PROCEDE POUR LA SEPARATION DE DERIVES DE TETRAHYDROPYRIMIDINE

PROCESS FOR THE SEPARATION OF TETRAHYDROPYRIMIDINE DERIVATIVES

Abrégé anglais

The invention relates to a process for the separation of
tetrahydropyrimidine derivatives, especially
2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acids
(ectoines)and 2-methyl,5-hydroxyl-1,4,5,6-
tetrahydropyrimidine-4-carboxylic acids (hydroxyectoines),
which are present together in aqueous solutions, especially
fermentation broths, by selective adsorption of one
component on zeolites.

Revendications

8
Process for the separation of tetrahydropyrimidine
derivatives
Claims
1. Process for the separation of tetrahydropyrimidines
present in aqueous solution from the corresponding
hydroxy compounds,
characterised in that
the aqueous solutions of these compounds are brought
into contact with an acidic zeolite having a modulus of
15 to 1000 at a pH of 1.0 to 7.0, the supernatant
liquid preferentially containing the hydroxy compounds
is separated after adsorption is complete and the
adsorbed derivatives are desorbed from the zeolites
with an aqueous solution adjusted to a pH of >8.0
optionally by adding a basic organic component.
2. Process according to claim 1,
characterised in that
a mixture of 2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic
acids (ectoines) of the general formula
<IMG>
wherein R1:H and R2:OH, present in aqueous solution, is
separated.

9
3. Process according to claims 1 and 2,
characterised in that
the supernatant liquid obtained after adsorption is
again brought into contact with one of the above
mentioned types of zeolite, the adsorbent is then
separated off and these steps are optionally repeated
one or more times.
4. Process according to claims 1 to 3,
characterised in that
a fermentation solution is used as the aqueous
solution.
5. Process according to claim 4,
characterised in that
before the adsorption the micro-organisms are at least
partly separated out of the fermentation solution.
6. Process according to claims 4 and 5,
characterised in that
before the adsorption the soluble proteins are at least
partly separated out of the fermentation solution.
7. Process according to one or more of claims 1 to 6,
characterised in that
acid zeolites of the types Y, DAY, mordenite,
dealuminised mordenite, ZSM-5, dealuminised ZSM-5, .beta. or
VPI-5 with a modulus of 15 to 1000 are used as
adsorbents.
8. Process according to claim 6,
characterised in that
zeolites of the ZSM5 type or mordenite are used in the
H-, ammonium or Na form.
9. Process according to one or more of claims 1 to 8 for
the separation of the tetrahydropyrimidine derivatives

10
mentioned above, especially according to formula (I),
characterised in that
it is combined with a cross-flow filtration, wherein
a) the charged zeolites in the form of suspensions are
made to flow past a porous surface/membrane,
b) a pressure difference being set up between the side
of the surface/membrane subject to the flow and the
opposite side, so that
c) part of the solution containing the hydroxy
compounds, completely or partially freed from the
adsorbed compounds, flowing over the
surface/membrane flows through the surface/membrane
transverse to the direction of flow (filtrate flow)
d) in a washing step the solution freed from the
adsorbed compounds, which contains the hydroxy
compounds, is separated off, and
e) the adsorbed compounds are then desorbed.
10. Process according to claim 9,
characterised in that
zeolites with an average particle diameter of 1 to
100 µm are used.
11. Process according to claims 9 and 10,
characterised in that
a trans-membrane pressure of 0.2 to 3 bar is
established.
12. Process according to claims 9 to 11,
characterised in that
ceramic or organic membranes/porous surfaces with
ultrafiltration or micro- or nanofiltration properties
are used.

11
13. Process according to one or more of the above claims,
characterised in that
the desorbed solution is subjected to adsorption and
desorption one or more times.
14. Process according to one or more of the above claims,
characterised in that
the process for the separation of compounds (A) is
combined with adsorption on cation exchangers or other
purification steps and the solutions obtained
therefrom, optionally repeatedly, by desorption.

Description

CA 02271519 1999-OS-12
1
Process for the separation of tetrahydropyrimidine
derivatives
The invention relates to a process for the separation of
tetrahydropyrimidine derivatives, especially 2-methyl-
1,4,5,6-tetrahydropyrimidine-4-carboxylic acids (ectoines)
and 2-methyl,5-hydroxyl-1,4,5,6-tetrahydropyrimidine-4-
carboxylic acids (hydroxyectoines), which are present
together in aqueous solutions, by selective adsorption of
one component on zeolites.
Tetrahydropyrimidine derivatives refer in particular to the
compounds known from EP-A1-0553884.
Structurally, ectoines are cyclic amino acid derivatives,
which belong to the class of so-called "compatible
solutes". They are also compatible with cytoplasm in high
concentration and stabilise the cell components in a medium
with low water activity. This action indicates a broad
field of application in medical and cosmetic sectors.
Owing to new biotechnological processes it has become
possible to culture halophilic eubacteria, e.g. of the
Halomonas species, and to cause these organisms to separate
out the ectoines into the medium surrounding them (T. Sauer
et al. GIT Fachz. Lab. 10/95).
Since both ectiones [sic] and hydroxyectoines are produced,
depending on the fermentation conditions, it is necessary
to separate these two components in the subsequent
processing steps in order to obtain the pure components.
This separation may take place for instance by extraction
with methanol, utilising the different solubilities of
ectoine and hydroxyectoine (T. Sauer and Erwin A. Galinski.
Biotechnology and Bioengineering, VOL. 57, NO 3, 1998).

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However, this process is expensive on an industrial scale
owing to the considerable quantities of methanol required.
The object of the invention is to provide an alternative
process which permits an effective separation of
tetrahydropyrimidine derivatives, especially ectoines and
hydroxyectoines, from an aqueous solution optionally
contained [sic] other organic and inorganic compounds
without the use of solvents.
The invention provides a process for the separation of
tetrahydropyrimidines present in aqueous solution from the
corresponding hydroxy compounds, especially of 2-methyl-
1,4,5,6-tetrahydropyrimidine-4-carboxylic acids (ectoines
(I)) 2-methyl,5-hydroxyl-1,4,5,6-tetrahydropyrimidine-4-
carboxylic acids (hydroxyectoines)(II) which are present
together in aqueous solutions, characterised in that the
aqueous solutions containing the two components are brought
into contact with a preferably acidic zeolite having a
modulus of 15 to 1000 at a pH of 1.5 to 7Ø This results
in a kinetically preferential adsorption of ectoine
compared with hydroxyectoine. By adjusting the contact time
between zeolite and solution to an optimum value to be
determined experimentally, a selective depletion of ectoine
from the starting solution occurs, while the hydroxyectoine
remains in solution initially and is adsorbed in
significant quantity only with prolonged contact times. The
ectoine derivatives bonded to the zeolite are desorbed from
the zeolite used by adjusting the pH to >8.0, especially
using an ammonium hydroxide solution. The solution may also
be adjusted to this pH value by adding another basic,
optionally organic component, especially lysine.
Depending on the required purity of the derivatives to be
isolated, the purification process may optionally be
carried out in multiple consecutive steps.

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The tetrahydropyrimidine derivatives to be separated
according to the invention are understood to be in
particular compounds of the formula
s , ~. .2
COOL C H COO"
CH
wherein R1: H and R2: OH.
The solutions from which these substances are separated are
generally aqueous or organic by nature.
Suitable zeolites for the adsorption of the compounds to be
separated according to the invention are those of the types
Y, DAY, mordenite, dealuminised mordenite, ZSM-5, '
dealuminised ZSM-5, (3 or VPIS and with a modulus of 10 to
1000, especially of 15 to 200, preferably of 15 to 45. The
ZSM-5 type is preferably used in the H-, ,ammonium- or Na
form.
The process generally takes place in a temperature range of
between 15 and 80°C, preferably 20 to 40° C.
The concentrations of the ectoines or hydroxyextoines [sic]
to be separated vary within the known solubility range. The
(molar) concentration ratios of the compounds to be
separated which are common in the prior art may be worked
up by the process according to the invention and provide
the desired product in the required purity. The adsorbents
are used in powder form, as mouldings or fixed on
substrates. The batch, fixed bed or cross-flow filtration

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processes, conducted continuously or in batches, are
available for the technical implementation of the process.
In an advantageous embodiment, the adsorption on zeolite is
combined with cross-flow filtration in which a solution
containing the tetrahydropyrimidine derivatives, optionally
freed from the biomass, is brought into contact with the
suspensions of an acidic zeolite for a period of time
appropriate to the adsorption kinetics and then
the hydroxytetrapyrimidine derivatives present in the
supernatant liquid are separated by cross-flow filtration,
in which
a) the charged zeolites in the form of suspensions are
made to flow past a porous surface/membrane,
b) a pressure difference being set up between the side of
the surface/membrane subject to the flow and the
opposite side, so that
c) part of the solution containing the hydroxy compounds,
completely or partially freed from the adsorbed
compounds, flowing over the surface/membrane flows
through the surface/membrane transverse to the
direction of flow (filtrate flow)
d) in a washing step the solution freed from the adsorbed
compounds, which contains the hydroxy compounds, is
separated off, and
e) the adsorbed compounds are then desorbed.
The latter takes place at an appropriate pH, especially at
a pH >8Ø
Fine particle size zeolite powders of the types mentioned
above with a particle diameter of 1 to 100 Vim, especially
2 to 20 Vim, are particularly suitable as adsorbents. The

CA 02271519 1999-OS-12
short intraparticle diffusion paths permit high adsorption
selectivities and thus high separation factors.
The purities achieved for hydroxyectoine in the supernatant
liquid (based on total ectoine) are 100% when using e.g.
5 ZSMS/28 after a contact time of 1 h (Example 1). In the
case of mordenite 30 the purities are approx. 60% after
24 h owing to slower kinetics (Example 2). However, they
can be increased with an appropriate combination of the
process steps.
To increase the purity, the solutions are transferred into
the purification process according to the invention one or
more times. A variant of the process according to the
invention consists in combining it with purification
processes known from the prior art by using organic cation
exchangers and other purification steps.
An appropriate adsorption and desorption step takes place
before and/or after a purification step by adsorption on
one of the zeolites used according to the invention, which
has been followed by desorption.

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Example 1
Adsorption of an ectione [sic]/hydroxyectoine mixture on
ZSMS /28
Time-related concentration curves for ectoine and
hydroxyectoine and the purities for hydroxyectoine (based
on total ectoine)
Starting solution composition
Volume of liquid [ml~ 30
Zeolite concentration [wt. %7 10
Ectoine concentration 1.5
Lg/11
Hydroxyectoine concentration 0.14
Lg/11
Other impurities [g/1] ca. 1
l.o0 loo.oo
0
0
o.so
O o Ectoine x.00
p O Hyd~oxyectoine
O '~~p Hydroxyectoine purity
O O
0.60 60.00
v
L
C/CO y,
0.40 ~ 40.00 c
0
m
0
~o
0.20 20.00
0
0.00 ~ 0. 6~:r Q ' ~ ~ 0.00
0 5 10 15 20 25
Time [hJ

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Example 2
Adsorption of an ectione [sic]/hydroxyectoine mixture on
mordenite 30
Time-related concentration curves for ectoine and
hydroxyectoine and the purities for hydroxyectoine (based
on total ectoine) in the supernatant liquid
Starting solution composition
Volume of liquid [ml] 30
Zeolite concentration [wt. ~] 10
Ectoine concentration 1.5
[g/11
Hydroxyectoine concentration 0.14
Ig/11 .
Other impurities [g/1] ca. 1
'.°° 0 0 0 '°°.°°
0 0
a Ectoine
O hlydroxyectoine O
O Hydroxyectoine pu~ty ,
0.80 80.00
0.60 ~ a ~ 80.00
O
a
CICo
a m
0.40 40.00 c
O
a
m
a x
0
a
0
0.20 C 20.00
0
00
a
0.00 , ~ 0.oa
0 5 10 15 20 25
Tlme (h]