Preparation of riboflavin-5'-phosphate (5'-FMN) and its sodium salt, and of riboflavin-4',5'-cyclophosphoric acid ester chloride as an intermediate

Riboflavin-4',5'-cyclophosphoric acid chloride of the formula I ##STR1## its preparation by reacting an alkali metal salt of riboflavin, in an aprotic solvent, with phosphorus oxychloride, its use for the preparation of riboflavin-5'-phosphate and of the sodium salt of riboflavin-5'-phosphate, and a process for the preparation of riboflavin-5'-phosphate or its sodium salt via the novel phosphoric acid ester chloride.

The present invention relates to riboflavin-4',-5'-cyclophosphoric acid 
ester chloride of the formula I 
##STR2## 
and to a process for the preparation of riboflavin-5'-phosphate 
(5'-flavin-mononucleotide and hence hereinafter referred to as 5'-FMN) and 
for the preparation of the commercial monosodium salt of 5'-FMN via the 
novel ester chloride of the formula I. 
5'-FMN is a compound which plays an important role as a coenzyme in various 
enzymatic reactions in a living organism and which is therefore used in 
the form of its salts, especially in the form of sodium 5'-FMN, as an 
additive for medicaments, foodstuffs and feeds. Sodium 5'-FMN is also used 
as a starting material for flavinadenine dinucleotide, which is used as a 
therapeutic agent to combat vitamin B.sub.2 deficiency. 
Industrially, sodium 5'-FMN is generally obtained by direct reaction of 
riboflavin with a phosphorylating agent, such as partially hydrolyzed 
phosphorus oxychloride, followed by treatment of the resulting 5'-FMN with 
sodium hydroxide solution. The selective phosphorylation of riboflavin is 
not entirely straightforward. Thus, for example, according to U.S. Pat. 
No. 2,610,177, a large excess of phosphorus oxychloride is used. According 
to C.A. 83 (1975), 7955ia, C.A. 83 (1975), 79549f (Japanese Preliminary 
Published Application 50/25 597) and C.A. 83 (1975), 79550z (Japanese 
Preliminary Published Application 50/25 598), a slight excess of 
phosphorus oxychloride in a solvent such as tetrahydrofuran, diethylene 
glycol dimethyl ether, monoethylene glycol dimethyl ether, triethyl 
phosphate, 1,2-dichloroethane or 1,2-dibromoethane is recommended. On 
repeating the examples we have found that under the stated conditions no 
5'-FMN at all was formed in many cases, whilst in others only extremely 
small amounts of 5'-FMN were obtainable. The high yields quoted in loc. 
cit. are presumably due to analytical problems. 
In certain cases, the phosphorylation is carried out in the presence of 
pyridine (cf. U.S. Pat. No. 2,111,491) or in the presence of acetonitrile 
(cf. Techn. Rapport No. 2715 (1979) by Frantz Kaufmann of Grindstedt 
Verket, Denmark). 
In all the known processes of preparation, a crude product which still 
contains substantial amounts of unconverted riboflavin as well as isomeric 
monophosphates and polyphosphates as byproducts is initially obtained. 
Hence, the 5'-FMN must be subjected to a technically complicated 
purification procedure, to give products which conform to the purity 
criteria of the U.S. and European pharmacopeia. For example, Chemical 
Engineering, Nov. 1954, pages 120 et seq. discloses that in one production 
process the 5'-FMN is concentrated by dissolving the isomer mixture in the 
form of monoammonium salts by repeated treatment with ethanolamine, and 
separating this solution from unconverted and undissolved riboflavin. 
The involved process steps and, in addition, the use of large amounts of 
phosphorus oxychloride relative to the riboflavin (vitamin B.sub.2) to be 
phosphorylated themselves show that such processes can represent a not 
insignificant effect on the chloride pollution of the effluent. The 
purification processes for vitamin B.sub.2 phosphate by absorption on a 
cellulose ion exchanger and elution with a sodium oxalate/oxalic acid 
buffer or ammonium formate/formic acid buffer (cf. Japanese Published 
Application 47/8836 and Japanese Published Application 47/8554) also do 
not make the process more economical and more environment-friendly, since, 
in industrial applications, excessively large amounts of buffer salts are 
employed. 
We have now found that, surprisingly, salts of 5'-FMN are obtained 
particularly advantageously if, contrary to the prior art, it is not the 
free riboflavin, but its metal salts, preferably the alkali metal salts, 
especially the potassium salt (II) of riboflavin which are employed for 
the phosphorylation. This first results in the 
riboflavin-4',5'-cyclophosphoric acid ester chloride of the formula I, 
which to the best of our knowledge has not previously been described in 
the literature, and which can be separated off in a crystallized form. 
This compound can then be hydrolyzed, with ring cleavage, under suitable 
conditions, and be converted, by partial neutralization with sodium 
hydroxide solution at pH 5.5, into the sodium salt of 5'-FMN. 
If the potassium salt is used, the reaction takes place in accordance with 
the following equation: 
##STR3## 
It was very surprising in the reaction of the alkali metal salts of 
riboflavin with phosphorus oxychloride or with an ester of phosphorus 
oxydichloride that the attack of the phosphorylating agent should occur in 
the 4',5'-position of the ribityl residue. It is known from the literature 
that the negative charge in the anion of riboflavin is localized in the 
heterocyclic rings, so that anyone skilled in the art would have expected 
the attack of the phosphorylating agent to take place at the position of 
highest charge density, namely in positions and 5 of the isoalloxazine 
ring and not at the remote 4',5'-position of the ribityl residue. 
Accordingly, the present invention not only relates to the 
riboflavin-4',5'-cyclophosphoric acid ester chloride of the formula I but 
also to a process for its preparation, wherein an alkali metal salt, 
especially the potassium salt, of riboflavin is reacted in a suitable 
aprotic solvent, at from 20.degree. to 50.degree. C., preferably at about 
30.degree.-45.degree. C., with from 1.2 to 3 moles of phosphorus 
oxychloride per mole of the alkali metal salt, and, where appropriate, the 
product which crystallizes out of the reaction mixture is isolated by 
filtration. 
Suitable aprotic solvents for the reaction are, in particular, linear or 
cyclic ethers, such as monoethylene glycol dimethyl ether, diethylene 
glycol dimethyl ether, triethylene glycol dimethyl ether, tetrahydrofuran 
or dioxane. 
The alkali metal salt used as the starting compound is obtained in a simple 
manner by dissolving the riboflavin in an equimolar amount of a dilute 
aqueous alkali metal hydroxide solution and causing the resulting salt to 
crystallize by dropwise addition of methanol. Filtration, washing with 
methanol and drying gives the alkali metal salt in almost quantitative 
yield. In the dried state, the alkali metal salt contains one molecule of 
water of crystallization and is just as stable as riboflavin itself. 
It is particularly advantageous to carry out the preparation of the 
phosphoric acid ester chloride of the formula I by introducing the alkali 
metal salt of riboflavin into a solution of about 2.8 moles of phosphorus 
oxychloride per mole of alkali metal salt (i.e. an approximately 1.8 molar 
excess) in a suitable aprotic solvent. After 2 hours' reaction at 
40.degree.-45.degree. C., more than 95% conversion has already been 
achieved. The crystalline riboflavin-4',5'-cyclophosphoric acid ester 
chloride formed can be isolated by filtration, or can be immediately 
hydrolyzed and isomerized to the desired 5'-FMN by adding water to the 
reaction mixture and heating the batch. The resulting 5'-FMN can then, if 
desired, be converted to the monosodium salt of 5'-FMN by partial 
neutralization. 
An intermediate isolation of the 5'-FMN is mostly unnecessary, since the 
sodium salt of 5'-FMN is obtained in the desired purity in a single 
process step by partial neutralization of the free acid. 
Accordingly, the present invention also relates to the use of 
riboflavin-4',5'-cyclophosphoric acid ester chloride of the formula I for 
the preparation of 5'-FMN by hydrolysis and isomerization, and to the 
preparation of the monosodium salt of 5'-FMN by hydrolysis, isomerization 
and partial neutralization. 
The invention further relates to the process described above, wherein, in 
order to prepare 5'-FMN or its sodium salt, the 
riboflavin-4',5'-cyclophosphoric acid ester chloride obtained, of the 
formula I, 
(a) is hydrolyzed to give riboflavin-4',5'-phosphoric acid ester which 
(b) is isomerized to give 5'-FMN and this 
(c) is reacted, if desired, with sodium hydroxide to give the monosodium 
salt of 5'-FMN. 
To carry out this process, the procedure followed is generally that to the 
reaction mixture containing the riboflavin-4',5'-phosphoric acid ester 
chloride of the formula I 
(a) there are rapidly added from 30 to 50, preferably from 32 to 35, moles 
of water per mole of phosphoric acid ester chloride, in the course of 
which the temperature rises to above 90.degree. C. and 
riboflavin4',5'-phosphoric acid ester is formed by hydrolysis, 
(b) the reaction mixture is kept for a further 5-15, preferably 8-12, 
minutes at from 80.degree. to 100.degree. C., preferably from 85.degree. 
to 90.degree. C., by introducing steam, in the course of which the 
riboflavin-4',5'-phosphoric acid ester formed is essentially isomerized to 
5'-FMN, 
(c) the isomerization is interrupted by addition of 68-100 moles of water 
to the reaction mixture and by the cooling which this causes, and, 
(d) if desired, for the preparation of the monosodium salt of 5'-FMN, the 
reaction mixture is brought to a pH of from 5.5 to 6 by means of sodium 
hydroxide. 
If isolated riboflavin-4',5'-cyclophosphoric acid ester chloride is used as 
starting compound, it is necessary 
(a) to introduce the latter into an amount of water, heated to 
80.degree.-95.degree. C., which suffices to effect dissolution, 
(b) to keep the reaction mixture for a further 5-15 minutes at 
80-100.degree. C. by introducing steam, to stop the isomerization by 
subsequent addition of 68-100 moles of water and, 
(d) if the preparation of the monosodium salt of 5'-FMN is desired, to 
bring the reaction mixture to a pH of 5.5-6 with sodium hydroxide. 
In the preparation, according to the invention, of 5'-FMN or of its 
monosodium salt it is necessary to ensure that the reaction mixture 
containing the riboflavin-4',5'-cyclophosphoric acid ester chloride 
reaches 
80.degree.-100.degree. C. as rapidly as possible and that it is kept at 
this temperature for the stated time, without intermediate cooling, since 
otherwise a product with unacceptably high riboflavin content is obtained. 
The reaction of the riboflavin-5'-phosphate with NaOH to give its 
monosodium salt is in general carried out at from 20.degree. to 50.degree. 
C., preferably at from 30.degree. to 40.degree. C. 
The invention further relates to the overall resulting elegant one-vessel 
process for the preparation 5 of pure riboflavin-5'-phosphate or of its 
monosodium salt, wherein 
(A) an alkali metal salt of riboflavin in a suitable aprotic solvent is 
reacted, at from 20.degree. to 50.degree. C., with from 1.2 to 3 moles of 
phosphorus oxychloride per mole of the alkali metal salt, 
(B) to the reaction mixture thus obtained, which contains the novel 
riboflavin-4',5'-cyclophosphoric acid ester chloride of the formula I, 
there are rapidly added from 30 to 50 moles of water per mole of ester 
chloride, in the course of which the temperature rises to above 90.degree. 
C., 
(C) the reaction mixture is kept at from 80.degree. to 100.degree. C. for a 
further 5-15 minutes by introducing steam, 
(D) thereafter from 68 to 100 moles of water are added to the reaction 
mixture and the riboflavin-5' 
phosphate which crystallizes out is isolated, or, if desired, 
(E) the reaction mixture obtained according to (D) is brought, at from 
20.degree. to 50.degree. C., preferably from 30.degree. to 40.degree. C., 
to a pH of from 5.5 to 6 by means of NaOH and the monosodium salt of 
riboflavin-5'-phosphate which crystallizes out, is isolated. 
The riboflavin-5'-phosphate obtained in the process according to the 
invention in general contains less than 6% of riboflavin and from 75 to 
80% of riboflavin-5'-phosphate and accordingly conforms to the purity 
requirements which apply in the pharmaceutical sector. Subsequent 
expensive purification operations are unnecessary. 
The riboflavin-4',5'-cyclophosphoric acid ester chloride of the formula I 
is a simply obtainable intermediate which offers a simple route to 
obtaining the desired product 5'-FMN, and its monosodium salt, in high 
purity.

EXAMPLE 1 
Preparation of riboflavin-4',5'-cyclophosphoric acid ester chloride 
60.12 g (0.392 mol) of phosphorus oxychloride were introduced into 180 ml 
of diethylene glycol dimethyl ether and 60 g (0.139 mol) of riboflavin 
potassium salt, in the form of fine powder, were added in portions, with 
stirring, during which the temperature rose to 30.degree. C. The reaction 
mixture was then heated to 45.degree. C. and stirred at that temperature 
for 2 hours. When the suspension had cooled to room temperature (RT), the 
product was filtered off with suction, under nitrogen, then washed with 
diethylene glycol dimethyl ether and subsequently with acetone, and 
finally dried under reduced pressure. 
The yield was 62.0 g, corresponding to 97.8% of theory. Analysis: 
Content of cyclic chloride, according to HPLC: 95% 
The molecule ion was measured by means of the FAB -MS method (cf. K. L. 
Rinehart in Science 218 (1982), 254). 
The molecular weight determination gave a figure of 456 g/mol, which 
corresponds to the title compound 
The product still contained potassium salts. 
EXAMPLE 2 
Preparation of riboflavin-5'-phosphate 
60 g (0.139 mol) of riboflavin potassium salt, as a fine powder, were 
introduced in portions into a mixture of 180 ml of diethylene glycol 
dimethyl ether and 60.12 g (0.392 mol)=36 ml of phosphorus oxychloride, 
and thereafter the reaction mixture was stirred for 2 hours at 45.degree. 
C. 75 g of water were then added rapidly at the same temperature, 
whereupon the temperature rapidly rose to 90.degree.-95.degree. C. It was 
kept at this value for from 10 to 15 minutes by introducing steam. During 
the subsequent dropwise addition of 170 ml of water, the reaction mixture 
was cooled slowly, in the course of which riboflavin-5'-phosphate began to 
precipitate as early as from 70.degree. to 80.degree. C. After a final 
stirring period of 2 hours at 20.degree.-25.degree. C., the product was 
filtered off with suction and the residue was washed first with a 
water/ethanol mixture (50:50 by volume) and then with a small amount of 
pure ethanol, and was subsequently dried at 75.degree. C. under reduced 
pressure. 
The yield was about 52 g corresponding to 82.0% of theory. Analysis: 
According to HPLC the product contained about 75-78% of 
riboflavin-5'-phosphate, about 9-11% of riboflavin-4'-phosphate, about 
5-7% of riboflavin-3'-phosphate and about 4-6% of free riboflavin. 
EXAMPLE 3 
Preparation of the sodium salt of riboflavin-5'-phosphate 
The procedure was initially as described in Example 2, but after hydrolysis 
and dropwise addition of 170 ml of water the reaction mixture was cooled 
to 30.degree. C. and the pH was brought to about 5.5, at from 30.degree. 
to 40.degree. C., by slow introduction of a 25% strength aqueous sodium 
hydroxide solution. After this pH had been reached, the reaction mixture 
was cooled to 20.degree. C. and the product was then immediately filtered 
off with suction, washed with a water/ethanol (50:50 by volume) mixture 
and with ethanol, and dried under reduced pressure at 75.degree. C. 
The yield was 54.5 g, corresponding to 82.1% of theory. 
Analysis: According to HPLC the product contained 9-11% of sodium 
riboflavin-4'-phosphate, 75-78% of sodium riboflavin -5'-phosphate and 
5-6% of unconverted riboflavin. 
Optical rotation: +37.3-+38.degree. 
Sodium content: about 5% 
pH of a 3% strength aqueous solution: 5-6.3. 
EXAMPLE 4 
A. 12 ml of phosphorous oxychloride were introduced into 100 ml of 
diethylene glycol dimethyl ether and 20 g (0.048 mol) of riboflavin 
potassium salt, as a fine powder, were added in portions, with stirring, 
whereupon the temperature rose to 30.degree. C. The reaction mixture was 
then heated to 35.degree. C. and stirred at this temperature for 3 hours. 
When the suspension had cooled to RT, the product was filtered off with 
suction, under N.sub.2, washed with 100 ml of diethylene glycol dimethyl 
ether and dried. According to HPLC analysis, the product contained 91% of 
riboflavin-4',5'-phosphoric acid chloride and only 1% of unconverted 
riboflavin 
B. 200 ml of water were heated to 75.degree.-85.degree. C. and the product 
obtained under A. was introduced, in portions, into the water at this 
temperature. The reaction mixture was then stirred for a further 15 
minutes at from 90.degree. to 95.degree. C. It was then cooled slowly and 
at 40.degree. C. was brought to pH 5.5 by means of 25% strength aqueous 
sodium hydroxide solution. When the desired pH had been reached, the 
reaction mixture was cooled to 20.degree. C. and the product was then 
immediately filtered off with suction, washed with a water/ethanol (50:50 
by volume) mixture and with ethanol and dried under reduced pressure at 
75.degree. C. The yield was 19 g. 
Analysis: According to HPLC, the product contained about 76% of 
riboflavin-5'-phosphate, about 9% of riboflavin4'-phosphate and about 5% 
of riboflavin.