Molybdenum Enzymes, Cofactors, and Model Systems - American

Chapter 6

Pterins, Quinoxalines, and Metallo-Ene-Dithiolates Synthetic Approach to the Molybdenum Cofactor Downloaded by UNIV OF CALIFORNIA SAN FRANCISCO on December 13, 2014 | http://pubs.acs.org Publication Date: July 26, 1993 | doi: 10.1021/bk-1993-0535.ch006

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Robert S. Pilato , K. Eriksen , M . A. Greaney , Y. Gea , E. C. Taylor , S. Goswami , L. Kilpatrick , T. G. Spiro , A. L. Rheingold , and Edward I. Stiefel 2

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Exxon Research and Engineering Company, Clinton Township, Route 22 East, Annandale, NJ 08801 Department of Chemistry, Princeton University, Princeton, NJ 08540 Department of Chemistry, University of Delaware, Newark, DE 19711

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The syntheses of the molybdopterin decomposition products, such as Form A , provide starting points for the total synthesis of Moco. Specifically, C(6) alkyne-substituted pterins similar to Form A react with metal polysulfides to yield the molybdenum-ene-dithiolate moiety of Moco. Syntheses are reported for the pterin-ene-dithiolate and quinoxaline-ene-dithiolate complexes (C H ) Mo- {S C [C(O)R]R'} where R = CH3, CH OSiPh tBu, and R' = N-pivaloyl-6-pterin, 2quinoxaline. Intermediates in the preparation are the unprecedented ene-1-thiolate-2-perthiolate (trithiolene) complexes. Reactions of the molybdenum ene-dithiolate complexes include the oxidation of Mo(IV) to Mo(V), reduction of the side-chain carbonyl, and transmetallation. The complexes (C H ) Mo-{S C [C(O)CH ]R'} have been ≥81% S enriched. Resonance Raman studies identify a υ(Mo-S) stretch at 350 cm-1, similar to a band in DMSO reductase. Fluorescence of the oxidized pterin is quenched in the metal ene-dithiolate and ene-1thiolate-2-perthiolate complexes. 5

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The proposed structure for Moco, the cofactor found in all the molybdoenzymes except nitrogenase, is shown infigure1. Thefirstcoordination sphere of molybdenum has been defined by EXAFS (Extended X-ray Absorption Fine Structure) (7-5) and all of the enzymes appear to possess thiolate ligation of molybdenum and (with the possible exception of dissimilatory nitrate reductase and formate dehydrogenase) either dioxo or oxo-sulfido ligation of the Mo(VI) state. The molybdopterin derivatives Form A , camMPT, Form Β and urothione, shown in figure 2 have been isolated and when combined with EXAFS results have led to the proposed Moco structure (4-7). The isolated C(6)-substituted pterins possess unsaturated carbon atoms α and β to the pterin ring. Sulfur functionalities are associated with these unsaturated carbons in camMPT, Form Β and urothione. The formation of these compounds is consistent with the decomposition of the proposed 4

Current address: Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742 0097-6156/93Α)535-0083$06.00/Ό © 1993 American Chemical Society

In Molybdenum Enzymes, Cofactors, and Model Systems; Stiefel, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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MOLYBDENUM ENZYMES, COFACTORS, AND MODEL SYSTEMS

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OP0 -

Downloaded by UNIV OF CALIFORNIA SAN FRANCISCO on December 13, 2014 | http://pubs.acs.org Publication Date: July 26, 1993 | doi: 10.1021/bk-1993-0535.ch006

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Figure 1. The proposed structure for Moco with Mo(VI).

Form Β

camMPT

Figure 2. The oxidative décomposition products of Moco and the Moco metabolite urothione.



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PILATO ET AL.

Pterins, Quinoxalines, and Metallo-Ene-Dithiolates

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1,2-ene-dithiolate Moco structure (see Rajagopalan, Κ. V., Chapter 3 and Meyer, O., Chapter 4, this volume). Moreover, the oxidative decomposition of quinoxalinesubstituted 1,2-ene-dithiolate molybdenum complexes has yielded thiophenes similar to Form Β and urothione (8). The structures of the molybdopterin derivatives; Form A, dephospho Form A, Form Β and urothione, have been confirmed by direct synthesis. The absolute configuration of Form A was determined by comparing the circular dichroism spectra of synthetic Form A (the S isomer) with enzyme isolated Form A (9-70). The synthesis of the S isomer of Form A is shown in scheme 1. Synthetic Strategy for Moco Our current research program is directed at the total synthesis of Moco and includes both synthetic organic and inorganic components (77). We have concentrated to date on the synthesis of the pterin substituted 1,2-ene-dithiolate portion of Moco, termed molybdopterin. A limited number of 1,2-ene-dithiolates can be isolated as the disodium salts and many of these contain electron-withdrawing groups (72-77). However, many 1,2-ene-dithiolates can be prepared as metal complexes where the 1,2ene-dithiolate is stabilized as a chelating ligand (as is suggested for Moco) (18-28). Synthetic routes to métallo-1,2-ene-dithiolate complexes include the reactions of metal complexes (containing labile ligands) with dithines (26), dithiolium salts (79,29) or ahydroxy ketones in the presence of P4S10 (17), as shown in equations 1, 2, and 3, respectively. Another interesting synthetically useful method for the preparation of dithiolenes is the reaction of a metal polysulfido complex with an alkyne as shown in equation 4 (8,11,18,21,22,24). The previously reported synthesis of C(6)-alkynesubstituted pterin derivatives (such as Form A) (9,70) should, in conjunction with the reaction shown in equation 4, allow die preparation of molybdenum 1,2-ene-dithiolate (dithiolene) complexes that approach the structure of Moco. A proposal for the total synthesis of Moco is shown scheme 2. N-Pivaloyl pterins are used to increase the solubility of the pterin ring in common organic solvents. The pivaloyl group can be removed by saponification of the amide without decomposing the pterin. The alkyne functionality is appended to the pterin in a Pd catalyzed coupling reaction (the reaction of C(6)-chloro-pivaloyl-pterin with a terminal alkyne). The subsequent reaction of the alkyne with a metal polysulfido complex should yield a métallo-1,2-ene-dithiolate. The dithiolene need not be a molybdenum complex since dithiolene metal exchange reactions are known and in theory can be used to introduce the [Mo02] or [Mo(0)(S)] fragmentonce the side-chain is complete and the pivaloyl protecting group removed. Our goal is to prepare biologically active materials. At any point in die synthesis the biological activity can be tested with the Nit-1 assay, the standard test for Moco activity (30,31). In selecting a metal polysulfido complex for the synthesis of molybdopterin (scheme 2) several factors must be considered. First, a robust metalframeworkis required that will allow subsequent reaction of the pterin and the appended side chain of the dithiolene complex. Second, it would be useful for the metal dichloride derivative of the framework to be relatively insoluble in common solvents to drive the metal chloride-metal-1,2-ene-dithiolate metathesis (used to introduce the [Μοθ2] or [Mo(0)(S)] functionalities) (76). Third, to simplify subsequent reactions and to better approximate the active site, the polysulfido complex should yield a mono-1,2ene-dithiolate complex upon reaction with die alkyne rather than die more common trisene-dithiolate complex. Finally, it would be advantageous to find a metal framework 2+

2+

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MOLYBDENUM ENZYMES, COFACTORS, AND MODEL SYSTEMS

(a) (b) (c) (d)

CBr , PPh (2 equiv), CH C1 ,0°CtoRT. n-BuLi (2 equiv), THF, -78°CtoRT. Pd(o-Ac) , (o-tolyl^P, Cul, Et N, CH CN, 100°C. 0.5 Ν HC1, dioxane, reflux. 4

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Scheme 1



6.

Pterins, Quinoxalines, and Metcdlo-Ene-DUhiolates

PILATO ET AL.

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L' L X

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RV^SN^R

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R - ^ ^ R '

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Molybdenum Enzymes, Cofactors, and Model Systems - American

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