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Home assignment for lecture 1& 2
1. a) With a schematic diagram describe how you choose a suitable microorganism (Class-1 or
GRAS) to act as the biocatalyst for a particular desired biotransformation?
b) What are the advantages and disadvantages of using whole-cell systems as opposed to isolated
enzymes for a specific biotransformation process?
Home assignment for lecture 3 & 4 (enzyme assay)
1. You have been asked by your supervisor to screen a particular enzyme from a huge
collection of biomaterials (plant homogenate or microbial type culture collection). You
have been provided with following five substrates required for specific enzyme assay.
Mention logically which substrate will you apply to screen for which enzymes and why?
You also need to mention which analytical technique is used in each case.
OH
N
O
CN
O
MeO
O
O
O
O
O
O
O
O
O
O 2N
O
Hint: Go through the lecture notes it should be answerable.
2. You have been provided with the following three substrates, can you think of designing
two enzyme assay experiment by using those substrates. Which enzymes will you assay
and what analytical methods you will use to run the experiment successfully?
MeO
OH
MeO
Non Fluorescent
CN
CHO
OH
Fluorescent (424nm)
MeO
Non Fluorescent
Hint: Think about alcohol oxidase and hydroxynitrile lyase activity.
Home assignment for lecture 5 & 6 (enzyme purification)
1. Sephadex G-75 has an exclusion limit of 80KD. If you tried to use this column material
to separate alcohol dehydrogenase (150 KD) from β-amylase (200KD), what would
happen?
Hint: Both the proteins would elute in the void volume together and will not be
separated.
2. Referring to the question above, could you separate β-amylase from bovine serum
albumin (66KD) using this column?
Hint: Yes, b-amylase will be in void volume and elute first, BSA will elute slowly.
3. What could be an advantage of using an anion exchange column based on quaternary
amines (i.e, resin-N+Et3) as opposed to a tertiary amine (resin-NH+Et2)?
Hint: With a quaternary amine, the column resin always has a net positive charge,
and you need not have to worry about the pH of your buffer altering the form of the
column. With a tertiary amine there is dissociable hydrogen and the resin might be
positive or neutrally charged depending on buffer pH.
4. Gel-Filtration chromatography is a useful method for removing salts, such as ammonium
sulphate, from protein solutions. Describe how such separation is accomplished?
Hint: A protein solution from an ammonium sulphate preparation is passed over a
gel-filtration column where the proteins of interest will elute in the void volume. The
salt being very small will move through the column slowly. In this way, the proteins
will leave the salt behind and exit the column without it.
5. Why is the order of separation based on size opposite for gel-filtration and gelelectrophoresis, even though they often use the same compound to form the matrix?
Hint: Suppose a polyacrylamide gel is used for gel-filtration, the larger proteins can
travel around the beads thereby having a shorter path to travel and elute first. With
electrophoresis, the proteins are forced to go through the matrix, so the larger one
travel more slowly due to friction.
6. A Gel Chromatography column of Bio-Gel- P 30 with a bed volume of 100mL is poured.
The elution volume of the protein hexokinase (96KD) on this column is 34mL. That of an
unknown protein is 50mL. What are the void volume of the column, the volume occupied
by the gel and the relative elution volume of the unknown protein? What would be the
rough molecular weight of the unknown protein?
Hint: Do it yourself.
7. How can gel-filtration chromatography be used to arrive at an estimate of the molecular
weight of a protein?
8. Why do most people elute bound proteins from an ion-exchange column by raising the
salt concentration instead of changing the pH?
Hint: Raising the salt concentration is relatively safe. Most proteins will elute this
way, and will be active too. If necessary salts can be removed by dialysis. Changing
the pH is enough to remove the charge, can cause denaturation.
9. From the following protein purification table calculate the missing values
Step
Protein (mg)
Activity(U)
Specific
Yield (%)
activity (U/mg)
Purification
factor
1
1200
800
?
-----
-----
2
600
600
?
?
?
3
200
400
?
?
?
4
30
300
?
?
?
Home assignments for lecture 8 & 9 (enzyme nomenclature & immobilization)
1. Describe different enzyme immobilization techniques in detail. Point out advantages and
disadvantages of those different techniques in tabular form.
2. Two enzymes have the following EC name EC 3.1.1.16 and EC 1.1.1.12. What type of
enzymes we are talking about?
Home assignment for lecture 10-13 (catalytic role of enzymes)
1. Other things being equal, what is a potential disadvantage of an enzyme having a
very high affinity for its substrate?
Hint: The ES complex will be in an “energy trough”, with consequentially
large activation energy to the transition state.
2. Amino acids that are far apart in the amino acid sequence of an enzyme can be
essential for its catalytic activity, what does this suggest about its active site.
Hint: Amino acids that are far apart in the amino acid sequence can be close
to each other in 3D due to protein folding and form the active site.
3. If only a few amino acid residues of an enzyme are involved in its catalytic
activity, why does the enzyme need such a large number of amino acids?
Hint: The overall protein structure is needed to ensure the correct
arrangement of amino acids in the active site.
4. Explain why mutating all three residues of trypsin’s catalytic triad has essentially
no greater effect on the enzymes catalytic rate enhancement than mutating only
Ser 195.
Hint: Trypsin is a serine protease.
5. The following three substrates are designed and tested as α-chymotrypsin mimic, predict
their relative rate of hydrolysis at pH = 4.0, at room temperature.
N
O
O
O
N
NH
O
O
N
NH
N
H
O
CO2H
Hint: see the lecture note
6. α-chloromethyl ketones are acting as chymotrypsin inactivator. When (2S)-Nacetyl-L-alanyl-L-phenylalanyl-α-chloroethane is treated with γ-chymotrypsin, its
activity was inhibited by the action of the chloroketone compound. The crystal
structure of the covalent adduct with the enzyme shows that the stereochemistry
of the inactivator is retained. Based on this information elucidate a detail
mechanism of the enzyme inactivation?
O
H
N
AcNH
O
Me
H
Cl
Ph
(2S)-N-acetyl-L-alanyl-L-phenylalanyl--chloroethane
Hint: see the lecture note
7. Hydroxynitrile lyase (HNL) is a plant enzyme which catalyzes the following
reaction. HNL is a flavoprotein having characteristic yellow color. When a HNL
solution (0.5mg/ml protein content) is treated with ; a) 10mM solution of sodium
dithionite the yellow color is gone but HNL exhibits its catalytic activity; b)
10mM solution of 2-iodoaceticacid is treated with HNL solution, it was observed
that catalytic activity of HNL is completely lost. Account for the two above
observations with proper reasoning?
OH
O
CN
HNL
H
+
HCN
Hint: a) Flavoproteins are yellow in color due to presence of FAD, treatment
of sodium dithionite reduces flavin. b) 2-iodo acetic acid is known to react
with cys- residue in protein (think about HNL active site).
8. Explain the following (concisely).
OH
O
HO
HO
HO
O
HO
OH
HO
HO
HO
O
O
OH
O
1
104
O
Relative rate of hydrolysis at pH = 6.0
Hint: The above two substrates are glycosidase mimic. Check the glycosidase
mechanism. It is a nice example of proximity effect in enzyme catalysis with
GABC.
Home assignment for lecture 15-20
1. How do you prepare both the enantiomers of the following compound from
ethylacetoacetate (EAA) by applying a biocatalytic route?
OH
CO2Me
Hint: Think about changing the size in both side of the prochiral ketone and then apply
Prelog’s rule.
2. In the following reaction what improvements will you perform to obtain better ee?
OH
O
Me
yeast
Me
ee = 69%
Reaction condition?
Same product with ee = 96%
3. How do you achieve the following transformation using a ketoreductase?
O
O
CO2Me
O
4. How do you achieve the following transformation using a ketoreductase?
O
S
OBn
CF3
CF3
5. How do you achieve the following transformation using a ketoreductase?
O
O
O
6. Provide a plausible mechanistic pathway (only the intermediates) for the following
enantioselective oxidation of meso-diols by HLADH.
HO
OH
O
O
Hint: Example of an EED reaction by HLADH
EED
HO
OH
HO
lactol oxidation
CHO
O
OH
O
O
lactol
7. Do a proper retrosynthetic disconnection (at least 3 different biocatalytic routes) for the
following molecule. Also provide what are the enzymes you will be using to construct the
stereocenter.
OH
OH
Hint: Think about lipase, ketoreductase and hydroxynitrile lyase mediated
biotransformation as main steps.
Home assignment for lecture 22
1. How do you achieve the asymmetric synyhesis of the following two enantiomeric
hydroxylated carbacycles by applying a chemo-enzymatic strategy?
OH
OH
(-)-Conduritol
OH
OH
OH
OH
OH
(+)-Conduritol
OH
2. How do you carry out the following transformation
Br
H
OHC
H
CO2Me
3. Explain the model for predicting the products in the enzymatic dihydroxylation reaction of the
following two single ring aromatic compounds.
I
I
Br
Me
4. Starting from toluene how do you prepare the following two chiral cyclopentenones.
CHO
Me
CHO
Me
5. How do you synthesize the following two enantiomeric diols from bromobenzene.
Br
Br
HO
OH
HO
OH
Hint: Answer of all the above can be found in Aldrichimica acta (1999, 32, 35). There are
numerous other related problems can be found in the above reference.
Home assignment for lecture 23-28 (hydrolases in synthesis)
1. Explain the Kazlauskas emperical rule for predicting the stereopreferences for EKR of
secondary alcohols catalyzed by lipases. How do you achieve the following transformation
using EKR strategy?
OH
OH
Hint: You can do it in two ways (EKR-Mitsunobu; ME-DKR)
2. How do you apply EHL (Aspergillus niger) for the synthesis of the (R)-nifenalol.
OH
H
N
O2N
(R)-Nifenalol
3. In the following EED reaction ee of the product acetate is often lower than expected.
Provide an explanation and suitable solution to overcome this.
Lipase
OAc
OH
R
R
OH
OH
vinyl acetate
Hint: substantial 1,3 migration.
4. Explain the following observation?
OH
OAc
CAL-B
vinyl acetate
OR
OR
R = Me; ee = 88%
R = Bn; ee = 96%
R = Tr; ee >99%
5. How do you synthesize the following structural scaffolds by using lipase
catalyzed EKR or DKR?
O
OH
O
O
OH
OH
O
Home assignment for lecture 29-30
1. In the following reaction sequence identify A, B and C with proper explanation.
HO
O
CN
+
Br
(R)-HNL
H
A
+
B
AgClO4
C
2. How do you synthesize the following lactone by HNL route?
OH
O
O
3. How do you synthesize Williams template (used in asymmetric alkylation) from
benzaldehyde by HNL route?
O
O
NZ
Williams template
4. How do you synthesize phenyl-glycine (enantiopure form) from benzaldehyde by HNL
route?
Hint: Answers (problems 1-4) can be found in Tetrahedron Asymmetry, 2003, 14, 147176. Other related problems also can be found.
5. How do you synthesize the following aziridine from benzaldehyde by HNL route?
H
CHO
N
Ph
Ph
Hint: J. Org. Chem, 2009, 74, 7548-7551.
6. How do you carry out the synthesis of the following tetrahydroisoquinoline derivative
from furfural by HNL route?
OH
O
CHO
N
H
OH
Hint: Tetrahedron, 2009, 65, 1919-1927.
7. How do you synthesize the following cyanohydrins in enantiopure form acyclic
precursors through HNL route?
OH
CN
O
Hint: Chem. Eur. J. 2008, 14, 11415-11422.
8. Asymmetric biocatalytic cyanohydrination of 2-butanone is unsuccessful for the
synthesis of following enantipoure hydroxy-acid. Can you suggest any alternate route
(HNL strategy) for the hydroxy-acid synthesis with high enantioselection?
O
HO CO H
HO CN
HbHNL
2
HCN
ee = 54%
HbHNL and other reagents
Two other precursors
Hint: Chem. Eur. J. 2007, 13, 3369-3376.