1、The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 13 Rearrangements,Rearrangements,Pericyclic Reactions - concerted reactions in which bonding changes occur via reorganization of electrons within a loop of interacting orbitals,Scheme 13.1,3,3 sigmatropic rearrangement,General form of the C
2、laisen rearrangement,Sigmatropic Rearrangements,Scheme 13.2,chorismate,prephenate,Chorismate Mutase-catalyzed Conversion of Chorismate to Prephenate,A step in the biosynthesis of Tyr and Phe in bacteria, fungi, plants,Required conformer for Claisen rearrangement (10-40% observed in solution from NMR
3、 spectrum),Conformation of Chorismate in Solution,chair-like TS,Evidence for Chairlike Transition State,Scheme 13.3,Stereochemical outcome if chorismate mutase proceeds via chair and boat transition states, respectively, during reaction with (Z)-9-3Hchorismate,To Determine the Position of the 3H,Sch
4、eme 13.4,Z-9- 3Hchorismate 20% 3H release E-9- 3Hchorismate 67% 3H release,Therefore, chair TS,Chemoenzymatic degradation of the prephenate formed from the chorismate mutase-catalyzed conversion of (Z)-9-3Hchorismate to determine the position of the tritium,Figure 13.1,2 inverse isotope effect on C-
5、4 (sp2 sp3); therefore not 1-3 (sp3 sp2),Five Hypothetical Stepwise Mechanisms for the Reaction Catalyzed by Chorismate Mutase,4,mechanism 5 excluded,mechanisms 1, 2, 5 excluded,16 mutants made to show neither general acid-base catalysis (mechanisms 1-3, 5) nor nucleophilic catalysis (mechanism 4) i
6、s important,Both are substrates,Function of the enzyme is to stabilize the chair transition state geometry,Conclusion: pericyclic,Oxy-Cope Rearrangement,Scheme 13.5,Cope,oxy-Cope,Neither observed yet by an enzyme, but a catalytic antibody has been raised,General form of Cope (A) and oxy-Cope (B) rea
7、ctions,Scheme 13.6,Oxy-Cope Rearrangement Catalyzed by an Antibody,hapten to raise the antibody,Scheme 13.7,2,3 Sigmatropic Rearrangement Catalyzed by Cyclohexanone Oxygenase,Scheme 13.9,boat like TS,4+2 Cycloaddition (Diels-Alder) Reaction,Scheme 13.10,solanopyrones,enzymatic exo : endo is 53 : 47,
8、in aqueous solution exo : endo is 3 : 97 (nonenzymatic),An Intramolecular Diels-Alder Reaction Catalyzed by Alternaria solani,Scheme 13.11,An Antibody-Catalyzed Diels-Alder Reaction,Hapten used,This hapten gives an antibody that makes only endo product,This hapten gives an antibody that makes only e
9、xo product,Rearrangements via a Carbenium Ion,Scheme 13.14,acid-catalyzed,acyloins,1,2 alkyl migration,An acid-catalyzed acyloin-type rearrangement,Scheme 13.15,Reactions Catalyzed by Acetohydroxy Acid Isomeroreductase,substrate,Kinetically-competent intermediate,Scheme 13.16,Proposed Acyloin-type M
10、echanism for Acetohydroxy Acid Isomeroreductase,intermediate,Cyclizations Sterol biosynthesis,Scheme 13.17,cholesterol,squalene,lanosterol,Conversion of squalene to lanosterol,Scheme 13.18,2,3-oxidosqualene-lanosterol cyclase,not isolated,17,protosterol,7 stereogenic centers,squalene 2,3-epoxidase,s
11、qualene,anti-Markovnikov (to get 6-membered ring),Isotope labeling shows the 4 migrations are intramolecular Covalent catalysis proposed to control stereochemistry,Initial Mechanism Proposed for 2,3-Oxidosqualene-lanosterol Cyclase,(128 possible isomers),only isomer formed,lanosterol,Evidence for 17
12、 Configuration,Scheme 13.19,no covalent catalysis needed,17,isolated,Use of 20-oxa-2,3-oxidosqualene to determine the stereochemistry at C-17 of lanosterol from the reaction catalyzed by 2,3-oxidosqualene-lanosterol cyclase,O instead of CH2,17,Further Support for Structure of Protosterol,Scheme 13.2
13、0,17,Use of (20E)-20,21-dehydro-2,3-oxidosqualene to determine the stereochemistry at C-17 of lanosterol from the reaction catalyzed by 2,3-oxidosqualene-lanosterol cyclase,Model Study for Stereospecificity and Importance of 17 Configuration,Scheme 13.21,17,17,90%,With the 17 isomer a mixture of C-2
14、0 epimers is formed,Chemical model for the conversion of protosterol to lanosterol,Evidence that the Cyclization Is Not Concerted,Scheme 13.22,Markovnikov addition,not when X=CH2,ring expansion,Mechanism proposed for the formation of the minor product isolated in the 2,3-oxidosqualene cyclase-cataly
15、zed reaction with 20-oxa-2,3-oxidosqualene,does not come from a concerted reaction,Vmax/Km for R = CH3, H, Cl138, 9.4, 21.9 pmol g-1h-1M-1,correlates with carbocation stabilization (CH3 Cl H),Evidence for Carbocation Intermediate,no reaction without methyls - suggests initial epoxide opening,Squalen
16、e Biosynthesis,farnesyl diphosphate,presqualene diphosphate,squalene,Squalene synthase-catalyzed conversion of farnesyl diphosphate to squalene via presqualene diphosphate,Scheme 13.23,Rearrangement of Presqualene Diphosphate to Squalene,Scheme 13.24,squalene,Mechanism proposed for the conversion of
17、 presqualene to squalene by squalene synthase,In the Absence of NADPH there is a Slow Hydrolysis Evidence for 13.56 and 13.57,Scheme 13.25,Mechanisms proposed for the squalene synthase-catalyzed hydrolysis of presqualene diphosphate to several different products in the absence of NADPH,Support for I
18、ntermediate 13.57,Scheme 13.26,dihydro-NADPH,Use of dihydro-NADPH to provide evidence for the formation of intermediate 13.57 in the reaction catalyzed by squalene synthase,unreactive NADPH to mimic bound NADPH,DNA Photolyase UV light causes DNA damage Reactions catalyzed by DNA photolyase and (6-4)
19、 photolyase,Scheme 13.27,visible h used as a substrate for photoreactivation,cyclobutane pyrimidine dimer,(6-4) photoproduct,both types carcinogenic, mutagenic,Rearrangements Via Radical Intermediates,reduced FADH-,N5,N10-methenyl H4PteGlun,8-OH-7,8-didemethyl-5-deazariboflavin,These act as photoant
20、ennae to absorb blue light and transmit to the FADH-,Other Cofactors Used by Photolyases,Scheme 13.28,EPR evidence,Mechanism Proposed for DNA Photolyase,Scheme 13.29,Proposed Mechanism for the Formation of the (6-4) Photoproduct,Scheme 13.30,Mechanism Proposed for (6-4) Photolyase,adenosylcobalamin,
21、(coenzyme B12),(vitamin B12),Coenzyme B12 Rearrangements,5-deoxyadenosyl,abbreviation for coenzyme B12,Conversion of Vitamin B12 to Coenzyme B12,Scheme 13.31,2nd known reaction at C-5 of ATP,Bioynthesis of coenzyme B12,B12r,B12s,Scheme 13.32,Light Sensitivity of the Co-C Bond of Coenzyme B12,Scheme
22、13.33,X is alkyl, acyl, or electronegative group,General Form of Coenzyme B12-Dependent Rearrangements,Figure 13.2,Three Examples of Coenzyme B12 Rearrangements,Scheme 13.34,(1R, 2R),(2S),No incorporation of solvent protons; therefore no elimination of water (enol would form),kH/kD = 10-12,Mechanism
23、 for Diol Dehydratase and Ethanolamine Ammonia-Lyase,Stereospecific conversion of (1R,2R)-1-2H-1-14Cpropanediol to (2S)-2-2H-1-14Cpropionaldehyde catalyzed by diol dehydratase,Stereospecific 1,2 migration of the pro-R H with inversion,R,R,Scheme 13.35,S,R,(1R, 2S),With the (1R, 2S) epimer, the pro-S
24、 H migrates; therefore stereochemistry at C-2 determines which C-1 H migrates,Stereospecific Conversion of (1R,2S)-1-2H-1-14Cpropanediol to 1-2H-1-14Cpropionaldehyde Catalyzed by Diol Dehydratase,Scheme 13.36,(2S)-1-18O,(2R)-1-18O,The same OH is eliminated (pro-R) regardless of which C-1 H migrates,
25、Stereospecificity of Elimination of Water,Diol dehydratase-catalyzed conversion of (2S)-1-18Opropanediol to 18Opropionaldehyde (A) and of (2R)-1-18Opropanediol to propionaldehyde (B),Therefore the C-1 H and the C-2 OH migrate from opposite sides giving inversion at both C-1 and C-2,Scheme 13.37,Cros
26、sover Experiment to Show that Diol Dehydratase Catalyzes an Intermolecular Transfer of a Hydrogen from C-1 to C-2,Therefore, hydrogen transfer is intermolecular,Figure 13.3,Time Course for Incorporation of Tritium from 1-3Hpropanediol into the Cobalamin of Diol Dehydratase,Scheme 13.38,no 3H here,1/
27、2 3H lost,all 3H retained,no 3H here,Reconstitution of the isolated 3H coenzyme B12 into apoenzyme with propanediol gives 2-3Hpropionaldehyde. All 3H transferred from 3H coenzyme B12,Determination of the Site of Incorporation of 3H into Coenzyme B12,Aerobic and anaerobic photolytic degradation of co
28、enzyme B12 to locate the position of the tritium incorporated from 1-3Hpropanediol in a reaction catalyzed by diol dehydratase,3H here,3H here,possible intermediate to equilibrate the C-5 protons,13.88 isolated with substrates that cannot rearrange,Synthesized (R,S)-5-3H Coenzyme B12 Transfers All 3
29、H to the Product Randomly,Coenzyme B12 is the hydrogen transfer agent.,Proposed Rationalization for EPR Spectrum of Co(II) + Carbon Radicals,Scheme 13.39,Formation of 5-deoxyadenosine, cob(II)alamin, and substrate radicals during coenzyme B12-dependent reactions,Scheme 13.40,Not clear if important,R
30、adicals observed in EPR spectrum,Mechanism(s) Proposed for Diol Dehydratase,The part shown in the dashed box is even more speculative than the rest of the mechanism,Scheme 13.41,Chemical Model Study for a Proposed Diol Dehydratase-catalyzed Rearrangement Involving a Co(III)-olefin -Complex,The trape
31、zoid represents the cobaloxime ligand,A Cobalt Complex Is Not Necessary,Scheme 13.42,The Fenton reaction as a model for a proposed diol dehydratase-catalyzed free radical rearrangement,(the cobalt complex is just to initiate the reaction by radical generation),Scheme 13.43,Another Chemical Model Stu
32、dy for a Proposed Diol Dehydratase-catalyzed Free Radical Rearrangement,Scheme 13.44,EPR confirms Co(II) + organic radical Crystal structures with and without substrates bound show the active site closes upon substrate binding - shields radical intermediates,Carbon Skeletal Rearrangements,Stepwise (
33、a) versus concerted (b) mechanisms for the methylmalonyl-CoA mutase-catalyzed generation of 5-deoxyadenosine, cob(II)alamin, and substrate radical,*,Co-C cleavage is 21 times faster with (CH3)MM-CoA than with (CD3)MM-CoA.,Therefore, Co-C and C-H cleavage are concerted.,Figure 13.4,Ab initio calculat
34、ions disfavor pathway e No concensus about the others,Six Possible Pathways for the Conversion of Methylmalonyl-CoA Radical to Succinyl-CoA Radical Catalyzed by Methylmalonyl-CoA Mutase,Converts ribonucleotides to deoxyribonucleotides,Ribonucleotide Reductase,Results are different from other coenzym
35、e B12 enzymes:,0.01-0.1% of 3H from 3-3HUTP is released,no 3H from 3-3HUTP found in adenosylcobalamin,no crossover between 3-3HUTP + ATP,3-3HUTP gives 3-3HdUTP,3H in 5-3Hadenosylcobalamin is washed out in the absence of substrate,adenosylcobalamin 5-deoxyadenosine + Co(II),By EPR formation of Co(II)
36、 corresponds to formation of 5-deoxyadenosine and the generation of a thiiyl radical (Cys-408),Scheme 13.45,rates of formation are identical; therefore, concerted reaction,Mechanism Proposed for Coenzyme B12-dependent Ribonucleotide Reductase,Scheme 13.46,regenerates active site for next cyclereduce
37、d by thioredoxin,electrons are transferred to active-site disulfide,The function of the cobalamin in this enzyme is to initiate the radical reaction by abstraction of H from Cys-408,Mechanism Proposed for Reducing and Reestablishing the Active Site of Coenzyme B12-dependent Ribonucleotide Reductase,
38、Figure 13.5,Other Ribonucleotide Reductases Use Other Radicals to Abstract a H from an Active Site Cys,Cofactors for class I (13.118), class III (13.119), and class IV (13.120) ribonucleotide reductases,Scheme 13.47,pro-R,pro-R,L-Lys,L-Lys,Requires PLP, SAM, 4Fe-4S, and a reducing agent,Reaction Cat
39、alyzed by Lysine 2,3-Aminomutase,Transfers 3-pro-R H of L-Lys to 2-pro-R of L-Lys with migration of 2-amino of L-Lys to C-3 of L-Lys,No exchange with solvent,With (S)-5-3Hadenosylmethionine, 3H ends up in both L-Lys and L-Lys,One equivalent of Met and 5-deoxyadenosine are formed with L-3-3HLys.,C-S
40、bond is stable, unlike C-Co bond,It appears that SAM is functioning like coenzyme B12,In the presence of a reducing agent, 4Fe-4S+ is observed in the EPR, which reduces SAM to Met and 5-deoxyadenosyl radical,1-6% of 3H ends up in SAM,Scheme 13.48,not observed in EPR,unique function for PLP,EPR detec
41、ts organic radicals; 13C label shows product radical 13.126 in EPR spectrum,Mechanism Proposed for Lysine 2,3-Aminomutase,Scheme 13.49,Model Study for New Function of PLP,Chemical model study to test the proposed rearrangement mechanism for lysine 2,3-aminomutase,stabilize -radical,To Get Evidence for Substrate Radical (13.124),Scheme 13.50,EPR detected,isolated,Lysine 2,3-aminomutase-catalyzed rearrangement of 4-thialysine to generate a more stable substrate radical,Evidence for Substrate Radical Formation,
