ORGANIC LETTERS
Solvolytic Enolization of Scytalone Gregory S. Basarab,*,§ Douglas B. Jordan,† and Ya-Jun Zheng‡
2000 Vol. 2, No. 11 1541-1544
E. I. DuPont de Nemours Central Research and DeVelopment, P.O. Box 80328, Experimental Station, Wilmington, Delaware 19880-0328, DuPont Pharmaceutical Company, Experimental Station, Wilmington, Delaware 19880-0400, and E. I. DuPont de Nemours Agricultural Products, Stine-Haskell Research Center, P.O. Box 30, Newark, Delaware 19714
[email protected] Received March 25, 2000
ABSTRACT
The major conformation of scytalone has an envelope shape with C3 forming the flap and the C3 hydroxyl in the equatorial position as determined by quantum mechanical calculations and corroborated by NMR. The C2 axial pro-R is slower to exchange with solvent than the equatorial pro-S hydrogen. Modeling the transition state for enolate formation points to a deprotonation through the flipped envelope conformation in which the C3-hydroxyl and the C2 pro-S hydrogen are axial.
Scytalone and vermelone (eq 1) are physiological substrates of scytalone dehydratase (SD), an enzyme of the fungal melanin biosynthetic pathway found in a number of pathogens.1 Blockage of fungal melanin biosynthesis by SD
inhibition effectively controls disease and has prompted fungicide design efforts.2 Kinetic measurements in conjunc§
DuPont Central Research and Development. DuPont Pharmaceutical Company. ‡ DuPont Agricultural Products. (1) (a) Howard, R. H.; Valent, B. Annu. ReV. Microbiol. 1996, 50, 491. (b) Bechinger, C.; Giebel, K.-F.; Schnell, M.; Leiderer, P.; Deising, H. B.; Bastmeyer, M. Science 1999, 285, 1896. (c) Chumley, F. G.; Valent, B. Mol. Plant-Microbe Interact. 1990, 3, 135. (d) Bell, A. A.; Wheeler, M. H. Annu. ReV. Phytopath. 1986, 24, 411. (2) (a) Jennings, L. D.; Wawrzak, Z.; Amorose, D.; Schwartz, R.; Jordan, D. B. Bioorg. Med. Chem. Lett. 1999, 9, 2509. (b) Basarab, G. S.; Jordan, D. B.; Gehret, T. C.; Schwartz, R. S., and Wawrzak, Z. Biorg. Med. Chem. Lett. 1999, 9, 1613. (c) Jordan, D. B.; Lessen, T.; Wawrzak, Z.; Bisaha, J. J.; Gehret, T. C.; Hansen, S. L.; Schwartz, R. S.; Basarab, G. S. Bioorg. Med. Chem. Lett.. 1999, 9, 1607. (d) Chen, J. M.; Xu, S. L.; Wawrzak, Z.; Basarab, G. S.; Jordan, D. B. Biochemistry 1998, 37, 17735. †
10.1021/ol0058590 CCC: $19.00 Published on Web 05/10/2000
© 2000 American Chemical Society
tion with X-ray crystallography have delineated a catalytic mechanism for the enzyme-catalyzed dehydration in which the substrate adopts a twist-boat conformation placing the C2 pro-R hydrogen and the C3-hydroxyl in axial orientations for facile removal.3 The scytalone carbonyl forms a hydrogen bond in the transition state with a water molecule that, in turn, forms hydrogen bonds with the hydroxyl groups of two tyrosine residues. This lowers the pKa of the CH2 R to the carbonyl, allowing a well-positioned histidine imidazole to remove the pro-R hydrogen. The same histidine residue is also well-positioned to deliver the abstracted proton to the leaving hydroxide at C3. This two-step sequence of enolization followed by the loss of hydroxide is thus characterized as an E1cb-like mechanism as described for other enzymes by Gassman and Gerlt.4 The SD-catalyzed dehydration reaction proceeds about 109 times faster than the nonenzyme-catalyzed reaction. (3) (a) Jordan, D. B.; Zheng, Y.-J.;. Lockett,B. A.; Basarab, G. S. Biochemistry, 2000, 39, 2276. (b) Basarab, G. S.; Steffens, J. J.; Wawrzak, Z.; Schwartz, R. S.; Lundqvist, T.; Jordan, D. B. Biochemistry 1999, 38, 6012. (c) Wawrzak, Z.; Sandalova, T.; Steffens, J. J.; Basarab, G. S.; Lundqvist, T.; Lindqvist, Y.; Jordan, D. B. Proteins: Struct., Funct. Genet. 1999, 35, 425. (d) Lundqvist, T.; Rice, J.; Hodge, C. N.; Basarab, G. S.; Pierce, J.; Lindqvist, Y. Structure (London) 1994, 2, 937. (4) (a) Gerlt, J. A.; Gassman, P. G. J. Am. Chem. Soc. 1993, 115, 11552. (b) Gerlt, J. A.; Gassman, P. G. Biochemistry 1993, 32, 11943. (c) Gerlt, J. A.; Gassman, P. G. J. Am. Chem. Soc. 1992, 114, 5928.
Figure 1. Four scytalone conformations: A, envelope conformation with axial C3 hydroxyl; B, envelope conformation with equatorial C3 hydroxyl; C, boat conformation with axial C3 hydroxyl; D, boat conformation with equatorial C3 hydroxyl.
It is of value to characterize the solvolytic dehydration of scytalone to understand constraints that the enzyme must render upon the substrate for catalysis. The scytalone C2 hydrogen atoms exchange for deuterium in D2O at 25 °C and neutral pH about 70-fold faster than the rate of dehydration, thus defining an E1cb mechanism.3a Consistent with this mechanism is the reported kinetic isotope effect for 2,2,4,4,5,7-hexadeuteoscytalone of 1.2 indicating that the energy barrier for hydroxide loss dominates that for proton abstraction.3b Stereoelectronic considerations dictate that both the hydrogen removed during enolization and the hydroxide eliminated are axially disposed, though not necessarily at the same time. Stereoselectivity in the deprotonation (and by microscopic reversibility, re-protonation) of scytalone should be influenced by the scytalone chiral center either through steric hindrance or through electronic control wherein the hydroxyl engenders a conformation that predisposes one of the C2 hydrogen atoms to the axial orientation. The absolute stereochemistry of scytalone at C3 has been determined to be R,5 and indeed, the C2 pro-S hydrogen (anti to the hydroxyl) exchanges 13-times faster than the C2 pro-R hydrogen.3a We report here that the major scytalone conformation in D2O places the pro-S hydrogen in an equatorial orientation.6 It was anticipated that the scytalone 1H NMR3a would reflect an averaging of the available conformations with one more heavily weighted. The C2 hydrogens separate in the NMR to δ ) 2.5-2.75 and 2.9-2.95 ppm, and the farther downfield resonance can be assigned to the predominately equatorial hydrogen.7 The ability of the neighboring C3 (5) Viviani, F.; Gaudry, M. Tetrahedron 1990, 46, 2827. (6) Scytalone was isolated from cultures of rsy- mutants of Magnaporthe grisea (ref 1c) via methods set out in the literature: Bell, A. A.; Stipanovic, R. D.; Puhalla, J. E. Tetrahedron 1976, 32, 1353. (7) (a) Abraham, R. J.; Ainger, N. J. J. Chem. Soc., Perkin Trans. 2 1999, 441-448. (b) Trimitsis, G. B.; Van Dam, E. M. J. Chem. Soc., Chem. Commun. 1974, 610. 1542
hydroxyl to deshield the axial hydrogen is not sufficiently large to invalidate the assignment.8 Similarly, the C4 hydrogens separate to δ ) 2.85-2.9 and 3.08-3.15 ppm with the farther downfield resonance being assigned as the equatorial hydrogen. Since the more downfield resonances at C2 and C4 have also been assigned anti to the hydroxyl from NOE studies,3a the predominate scytalone conformation seen in solution must be an envelope shape for the saturated ring with C3 forming the flap of the envelope (Figure 1B). The remaining five carbon atoms of the ring are nearly coplanar, and the C3 hydroxyl lies in an equatorial orientation. The NMR coupling constants support this assignment as a larger J ) 6-7 Hz occurs between the C3 hydrogen and each of the pro-R hydrogens at C2 and C4 versus a smaller J ) 3-4 Hz between the C3 hydrogen and each of the pro-S hydrogens. Having the C3 hydroxyl equatorial produces a trans-diaxial orientation between the C3 hydrogen and the pro-R hydrogens at C2 and C4. We applied density functional theory to the scytalone ground state and the transition state of its deprotonation to understand the trajectory for enolization. The ground-state calculations were carried out using the Gaussian 98 program.9 Geometrical optimizations were performed with the standard 6-31G(d) basis set using a hybrid density functional theory (8) Lemieux, R. U.; Stevens, J. D. Can. J. Chem. 1965, 43, 2059. (9) Gaussian 98. Revision A.5, Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.; Stratmann, R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.; Martin, R. L..; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; M. W.; Wong, Andres, J. L.; Gonzalez, C.; Head-Gordon, M.; Replogle, E. S.; Pople, J. A. Gaussian, Inc.: Pittsburgh, PA, 1998. Org. Lett., Vol. 2, No. 11, 2000
method (B3LYP);10 it has been shown recently that B3LYP offers a reasonable treatment of intramolecular hydrogen bonding interactions such as in scytalone.11 Transition state searches were carried out at the B3LYP/6-31G(d) level of theory, and the solvent effect was included in the calculation with a self-consistent reaction field method as in Jaguar 3.5.12 Specifically, the Poisson-Boltzmann solver was used. The dielectric constant of water (80.37) and a probe size of 1.4 Å were used in these calculations. This solvation model was previously applied to a reaction pathway calculation in solution and was shown to give reasonable results.13 Two lower energy ground state conformations of scytalone in the gas phase were identified (Table 1). Previous work
Table 1. Calculated Relative Energies for Scytalone Conformations (kcal/mol): Gas Phase and Solution conformer (with respect to the H-C-O-H angle)
gas phase
solution
gas phase
solution
anti gauche 1 gauche 2
0.2 1.0 0.8
0.7 1.8 1.0
0a -0.1 -0.4
0 -0.05 0.3
conformation A
conformation B
a The total electronic energy for the anti conformer (-687.98479 Hartree) is used as the reference.
had delineated these two conformations in reference to having a water molecule induce enolization by donating a hydrogen atom to the scytalone carbonyl.14 The two conformations can be described as envelope shapes with the C3 scytalone carbon forming the envelope flap and the C3 hydroxyl in either an axial or equatorial position (Figures 1A and 1B). At least three orientations of the hydrogen atom on the C3 hydroxyl group for each conformation are possible, and the most stable orientation for each was determined. The calculated energies suggest that the equatorial and axial scytalone conformations are close in energy with the former favored by 0.6 kcal/mol. The polarized continuum solvent model (PCM) developed by Tomasi and co-workers15 also favors the equatorial conformation (Figure 1B) of scytalone in aqueous solution (by 0.7 kcal/mol) at the B3LYP/6-31G(d) level of theory. In addition to the two envelope conformations, we examined the scytalone boat conformation in which both the C3 hydroxyl and the C2 pro-R hydrogen are eclipsed (both are in axial positions, see Figure 1C). According to the B3LYP/6-31G(D) calculations, this con(10) (a) Becke, A. D. J. Chem. Phys. 1993, 98, 5648-5652. (b) Lee, C.; Yang, W.; Parr, R. G. Phys. ReV. B 1988, 37, 785. (11) Schiott, B.; Iversen, B. B.; Madsen, G. K. H.; Bruice, T. C. J. Am. Chem. Soc. 1998, 120, 12117. (12) (a) Tannor, D. J.; Marten, B.; Murphy, R.; Friesner, R. A.; Stikoff, D.; Nicholls, A.; Ringnalda, M.; Goddard, W. A., III; Honig, B. J. Am. Chem. Soc. 1994, 116, 11875. (b) Jaguar 3.5. Schrodinger, Inc.: Portland, OR, 1998. (13) Zheng, Y.-J.; Ornstein, R. L. J. Am. Chem. Soc. 1997, 119, 648. (14) Zheng, Y.-J.; Bruice, T. C. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4158. (15) (a) Miertus, S.; Scrocco, E.; Tomasi, J. Chem. Phys. 1981, 55, 117129. (b) Tomasi, J.; Persico, M. Chem. ReV. 1994, 94, 2027. (16) Allinger, N. L. J. Am. Chem. Soc. 1977, 99, 8127. Org. Lett., Vol. 2, No. 11, 2000
formation is 6.7 kcal/mol higher in energy than the envelope conformation with an equatorial C3 hydroxyl group (the anti conformer). As a comparison, there is a 6.5 kcal/mol difference between the cyclohexane chair and boat conformations.16 There are fewer vicinal eclipsing interactions in the scytalone boat relative to the cyclohexane boat, but the fusion of the aromatic ring adds a van der Waals repulsion between the nearly coplanar C4 pro-R and C5 hydrogens. Furthermore, the C1 pucker of the boat conformation misaligns the carbonyl out of plane for intramolecular hydrogen bonding with the C8 hydroxyl. Though this eliminates the boat conformation from consideration for the solvolytic enolization, a slight twist of the boat from a perfect 0° eclipsing dihedral angle affords a lower energy conformation, but not one that is well populated by the NMR analysis. It is this conformation that is relevant to the enzymecatalyzed dehydration. Attempts to locate a local minimum without a torsional constraint failed and instead afforded the envelope conformation of Figure 1A. Neither was there a local minimum identified for the alternate boat conformation wherein the C3 hydroxyl is equatorial (Figure 1D). This latter conformation is of interest since it places the more rapidly exchanged C2 pro-S hydrogen in an axial orientation. Both the theoretical ground state models and the NMR assignments for scytalone indicate a preference for the envelope conformation having the C3 hydroxyl in the equatorial orientation where the C2 and C4 hydrogen atoms anti to the hydroxyl are equatorial (Figure 1B). On the basis of mass action considerations alone, the exchange rate in aqueous media for the axial pro-R C2 hydrogen would be faster. But this is not the case as the hydrogen with the predominant equatorial orientation is more rapidly exchanged. Flipping the pucker of the solution conformation to the envelope in which the hydroxyl is axial (Figure 1A) would position the C2 pro-S hydrogen into the axial orientation for favorable removal. Enolization from this conformation is well situated for the slower elimination halfreaction, as the C3 hydroxyl is also axially orientated. Three transition states for the enolization by hydroxide were located. The enolization transition state for the conformation with an axial C3 hydroxyl has the lowest energy resembling the substrates scytalone and hydroxide (Figure 2A). The breaking C-H and forming O-H distances are 1.22 and 1.52 Å, respectively; the O-H-C angle is 164°. A second transition state is 1.9 kcal/mol higher in energy and corresponds to deprotonation of the conformation with an equatorial C3 hydroxyl (Figure 2B). The transferred proton lies 1.29 and 1.35 Å from the scytalone C2 carbon and the hydroxide oxygen, respectively, reflecting a later transition state. The third transition state (Figure 2C), which involves the deprotonation of an equatorial hydrogen, is 10 kcal/mol higher in energy and can be discounted from contributing to the course of the enolization. It is more product-like than the previous two with O-H and C-H bond distances of 1.40 and 1.24 Å, respectively. The transition state calculations for enolization show that the course for removal of the axially orientated pro-S C2 hydrogen by hydroxide (Figure 2A) is lower in energy than 1543
Figure 2. Three calculated transition states and relative energies for scytalone enolate formation in water at the B3LYP/6-31G(d) level of theory with a SCRF solvation model: A, axial hydroxide approach to the scytalone pro-S hydrogen; B, axial hydroxide approach to the scytalone pro-R C2 hydrogen; C, equatorial hydroxide approach to the scytalone pro-S hydrogen.
that for removal of the axially oriented pro-R hydrogen (Figure 2B). The activation energy in proceeding from the envelope in Figure 1A to the transition state in Figure 2A is 1.1 kcal/mol lower than that when proceeding from the envelope in Figure 1B to the transition state in Figure 2B. This correlates well with the observed 13-fold selectivity (∆G ) 1.5 kcal/mol) for removal of the scytalone pro-S over the pro-R hydrogen. The earlier timing and lower energy of activation in the transition state for removal of the pro-S hydrogen can be explained, in part, by the solvation sphere around the C3 hydroxyl. The solvent hydroxide and C3 hydroxyl are antiperiplanar about as far from one another
1544
as possible for the Figure 2A transition state. In contrast, the approach of the hydroxide is only 48° offset from the C3 hydroxyl in the Figure 2B transition state. To accelerate enolization for the dehydration of scytalone, SD must select a higher energy twist-boat conformation relative to the ground state conformation represented in Figure 1B. It does so through strategic positioning of His85 and His110 relative to the elements of water that are lost during the dehydration. Catalysis is also promoted by meshing the pKa of the C2 pro-R hydrogen that is removed with that of His85. The pKa of the former is lowered by protonation of the scytalone carbonyl, and the pKa of the latter is raised by the presence of an aspartate residue backside to the imidazole. One might speculate that the hydrophobic residues in the SD active site offer a measure of shape recognition for the boat conformation. In the event, nature has engineered a (4 × 107)-fold rate acceleration for the SD-catalyzed enolization3a from a less populated conformation invoking a change in stereoselectivity. When comparing the solvolytic and SD-catalyzed enolization rates for the leaving pro-R hydrogen, there is a (5 × 108)-fold acceleration. By removing the pro-R hydrogen from the scytalone twist-boat conformation, the enzyme has a proton readily positioned to accelerate subsequent hydroxide loss. Hence, SD improves catalytic efficiency through its recognition of the scytalone boat conformation rather than the lower energy envelope with an economy of proton shuttling functionality. In summary, we have established that in water the C2 pro-S hydrogen of scytalone exchanges more rapidly than the C2 pro-R hydrogen with a transition state that is not dictated by the solution conformation. We believe instead that the transition state follows a course that minimizes the repulsion between the solvent spheres surrounding the base that removes the C2 hydrogen atom and the C3 hydroxyl that is ultimately eliminated in the dehydration. OL0058590
Org. Lett., Vol. 2, No. 11, 2000
