Note: Descriptions are shown in the official language in which they were submitted.
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Separation of phosphoric acid from aqueous solutions
of thiamine phosphates
Cocarboxylase (also referred to as thiamine pyro
phosphate or thiamine diphosphate) of the formula I is a
prosthetic group and, together with specific proteins,
forms several enzymes which catalyze a number of impor-
tant reactions in the metabolism of the human and animal
organism.
NH 2
N~ N CH3 0 0
C H 3~ I ~5~0-IP--0-IP--Oe ( I J
OH OH
Under certain conditions, for example where there
is enzyme insufficiency or oxygen deficiency in the
tissue, the mechanism of phosphorylation of vitamin B1 to
cocarboxylase may be disturbed. In such cases, therapy
with cocarboxylase is of just as much interest as in
diabetic acidosis, certain forms of peripheral vascular
disorders, neuralgia or herpes zoster.
Since the isolation of the coenzyme of cocarbox-
ylase from yeast in 1937, there has been no lack of
attempts to develop an advantageous process for the
preparation thereof. A summary of known processes
appears in the description of DE-A-10 85 527. All indus-
trially relevant syntheses of cocarboxylase which have
been described start from thiamine, which is reacted with
a very wide range of phosphorylating agents . The most
freuqently used phosphorylating agent is highly concen-
trated phosphoric acid. As described in DE-A-10 85 527,
highly concentrated orthophosphoric acid contains only
about 25~ of pyrophosphoric acid, so that cocarboxylase
can in theory be formed in a yield of not more than 25~.
The yield can only be increased by partially hydrolyzing
one of the higher thiamine polyphosphates formed as a by-
product, without simultaneously degrading the cocarbox-
ylase itself.
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The principal difficulty in the preparation of
cocarboxylase is therefore in isolating the cocarboxylase
in a very advantageous manner from the mixture of phos-
phoric acid and the various thiamine phosphates, which
mixture is obtained in the phosphorylation of thiamine.
Because of the sensitivity of the thiamine phosphates to
acid, it has proven advantageous for this purpose to
separate off the excess phosphoric acid as far as pos-
~ible before the actual isolation of the esters.
In the prior art, the phosphoric acid is general-
ly separated off by repeated precipitation of the thiam-
ine phosphates with solvents, such as methanol or
acetone, and separation of the said phosphates from the
aqueous solution containing phosphoric acid. The amounts
of solvents required for this purpose are considerable.
Moreover, complete removal of the phosphoric acid is not
possible by this procedure.
Another possible method is the treatment of the
phosphoric acid-containing thiamine phosphate solutions
with basic ion exchangers. The disadvantage here is that
very large amounts of ion exchangers are required and
that the thiamine phosphates are obtained in highly
dilute solution as a result of the treatment with ion
exchangers.
We have found that the excess phosphoric acid can
be completely separated off by liquid-liquid extraction
of the thiamine phosphate solutions obtained in the phos-
phorylation of thiamine, with a mixture of a suitable
water-insoluble tertiary amine and a water-immiscible
solvent of moderate polarity. In this procedure, the
phosphoric acid is converted with the water-insoluble
amine into the corresponding salt, which is then ex-
tracted by the water-immiscible solvent. It was very
surprising that, in this extraction of the phosphoric
acid, the thiamine phosphates, which are also capable of
salt formation with amines, are not extracted but remain
virtually quantitatively in the aqueous solution.
3 20 1 29 28
The present invention therefore relates to a process for
separating phosphoric acid from aqueous solutions of
thiamine phosphates which are obtained in the
phosphorylation of thiamine, with or without subsequent
partial hydrolysis, wherein the phosphoric acid is
converted with a virtually water-insoluble or only slightly
water-soluble tertiary amine selected from the group
consisting of an aliphatic or aliphatic-cycloaliphatic
tertiary amine of 8 to 40 carbon atoms and an aliphatic-
araliphatic tertiary amine of 9 to 36 carbon atoms into the
corresponding salt and the latter is extracted with a
virtually water-immiscible or only slightly water-miscible
solvent of moderate polarity selected from the group
consisting of an aliphatic or cycloaliphatic alcohol of 4
to 8 carbon atoms, an ether of 4 to 12 carbon atoms, a
ketone of 5 to 8 carbons atoms and a hydrocarbon of 6 to 10
carbon atoms.
The novel process is particularly advantageously
carried out by a method in which the phosphoric acid is
extracted with a mixture of a virtually water-insoluble or
only slightly water-soluble tertiary amine and a virtually
water-immiscible or only slightly water-miscible solvent of
moderate polarity.
The extracting agent can be regenerated by simple
back-extraction with aqueous alkaline solutions (eg. NaOH
or KOH) . After the extraction, the thiamine phosphates are
present in the aqueous phase and can be separated in a
conventional manner by ion exchange chromatography.
Since the excess phosphoric acid can be virtually
completely removed by the extraction, substantially smaller
amounts of ion exchangers are required here.
,.r,
3a 2 0 1 2 9
Observations to date have shown that suitable
tertiary amines for the novel process are in principle all
tertiary amines which have little or no water solubility,
so that, provided that they cannot react in other ways with
the reactants owing to functional groups, their chemical
nature is unimportant. Examples are aliphatic tertiary
amines of, in total, 8 to 40, preferably 12 to 36, carbon
atoms, especially trioctylamine, trihexilamine and
tridodecylamine,
aliphatic-cycloaliphatic tertiary amines, such as N,N-
dimethylcyclohexylamine and
aliphatic-araliphatic tertiary amines, such as N,N-
30
_ ._.,~,
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dimethylbenzylamine.
The readily obtainable and therefore cheap amines
trioctylamine and tridodecylamine are particularly advan-
tageously used.
The tertiary amines are generally used in the
amounts required for salt formation. In a particularly
advantageous procedure, salt formation is effected
directly in the presence of the virtually water-
immiscible solvent of moderate polarity which is used for
the extraction. In practice, this means that the phos-
phoric acid is extracted with a mixture of the water-
insoluble or only slightly water-soluble amine and the
water-immiscible or only slightly water-miscible solvent.
Mixtures which contain the amine in amounts of from 10 to
80, preferably from 40 to 70, $ by weight, based on the
solvent, are used for this purpose. If mixtures which
contain only small amounts of amine are used, either
large amounts of the amine/solvent mixture must be used
or the extraction process must be repeated several times .
If an amine/solvent mixture which contains large amounts
of amine is employed, a single extraction process may be
sufficient.
Suitable water-immiscible or only slightly water
miscible solvents of moderate polarity are essentially
those solvents which have an ET value of about 50 to about
kcal/mol (from 211 to 125 kJ/mol) (cf. Chr. Reichardt,
Solvent Effects in Organic Chemistry, Verlag Chemie,
1979, especially pages 242-245). Examples are alcohols
of 4 to 8 carbon atoms, ethers, such as diethyl ether,
30 methyl tert-butyl ether, diphenyl ether or di-n-butyl
ether, and ketones of 5 to 8 carbon atoms, such as di-
ethyl ketone, methyl isobutyl ketone, acetophenone or
cyclohexanone, and hydrocarbons, such as toluene. Ethyl
acetate and halohydrocarbons can in principle also be
used but are not very advantageous for reasons relating
to process engineering.
Methylisobutylcarbinol, 3-ethylpentanol, 1-
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hexanol, methylcyclohexanol, methyl isobutyl ketone and
methyl tert-butyl ether are particularly advantageously
used.
The solvents are used in general in amounts of
from 1 to 4, preferably from 1.5 to 2, kg per kg of the
phosphoric acid to be extracted. In the mixture with the
tertiary amine, it is present in amounts of from 20 to
90, preferably from 30 to 60, ~ by weight, based on the
tertiary amine.
If the mixture of tertiary amine and the solvent
is not used, ie. if the tertiary amine is added to the
phosphoric acid-containing aqueous solution of thiamine
phcsphates in the absence of the water-immiscible sol-
vent, the resulting salt may be obtained in greasy form
and may therefore present difficulties in the subsequent
extraction.
The extraction of the phosphoric acid is complete
when the aqueous solution has a pH of from 3 to 4, in
particular from 3.2 to 3.4.
The novel process is suitable for separating
phosphoric acid from phosphoric acid-containing aqueous
solutions of thiamine phosphates which are obtained in
the phosphorylation of thiamine.
The composition of the mixture of thiamine phos
phates which is obtained in the phosphorylation of
thiamine is dependent on the amount of thiamine intro
duced into the phosphoric acid. At low thiamine/phos
phoric acid ratios, virtually 50~ of higher thiamine
phosphates are formed, whereas the content of thiamine
monophosphate increases to above 60~ at high thiamine/-
phosphoric acid ratios.
The thiamine phosphates are hydrolyzed in acidic
solution. The diphosphates, triphosphates and higher
phosphates are much more unstable to aqueous acids than
thiamine monophosphate. At a pH of less than 1, which is
reached when the crude phosphorylation product is dis-
solved in water, the rate of hydrolysis of the higher
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phosphates is relatively high.
If the crude phosphorylation product has a high
content of higher thiamine phosphates, it is advisable
first to subject the mixture obtained in the phosphoryla-
tion to partial hydrolysis. For this purpose, the crude
thiamine phosphate obtained in the phosphorylation is
generally heated in water at a pH of from 0.5 to 3,
preferably from 0.5 to 1.5, to 30-100°C, preferably 50-
80°C. The most advantageous conditions can be determined
in each case by investigating the mixture by HPLC. The
longer the crude thiamine phosphate is heated in water,
the greater will be the content of thiamine monophos-
phate. By prolonged partial hydrolysis, ie. by heating
the crude thiamine phosphate mixture in water for about
2-3 hours, it is possible, if desired, to obtain aqueous
phosphoric acid-containing solutions which contain pre-
dominantly thiamine monophosphate and from which the
thiamine monophosphate likewise required can be obtained
in a simple manner after the removal, according to the
invention, of the phosphoric acid.
After the extraction of the excess phosphoric
acid, the thiamine phosphates can be isolated in a con-
ventional manner.
Cocarboxylase and thiamine monophosphate are
advantageously isolated by a method in which a major part
of the thiamine monophosphate is precipitated as crystal
line thiamine phosphate from the thiamine phosphate solu
tioin, freed from phosphoric acid by extraction, by the
addition of a lower alcohol or acetone, and carboxylase
is obtained from the remaining solution having a higher
cocarboxylase content in a conventional manner by ion
exchange chromatography, after the organic solvent has
been distilled off.
The isolation of the thiamine phosphates is des
cribed in more detail in, for example, the abovementioned
DE-A-10 85 527.
Any desired salts of the cocarboxylase can also
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be prepared in a conventional manner from the cocarbox-
ylase tetrahydrate obtained in the isolation step.
With the aid of the novel process, cocarboxylase
can be isolated in a simpler and more advantageous manner
from the mixtures of orthophosphoric acid and various
thiamine phosphates, which mixtures are obtained in the
phosphorylation of thiamine.
Another advantage of the thiamine phosphate solu
tion obtained by complete removal of phosphoric acid by
extraction is that a major part of the thiamine mono
phosphate can be obtained directly as crystalline thiam-
ine monophosphate (saleable commercial product) by the
addition of an organic solvent, such as methanol, ethanol
or acetone. The remaining solution having a higher co-
carboxylase content can be purified in a conventional
manner by ion exchange chromatography after the organic
solvent has been distilled off. Owing to the lower
thiamine monophosphate content of this solution, substan-
tially smaller amounts of ion exchangers are required.
By prolonged partial hydrolysis of the solution,
obtained by phosphorylativn of thiamine, of thiamine
phosphates, subsequent extraction of the phosphoric acid
and addition of an organic solvent, such as methanol,
ethanol or acetone, virtually the entire content of
thiamine phosphates can be isolated in a simple manner in
the form of crystalline, saleable thiamine monophosphate.
The Examples which follow illustrate the novel
process.
The Examples are preceded by some explanations of
the analysis used for the thiamine phosphates.
The analysis of the thiamine phosphorylation
products was carried out by HPLC on a reverse phase
silica gel column (cf . M. Kumura et al . , J. Chromatog-
raphy, 332 (1985), 181-188).
Column . RP 18.7 um, 250 x 8 mm
Mobile phase . 997 ml of 0.2 M NaHZP04 buffer in Hz0
3 ml of acetonitrile
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Pressure . 115 bar
For the detection of thiamine and the phosphor-
ylation products, measurements were made with a W
detector at a = 235 nm, and phosphoric acid was detected
by means of a downstream RI detector.
The phosphorylation product ratios were deter-
mined from the W integrator printout and did not corres-
pond to the molar yields of the various phosphorylation
reaction products. These can be obtained by incorporat-
ing the various extinction coefficients of the individual
phosphate species, but this is not necessary for relative
comparisons of the phosphorylation reactions.
EXAMPLE 1
A) Phosphorylation of thiamine chloride hydrochloride
200 g (0.59 mol) of thiamine chloride hydro
chloride were stirred with 250 g of orthophosphoric acid
for 1 hour (h) at 100°C. The mixture was heated to 120°C,
after which 200 g of phosphorus pentoxide were added, so
that the evolution of HC1 gas which occurred remained
under control. After reaction for a further 15 minutes,
the reaction mixture was allowed to cool. 544 g of a
phosphoric acid-containing thiamine phosphate mixture
were obtained, the said mixture having the following com-
position according to HPLC:
33.7 of cocarboxylase,
37.2 of thiamine monophosphate,
15.0 of thiamine triphosphate and
13.1 of thiamine tetraphosphate.
B) Partial hydrolysis of the thiamine phosphate mixture
The crude phosphate (544 g) which had solidified
to a glassy material was dissolved in 1,088 ml of water
and the solution was then heated at 70°C for 1 h and then
investigated by HPLC. It had the following composition:
61.4 of thiamine monophosphate,
31.5 of cocarboxylase and
2.6~ of thiamine triphosphate.
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9
C) Extraction of the phosphoric acid
The solution was cooled and then extracted with
twice 1 1 of a mixture of methylisobutylcarbinol contain-
ing 75~ by weight of tri-n-octylamine (extracting agent
can be recycled) in order to remove the principal amount
of free phosphoric acid. 907 g of a crude phosphate
solution (pH 3.3) were obtained, the said solution having
the following composition:
66.9 of thiamine monophosphate,
30.2$ of cocarboxylase and
1.5$ of thiamine triphosphate.
D) Separation of the thiamine phosphates by means of
ion exchangers .
The thiamine phosphate solution (907 g) obtained
after extraction of the crude phosphate was separated
over 2 ion exchange columns. The columns contained:
1) 400 ml of Amberlite,*IRA 93 (weakly basic, OH form)
2) 3,000 ml of Lewatit IR-120 (strongly acidic, H+
form).
The solution emerging from the weakly basic ion
exchanger (IRA 93) was fed directly to the acidic ion
exchanger Lewatit*IR-120. After a first fraction of 300
ml, 1.2 1 of a cocarboxylase solution having the follow-
ing composition were obtained:
99.5 of cocarboxylase and
0.5~ of thiamine monophosphate.
After the cocarboxylase solution ( 1. 2 1 ) had been
evaporated down to about 100 ml under about 40 mbar and
400 ml of methanol had been added, 54 g (18$, based on
thiamine.HCl used) of cocarboxylase tetrahydrate were
obtained.
E) Regeneration of the ion exchangers and isolation of
thiamine orthophosphoric acid bishydrochloride
a) The weakly basic ion exchanger (Amberlite TRA 93)
was regenerated with 1.0 1 of a 1.5$ strength
aqueous NaOH solution and, after being washed
neutral with 3 1 of HzO, could be used for further
* (Trademarks)
zo~z92s
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separation.
b) The acidic ion exchanger IR-120 was regenerated with
1 of a 10~ strength aqueous HC1 solution and,
after being washed neutral with 20 1 of HzO, could
5 be used for further separations. The regeneration
solution (5 1) containing hydrochloric acid was
evaporated down to 400 ml (under about 40 mbar).
The addition of 2 1 of MeOH gave 153 g (57~, based
on thiamine hydrochloride used) of thiamine ortho-
phosphoric acid bishydrochloride, which can be used
instead of thiamine chloride hydrochloride for
further phosphorylations.
Phosphoric acid was removed virtually quantita
tively by extraction in a similar manner from aqueous
solutions of thiamine phosphates which had been prepared
by phosphorylation of thiamine, with or without subse-
quent partial hydrolysis:
a) by extraction 3 times with a mixture consisting of
40~s by weight of tri-n-dodecylamine and 60$ by
weight of methyl tert-butyl ether;
b) by extraction twice with a mixture consisting of 50$
by weight of tri-n-octylamine and 50~ by weight of
1-hexanol;
c) by extraction 3 times with a mixture consisting of
60~ by weight of tri-n-hexylamine and 40~ by weight
of 3-ethylpentanol and
d) by extraction 3 times with a mixture consisting of
40~ by weight of tri-n-butylamine and 60$ by weight
of methyl isobutyl ketone.
EXAMPLE 2
200 g of thiamine chloride hydrochloride were
phosphorylated with 250 g of orthophosphoric acid simi-
larly to Example lA, the resulting thiamine phosphate
mixture was partially hydrolyzed similarly to Example 1B
and the resulting solution was extracted, similarly to
Example 1C, with twice 1 1 of a mixture of methyl iso-
butylcarbinol and 75~ by weight of tri-n-octylamine.
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1,100 ml of the extracted solution contained
318 g of dry substance having the following composition:
72.7 of thiamine monophosphate,
23.4$ of cocarboxylase and
1.0~ of thiamine triphosphate.
1,100 ml of ethanol were added to the 1,100 ml of
the extracted crude phosphate solution at 50°C and the
resulting mixture was cooled to room temperature in the
course of 1 h. It was then left to stand for about a
further 30 min at room temperature, after which the pre-
cipitated crystals were filtered off.
The crystals (180 g of dry substance) contained:
98.0 of thiamine monophosphate and
1.5~ of cocarboxylase.
The crystals were dried at 40°C under reduced
pressure. They can then be used as such or recycled to
the phosphorylation reaction.
The mother liquor (137 g of dry substance) con-
tained:
35.8 of thiamine monophosphate,
53.5 of cocarboxylase and
2.3~ of thiamine triphosphate.
The mother liquor was distilled under reduced
pressure at from 30 to 40°C to separate off the ethanol.
The residue obtained was purified by ion exchange chroma
tography over 350 ml of Amberlite IRA 93 (weakly basic;
OH form) and Lewatit IR-120 in a known manner.
1,000 ml of a cocarboxylase-containing solution
were obtained, the said solution giving 54 g of cocarbox
ylase tetrahydrate after evaporation and crystallization.
EXAMPLE 3
200 g of thiamine chloride hydrochloride were
phosphorylated with a mixture of 250 g of orthophosphoric
acid and 200 g of P205 similarly to Example lA, the
resulting thiamine phosphate mixture was partially
hydrolyzed by heating at 70°C for 3 1/2 hours and the
solution obtained was extracted, similarly to Example 1C,
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with twice 1 1 of a mixture of methylisobutylcarbinol and
75~s by weight of tri-n-octylamine.
1,100 ml of the extracted solution contained
318 g of dry substance having the following composition:
94~ of thiamine monophosphate and
3~s of cocarboxylase.
1,100 ml of ethanol were added to the 1,100 ml of
the extracted crude phosphate solution at 50°C and the
resulting mixture was cooled to room temperature in the
course of 1 h. It was then left to stand for about 30
minutes at room temperature and the precipitated crystals
were then filtered off.
The crystals (240 g of dry substance) contained:
99.6 of thiamine monophosphate.
The crystals were dried at 40°C under reduced
pressure. They can be used as such.
