Synthesis and study of a 6-amino-5-oxo-3H,5H-uracil and derivatives. The structure of an intermediate proposed in mechanisms of flavin and pterin oxygenases

The possibility that the spectrum of intermediate two, seen in the course of reaction of flavoenzyme phenol hydroxylases, may be attributable to iminol isomers of a flavin-derived 6-arylamino-5-oxo(3H,5H)uracil (flavin mixed-function oxidase/hydroxylation) ALBERT WESSIAK, J. BARRY NOAR, AND THOMAS C. BRUICE* Department of Chemistry, University of California, Santa Barbara, CA 93106 Contributed by Thomas C. Bruice, September 19, 1983 ABSTRACT A commonly held view of the mechanism of flavin mixed-function oxidases is that enzyme-bound 4a-hydroperoxyflavin (4a-FIHOOH) undergoes ring opening to provide a carbonyl oxide (IV), which, after transferring an oxene equivalent to substrate, yields a 6-arylamino-5-oxo(3H,5H)-uracil (I). The latter is then thought to undergo ring closure to form a 4a-hydroxyflavin (4a-FIHOH), which by loss of water yields flavin (scheme I). A close structural analogue of I (i.e., III) has been synthesized. Comparison of the spectra of III (and II), taken in solvents of widely differing dielectric constants and in a strongly basic medium, with those of the intermediate(s) observed to be formed in time between 4a-FIHOOH and 4a-FIHOH has shown that the enzyme-bound intermediate(s) does not resemble spectrally I nor its iminol tautomers.

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confidence: 99%

“…We have recently succeeded in preparing II (36,37). The UV/visible spectrum of II (Xmax 342 nm; E, 7,100 M-1 cm-1; acetonitrile) was found to be quite different from that of the enzyme-bound intermediate postulated to be I.…”

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confidence: 99%

The possibility that the spectrum of intermediate two, seen in the course of reaction of flavoenzyme phenol hydroxylases, may be attributable to iminol isomers of a flavin-derived 6-arylamino-5-oxo(3H,5H)uracil.

Wessiak¹,

Noar²,

Bruice³

1984

“…It is generally accepted that flavin-4a-hydroperoxides form in the active sites of flavindependent mixed-function oxidases when reduced flavin reacts with molecular oxygen (6)(7)(8)(9)(10)(11)(12). It was determined that the monooxygen transfer involving 4a-hydroperoxyflavins does not occur from a carbonyl oxide formed on ring opening of the isoalloxazine ring between C4 and C4a (13,14), as originally proposed (1), but by nucleophilic attack on the terminal oxygen of the flavin 4a-hydroperoxide (15). Dioxygen transfer from the oxygen anion 4a-FlEt-OO Ϫ is known in model reactions (3,(16)(17)(18)(19)(20), but there is no evidence for the existence of a flavin dioxygenase enzyme.…”

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confidence: 99%

Theoretical investigation of the [1,2]-sigmatropic hydrogen migration in the mechanism of oxidation of 2-aminobenzoyl-CoA by 2-aminobenzoyl-CoA monooxygenase/reductase

Torres

Bruice

1999

Self Cite

The flavin hydroperoxide at the active site of the mixed-function oxidase 2-aminobenzoyl-CoA monooxygenase͞reductase (Azoarcus evansii) transfers an oxygen to the 5-position of the 2-aminobenzoyl-CoA substrate to provide the alkoxide intermediate II ؊ . Hydrogen migration from C5 to C6 follows this monooxygenation. The nature of the monooxygenation intermediate and plausible competing reactions leading to hydrogen migration have been considered. Ab initio molecular orbital theory has been used to calculate structures and electron distributions in intermediate and transition state structures. Electrostatic potential surface calculations establish that the transition state and product, associated with the C5 to C6 hydrogen transfer, are stabilized by electron distribution to the benzoyl-CoA thioester carbonyl oxygen. This is not so for the transition state and product associated with hydrogen transfer from C5 to C4. The activation energy for the 5,6-shift is 2.5 kcal͞mol lower than that for the 5,4-shift. In addition, the product of the hydrogen 5,6-shift is more stable than is the product of the hydrogen 5,4-shift, by Ϸ6 kcal͞mol. These results explain why only the shift of hydrogen from C5 to C6 is observed experimentally. Oxygen transfer and hydrogen migration almost coincide in the gas phase (activation energy of Ϸ0.6 kcal͞mol, equivalent to a single bond vibration). Enzymatic formation of alkoxide II ؊ requires its stabilization; thus, the rate constant for its breakdown would be slower than in the gas phase. O xygenase enzymes are separated into two classes: dioxygenases and mixed-function oxidases. Dioxygenases transfer both oxygens from molecular oxygen to the substrate to provide product. Mixed-function oxidases transfer one oxygen from molecular oxygen to the substrate, and the remaining oxygen is incorporated into a water molecule. For flavin mixed-function oxidases, Hamilton proposed that oxygen reacts with enzymebound dihydroflavin to provide a 4a-hydroperoxyflavin (4a-FlH-OOH) (1). Kemal and Bruice synthesized a 4a-hydroperoxide [4a-hydroperoxy-5-ethyl-3-methyllumiflavin (4a-FlEt-OOH)] by reaction of hydrogen peroxide with the flavinium cation, N(5)-ethyl-3-methyl-1,5-dihydrolumiflavin (2). These studies made it possible to explore oxygen transfer from 4a-hydroperoxides to substrates (3). The reaction of 1,5-dihydroflavins to form 4a-hydroperoxideds has been shown to involve O 2 . and a flavin radical as intermediates (4, 5). It is generally accepted that flavin-4a-hydroperoxides form in the active sites of flavindependent mixed-function oxidases when reduced flavin reacts with molecular oxygen (6-12). It was determined that the monooxygen transfer involving 4a-hydroperoxyflavins does not occur from a carbonyl oxide formed on ring opening of the isoalloxazine ring between C4 and C4a (13, 14), as originally proposed (1), but by nucleophilic attack on the terminal oxygen of the flavin 4a-hydroperoxide (15). Dioxygen transfer from the oxygen anion 4a-FlEt-OO Ϫ is known in model reactions (3,(16)(17)(1...

“…The 4a,5-ringopened isoalloxazines (6-{[2-(dimethylamino)-4,5-dimethylphenyl]methylamino}-5-oxo-3H,5H-uracils) Ha (R3 = CH3) and lIb (R3 = H) were synthesized (14)(15)(16) to compare their spectral properties to those of enzyme-bound intermediates, observed during the hydroxylation reaction of flavindependent phenolic hydroxylases (17). Pyrimidopteridines (2,3,4,6,7,8,9 -heptahydro -2,4,6,8 -tetraoxopyrimido -5, -4g-pteridines), III (R3 or R7 = H or CH3), were designed to provide flavin cofactor analogs with more negative redox potentials than the native FMN of FAD (18).…”

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confidence: 99%

“…The UV/visible spectra of the compounds II free in solution and those of enzyme-bound intermediates turned out to be quite dissimilar (14)(15)(16). It therefore became of interest to determine whether the effect of apoprotein interactions on the physical properties of compounds II might account for the differences.…”

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confidence: 99%

Use of riboflavin-binding protein to investigate steric and electronic relationships in flavin analogs and models.

Wessiak¹,

Schopfer²,

Yuan³

et al. 1984

We have examined the affinity of two recently synthesized flavin analogs for the isoalloxazine binding site of riboflavin-binding protein (RBP). The results showed that pyrimidopteridines could bind to . This suggested that, at the FMN or FAD level, these analogs might also bind to other apoflavoproteins, thereby providing a high potential probe for flavin enzymology. In contrast, 4a,5-ringopened isoalloxazines did not bind to RBP. However, 1,10a-ring-opened flavins bind with considerable avidity (Kd about 40 nM). Evidence is presented which indicates that the 4a,5-ring-opened species adopted a nonplanar configuration which, in turn, was responsible for the lack of affinity to RBP. Steric and electronic consequences of a 4a,5 ring opening are discussed in relation to flavin-dependent phenolic hydroxylases.Riboflavin-binding protein (RBP) is a protein of small molecular weight, whose primary physiological function is storage of vitamin B2 (riboflavin, formula I in Scheme 1) (1). Similar to other flavoproteins (2), its "active site" is able to bind not only the native "cofactor" but also various analogs in which the isoalloxazine nucleus of riboflavin has been modified (3-8). In contrast to other flavoproteins, in which the N(10) side chain of the cofactor (ribityl phosphate in case of FMN, ribityl diphosphate adenosyl for FAD) has to be highly preserved to accomplish binding and catalytic function, RBP tolerates modifications of the ribityl side chain. It therefore binds a variety of molecules that resemble flavins in their steric and electronic characteristics (3,4,6,9). Due to that feature, the isoalloxazine binding site of RBP has been well characterized by physical methods and a variety of probes (10-13). It is considered to be hydrophobic, and a tyrosine and a tryptophan are thought to be part of it. Their interaction with flavins and other probes is considered to be responsible for the fluorescence quenching of bound molecules. "Sandwiching" the flavin between them may account for the preferential binding of molecules with flat geometries. Weak binding of negatively charged molecules is ascribed to the presence of a negative charge in the flavin-binding site of the protein.Recently, two additional types of flavin models have become available for investigation (Scheme 1). The 4a,5-ringopened isoalloxazines (6-{[2-(dimethylamino)-4,5-dimethylphenyl]methylamino}-5-oxo-3H,5H-uracils) Ha (R3 = CH3) and lIb (R3 = H) were synthesized (14-16) to compare their spectral properties to those of enzyme-bound intermediates, observed during the hydroxylation reaction of flavindependent phenolic hydroxylases (17). Pyrimidopteridines (2,3,4,6,7,8,9 -heptahydro -2,4,6,8 -tetraoxopyrimido -5, -4g-pteridines), III (R3 or R7 = H or CH3), were designed to provide flavin cofactor analogs with more negative redox potentials than the native FMN of FAD (18). This would enable us to gain mechanistic insights by studying the reverse reactions of flavoenzymes with high redox potentials.The UV/visible spectra of the compounds II...