowadays, this technique is extensively applied to determine thermodynamic parameters of biomolecular binding equilibria. In addition, ITC has been demonstrated to be able of directly measuring kinetics and thermodynamic parameters (kcat, KM, &#916;H) of enzymatic reactions, even though this application is still underexploited. As heat changes spontaneously occur during enzymatic catalysis, ITC does not require any modification or labeling of the system under analysis and can be performed in solution. Moreover, the method needs little amount of material. These properties make ITC an invaluable, powerful and unique tool to study enzyme kinetics in several applications, such as, for example, drug discovery.
In this work an experimental ITC-based method to quantify kinetics and thermodynamics of enzymatic reactions is thoroughly described. This method is applied to determine kcat and KM of the enzymatic hydrolysis of urea by Canavalia ensiformis (jack bean) urease. Calculation of intrinsic molar enthalpy (&#916;Hint) of the reaction is performed. The values thus obtained are consistent with previous data reported in literature, demonstrating the reliability of the methodology.

Isothermal titration calorimetry measures heat flow released or absorbed in chemical reactions. This method can be used to quantify enzyme-catalysis. In this paper, the protocol for instrumental setup, experiment running, and data analysis is generally described, and applied to the characterization of enzymatic urea hydrolysis by jack bean urease.

Isothermal titration calorimetry (ITC) is a well-described technique that measures the heat released or absorbed during a chemical reaction, using it as ...

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of trifluoromethoxylated aromatic compounds remains a formidable challenge in organic synthesis. Conventional approaches often suffer from poor substrate scope, or require use of highly toxic, difficult-to-handle, and/or thermally labile reagents. Herein, we report a user-friendly protocol for the synthesis of methyl 4-acetamido-3-(trifluoromethoxy)benzoate using 1-trifluoromethyl-1,2-benziodoxol-3(1H)-one (Togni reagent II). Treating methyl 4-(N-hydroxyacetamido)benzoate (1a) with Togni reagent II in the presence of a catalytic amount of cesium carbonate (Cs2CO3) in chloroform at RT afforded methyl 4-(N-(trifluoromethoxy)acetamido)benzoate (2a). This intermediate was then converted to the final product methyl 4-acetamido-3-(trifluoromethoxy)benzoate (3a) in nitromethane at 120 &#176;C. This procedure is general and can be applied to the synthesis of a broad spectrum of ortho-trifluoromethoxylated aniline derivatives, which could serve as useful synthetic building blocks for the discovery and development of new pharmaceuticals, agrochemicals, and functional materials.

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

An operationally simple procedure for the synthesis of ortho-trifluoromethoxylated aniline derivatives via a two-step sequence of O-trifluoromethylation of N-aryl-N-hydroxyacetamide followed by thermally induced intramolecular OCF3-migration is reported.

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins with the synthesis of a hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) homopolymer using reversible addition-fragmentation chain-transfer (RAFT) polymerization. Under mild visible light irradiation (&#955; = 460 nm, 0.7 mW/cm2), this macro-chain transfer agent (macro-CTA) in the presence of a ruthenium based photoredox catalyst, Ru(bpy)3Cl2 can be chain extended with a second monomer to form a well-defined block copolymer in a process known as Photoinduced Electron Transfer RAFT (PET-RAFT). When PET-RAFT is used to chain extend POEGMA with benzyl methacrylate (BzMA) in ethanol (EtOH), polymeric nanoparticles with different morphologies are formed in situ according to a polymerization-induced self-assembly (PISA) mechanism. Self-assembly into nanoparticles presenting POEGMA chains at the corona and poly(benzyl methacrylate) (PBzMA) chains in the core occurs in situ due to the growing insolubility of the PBzMA block in ethanol. Interestingly, the formation of highly pure worm-like micelles can be readily monitored by observing the onset of a highly viscous gel in situ due to nanoparticle entanglements occurring during the polymerization. This process thereby allows for a more reproducible synthesis of worm-like micelles simply by monitoring the solution viscosity during the course of the polymerization. In addition, the light stimulus can be intermittently applied in an ON/OFF manner demonstrating temporal control over the nanoparticle morphology.

This article describes a process for producing polymeric self-assembled nanoparticles using visible light mediated dispersion polymerization. Using low energy visible light to control the polymerization allows for the reproducible formation of self-assembled worm-like micelles at high solids content.

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins ...

Palladium N-Heterocyclic Carbene Complexes: Synthesis from Benzimidazolium Salts and Catalytic Activity in Carbon-carbon Bond-forming Reactions

Detailed and generalized protocols are presented for the synthesis and subsequent purification of four palladium N-heterocyclic carbene complexes from benzimidazolium salts. Detailed and generalized protocols are also presented for testing the catalytic activity of such complexes in arylation and Suzuki-Miyaura cross-coupling reactions. Representative results are shown for the catalytic activity of the four complexes in arylation and Suzuki-Miyaura type reactions. For each of the reactions investigated, at least one of the four complexes successfully catalyzed the reaction, qualifying them as promising candidates for catalysis of many carbon-carbon bond-forming reactions. The protocols presented are general enough to be adapted for the synthesis, purification and catalytic activity testing of new palladium N-heterocyclic carbene complexes.

Palladium N-Heterocyclic Carbene Complexes: Synthesis from Benzimidazolium Salts and Catalytic Activity in Carbon-carbon Bond-forming Reactions

Detailed and generalized protocols are presented for the synthesis and subsequent purification of four palladium N-heterocyclic carbene complexes from benzimidazolium salts. The complexes were tested for catalytic activity in arylation and Suzuki-Miyaura reactions. For each reaction investigated, at least one of the four complexes successfully catalyzed the reaction.

Detailed and generalized protocols are presented for the synthesis and subsequent purification of four palladium N-heterocyclic carbene complexes from ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise &#945;-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial &#946;-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man5GlcNAc2 (Man5) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

Enzymatic Synthesis of Epoxidized Metabolites of Docosahexaenoic, Eicosapentaenoic, and Arachidonic Acids

The epoxidized metabolites of various polyunsaturated fatty acids (PUFAs), termed epoxy fatty acids, have a wide range of roles in human physiology. These metabolites are produced endogenously by the cytochrome P450 class of enzymes. Because of their diverse and potent biological effects, there is considerable interest in studying these metabolites. Determining the unique roles of these metabolites in the body is a difficult task, as the epoxy fatty acids must first be obtained in significant amounts and with high purity. Obtaining compounds from natural sources is often labor intensive, and soluble epoxide hydrolases (sEH) rapidly hydrolyze the metabolites. On the other hand, obtaining these metabolites via chemical reactions is very inefficient, due to the difficulty of obtaining pure regioisomers and enantiomers, low yields, and extensive (and expensive) purification. Here, we present an efficient enzymatic synthesis of 19(S),20(R)- and 16(S),17(R)-epoxydocosapentaenoic acids (EDPs) from DHA via epoxidation with BM3, a bacterial CYP450 enzyme isolated originally from Bacillus megaterium (that is readily expressed in Escherichia coli). Characterization and determination of purity is performed with nuclear magnetic resonance spectroscopy (NMR), high-performance liquid chromatography (HPLC), and mass spectrometry (MS). This procedure illustrates the benefits of enzymatic synthesis of PUFA epoxy metabolites, and is applicable to the epoxidation of other fatty acids, including arachidonic acid (AA) and eicosapentaenoic acid (EPA) to produce the analogous epoxyeicosatrienoic acids (EETs) and epoxyeicosatetraenoic acids (EEQs), respectively.

We present a method useful for large-scale enzymatic synthesis and purification of specific enantiomers and regioisomers of epoxides of arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) with the use of a bacterial cytochrome P450 enzyme (BM3).

The epoxidized metabolites of various polyunsaturated fatty acids (PUFAs), termed epoxy fatty acids, have a wide range of roles in human physiology. These ...