A method is disclosed for preparing functionalized adenine derivatives, said method comprising the step of reacting a compound which contains an adenine nucleus which has been halogen substituted in the 8-position, with a compound having the general formula N.sup.+- S--(CH.sub.2).sub.n --CO0.sup.- M.sup.+ wherein M.sup.+ is the ion of an alkali metal and n is an integer, the reaction being carried out in a polar aprotic solvent. Procedures for preparing macromolecularized adenine compounds are also indicated by reacting a functionalized adenine derivative with a polymer which has at least one primary or secondary aminic group in its structure.

This invention relates to a method for the preparation of functionalized 
adenine derivatives and to the products obtained thereby. More detailedly, 
the present invention relates to a method for the preparation, starting 
from compounds which contain an adenine nucleus having a halogen atom in 
the 8-position, of functionalized adenine derivatives carrying in said 
position an omega-carboxylic side chain. The starting materials for said 
method can be obtained, with conventional methods, by halogenating 
compounds which contain the adenine nucleus, such for example 
nicotinoylamine adenine dinucleotide (NAD.sup.+), nicotinoylamide-adenine 
dinucleotide phosphate (NADP.sup.+), adenosine triphosphate (ATP), 
adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine. 
The majority of these compounds have an outstanding importance in 
biochemistry and their functionalization widens their field of 
application. 
For example, in the case of NAD.sup.+, but these considerations hold good 
also for the other members, its functionalized derivatives can be used, 
after having been attached by a covalent bond to macromolecules which are 
either water-soluble or water-insoluble, as non-diffusible coenzymes, or 
in affinity chromatography. Thus, in the case of attachment to 
water-soluble macromolecules, they can be used as non diffusible, 
water-soluble macromolecularized coenzymes. These permit the widening of 
the field of application of the known enzymic systems in which the enzyme 
is physically embedded in insoluble porous structures, such as fibers, 
polyacrylamide gel, microcapsules, etc. which are impervious to 
macromolecules. As a matter of fact, by physically embedding, together 
with the enzyme or a polyenzymic system also the water-soluble 
macromolecularized coenzyme, both the enzyme and the coenzyme remain in 
close contact and the scattering of the latter towards the outside of the 
occluding structure is prevented, while with the natural coenzyme this 
cannot be done on account of the low molecular weight of the latter. In 
the case of attachment of water-insoluble macromolecules they can be used 
for affinity chromatography or for enzymic reactions in a heterogeneous 
phase, it being possible to recover the coenzyme. 
According to the present invention, the above mentioned derivatives which 
have been functionalized in the adenine nucleus are obtained by reacting 
the corresponding starting compound which has been halogen-substituted at 
the C.sub.8 of the same nucleus, with the di-salt of an 
omega-mercaptocarboxylic acid having the general formula: 
EQU M.sup.+- S--(CH.sub.2).sub.n --COO.sup.- M.sup.+ 
wherein M.sup.+ is the ion of an alkali metal and n is an integer. 
The reaction is conducted in aprotic polar solvents (such as 
hexamethyl-phosphotriamide, dimethyl sulphoxide and dimethylformamide) at 
a temperature ranging from +20.degree. C. to +60.degree. C., preferably at 
room temperature, and under anhydrous conditions. 
The reaction causes a substitution of the halogen in the 8-position of the 
adenine nucleus in the manner shown by the formula: 
##STR1## 
wherein n and M.sup.+ have the meanings as indicated above, X is a halogen 
and R is the non-adenine residue of the compound. 
The salt of the as-obtained carboxyl derivative is then converted during 
progress of the processing, into its corresponding free acid. 
The method is absolutely general, but in the following portion of the 
disclosure, reference will be had to the reaction of the sodium bi-salt of 
the 3-mercaptopropionic acid with the derivatives of NAD.sup.+ and 
NADP.sup.+ which have been brominated at the 8-position carbon of the 
adenine nucleus of adenosine, with the aim of illustrating the methods 
which are required for carrying out said process. 
It will be anyhow apparent, on reading the following, that anyone skilled 
in the art will be able to obtain functionalized adenine derivatives of 
the kind referred to above, starting from any 8-halogen adenine substrate 
by merely adapting the working conditions to the nature of the starting 
compound, without departing from the scope of the present invention. 
This invention is also concerned with the preparation of macromolecularized 
adenine derivatives and the products obtained thereby, these latter 
containing one or more units having the general formula: 
##STR2## 
wherein n is an integer, and the radical 
##STR3## 
can be obtained from any compound having adenine nucleus, such as, for 
example, nicotinoylamide-adenine dinucleotide, nicotinoylamide-adenine 
dinucleotide phosphate, adenosine monophosphate, cyclic adenosine 
monophosphate, adenosine biphosphate, adenosine triphosphate, adenosine, 
adenine and wherein the nitrogen atom bound to the CO group is a part of a 
compound having a high molecular weight and which is either water-soluble 
or water-insoluble and contains one or more primary or secondary aminic 
groups (for example: polylysine, omega-aminoalkylpolyacrylamides, 
polysaccharide esters of omega-aminoalkylcarbamic acids, polyvinylamine, 
omega-aminoalkyl esters, or omega-aminoalkyl amides of the polyglutamic 
acid, aminoalkylsilanized glass microspheres, polyethyleneimine and 
others). Such functionalized compounds can react with at least one polymer 
having at least a primary or secondary aminic group, to give the 
macromolecularized adenine derivatives mentioned above, in the presence of 
a water-soluble carbodiimide (for example 
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride) or an 
insoluble one (for example: N,N'-dicyclohexylcarbodiimide) as a condensing 
agent. 
The condensation reaction between the carboxyl group of the functionalized 
adenine derivative and the aminic group of the macromolecule to give the 
amidic bond, is carried out in an aqueous environment or in a mixture of 
water and a water-soluble organic solvent (for example: pyridine, 
tetrahydrofuran, dioxan and others) at a temperature comprised between 
+5.degree. C. and +50.degree. C.; and preferably at room temperature. 
The macromolecularized adenine derivatives which are the subject-matter of 
the present invention, have a number of applications. 
For example, in the case of the macromolecularized derivatives of the 
nicotinoylamide-adenine dinucleotide (NAD), but the same is true of the 
other macromolecularized derivatives, they find application in affinity 
chromatography or as active, non-diffusible coenzymes. 
Thus, in the case of attachment to water-soluble macromolecules, they can 
be used as non-diffusible, water-soluble macromolecularized coenzymes. 
These permit the widening of the field of application of the known enzymic 
systems in which the enzyme is embedded physically in insoluble 
structures, such as fibers, polyacrylamide gels, microcapsules and others, 
which are impervious to the macromolecules. As a matter of fact, by 
physically embedding together with the enzyme, or the polyenzymic system, 
also the water-soluble macromolecularized coenzyme, both the enzyme and 
the coenzyme remain in close contact and the scattering of the coenzyme 
towards the outside of the occluding structure is prevented, whereas, with 
the natural coenzyme, this cannot be done due to its low molecular weight. 
In the case of attachment to water-insoluble macromolecules, they can be 
used for affinity chromatography or for enzymic reactions in a 
heterogeneous phase, it being possible to recover the coenzyme.

EXAMPLE No. 1 
Preparation of the 8-(2-carboxyethylthio)adenosine. 
To a solution of 380 milligrams (1.1 millimol) of 8-bromoadenosine in 5 mls 
of anhydrous phosphotriamide there are added with stirring at room 
temperature and in an anhydrous atmosphere (nitrogen), 633 milligrams (4.2 
millimols) of the sodium disalt of 3-mercaptopropionic acid (obtained by 
treating at room temperature the mercaptoacid with a stoichiometric amount 
of sodium hydride in anhydrous tetrahydrofuran and subsequent withdrawal 
of the solvent under vacuum). 
After 16 hours of stirring at room temperature, the mixture is filtered and 
the filtrate is supplemented with 15 mls of water and extracted several 
times with chloroform until hexamethylphosphotriamide has been discharged. 
The aqueous solution adjusted to a pH 8.5 with diluted HCl, is 
chromatographed on DOWEX-1 (HCOO.sup.-) eluting with a gradient of formic 
acid in water. The fractions with .lambda..sub.max 282 mm are combined and 
freeze-dried to give 327 milligrams of 8-(2-carboxyethylthio)-adenosine. 
The product proves to be pure at thin layer analysis (silica gel with a 
fluorescence indicator; eluent; isopropanol--water--32% ammonia in the 
volume ratios of 7:2:1; visualization of the spot by a UV lamp at 254 nm; 
Rf=0.56) and also at the high-voltage electrophoresis (Whatman paper 3 MM 
11.times.57 cm; electrolyte 0.02 M ammonium acetate pH 5.0, potential 5000 
V during 40 mins., visualization of the spot with a UV lamp at 254 nm; the 
mobility of the product towards the anode is in agreement with the 
presence of the carboxyl group whereas adenosine migrates towards the 
cathode). 
The UV spectrum in 0.1 M NaOH exhibits peak absorbance at 282 nm whereas 
that of 8-bromoadenosine is at 263 nm. 
The .sup.1 H NMR in NaO.sup.2 H shows, in addition to the signals relative 
to adenosine with the exclusion of that of the proton at the 8-position, 
those relative to the protons of the side chain: 2.88 (2H, t; CH.sub.2 
COO) and 3.86 (2H, t; CH.sub.2 S). 
Also the mass spectrum confirms the structure attributed to the product 
(m/e 239, 221, 192, 167). 
EXAMPLE No. 2 
Preparation of the nicotinoylamide-8-(2-carboxyethylthio)adenine 
dinucleotide. 
To 118 milligrams (160 micromols) of nicotinoylamide-8-bromoadenine 
dinucleotide dissolved in 2 mls of anhydrous dimethylsulphoxide there are 
added with stirring at room temperature and under an anhydrous atmosphere 
(nitrogen), 98.4 milligrams (656 micromols) of the sodium disalt of the 
3-mercaptopropionic acid (prepared as described in Example 1). 
After 16 hours of stirring at room temperature, the mixture is filtered and 
ten volumes of acetone are added to the filtrate. 
The obtained precipitate, separated by centrifugation and washed with 
acetone, is dried in a vacuo and dissolved in 15 mls of 0.1 M HCl. The 
solution, as adjusted to a pH of 7.5 with diluted soda, is chromatographed 
on DOWEX-1 (HCCO.sup.-) by eluting with a gradient of formic acid in 
water. 
The chromatographic fractions with .lambda..sub.max 276.5 are combined and 
freeze-dried to give 90 milligrams of nicotinoylamide 
8-(2-carboxyethylthio)adenine dinucleotide. The product proves to be pure 
at the thin layer analysis silica gel with fluorescence indicator; eluent; 
isobutyric acid--water--32% ammonia in the volume ratios of 66:33:1.7; 
visualization of the spot with an UV lamp at 254 nm; Rf 0.31), and at 
high-voltage electrophoresis (3 MM Whatman paper 11 by 57 cm; electrolyte: 
0.02 M ammonium acetate, pH 5.0, potential 5,000 V during 30 minutes; 
visualization of the spot by UV lamp at 254 nm; mobility towards the anode 
greater than that of NAD.sup.+, consistently with the presence of the 
carboxyl group). 
The Ultraviolet spectrum in a solution of pyrophosphate buffer, pH 8.7, 
shows a peak at 276.5 nm which, by enzymic reduction with alcohol 
dehydrogenase from yeast, passes to 282 nm with the appearance of a new 
peak at 340 nm which is characteristic of the reduced nicotinoylamide 
nucleus. 
The .sup.1 H NMR in .sup.2 H.sub.2 O shows, in addition to the signals 
relative to NAD.sup.+ with the exclusion of that of the proton attached to 
the carbon in the 8-position of the adenine nucleus, those relative to the 
protons of the side chain: .delta.2.88 (2H, t; CH.sub.2 COO) and 3.86 (2H, 
t; CH.sub.2 S). Also the mass spectrum is in agreement with the structure 
attributed to the product (m/e 221, 192, 167). 
EXAMPLE No. 3 
Preparation of the nicotinoylamide-8-(2-carboxyethylthio)adenine 
dinucleotide phosphate. 
To 50 milligrams (57.6 micromols) of nicotinoylamide-8-bromoadenine 
dinucleotide phosphate dissolved in 1 ml of anhydrous dimethylsulphoxide, 
there are added with stirring at room temperature and in an anhydrous 
atmosphere (nitrogen), 36 milligrams (240 micromols) of the sodium disalt 
of the 3-mercaptopropionic acid (prepared as described in Example 1). 
After 16 hours of stirring at room temperature, the mixture has been 
filtered and the filtrate is supplemented with ten volumes of acetone. The 
as-obtained precipitate, separated by centrifugation and washed with 
acetone, is dried in a vacuo and dissolved in 10 mls of 0.1 M HCl. 
The solution, adjusted to pH 7.5 with diluted soda, is chromatographed on 
DOWEX-1 (Cl.sup.-) by eluting with a gradient of CaCl.sub.2 in water which 
has a pH of 3 by addition of HCl. The chromatographic fractions containing 
the expected product are combined, concentrated to a small volume, and 
desalified by gel-filtration on Sephadex G-10 by eluting the product with 
water. 
The characterization of the nicotinoylamide-8-(2-carboxyethylthio)adenine 
dinucleotide phosphate is carried out similarly to what has been described 
in Example 2 for the corresponding derivative of the NAD.sup.+ 
(glucose-6-phosphate dehydrogenase has been used for the enzymic 
reduction). 
EXAMPLE No. 4 
Preparation of the 
nicotinoylamide-8-(polyethyleneimine-3-carbonylethylthio)adenine 
dinucleotide. 
##STR4## 
To 0.76 mls of an aqueous solution of polyethyleneimine (33% conc. 
weight/volume) adjusted to a pH of 5 with conc.HCl, there are added 39 
milligrams of nicotinoylamide-8-(carboxyethylthio)adenine dinucleotide 
##STR5## 
dissolved in 0.5 ml of water and 40 milligrams of 
N-ethyl-N'-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride dissolved 
in 0.5 mls of water. 
The reaction mixture, adjusted to a pH of 4.8 by addition of 2 M HCl is 
stirred at room temperature during 48 hours maintaining the pH at 4.8 
during the first three hours by addition of 0.1 M HCl. The reaction 
mixture diluted with water to 20 mls is then transferred to a 
centrifugation tube and precipitated with 20 mls of 1 M phosphate buffer 
at a pH of 6. Centrifugation for 10 minutes is effected at 39,000 g and 
the solution is stripped of the polymeric precipitate by decantation. 
In order further to purify the polymer, the latter is dissolved in 4 mls of 
a 2 M solution in NaCl and 0.05 M in acetate buffer at a pH of 5.5 and the 
solution thus obtained is supplemented by 16 mls of water and precipitated 
again with 20 mls of 1 M phosphate buffer at pH 6 and centrifuged during 
10 minutes at 39,000 g by collecting the polymer by decantation. 
Such a purification procedure is repeated for at least four times. 
The product redissolved in a 2 M solution in NaCl and 0.5 M in acetate 
buffer at pH 5.5, is dialized against portions of 11 of 1.10.sup.-4 M HCl 
during four days, changing the solution every day. 
By freeze drying the residue of the dialysis, there are obtained 223 
milligrams of the polymeric derivative of NAD, .lambda..sub.max 276.5 nm. 
The determination of the total NAD bound to the polymer is carried out by 
the measurement of the absorbance at 276.5 nm by assuming an extinction 
coefficient equal to that of the NAD derivative II (.epsilon.18800 
M.sup.-1 cm.sup.-1). 
The determination of the coenzymically active NAD bound to the polymer is 
effected by quantitative enzymic reduction with alcohol dehydrogenase from 
yeast in a 0.1 M tris buffer at pH 9 in the presence of 0.2 M ethanol and 
0.5 M semicarbazide hydrochloride. 
From the spectrophotometric measure at 340 nm of NADH derivative which has 
been formed, the result is that 115 micromols of enzymically reducible NAD 
are bound to each gram of the polymer, and correspond to 90% of the total 
NAD bound to the polymer. 
The thus obtained macromolecularized NAD is coenzymically active with 
several dehydrogenase. For example, with alcohol dehydrogenase from yeast, 
the speed of enzymic reduction of the macromolecularized NAD is 50% 
relative to that of the natural coenzyme. The determination is carried out 
in the following incubation mixture (1.0 ml): tris. HCl, 83 micromols, 
ethanol 166 micromols, semicarbazide. HCl, 42 micromols; coenzyme 0.1 
micromol (expressed as bound and enzymically reducible NAD); enzyme 0.2 
micrograms; pH 9.0; incubation temperature 25.degree. C. The reduction 
speed is determined by the increase of the absorbance at 340 nm. 
EXAMPLE 5 
Preparation of the 
nicotinoylamide-8-(polylysine-2-carbonylethylthio)adenine dinucleotide 
##STR6## 
To 50 milligrams of polylysine hydrobromide having a mol.wt. of about 
50,000 and dissolved in 0.5 mls of water, there are added 40 milligrams of 
nicotinoylamide-8-(carboxyethylthio)adenine dinucleotide. 
##STR7## 
dissolved in 0.5 ml of water, and 40 milligrams of 
N-ethyl-N'-(3-dimethylaminopropyl)carbo diimide hydrochloride dissolved in 
0.5 ml of Water. 
The reaction mixture, adjusted to a pH of 4.7 with 0.1 M NaOH is stirred at 
room temperature during 24 hours while maintaining the pH at 4.7 during 
the first three hours by addition of 0.1 M HCl. 
Under the same conditions there have been added 40 additional milligrams of 
carbodiimide in 0.5 ml of water and stirring is continued during 24 
additional hours. 
The reaction mixture diluted with water to a volume of 15 mls is then 
transferred to a centrifugation tube and precipitated with 10 mls of 0.15 
M phosphate buffer at a pH of 6. 
Centrifugation is carried out during ten minutes at 19,000 g and the 
solution of the polymeric precipitate is decanted. 
To further purify the polymer, the latter is dissolved in 1 ml of a 2 M 
solution in NaCl and 0.05 M in acetate buffer at pH 5.5 and the thus 
obtained solution is supplemented with 15 mls of water and precipitated 
again with 10 mls of 0.15 M, pH 6, pyrophosphate buffer and centrifuged 
during 10 minutes at 39,000 g. collecting the polymer by decantation. 
Such a purification procedure is repeated for at least four times. The 
product, redissolved in a 2 M solution in NaCl and 0.05 M in pH 5.5 
acetate buffer, is dialized during 48 hours against 500 mls of a 3 M 
solution in NaCl. 
Dialysis is then carried out against 2-mol portions of 1.10.sup.-4 M HCl 
during four days, the solution being daily renewed. 
By freeze-drying the residue of the dialysis, there are obtained 34 
milligrams of the polymeric derivative of NAD, .lambda..sub.max 276.5. 
The determination of the total NAD bound to the polymer is carried out by 
the measure of the absorbance at 276.5 nm, assuming an extinction 
coefficient equal to that of the derivative II of NAD (.epsilon.18,800 
M.sup.-1 cm.sup.-1). The determination of the coenzymically active NAD 
bound to the polymer is carried out by quantitative enzymic reduction with 
alcohol dehydrogenase from yeast in a 0.1 M tris buffer at pH 9 in the 
presence of 0.2 M ethanol and 0.005 M semicarbazide hydrochloride. 
From the spectrophotometric measure at 340 nm of the as-formed NADH 
derivative, it appears that 157 micromols of enzymically reducible NAD are 
bound to each gram of polymer, corresponding to 90% of the total NAD bound 
to the polymer. 
The thus obtained macromolecularized NAD is coenzymically active with 
several dehydrogenases. 
EXAMPLE 6 
Preparation of the 
nicotinoylamide-8-(aminohexyl-sepharose-2-carbonylethylthio)adenine 
dinucleotide. 
##STR8## 
500 milligrams of aminohexyl sepharose 4B are allowed to swell with a 0.5 M 
NaCl solution, then washed with 200 mls of 0.5 M NaCl and then with water. 
To the as-obtained gel, slurried in 2 mls of water, there are added 40 
milligrams of nicotinoylamide-8-(carboxyethylthio)adenine dinucleotide. 
##STR9## 
dissolved in 0.5 ml of water and the pH is adjusted to 4.7 with 1 M NaOH. 
To the slurry stirred at room temperature with a mechanical stirrer, there 
are added 40 milligrams of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide 
hydrochloride dissolved in 0.2 ml water and stirring is continued during 
24 hours while maintaining the pH at 4.7 during the first three hours by 
adding 0.1 M HCl. 
Under the same conditions there are added at 24-hour intervals, three 
additional increments of the carbodiimide solution (40 milligrams in 0.2 
ml of water) for a total reaction time of 96 hours. The gel is then 
filtered and washed with a 1 M solution in NaCl and 1.10.sup.-4 M in HCl 
until the disappearance of the UV absorption (not bound NAD) is 
experienced. There are thus obtained 1.8 ml of the polymeric derivative of 
NAD in the form of a moist gel, .lambda..sub.max 276.5 nm. 
The UV spectrum has been obtained by suspending the gel in an aqueous 50% 
solution of sucrose (weight/weight) which delays the settling of the gel. 
The determination of the total NAD bound to the polymer is carried out by 
the measurement of the absorbance at 276.5 nm, assuming an extinction 
coefficient equal to that of the derivative II of NAD (.epsilon.18,800 
M.sup.-1 cm.sup.-1). The determination of the coenzymically active NAD 
bound to the polymer is carried out by quantitative enzymic reduction with 
alcohol dehydrogenase from yeast by suspending the gel in a 50% aqueous 
solution of sucrose (weight/weight) containing 0.1 M tris.HCl buffer, 0.2 
M ethanol and 0.5 M semicarbazide hydrochloride, adjusted to a pH of 9. 
From the photometric readings at 340 nm of the NADH derivative, it appears 
that 21 micromols of enzymically reducible NAD are bound to each gram of 
the dry product, that which corresponds to 80% of the total NAD bound to 
the polymer. 
EXAMPLE 7 
Preparation of the 
nicotinoylamide-8-(polyethyleneimine-2-carbonylethylthio) adenine 
dinucleotide phosphate. 
##STR10## 
To 0.38 ml of a 33% aqueous solution of polyethyleneimine (weight/volume) 
adjusted to a pH of 5 with conc. HCl, there are added 20 milligrams of 
nicotinoylamide-8-(carboxyethylthio)adenine dinucleotide phosphate 
##STR11## 
dissolved in 0.5 ml of water and 20 milligrams of 
N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride dissolved in 
0.2 ml of water. The reaction mixture, adjusted to a pH of 4.8 by addition 
of 2 M HCl, is stirred at room temperature during 24 hours by maintaining 
the pH at 4.8 during the first 3 hours by addition of 0.1 M HCl. Under the 
same conditions there are added 20 additional milligrams of cabodiimide in 
0.2 ml of water and stirring is continued during 24 additional hours. The 
reaction mixture, dilute with water to a volume of 10 mls, is then 
transferred to a centrifugation tube and precipitated with 10 mls of 1 M 
phosphate buffer at a pH of 6. Centrifugation is carried out during 10 
minutes at 39,000 g and the solution is stripped of the polymeric 
precipitate by decantation. To further purify the polymer, the latter is 
dissolved in 2 mls of 2 M solution in NaCl and 0.05 M in pH 5.5 acetate 
buffer and the solution thus obtained is supplemented with 8 mls of water 
and precipitated again with 10 mls of 1 M, pH 6 phosphate buffer and 
centrifuged during 10 minutes at 39,000 g, the polymer being collected by 
decantation. Such a purification procedure is repeated for at least 4 
times. The product, redissolved in a 2 M solution in NaCl and 0.05 M in pH 
5.5 acetate buffer, is dialized against 1-liter portions of 1.10.sup.-4 M 
HCl during four days, the solution being renewed daily. By freeze-drying 
the residue of the dialysis there are obtained 83 milligrams of the 
polymeric derivative of the NADP, .lambda..sub.max 276.5 nm. The 
determination of the total NADP bound to the polymer is carried out by 
measuring the absorbance at 276.5 nm, assuming an extinction coefficient 
equal to that of the derivative II of NAD (.epsilon.18,000 M.sup.-1 
cm.sup.-1). 
The determination of the coenzymically active NADP bound to the polymer is 
carried out by quantitative enzymic reduction with glucose-6-phosphate 
dehydrogenase from yeast in 86.3 nM triethanolamine buffer, pH 7.6, 
containing 6.7 mM MgCl.sub.2 and 1.2 mM glucose-6-phosphate. From the 
spectrophotometric measure at 340 nm of NADP derivative as formed, it 
appears that 23 micromols of enzymically reducible NADP are bound to each 
gram of polymer, that which corresponds to 20% of the total NADP bound to 
the polymer. 
The thus obtained macromolecularized NADP is coenzymically active with 
several dehydrogenase such as for example 6-phosphogluconate dehydrogenase 
and L-glutamate dehydrogenase.