cases 2

Dehydrogenase

An enzyme that catalyses reversible oxidation or reduction by removing or adding a hydride (H⁻) and a proton (H⁺)

Chemoselectivity in dehydrogenases

Different dehydrogenases act on specific substrate classes — alcohol dehydrogenases (ADHs), aldehyde dehydrogenases, and amine dehydrogenases each target distinct functional groups

Enantioselectivity in dehydrogenases

The enzyme positions the substrate so hydride is delivered to only one face of the carbonyl, controlling the stereochemistry of the alcohol product

NAD⁺/NADH/NADPH

Nicotinamide cofactors used by dehydrogenases to carry hydride, since amino acid side chains cannot transfer hydride directly — the reactive site is the nicotinamide heterocycle ring

Nicotinamide hydride transfer mechanism

The enzyme positions the nicotinamide ring under the substrate carbonyl → hydride transfers from substrate to cofactor forming an aromatic stabilised ring → an acidic residue donates a proton → product formed

Montelukast chemical synthesis (DIP chloride)

A chiral boron reagent used at −20°C in THF giving 94% ee (99% after crystallisation) — but toxic, moisture sensitive, produces waste, and requires extra purification

Montelukast enzymatic synthesis (KRED)

A ketoreductase converts the ketone to a chiral alcohol using NADPH at room temperature in aqueous solvent, with cofactor recycling via isopropanol → acetone, giving very high enantioselectivity with catalytic enzyme loading (3–5 wt%)

NADPH cofactor recycling

Isopropanol is oxidised to acetone to regenerate NADPH consumed during ketoreductase reactions — essential for industrial viability

PMI (Product Mass Intensity)

Total input mass divided by product mass — a measure of process greenness where a lower value indicates less waste

L-amino acid oxidase

A FAD-dependent enzyme from cobra venom that oxidises L-amino acids to imines, producing H₂O₂ as a toxic byproduct that damages cells

FAD (flavin adenine dinucleotide)

Cofactor used by L-amino acid oxidase — accepts two hydrogens from the substrate and is reoxidised by molecular oxygen, generating H₂O₂

7-ACA (7-aminocephalosporanic acid)

The core scaffold for semisynthetic cephalosporin antibiotics, produced from cephalosporin C via either chemical or enzymatic routes

Cephalosporin C chemical route problems

Requires multiple protection steps, harsh reagents (PCl₅, dichloromethane, dimethylaniline), toxic solvents, and complex multi-step synthesis

Enzymatic route to 7-ACA

Three steps: D-amino acid oxidase converts amino group to keto acid → H₂O₂ causes spontaneous decarboxylation → glutaryl-7-ACA acylase (GAC) hydrolyses the amide to release 7-ACA

Glutaryl-7-ACA acylase (GAC) mechanism

Serine nucleophile attacks the amide → forms an enzyme–ester intermediate → water hydrolyses the intermediate → 7-ACA product released

D-amino acid oxidase

Enzyme used in the first step of the enzymatic 7-ACA route, oxidatively deaminating the cephalosporin C side chain to a keto acid

Enzyme immobilisation

Technique allowing industrial enzymes to be physically retained and reused across multiple reaction cycles, improving cost efficiency

Aldolase

An enzyme catalysing aldol or retro-aldol reactions, forming or breaking C–C bonds via enolate or enamine intermediates

Cofactor recycling — why it matters

Stoichiometric cofactor use would be prohibitively expensive industrially — recycling systems (e.g. isopropanol for NADPH) allow catalytic cofactor loading

Chemical vs enzymatic montelukast comparison

Enzymatic route uses catalytic reagent loading, room temperature, aqueous solvent, gives higher enantioselectivity, higher yield, and lower PMI versus the chemical route requiring stoichiometric reagent, −20°C, and THF

Chemical vs enzymatic 7-ACA comparison

Enzymatic route has slightly lower yield but higher mass efficiency, lower solvent waste, lower energy usage, safer reagents, and reusable immobilised enzymes