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Molecules 2001, 6
4,5-dimethyl-2-phenylazo-N-ribityl 5'phosphate-bis[2-(methylsulfonyl)ethyl]-aniline (5)
Compound 1 (5.0 g) was dissolved in absolute pyridine (100 mL) and the phosphorylating agent
(7.5 g) was added. The clear solution was then stirred at room temperature. After one hour about 50%
of the starting material was converted to the product as evidenced by tlc (Silica Gel Plastic Plates,
Kodak no. 13181, mobile phase: n-butanol/EtOH/H20=50/15/35). At this point additional
phosphorylating agent (3.0 g) was added. After a total reaction time of 3.5 h the reaction was almost
completed. It was noted that by-product(s) formed after longer reaction times, therefore the reaction
was quenched after 3.5 h by pouring the reaction mixture into 1 M pH 7 phosphate buffer (600 mL),
containing ice. The small amount of precipitate formed, some residual starting material, was removed
by filtration and the clear solution several times extracted with n-butanol. The collected organic phase
was then evaporated to dryness under reduced pressure. The residue was dissolved in ethyl acetate and
chromatographed on a Kieselgel 60 column (7 x 15 cm). Elution with ethyl acetate gave a small
amount of an unidentified by-product and residual starting material together with a small amount of the
aforementioned secondary product. The main product was eluted with ethyl acetate-10% methanol.
The solution of the main fraction was then evaporated to dryness under reduced pressure and the
residue dissolved in ethyl acetate, and again freed from solvent by evaporation. This procedure was
repeated until a crystalline product was obtained. The yield of 5 was 6.5 g (72 %). The product should
1
be stored under dry conditions (hygroscopic!). H-NMR (DMSO): 8.95 (1 H, s, broad), NH; 7.81 (1
H), 7.75 (1 H), 7.25 (1 H), 7.50 (1 H), 7.47 (1 H), 7.41 (1 H, d), 6.72 (1 H, d), aromatic protons;
5.7-5.0 (7 H, multiplet), ribityl side-chain; 4.01 (2 H, doublet), 3.62 (2 H, doublet), O-CH2-CH2-SO2;
3.00 (3 H), 2.97 (3 H), SO2CH3; 2.31 (2H), NH-CH2; 2.25 (3 H, s), CH3 (5α); 2.20 (3 H, s), CH3 (4α).
13C-NMR (DMSO; proton-decoupled): 156.2 (s), C(2); 146.7 (s), C(1), 146.2 (s), C(1’); 138.2 (s),
C(4); 133.4 (d), C(4’); 133.1 (d), C(2’,6’); 126.9 (s), C(5 ); 125.4 (d), C(3’,5’); 117.1 (s), C(3); 117.0
(s), C(6); 76.6 (d), CH2SO2*; 74.1 (d), OCH2*; 70.1 (d), 1ε*; 67.0 (d), 1δ ; 62.5 (d), 1γ*; 59.0 (d),
1ζ*; 49.3 (d), SO2CH3; 48.6 (d), SO2CH3; 46.1 (d), NHCH2; 24.1 (s), CH3 C(5α); 22.1 (s), CH3 C(4α);
(Note: * indicates tentative assignments). 31P-NMR: 2.6 (d); 2.7 (d); syn and anti forms.
7,8-dimethyl-10-ribityl-5'phosphate-bis(2-(methylsulfonyl)ethyl)-3,4-dihydro-isoalloxazine (6)
Compound 5 (0.5 g) was suspended in glacial acetic acid (15 mL) and dissolved by warming, then
Pd/C was added and the compound hydrogenated under atmospheric pressure. After completion of the
hydrogenation reaction the colorless solution was filtered under anaerobic conditions into a 250 mL
round bottom flask and the solvent evaporated under reduced pressure to a small volume of about 3-4
mL. The residual solution was then flushed with nitrogen while isodialuric acid (2, 0.3 g), dissolved in
warm MeOH (3mL) was added. To the mixture was then added ethyl acetate (25 mL) and the mixture
flushed with nitrogen to provide an anaerobic solution. The solution was sealed with a rubber stopper
and the reaction mixture stirred over night at room temperature and protected from light. The
precipitate formed was filtered, washed with ethyl acetate and dried with ether. The yield of crude