Biotransformations

Question 1

Types of hydrolytic enzymes

Answer 1

Lipases
Esterases
Proteases

Question 2

PPL

Answer 2

Porcine pancreatic lipase

Question 3

PCL

Answer 3

Pseudomonas cepacia lipase

Question 4

PSL

Answer 4

Pseudomonas species lipase

Question 5

CAL-B

Answer 5

Candida Antarctica lipase B

Question 6

ACE

Answer 6

Acetylcholine esterase

Question 7

PLE

Answer 7

Pig liver esterase

Question 8

CE

Answer 8

Cholesterol esterase

Question 9

Examples of proteases

Answer 9

Chymotrypsin
Thermolysin
Carboxypeptidase A

Question 10

Alpha-chymotrypsin

Answer 10

Selectively hydrolyses amide bonds where the R group on the carboxylic acid portion of the molecule is aromatic

Question 11

Trypsin R group

Answer 11

Polar

Question 12

Elastase

Answer 12

Small, uncharged group

Question 13

Alpha-chymotrypsin mechanism

Answer 13

Serine protease

Catalytic triad = Asp(102), His(57), Ser(195)

Question 14

Xenobiotic substrate

Answer 14

Not encountered in a biological environment

Question 15

Disadvantages of kinetic resolution

Answer 15

Max yield of product is 50 % (at 100 % ee)

But often the isolate yield and enantioselectivity are lower than this

Question 16

Desymmetrisation reaction

Answer 16

100 % yield

Achiral –> single enantiomer

Question 17

tBu group

Answer 17

Often inert to enzymes
Very bulky
Very few examples of natural products containing a tBu group
Can exploit this inertness in kinetic resolutions, for example tBu esters won’t be affected by enzymes

Question 18

Why is an aqueous buffer solution required for large scale ester hydrolyses?

Answer 18

Production of a carboxylic acid will lower the pH of the solution
Enzymes are pH-sensitive so a low pH may compromise their activity

Question 19

Irreversible transesterification

Answer 19

Use a vinyl alcohol (its ester is often referred to as the ‘acyl donor’)
Can undergo keto-enol tautomerisation
Can drive reaction to completion

Question 20

Mitsunobu inversion

Answer 20

Can use to obtain the opposite enantiomer of the alcohol

Question 21

Dynamic kinetic resolution

Answer 21

Can take place when the 2 enantiomers of the starting material are in dynamic equilibrium
Enzyme will only react with one enantiomer
In this way, both enantiomers of the starting material are converted into one enantiomer of the product

Question 22

Criteria for a dynamic kinetic resolution

Answer 22

1. Racemisation of starting material
2. Stereoselective catalyst that only reacts with one enantiomer
3. Reaction conditions do not induce racemisation of the product or interfere with/prevent enzyme function

Question 23

Why are cyanohydrins good substrates for DKRs with enzymes?

Answer 23

They can be racemised in the presence of base

Draw

Question 24

Rationale behind using a basic resin

Answer 24

Enzyme incompatible with bases in solution

Use of a basic resin minimises interaction with the enzyme

Question 25

Oxazolinones

Answer 25

Draw

Question 26

Why are oxazolinones easy to racemise, even at neutral pH?

Answer 26

The anion formed upon deprotonation is a 6pi aromatic system

Question 27

Why are "whole cell" organic reactions difficult to set up?

Answer 27

Yeast need to grow in water but the SM or product may have low water solubility Yeast often only tolerate low concentrations of SM/product, so need to use several litres of water per gram of substrate Often difficult to isolate the product at the end of the reaction - have to extract from all the water and get rid of lots of impurities

Question 28

Fumarase

Answer 28

Selectively adds water to fumaric acid, leading to the formation of malic acid as a single enantiomer

Question 29

Hydration of nitriles

Answer 29

Nitrile group can be hydrolysed to a primary amide using various bacteria e.g. rhodococcus butanica

Question 30

Epoxide hydrolase

Answer 30

Hydrolysis of epoxides

Question 31

Alcohol dehydrogenase

Answer 31

Can catalyse the conversion of ketones to alcohols | Uses NADH as a cofactor

Question 32

Balance between NADH/NAD+

Answer 32

``` NADH = neutral, non-aromatic NAD+ = charged, aromatic ```

Question 33

How to regenerate NADH (use it catalytically)

Answer 33

Use a second enzyme (formate dehydrogenase) and formate as a cheap reductant

Question 34

Leucine dehydrogenase

Answer 34

Catalyses the conversion of a ketone into an amine in the presence of ammonium salts Occurs via the imine, then enzyme-catalysed reduction of the imine to the amine

Question 35

Oxidation reactions that can be achieved by biotransformations

Answer 35

1. Biotransformation equivalent of the Baeyer-Villager reaction (achiral ketone to chiral lactone using mono-oxygenase) 2. Enantioselective oxidation of sulphides into sulphoxides using Aspergillus niger (S = stereocentre)

Question 36

Mitsunobu inversion reagents

Answer 36

PPh3 | DEAD (diethyl azodicarboxylate)