1',2'-Diacyl-(6R,S)-5,6,7,8-tetrahydro-L-biopterin and process for preparing the same

A novel compound, 1',2'-diacyl-(6R,S)-5,6,7,8-tetrahydro-L-biopterin which is prepared by catalytical hydrogenation of a 1',2'-diacyl-L-biopterin in a solvent in the presence of a catalyst. The 1',2'-diacyl-L-biopterin is prepared from a 1,1-dialkylsulfonyl-L-rhamnose through an acyl derivative of 5-deoxy-L-arabinose and a hydrazine derivative of tetrahydro-L-biopterin without isolating the intermediate products. The 1', 2'-diacyl-(6R,S)-5,6,7,8-tetrahydro-L-biopterin can be used for treatment of atypical phenylketonuria or dihydropterin-reductase deficiency and can readily cross the blood brain barrier without neurotransmitter precursors.

BACKGROUND OF THE INVENTION 
The present invention relates to a novel compound, 
1',2'-diacyl-(6R,S)-5,6,7,8-tetrahydro-L-biopterin and a novel compound, 
1',2'-diacyl-L-biopterin, and processes for preparing them. 
It has been found recently that (6R,S)-5,6,7,8-tetrahydro-L-biopterin and 
7,8-dihydro-L-biopterin can be successfully used for a treatment of 
patients with atypical phenylketonuria (hereinafter referred to as PKU) or 
with dihydropterin-reductase deficiency [A. B. Schircks, M. Viscontini and 
J. Schaub, Lancet, 1979, 131; H.-Ch. Curtius, A. Niederwieser, M. 
Viscontini, A. Otten, J. Schaub, S. Scheibenreiter and H. Schmidt, Clin. 
Chim. Acta, 93, 251 (1979)]. 
Though both compounds can conduct an enzymatic hydroxylation of 
L-tryptophane and L-tyrosine to 5-hydroxytryptophane and DOPA, 
respectively, in the brain, they have difficulties to cross the blood 
brain barrier. Therefore neurotransmitter precursors must be given with 
those compounds during the treatment of both deficiency-diseases. 
The present invention has been completed on the basis of the fact that 
lipophile substances can more readily cross the brain barrier than 
compounds which possess a greater polarity as 
(6R,S)-tetrahydro-L-biopterin with its amphoteric nucleus and its free 
sugar chain. 
An object of the present invention is to provide a novel compound, 
1',2'-diacyl-(6R,S)-5,6,7,8-tetrahydro-L-biopterin having a low polarity 
and the same effect as (6R,S)-5,6,7,8-tetrahydro-L-biopterin in treatment 
of atypical PKU and dihydropterin-reductase deficiency without 
neurotransmitter precursors. 
Another object of the invention is to provide a process for preparing the 
compound by acylating the free hydroxy groups of the side chain of 
(6R,S)-tetrahydro-L-biopterin. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, there is provided a novel 
compound, 1',2'-diacyl-(6R,S)-5,6,7,8-tetrahydro-L-biopterin having the 
general formula (I): 
##STR1## 
wherein R.sup.1 and R.sup.2 are the same or different and each is an acyl 
group. 
In accordance with the present invention, there is also provided a process 
for preparing the compound of the general formula (I) by catalytically 
hydrogenating a 1',2'-diacyl-L-biopterin having the general formula (II): 
##STR2## 
wherein R.sup.1 and R.sup.2 are as defined above, in a solvent in the 
presence of a catalyst. 
Furthermore, there is provided a novel compound, 1',2'-diacyl-L-biopterin 
having the general formula (III): 
##STR3## 
wherein R.sup.3 and R.sup.4 are the same or different and each is an acyl 
group having 3 or more carbon atoms. 
1',2'-diacyl-L-biopterin of the general formula (III) can be prepared by 
converting L-rhamnose to an acyl derivative of 
5-deoxy-L-arabinose-phenylhydrazone through a 
1,1-dialkylsulfonyl-L-rhamnose, reacting the obtained acyl derivative with 
2,4,5-triamino-6-hydroxy-pyrimidine to obtain a hydrazine derivative of 
tetrahydro-L-biopterin and oxidizing the hydrazine derivative, without 
isolating the intermediate products in the course of reaction from the 
1,1-dialkylsufonyl-L-rhamnose to the 1',2'-diacyl-L-biopterin. 
The compound having the general formula (I) can be used for treatment of 
atypical PKU or dihydropterin-reductase deficiency and can readily cross 
the blood brain barrier without neurotransmitter precursors.

DETAILED EXPLANATION 
The acyl groups represented by R.sup.1 and R.sup.2 in the general formulas 
(I) and (II) are protective groups of the hydroxyl groups of 
(6R,S)-5,6,7,8-tetrahydro-L-biopterin. 
The acyl group has preferably 1 to 10 carbon atoms, in particular 3 to 10 
carbon atoms. Preferable acyl group is represented by the general formula 
R.sup.5 CO--wherein R.sup.5 is hydrogen or a hydrocarbon residue having 1 
or more carbon atoms, in particular 2 to 9 carbon atoms. Preferable 
examples of the hydrocarbon residue represented by R.sup.5 are, for 
instance, a linear or branched alkyl group having 1 or more carbon atoms, 
preferably 2 to 9 carbon atoms, which is either saturated or unsaturated; 
a substituted or unsubstituted phenyl group represented by the general 
formula 
##STR4## 
wherein R.sup.6, R.sup.7, R.sup.8, R.sup.9 and R.sup.10 are hydrogen or a 
linear or branched alkyl group wherein the combined number of carbon atoms 
is R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10 is preferably not more 
than 3; a substituted or unsubstituted benzyl group represented by the 
general formula 
##STR5## 
wherein R.sup.11 and R.sup.12 are hydrogen, methyl or ethyl wherein the 
combined number of carbon atoms R.sup.11 and R.sup.12 is preferably not 
more than 2; and a substituted or unsubstituted arylalkyl group 
represented by the general formula 
##STR6## 
wherein R.sup.13 is hydrogen or methyl group. Among the above acyl groups, 
formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl and 
benzoyl are most preferable. It is preferable that R.sup.1 and R.sup.2 are 
the same. 
The compound of the general formula (I) has two diastereomers, i.e. 
1',2'-diacyl-(6R)-5,6,7,8-tetrahydro-L-biopterin and 
1',2'-diacyl-(6S)-5,6,7,8-tetrahydro-L-biopterin which are diastereomeric 
at the 6 position. The compound of the present invention includes the two 
diastereomers and a mixture thereof. 
The compound of the present invention can be readily prepared by means of 
catalytic hydrogenation of the compound of the general formula (II) in a 
suitable solvent in the presence of a catalyst. 
Examples of the catalyst are, for instance, Pt, Ni, Cr, Pd, Rh, and the 
like. 
Examples of the solvent are, for instance, a solvent in which the compound 
(II) is soluble, such as trifluoroacetic acid, methanol, ethanol, propanol 
(1) and propanol (2), a conc. HCl, an acidic water, a basic water, or the 
like and a solvent in which the compound (II) is insoluble, but the 
compound (I) is soluble, such as acetic acid. 
When the solvent which dissolves the compound (II) is employed, the process 
of the present invention can be carried out in a similar manner to the 
known process for preparing tetrahydro-L-biopterin by hydrogenating 
L-biopterin in trifluoroacetic acid in the presence of Pt [Bernard 
Schircks, Jost H. Bieri and Max Viscontini, Helvetica Chimica Acta, 61(7), 
2731 (1978)]. 
In case where the compound (I) is used as an active ingredient of a 
pharmaceutical composition, it is not preferable that any solvent which is 
not pharmacologically acceptable remains. In such view point, the 
employment of acetic acid as a solvent is practically useful. The 
catalytical hydrogenation of L-biopterin can also be carried out by using 
acetic acid as a solvent. 
As a result of the catalytical hydrogenation, a mixture of 
1',2'-diacyl-(6R)-5,6,7,8-tetrahydro-L-biopterin and 
1',2'-diacyl-(6S)-5,6,7,8-tetrahydro-L-biopterin is, in general, obtained 
in a proportion of about 1:1. The mixture can be resolved, for example, by 
means of high pressure liquid chromatography which is adapted to a 
resolution of (6R,S)-tetrahydro-L-biopterin [J. Biol. Chem., 253, 1593 
(1978)]. 
The compound (II) can be prepared by a similar process to the process for 
preparing 1',2'-diacetyl-L-biopterin [Bernard Schircks, Jost H. Bieri and 
Max Viscontini, Helvetica Chimica Acta, 60(1), 211 (1977)]. According to 
the process, L-rhamnose hydrate is reacted with ethanethiol. The obtained 
L-rhamnose-diethylmer-captal is converted to 5-deoxy-L-arabinose through 
1,1-diethylsulfonyl-L-rhamnose. 5-Deoxy-L-arabinose is reacted with 
phenylhydrazine to obtain 5-deoxy-L-arabinose-phenylhydrazone. 
5-Deoxy-L-arabinose-phenyl-hydrazone is reacted with an acylating agent. 
The obtained 2,3,4-triacyl-5-deoxy-L-arabinose-phenylhyrazone is reacted 
with 2,4,5-triamino-6-hydroxy-pyrimidine dihydrochloride, and then 
oxidized with an oxidizing agent such as iodine to obtain a 
1',2'-diacyl-L-biopterin. L-Biopterin can be obtained by deacylation of 
the 1',2'-diacyl-L-biopterin. 
The above-mentioned process, however, is not suitable for industrial 
processes because large amount of solvent is required and the total yield 
of 1',2'-diacyl-L-biopterin is low due to its complicated procedures. 
The inventor has found the fact that when the the course of reaction from 
the 1,1-dialkylsulfonyl-L-rhamnose to the 1',2'-diacyl-L-biopterin, if 
necessary, to L-biopterin is carried out without isolating the 
intermediate products, that is to say, in one pot synthesis, it is 
possible to improve the total yield, to reduce the amount of the solvent 
and to simplify the procedures. The improved process of the present 
invention is quite suitable for industrial processes. As a mercaptal, 
methylmercaptal and propanethiol may be employed in addition to 
ethanthiol. In case of employing methylmercaptal and propanethiol, the 
respective starting compound of the improved process is 
1,1-dimethylsulfonyl-L-rhamnose and 1,1-dipropylsulfonyl-L-rhamnose. 
The improved process of the present invention can also be adapted to a 
production of L-biopterin and to productions of monapterins and neopterins 
which have different side chains at the 6 position. 
Among the compounds (II), the 1',2'-diacyl-L-biopterin having the general 
formula (III), that is to say, the acyl group of which has 3 or more 
carbon atoms is a novel compound. 
The compound (I) of the present invention can be isolated from a reaction 
mixture in a form of an inorganic salt such as a hydrochloride, a sulfate 
or a phosphate, an organic salt such as an acetate, an oxalte, or a 
complex salt. 
The present invention is more specifically described and explained by means 
of the following Examples. It is to be understood that the present 
invention is not limited to the Examples and various changes and 
modifications may be made in the invention. 
EXAMPLE 1 
Preparation of 1',2'-diacetyl-(6R,S)-5,6,7,8-tetrahydro-L-biopterin 
After reducing 350 mg. of PtO.sub.2 by a usual method with H.sub.2 in 50 
ml. of trifluoroacetic acid, 1 g. of pure 1',2'-diacetyl-L-biopterin was 
added to the obtained suspension. Then, H.sub.2 was passed into the 
suspension. After 40 min. the rate of H.sub.2 -uptake was slowed down, and 
the H.sub.2 -uptake was stopped after 45 min. The catalyst was quickly 
filtered and a colorless filtrate was frozen in liquid N.sub.2. A cold 
mixture of 20 ml. of ether was added thereto. While melting the frozen 
solution at room temperature, 
1',2'-diacetyl-(6R,S)-5,6,7,8-tetrahydro-L-biopterin.multidot.2HCl was 
separated as a white powder. The powder was centrifuged, washed with 
acetonitrile and ether, dried over KOH in desiccator, and then dried under 
reduced pressure (0.01 Torr) at 60.degree. C. for 15 hours. Yield: 1.1 g. 
The obtained 1',2'-diacetyl-(6R,S)-5,6,7,8-tetrahydro-L-biopterin was 
analysed by means of .sup.1 H-NMR (d.sub.5 -pyridine) and .sup.13 C-NMR 
(D.sub.2 O ). 
The .sup.1 H-NMR-spectrum charts are shown in FIG. 1 and FIG. 2. 
FIG. 1 and FIG. 2 showed distinctly that the obtained compound was a 
diastereomeric mixture of 
1',2'-diacetyl-(6R)-5,6,7,8-tetrahydro-L-biopterin and 
1',2'-diacetyl-(6S)-5,6,7,8-tetrahydro-L-biopterin in a proportion of 
about 1:1. 
EXAMPLE 2 
Preparation of (6R,S)-5,6,7,8-tetrahydro-L-biopterin 
10 mg. of PtO.sub.2 were stirred with 30 ml. of pure acetic acid at room 
temperature in the atmosphere of hydrogen for about 10 minutes until the 
catalyst was completely reduced and saturated with hydrogen. 500 mg. of 
pure L-biopterin were then added. The L-biopterin was not soluble in 
acetic acid and a suspension was obtained. It was stirred in the 
atmosphere of hydrogen until all the L-biopterin became into solution. It 
took about 5 hours. The obtained solution was then filtered from the 
catalyst and the filtrate was cooled in a freezing mixture until it 
solidified. Then a solution of 9 ml. of methanol, 90 ml. of ether and 1 
ml. of 12 N HCl was added to the solidified mixture and the whole flask 
was allowed to come to room temperature. The precipitated insoluble 
tetrahydrobiopterin.multidot.2HCl was filtered and washed with ethanol and 
then ether until became free from excess HCl and dried under reduced 
pressure. The crude tetrahydrobiopterin was crystallized with acetic acid. 
For the elimination of both solvents it was further dried in vacuo (0.01 
Torr) at 60.degree. C. for 16 hours. Yield: about quantitative. 
The following Table 1 shows the different times which are required for the 
complete catalytic reduction of L-biopterin by using several varieties of 
amount of biopterin, catalyst or acetic acid. 
TABLE 1 
______________________________________ 
Amount of 
Amount of 
Amount of solvent Time for 
biopterin 
catalyst (Acetic acid) 
complete reduction 
(mg.) (mg.) (ml.) (hours) 
______________________________________ 
100 40 6 1.5 
100 20 6 1.5 
100 10 6 2.0 
200 10 12 3.0 
300 10 18 4.0 
400 10 24 4.5 
500 10 30 5.0 
1000 20 60 8.0 
______________________________________ 
EXAMPLE 3 
Preparation of 1',2'-dibutyryl-(6R,S)-5,6,7,8-tetrahydro-L-biopterin 
50 mg. of PtO.sub.2 were stirred with pure 25 ml. of acetic acid at room 
temperature in the atmosphere of hydrogen for about 10 minutes until the 
catalyst was completely reduced and saturated with hydrogen. 500 mg. of 
1',2'-dibutyryl-L-biopterin were added. Dibutyryl-L-biopterin was not 
soluble in acetic acid and a suspension was obtained. It became a solution 
while a tetrahydro-derivative was produced by means of hydrogenation with 
stirring. The clear solution was obtained after 5 hours. The obtained 
solution was filtered from the catalyst and cooled in a freezing mixture 
(ice and salt). After freezing, a solution of 1 ml. of 12 N HCl, 9 ml. of 
methanol and 300 ml. of ether was added and the whole flask was allowed to 
come to room temperature. The insoluble 
dibutyryl-tetrahydro-L-biopterin.multidot.2HCl was filtered, washed with 
ethanol and then ether until became free from HCl and dried in vacuo (0.01 
Torr) at 60.degree. C. for 16 hours for the elimination of the solvents. 
Yield: about quantitative. 
The obtained 1',2'-dibutyryl-(6R,S)-5,6,7,8-tetrahydro-L-biopterin was 
analyzed by means of .sup.1 H-NMR (1 N DCl). 
The .sup.1 H-NMR-spectrum cart is shown in FIG. 3: .sup.1 H-NMR-spectrum 
analysis (the values of the chemical shifts are expressed in .delta.): 
(recorded in CDCl.sub.3). 
4.33-3.66 (m, H.sub.2 --C (7), H--C (6); H--C (1'); H--C (2')); 2.66 (m, 
2.times.H.sub.2 C--(CH.sub.2 --CH.sub.3)); 1.90 (m, 2.times.H.sub.2 
C--(CH.sub.3)); 1.60 (m, H.sub.3 --C (3')); 1.20 (t, H.sub.3 
--C--(CH.sub.2)). 
FIG. 3 shows distinctly that the obtained compound was a diastereomeric 
mixture of 1',2'-dibutyryl-(6R)-5',6',7',8'-tetrahydro-L-biopterin and 
1',2'-dibutyryl-(6S)-5',6',7',8'-tetrahydro-L-biopterin in a proportion of 
about 1:1. 
EXAMPLE 4 
One pot synthesis of 1',2'-dibutyryl-L-biopterin 
A suspension of 14 g. (42.1 mmol.) of diethylsulfonyl-L-rhamnose in 120 ml. 
of water was treated with 4 N NH.sub.4 OH while stirring until the pH of 
the solution became 9 to 10. After 14 hours standing with stirring from 
time to time, the precipitate of diethylsulfonyl-methane was filtered off 
and the filtrate was dried in vacuo at 40.degree. C. The residue was 
dissolved in 80 ml. of absolute methanol. After adding 5 g. (46 mmol.) of 
pure phenylhydrazine, the solution was kept at room temperature for 1 hour 
and then dried in vacuo at 40.degree. C. The residue was washed two or 
three times with ether (50 ml. each time) and dried. The dried residue was 
dissolved in 35 ml. of pyridine and the solution was cooled. To the 
ice-cold solution of 0.degree.-5.degree. C. 35 ml. of butyric anhydride 
were slowly added. After adding completely, the reaction mixture was 
allowed to stand in the ice-bath for 10 min. and then it was kept at room 
temperature for 5 hours. After 200 ml. of methanol were added to the 
solution, the solution was kept at room temperature for 10-15 hours 
(overnight). A solution of 1.0 g. of sodium dithionite (Na.sub.2 S.sub.2 
O.sub.4) and 12.5 g. of sodium acetate.multidot.3H.sub.2 O dissolved in 
300 ml. of water, and a suspension of 12.0 g. of 
6-hydroxy-2,4,5-triamino-pyrimidine sulfate.multidot.H.sub.2 O in 500 ml. 
of water were then added successively to the methanol/pyridine solution. 
The whole reaction mixture was closed under nitrogen and stirred at 
35.degree.-40.degree. C. for 20 hours to give an homogeneous, reddish 
brown solution. In the resulting solution the obtained tetrahydrobiopterin 
derivative was oxidized by adding 25 g. of iodine dissolved in 300 ml. of 
methanol. The excess of iodine, if any, was removed with sodium 
thiosulfate. If no excess was present, a small quantity of iodine solution 
was added in order to complete the oxidation of the tetrahydrobiopterin 
derivative. During the oxidation a fine brown crystalline precipitate was 
obtained. 
The resulting oxidized suspension was concentrated to 50 ml. in vacuo at 
40.degree. C. and filtered. The insoluble fraction was washed with water, 
40 ml. of cold ethanol and finally with ether. The residue was then 
recrystallized from hot ethanol with active charcoal and the crystal was 
filtered while heating. 
After cooling, yellowish precipitate of 1',2'-dibutyryl-L-biopterin was 
obtained in the filtrate. It was filtered, washed with ether and dried to 
give 10.3 g. of 1',2'-dibutyryl-L-biopterin. Yield: 65%. 
The obtained 1',2'-dibutyryl-L-biopterin was analyzed by means of several 
analytical methods. The .sup.1 H-NMR-spectrum chart is shown in FIG. 4. 
Rf (3% NH.sub.4 Cl/H.sub.2 O, on cellulose)=0.36 [.alpha.].sub.589.sup.220 
=-74.3.+-.3.degree. (C=0.8, 1 N HCl) 
The optical rotation increased with the time, perhaps because the butyrate 
ester is hydrolyzed in an acidic solution. .sup.1 H-NMR-spectrum analysis 
(.delta.: p.p.m.): (90 MHz, in DCl) 9.33 (s, H--C (7); 6.5 (d, H--C (1')); 
5-6 (m, H.sub.2 O, H--C (2')); 3-2.66 (m, 2.times.H.sub.2 C--(CH.sub.2 
--CH.sub.3)); 2.30-1.8 (m, 2.times.H.sub.2 C--(CH.sub.3)); 1.66 (d, 
H.sub.3 --C (3')); 1.33 (t, H.sub.3 C--(CH.sub.2)). 
Elemental analysis for C.sub.17 H.sub.23 N.sub.5 O.sub.5 
Calc. (%): C 54.11, H 6.1, N 18.56. 
Found. (%): C 54.09, H 6.99, N 18.99 
EXAMPLE 5 
One pot synthesis of 1',2'-diacetyl-L-biopterin 
A suspension of 14 g. (42.1 mmol.) of diethylsulfonyl-L-rhamnose in 120 ml. 
of water was treated with 4 N NH.sub.4 OH while stirring until the pH of 
the solution reached 9 to 10. After 14 hours-standing with stirring from 
time to time, the precipitate of diethylsulfonyl-methane was filtered off 
and the filtrate was dried in vacuo at 40.degree. C. The residue was 
dissolved in 80 ml. of absolute methanol. After adding 5 g. (46 mmol.) of 
pure phenylhydrazine, the solution was kept at room temperature for 1 hour 
and then dried in vacuo at 40.degree. C. The residue was washed two or 
three times with ether (50 ml. each time) and dried. The dried residue was 
dissolved in 35 ml. of pyridine and the solution was cooled. To the cooled 
solution of 0.degree.-5.degree. C. 35 ml. of acetic anhydride were slowly 
added. After adding completely, the reaction mixture was allowed to stand 
in the ice-bath for 10 min. and then it was kept at room temperature for 5 
hours. After 200 ml. of methanol were added to the solution, the solution 
was kept at room temperature for 10-15 hours (overnight). A solution of 
1.0 g. of sodium dithionite (Na.sub.2 S.sub.2 O.sub.4) and 12.5 g. of 
sodium acetate.multidot.3H.sub.2 O dissolved in 300 ml. of water, and a 
suspension of 12.0 g. of 
6-hydroxy-2,4,5-triamino-pyrimidine-sulfate.multidot.H.sub.2 O in 500 ml. 
of water were then added successively to the methanol/pyridine solution. 
The whole reaction mixture was closed under nitrogen and stirred at 
35.degree.-40.degree. C. for 20 hours to give an homogeneous, reddish 
brown solution. In the resulting solution the obtained tetrahydrobiopterin 
derivative was oxidized by adding 25 g. of iodine dissolved in 300 ml. of 
methanol. The excess of iodine, if any, was removed with sodium 
thiosulfate. If no excess was present, a small quantity of iodine solution 
was added in order to complete the oxidation of the tetrahydrobiopterin 
derivative. During the oxidation a fine brown crystalline precipitate was 
obtained. 
The resulting oxidized solution was then concentrated to about 100 ml. and 
filtered after cooling in the refrigerator for some hours. The filtered 
crude diacetylbiopterin was washed with 50 ml. of cold water, 100 ml. of 
cold ethanol, 100 ml. of ether and dried. The dried residue was dissolved 
in about 1200 ml. of boiling water and discolored with about 0.5 g. of 
active charcoal. The solution was filtered while heating, the charcoal bed 
was washed with 50 ml. of boiling water, the filtrate was allowed to cool 
to room temperature and then kept to 0.degree.-5.degree. C. for 10 hours. 
The crystalline diacetyl-L-biopterin was filtered, washed twice with 50 ml. 
of ethanol, then ether and dried to give 8.1 g. of crystalline 
1',2'-diacetyl-L-biopterin. Yield: 60%. 
EXAMPLE 6 
One pot synthesis of L-biopterin 
A suspension of 14 g. (42.1 mmol.) diethyl-sulfonyl-L-rhamnose in 120 ml. 
of water was treated with 4 N NH.sub.4 OH while stirring until the pH of 
the solution reached 9 to 10. After 14 hours standing with stirring from 
time to time, the precipitate of diethylsulfonyl-methane was filtered off 
and the filtrate was dried in vacuo at 40.degree. C. The residue was 
dissolved in 80 ml. of absolute methanol. After adding 5 g. (46 mmol.) of 
pure phenylhydrazine, the solution was kept at room temperature for 1 hour 
and then dried in vacuo at 40.degree. C. The residue was washed two or 
three times with ether (50 ml. each time) and dried. The dried residue was 
dissolved in 35 ml. of pyridine and the solution was cooled. To the 
ice-cooled solution of 0.degree.-5.degree. C. 35 ml. of acetic anhydride 
were slowly added. After adding completely, the reaction mixture was 
allowed to stand in the ice-bath for 10 min. and then it was kept at room 
temperature for 5 hours. After 200 ml. of methanol were added to the 
solution, the solution was kept at room temperature for 10-15 hours 
(overnight). A solution of 1.0 g. of sodium dithionite (Na.sub.2 S.sub.2 
O.sub.4) and 12.5 g. of sodium acetate.multidot.3H.sub.2 O dissolved in 
300 ml. of water, and a suspension of 12.0 g. of 
6-hydroxy-2,4,5-triamino-pyri-midine-sulfate H.sub.2 O in 500 ml. of water 
were then added sucessively to the methanol/pyridine solution. The whole 
reaction mixture was closed under nitrogen and stirred at 
35.degree.-40.degree. C. for 20 hours to give an homogeneous, reddish 
brown solution. In the resulting solution the obtained tetrahydrobiopterin 
derivative was oxidized by adding 25 g. of iodine dissolved in 300 ml. of 
methanol. The excess of iodine, if any, was removed with sodium 
thiosulfate. If no excess was present, a small quantity of iodine solution 
was added in order to complete the oxidation of the tetrahydrobiopterin 
derivative. During the oxidation a fine brown crystalline precipitate was 
obtained. 
The resulting oxidized solution was concentrated in vacuo to about 100 ml. 
150 ml. of ethanol and 250 ml. of 14 N NH.sub.4 OH were added to the 
concentrate. The resulting mixture was kept at 50.degree. C. for one hour. 
The deacetylated biopterin solution was then evaporated to dryness in 
vacuo at 40.degree. C., the residue was taken up with 100 ml. of methanol 
and filtered. 
The crude biopterin was washed with 50 ml. of cold water and 200 ml. of 
ethanol. And then, without drying, it was dissolved in 1400 ml. of boiling 
water with the smallest quantity of active charcoal to discolorize it. 
After hot filtration, the solution was allowed to cool to room temperature 
and then kept at 5.degree. C. for 10 hours. The crystalline biopterin was 
filtered, washed with cold water, ethanol, ether and then dried in vacuo 
(0.01 Torr) at 40.degree. C. for 14 hours until became free from solvents 
to give 6 g. of L-biopterin. Yield: 60%.