Deazaflavines

The UV spectra have been used in studies of protonation and related covalent hydration, structural assignments and tautomerism (see appropriate Sections), as well as in studies of bridgehead addition to 5-deazapterins (79MI21500, 78TL2271) and related 5-deazaflavin derivatives (80JA1092). 

Oxidative substitutions at ring junction positions in various tetrahydro-5-deaza-pterins (79JA6068) and -flavins (77JA6721) have been studied, e.g. to give (13), and the oxidation-reduction reactions of 5-deazaflavins (e.g. 78CL1177, 80CPB3514) across the 1,5-positions, e.g. (19) (20), are involved in their co-enzymic role in enzymic oxidations (see Section... 

The 5-chloro in 5-deazaflavins is reactive (7SJHC181), but not the 8-chloro, in contrast to the flavin case (79LA1802), and 4-chloropyrimido[4,5-Z>]quinolinium salts are readily hydrolyzed (76JCS(Pl)l3l). 

The NMR spectra of some derivatives of the benzo fused pyrido[3,4-f ]quinoxaline (1-deazaflavin) (391) system have been recorded (74JCS(P1)1965). 

IR spectroscopy has also been used in structural problems in 2- and 3-hydroxypyrido[3,4-f ]pyrazines (63JCS5156), in 8-oxopyrido[2,3-f ]pyrazine-7-acids (73MI21501) and in the pyrido[3,4-f ]quinoxaline field (74JCS(P1)1965). IR spectra were recommended for the distinction of isomeric products in the Isay reaction (Section 2.15.15.6.1) (71TH21500) UV spectra were not satisfactory. The Raman spectra of a number of 1- and 3-deazaflavin analogues have been recorded and discussed (80BBA(623)77). 

The UV spectra of 1- and 3-deazaflavins have been measured 74JCS(P1U965, 78B1942), and used in the latter instance in a study of their interaction with enzymes 79B3635). 

No systematic study of the mass spectra of pyridopyrazines has been noted, but those of 2,3-dialkyl and 2,3-diaryl derivatives have been recorded 750MS97), and mass spectrometry has been used in the elucidation of problems in the reactions of pyrido[2,3-f ]pyrazines with amide ion (including use of and derivatives) (79JHC305), and of pyrido[2,3-f ]pyrazinium salts with indoles (78ZOR431). The mass spectra of some 1-deazaflavins have been recorded (74JCS(P1)1965). 

The values have been measured by a rapid reaction method, as have those for a series of 5-hydroxy and 8-hydroxy analogues of oxine (e.g. 54JCS505, 66JCS(B)436), and for other deazaflavins and pyridopyrazines (78B1942, 68JOC2393). 

Oxo substituents are, in a few cases, methylated with diazomethane to give methoxy derivatives in addition to the major iV-methylation, and oxo groups in the 3-deazaflavins have been acylated. 

Apart from the nuclear bromination observed (Section 2.15.13.1) in the attempted radical bromination of a side-chain methyl group leading to (396), which may or may not have involved radical intermediates, the only other reaction of interest in this section is a light-induced reduction of certain hydroxypyrido[3,4-f)]pyrazines or their 0x0 tautomers analogous to that well-known in the pteridine field (63JCS5156). Related one-electron reduction products of laser photolysis experiments with 1 -deazaflavins have been described (79MI21502). 

Deazaflavin, 5-amino-synthesis, 3, 222 5-Deazaflavin, tetrahydro-oxidative substitutions, 3, 205 Deazaflavinium salts synthesis, 3, 228 5-Deazaflavinoids, 3-aryl-synthesis, 3, 219 Deazaflavins... 

Reactions of anilinouracils and CDI lead to 5-deazaflavins, as shown in the following scheme 11501... 

In analogy to this reaction, a substituted anilinouracil with CDI in trifluoroacetic acid was shown to yield the 5-deazaflavin with a trifluoromethyl group in the 5-position [151]... 

Scheme 2 Mechanism of repair of cyclobutane pyrimidine dimers (CPD) by a CPD photolyase. 8-HDF 8-hydroxy-5-deazaflavin, ET electron transfer. FADH reduced and de-protonated flavin-coenzyme...

Flavin Coenzymes.—5-Deazaflavin-adenine dinucleotide (2) can be prepared from the 5-deazaFMN,21 using a FAD pyrophosphorylase from rat liver.22 When the apoprotein of D-amino-acid oxidase from pig kidney is reconstituted with (2), no oxidation of D-alanine is observed, although the flavin chromophore in the reconstituted enzyme is reduced on the addition of DL-amino-acids.22 This has been interpreted as indicating that hydrogen transfer from the amino-acid to (2) can still... 

Reduction of A. vinosum hydrogenase in an ETIR cuvette by illumination in the presence of deazaflavin... 

Figure 1.11 Reduction of A. vinosum hydrogenase in an FTIR cuvette by illumination in the presence of deazaflavin. An Ar-flushed solution of enzyme in the ready state, supplemented with deazaflavin and EDTA, was illuminated with white light for periods of about 4 min. After each illumination a spectrum was recorded. Reduction proceeds from the front to the back. Using the overview in Fig. 7.6, one can easily identify the several states of the enzyme by looking at the v(CO) frequency. Adapted from (Pierik et al. 1998a).

Alex, L. A., Reeve, J. N., Orme-Johnson, W. H. and Walsh, C. T. (1990) Cloning, sequence determination, and expression of the genes encoding the subunits of the nickel-containing 8-hydroxy-5-deazaflavin reducing hydrogenase from Methanobacterium thermoautotrophicum delta H. Biochemistry, 29, 7237-44. 

Michel, R., Massanz, C., Kostka, S., Richter, M. and Fiebig, K. (1995) Biochemical characterization of the 8-hydroxy-5-deazaflavin-reactive hydrogenase from Methanosarcina barkeri Fusaro. Eur. J. Biochem., 233, 727-35. 

Muth, E., Morschel, E. and Klein, A. (1987) Purification and characterization of an 8-hydroxy-5-deazaflavin- reducing hydrogenase from the archaebacterium Methanococcus voltae. Eur. J. Biochem., 169, 571-7. 

Fig. 7. PTIR spectra of recombinant R/jodreius NPl. (a) Ferric NPl (3.0 mM) exchanged into D2O (50 mM citrate/NaOD, measured pH 5.6), path length 13 xm. (b) Perrous-CO derivative (6.7 mM) in 50 mM Tris, 50 mM EDTA, 5 mM Na2S204, pH 8, path length 56 p,m (>90% CO complex), (c) Ferric-NO derivative (3.0 mM) in buffer identical to that of (a), path length 13 p,m (73% NO complex), (d) Ferric-NO derivative (11.0 mM) in buffer identical to that of (b) with 1 mM deazaflavin rather than 5 mM Na2S204, but not illuminated. The path length of the IR cell was 13 p,m (95% NO complex). AH spectra were recorded at a resolution of 2 cm and are averages of 800 scans. Reproduced with permission from Ref. 49).

By this time it was demonstrated that the [3Fe-4S]W+ form of aconitase is inactive, while the [4Fe-4S]2+ form is active. How is the activity of the enzyme affected by the oxidation state of the [4Fe-4S] cluster Because the active enzyme contains a [4Fe-4S]2+ cluster, either the 3+ or 1+ oxidation states may also be stable. The 3+ state is unstable since oxidation of the [4Fe-4S]2+ resulted in the immediate loss of a ferrous ion and conversion to a [3Fe-4S]i+ cluster (46,47). However, reduction of active aconitase by sodium dithionite or photoreduction in the presence of deazaflavin produced in high yields an EPR signal characteristic for [4Fe-4S]l+ clusters (47). When active enzyme within an anaerobic assay cuvette was photoreduced, the activity of the enzyme dropped to 1/3 of its initial value. Further photoreduction resulted in cluster destruction. Then, if the enzyme is reoxidized with air, the activity returned to its original value. This demonstrated that the redox state of the cluster can modulate the enzyme activity. A scheme summarizing the cluster interconversions and various redox states of the Fe-S cluster of aconitase is shown below. 

Figure 7. EPR absorption derivative spectra of photoreduced active aconitase. Enzyme ( 5 mg/ml) in 100 mM Hepes, pH 7.5, plus 5 pM deazaflavin and 10 mM potassium oxalate was photoreduced in the presence of either A) 10 mM tricarb-allylate, B) 1 mM citrate, or C) 10 mM rrans-aconitate. The numbers above each spectrum are the g-values of prominent features. Experimental conditions for obtaining EPR spectra were 13 K, 1 milliwatt microwave power, 0.8 mT modulation amplitude, and 9.24 GHz microwave frequency.

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