1816 J. Am. Chem. Soc., Vol. 119, No. 8, 1997
Pieper et al.
18.6 (2-Me). High-resolution FAB-MS: calcd for C10H11FNO4 (M +
137.2 (C-5), 128.7 (C-4′, d, J ) 9.8), 63.0 (C-5′), 18.8 (2-Me). High-
resolution FAB-MS: calcd for C8H10FN2O2 (M + 1)+ 185.0726, found
185.0717.
1)+ 228.0672, found 228.0681.
3-Deoxy-3-fluoropyridoxine (17). Compound 16 (0.75 g, 3.30
mmol) was dissolved in anhydrous ether (8 mL) under nitrogen. To
this solution, kept in an ice bath, was added dropwise 13.5 mL of 1.0
M diisobutylaluminum hydride (4.1 equiv). The reaction was stirred
at 0 °C and quenched with an ammonium chloride solution after it
was judged to be complete by TLC (ca. 1 h). The quenched solution
was stirred vigorously, and some salts precipitated out. The pH of the
water layer was neutralized with 1 N HCl, and the resulting mixture
was thoroughly extracted with ethyl acetate. The combined organic
extracts were dried, concentrated, and purified by flash chromatography
(80% EtOAc/hexanes) to give a white solid: TLC (EtOAc): Rf ) 0.2.
The yield was 46%. Mp: 127-128 °C. 1H NMR (CDCl3): δ 8.32
(1H; s; 6-H), 4.84 (2H; d, J ) 1.6; 4′-CH2), 4.77 (2H; s; 5′-CH2), 2.52
(3H; d, J ) 3.2; 2-Me). 13C NMR (CD3OD): δ 158.8 (C-3, d, J )
256.6), 148.4 (C-2, d, J ) 19.6), 146.0 (C-6, d, J ) 5.9), 138.5 (C-5),
137.0 (C-4, d, J ) 13.4), 61.5 (C-5′), 55.9 (C-4′, d, J ) 7.6), 18.8
(2-Me). High-resolution FAB-MS: calcd for C8H11FNO2 (M + 1)+
172.0773, found 172.0760. A minor compound was also isolated
(eluted with 50% EtOAc/hexanes) which was identified as 2,5-di-
methyl-3-fluoro-4-(hydroxymethyl)pyridine in ca. 15% yield. TLC
(EtOAc): Rf ) 0.44. 1H NMR (CDCl3): δ 8.07 (1H; s; 6-H), 4.73
(2H; d, J ) 1.3; 4′-CH2), 2.44 (3H; d; J ) 3.0; 2-Me) 2.36 (3H; s;
5-Me). 13C NMR (CDCl3): δ 156.3 (C-3, d, J ) 252.7), 145.4 (C-6,
d, J ) 5.8), 144.2 (C-2, d, J ) 19.5),* 133.1 (C-4, d, J ) 12.2),*
132.0 (C-5), 55.1 (C-4′, d, J ) 5.9), 17.6 (2-Me), 15.3 (5-Me). High-
resolution FAB-MS: calcd for C8H11FNO (M + 1)+ 156.0825, found
156.0815.
3-Deoxy-3-fluoropyridoxal (18). To a mixture of dry pyridinium
chlorochromate (PCC; 25.3 mg, 0.12 mmol), sodium acetate (19.2 mg,
0.23 mmol), and powdered 3 Å molecular sieves (40 mg) in dry
methylene chloride (8 mL) was added a suspension of compound 17
(20 mg, 0.12 mmol) in 2 mL of methylene chloride. The reaction was
stirred at room temperature for 1 h and spiked with a small amount of
PCC to ensure complete oxidation. After being stirred for another 30
min, an equal volume of anhydrous ether was added, and the resulting
mixture was stirred vigorously for an additional 30 min. The solution
was then filtered through silica gel and washed extensively with ether.
The combined filtrates were evaporated to dryness to give the desired
product 18, along with a smaller amount of the 5′-oxidized product, as
a white solid. The overall yield was 85%. Product 18 exists as a
mixture of the aldehyde and the corresponding hemiacetal form in
solution, whereas the 5′-oxidized byproduct is mainly in the hemiacetal
form. Since the components of this mixture have similiar Rf values, it
was difficult to fully purify the desired product 18. Compound 18 in
its aldehyde form are as follows. 1H NMR (CDCl3): δ 10.13 (1H; d,
J ) 2.3; 4′-H), 8.75 (1H; s; 6-H), 4.88 (2H; s; 5′-CH2), 2.63 (3H; d, J
) 3.3; 2-Me). 13C NMR (CDCl3): δ 193.1 (C-4′), 160-130 (Ar C’s),
54.2 (C-5′, d, J ) 7.2), 19.3 (2-Me). 1H NMR (CDCl3) of the
hemiacetal form of compound 18: δ 8.26 (1H; s; 6-H), 6.65 (1H; d, J
) 1.9; 4′-H), 5.30, 5.05 (1H each; ABq, J ) 13.3; 5′-CH2),* 2.56 (3H;
d, J ) 3.0; 2-Me). 1H NMR (CDCl3) of the hemiacetal form of the
5′-oxidized product: δ 8.38 (1H; s; 6-H), 6.57 (1H; s; 5′-H), 5.27,
5.09 (1H each; ABq, J ) 13.3; 4′-CH2),* 2.56 (3H; d, J ) 3.0; 2-Me).
13C NMR (CDCl3) of the mixture of both hemiacetals: δ 160-130
(Ar C’s), 100.4, 99.8 (C-4′), 70.4, 68.8 (C-5′), 17.4 (2-Me’s). High-
resolution FAB-MS of 18; calcd for C8H9FNO2 (M + 1)+ 170.0617,
found 170.0609.
3-Deoxy-3-fluoropyridoxamine (20). To a solution of oxime 19
(100 mg, 0.54 mmol) in methanol (10 mL) was added 10% palladium
on activated carbon (50 mg). The resulting mixture was stirred under
hydrogen at room temperature for 5 h. The catalyst was removed by
filtration and the filtrate concentrated to dryness. The desired product
was isolated in 88% yield. TLC (40% MeOH/CHCl3): Rf ) 0.41. 1H
NMR (CD3OD): δ 8.26 (1H; s; 6-H), 4.75 (2H; s; 5′-CH2), 3.99 (2H;
d; J ) 1.4; 4′-CH2), 2.54 (3H; d, J ) 3.1; 2-Me). 13C NMR
(CD3OD): δ 159.3 (C-3, d, J ) 253.0), 148.7 (C-2, d, J ) 19.7), 146.6
(C-6, d, J ) 6.0), 139.6 (C-4, d, J ) 13.4), 138.0 (C-5), 62.0 (C-5′),
37.6 (C-4′, d, J ) 5.0), 18.9 (2-Me). High resolution FAB-MS: calcd
for C8H12FN2O (M + 1)+ 171.0855, found 171.0935.
3-Deoxy-3-fluoropyridoxamine 5′-Phosphate (8). To compound
20 (30 mg, 176 µmol) was added 10 times its weight of anhydrous
phosphoric acid under nitrogen, and the mixture was heated to 100 °C
for 24 h. Nine volumes of absolute ethanol was added slowly with
stirring to the cooled reaction mixture to yield a white precipitate, which,
after being collected and washed successively with absolute ethanol
and ether, was dissolved in a minimal amount of water and brought to
about pH 6 with concentrated ammonium hydroxide. This mixture
was applied to the top of an Amberlite XE-64 column (a fine mesh,
weak cation exchange resin) in the washed free acid form. The effluent
fractions from the column, on elution with water, were examined by
TLC. Fractions of the desired product were combined and lyophilized
to give a white solid in 43% yield. TLC (1-propanol:NH4OH:H2O )
6:3:1): Rf ) 0.45. Mp: 205-206 °C. 1H NMR (D2O): δ 8.40 (1H;
s; 6-H), 5.10 (2H; d, J ) 7.4; 5′-CH2), 4.47 (2H; d, J ) 1.0; 4′-CH2),
2.59 (3H; d, J ) 3.0; 2-Me). 13C NMR (D2O): δ 159.6 (C-3, d, J )
256.6), 150.9 (C-2, d, J ) 19.1), 147.5 (C-6, d, J ) 5.7), 134.8 (C-5,
d, J ) 7.3), 131.1 (C-4, d, J ) 13.4), 65.4 (C-5′, d, JC-P ) 4.9), 36.2
(C-4′, d, JC-F ) 5.1), 19.5 (2-Me). 19F NMR (D2O as solvent, CFCl3
as external standard): δ -125.9 (d, J ) 2.7). 31P NMR (D2O as solvent,
85% H3PO4 as the external standard): δ 0.84 (t, J ) 7.4). High-
resolution FAB-MS: calcd for C8H13FN2O4P (M + 1)+ 251.0596, found
251.0586.
[4′-3H]-3-Deoxy-3-fluoropyridoxamine (21). To a mixture of
sodium borohydride (19 mg, 0.5 mmol) and titanium(IV) chloride (54.9
µL, 0.5 mmol) in dry 1,2-dimethoxyethane (DME, 6 mL) at 0 °C was
added [3H]NaBH4 (1 mmol, 100 mCi), followed by compound 19 (100
mg, 0.538 mmol) in DME (20 mL). After stirring for 4 h, additional
titanium(IV) chloride (295 µL, 2.69 mmol) and sodium borohydride
(204 mg, 5.38 mmol) in DME (10 mL) were added to drive the reaction
to completion. The reaction was quenched 17 h later with water, and
the solution was neutralized with an ammonium hydroxide solution.
The product was extracted with ether, and the combined organic extracts
were dried, concentrated, and purified by flash chromatography (40%
MeOH/CHCl3) to give 21 as a white solid in 52% yield. The specific
activity of tritium of this sample was 1.98 mCi/mmol.
[4′-3H]-3-Deoxy-3-fluoropyridoxamine 5′-Phosphate (22). The
labeled compound 21 (38 mg, 0.22 mmol) was mixed with the unlabeled
20 (48 mg, 0.28 mmol) and then reacted with anhydrous phosphoric
acid (1.5 g) under nitrogen. After heating at 100 °C for 36 h, the
mixture was cooled and treated with absolute ethanol (1.5 mL). The
resulting precipitate was collected by filtration, washed with ether and
absolute ethanol, and redissolved in a minimal amount of water. This
solution was brought to pH 6 with concentrated ammonium hydroxide
and purified by an Amberlite XE-64 column. Fractions containing the
desired product were combined and lyophilized to give 22 as a white
solid (39 mg) in 31% yield. The specific activity (SA) of tritium of
this sample was 0.89 mCi/mmol.
3-Deoxy-3-fluoropyridoxal Oxime (19). To a suspension of 18
(20 mg, 0.12 mmol) in water (3 mL) were added sodium acetate (12.9
mg, 0.16 mmol) and hydroxylamine hydrochloride (12.3 mg, 0.18
mmol). This mixture was heated to 100 °C for a few minutes until the
solids dissolved and then cooled to room temperature, during which
the oxime products precipitated. The precipitates were collected,
washed with water, and dried. Two oxime products were formed, and
they were separable by flash chromotography (60% EtOAc/hexane).
The desired 4′-oxime product 19 was isolated in 78% yield. Mp: 208-
210 °C dec. TLC (EtOAc): Rf ) 0.35. 1H NMR (CD3OD): δ 8.46
(1H; s; 4′-CH), 8.45 (1H; s; 6-H), 4.83 (2H; s; 5′-CH2), 2.55 (3H; d, J
) 3.2; 2-Me). 13C NMR (CD3OD): δ 158.3 (C-3, d, J ) 258.6), 148.3
(C-2, d, J ) 18.5),* 145.9 (C-4, d, J ) 6.7),* 144.0 (C-6, d, J ) 5.7),
GC-MS Assay. The E1-E3 product formation was detected by a
previously reported GC-MS procedure.4b A few minor modifications
included (1) the enzymes were removed by placing the solution is a
microcon 10 microconcentrator (Amicon, Beverly, MA) and spinning
at 13000g for 30 min; (2) the crude products were not purified by
HPLC, and (3) the amounts of reagents used in the derivatization were
cut in half. The same procedure was also repeated using F-PMP (0.5
mg, 2.0 µmol) in place of PMP. Incubations with denatured E1 and/or
E3 under identical conditions served as the controls.