Home assignment for lecture 1& 2
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- Hector Roberts
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1 ome 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?
2 ome 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. ntion 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. C 2 int: 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? on Fluorescent Fluorescent (424nm) C on Fluorescent int: Think about alcohol oxidase and hydroxynitrile lyase activity. C
3 ome 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? int: 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? int: 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- + Et 3 ) as opposed to a tertiary amine (resin- + Et 2 )? int: With a quaternary amine, the column resin always has a net positive charge, and you need not have to worry about the p 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 p. 4. Gel-Filtration chromatography is a useful method for removing salts, such as ammonium sulphate, from protein solutions. Describe how such separation is accomplished? int: 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? int: 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? int: Do it yourself. 7. ow 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 p? int: 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 p is enough to remove the charge, can cause denaturation.
4 9. From the following protein purification table calculate the missing values Step Protein (mg) Activity(U) Specific activity (U/mg) Yield (%) Purification factor ? ??? ??? ???
5 ome 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 and EC What type of enzymes we are talking about?
6 ome assignment for lecture (catalytic role of enzymes) 1. ther things being equal, what is a potential disadvantage of an enzyme having a very high affinity for its substrate? int: 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. int: 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? int: 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. int: 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 p = 4.0, at room temperature. C 2 int: see the lecture note 6. α-chloromethyl ketones are acting as chymotrypsin inactivator. When (2S)-acetyl-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? Ac Ph Cl (2S)--acetyl-L-alanyl-L-phenylalanyl- -chloroethane
7 int: see the lecture note 7. ydroxynitrile lyase (L) is a plant enzyme which catalyzes the following reaction. L is a flavoprotein having characteristic yellow color. When a L solution (0.5mg/ml protein content) is treated with ; a) 10mM solution of sodium dithionite the yellow color is gone but L exhibits its catalytic activity; b) 10mM solution of 2-iodoaceticacid is treated with L solution, it was observed that catalytic activity of L is completely lost. Account for the two above observations with proper reasoning? C L + C int: 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 L active site). 8. Explain the following (concisely) Relative rate of hydrolysis at p = 6.0 int: The above two substrates are glycosidase mimic. Check the glycosidase mechanism. It is a nice example of proximity effect in enzyme catalysis with GABC.
8 ome assignment for lecture ow do you prepare both the enantiomers of the following compound from ethylacetoacetate (EAA) by applying a biocatalytic route? C 2 int: 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? yeast ee = 69% Reaction condition? Same product with ee = 96% 3. ow do you achieve the following transformation using a ketoreductase? C 2 4. ow do you achieve the following transformation using a ketoreductase? S Bn CF 3 CF 3 5. ow do you achieve the following transformation using a ketoreductase? 6. Provide a plausible mechanistic pathway (only the intermediates) for the following enantioselective oxidation of meso-diols by LAD.
9 int: Example of an EED reaction by LAD EED C lactol oxidation 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. lactol int: Think about lipase, ketoreductase and hydroxynitrile lyase mediated biotransformation as main steps.
10 ome assignment for lecture ow do you achieve the asymmetric synyhesis of the following two enantiomeric hydroxylated carbacycles by applying a chemo-enzymatic strategy? (-)-Conduritol (+)-Conduritol 2. ow do you carry out the following transformation Br C C 2 3. Explain the model for predicting the products in the enzymatic dihydroxylation reaction of the following two single ring aromatic compounds. I I Br 4. Starting from toluene how do you prepare the following two chiral cyclopentenones. C C 5. ow do you synthesize the following two enantiomeric diols from bromobenzene. Br Br
11 int: 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.
12 ome assignment for lecture (hydrolases in synthesis) 1. Explain the Kazlauskas emperical rule for predicting the stereopreferences for EKR of secondary alcohols catalyzed by lipases. ow do you achieve the following transformation using EKR strategy? int: You can do it in two ways (EKR-Mitsunobu; ME-DKR) 2. ow do you apply EL (Aspergillus niger) for the synthesis of the (R)-nifenalol. 2 (R)-ifenalol 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 Ac R R vinyl acetate int: substantial 1,3 migration. 4. Explain the following observation? CAL-B Ac R vinyl acetate R R = ; ee = 88% R = Bn; ee = 96% R = Tr; ee >99% 5. ow do you synthesize the following structural scaffolds by using lipase catalyzed EKR or DKR?
13 ome assignment for lecture In the following reaction sequence identify A, B and C with proper explanation. C + Br (R)-L A + B AgCl 4 2. ow do you synthesize the following lactone by L route? C 3. ow do you synthesize Williams template (used in asymmetric alkylation) from benzaldehyde by L route? Z Williams template 4. ow do you synthesize phenyl-glycine (enantiopure form) from benzaldehyde by L route? int: Answers (problems 1-4) can be found in Tetrahedron Asymmetry, 2003, 14, ther related problems also can be found. 5. ow do you synthesize the following aziridine from benzaldehyde by L route? C Ph Ph int: J. rg. Chem, 2009, 74, ow do you carry out the synthesis of the following tetrahydroisoquinoline derivative from furfural by L route?
14 C int: Tetrahedron, 2009, 65, ow do you synthesize the following cyanohydrins in enantiopure form acyclic precursors through L route? C int: Chem. Eur. J. 2008, 14, Asymmetric biocatalytic cyanohydrination of 2-butanone is unsuccessful for the synthesis of following enantipoure hydroxy-acid. Can you suggest any alternate route (L strategy) for the hydroxy-acid synthesis with high enantioselection? bl C C 2 C ee = 54% bl and other reagents Two other precursors int: Chem. Eur. J. 2007, 13,
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