Evidence for Enzymatic Catalysis of the Diels-Alder Reaction in Nature

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Background uncatalyzed cyclization of III competes with enzymatic reaction, ..... Propose a mechanism for the acid catalyzed rearrangement of cinenic acid. O.
Evidence for Enzymatic Catalysis of the Diels-Alder Reaction in Nature

Carmen Drahl Sorensen Group Organic Supergroup Literature Presentation July 2005

Does Nature Know the Diels-Alder Reaction? i. 8π conrot. ii. 6π disrot. iii. π4s + π2s

Ph

H HH H

H H

CO2R

CO2H

representative member of enediandric acids Ph

CO2

[3, 3] sigmatropic rearrangement CO2

CO2

O2 C

O

chorismate mutase

OH

Laschat, S. Angew Chem. Int. Ed. 1996, 35, 289. Nicolaou, K. C.; Sorensen, E. J. in Classics in Total Synthesis, 1996, 265-267.

OH

What is an Enzyme? Thermodynamic Review Transition State ‡

∆G‡S->P ∆G‡P->S ∆G'°

S

Free Energy, G

Free Energy, G

Transition State ‡

∆G‡uncat ‡ ES S

P Reaction Coordinate

∆G'° = −RT ln K'eq

∆G‡cat

EP

P Reaction Coordinate

k = Ae -∆G‡/RT

Catalysts do NOT affect reaction equilibria. Catalysts enhance reaction rates by lowering activation energies.

Nelson, D. L.; Cox, M. M. in Lehninger Principles of Biochemistry, 3rd ed. 2000, 246-250.

Solanapyrone A O S O

S

S OCH3

O

180 °C, sealed tube toluene, 1 h, 71%

S

O

S OCH3

O

S

O +

O OCH3

O H

O

H

OCH3

H H H

H 2 endo : 1 exo

solanapyrone A

Solanapyrone A: decalin polyketide phytotoxin produced by the pathogenic fungus Alternaria solani, causal organism of potato early blight disease Solanapyrone A inhibits DNA polymerase β and γ. The first synthesis of (±)-solanapyrone A was achieved through an intramolecular Diels-Alder reaction, which fueled speculation about its assembly in Nature.

Ichihara, A.; Tazaki, H.; Sakamura, S. Tet. Lett. 1983, 24, 5373 Ichihara, A.; Miki, M.; Tazaki, H.; Sakamura, S. Tet. Lett. 1987, 28, 1175. Mizushina, Y., et al. J. Biol. Chem. 2002, 277, 630-638.

Solanapyrone D and a Proposed Biosynthesis OCH3

O O

OCH3

O O O

O

exo

endo

O

O

O

OCH3

O

O

OCH3 O

H

H solanapyrone A (MAJOR)

H The proposed triene precursor contains no chiral centers. Solanapyrones A and D are found in nature in 100% ee.

H solanapyrone D

Oikawa, H.; Yokota, T.; Ichihara, A.; Sakamura, S. J. Chem. Soc. Chem. Comm. 1989, 1284. Oikawa, H.; Suzuki, Y.; Naya, A.; Katayama, K.; Ichihara, A. J. Am. Chem. Soc. 1994, 116, 3605.

Solanapyrones: Feeding Experiment Summary O

CHO

8

D

D

O

O

OCH3

13

2 [CH3- C]met O

D

D

D

O

O

OCH3 O

D D

H

D D

D

D

H solanapyrone A

R

R Michael

O

retention of D at C5!

O

O

X solanapyrone A

O R=

Aldol O

O

O

R

OCH3

R

Diels Alder

O

Oikawa, H.; Yokota, T.; Abe, T.; Ichihara, A.; Sakamura, S.; Yoshizawa, Y.; Vederas, J. C. J. Chem. Soc. Chem. Comm. 1989, 1282.

Biosynthesis: Summary of Feeding Experiments OH O 8 acetate + 2 C1-Met

OCH3

O [O]

O

prosolanapyrone I I

OCH3 [O]

prosolanapyrone II II

prosolanapyrone III * III Intramolecular Diels-Alder

O

O

OCH3 O

OCH3

OCH3 O

O

O

O

O

O

O HO

O

OCH3

HO

O H

+

O

H

+2H

+2H

H

H

OCH3 O H

H

H

solanapyrone A

solanapyrone D

solanapyrone B

H solanapyrone E

Oikawa, H.; Suzuki, Y.; Naya, A.; Katayama, K.; Ichihara, A. J. Am. Chem. Soc. 1994, 116, 3605. * Feeding experiments with III inconclusive. III underwent spontaneous endo cyclization in aqueous conditions.

Solution Reactivity of Prosolanapyrones R O

OCH3 O

R = CH3 (I) R = CH2OH (II)

substrate I I II II II II III III III III

solvent PhCH3 H 2O PhCH3 CHCl3 CH3CN H 2O PhCH3 CHCl3 CH3CN H 2O

temperature (°C) 180 30 110 110 110 30 110 110 110 30

time (h) 48 168 48 2 24 48 1 1 1 3

yield (%) 12 7 55 7 71 19 68 64 82 62

SM recovery (%) 11 93 2 91 18 81 27 28 10 28

R = CHO (III)

Endo-selectivity increases with increasing solvent polarity. Rate depends on the oxidation level of the pyrone substituent.

Oikawa, H.; Kobayashi, T.; Katayama, K.; Suzuki, Y.; Ichihara, A. J. Org. Chem. 1998, 63, 8748.

endo /exo 1.9 > 10 2.2 3.6 5.6 20 2.7 3.4 4.4 23

Solanapyrone Synthase: Improved Exo Selectivity O

R O

OCH3 O

O

O

OCH3

O

O

OCH3 O

H

+

H

R = CH3 (I) R = CH2OH (II) R = CHO (III)

substrate II II III III III

H

H

solanapyrone D (endo)

solanapyrone A (exo)

conditions control + extract control + extract + denatured extract

yield (%) 0 19 15 25 10

SM recovery (%) 100 75 + 6 % of III 85 75 90

endo :exo n/a 0.176 32.3 0.887 32.3

ee (%) 99 92

Crude enzyme preparation oxidized II to III. No reaction of II in absence of O2(g). Background uncatalyzed cyclization of III competes with enzymatic reaction, hence lower ee when III is used as starting material. Enzyme not yet isolated; further purification of the enzyme(s) responsible is in progress. Oikawa, H.; Katayama, K.; Suzuki, Y.; Ichihara, A. J. Chem. Soc. Chem. Comm. 1995, 1321. Katayama, K.; Kobayashi, T.; Oikawa, H.; Honma, M.; Ichihara, A. Biochim. et Biophys. Acta, 1998, 387. An enantioselective synthesis of solanapyrone A utilized this crude enzyme for the IMDA: Oikawa, H.; Kobayashi, T.; Katayama, K.; Suzuki, Y.; Ichihara, A. J. Org. Chem. 1998, 63, 8748.

Lovastatin HO

O O

O O

lovastatin (closed form)

HO O

HO

COOH OH

O O

O

lovastatin (open form)

dihydromonacolin L

Lovastatin (mevinolin) is produced by fermentation of the fungal strain Aspergillus terreus, and has also been isolated from Monascus ruber. The lactone opened form is a potent inhibitor of the liver enzyme HMG-CoA reductase, which reduces HMG-CoA to mevalonate, the rate limiting step in cholesterol biosynthesis. Prescribed as Mevacor (Merck) to lower cholesterol and fats in blood. No bicyclic precursor less oxidized than dihydromonacolin L has been reported. Alberts, A. W., et al. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 3957. Endo, A. J. Antibiot. 1979, 32, 852. Endo, A. Trends Biochem. Sci. 1981, 6, 10. Kennedy, J.; Auclair, K.; Kendrew, S. G.; Park, C.; Vederas, J. C.; Hutchison, C. R. Science 1999, 284, 1368.

Lovastatin: Feeding Experiment Summary D O

9+2 D

D

O

2 [CH3-13C]met

O D

O D D

D

HO

O O

O* D D H3 C D

D

D

O

O

D

O D D

H3C

Diels-Alder proposed as key biogenesis step: O

SR

O

O

SR lovastatin

H

Chan, J. K.; Moore, R. N.; Nakashima, T. T.; Vederas, J. C. J. Am. Chem. Soc. 1983, 105, 3334. Moore, R. N.; Bigam, G.; Chan, J. K.; Hogg, A. M.; Nakashima, T. T.; Vederas, J. C. J. Am. Chem. Soc. 1985, 107, 3694. Yoshizawa, Y.; Witter, D. J.; Liu, Y. Q.; Vederas, J. C. J. Am. Chem. Soc. 1994, 116, 2693.

Lovastatin: Attempted Laboratory Cyclizations O

O

O

X

X

X

X endo

O

exo

X

O

O

X O a, X = S(CH2)2NHAc b, X = OEt c, X = OH

endo

O

X

X exo

X

O

H

H

H

H

H

H

H

H

lovastatin

Not observed. Is an enzyme needed to stabilize pseudoaxial Me?

Witter, D .J.; Vederas, J. C. J. Org. Chem. 1996, 61, 2613.

Thermal: (EtAlCl2):

1 9 19

: : :

X

1 a,b,c 1 b 1 a

Solution Reactivity of Lovastatin Precursor O

X

O H

a, X = S(CH2)2NHAc b, X = OEt c, X = OH

substrate a b c a b b b

conditions PhCH3 PhCH3 PhCH3 EtAlCl2 + PhCH3 EtAlCl2 + PhCH3 CHCl3 H2O:CH3CN:MeOH (5:5:1)

X

O +

H

endo

exo

Witter, D .J.; Vederas, J. C. J. Org. Chem. 1996, 61, 2613.

(Transition states for pseudoequatorial methyl)

H

H

temperature (°C) 160 160 160 23 23 22 28

X

time (h) 96 96 96 3 3 240 48

yield (%) 81 72 83 80 58 50 50

SM recovery (%) n/a 6 n/a n/a n/a n/a n/a

endo /exo 1 1 1 9 19 n/a n/a

Lovastatin: Attempted Feeding Experiment HO O

S

O

O

NHAc X

O

O

lovastatin (closed form)

Feeding this 13C labeled substrate to Aspergillus terreus resulted in no formation of carbon-carbon coupled lovastatin or precursors. Presumably, the substrate is catabolized before it can undergo cycloaddition.

Witter, D .J.; Vederas, J. C. J. Org. Chem. 1996, 61, 2613.

LNKS Enzyme Affords Correct Stereochemistry O

X

O

X

O

H

O

H +

a, X = S(CH2)2NHAc b, X = OEt

X

X

H +

H

H

H

1

2

3 natural product's stereochemistry

Stereochemistry found in the natural product (3) is only obtainable in the presence of purified LNKS enzyme. The enzyme may stabilize the transition state through van der Waals or other contacts. kcat = 0.073 ± 0.001 min-1. Nonenzymatic cyclization competes with catalysis. Auclair, K.; Sutherland, A.; Kennedy, J.; Witter, D. J.; Van den Heever, J. P.; Hutchinson, C. R.; Vederas, J. C. J. Am. Chem. Soc. 2000, 122, 11519.

Macrophomate: Benzoate from a Pyrone O - 2CO2; - H2O

O

OCH3

O

OCH3 O

HO2C

O

O O

O

O macrophomic acid

Isolated from Macrophoma commelinae fungus, which causes spots on the leaves of the Asiatic dayflower. The benzoate macrophomate is made by an unusual multistep transformation from a 2-pyrone. This type of aromatic compound is typically biosynthesized via the shikimate or polyketide pathway. http://www.sycamoreisland.org

Sakurai, I.; Suzuki, H.; Miyaijima, K.; Akiyama, S.; Simizu, S.; Yamamoto, Y. Chem. Pharm. Bull. 1985, 33, 5141. Oikawa, H.; Yagi, K.; Watanabe, K.; Honma, M.; Ichihara, A.; Chem. Commun. 1997, 97.

Macrophomate: Summary of Biosynthetic Studies OH O

OH

R1 2 R

OH

* CO2H

HO H2N

O

oxaloacetate

Me H2N

H

- CO2, -H2O OCH3 O

O OPO32phosphoenolpyruvate O

^ CO2H

O

HO

O C

+

O

H

[1-13C]-L-serine

O

O O pyruvate

5-acetyl-4-methoxy6-methyl-2-pyrone

^

*

glycerol 1 R , R2 = H or D

O

O

O

R2 OCH3 O macrophomic acid

Oxaloacetate was determined to be the most likely three-carbon donor by isotope incorporation.

[1-13C]-L-alanine

Oikawa, H.; Yagi, K.; Watanabe, K.; Honma, M.; Ichihara, A. Chem. Commun. 1997, 97.

Michael/Aldol or Diels-Alder? O O O

O

- CO2

O

O O

OCH3 O

O

O

O

O

OCH3 O

O

O

O

O

- CO2 O

O O

O O

H3CO O

H3CO O

O

O

OH H CO2 H

- CO2, H2O

O HO

OCH3

O

Watanabe, K.; Mie, T.; Ichihara, A.; Oikawa, H.; Honma, M. J. Biol. Chem. 2000, 275, 38393.

A Bicyclic Inhibitor of Macrophomate Synthesis O O O

O O

+

i. oxaloacetate decarboxylation ii. Diels-Alder reaction

O OCH3

O

O MeO

cell-free extract M. commelinae

O O

O

O

OH H CO2 H

proposed transition state for Diels-Alder reaction

O MeO

O

O HO

OCH3

O

Oikawa, H.; Yagi, K.; Watanabe, K.; Honma, M.; Ichihara, A. Chem. Commun. 1997, 97.

Biomimetic Synthesis of Pyrenochaetic Acid A Pyrenochaeta terrestris or Macrophoma commelinae

OCH3

O

HO2C

OCH3

HO2C

OCH3

O O

O

pyrenocine A

O

pyrenochaetic acid A

macrophomic acid

O O

OCH3

O

+

O

180-200 °C, sealed tube OEt

neat, 17 h, 84%

O

X

OMe

Y

O

O

Product 1 closely resembles macrophomic acid.

-CO2 X

OCH3

Y O Sato, H.; Konoma, K.; Sakamura, S. Agric. Biol. Chem. 1979, 43, 2409. Sato, H.; Konoma, K.; Sakamura, S. Agric. Biol. Chem. 1981, 45, 1675. Ichihara, A.; Murakami, K.; Sakamura, S. Tetrahedron 1987, 43, 5245.

product 1: X = CO2Et; Y = H product 2: X = H; Y = CO2Et 1:2 ratio = 2:3

Crystal Structure of Macrophomate Synthase Structure 1.7 Å in complex with pyruvate and Mg2+. MW = 36 kDa. Hexameric functional unit associated by hydrophobic interactions. Each protomer is an 8-stranded β-barrel containing an octahedrally coordinated Mg2+ ion. Magnesium was known at the time to be necessary for oxaloacetate decarboxylation.

Ose, T.; Watanabe, K.; Mie, T.; Honma, M.; Watanabe, H.; Yao, M.; Oikawa, H.; Tanaka, I. Nature 2003, 422, 185. Berman, H. M. et al. The Protein Data Bank. Nucl. Acids Res. 2000, 28, 235. Image rendered with Deep View / Swiss PDB Viewer. http://www.expasy.org/spdbv/

Macrophomate Synthase Active Site The Mg2+ ion stabilizes the pyruvate enolate. Enzyme kcat= 0.60±0.02 s-1 Arg101 and Tyr169 are thought to bind pyrone. Mutants lose MPS activity while retaining decarboxylase activity. Steric congestion of peptide backbone allows access to one face of enolate. Product inhibition is avoided by second decarboxylation and dehydration.

Ose, T., et al. Nature 2003, 422, 185. Watanabe, K.; Oikawa, H.; Yagi, K.; Ohashi, S.; Mie, T.; Ichihara, A.; Honma, M. J. Biochem. 2000, 127, 467. Berman, H. M. et al. The Protein Data Bank. Nucl. Acids Res. 2000, 28, 235. Image rendered with Deep View / Swiss PDB Viewer. http://www.expasy.org/spdbv/

"A Bucket of Cold Water" O

O

O O O H3CO O

17.7 kcal/mol

Crystal structure provided the basis for a QM/MM/ Monte Carlo calculations to investigate the energetics of both possible pathways. The Diels-Alder transition state is significantly less stable than both Michael and Aldol transition states.

12.1 kcal/mol

O

O O

OCH3 O

O

O O

O

OCH3 O

O O

O

Guimarães, C. R. W.; Udier-Blagovic, M.; Jorgensen, W. L. J. Am. Chem. Soc. 2005, 127, 3577. Wilson, E. Chem. Eng. News. 2005, 83(18), 38.

Diels-Alderases: Summary — Each of the three putative Diels-Alderases catalyzes one or several reactions prior to the cyclization step. solanapyrone synthase: oxidation lovastatin nonaketide synthase: polyketide chain formation macrophomate synthase: decarboxylation —General strategy appears to be entropy trapping of the substrate in the correct conformation to facilitate a [4+2] cycloaddition. —"Proof that the proteins are accelerating the rates of the pericyclic Diels-Alder reaction remains to be rigorously established". (RNA Diels-Alderases have shown up to 20,000 fold rate enhancement). —"Calculations as well as work with mutants and inhibitors will have to clarify to what extent the Diels-Alder reaction in the enzyme active site of macrophomate synthase does indeed follow a concerted... pathway."

Pohnert, G. ChemBioChem 2003, 4, 713. Stocking, E. M.; Williams, R. M. Angew. Chem. Int. Ed. 2003, 42, 3078. Pitt, J. N.; Ferré-D'Amaré, A. R. Nat. Struct. Mol. Biol. 2005, 12, 206.

Kinetic Isotope Effect and Concerted Mechanisms Kinetic Isotope Effect: difference in reaction rate when an atom is replaced by its isotope

En = hν (n + ½) ν = 1/2π sqrt(k/µ) R-H R-D

Carey, F. A.; Sundberg, R. J. in Advanced Organic Chemistry, 4th ed. 2000, 222-225.

Kinetic Isotope Effect and Stepwise Mechanisms Isotopic fractionation at the bond-making site measured as a function of the isotope at the bond-breaking site can be used to test for concertedness of enzymatic mechanism. Example: the enzyme proline racemase.

B-H'

H"

B

B-H'

B-H"

B

H'

gain D'

gain D' lose H"

2-1H - D-proline

gain H'

L-proline

B-H"

lose D" gain H'

2-2H - D-proline

Belasco, J. G.; Albery, W. J.; Knowles, J. R. J. Am. Chem. Soc. 1983, 105, 2475.

L-proline

Kinetic Isotope Effect and Concerted Mechanisms Isotopic fractionation at the bond-making site measured as a function of the isotope at the bond-breaking site can be used to test for concertedness of enzymatic mechanism.

B-H'

H"

B

B

H'

B-H"

lose D", gain D' lose D", gain H' lose H", gain D' lose H", gain H'

D-proline

L-proline

Belasco, J. G.; Albery, W. J.; Knowles, J. R. J. Am. Chem. Soc. 1983, 105, 2475.

Key Papers: Diels-Alder Reactions in Biology Biosynthetic Diels-Alder Reactions (established and speculated) — Stocking, E. M.; Williams, R. M. Angew. Chem. Int. Ed. 2003, 42, 3078. — Oikawa, H.; Tokiwano, T. Nat. Prod. Rep. 2004, 21, 321. — Oikawa, H. Bull. Chem. Soc. Jpn. 2005, 78, 537.

Antibody Diels-Alder Catalysis — Hilvert, D.; Hill, K. W.; Nared, K. D.; Auditor, M. M. J. Am. Chem. Soc. 1989, 111, 9261. — Romesberg, F. E.; Spiller, B.; Schultz, P. G.; Stevens, R. C. Science 1998, 279, 1929. — Heine, A., et al. Science 1998, 279, 1934.

RNA Diels-Alder Catalysis — Tarasow, T. M.; Tarasow, S. L.; Eaton, B. E. Nature 1997, 389, 54. — Seelig, B.; Jäschke, A. Chem. Biol. 1999, 6, 167.

Problem: Mechanism? Propose a mechanism for the acid catalyzed rearrangement of cinenic acid.

H3C H3C

O

CH3 OH O

conc. H2SO4

CH3 H3C

O

CH3 CO2H

Meinwald, J.; Hwang, H. C.; Christman, D.; Wolf, A. P. J. Am. Chem. Soc. 1960, 82, 483.

Problem: Mechanism? Propose a mechanism for the acid catalyzed rearrangement of cinenic acid. H3C H3C

+

H

H3C H3C

O

CH3 OH2 O

conc. H2SO4

CH3 H3C

O

CH3 CO2H

By decarboxylation, cinenic acid can rid itself of a 1,3 diaxial interaction (either between 2 methyls or a methyl and a carboxyl).

CH3 O

O

+ H2O

H3C H3C

- H2O

H3C H3C

O

CH3 OH O

O + CO H3C H3C

- CO H3C H3C

CH3 O

H3C H3C

CH3 O

Meinwald, J.; Hwang, H. C.; Christman, D.; Wolf, A. P. J. Am. Chem. Soc. 1960, 82, 483.

O

CH3

O

CH3