Name: enzymatic cascade reactions for the synthesis of chiral amino alcohols from l-lysine
Uploaded: 2018-02-16T12:30:00.000+00:00
Duration: 9 min 14 s
Description: Watch this Scientific Journal Video about Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine at JoVE.com

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/science-education/5680/nuclear-magnetic-resonance-nmr-spectroscopy">https://www.jove.com/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 re...

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/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/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/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>(5&lt;em&gt;S&lt;/em&gt;)-hydroxy-L-lysine</td><td>Sigma Aldrich</td><td>GPS NONH</td><td>Out sourcing</td></tr><tr><td>&amp;alpha;-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&amp;micro;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&amp;nbsp;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&amp;uuml;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 &amp;micro;L</td><td>Eppendorf</td><td>3121000031</td><td></td></tr><tr><td>Micropipette 100 &amp;micro;L</td><td>Eppendorf</td><td>3121000074</td><td></td></tr><tr><td>Micropipette 500 &amp;micro;L</td><td>Eppendorf</td><td>3121000112</td><td></td></tr><tr><td>Micropipette 1000 &amp;micro;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 th...

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

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical probe that targets the enzyme&#39;s active sites. A reporter tag introduced into the probe allows for the detection of the labeled enzyme by in-gel fluorescence scanning, protein blot, fluorescence microscopy, or liquid chromatography-mass spectrometry. Here, we describe the preparation and use of the compound ARN14686, a click chemistry activity-based probe (CC-ABP) that selectively recognizes the enzyme N-acylethanolamine acid amidase (NAAA). NAAA is a cysteine hydrolase that promotes inflammation by deactivating endogenous peroxisome proliferator-activated receptor (PPAR)-alpha agonists such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). NAAA is synthesized as an inactive full-length proenzyme, which is activated by autoproteolysis in the acidic pH of the lysosome. Localization studies have shown that NAAA is predominantly expressed in macrophages and other monocyte-derived cells, as well as in B-lymphocytes. We provide examples of how ARN14686 can be used to detect and quantify active NAAA ex vivo in rodent tissues by protein blot and fluorescence microscopy.

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Here, we describe the preparation and use of an activity-based probe (ARN14686, undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl]carbamate) that allows for the detection and quantification of the active form of the proinflammatory enzyme N-acylethanolamine acid amidase (NAAA), both in vitro and ex vivo.

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

Biochemistry

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this procedure amino functionalized 'lysine' and guanido functionalized 'arginine' peptoid monomers can be included within the same peptoid sequence. This procedure uses on-resin (N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) or Dde protection, orthogonal conditions to the Boc protection of lysine monomers. Subsequent deprotection allows an efficient on-resin guanidinylation reaction to form the arginine residues. The procedure is compatible with the commonly used submonomer method of peptoid synthesis, allowing simple peptoids to be made using common laboratory equipment and commercially available reagents. The representative synthesis, purification and characterization of two mixed peptoids is described. The evaluation of these compounds as potential anti-infectives in screening assays against Leishmania mexicana is also described. The protozoan parasite L. mexicana is a causative agent of cutaneous leishmaniasis, a neglected tropical disease that affects up to 12 million people worldwide.

A protocol to synthesize peptoids with mixed cationic functionality in the same sequence is presented (lysine- and arginine-type monomers). Subsequent testing of these compounds against Leishmania mexicana, the protozoan parasites that cause cutaneous leishmaniasis, is also described.

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this ...

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide sequences. Synthesis of some unnatural amino acids often includes the use of a precursor consisting of a Schiff-base stabilized by a nickel cation. Unnatural side-chains can be installed on an amino acid backbone found in this Schiff-base complex. The resulting unnatural amino acid can then be isolated from this complex using hydrolysis of the Schiff-base, typically by employing reflux in strongly acidic solution. These highly acidic conditions may remove acid-labile side-chain protecting groups necessary for the unnatural amino acids to be used in microwave-assisted solid-phase peptide synthesis. In this work, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff base complex. Hydrolysis conditions presented in this work are suitable for retention of acid-labile side-chain protecting groups and may be adaptable to a variety of unnatural amino acid substrates.

Here, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff-base complex. Hydrolysis conditions presented here are suitable for use when retention of acid-labile side-chain protecting groups is required. This technique may be adaptable to a variety of unnatural amino acid substrates.

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide ...

Highly Stereoselective Synthesis of 1,6-Ketoesters Mediated by Ionic Liquids: A Three-component Reaction Enabling Rapid Access to a New Class of Low Molecular Weight Gelators

In organic chemistry ionic liquids (ILs) have emerged as safe and recyclable reaction solvents. In the presence of a base ILs can be deprotonated to form catalytically active N-Heterocyclic Carbenes (NHCs). Here we have used ILs as precatalysts in the addition of &#945;,&#946;-unsaturated aldehydes to chalcones to form 1,6-ketoesters, incorporating an anti-diphenyl moiety in a highly stereoselective fashion. The reaction has a broad substrate scope and several functional groups and heteroaromatics can be integrated into the ketoester backbone in generally good yields with maintained stereoselectivity. The reaction protocol is robust and scalable. The starting materials are inexpensive and the products can be obtained after simple filtration, avoiding solvent-demanding chromatography. Furthermore, the IL can be recycled up to 5 times without any loss of reactivity. Moreover, the 1,6-ketoester end product is a potent gelator in several hydrocarbon based solvents. The method enables rapid access to and evaluation of a new class of low molecular weight gelators (LMWGs) from recyclable and inexpensive starting materials.

Ionic liquids (ILs) mediate fast, simple and cheap access to 1,6-ketoesters in high diastereoselectivities and good yields. The reaction protocol is robust and the 1,6-ketoesters can be obtained in gram scale after a simple filtration protocol. Moreover, the 1,6-ketoesters are potent gelators in hydrocarbon solvents.

In organic chemistry ionic liquids (ILs) have emerged as safe and recyclable reaction solvents. In the presence of a base ILs can be deprotonated to form ...

Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene hydrothiolation. In addition, this protocol describes the preparation of vinyl-sulfide-tethered peptides using in-solution intramolecular thiol-yne hydrothiolation between amino acids that possess alkene/alkyne side chains and cysteine residues at i, i+4 positions. Linear peptides were synthesized using a standard Fmoc-based solid-phase peptide synthesis (SPPS). Thiol-ene hydrothiolation is carried out using either an intramolecular thio-ene reaction or an intermolecular thio-ene reaction, depending on the peptide length. In this research, an intramolecular thio-ene reaction is carried out in the case of shorter peptides using on-resin deprotection of the trityl groups of cysteine residues following the complete synthesis of the linear peptide. The resin is then set to UV irradiation using photoinitiator 4-methoxyacetophenone (MAP) and 2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone (MMP). The intermolecular thiol-ene reaction is carried out by dissolving Fmoc-Cys-OH in an N,N-dimethylformamide (DMF) solvent. This is then reacted with the peptide using the alkene-bearing residue on resin. After that, the macrolactamization is carried out using benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 1-hydroxybenzotriazole (HoBt), and 4-Methylmorpholine (NMM) as activation reagents on the resin. Following the macrolactamization, the peptide synthesis is continued using standard SPPS. In the case of the thio-yne hydrothiolation, the linear peptide is cleaved from the resin, dried, and subsequently dissolved in degassed DMF. This is then irradiated using UV light with photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA). Following the reaction, DMF is evaporated and the crude residue is precipitated and purified using high-performance liquid chromatography (HPLC). These methods could function to simplify the generation of thioether-tethered cyclic peptides due to the use of the thio-ene/yne click chemistry that possesses superior functional group tolerance and good yield. The introduction of thioether bonds into peptides takes advantage of the nucleophilic nature of cysteine residues and is redox-inert relative to disulfide bonds.

We present a protocol for the construction of thioether/vinyl sulfide-tethered helical peptides using photo-induced thiol-ene/thiol-yne hydrothiolation.

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene ...

Efficient Construction of Drug-like Bispirocyclic Scaffolds Via Organocatalytic Cycloadditions of &#945;-Imino &#947;-Lactones and Alkylidene Pyrazolones

Bispirocyclic scaffolds are one of the important structural subunits in many natural products that exhibit diverse and attractive biological activities. Recently, we have developed an efficient organocatalytic strategy, which provides facile access to a variety of enantiomerically enriched bispiro[&#947;-butyrolactone-pyrrolidin-4,4&#39;-pyrazolone] skeletons. In this paper, we demonstrate a detailed protocol for the asymmetric synthesis of drug-like bispirocyclic compounds with two spirocyclic carbon centers via an organocatyltic 1,3-dipolar cycloaddition reaction. Spirocyclization synthons &#945;-imino &#947;-lactones and alkylidene pyrazolones are prepared first, which are then subjected to a cycloaddition reaction in the presence of a bifunctional squaramide organocatalyst to afford the desired bispirocycles in high yields and excellent stereoselectivities. Chiral high-performance liquid chromatography (HPLC) is carried out to determine the enantiomeric purity of the products, and the d.r. value is examined by proton nuclear magnetic resonance (1H NMR). The absolute configuration of the product is assigned according to an X-ray crystallographic analysis. This synthetic strategy allows scientists to prepare a diversity of bispirocyclic scaffolds in high yields and excellent diastereo- and enantioselectivities.

Enantiomerically enriched bispiro[&#947;-butyrolactone-pyrrolidin-4,4&#39;-pyrazolone] skeletons are asymmetrically synthesized through a simple organocatalytic 1,3-dipolar cycloaddition reaction.

Bispirocyclic scaffolds are one of the important structural subunits in many natural products that exhibit diverse and attractive biological activities. ...

Preparation of Enantiopure Non-Activated Aziridines and Synthesis of Biemamide B, D, and epiallo-Isomuscarine

Nitrogen-containing heterocycle aziridines are synthetically very valuable for the preparation of azacyclic and acyclic molecules. However, it is very difficult and laborious to make aziridines in optically pure forms on a large scale to apply asymmetric synthesis of aza compounds. Fortunately, we successfully achieved both enantiomers (2R)- and (2S)-aziridine-2-carboxylates with the electron-donating &#945;-methylbenzyl group at the ring nitrogen as non-activated aziridines. These starting aziridines have two distinct functional groups-highly reactive three-membered ring and versatile carboxylate. They are applicable in ring-opening or ring-transformation with aziridine and in functional group transformation to others from carboxylate. Both of these enantiomers were utilized in the preparation of biologically important amino acyclic and/or aza-heterocyclic compounds in an asymmetric manner. Specifically, this report describes the first expedient asymmetric synthesis of both enantiomers of 5, 6-dihydrouracil-type marine natural products biemamide B and D as potential TGF-&#946; inhibitors. This synthesis consisted of regio- and the stereoselective ring-opening reaction of aziridine-2-carboxylate and subsequent formation of 4-aminoteterahydropyrimidine-2,4-dione. One more example in this protocol dealt a highly stereoselective Mukaiyama reaction of aziridine-2-carboxylate and silyl enol ether, following intramolecular aziridine ring-opening to provide easy and facile access to (-)-epiallo-isomuscarine.

Preparation of Enantiopure Non-Activated Aziridines and Synthesis of Biemamide B, D, and epiallo-Isomuscarine

In this study, we prepare both enantiomers of aziridine-2-carboxylate, which are used in the asymmetric synthesis of alkaloids, including biemamide B and D, and (-)-epiallo-isomuscarine.

Nitrogen-containing heterocycle aziridines are synthetically very valuable for the preparation of azacyclic and acyclic molecules. However, it is very ...

Organocatalysis

Source: Vy M. Dong and Faben Cruz, Department of Chemistry, University of California, Irvine, CA
This experiment will demonstrate the concept of organocatalysis by illustrating the proper setup of a reaction that utilizes enamine catalysis. Organocatalysis is a form of catalysis that uses substoichiometric amounts of small organic molecules to accelerate reactions. This type of catalysis is complementary to other forms of catalysis such as transition metal or biocatalysis. Transition metal catalysis involves transition metals as catalysts and biocatalysis uses enzymes as catalysts. Some advantages of organocatalysis include the low toxicity and cost of the organocatalysts in comparison to many metal catalysts. In addition, most organocatalysts are not sensitive to air and moisture, unlike metal catalysts. In contrast to enzymes found in living organisms, the small molecules that act as organocatalysts are typically easy to access. Furthermore, organocatalysis offers complementary and new reactivity not observed with other forms of catalysis.

Organocatalysis: Principles and Applications

Source: Vy M. Dong and Faben Cruz, Department of Chemistry, University of California, Irvine, CA
This experiment will demonstrate the concept of ...

JoVE Science Education

Organic Chemistry II

Acid Halides to Esters: Alcoholysis

Alcoholysis is a nucleophilic acyl substitution reaction in which an alcohol functions as a nucleophile. Acid halides react with alcohol to produce esters. The mechanism proceeds in three steps:
<ol>
	<li>First, the alcohol acts as a nucleophile and attacks the acyl carbon to form a tetrahedral intermediate.</li>
	<li>Next, the carbonyl re-forms with the loss of a chloride ion.</li>
	<li>Lastly, the positively charged intermediate loses a proton to give an ester as the final product along with H3O+, making HCl an overall byproduct of the reaction.</li>
</ol>
<img alt="Organic reaction mechanism diagram, nucleophilic substitution process displaying molecular transition." src="/files/ftp_upload/12362/12362_PT_Figure_1.svg" style="text-align: center; height: 200px;" data-altupdatedat="1747908711000" data-alt="Figure1">
Pyridine is used as a base to neutralize the acidic reaction mixture.
Alcoholysis of sulfonyl chlorides follows a similar pattern and forms sulfonic acid esters.
<img alt="Chemical reaction diagram; synthesis process; reaction of acyl chloride with alcohol; ester formation." src="/files/ftp_upload/12362/12362_PT_Figure_2.svg" style="text-align: center; height: 75px;" data-altupdatedat="1747908711000" data-alt="Figure2">

Alcoholysis is a nucleophilic acyl substitution reaction in which an alcohol functions as a nucleophile. Acid halides react with alcohol to produce ...

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

Summary

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Chapters in this video

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

Highly Stereoselective Synthesis of 1,6-Ketoesters Mediated by Ionic Liquids: A Three-component Reaction Enabling Rapid Access to a New Class of Low Molecular Weight Gelators

Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation

Efficient Construction of Drug-like Bispirocyclic Scaffolds Via Organocatalytic Cycloadditions of α-Imino γ-Lactones and Alkylidene Pyrazolones

Preparation of Enantiopure Non-Activated Aziridines and Synthesis of Biemamide B, D, and epiallo-Isomuscarine

Organocatalysis

Acid Halides to Esters: Alcoholysis