9-(AMINOALCOYL)-8-HYDROXYADENINES ET METHODE DE PREPARATION

9-(AMINOALKYL)-8-HYDROXYADENINES AND METHOD OF THEIR PREPARATION

Abrégé anglais

ABSTRACT OF THE DISCLOSURE:
The present invention relates to new compounds of
general formula
<IMG> (I)
where R1 and R3 is hydrogen, alkyl, or the hydroxyalkyl
group, R2 hydrogen or the hydroxy group, X is H or
CO(CH2)6R4, n is 0 or 1, and R4 is the residue of a suitable
support e.g. a macromolecular dextran gel. The present
invention also provides a method of preparation of these
compounds, i.e. of 9-(aminoalkyl)-8-hydroxy-adenines or
derivatives thereof. The above compounds may serve as
ligands for the preparation of modified polymerous dextran
gels for affinity chromatography of S-adenosyl-L-homocysteine
hydrolase.

Revendications

The embodiments of the invention in which an
exclusive property or privilege is claimed are defined as
follows:
1. A compound of general formula (I)
(I)
<IMG>
where R1 and R3, independently, are a hydrogen atom, an
alkyl group of 1 to 4 carbon atoms, or a hydroxyalkyl group
with one to four carbons in the alkyl moiety, R2 is a
hydrogen atom or an hydroxy group, X is H or CO(CH2)6R4 and
R4 is the residue of a macromolecular dextran gel, and n is
0 or 1.
2. A compound of general formula (Ia)
<IMG> (Ia)
where R1 and R3, independently, are a hydrogen atom, an
alkyl group of 1 to 4 carbon atoms, or a hydroxylalkyl
group with one to four carbons in the alkyl moiety, R2 is a
19

hydrogen atom or an hydroxy group, and n is 0 or 1.
3. Method of preparation of compounds of general
formula (Ia) as defined in claim 2, according to which
9-(hydroxyalkyl)adenine of general formula (II)
(II)
<IMG>
where R1, R2, R3 and n are as defined above, is made to
react with bromine in aqueous solution or suspension, at a
temperature of 0 to 30°C and the 9-(hydroxyalkyl)-8-bromo-
adenine of general formula (III)
<IMG> (III)
where R1, R2, R3 and n are as defined above is heated at a
temperature of 80 to 150°C with an aqueous 20 to 30% (by
weight) solution of ammonia at a weight ratio of 1:10 to 1:40.
4. A compound of general formula (Ib)

<IMG> (Ib)
where R1 and R3, independently, are a hydrogen atom, an
alkyl group of 1 to 4 carbon atoms, or a hydroxyalkyl group
with one to four carbons in the alkyl moiety, R2 is a
hydrogen atom or an hydroxy group, X is CO(CH2)6R4 and R4
is the residue of a macromolecular dextran gel, and n is 0
or 1.
5. Method of preparation of a compound of general
formula (Ib) as defined in claim 4, according to which a
dextran gel with 6-carboxyhexyl groups, is made to react
with a 9-aminoalkyl-8-hydroxyadenine of general formula (Ia)
as defined in claim 2, at a ratio of 1 to 10 molar equiva-
lents in terms of carboxyl groups of the dextran gel, and in
the presence of one or more salts of soluble carbodiimides
of general formula (IV)
<IMG> (IV)
where R5, R6 and R7 are methyl groups, or R5 and R6 together
are (-CH2CH2)2O and R7 is a methyl or ethyl group, R8 is a
cyclohexyl or ethyl group, Y is a chloride or p-toluene-
sulfonate anion and m is 2 to 4, in aqueous solution at pH 5
to 6 and temperatures of 0 to 30°C; after completion of the
reaction the insoluble gel is filtered off, washed with water
and then a neutral buffer solution.
21

6. A method for the isolation of S-adenosine-L-
homocysteine hydrolases from extracts of biological materials,
consisting in adsorption of the crude or partly purified
extract of biological material batch-wise or in a column to
an affinity support of general formula (Ib) as defined in
claim 4, and after washing the support with buffered solu-
tions of neutral electrolytes, in displacing the S-adenosyl-
L-homocysteine hydrolases by a small volume of adenosine
solutions 0.01 to 1.0 mmol.1-1 in an aqueous buffer at
temperatures of 0 to 20°C, and in subsequent removal of
adenosine from the eluate by gel filtration or dialysis.
7. Method of preparation of a compound of general
formula (I)
(I)
<IMG>
where R1 and R3, independently, are a hydrogen atom, an alkyl
group of 1 to 4 carbon atoms, or a hydroxyalkyl group with one
to four carbons in the alkyl moiety, R2 is a hydrogen atom or
an hydroxy group, X is H or CO(CH2)6R4 and R4 is the residue
of a macromolecular dextran gel, and n is 0 or 1, character-
ized in that
(a) when X is H, a 9-(hydroxyalkyl)adenine of
general formula (II)
<IMG> (II)
22

where R1, R2, R3 and n are as defined above is made to
react with bromine in aqueous solution or suspension, at a
temperature of 0 to 30°C and the 9-(hydroxyalkyl)-8-
bromoadenine of general formula (III)
<IMG> (III)
where R1, R2, R3 and n are as defined above is heated at a
temperature of 80 to 150°C with an aqueous 20 to 30% (by
weight) solution of ammonia at a weight ratio of 1:10 to
1:40, and
(b) when X is CO(CH2)6R4, a dextran gel with
6-carboxyhexyl groups, is made to react with a 9-aminoalkyl-
8-hydroxyadenine of general formula (I) where X is H at a
ratio of 1 to 10 molar equivalents in terms of carboxyl
groups of the dextran gel, and in the presence of one or
more salts of soluble carbodiimides of general formula (IV)
<IMG>
(IV)
where R5, R6 and R7 are methyl groups, or R5 and R6 together
are (-CH2CH2)2O and R7 is a methyl or ethyl group, R8 is a
cyclohexyl or ethyl group, Y is a chloride or p-toluene-
sulfonate anion and m is 2 to 4, in aqueous solution at pH 5
to 6 and temperatures of 0 to 30°C; after completion of the
reaction the insoluble gel is filtered off, washed with water
and then a neutral buffer solution.
23

8. A compound of general formula (Ib)
<IMG>
where R1 and R3, independently, are a hydrogen atom, an
alkyl group of 1 to 4 carbon atoms, or a hydroxyalkyl group
with one to four carbons in the alkyl moiety, R2 is a
hydrogen atom or an hydroxy group, X is CO(alk)R4, alk is a
suitable alkylene chain and R4 is the residue of a macro-
molecular dextran gel, and n is 0 or 1.
9. Method of preparation of a compound of general
formula (Ib) as defined in claim 8, according to which a
dextran gel with .omega.-carboxyalkyl groups, is made to react with
a 9-aminoalkyl-8-hydroxyadenine of general formula (Ia) as
defined in claim 2 at a ratio of 1 to 10 molar equivalents
in terms of carboxyl groups of the dextran gel, and in the
presence of one or more salts of soluble carbodiimides of
general formula (IV)
<IMG> (IV)
where R5, R6 and R7 are methyl groups, or R5 and R6 together
are (-CH2CH2)2O and R7 is a methyl or ethyl group, R8 is a
cyclohexyl or ethyl group, Y is a chloride or p-toluene-
sulfonate anion and m is 2 to 4, in aqueous solution at pH 5
to 6 and temperatures of 0 to 30°C; after completion of the
24

reaction the insoluble gel is filtered off, washed with
water and then a neutral buffer solution.
10. Method of preparation of a compound of general
formula (I)
<IMG> (I)
where R1 and R3, independently, are a hydrogen atom, an alkyl
group of 1 to 4 carbon atoms, or a hydroxylalkyl group with
one to four carbons in the alkyl moiety, R2 is a hydrogen atom or
an hydroxy group, X is a H or CO(alk)R4, alk is a suitable
alkylene chain, and R4 is the residue of a macromolecular
dextran gel, and n is 0 or 1, characterized in that
(a) when X is H, a 9-(hydroxyalkyl)adenine of
general formula (II)
<IMG> (II)
where R1, R2, R3 and n are as defined above, is made to
react with bromine in aqueous solution or suspension, at a
temperature of 0 to 30°C and the 9-(hydroxyalkyl)-8-
bromoadenine of general formula (III)

<IMG> (III)
where R1, R2, R3 and n are as defined above is heated at a
temperature of 80 to 150°C with an aqueous 20 to 30% (by
weight) solution of ammonia at a weight ratio of 1:10 to
1:40, and
(b) when X is CO(alk) R4, a dextran gel with
.omega.-carboxyalkyl groups, is made to react with a 9-aminoalkyl
8-hydroxyadenine of general formula (I) where X is H at a
ratio of 1 to 10 molar equivalents in terms of carboxyl
groups of the dextran gel, and in the presence of one or
more salts of soluble carbodiimides of general formula (IV)
<IMG> (IV)
where R5, R6 and R7 are methyl groups, or R5 and R6 together
are (-CH2CH2)2O and R7 is a methyl or ethyl group, R8 is a
cyclohexyl or ethyl group, Y is a chloride or p-toluene-
sulfonate anion and m is 2 to 4, in aqueous solution at pH 5
to 6 and temperatures of 0 to 30°C; after completion of the
reaction the insoluble gel is filtered off, washed with water
and then a neutral buffer solution.
11. A compound of general formula (I)
26

<IMG> (I)
where R1 and R3, independently are a hydrogen atom, an alkyl
group of 1 to 4 carbon atoms, or a hydroxyalkyl group with
one to four carbons in the alkyl moiety, R2 is hydrogen or
the hydroxy group, X is H or the residue of a suitable
polymer support and n is 0 or 1.
12. A compound of general formula (I)
<IMG> (I)
where R1 and R3, independently, are a hydrogen atom, an
alkyl group of 1 to 4 carbon atoms, or a hydroxyalkyl group
with one to four carbons in the alkyl moiety, R2 is hydrogen
or the hydroxy group, X is the residue of a suitable
polymer support and n is 0 or 1.
13. A method for the isolation of S-adenosine-L-
homocysteine hydrolases from extracts of biological materials,
consisting in adsorption of the crude or partly purified
extract of biological material batch-wise or in a column to
an affinity support of general formula (I) as defined in
27

claim 12, and after washing the support with buffered
solutions of neutral electrolytes, in displacing the S-
adenosyl-L-homocysteine hydrolases by a small volume of
adenosine solutions 0.01 to 1.0 mmol.1-1 in an aqueous
buffer at temperatures of 0 to 20°C, and in subsequent
removal of adenosine from the eluate by gel filtration or
dialysis.
14. A compound of general formula (I)
<IMG> (I)
where R1 and R3, independently, are a hydrogen atom, an
alkyl group or a hydroxyalkyl group, R2 is a hydrogen
atom or an hydroxy group, X is H or is the residue of a
suitable dextran gel, sepharose or agarose support, and
n is 0 or 1.
28

Description

-- 1 --
~2~
The present invention relates to new compounds of
general formula I
NH2
S
~ N ~ (I)
R -CH-(CHR ) CH(R3)NHX
where Rl and R3, independently, are a hydrogen atom, an
alkyl group , an hydroxyalkyl group
R is hydrogen or an hydroxy group, X is H or is the
residue of a suitable support, n is 0 or 1. X may be
CO(CH2)6R where R is the residue of a macromolecular
dextran gel. The support may be organic or inorganic.
Alkyl groups or moieties thereof may have ~rom
1 to 4 carbon atoms, i.e. for Rl and R3.
An efficient purification and isolation of
enzymes can be carried out by the so-called affinity chro~
matography based on strong specific and reversible bonding
of the enzyme to a polymer support with immobilized low
molecular weight ligands having the charac-ter of modified
substrates, products, or inhibitors of the corresponding
enzyme.
One of the important enzymes which ccntrols the
basic metabolic functions of the living organism is S-
adenosyl-L-homoc~steine hydrolase (SAH-hydrolase). This
enzyme is present in all kinds of eukaryo-tic cells and its
level may be related -to the development of some metabolic
disorders. Data on the relationship between this enzyme
and the growth of experimental tumors in animals have been
r ~

- 2 ~ 48
reported tGoN~ Orlov, J.V. Bukin: Voprosy med. chim. 26,
699 (1980)). This finding stimulated interest in methods
of isolation/ purification, and determination of the level
of this enzyme in samples of biological material. The SAH-
hydrolases are very unstable and loose their enzymaticactivity in the course of procedures routinely used for the
isolation of proteins (fractionation by precipitation, ion
exchange chromatography, gel filtration). Methods which
permit a rapid and specific isolation of these enzymes from
crude biological extracts are the most suitable ones; such
a method is, e.g. affinity chromatography.
Affinity chromatography as a method of isolation
of SAH-hydrolase on a support with immobilized 8-(3-amino-
propylamino)adenosine has been reported (E.O. Kajander,
A.M. Raina: Biochem. J. 193, 503 (1981)); this method,
however, is not very e~ficient. A procedure based on the
use of polymer supports with covalently bonded 9-~RS)-(3(2)-
aminopropylamino-2(3)-hydroxypropyl)-8-hydroxyadenine (a.c.,
PV 8920-82) has also been described; this procedure is very
~ efficient yet it requires a ligand which can be prepared by
a method which is technically relatively demanding (a.c.
PV 8919-82).
These drawbacks can be eliminated by usin~ the
subject of this invention, the new compound of formula I
described above.
The present invention also relates to a method of
preparation of compounds of general fcrmula I, where X is
H~9-(aminoalkyl)-8-hydroxyadenine). According to this
method 9-(hydroxyalkyl)adenine of general formula II,
NH2
(II)
Rl-CH-(CHR )nCH(R3)OH

- 3 ~ 8
where Rl through R3 and n are the same symbols as in formula
I, is allowed to react with a halide (e.g. bromine) in
aqueous solution or suspension at a temperature of 0 to 30C
and the 9-(hydroxyalkyl)-8-bromoadenine formed of general
formula III,
NH2
~ ~ ~ -hal (e.g. Br) (III)
1 1 2 3
R -CH-(CHR )n-CH(R )-OH
where Rl through R3 and n are the same symbols as in formula
I, is heated with an aqueous 20 to 30% (by weight) solution
of ammonia at a weight ratio of 1:10 to 1:40 at a tempera-
ture of 80 to 150C.
The present invention also relates to a method of
preparat.ion of compounds of general formula I, where X is
CO(alk)R4, alk being a suitable alkylene chain e.g. (CH2)6
and R is the same symbol as in formula I above. According
to this method a dextran gel bearing an ~-carboxyalkyl, to
advantage a 6-carboxyhexyl group, is allowed to react with
9-aminoalkyl-8-hydroxyadenine of general formula I, where X
is H, the ratio being 1 to 10 molar equivalents per one
c ~ ~e

4 -
carbodiimides of general formula IV,
R5
X6_ N~-(CH2)mN=C=N~R8.Y (IV)
R7
where R~ through R7 are methyl groups, or R5 and R6 a~e
together (-CH2CH2)20 and R7 the methyl or ethyl group, R8 the
cyclohexyl or ethyl group, Y the chloride or p-toluenesul-
phonate anion~ and m is 1 to 4. The reaction is allowed
to proceed in an aqueous so].ution at pH 5 to 6 and tempera-
tures of 0 to 30 C and after completion of the reaction the
insoluble gel is filtered off and washed with water and 501-
utions of neutral buffers.
Still another subject of the inve~tion is the use of the
affinity support according to the invention for the i~olation
of S-adenosyl-L-homocy~Rine hydrolases from e~tracts o~ bio-
logical materials, consisting in the ad~orption of the crude
or p~rtly purified extract of the biological material either
batchwise or in a column to the af~inity 3upport of general
formula I, where X is Co(CH2)6R4; after washing of the ~up-
port with aquQous buffered solution~ of neutral electrolytes
the S-adenosyl-L-homocysteine hydrola6es are ~pecifically
displaced by a small volwme of 0.01 to 1~0 mmol.l 1 adeno~ine
solution in an aqueous buffer at temperature~ of 0 to 20 C
and subsequently adenosine is removed from the effluent by
gel ~iltration or dialysis.
The advantage of the preparation of compounds o~ formula I,
where X is H~ according to the invention consists in the
fact tha-t the replacement of the substituent in position ~ of

- 5 - ~.22~
the compounds of formula III and the exchange of the hydroxyl
group in their side chain for an amino group may proceed in
one reaction step and under extraordinarily simple conditions.
The condition necessary for this reaction to proceed i8 the
possibility o~ formation of cyclic intermediate~ (A.Hol~
Collect~Czech.Chem.Commun. 48, 1910 (1983) ) and at the seme
time is limited to the preparation of ~uch compound3 of for
mula I~ where X is H, in which th~ a~ino group of the sid~
chain is i~ alpha or beta po~ition with respect to the carbon
atom binding the adenine ring.
The starting compounds o~ general formula II ~re gensr~lly
well accessible, e.g. by alkylations of ad~nine t~.Hol~,
Collect~Czech~Chem.Commun. 43~ 3103 (1978); 43~ 3444 (1978);
43, 2054 (1978); 44~ 593 ~1979); 47, 173 (1982) ). Th~ reactio~
o~ these compounds with bromine e~ily proceed~ i~ an aqueou~
medium~ both homogeneous and heterogeneou~7 with a small ex-
c~ss (20 to 50~) of bromine and yield~ hydrobromide~ o~ ~or-
mula III as the only reaction products. These compounds can
be reacted with ammonia directly; it i~ howevor~ advantageous
to isolate the compounds of formula III thu~ alimi~ating the
ha2ard of formation of colored contaminants in the pr~duct~
which are removable with difficulties only. This isolation i~
carried out after evaporation of water from the reaction mi~-
ture in vacuo (e.g. 2 to 2.5 kPa at temperatures o~ 30 to 50 C~
by careful and e~act neutralization of the reaction mixtur~
with concentrated tO.5 to 4.0 mol~ aqueou~ solution~ o~
alkaline hydroxides (with lithium hydroxide to adv~ntage) thus
precipitating the very little soluble product of formula III
in most cases. I~ compound~ o~ formula III are more or les~

~2~ 8
-- 6
soluble in water they can be easily extracted from the dry
evaporate of the neutralized reaction mixture with chloro-
form or its mixtures with methanol or ethanol; they can be
isolated from the neutralized solution of the reaction mix-
ture to advantage also by deionization on a cation exchangerwhich is washed with water and subsequently weakly alkalized
by the addition of a volatile amine (ammonia to advantage)
and the compound of formula III is obtained after evaporation
of this effluent.
The reaction of the compounds of formula III is
carried out to advantage by heating with concentrated aqueous
ammonia, in suspension or solution, without external stirring.
Since the pressure in the closed reaction vessel does not
rise to high values at the temperatures used, ~imple common,
low-pressure reaction vessels, resistant to aqueous ammonia,
or glass thick-walled reactors can be used for the reaction.
The control of the course of the reaction and of
the purity of the reaction products is carried out to advan-
tage by paper chromatography or thin-layer chromatography on
silica gel, in both cases in the system 80~ aqueous 2-propanol-
concentrated aqueous ammonia (9:1); the detection is performed
in ultraviolet light and the compounds of formula I, where
X is H, are detected by spraying the chromatogram with nin-
hydrin (violet spots).
The reaction yields generally compounds of formula
I, where X is H, as the only reaction products. The evapora-
tion of the reaction mixture affords a mixture of the product
or its hydrobromide with ammonium bromide. The compound of
formula I, where X is H, can be obtained in pure state by
chromato-

graphy, e.g. on silica gel or cellulose in the mixture 80%
aqueous 2-propanol-con. aqueous ammonia (9:1) or by chroma-
tography on octadecylsilica gel in water.
The binding of compounds of formula I, where X is
H to suitable supports may be effected by any suitable
mechanism, e.g. between the amino group and a suitable reac-
tive functional group on a suitable (polymer) support, e.g.
carboxyl group, acylhalide group, aldehyde group, etc.
The binding of compounds of formula I, where X is
H, to polymer supports may,for example, be effected by
formation of an amide linkage, i.e. an amide bond between the
compound and the carboxyl functions of the-polymer support.
Such a support may be a dextran gel (e.g. Sepharose, Agarose)
and also cellulose or other polymer material which has been
modified in advance to contain free carboxyl groups attached
to the polvmer matrix through sufficiently long, e.g. poly-
methylene (hexamethylene) chains. Since the amide bond formed
is chemically stable and resistant to the action of most
enzymes the polymer materials thus prepared of formula I,
where x is CO(CH2)6R , are stable for several months at tem-
peratures of 0 to 10C in water or, to advantage, in satu-
rated solutions of sodium or potassium chloride. The reaction
in the presence of so-called soluble carbodiimides of general
formula IV, by which the condensation is effected, takes
place under mild conditions which do not deteriorate the
structure of the polymer matrix.
The polymer supports of formula I, where x is
CO(CH2)6R , show a high affinity for SAH-hydrolases. They
can be used therefore for specific concentration of these
enzymes during their isolation from various crude or only
partly purified extracts of various biological materials.
Thus, e.g. compounds of formula I, where x is CO(CH2)6R , can
be added to advantage to a dilute solution till its enzymatic
activity disappears; subsequently the polymer support is
washed to remove contaminating proteins and other components
not adsorbed (dyes

etc.) with solutions of neutral electrolytes of increasing
ionic strength~ to advantage at O to 10 C. The SAH-hydrolase
is subsequently eluted from the support by dilute solutions
of adenosine which is a substrate or~ alternatively, an in-
hibitor of this enzyme. The consumption of adeno~ine ~or this
purpose i8 minimal. The whole isolation proc~dure can be
carried out equally well batchwi SQ with filtration aftar each
elution step or on a column of the above support. Adenosine
is removed from the effluent containing the purified enzyme
by dialysis~ ultrafiltration, or g~l filtration (e.gO on
SeE)hadex ~ or Biogel ~) ) .
This procedure of isolation of SAH-hydrolases is not
very demanding as regards time and material and yields vory
pure, entirely or almost entirely homogeneous proteins (ac-
cording to gel electrophoresis). It is therefore espccially
suited for rapid analyses of biological. materials, tissue
extracts etcO in those cases where a small quanti~y only of
the preparation is available which could not be obta~ned by
other isolation procedures. The procedure can be also u~ed
for preparative-scale isolations ~hen larg~r quantities of
the homogeneous proteins are to be isolated.
S-Adenosyl-L-homocysteine hydrolase is o~ conside~able
preparative importance from this viewpoint: using this enzyme
S-adenosyl-L-homocysteine can easily be prepared ~rom adeno-
sine and L-homocysteine~ at low cost in an aqueous medium in
a high yield, with the possibility of adenosine regenera-tion
(Chabannes B., Charit A., Cronenberger L~, Pach~co H : Prep.
Biochem. 12, 195 (1982) ). Since S-adenosyl-L-homocysteine
is the starting material for S-adenosyl-L-methionin~used as
. !

~2~1L48
g
a drug, a simple purification of SAH-hydrolases, especially
the removal of contaminating enzymes degrading adenosine,
is of technical importance.
The general procedure of compounds of formula I
according to the invention is given below and their use for
the isolation of SAH-hydrolases is illustrated by additional
examples which in no way limit its scope.
EXAMPLE 1:
The solution of 1 ml of bromine in 150 ml of water
is treated with 10 mmol of compound of formula II and the
mixture is stirred in a closed vessel at a temperature of
18 to 24 C for 16 to 24 h. The suspension is then evapo-
rated at 40 C/kPa to dryness and the residue is dissolved
in 100 ml of water. The solution is neutralized to pH 7.0
(6.95 to 7.05) with stirring using a pH-meter and 4 mol.1 1
sodium or lithium hydroxide and subsequently cooled down to
0 C for 1 to 2 h. The separated product of formula III is
filtered off, washed with water (200 ml), acetone (100 ml)
and ether (lO0 ml), and dried at 10 to 15 Pa over phosphorus
pentoxide. The yields and properties of compounds of formula
III prepared by this procedure are given in Table l at page
15.
The suspension of 5 mmol of the compound of formula
III in 50 ml of concentrated aqueous ammonia (25 to 29~ NH3)
is heated in a steel pressure vessel at 100 to 110 C for 2
to 8 h. After cooling the clear pink solution is evaporated
at 40 C/2 kPa to dryness, the residue is dissolved in 20 ml
of a mixture of ~0~ aqueous 2-propanol and conc. aqueous
ammonia (9:1) and applied to a column (80 x 4 cm) of micro-
crystalline
~. /
,~

- l o -
~ 2Z~
cellulose in the same system. The column is eluted (at a rate
of 20 ml/h) by the same system and the fractions (20 ml) are
analyzed by paper chromatography in the same system. The pro-
duct-containing fractions are pooled9 taken to dryness at
40 C/2 kPa~ the residue is evaporated with ethanol ~2 x 20 ml)
under the same conditions~ and the residue is crystallized
from methanol with the addition o~ ether till turbidity appears.
The obtained compound of formula I~ where X is H~ is filter-
ed off, washed with ether, and dried at 10 to 15 Pa over 80-
dium hydroxide or natron calk. The mother liquors contain an
additional amount of the compound in th~ ~orm oP bicarbonat~
which can be repeatedly subjected to the same chrom~tography
or precipitated with ether and isolated as the bicarbonate.
The yield and the properties of the compounds thus prepared
of formula I, where X is H, are given in Table 2 at page 16.
Exnmple 2
The compound of formula II (10 mmol) is reacted with brom-
ine and treated as described under Example 1. In ca~e that
the product of ~ormula III is water-soluble and does not pr~-
cipitate from water the following procedure lS used:
A neutral aqueous solution of the dry residue of th~ r~-
action mixture is applied to a column t200 to 250 ml) of a
cation exchanger (Dowex~50 to advantage) in acid form and th~
column is washed with 1 1 of water. The ion ~xch~nger is then
suspended in 400 ml of water and treated with stirring with
dilute (1:2, vol/vol) aqueous ammonia so that the pH is kept
below 8 until this value remains constant ~or 30 minr The
suspension is ~iltered off and the ion exchanger is washed

with boiling water (0.5 to 1.0 1). The filtrate is
evaporated at 40 C/2 kPa and the dry residue is crystal-
lized from ethanol or 80% aqueous ethanol. The product
is filtered off and dried at 10 to 15 Pa over phosphorus
pentoxide. The properties and yields of the compounds
thus prepared of formula III are given in Table 1 at page
15. The subsequent procedure is the same as that described
under Example 1.
EXAMPLE 3:
The dextran gel slurry (200 ml), e~g. CH-Sepharose
(a registered trade mark of the product of Pharmacia, Uppsala,
Sweden), where CH stands for the gel with modified carboxyl-
hexyl groups) is washed stepwise with 0.1 mol.l sodium
bicarbonate (5 litres) and water (4 litres). The compound
of formula I, where X is H, is added to a suspension of 10
ml of the gel slurry in 30 ml of water (usually a two- to
three-fold excess in terms of the carboxylate capacity of
the support). The pH of the mixture is adjusted to pH
5.0 by 2 mol.l 1 hydrochloric acid with magnetic stirring
using a pH-meter and the first portion is added of the com-
pound of formula IV (usually a 2.5-fold excess with respect
to the compound of formula I, where X is H). The suspension
is stirred and the pH is maintained at 5.0-5.5 by hydrochloric
acid (2 mol-l 1). The same portion of the compound of for-
mula IV is added 30 min later and the pH of the mixture is
maintained in the same manner until it remains constant.
The pH is adjusted to 5.0 and the suspension is gently
shaken for 15 to 24 h at room temperature (18 to 25 C).
Subsequently the suspension is filtered off, the gel is
washed with water (500 ml) and suspended in 20 ml of 0.5
mol.l 1 solution of 2-aminoethanol hydrochloride (pH 5.0).
Subsequently the third, same portion of the compound of
formula IV is added and the pH is again maintained at 5.0-

- 12 - ~22~8
5.5 by adding 2 mol.l l hydrochloric acidO The suspension
is gently shaken for additional 3.5 h, filtered off after-
wards, and washed stepwise with water (1 liter) and saturated
solution of potassium chloride. The support thus prepared
of formula I, where X is CO(CH2)6R , is stored in saturated
solution of potassium chloride containing 0.02% of sodium
azide at a temperature of 0 to 4 C. Specific data on the
affinity support types of formula I, where X is CO(CH2)6R ,
obtained in this manner and on the mutual ratios of reactants
used are given in Table 3 at page 17.
EXAMPLE 4:
The isolation of SAH-hydrolase from rat liver is
carried out with a partly purified concentrate of this
enzyme (Votruba I., Holy, A.: Collect. Czech. Chem. Commun.
45, 3039 (1980)) in 0.1 mol.1 1 sodium chloride having a
specific activity of 0.9 EU (EU stands for international
units of enzyme activity) per mg of protein. This enzyme
preparation is diluted 1:4 with 0.01 mol.1 1 Sorensen
potassium phosphate buffer, pH 7.37, containing 0.001 mol.1 1
dithiothreitol. The whole procedure is carried out at 0 C.
The support (0.6 ml) of formula I, where X is CO(CH2)6R , is
added to 1 ml of the dilute enzyme solution and the suspen-
sion is gently shaken 20 min at 0 C; it is then centrifuged
and the sediment shaken stepwise for 20 min with the following
solutions (1 ml of each):
_,~

- 1~4~- ~2~
G.01 mol.l 1 ~orensen phosphate buffer~ p~ 7.37~ same bu~er
yet 0.2 mol.l 1, 1 mol.l 1 potassium chloride, 1.5 mol.l 1
potassium chloride. 2.0 mol.l 1 potassium chloride~ 0.025
molOl 1 adenosine in 0.75 mol.l 1 potassium chloride. All
buffers contain 0.001 mol.l 1 dithiothreitol. The enzymatic
activity of ShH-hydrolase is determined in all eluates by the
procedure described under Example 5. The individual eluate~
are obtained by centrifugation of the suspen~ion. The pro-
perties of the supports used in this example and the yields
of SA~-hydrolase isolated from the last elua~e are summarized
in Table 4 at page 18.
Example 5
The wet mass (100 g) of a suspension culture of Nicotiana
tabacum cells is washed on a glass filter with 0.01 mol.l 1
potassium hydrogenphosphate, pH 7.4 ~2 liters~ and the pellet
is triturated in liquid nitrogen in four portions with 15 g
each of A1203 (alumina 305). The material obtained is suspen-
ded in 200 ml of the same bu~er also containing 1 mmol.l 1
dithiothreitol and 14 g o~ polyvinylpyrrolidone. The extract
is centrifuged 40 min at 27 000 g and the supernatant i~
treated ~ith slow stirring at 4C with ammonium sulfate added
to 80% saturation. The suspension is centri~uged 25 min at
30 000 g and then dissolved in the above buffer containing
dithiothreitol (100 ml). A total volume of 10 ml o~ the affin-
ity support (No 3, Table 3) is added successively with stir-
ring. The mixture is stirred 30 min at 30 C, filtered of~
ar.d then washed stepwise with 40-ml portions of the first five
washing buffers described under Example 4~ The support is

2~
then transfered to a column (0.~ x 20 cm) which is eluted
at 3 C by 40 ml of 0.25 rnmol.l 1 adenosine in 0O75 mol.l71
potassium chloride containing 1 mmol.1 1 dithiothreitol. The
effluent is chromatographed on a column (1.20 x 60 cm) of
Sephadex G-25 in 0.01 mol.l 1 potassium hydrogenphosphnto,
p~ 7.4, containing 1 mmol.l 1 dithiothreitol. The enzymatic
activity of the fractions (2 ml) is assayed as described
under ~xample 6. The frac-tions containing enzymatic activity
are pooled and ~lycerol is added to the final concentration
of 20% (by vol.). The yield is 238 ~g of electrophoretically
pure SAH-hydrolase protein of specific activity 72~7 ncat.mg 1.
~xample 6
The e~zymatic activity of SAH-hydrolase is determined in
100 ~1 of a solution which is 0.1 mol.l 1 in potassium hydro-
genphosphate, pH 7.37, 0.003 mol.1 1 in dithiothreitol~ 3~75
x 10 3 mol-l 1 in L-homocysteine~ and 2.5 x 10 5 mol.l 1 in
adenosine. The solution assayed (50 ~1) i9 added9 the mi~ture
is incubated 10 min Qt 37 C~ and a 10- (ul sample of the
mixture is separated on a column of Separon SI C18 (5~u) (3.3
x 150 mm) eluted by 0.01 mol-l 1 potassium dihydrogenphos-
phate9 pH 2.8, containing 10 volume per cent of methanol.
The flow rate is 0.4 ml/min~ the effluent is monitored at
254 nm. The elution profile is continuously recorded in a
recording UV-detector and the peak areas of adenosine and
S-adenosyl-L-homocys-teine, whose positions are determined
in advance in runs with st~ndards, are evaluated by
planimetry. The number of enzyme activi-ty units is calculated
irom the formula

~ ~20~
- 14 -
EU = 0.5 x 10 K
where 1 EU is the quantity of enzyme converting 1 umol of
substrate in 1 min and K the conversion of adenosine into
S-adenosyl-L-homocysteine expressed in per cent. This
value corresponds to the quantity of enzyme in 50~ul of the
mixture assayed.
.,~

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~22()~
Table 4 Isolation of rat liver S-adenosyl-L-homo-
cysteine hydrolase by affinity chromatography according to
Example 4 (from 50 EU of enzymatic activity applied)
Support (a) Losses due toYield of pure
nonspecific enzyme
desorption
(%) (%)
1 2.5 32
2 0 39
3 0 40
4 15 29
7 0 33
(a) Designation according to Table 3