David Miller Flashcards
What is a biocatalyst?
- Anything of biological origin that catalyzes a process.
- Whole cell (brewing beer)
- Cell lysate or multicomponent assembly
- Purified protein – enzyme
- Rarely – nucleic acid – e.g. Ribozymes
Discuss the advantages/disadvantages of biocatalysts vs. conventional catalysts
Advantages
Disadvantages
Exquisite Reaction selectivity – often no need for protection chemistry
Efficiency – typical conventional catalyst 0.1 mol%, enzyme 10-4-10-3 mol%
Environmentally benign – work at room temp, pH 7 in water. Also use sustainable feedstocks
Low toxicity – little or no use of heavy metals in biocatalysis
Pathway catalysis – multistep chemistry possible with one catalyst
Stability – narrow range of operating conditions
Complexity and scale of preparation - proteins can be very expensive to produce
Specificity/selectivity – advantage – also a disadvantage – enzymes can have a very narrow substrate specificity
Co-substrate limitations – cofactor requirements can lead to complications
Aqueous environment - water has a high boiling point – organics tend not to be soluble
Inhibition – inhibition is nearly always an issue when biocatalysts are used
Allergenic
What is the typical reaction catalysed by subtilisin (serine protease)?
- Cuts at the aromatic amino acid!
- Can harness this to perform kinetic resolution reactions, desymmetrisations and regioselective reactions.
- Uses the inherent enantioselectivity of subtilisin and other proteases, esterases and lipases.
- General family of enzymes termed hydrolases.
Draw the mechanism of Serine Protease
- Catalytic serine (Ser) attacks backbone carbonyl of peptide
- Substrate creating a tetrahedral transition state stabilised by the oxyanion hole residues Asn and Ser.
- Collapse of the transition state causes the amino half of the substrate to leave the active site, the carboxyl half remains attached as an acyl enzyme intermediate.
- Finally, an acquired H2O attacks the acyl enzyme intermediate.
- This creates a second – oxyanion hole stabilised transition state which then collapses releasing the final product.
Discuss the some of the commonly employed enzymes in organic synthesis
Do they have the same mechansims?
All follow the same mechanism
All of these enzymes use an active site serine (or cysteine) and form an acyl-enzyme intermediate. So the same principles of enzyme, substrate and medium manipulation apply.
- Subtilisin, a-chymotrypsin, trypsin… serine proteases
- Papain – cysteine protease
- Porcine Pancreatic Lipase (PPL) – Lipase
- Pigs Liver Esterase (PLE) – Esterase
- PPL and PLE are generally applicable chiral esterase enzymes with broad substrate specificity.
- and many, many more.
What are sime examples of processes carried out by hydrolases?
Ester hydrolysis – Enzyme will carry out the hydrolysis of ester bonds in a regioselective
and enantioselective manner under mild reaction conditions.
Such hydrolyses can equally be carried out by K2CO3 in MeOH (or similar) - much cheaper
reagents – so the focus using enzymes has to be on their selectivity and the mild reaction conditions otherwise their use is not justified.
- Typical substrates* for enzyme catalysed ester hydrolysis.
- * = (pro)chiral center – if reacted, a chiral center is generated*
- We need a proton on the chiral centre
- Don’t remember these reactions, we will be given unknown examples in the exam
What is kinetic resolution?
- Resolution of racemates and desymmetrisation of prochiral or meso compounds are all examples of kinetic resolutions.
- Enantioselectivity is dependent upon facial selectivity of enzyme for the transition state.
- Maximum yield/conversion in this case of 50%.
- Alternative reaction is just slower – so must fine tune reaction conditions to maximize e.e.
- Desymmetrisations do not suffer from 50% yield limitation
- In ideal cases enzymes are so good at recognizing one enantiomer that the other does not get converted at all.
- In practice, unfortunately, the rate of conversion of the ‘wrong’ enantiomer is not zero but finite. This has some consequences:
- The rate of turnover of each enantiomer varies with degree of conversion – ratio of two substrates is not constant with time.
- Optical purity of both substrates and products becomes a function of the extent of conversion.
What is dynamic kinetic resolution? (DKR)
- This is a process that is useful for obtaining 100% conversion in resolution of racemates.
- Requires an extra step that re-racemises the substrate as the reaction progresses
Give some examples of DKR:
*OFTEN IN EXAMS - LECTURE . 1 1hr32
- Racemisation of amines: Amines can be oxidized on palladium surface. H2 is lost to surface generating a planar imine. Reverse reaction delivers H2 to either face of imine and so racemises the amine.
R
Acyl donor
Lipase
E
Et
n-C3H7
n-C5H11
cyclo-C6H11
Ph
Ph
MeOCH2
Ethyl methoxyacetate
Ethyl methoxyacetate
Ethyl acetate
Ethyl methoxyacetate
Ethyl methoxyacetate
Ethyl acetate
Ethyl methoxyacetate
PSL
PSL
CAL
PSL
PSL
CAL
PSL
~8
>100
>100
>100
>100
>100
>100
PSL = Pseudomonas sp. Lipase, CAL = Candida arctica lipase
This is often in exams; 1:32:00 of Lecture 1.
R
Lipase
Configuration
e.e. %
Ph
CH3SCH2CH2
(CH3)2CHCH2
3-indolyl-CH2
PhCH2
Ph
CH3SCH2CH2
PhCH2
PPL
PPL
PPL
PPL
PPL
- Aspergillus sp.*
- Aspergillus sp.*
- Aspergillus sp.*
L
L
L
L
L
D
D
D
76
80
87
98
>99
80
83
>99
Show some examples of how enzymes can achieve different regio- and enantio-selectivities
Show how desymmetrisation of prochiral or meso compounds can occur using an enzyme. What enzyme is this?
Esterases can cause desymmetrisation
- Note the change in selectivity as the 6-ring is used as substrate
Show how esterases can cause a
- Change of selectivity through substrate alteration:
Show how esterases can lead to a desymmetrisation through:
- Change of selectivity through solvent engineering
In some cases reversal of enantioselectivity can be achieved through change in solvent.
We need to understand and interpret these tables, not reproduce them.
Alternative reactions catalysed by hydrolase enzymes
- So far we’ve mostly only met the use of hydrolases in their ‘natural function’ i.e. hydrolysis.
- But they can be used in organic solvents to do synthesis.
Draw the meachnsim for Serine Hydrolase: (again)
Water is a good nucleophile for attacking the acyl enzyme intermediate, but can we use alternative nucleophiles or even get the reaction to work in reverse…? Use of alcohols or amines and other nucleophiles would all expand the reactivity of this protease if we could get them to work.
Discuss the alternate reactivity of proteases compared to hyrolases:
The enzyme solution has [H2O] ~ 56 mol dm-3. Any alternative nucleophile must be able to compete with this. No chance. So, the alternative is to use enzymes in solvents other than water – Organic solvents.
Can we use enzymes in organic solvents?
- Water is an essential component of enzyme structure and often reactivity – nearly all enzymes need some water to work.
- Experiments have shown that often enzymes can be made to work in extremely hydrophobic environments.
- Remember that the inside of a cell can be very hydrophobic, and many enzymes work whilst inside or bound to hydrophobic membranes.
- Water, although stabilizing to enzymes in many ways also participates in most of the reactions leading to their decomposition.
- Organic solvents have some big advantages over water.
- Water is a poor solvent for organic molecules.
- Water is very reactive – hydrolysis of products may occur – it is a mild acid/base, also a good nucleophile. Other side reactions such as racemization or polymerization can also occur.
- Very difficult to remove due to high heat of vaporization and boiling point.
How much water is needed for enzyme activity to be retained?
We would not be able to know examples. Would have to conduct an experiment to find out.
- Some. Completely anhydrous solvents do not work.
- Answer depends upon the enzyme in question.
- a-chymotrypsin has been shown to work with as little as 50 water molecules per enzyme molecule – less than a monolayer.
- Subtilisin is similar in its water needs as are various lipases and esterases.
- Other enzymes are much greedier – polyphenol oxidase – a commonly used oxidase enzyme in synthesis requires 3.5 x 107 molecules of water per molecule of enzymes!
- Note that water crucially bound up with an enzyme for structural purposes differs from bulk solvent water in many ways and should be regarded as part of the enzyme in effect.
What are advantges of using enzymes in organic solvents?
- Overall yields better - no extractive step required. Enzymes insoluble and so precipitate –removed by simple filtration.
- Solubility of substrates improves reaction rates.
- Microbial contamination negligible.
- Water caused side reactions are reduced.
- Enzymes are generally more stable in organic media
- e.g. Porcine pancreatic lipase (PPL) is stable for 1 hr at 100 °C in organic solvent but is rapidly degraded in water at this temperature (denatures).
- Water facilitates greater enzyme movement due to interchangeable H-bonding (enzyme- enzyme v. enzyme-water) – so greater rigidity in organic solvents can allow us to modulate reactivity by solvent engineering.
- Greatest advantage – thermodynamic equilibria can be shifted.
- Hydrolase enyzmes (proteases, lipases, esterases) favour hydrolysis in water. In organic solvents the reverse is true – synthesis is favoured.
- Therefore proteases and lipase can be used to synthesise, esters, lactones, amides and peptides with the tight regio- and stereocontrol characteristic of enzymes.
- In the presence of water these reactions may not happen at all.
Discuss the effect of solvent choice (organic or aqueous) on the reaction with Sutilin:
Discuss the effect organic vs aqueous solvent for different reaction systems with different acyl enzyme intermediates:
- System containing lots of water –> one molecule lost from solvent - no real difference.
- System with little water –> equilibrium will shift to the left as addition of water to the solvent will be favoured i.e. Le Chatelier’s principle.
- (caution – released water can be a problem and so acyl transfer is often used as an alternative
- i.e. ester instead of carboxylic acid used as cosubstrate in reverse reaction)
So if we use an alternative nucleophile in place of water we can achieve a variety of reactions.
Show how alternate substrates of hydrolases can be used:
How can we chnage the gylcine? OH groups?
All are preparative reactions that have been achieved using hydrolases in organic solvents.
Alternative substrates of hydrolases. We can change the glycine of the crystal structure shown in Lecture one. Gly166 can we changed to a carboxylate to bind to a NH3-R, or alternatively can be modified to an amide to bind to carboxylates. These substrates show strong regioselectivity. We can use these substrates and selectively bind. We can modify these structures
- Modern advances have allowed very complex alcohols to be used as the nucleophile that attacks the acyl-enzyme intermediate of subtilisin. The arrow indicates the alcohol that is acylated.
- Engineering of enzymes can enable a greater range of substrates and modify specificity.
What is acyl transfer or trans-esterification? Why is it useful?
Discuss how the following solution (Use an excess of the acyl transfer reagent) helps the equilibrium problems for acyl transfer or trans-esterification?
- Use an excess of the acyl transfer reagent. May be expensive and there are better solutions
Discuss how the following solution (changing the nucleophile) helps the equilibrium problems for acyl transfer or trans-esterification?
Make R4OH a very poor nucleophile.
Racemic alcohol made and we do a trans esterification reaction. This is kinetic resolution. The long-chain alcohol is a poor nucleophile as it is the wrong stereochemistry and the trichloro ethanol is a poor alcohol. Electron withdrawing chlorides make trichloro ethanol a poor nucleophile Hence its ability to attack the acyl-enzyme intermediate is not good.
Discuss how the following solution (using enol ester or anhydride) helps the equilibrium problems for acyl transfer or trans-esterification?
Discuss equilibrium properties of amination of esters
- Once we have an amine, we don’t need to worry as much about the backwards reaction because they are less reactive
- Amination of esters does not suffer the same equilibrium problem as with transesterification (amides are more thermodynamically stable than esters)
- Again different enzymes can achieve different selectivities
- Ethanol does not compete with diamine. The diamine is much better nucleophile.
- Note the Chemoselectivity. In absence of enzyme SN2 substitution of chloride would compete.
- Amination of esters provides an alternative to transesterification.
- Below example shows enhanced enantioselectivity for NH3 over H2O as a nucleophile.
- (although this is not a general rule)
Discuss the reaction of hydrolysis of epoxides briefley:
Discuss properties of reductases:
Reduction reactions are catalyzed by dehydrogenase enzymes
- Often use NAD+; don’t need to know the whole of the structure, just the nicatinamide ring
- Reduction is the reverse reaction.
- NAD(P)H is a complex water soluble only cosubstrate and needs recycling.
- Because of this recycling problem whole cell systems such as Baker’s Yeast are often used
Discuss the process of recycling NADH:
Discuss the stereochemistry of dehydrogenase reduction:
*EXAM*
Above reaction is PRELOG’S RULE positive. We could be asked about this in the exam! S=small, L=large. Won’t ask for stereochemistry. Hydride is delivered from top face (from NADH/NADPH) if Prelog positive and if small on left, large on the right. Therefore the OH is down.
Dehydrogenase
Specificity
Cofactor
Yeast-ADH
Horse-liver-ADH
Thermoanaerobium brockii-ADH
Hydroxysteroid-DH
- Candida parapsilosis-*ADH
- Lactobacillus kefir*-ADH
- Mucor javanicus-*ADH
- Pseudomonas sp.*-ADH
Prelog
Prelog
Prelog*
Prelog
Prelog
Anti-Prelog
Anti Prelog
Anti-Prelog
NADH
NADH
NADPH
NADH
NADH
NADPH
NADPH
NADH
* - Anti-Prelog when small aldehydes and ketones are the substrates
What are preferred substrate sizes for dehydrogenases?
Give some examples reactions of dehydrogenases:
Make sure we draw with small group on the left and large group on the right. Not a resolution reaction as we do not start with a mixture.
Desymmetrisation of prochiral or meso diols by dehydrogenase oxidation is a reliable method of for the production of chiral lactones.
Discuss the use of oxygenase enzymes:
Here there is a real advantage over traditional solution phase synthetic organic chemistry. Complex enzyme chemistry with radicals. Direct insertion of molecular oxygen, selectively into organic substrates is very rare. Where such reactions do exist there is little ability to do regioselctive and stereoselective chemistry. So enzymes that do this offer a rare opportunity for the synthetic chemist. There is little alternative but to use biocatalysis for this type of chemistry.
- Monoxygenases
- Incorporate one oxygen atom from O2.
- Other oxygen is reduced to H2O by a cofactor such as NADH.
- Substrate + H2-Donor + O2 –> Substrate-O + Donor + H2O
-
2. Dioxygenases
- Incorporate both oxygen atoms of O2 into the substrate.
- This is done through formation of an intermediate peroxy- species.
- Substrate + O2 –> Substrate-O2
- O2 is a triplet state and so reaction with singlet state organics is spin forbidden. So a cofactor and metal ion is required, these can be various.
- NADH is only used for recycling purposes
- We can be examined on FAD!
- Don’t need to remember structure of haem, just need to know they contain iron
Give some uses of oxygenase enzymes:
What is dihydroxylation of aromatics?
- This reaction is of famous synthetic utility – it proceeds via a dioxygenase enzyme followed by a reductase.
- Has been employed on an industrial scale. 1 has been used as a chiral synthon for the preparation of many sugar based natural products, analogues and drugs.
Uses of oxygenase enzymes:
Discuss what Bayer-Villiger Oxidations are:
- Show mechanism
- Name required cofactor
- Show how R group affects product
Small R groups, S isomer. Enzyme active site controls which R group migrates.
R
Configuration
e.e. %
CH3O
S
75
Et
S
>98
n-Pr
S
>98
t-Bu
S
>98
n-Bu
R
52
Discuss use of Bayer Villager for monoamine oxidases. Show the reaction mechansim for this reaction:
- These are enzymes that transform amines into imines using FAD as cofactor
- However the wild-type (natural) enzymes have limited substrate tolerance…
- For example Monamine Oxidisase N from Aspergillus Niger (MAO-N) can only turn over a small range of substrates
- Mechanism of action – use an FAD prosthetic group to oxidise the amine substrate – O2 regenerates the prosthetic group
- Important for drug metabolism. Normally have poor substrate specificity. If we put an R group on the precursors, we generate a chiral center and we can therefore get enzyme generated specificity.
- But we want enzymes that can turn over complex amines – in enantioselective manner
- The enzyme doesn’t really need a recycling agent but in principle it is not a clean reaction.
- To do this we must engineer the enzymes to tolerate a much broader (bulkier) range of amines
- Approximately 40-45% of drug candidates contain a chiral amine somewhere in their structure
- Natural product alkaloids nearly always contain a nitrogen atom in a chiral environment.
- Can we engineer MAOs to use biocatalysis to prepare these important small molecules?
What is the selection/screening process required for directed evolution? (Monoamine oxidases)
Diversity in gene generated through incubation in E. coli XL-1 red strain
Positive hits identified with colorimetric assay
Only brown spots are peroxide producing colonies. We get the opposite amine (bottom one is S). We have learnt this mechanism. NaBH3CN reverses the previous reaction. Depending on which stereoselective MAO is used, the amine will accumulate.
Discuss deracemisation of chiral amines:
R-selective MAO will re-oxidise the racemic amine produced but preferentially the (R)-isomer – hence (S)-amine will accumulate. Vice versa for the (S)-selective MAO.
Discuss how wild-types of MAOs were adapted:
Wild-types were adapted and adapting MAO-N mutants were generated:
What are C-C bond forming enzymes?
- What reactions do they catalyse?
- What can enzymes solve?
Three main classes of C-C bond forming reaction used in biocatlysis
- Aldol reactions.
- Acyloin/Benzoin condensation reactions.
- Michael type additions.
- Will only mention aldol reactions here. Use of enzymes can solve:
- Regioselectivity
- Enantioselectivity
Discuss enzyme catalysed aldol reactions:
There are four types of aldolase enzyme that are classified according to the donor nucleophile. In order to trap within cells, they are phosphorylated to give a negative charge so they become trapped within the cell. It’s the donor nucleophile (enol form that reacts) gives the name. There is strict donor specificity.
On the last type 4 examples, two stereoisomers formed. Most natural aldolases are used for synthesis/breakdown of sugars. Hence this is also their synthetic utility for biocatalysis in the lab.
Discuss the significance of (+)-exo-brevicomin:
Outline the mevalonate pathway for the production of terpenoids:
(C5H8)n
Need to be able to discuss the below mechanism in the exam
- 2 CoA undergo Claisen condensation
- Undergoes hydrolysis of CoA (34min)
- Reduced by 2 equivalents of 2NADPH to give Mevalonate
- Hydrolyzed and phosphorylated multiple times
- Decarboxylation to give Dimethyallyl pyrophosphate
- Isomerase to geranyl pyrophosphate
- Adding IPP adds one terpene unit each time
Increasing numbers of sesquiterpene synthase crystal structures have been solved. Each enzyme has an alpha-helix barrel with a very hydrophobic active site. A Mg(II) ion is present at the top of this barrel. The intermediates contain carbocations of very similar energies, so it is interesting how the enzymes can exploit such differences in the structures of the products. They make vastly different compounds.
Draw the mechanims of formation of Aristolochene from FPP:
Carbocationic cyclisation mechanisms: Mechanism of Aristolochene Synthase from Penicillium roqueforti (PR-AS).
The first step always requires magnesium ions stabilizing the OPP group of FPP. We can use 13C to label FPP to study the reaction. When we are being challenged with these problems, look for the dimethyl and work back from there. We need to redraw FPP in a conformation that allows for the folding to gain our product. Don’t worry about stereochemistry for these reactions. Additionally we can label with deuterium to study the reaction.
- FPP folded, releasing PPi and carbocation (at pH 7 in aqueous conditions!)
- Proton release, ring closure and further protonation gives bicyclic compound
- Hydride and alkyl shift rearrangement to give final carbocation
- Proton loss to give final compound
- If we drew this mechanism again however with fluorine beta-to the OPP in step 1 (alpha to the carbon that is attacked), in the third step (-) gemacrene A, we would have a fluorine on the other side of the double bond that is attacking the other alkene. This is a problem because the next step forms a carbocation, but we know fluorine is electronegative and destabilizes this carbocation so this step would not be favorable and therefore we would end up with an accumulation of germacrene A as the next step is too high in energy.*
- Remember that a secondary carbocation with an alpha fluorine is unstable, however a tertiary carbocation where one R group is a fluorine is isoelectronic to a carbonyl with a positively charged hydrogen attached. This is because like oxygen, fluorine in this conformation can donate a lone pair to the carbocation stabilizing it.*
- Mechanism: Site directed mutagenesis, FPP (substrate) analogues
- Synthetic Biology: production of unnatural sesquiterpene analogues
Discuss general enzymes and their products:
Why do we modify FPP?
* insert mechanism*
- Mechanistic probes for sesquiterpene synthases
- Substrates for enzymes will generate novel (bioactive?) sesquiterpenoids
- Fluorine atoms are similar sized to protons so it doesn’t interfere with binding but has a powerful effect electronically.
How can wer use 12,13DiF-FPP – probe for mechanism of PR-AS
We only know whether this is true if we do an experiment and it was found that there was a competitive inhibitor. A series of lines at the y-axis shows competitive inhibition. We also see no products by GC-MS. It is therefore pathway B of the mechanism above and is Sn2 like.
10-membered rings exhibit flexibility so NMR was run at different temperatures. At low temperature the two peaks required were assigned. 1D and 2D NMR helped to assign the compound.
Discuss some F-FPP analogues and PR-AS
Show the mechanism of d-cadinene production from FPP:
- There is a z-double bond however previously there was only E. This means an intermediate has to go through an allylic step which is unfavourable.
Show the two differtent routes of 1,10 ring closures of FDP leading to delta-cadinene:
What is (S)-Germacrene D synthase? How is it made?
(S)-Germacrene D – aphid alarm pheromone produced by several organisms including Solidago canadensis
- Overview of project – transform various FPP analogues with germacrene D synthase (GDS) and in collaboration with Rothamsted research these will be tested for activity with the insects.
- (S)-Germacrene D synthase – production of germacrene D analogues*
- Yields up to 76% have been obtained – compounds substituted at C6 and C14 retain
some semiochemical activity. Olfactory sensors often have stringent SAR requirements.
Catalytic mechanism of Amorphadiene Synthase (ADS)
What is the Amorphadiene and artemisinin biosynthetic pathway
- Just be aware that alkyl to oxygen-containing need oxygenases so yeast, plants that support p450. Bacteria can’t do this.
Exam question:
Can use organisms to manufacture proteins and scout faesible chemistry.
- PCR makes copies of genes of interest. Know the stages
Need to identify the gene of interest first and know antibiotic resistance is required
- Gene insertion: assembly method required in a self-replicating system. Need to use selectable marker and polycloning site with lac operon. This could be induced with IPTG
- Recombiant DNA technology
(i) Site directed mutagenesis - one nuclotide at a time, slow and relies on assumptions
(ii) Directed evolution: diversification, mutate wild type with gene (error prone PCR)
- conduct selection/screening to rapidly assess samples
- take positive mutants further,
- gain enzymes of different substrate selectivity
XL1-red lacks several DNA repair pathways
Colourimetric pathway
Exam question:
Draw
Exam question:
