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Description  |
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SUMMARY OF THE INVENTION
The present invention relates to novel derivatives of polyamides and
especially of nylon-type polyamides. It relates more particularly to
polyamides to which there have been bonded certain side-chains through
which it is possible to attach to the backbone of the polymer desired
functional groups, molecules imparting certain desired properties or
biologically active materials, so as to result in biologically active,
carrier-bound molecules. The invention further relates to a novel method
for attaching to polyamides, especially to nylon-type polymers, desired
side-chains, without substantially changing the average molecular weight
distribution of the starting polymer. The invention also relates to a
four-component reaction whereby desired side-chains are introduced into a
polyamide polymer, said reaction involving the amine and carboxyl groups
of the partially hydrolyzed polymer, an aldehyde and an isocyanide. Other
and further aspects of the invention will become apparent hereinafter.
BACKGROUND OF THE INVENTION
In recent years biologically active materials, and especially enzymes, have
been chemically and physically bonded to certain carriers, retaining the
greater part of the biological activity. Such carrier bound enzymes are
used repeatedly, can be filtered off easily from the substrate and are
finding a wide-scale use in various applications in industry and research.
Supports used hitherto are certain synthetic polyamides, certain natural
materials such as cellulose or derivatives thereof, other polysaccharides,
polyacrylamide and the like.
Polyamides would of course have many advantageous properties as compared
with the materials used hitherto, but due to the rather inert character of
same as regards chemical reactivity, these have not been used for this
purpose. Readily available polyamides have only terminal carboxy and amino
groups available for such bonding and these are not adequate. Recently
procedures have been described by which the binding capacity of nylons can
be increased by mild hydrolysis, see Sundaram et al., (1970) FEBS Letters
10, 325 and Inman et al., (1972) Biochem.J. 129, 255. Such hydrolysis
results in the partial breakup of the polymer into fragments of lower
molecular weight, and this is a serious drawback.
THE PRESENT INVENTION
According to the present invention, a polyamide polymer such as any of the
commercially available nylons, is subjected to controlled hydrolysis, so
as to increase the number of free carboxyl and amino groups, especially on
the surface of the polymer, by a factor of from about 2 to 5, as
determined titrimetrically. The polymer can be used in any convenient
form, such as powder, fiber, film, membrane, sheet, web of fibers, etc.
Most experiments were carried out with polymer powder, but experiments
have shown that the process is applicable in a similar manner to all the
other forms of the polymer. After this step of controlled hydrolysis,
there is effected a four-component reaction involving the carboxyl and
amino groups of the polymer, which can be schematically designated as
--CONH--A--COOH H.sub.2 N--A--CONH--B--CONH--A--
with an aldehyde R.sub.2 CHO and with an isonitrile R.sub.3 -N.tbd.C
resulting in the formation of a side chain-substituted polymer of the
general formula
##EQU2##
The reaction can be represented by the following formulas:
##EQU3##
wherein the different moieties can be represented as follows:
##EQU4##
The derivatized polyamide is obtained by effecting first a controlled
hydrolysis, followed by a reaction with an aldehyde and an isonitrile:
##EQU5##
The preparation of MDA-nylon is according to the following reaction:
##SPC1##
wherein R.sub.2 and R.sub.3 are the residues of the aldehyde and of the
isonitrile, respectively. A and B are hydrocarbon-backbone residues of
--(CH.sub.2).sub.n -- where n is an integer. When R.sub.3 is a reactive
functional group, this can be further reacted so as to chemically bind to
the polymer backbone any desired substituent, this term including
biologically active molecules, or chromophores. R.sub.2 and R.sub.3 are
any residues of aldehydes and isocyanides.
According to a preferred embodiment of the present invention, there is used
a diisocyanoalkane, such as for example 1,6-diisocyanohexane, resulting in
the formation of a side chain having a terminal isocyano group, which in a
further reaction is again reacted with a suitable aldehyde, carboxyl-group
and amino group to chemically bind a suitable group to the end of this
side-chain. By using nylon-6, acetaldehyde and 1,6-diisocyanohexane, a
derivative of nylon designated as PIN-nylon is obtained, as set out in
detail hereinafter, which is advantageously used for the chemical binding
of various substituents, molecules, etc. as will be described in detail
hereinafter.
As is well known, the chemistry of the various polyamides is very similar,
especially as regards the reactivity of partially hydrolyzed polymers
having reactive carboxyl and amino groups. In view of this the invention
is illustrated mainly with reference to nylon 6, it being clearly
understood that this is by way of example only and that the present
invention is applicable to other polyamides with no, or only minor changes
of procedure, as will be self-evident to any person versed in the art.
MATERIALS AND METHODS
Preparation 1: Isocyanides
The general synthetic procedure adopted was based on the conversion of a
primary amine into the appropriate N. Alkyl formamide by treatment with
ethyl formate, followed by dehydration to the isocyanide (Ugi I, Fetzer U,
E Holzer U, Kupfer H. and Offerman K - Angew Chem. Int. Ed. 4 472 (1965)).
a. N-formyl cyclohexylamine was prepared as described by Ugi (Ugi I (1961)
Organic Synthesis 41 13-14).
Ir spectrum in chloroform .nu..sub.max : 1460 (alicyclic-CH.sub.2 -) 1680
(amide CO) 2860, 2940 (aliphatic-CH.sub.2 -) 3420 (-CH-), 3700 (amide NH).
b. Cyclohexyl isocyanide was prepared from the formamide by dehydration
with cyanuric chloride according to Wittman (R. Wittman, (1961) Angew,
Chem. 73 219).
Ir spectrum in chloroform .nu..sub.max : 1460 (alicyclic CH.sub.2) 2170
(N.tbd.C), 2860, 2950 (aliphatic CH.sub.2) 3420 (CH)cm.sup..sup.-1.
c. N,N' diformyl -- (1,6-diaminohexane) was prepared essentially as
described by Moffat et al (J. Moffat, M. V. Newton and G. J. Papenmeier
(1962) J.Org.Chem. 27, 4058). Mt.Pt. 105.degree.C.
Ir spectrum in chloroform, .nu..sub.max : 1680 (amide CO), 2840,2920
(aliphatic CH)cm.sup..sup.-1.
d. 1,6-diisocyanohexane was prepared by dehydration of
N,N'-diformyl-(1,6-diaminohexane) with p-toluene sulfonyl chloride,
essentially as described by Heztler and Covey (N. R. Heztler and E. J.
Corey (1918), J. Org. Chem. 23 1222).
Ir spectrum in chloroform: 1440 (aliphatic CH.sub.2), 2130 (N.tbd.C), 2840,
2920 (aliphatic CH.sub.2). NMR in CDCl.sub.3 : Hydrogen ratio 2:1
(corresponding to 8:4); .alpha.-CH.sub.2 :.tau. = 6.70 (tt); J.sub.NH =
2cps; .beta.-CH.sub.2 :.tau. = 8.36 (multiple splitting); .gamma.-CH.sub.2
:.tau. = 8.52 (multiple splitting) (Kurtz et al, 1961).
e. p-Nitrophenyl isocyanide
p-nitroaniline (5 gms; 0.036 mole) and potassium-t-butoxide (20 gms; 0.18
mole) were dissolved with stirring in t-butanol (1 liter). Chloroform (8
ml: 0.07 mole) was then added dropwise and the reaction allowed to proceed
with stirring for 1 hr. Benzene (200 ml) and water were added until phase
separation occured. The benzene phase was separated. The aqueous phase was
extracted with two 100 ml portions of benzene. The combined benzene
extract was washed with water and concentrated to a final volume of about
100 ml. in a rotatory evaporator. The concentrated benzene solution was
passed through an activated-alumina column (2.5 .times. 25 cm), the
yellow-orange fractions collected and evaporated to dryness. The yellowish
brown solid (3.5 gms; 60% yield) was stored in a closed vessel, at
-5.degree..
IR in chloroform:.nu. max: 2130, 1680, 1610, 1580, 1370, 1330
cm.sup..sup.-1.
f. Preparation of succinyltrypsin
Trypsin (250 mg) was dissolved in cold half-saturated sodium acetate (6
ml), and the solution adjusted to pH 8. Succinic anydride (600 mg) was in
the course of 1 hr. to the ice-cooled enzyme solution the pH being
maintained at 8 with an automatic titrator using 2N NaOH as titrant. The
reaction mixture was exhaustively dialyzed against distilled water at
4.degree. and lyophilized (net weight of lyophilized powder 235 mg).
Determination of the free amine nitrogen by the Van Slyke method indicated
that 13 out of the 14 lysyl residues of trypsin had been succinylated. The
specific activity of the succinyl trypsin sample was 20 esterase units/mg
as compared with 35 esterase units/mg for native trypsin.
ASSAY METHODS
Preparation 2: Nylon-6 powder
Stage A: Preparation
Commercial Nylon-6 pellets (30 gms) were suspended in a 20% solution of
anhydrous CaCl.sub.2 in methanol (1 liter) and stirred at room temperature
until a homogeneous, extremely viscous solution was obtained. (K. Fabel,
Kunststoffe 37, 197 (1947)). The nylon solution was added dropwise with
strong stirring into large excess of water (at least 8 liters water per
liter nylon solution); the powder was separated on a suction filter,
washed with water (1 liter) resuspended in water (5 liters) and
homogenized with an Ultraturax Homogenizer (janke & Kunkel KG, Staufen
i.Br.) The fine powder was separated, washed again with water, ethanol and
ether and air dried. Traces of solvent and moisture were removed in a
vacuum dessicator over phosphorous pentoxide. The mean diameter of the
nylon powder particles as determined by examination under the microscope
was 0.5-1.mu. . The mean carboxyl-content of the Nylon powders,
determined titrimetrically, was about 25.mu. moles per gm. dry nylon
powder.
Stage B: Determination of the Carboxyl Content of Nylon Powders
The carboxyl content of the nylon powder samples (50 mg) was determined by
anhydrous titration with sodium methoxide (Patchornik A. and
Ehrlich-Rogozinski S. (1959) Anal.Chem.31 985).
ASSAY METHODS
The enzymic activities of trypsin, succinyl trypsin and papain and of their
water insoluble derivatives were determined at 25.degree. by the pH-stat
method (K. A. Walsh and P. E. Wilcox (1970) Methods Enzymol.19 31). The
substrate solutions (5 ml) were 1.5 .times. 10.sup..sup.-2 M
benzoyl-L-arginine ethylester 0.05M KCl for trypsin and succinyl trypsin
(Laskowski M. (1955) Methods Enzymol.2 26) and 0.05M benzoyl-L-arginine
ethylester 0.005 crystein, 0.002 EDTA for papain (Smith E. L. and Parker
M. J. (1958) J. Biol. Chem. 233 1387). The titrant was 0.1N NaOH. The
assays were carried out at pH 8 for trypsin, at pH 9 for succinyl trypsin,
et pH 9.5 for immobilized trypsin and succinyl trypsin at pH 6.5 for
papain and at pH 7 for immobilized papain. One unit of esterase activity
was defined as that amount of enzyme which catalyzed the hydrolysis of
1.mu. mole of substrate per min under the specified assay conditions. The
specific activities of the native enzyme samples used were as follows:
trypsin -32.5 esterase units/mg; succinyltrypsin - 20 esterase units/mg;
papain -16 esterase units/mg.
The enzyme activity of crystalline pepsin and of the immobilized pepsin
derivatives were determined at 37.degree. by the hemoglobin digestion
method (Anson M. L. 1938; J.Gen.Physiol. 22,79). The reaction mixtures
containing immobilized enzyme were stirred magnetically to ensure
effective mixing of the reagents.
EXAMPLE 1
Controlled Hydrolysis of Nylon Powders
Nylon-6 powder (10 gms) was suspended in 3N HCl (300 ml) and stirred at
room temperature (20.degree.) for the desired amount of time. The powder
was separated on a suction filter, washed exhaustively with water,
ethanol, ether and air dried. Traces of solvent and moisture were removed
in a vacuum dessicator over phosphorous pentoxide and the powders stored
in closed vessels.
The dependence of the carboxy content of nylon-6 powder on the time of
hydrolysis is summarized in the attached Table, for a typical set of
experiments. The Nylon-6 samples routinely used were hydrolyzed for 4
hours.
______________________________________
Controlled Hydrolysis of Nylon-6 Powders
______________________________________
Time of hydrolysis (hours)
0 2 4 6 17 21 24
Carboxyl content (.mu.moles/gm)
26 55 65 70 91 96 104
______________________________________
In a similar manner a partial hydrolysis was effected with nylon-6,6 in
powder form and with nylon-11 (Rilsan;TM), the results being as follows:
Nylon 6,6; unhydrolysed -- 30.mu. moles/gm; 4 hrs. hydrolysis -- 70/80.mu.
moles/gm. Nylon 11: unhydrolyzed -- 10.mu. moles/gm; 4 hrs. hydrolysis --
35-40.mu. moles/gm; 7 hrs. hydrolysis -- 60-70.mu. moles/gm.
Similar experiments were carried out with nylon fibers, with thin sheets of
nylon-6, nylon netting, etc. The reaction was carried out under similar
conditions and also in these cases an increase of titrimetrically
determinable carboxyl groups by a factor of from about 2 to 5 as compared
with untreated material, was attained.
EXAMPLE 2
Attachment of side-chains to partially hydrolyzed nylon
Partially hydrolyzed nylon powder, 50 mg (sample hydrolyzed for 4 hrs; mean
carboxyl content 62.5.mu. moles/gm) was suspended in 2 ml isopropanol. The
suspension was stirred and 0.5 ml of an aldehyde and 0.2 ml of an
isocyanide were added and the reaction allowed to proceed at room
temperature with stirring for 24 hours. The nylon powder was separated on
a suction filter, washed with ether (50 ml) and air-dried. The dry nylon
powder was transferred quantitatively to a stoppered vial, and the
carboxyl content determined as described in the experimental section. The
results are summarized in the following Table.
Similar results were obtained with Nylon-11 powder (Rilson Powder) and with
Nylon 6,6.
FOUR COMPONENT CONDENSATION REACTIONS WITH PARTIALLY HYDROLYZED NYLON
POWDERS
Reaction Mixture Carboxyl Content
Exp.
No.
Nylon Powder
Aldehyde
Isocyanide .mu.moles/gm.
% of Reference
__________________________________________________________________________
1. Non-hydrolyzed
-- -- 24.2 100
2. Partially hydrolyzed
-- -- 62.5
3. Partially hydrolyzed
Acetaldehyde
Cyclohexyl isocyanide
21.4 134.3
4. Acetaldehyde
1,6-diisocyanohexane
16.9 27.1
5. Benzaldehyde
Cyclohexyl isocyanide
54.0 86.5
6. Glutaraldehyde
Cyclohexyl isocyanide
60.0 96.0
7. Acetaldehyde
-- 56.6 90.5
8. Benzaldehyde
-- 55.0 88
9. Glutaraldehyde
-- 59.2 94.2
10. -- Cyclohexyl isocyanide
52.3 83
11. -- 1,6-diisocyanohexane
56.5 90.5
__________________________________________________________________________
EXAMPLE 3
Preparation of Polyisonitrile Nylon (PIN-nylon)
Partially hydrolyzed nylon-6 powder, 2 gms (sample hydrolyzed for 4 hours;
mean carboxyl content 60-65.mu. moles/gm.) was suspended in isopropanol
(80 ml); acetaldehyde (20 ml) was then added, followed by
1,6-diisocyanohexane (8 ml) and the reaction allowed to proceed in a
closed vessel for 24 hours with stirring at room temperature. The
acetaldehyde was pipetted with a precooled pipette, to prevent formation
of bubbles. The PIN-nylon powder was separated on a suction filter, washed
with isopropanol (50 ml) and then with ether (200 ml) and air dried.
Traces of solvent were removed in a vacuum dessicator over phosphorous
pentoxide. The PIN-nylon powder was stored at -5.degree. in a dark
stoppered vial over silica gel. The coupling capacity of PIN-nylons were
25-50.mu. moles/gm. (see Example 6).
EXAMPLE 4
Preparation of MDA-nylon
p,p'-diaminodiphenylmethane, MDA (2 gms, 0.01 mole) was dissolved in 160
ml. 100 propanol cooled over ice and 4 ml acetaldehyde were then added.
PIN-nylon powder (2 gms) was suspended in 160 ml isopropanol, 2 ml glacial
acid (0.033 moles) were added, followed by the MDA-acetaldehyde solution.
The reaction was allowed to proceed in a closed vessel for 24 hours. with
stirring at room temperature. The MDA-nylon was separated on a suction
filter, washed with ethanol and then with ether and air dried. The
coupling capacity of the MDA-nylon was 20.mu. moles/gm. (see Example 12).
EXAMPLE 5
p-Nitrophenyl nylon
p-nitrophenyl isocyanide (200 mg; 0.00135 mole) was dissolved in 2 ml
acetone, cooled over ice and 2 ml. acetaldehyde were added. Partially
hydrolyzed nylon-6 powder (100 mg) was then added and the reaction mixture
stirred overnight at room temperature in a tightly closed vessel. The
yellow powder was washed with acetone, ether and air-dried. The
p-nitrophenyl content estimated from the amount of p-nitroaniline,
obtained following total acid hydrolysis of p-nitrophenyl-nylon was 30.mu.
mole/gm.
EXAMPLE 6
Coupling of Trypsin to PIN-nylon
PIN-nylon (50 mg) was suspended in 2 ml cold 0.1M phosphate 0.5M Sod.
Acetate pH 8. A cold aqueous solution of trypsin (6 mg in 1 ml) was then
added, followed by 0.1 ml acetaldehyde. The reaction mixture was left
stirring overnight at 4.degree.. The insoluble enzyme derivative was
separated on a filter, washed with water, IM KCl 0.1M in NaHCO.sub.3 and
again with water, resuspended in water (4 ml) and stored at 4.degree.. The
recovery of immobilized enzymic activity was 35%.
EXAMPLE 7
Succinyltrypsin-nylon
PIN-nylon (50 mg) was suspended in 1.0 ml cold 0.1 M Tris buffer pH 7.0. A
cold solution of succinyltrypsin in the same buffer (2-10 mg in 1 ml) was
added, followed by 0.1 ml acetaldehyde. The reaction mixture was left
stirring overnight at 4.degree., washed and resuspended in water as
described for trypsin-nylon.
Maximal recoveries of immobilized enzymic activity (30-40% of the amount
added to the reaction mixture) were obtained for succinyltrypsin with 4-5
mg enzyme per 100 mg support.
EXAMPLE 8
Pepsin-nylon
PIN-nylon (50 mg) was suspended in 2 ml cold 0.1M Tris adjusted to pH 5 and
2-10 mg pepsin added. After the enzyme had dissolved 0.1 ml
acetaldehyde.sup.2 was added. The reaction mixture was left stirring
overnight at 4.degree.. The insoluble pepsin derivative was washed with
0.01 N HCl, resuspended in 0.001N HCl (3-4 ml) and stored at 4.degree..
EXAMPLE 9
Coupling of low molecular weight amines to PIN-nylon
PIN-nylon (50 mg) was suspended in 2 ml 0.1M phosphate 1M Sodium Acetate pH
8. An aqueous solution of benzoylglycyl-lysine (hippuryllysine) (5 mg in 1
ml) was then added followed by 0.1 ml acetaldehyde. The reaction mixure
was left stirring overnight at room temperature. The insoluble
benz-gly-lys-nylon conjugate was exhaustively washed with water, ethanol,
ether and air-dried. The glycine content of benz-gly-Lys-nylon as
determined by amino acid analysis of an acid hydrolyzate, was about 25.mu.
mole/gm.
EXAMPLE 10
Diazotization of MDA-nylon
MDA-nylon (100 mg) was suspended in cold 0.2N HCl (7 ml) and aqueous sodium
nitrite (25 mg in 1 ml) added dropwise. The reaction mixture was stirred
for 30 min. over ice; the red brown diazotized MDA-nylon was separated on
a suction filter, washed with cold water and finally with cold 0.1M
phosphate, pH 8, resuspended in the same buffer and used directly in the
coupling experiments.
EXAMPLE 11
Coupling of papain to MDA-Nylon
An aqueous solution of papain (10 ml 1-1.3 mg protein per ml) was added to
a magnetically stirred suspension of diazotized MDA-nylon (Example 10) in
0.1M phosphate buffer pH 8 (100 mg in 6 ml). The reaction mixture was left
stirring overnight at 4.degree.. The insoluble MDA-nylon papain conjugate
was separated by filtration, washed with water, 1M KCl and again with
water, resuspended in water (5 ml) and stored at 4.degree.. The recovery
of immobilized enzymic activity was about 40%.
EXAMPLE 12
Coupling of p-bromophenol to MDA-nylon
An aqueous solution of p-bromophenol (50 mg dissolved in 3 ml water by the
dropwise addition of 2N NaOH) was added to a diazotized MDA-nylon sample
(50 mg) suspended in cold 0.1M phosphate buffer pH 8 (6 ml). The reaction
mixture was left stirring overnight at 4.degree.. The red-brown
p-bromophenol-MDA-nylon conjugate was separated on a funnel, washed with
0.05 M carbonate buffer pH 10.5 or water brought to the same pH, then with
deionized water and finally with methanol and ether and dried in vacuum
over phosphorous pentoxide. The diazotization capacity of MDA-nylon could
be estimated from the bromine content of the p-bromophenol conjugate.
EXAMPLE 13
Dyeing of MDA-nylon
Diazotized MDA-nylon (nylon 6) (50 mg) was suspended in cold 0.1M phosphate
buffer pH 8(6 ml). A saturated aqueous solution of chromotropic acid
sodium salt (4,5-dihydroxy-2,7-naphthalene disulfonic acid; 1 ml) was
added and the reaction mixture left stirring overnight at 4.degree.. The
chromotripic acid/MDA-nylon conjugate was washed with water, methanol,
ether and air-dried.
Analogous procedures were employed to couple diazotized MDA-nylon with
2-naphthol-3,6-disulfonic acid and with pyrogallol
(1,2,3-trihydroxybenzene). The colours of the azo derivatives thus
obtained were as follows: Chromotropic acid-MDA-nylon . . . violet
2-naphthol-3,6-disulfonic acid/MDA-nylon . . . pink pyrogallol/MDA-nylon .
. . light yellow.
Examples 3 to 13 were repeated with other types of nylon, namely with nylon
11 and with nylon 6--6, and similar results were obtained.
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