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

Summary

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.

Abstract

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 >95%).

Introduction

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 stereoselectivity^1,2,3. Moreover, as biocatalytic reactions are conducted in similar conditions, it is therefore feasible to perform cascade reactions in a one-pot fashion^4,<....

Protocol

1. Enzyme Preparation

1. Express and purify proteins as previously described²⁶.
   NOTE: Recombinant proteins were obtained with the following final concentrations: αKAO from Catenulispora acidiphila, UniProtKB ID: C7QJ42 (KDO1), 1.35 mg/mL; αKAO from C. pinensis, UniProtKB ID: C7PLM6 (KDO2), 2.29 mg/mL; PLP-DCs from S. rumirantium, UniProtKB ID: O50657 (LDC_Srum), cell free extract with total enzyme at 12.44 mg/mL; PLP-DC from C. pinensis, UniProtKB ID: C7PLM7 (DC_Cpin), 2.62 mg/mL.

2. Preparation of Solutions

Results

We have previously reported the synthesis of mono- and di-hydroxy-L-lysines by diastereoselective enzymatic hydroxylation catalyzed by dioxygenases of the iron(II)/αKAO family (Figure 1)¹⁶. To optimize the protocol of the entire cascades presented here, which combine one or two hydroxylation steps catalyzed by an αKAO followed by a decarboxylation step catalyzed by a PLP-DC, the reaction conditions were adjusted to satisfy the requirem.......

Discussion

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.......

Materials

List of materials used in this article

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