[148] Chemical synthesis of flavin coenzymes

458

FLAVINS AND DERIVATIVES

[148]

The riboflavin analogs described are converted under mild conditions by acid hydrolysis or air oxidation to their parent compound riboflavin (VIII). The derivatives with a univalent functional group in position 2 of the isoMloxazine ring are much more basic and therefore protonated at a higher pH than for example 2-thioriboflavin (see the table). ~,7 The effect of pH on these derivatives thus distinguishes them from other derivatives with similar spectra (see the table). e K. H. Dudley, A. Ehrenberg, P. Hemmerich,and F. Mtiller, Helv. Chim. Acta 47, 1354 (1964). 7F. Mtiller, W. Walker, and P. Hemmerich, Hdv. Chim. Acta 49, 2365 (1966).

[148]

By

C h e m i c a l S y n t h e s i s of F l a v i n C o e n z y m e s WERNER F6RY and DONALD B. MCCORMICK

General Techniques and Materials

All liquid reagents should be distilled before use. Pyridine is refluxed over sodium hydroxide for 4 hours and stored over sodium hydroxide. Aqueous solutions are concentrated under reduced pressure at low temperature (30 ° or less). For separation of the flavin mononucleotides and flavin adenine dinucleotides, the anion exchanger DEAE-cellulose is used in its chloride form. The columns are prepared by packing a thin slurry of the anion-exchange cellulose. Thin-layer chromatograms are run on MN Silica Gel S as stationary phase and developed in suitable solvents (noted at appropriate places in the text). Spots are detected by visual examination under ultraviolet light. All syntheses involving flavin compounds are carried out in a darkened room. 2-Thioriboflavin 5'-Phosphate 1 Principle. 1-D-l'-Ribitylamino-3,4-dimethyl-f-phenylazobenzene 5'phosphate 2 (I) is condensed with 2-thiobarbituric acid (II) to give 2-thioriboflavin 5'-phosphate (III) isolated as the monosodium salt. 2-Thioriboflavin 5'-Phosphate. 2-Thiobarbituric acid (II) (3.8 g, 27 millimoles) is suspended in 250 ml of a mixture of acetic acid-n-butyl alcohol (1:4). The monosodium salt of the monophosphoric acid ester of 1-D-l'-ribitylamino-3,4-dimethyl-f-phenylazobenzene2 (I) (6.17 g, 13.4 millimoles) is added, and the reaction mixture is stirred under nitrogen

1W. FSry and P. Hemmerich, Hdv. Chim. Acta 50, 1766 (1967). 2L. A. Flexser, U. Montclair, and W. G. Farkas, U.S. Patent 2,610,176 (1952).

[148]

CHEMICAL SYNTHESIS OF FLAVIN CO:ENZYMES

459

CH2OPO3H2

(HO(~H)3

H

~b/N

O (II)

(1) CH2OPOIH2 l (HO~H)3

H3C~ - . . . N . - 4 - ~

NH O

(Ill)

at 530-55 ° for 36 hours. The mixture is kept at room temperature overnight; the dark red precipitate formed is collected on a filter and washed with a mixture of n-butyl alcohol-acetic acid (99:1), acetone, and ether. The crude product is suspended in dimethyl sulfoxide (150 ml) at 50o-60 ° and filtered; the filtrate is poured into vigorously stirred acetone (600 ml). The precipitate is collected on a filter, washed with acetone, and dried (4.4 g). This crude product is suspended in water (50 ml) and adjusted to pH 7.0 with 0.1 M sodium hydroxide; the slightly turbid solution is treated with decolorizing carbon and filtered. The filtrate is adjusted to pH 4.5 with 2 M acetic acid, treated again with decolorizing carbon, and filtered into ethyl alcohol (250 ml). The precipitate is kept for 1 hour at 0 ° and then collected on a filter, washed with 80% aqueous ethyl alcohol, anhydrous ethyl alcohol, and ether to give 2.7 g (31%) of (III). The product has one Xm~xat 494 mr, (pH 7) with an eof 20,800 andis chromatographically homogeneous in acetonitrile-water (7:3) and n-butyl alcohol-acetic acidwater (3 : 1 : 1). 2-fl-Hydroxyethyliminoriboflavin 5'-Phosphate 1

Principle. S-Methyl-2-thioriboflavin 3 (IV) is phosphorylated by monochlorophosphoric acid. Without further purification, the intermediate F. Mtiller and P. Hemmerich, Helv. Chim. Acta 49, 2352 (1966).

460

FLAVINSAND DERIVATIVES

VH2OPOaH2 (HO~H)3 CH2 J HsC~ T ~ N"~N ["~SCHa

CH2OH (HOCH) 3 I

H3C~ T ~ N " ~ N

[148]

["~ SCH3 O

0

(iv)

(V)

CH2OPOaH2 I (HOCH)3

HaC~ . / ~ N / - ~

N O

(vi)

S-methyl-2-thioribofiavin 5'-phosphate (V) is reacted with/~-hydroxyethylamine to give 2-/%hydroxyethyliminoriboflavin 5'-phosphate (VI). S-Methyl-2-Thioriboflavin 5'-Phosphate. 1 Water (6.9 g, 37 millimoles) is added dropwise during 2-3 hours at 0 ° under nitrogen to phosphorus oxychloride (28.9 g, 18 millimoles) in a three-necked flask provided with condenser and drying tube. The reaction mixture is stirred for 2 hours at room temperature and then allowed to stand overnight. The mixture is stirred until all gas evolution has ceased. S-Methylriboflavin 3 (IV) (2 g, 4.7 millimoles) is added, and the mixture is stirred under nitrogen for 8-9 hours at room temperature. The solution is added dropwise to stirred anhydrous ether (500 ml) within 2-3 minutes. The temperature is maintained at 5°-10 ° by external cooling. The suspension formed is stirred for 15 minutes at room temperature; the crude material is collected on a fritted Btichner funnel and washed with anhydrous ether (100 ml). The hygroscopic precipitate is dried in vacuo at room temperature and treated for 15-20 minutes in 30 ml of a mixture of dioxane-water (6:1). The solution is treated with decolorizing carbon and filtered into ether (700 ml). The oily precipitate folmed is dissolved in minimal amounts of methyl alcohol and precipitated with ether. This procedure (dissolving in methyl alcohol and precipitating with ether) is repeated twice more. The crude (V) is dissolved in methyl Mcohol and evaporated several times from mixture of benzene-ethyl alcohol. The residue is used in the following synthesis without further purification.

[148]

CHEMICAL SYNTHESIS OF FLAVIN COENZYMES

461

2-fl-Hydroxyethylirninoriboflavin 5P-Phosphate. 1 The crude S-methyl2-thioriboflavin 5P-phosphate (V) is dissolved in anhydrous methyl alcohol (30 ml) and reacted with fl-hydroxye~hylamine (30 ml) for 90 minutes at 50° under nitrogen in a three-necked flask provided with condenser and drying tube. Acetone (250 ml) is added to the reaction mixture, which is kept 1 hour at 0% The crude material (1.8 g) is filtered and washed with acetone. The filtered residue is suspended in water (100 ml), and the suspension is adjusted to pH 8 with 1 M sodium hydroxide, treated with decolorizing carbon, and filtered. The filtrate is applied to a column (80 X 2.5 cm) of DEAE-cellulose (chloride). The nonphosphorylated flavins (riboflavin, fl-hydroxyethyliminoriboflavin) are eluted with water (2 liters). The column is then eluted by using a linear gradient with water (2 liters) in the mixing chamber and 0.8 M lithium chloride (2 liters) in the reservoir. The flow rate should be 2-2.5 ml/min, 20-ml fractions being collected. The elution of fiavin mononucleotide is followed spectrophotometrically at 451 m~. The fractions that contain 2-~-hydroxyethyliminoriboflavin 5'-phosphate (Fig. 1) are combined and concentrated at 30 ° in vacuo until a small precipitate is formed. The suspension is adjusted to pH 5 with 0.1 M perchloric acid, and the solution is treated with decolorizing

I00

75 ~t

5O

5 T

25

..A

J

Fr

\

i"

0.2 0.1

I

2 5 Liters of effluent

4

FIG. 1. Elution p a t t e r n from DEAE-cellulose of a reaction mixture containing: (I) riboflavin; (II) fl-hydroxyethyliminoriboflavin; (III) riboflavin 5'-phosphate; (IV) fl-hydroxyethylriboflavin 5~-phosphate. Compounds are eluted from an 80 X 2.5 cm column of DEAE-cellulose (chloride) with a linear gradient from 2 liters of water to 2 liters of 0.8 M LiC1. Fractions of 20 ml are collected, and aliquots are diluted appropriately with water for measurements of A451.

462

FLAVINS AND DERIVATIVES

[148]

carbon and filtered into acetone (250 ml). A liquid-liquid phase separation is removed b y dropwise addition of methyl alcohol. The precipitate obtained is k e p t for 1 hour at 0 °, collected on a filter, washed with acetone and ether, and dried i n vacuo to give 0.6 g (20%) of (VI). The product has one )kmax at 451 mt~ (pH 7) with an e of 17,300 and is chromatographicMly homogeneous in acetonitrile-water (7:3) and n-butyl alcohol-acetic acidwater (3 : 1 : 1). The phosphorylation procedure for the preparation of N-3-methylriboflavin 5'-phosphate 1 from N-3-methylriboflavin 1,4 is the same but for the following modifications: T h e phosphorylation reaction requires 36 hours. The hygroscopic precipitate (mixture of mono- and polyphosphate, see under S-methyl-2-thioribofiavin 5'-phosphate, p. 460) is treated with 68 ml of a mixture of dioxane-water (10.3:1) at room t e m p e r a t u r e overnight. Flavin

8-Bromoadenine

Dinucleotide

~

P r i n c i p l e . 8-Bromoadenosine 5'-phosphoromorpholidate is reacted with tri-n-octylammonium F M N to give flavin 8-bromoadenine dinucleotide (VII).

- Brom

FMN

5

1.5

o

o

1.0

<

IJ IJ

0.5

I

I I

FS-BromoAD f Rof,o,,n 2

I

5

Liters of effluent

FIG. 2. Elution pattern from DEAE-cellulose of a reaction mixture containing F8bromoAD. Compounds are eluted from a 40 X 2.5 cm column of DEAE-cellulose (chloride) with a linear gradient from 2 liters of 0.003 N HC1 to 2 liters of the same plus 0.1 M LiC1. Fractions of 100 ml are collected, and aliquots are diluted 10-fold with water for measurements of A450 (dashed line) and Au0 (solid line). 4 p. IIemmerich, Hdv. Chim. Acta 47, 464 (1964). 5 D. B. McCormick and G. E. Opar, J. Med. Chem. 12, 333 (1969).

[148]

CHEMICAL SYNTHESIS OF FLAVIN COENZYMES

463

Flavin 8-Bromoadenine Dinucleotide. 8-Bromoadenosine 5'-phosphoromorpholidate (0.39 g, 0.5 millimole) is condensed with tri-n-octylammonium FMN in anhydrous pyridine (100 ml) plus dimethyl formamide (5 ml) by a modification of the method for FAD synthesis by Moffatt and Khorana. 6,7 After 1 week at room temperature, the pyridine is evaporated off, the residue is dissolved in water (25 ml), and the solution is extracted twice with ether (25 ml). The aqueous phase is carefully neutralized with 1 M ammonium hydroxide and the solution is poured over a column (40 × 2.5 cm) of DEAE-cellulose (chloride). The small amount of riboflavin is washed through with water (2 liters), and the column is then eluted by using a linear gradient with 0.003 M hydrochloric acid in the mixing chamber and 0.1 M lithium chloride in 0.003 M hydrochloric acid (2 liters) in NH2

OH OH I I CH20--P--O-- P--O--CH 2 / 0 ~ i It II I// ~ (HOCH)3 0 O ~ / o.

I I

oH

o (Vll) NH2 o . o .

'

CH2O--~--o--~--O--CH2 o.. \ N ~ " N / /

i (HOCH)s

II 0

tt 0

I7/ ~

~1 /

~H2 H3C . ~ . . . . v / N ......,~N~

~

O

OH

OH

o (VIII) 6 j. G. Moffatt and H. G. Khor~na, J. Am. Chem. Soc. 83, 649 (1961). 7 j. G. Moffatt and H. G. Khorana, J. Am. Chem. Soc. 80, 3756 (1958).

464

FLAVINS AND DERIVATIVES

[148]

the reservoir. The elution is followed spectrophotometrically at 260 and 450 m~. The FS-bromoAD, which exhibits a 260/450 ratio near 3.6, is eluted after 8-bromo-5'-AMP and F M N (Fig. 2), the pH adjusted to 6 with 0.1 M lithium hydroxide, and the solution lyophilized. The residue is stirred in methyl alcohol (20 ml) and the dilithium salt of FS-bromoAD precipitated with acetone (200 ml) plus ether (20 ml). The precipitate is collected by centrifugation and washed twice more to remove all lithium chloride by resuspending in methyl alcohol (3 ml) and reprecipitating with acetone (30 ml) plus ether (3 ml). This material is dissolved in water (10 ml), filtered, and lyophilized to give 0.13 g (28%) of (VII). The syntheses of 2-morpholino-2-deoxy FAD 1 (VIII) from 2-morpholino-2-deoxyriboflavin 5'-phosphate and adenosine 5'-phosphoromorpholidate 6 is the same but for the following modifications: The mixture of 2-morpholino-2-deoxy F M N and adenosine 5'-phosphoromorpholidate in the pyridine-dimethyl formamide solvent is kept 1 week at 40 °. The FAD derivative is eluted by using a linear gradient with 0.01 M lithium chloride in 0.01 M lithium acetate (pH 5.5, 2 liters) in the reservoir. The 2-morpholino-2-deoxy FAD exhibits a 260/464 ratio near 1.2 and an RI ratio of F M N / F A D near 3.2 on thin-layer chromatography with n-butyl alcohol acetic acid-water (2: l : 1). Comments The above syntheses exemplify methods useful for obtaining derivatives of flavin coenzymes in which either isoalloxazine or adenyl moieties can be variously substituted. In particular, the 2-thio and S-methyl-2-thio analogs are useful as intermediates for the formation of other 2-substituted flavins, as these functions can be displaced with appropriate nucleophiles. 1,~