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Preparation of polymers with luminescent markers
PREPARATION OF POLYMERS WITH LUMINESCENT MARKERS* M. G. KRAKOVYAK,YE. V. A~UFRIEVA and S. S. SKOttOKI-IODOV High Molecular Weight Compounds Institute, U.S.S.R. Academy of Sciences
(Received 4 November 1968)
POLYMERS tO which chromophore groups have been covalently added are most frequently used for studying certain properties of natural and synthetic macromolecules and the mechanism of their formation [1-4]. The relaxation behaviour of polymers with luminescent groups may be studied by the polarized luminescence method. The latter is repeatedly used with success in studying rapid relaxation processes w h e n the periods of relaxation and luminescence are comparable; moreover it enables us to study the intramolecular motion of macromolecules in solution and in the mass. The advantages of the method include the possibility of experiments in water and other polar solvents; this along with the high sensitivity of the method facilitates work with low concentrations of the polymer in solution. The polarized luminescence method [1, 4] involves the use of luminescent markers covalently added to the macromolecules and possessing certain optical properties. The number of these specially added groups must be sufficient for purposes of spectral analysis and small enough to prevent distortion of the properties of the macromolecules. Several methods of preparing macromolecules with chemically added luminescent groups have been described [2, 5], but these are mainly applicable to polymers that have NH bonds. Our aim was to develop a convenient and general method of adding luminescent groups to polymers containing carboxyl groups. In a previous study [4] we prepared polyacrylic and polymethacrylic acids with covalently added luminescent groups. However the method used for their preparation involves polymer analogue conversions of the corresponding polyacryloyl chlorides, and this limits its usefulness. The chemical addition of luminescent groups to polymers containing carboxyl groups may be realized through specially selected aliphatic diazo compounds (diazoalkanes) interacting with COOH groups of the polymer. The reaction of diazoalkanes with earboxylic acids proceeds by the scheme [6] ® CI-I--N-- N R
@ H
)
- ~ ® R'cooeJ--~RCH,OCOR ' ~CI-I2--N ~ N ~C H ~ ® [ ] R COOH~_-~RCH2OCOR, + i_I R R e
* Vysokomol. soyed. A l l : No. 11, 2499-2504, 1969. 2842
Preparation of polymers with luminescent markers D 07
2843
./~
liI i,"i /:i /\I? i [lis
\. I. •_ - .
-,\
280
/t
i
\
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~ / ~
~ ~. ~.~..
320
360
400
440
,t, m/z
Fro. 1. Absorption spectra of solutions in methanol: 1--9-diazomethylanthracene (II); 2--9-anthrylaeetoxymethane model compound (c =7.5 × l0 -5 mole/l.) and 3--PMAA containing one 9-anthrylaeetylhydroxymethane group per 1400 polymer units (%olymer=8"5 mg/ml). and under mild conditions in an inert medium the above reaction yields esters in practically quantitative yield. The only by-product is nitrogen. Only a small number o£ papers has been published by authors studying the reactions of aliphatic diazo compounds with polymers possessing functional groups (generally - - O H or COOH). These papers m a y be divided into three groups according to the manner in which the reactions were carried out: 1) reactions under homogeneous conditions (e.g. the methylation o£ carboxyl groups in methyl methacrylate-methacrylic acid copolymers soluble in aromatic hydrocarbons, conducted in the presence of diazomethane [7]); 2) reactions at the interface o£ the solid and liquid phases (mcthylation of polymethacrylic acid caused by diazomethane in a hydrocarbon solvent [8], preparation of cellulose ethers by means of diazoalkanes [9]); 3) reactions at the interface of two liquid phases (methylation o£ ribonucleic and desoxyribonucleic acids in aqueous solution using and ether solution o£ diazomethane [10]). The reagents used for the addition of luminescent groups to carboxylated macromolecules were diazomethyl-a-naphthylketone (I) and 9-diazometh~lanthracene (II). CHN2 CHN,, c=o
I
g\/~/%
II
2844
M. G. K~AKO'VY.CK et al.
The choice of reagents depended on the optical properties of the luminescent marker and on the relative stability of the diazoalkanes. The experiments showed that at room temperature the hydrocarbon solution I reacts very slowly with an aqueous solution of polymethacrylic acid (PM_&A); it was only at 50-55 ° that it was found possible b y increasing the content of I (1 mole to 8 basic moles PMAA) and the reaction time to obtain PMAA containing one ~-ketonaphthyl group per 300-400 elementary units. D 0"8:
! II
0.6
O.Zl
0.2
350
z~50
550 ;~ , m #
Fro. 2
I
I
i
i
260
300
3~0
380
FIG. 3
FIG. 2. Luminescence spectra: /--solution of 9-diazomethylanthracene (II) and 2 - 9 . anthrylacetoxymethane in benzene (c 27.5 × 10-6 molefl.); 3--aqueous solution of PMAA containing one 9-anthrylacylhydroxymethane group per 1400 polymer units (%olymer=0"5 mg/ml). Fro. 3. Absorption spectra of solutions in methanol: 1--methyl-~-naphthylketone model compound, (o=l×10 -~ gfl.); 2--PMAA prepared in blank experiment (Cvolymer=3g/1.); 3--PMAA sample from main experiment (£:polymer=3 g/1.). This relatively low reactivity of I is due to the effect of the carbonyl group which reduces the electron density at the diazo carbon atom and lessens the nueleophilic nature of the diazoalkane. It was found that unlike I solution II reacts readily with different carboxylated compounds under mild conditions. The reaction of II with carboxylated polymers leading to the formation of maeromolecules having luminescent 9-anthrylacylhydroxymethane groups may be brought about b y different methods, the choice depending on the properties of the polymer. Where the polymer and solution of I I dissolve in a common
Preparation of polymers with luminescent markers
2845
s o l v e n t t h e h o m o g e n e o u s m e t h o d s is t h e m o s t convenient. I n this case w i t h t h e P M A A / I I ratios i n v e s t i g a t e d 30-50~o of I I r e a c t s with t h e c a r b o x y l g r o u p s o f the polymer. I n t h e case o f w a t e r - s o l u b l e p o l y m e r s we m a y use one of t h e h e t e r o p h a s e m e t h o d s . B y a d d i n g solution of I I to a n a q u e o u s solution of t h e p o l y m e r , while stirring, in a s o l v e n t (dioxane, alcohols, etc.) miscible w i t h w a t e r we o b t a i n a t h i n suspension of I I a n d t h e r e a c t i o n proceeds a t t h e solid-liquid interface. T h e r e a c t i o n a t t h e i n t e r f a c e t a k e s place on m i x i n g a n a q u e o u s solution of t h e p o l y m e r a solution of I I in a w a t e r - i m m i s c i b l e solvent (ether, s a t u r a t e d a n d a r o m a t i c h y d r o c a r b o n s , etc.). I n t h e case of h e t e r o p h a s e m e t h o d s a considerable p o r t i o n of I I is used u p in side reactions, a n d so t h e q u a n t i t a t i v e ratio of the r e a g e n t s m u s t first be d e t e r m i n e d e x p e r i m e n t a l l y in order to o b t a i n p o l y m e r s w i t h a g i v e n n u m b e r of c h r o m o p h o r e groups. T h e r e m o v a l of t h e u n r e a c t e d I I a n d of low m o l e c u l a r weight p r o d u c t s of conversion of I I is effected b y r e p e a t e d r e p r e c i p i t a t i o n of t h e p o l y m e r b y m e a n s of dialysis or b y a d s o r p t i o n - c h r o m a t o g r a p h i c m e t h o d s . T h e a b s o r p t i o n a n d luminescence s p e c t r a of solutions of m a c r o m o l e c u l e s possessing 9 - a n t h r y l a c y l h y d r o x y m e t h a n e g r o u p s are in good a g r e e m e n t w i t h t h o s e o f t h e m o d e l c o m p o u n d ( 9 - a n t h r y l a c e t o x y m e t h a n e ) , a n d differ g r e a t l y f r o m t h e s p e c t r a of t h e initial diazo c o m p o u n d (II) (Figs. 1 a n d 2). A n o t h e r criterion for t h e a d d i t i o n o f l u m i n e s c e n t g r o u p s to t h e m a c r o m o l e c u l e s is a high degree of l u m i n e s c e n c e p o l a r i z a t i o n (10%) in a low-viscosity m e d i u m ~vhere t h e r e is o n l y slight p o l a r i z a t i o n of t h e luminescence of 9 - a n t h r y l a c e t o x y m e t h a n e
(~1%). EXPERIMENTAL *
Polyacrylic (PAA) and po]ymethacrylic acids obtained by radical polymerization of the corresponding monomers [11, 12] were purified by repeated reprecipitation from methanol by ether or ethyl acetate, hiazo compound I was synthesized by reacting diazomethane [13] and a-naphthoic acid chloride in ether solution by the method described in [14]. After double recrystallization from benzene compound I had m.p. 53.5-54.5 ° (m.p. 55 ° according to data in [4]). The position of the bands in the I R spectrum of compounds I prepared by us is in good agreement with data in [15]. Reaction of I with PI~IAA. Main experiment. A mixture of solutions of 0.1 g (0.0012 mole) PMAA in 10 ml H.~O and 0.03 g (0'00015 mole) of I in 8 ml of hexane was stirred at 50-55 ° for 14 hr. The aqueous layer was separated and after five-fold washing with ether the water was evaporated with a rotary evaporator. The resulting polymer was thrice reprecipitated from methanol with ethyl acetate. The UV spectrum of this PMAA sample (Fig. 3, curve 3) has a band with imax =304 m/2 characteristic for the a-ketonaphthyl group (Fig. 3, curve 1). Blank experiment. A reaction mixture of the same composition as in the main experiment was stirred rapidly for 1 rain, after which the PM.AA was separated and purified as in the main experiment. The UV spectrum of the PMA.A is shown in Fig. 3 (curve 2). Determination of number of a-ketonaphthyl chromophore group~ added to P M A A . The
number of g-ketonaphthyl groups covalently added to PMAA macromolecules was calculated * T. V. Shevelevaya, 1~. P. Novozhilova and S. G. Telichkina participated in this part, of the work.
2846
NI. G. Kmcxov-rax et al.
b y means of UV spectroscopy. Spectroscopic analysis of the P M A ~ sample obtained i n the blank experiment was carried out in order to determine the absorption due not to the added chromophore groups, b u t to the molecules of compounds containing a ketonaphthyl group (diazomethyl-a-naphthylketone or its low molecular weight conversion products) from which the polymer could not be liberated. The n u m b e r of a-ketonaphthyl groups eovalently added to PMAA in methanol solution was calculated from the equation e =JD/ed, where c is the concentration of a-ketonaphrhyl groups (mole/L); D is the optical density: ,~Ar~-~a0ar~bas.-__~a04r~b~nk., S is the molar absorption coefficient; d is the cell length, era. The value e~8,~26300 was determined from the UV spectrum of a methyl-g-naphthylketone solution in methanol [16] measured under identical conditions, the latter being used as model compound. The measurements carried out in this way showed that the PMAA samples obtained b y the method in question have a single chromophore group per 300-400 polymer units. 9-Diazomethylanthracene was obtained b y oxidation of 9-anthraldehyde hydrazone following the procedure in reference [17]. The ~ spectrum is shown in Fig. 1 (curve 1). The n u m b e r of 9-anthrylacylhydroxymethane groups in the polymers was determined b y U-V spectroscopy by the same method as that (see above) used for the polymers containing a-ketonaphthyl groups. As a model we used 9-anthrylaeetoxymethyl obtained by reacting I I with acetic acid [17]. For the given chromophore group in methanol e3,4 determined b y means of 9-anthrylacetoxymethane for A=384 m/l is equal to 7500. Preparation of polymers with luminescent groups eovalently added. The following are examples of experiments in the preparation of polymers possessing luminescent markers added b y different methods. Preparation of P A A with 9-anthrylacylhydroxymethane markers (PAA-A). To a solution of 1,5 g (0.021 base-mole) of PAA in 30 ml of methanol were added, while stirring (not with magnetic stirrers) at room temperature, 9 ml (4.13 × 10 -5 mole) of I I in 4-5 ml of purified dioxane (calculating for one molecule of I I per 500 PAA units). After 2 hr the polymer was precipitated with ether, separated, carefully washed with ether, again reprecipitated from methanol with ether, and then dried. The resulting PAA-A sample had one luminescent group per 1500 polymer units, i.e. N 3 0 ~ of the total amount of I I reacted with PAA. Preparation of P M A A with 9.anthrylacylhydroxymethane markers ( P M A A - A ). To a solution of 0.1 g PMA2k (1.16 × 10 -3 base-mole) in 3 m m of water were added at room temperature, while stirring, 0'5 ml of I I (2"3 × 10 -8 mole) in 0.25 ml of purified dioxane. Waterinsoluble yellowish particles appeared almost immediately in the reaction mixture, a n d remained until the end of the experiment. After 1.5 hr the reaction mixture was filtered, the solution was washed five times with water, and the polymer was separated by lyophilic drying. The PMAA-A sample obtafined in this experiment had a U-V spectrum characteristic of the 9-anthrylacetoxymethane model compound, and had one lurninescent marker per 10,000 polymer units.
Preparation of polymethyl methacrylate with 9-anthrylacylhydroxymethane markers (P M M AA). PMMA-A was obtained through the methylation of PMAA-A in the presence of diazomethane in toluene, using the procedure described in [8]. PMMA-A was double-reprecipitared from toluene with petroleum ether, and from chloroform with methanol. The same method m a y be used in the preparation of other polyalkyl acrylates and polyalkyl methacrylares with covalently added luminescent markers.
Preparation of polyglutamic acid with 9-anthrylacylhydroxymethane markers (PGA-A). To a solution of 12 mg of the sodium salt of PGA in 3 ml of water was added a solution of 0.3 mg of I I in 3 ml of ether, and the mixture was stirred for 1.5 hr at room temperature. The aqueous layer was separated and washed five times with ether. PGA-A formed by ly ophilic (h'ying contained one 9-anthrylacylhydroxymetlmne group per 10,000 polymer units.
Preparation of polymers with luminescent markers
2847
I t is t h e r e f o r e possible w i t h t h e help of t h e p r o p o s e d m e t h o d to a d d l u m i n e s cent m a r k e r s to different c a r b o x y l a t e d p o l y m e r s . T h e a d v a n t a g e s of t h e m e t h od in question include t h e m i l d r e a c t i o n conditions, a n d t h e s h o r t r e a c t i o n t i m e e n a b l i n g t h e " l a b e l s " t o be i n t r o d u c e d into u n s t a b l e s u b s t a n c e s , p a r t i c u l a r l y biological ones. M o r e o v e r t h e 9 - a n t h r y l a c y l h y d r o x y m e t h a n e l u m i n e s c e n t g r o u p f o r m e d as a result of t h e r e a c t i o n has optical p r o p e r t i e s f a c i l i t a t i n g t h e m e a s u r e m e n t s , i.e. a n intense l u m i n e s c e n t b a n d in t h e visible region o f t h e s p e c t r u m , possessing a v e r y v a r i a b l e s t r u c t u r e a n d a h i g h degree o f polarization. CONCLUSIONS
(1) A m e t h o d for t h e p r e p a r a t i o n of c a r b o x y l a t e d p o l y m e r s w i t h c o v a l e n t l y a d d e d l u m i n e s c e n t groups h a s b e e n proposed. T h e m e t h o d is b a s e d on t h e reaction of 9 - d i a z o m e t h y l a n t h r a c e n e w i t h C O O H g r o u p s of t h e m a c r o m o l e c u l e s . (2) P o l y a c r y l i c , p o l y m e t h a c r y l i c a n d p o l y g l u t a m i c acids a n d s o m e of t h e i r esters ( p o l y m e t h y l m e t h a c r y l a t e , p o l y b u t y l m c t h a c r y l a t c ) possessing t h e lumin e s c e n t m a r k e r s h a v e b e e n synthesized. (3) Some optical characteristics ( a b s o r p t i o n a n d fluorescent spectra, polarization of luminescence) of t h e 9 - a n t h r y l a e y l h y d r o x y m e t h a n e g r o u p s a d d e d to the polymers have been investigated. Translated by R. J. A. HENDRY REFERENCES
1. G. WEBER, Biochem. J. 52: 145, 1952 2. G. KEMERER, Polymer Chem. and Technol., No. 3, 26, 1966 3. M. G. KRAKOVYAK, Ye. V. ANUFRIEVA and S. S. SKOROKHODOV, Vysokomol. soyed. 8: 1681, 1966 (Translated in Polymer Sci. U.S.S.R. 8: 10, 1853, 1966) 4. Ye. V. ANUFRIEVA, M. V. VOL'KENSHTEIN, M. G. KRAKOVYAK and T. V. SHEVlWLEVA, Dokl. AN SSSR 182: 361, 1968 5. R. F. STEINER and H. EDELHOCH, Chem. Revs. 62: 457, 1962 6. H. Z0LLINGER, Azo- and diazochemistry of aliphatie and aromatic compounds, New York-London, 1961 7. Yu. N. PANOV, Thesis, 1967 8. A. KATCHALSKY and H. EISENBERG, J. Polymer Sei. 6: 145, 1951 9. M. Ya. PORMALE, Ye. A. PLISKO and S. N. DANILOV, Zh. prikl, khimii 39: 2310, 1966 10. E. KRICK and P. EMMELOT, Bioehim et biophys, aeta 91: 59, 1964 11. S. NEWMAN, W. R. KRIEGBAUM, $. L'AUGIER and P. FLORY, J. Polymer Sci. 14: 451, 1954 12. T. N. NEKRASOVA, O. B. PTITSYN and M. S. SHIKANOVA, Vysokomol. soyed. AI0: 627, 1968 (Translated in Polymer Sei. U.S.S.R. 10: 7, 1771, 1968) 13. Syntheses of Organic Compounds, ed. by Kazanskii, No. 2, p. 174, 1949 14. H. SCHUBERT and L. SELISKO, J. Pract. Chem. 16: 1, 1962 15. P. YATES, B. L. SHAPIRO, N. YODA and L. FUGGER, J. Amer. Chem. Soc. 79: 5756, 1957 16. V. BADDELEY, J. Chem. Soe., 99, 1947 17. T. NAKAYA, T. TOMOMOTO and M. IMOTO, Bull. Chem. Soc. Japan 40: 691, 1967
(Received 4 November 1968)
POLYMERS tO which chromophore groups have been covalently added are most frequently used for studying certain properties of natural and synthetic macromolecules and the mechanism of their formation [1-4]. The relaxation behaviour of polymers with luminescent groups may be studied by the polarized luminescence method. The latter is repeatedly used with success in studying rapid relaxation processes w h e n the periods of relaxation and luminescence are comparable; moreover it enables us to study the intramolecular motion of macromolecules in solution and in the mass. The advantages of the method include the possibility of experiments in water and other polar solvents; this along with the high sensitivity of the method facilitates work with low concentrations of the polymer in solution. The polarized luminescence method [1, 4] involves the use of luminescent markers covalently added to the macromolecules and possessing certain optical properties. The number of these specially added groups must be sufficient for purposes of spectral analysis and small enough to prevent distortion of the properties of the macromolecules. Several methods of preparing macromolecules with chemically added luminescent groups have been described [2, 5], but these are mainly applicable to polymers that have NH bonds. Our aim was to develop a convenient and general method of adding luminescent groups to polymers containing carboxyl groups. In a previous study [4] we prepared polyacrylic and polymethacrylic acids with covalently added luminescent groups. However the method used for their preparation involves polymer analogue conversions of the corresponding polyacryloyl chlorides, and this limits its usefulness. The chemical addition of luminescent groups to polymers containing carboxyl groups may be realized through specially selected aliphatic diazo compounds (diazoalkanes) interacting with COOH groups of the polymer. The reaction of diazoalkanes with earboxylic acids proceeds by the scheme [6] ® CI-I--N-- N R
@ H
)
- ~ ® R'cooeJ--~RCH,OCOR ' ~CI-I2--N ~ N ~C H ~ ® [ ] R COOH~_-~RCH2OCOR, + i_I R R e
* Vysokomol. soyed. A l l : No. 11, 2499-2504, 1969. 2842
Preparation of polymers with luminescent markers D 07
2843
./~
liI i,"i /:i /\I? i [lis
\. I. •_ - .
-,\
280
/t
i
\
;I
IX
~ / ~
~ ~. ~.~..
320
360
400
440
,t, m/z
Fro. 1. Absorption spectra of solutions in methanol: 1--9-diazomethylanthracene (II); 2--9-anthrylaeetoxymethane model compound (c =7.5 × l0 -5 mole/l.) and 3--PMAA containing one 9-anthrylaeetylhydroxymethane group per 1400 polymer units (%olymer=8"5 mg/ml). and under mild conditions in an inert medium the above reaction yields esters in practically quantitative yield. The only by-product is nitrogen. Only a small number o£ papers has been published by authors studying the reactions of aliphatic diazo compounds with polymers possessing functional groups (generally - - O H or COOH). These papers m a y be divided into three groups according to the manner in which the reactions were carried out: 1) reactions under homogeneous conditions (e.g. the methylation o£ carboxyl groups in methyl methacrylate-methacrylic acid copolymers soluble in aromatic hydrocarbons, conducted in the presence of diazomethane [7]); 2) reactions at the interface o£ the solid and liquid phases (mcthylation of polymethacrylic acid caused by diazomethane in a hydrocarbon solvent [8], preparation of cellulose ethers by means of diazoalkanes [9]); 3) reactions at the interface of two liquid phases (methylation o£ ribonucleic and desoxyribonucleic acids in aqueous solution using and ether solution o£ diazomethane [10]). The reagents used for the addition of luminescent groups to carboxylated macromolecules were diazomethyl-a-naphthylketone (I) and 9-diazometh~lanthracene (II). CHN2 CHN,, c=o
I
g\/~/%
II
2844
M. G. K~AKO'VY.CK et al.
The choice of reagents depended on the optical properties of the luminescent marker and on the relative stability of the diazoalkanes. The experiments showed that at room temperature the hydrocarbon solution I reacts very slowly with an aqueous solution of polymethacrylic acid (PM_&A); it was only at 50-55 ° that it was found possible b y increasing the content of I (1 mole to 8 basic moles PMAA) and the reaction time to obtain PMAA containing one ~-ketonaphthyl group per 300-400 elementary units. D 0"8:
! II
0.6
O.Zl
0.2
350
z~50
550 ;~ , m #
Fro. 2
I
I
i
i
260
300
3~0
380
FIG. 3
FIG. 2. Luminescence spectra: /--solution of 9-diazomethylanthracene (II) and 2 - 9 . anthrylacetoxymethane in benzene (c 27.5 × 10-6 molefl.); 3--aqueous solution of PMAA containing one 9-anthrylacylhydroxymethane group per 1400 polymer units (%olymer=0"5 mg/ml). Fro. 3. Absorption spectra of solutions in methanol: 1--methyl-~-naphthylketone model compound, (o=l×10 -~ gfl.); 2--PMAA prepared in blank experiment (Cvolymer=3g/1.); 3--PMAA sample from main experiment (£:polymer=3 g/1.). This relatively low reactivity of I is due to the effect of the carbonyl group which reduces the electron density at the diazo carbon atom and lessens the nueleophilic nature of the diazoalkane. It was found that unlike I solution II reacts readily with different carboxylated compounds under mild conditions. The reaction of II with carboxylated polymers leading to the formation of maeromolecules having luminescent 9-anthrylacylhydroxymethane groups may be brought about b y different methods, the choice depending on the properties of the polymer. Where the polymer and solution of I I dissolve in a common
Preparation of polymers with luminescent markers
2845
s o l v e n t t h e h o m o g e n e o u s m e t h o d s is t h e m o s t convenient. I n this case w i t h t h e P M A A / I I ratios i n v e s t i g a t e d 30-50~o of I I r e a c t s with t h e c a r b o x y l g r o u p s o f the polymer. I n t h e case o f w a t e r - s o l u b l e p o l y m e r s we m a y use one of t h e h e t e r o p h a s e m e t h o d s . B y a d d i n g solution of I I to a n a q u e o u s solution of t h e p o l y m e r , while stirring, in a s o l v e n t (dioxane, alcohols, etc.) miscible w i t h w a t e r we o b t a i n a t h i n suspension of I I a n d t h e r e a c t i o n proceeds a t t h e solid-liquid interface. T h e r e a c t i o n a t t h e i n t e r f a c e t a k e s place on m i x i n g a n a q u e o u s solution of t h e p o l y m e r a solution of I I in a w a t e r - i m m i s c i b l e solvent (ether, s a t u r a t e d a n d a r o m a t i c h y d r o c a r b o n s , etc.). I n t h e case of h e t e r o p h a s e m e t h o d s a considerable p o r t i o n of I I is used u p in side reactions, a n d so t h e q u a n t i t a t i v e ratio of the r e a g e n t s m u s t first be d e t e r m i n e d e x p e r i m e n t a l l y in order to o b t a i n p o l y m e r s w i t h a g i v e n n u m b e r of c h r o m o p h o r e groups. T h e r e m o v a l of t h e u n r e a c t e d I I a n d of low m o l e c u l a r weight p r o d u c t s of conversion of I I is effected b y r e p e a t e d r e p r e c i p i t a t i o n of t h e p o l y m e r b y m e a n s of dialysis or b y a d s o r p t i o n - c h r o m a t o g r a p h i c m e t h o d s . T h e a b s o r p t i o n a n d luminescence s p e c t r a of solutions of m a c r o m o l e c u l e s possessing 9 - a n t h r y l a c y l h y d r o x y m e t h a n e g r o u p s are in good a g r e e m e n t w i t h t h o s e o f t h e m o d e l c o m p o u n d ( 9 - a n t h r y l a c e t o x y m e t h a n e ) , a n d differ g r e a t l y f r o m t h e s p e c t r a of t h e initial diazo c o m p o u n d (II) (Figs. 1 a n d 2). A n o t h e r criterion for t h e a d d i t i o n o f l u m i n e s c e n t g r o u p s to t h e m a c r o m o l e c u l e s is a high degree of l u m i n e s c e n c e p o l a r i z a t i o n (10%) in a low-viscosity m e d i u m ~vhere t h e r e is o n l y slight p o l a r i z a t i o n of t h e luminescence of 9 - a n t h r y l a c e t o x y m e t h a n e
(~1%). EXPERIMENTAL *
Polyacrylic (PAA) and po]ymethacrylic acids obtained by radical polymerization of the corresponding monomers [11, 12] were purified by repeated reprecipitation from methanol by ether or ethyl acetate, hiazo compound I was synthesized by reacting diazomethane [13] and a-naphthoic acid chloride in ether solution by the method described in [14]. After double recrystallization from benzene compound I had m.p. 53.5-54.5 ° (m.p. 55 ° according to data in [4]). The position of the bands in the I R spectrum of compounds I prepared by us is in good agreement with data in [15]. Reaction of I with PI~IAA. Main experiment. A mixture of solutions of 0.1 g (0.0012 mole) PMAA in 10 ml H.~O and 0.03 g (0'00015 mole) of I in 8 ml of hexane was stirred at 50-55 ° for 14 hr. The aqueous layer was separated and after five-fold washing with ether the water was evaporated with a rotary evaporator. The resulting polymer was thrice reprecipitated from methanol with ethyl acetate. The UV spectrum of this PMAA sample (Fig. 3, curve 3) has a band with imax =304 m/2 characteristic for the a-ketonaphthyl group (Fig. 3, curve 1). Blank experiment. A reaction mixture of the same composition as in the main experiment was stirred rapidly for 1 rain, after which the PM.AA was separated and purified as in the main experiment. The UV spectrum of the PMA.A is shown in Fig. 3 (curve 2). Determination of number of a-ketonaphthyl chromophore group~ added to P M A A . The
number of g-ketonaphthyl groups covalently added to PMAA macromolecules was calculated * T. V. Shevelevaya, 1~. P. Novozhilova and S. G. Telichkina participated in this part, of the work.
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NI. G. Kmcxov-rax et al.
b y means of UV spectroscopy. Spectroscopic analysis of the P M A ~ sample obtained i n the blank experiment was carried out in order to determine the absorption due not to the added chromophore groups, b u t to the molecules of compounds containing a ketonaphthyl group (diazomethyl-a-naphthylketone or its low molecular weight conversion products) from which the polymer could not be liberated. The n u m b e r of a-ketonaphthyl groups eovalently added to PMAA in methanol solution was calculated from the equation e =JD/ed, where c is the concentration of a-ketonaphrhyl groups (mole/L); D is the optical density: ,~Ar~-~a0ar~bas.-__~a04r~b~nk., S is the molar absorption coefficient; d is the cell length, era. The value e~8,~26300 was determined from the UV spectrum of a methyl-g-naphthylketone solution in methanol [16] measured under identical conditions, the latter being used as model compound. The measurements carried out in this way showed that the PMAA samples obtained b y the method in question have a single chromophore group per 300-400 polymer units. 9-Diazomethylanthracene was obtained b y oxidation of 9-anthraldehyde hydrazone following the procedure in reference [17]. The ~ spectrum is shown in Fig. 1 (curve 1). The n u m b e r of 9-anthrylacylhydroxymethane groups in the polymers was determined b y U-V spectroscopy by the same method as that (see above) used for the polymers containing a-ketonaphthyl groups. As a model we used 9-anthrylaeetoxymethyl obtained by reacting I I with acetic acid [17]. For the given chromophore group in methanol e3,4 determined b y means of 9-anthrylacetoxymethane for A=384 m/l is equal to 7500. Preparation of polymers with luminescent groups eovalently added. The following are examples of experiments in the preparation of polymers possessing luminescent markers added b y different methods. Preparation of P A A with 9-anthrylacylhydroxymethane markers (PAA-A). To a solution of 1,5 g (0.021 base-mole) of PAA in 30 ml of methanol were added, while stirring (not with magnetic stirrers) at room temperature, 9 ml (4.13 × 10 -5 mole) of I I in 4-5 ml of purified dioxane (calculating for one molecule of I I per 500 PAA units). After 2 hr the polymer was precipitated with ether, separated, carefully washed with ether, again reprecipitated from methanol with ether, and then dried. The resulting PAA-A sample had one luminescent group per 1500 polymer units, i.e. N 3 0 ~ of the total amount of I I reacted with PAA. Preparation of P M A A with 9.anthrylacylhydroxymethane markers ( P M A A - A ). To a solution of 0.1 g PMA2k (1.16 × 10 -3 base-mole) in 3 m m of water were added at room temperature, while stirring, 0'5 ml of I I (2"3 × 10 -8 mole) in 0.25 ml of purified dioxane. Waterinsoluble yellowish particles appeared almost immediately in the reaction mixture, a n d remained until the end of the experiment. After 1.5 hr the reaction mixture was filtered, the solution was washed five times with water, and the polymer was separated by lyophilic drying. The PMAA-A sample obtafined in this experiment had a U-V spectrum characteristic of the 9-anthrylacetoxymethane model compound, and had one lurninescent marker per 10,000 polymer units.
Preparation of polymethyl methacrylate with 9-anthrylacylhydroxymethane markers (P M M AA). PMMA-A was obtained through the methylation of PMAA-A in the presence of diazomethane in toluene, using the procedure described in [8]. PMMA-A was double-reprecipitared from toluene with petroleum ether, and from chloroform with methanol. The same method m a y be used in the preparation of other polyalkyl acrylates and polyalkyl methacrylares with covalently added luminescent markers.
Preparation of polyglutamic acid with 9-anthrylacylhydroxymethane markers (PGA-A). To a solution of 12 mg of the sodium salt of PGA in 3 ml of water was added a solution of 0.3 mg of I I in 3 ml of ether, and the mixture was stirred for 1.5 hr at room temperature. The aqueous layer was separated and washed five times with ether. PGA-A formed by ly ophilic (h'ying contained one 9-anthrylacylhydroxymetlmne group per 10,000 polymer units.
Preparation of polymers with luminescent markers
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I t is t h e r e f o r e possible w i t h t h e help of t h e p r o p o s e d m e t h o d to a d d l u m i n e s cent m a r k e r s to different c a r b o x y l a t e d p o l y m e r s . T h e a d v a n t a g e s of t h e m e t h od in question include t h e m i l d r e a c t i o n conditions, a n d t h e s h o r t r e a c t i o n t i m e e n a b l i n g t h e " l a b e l s " t o be i n t r o d u c e d into u n s t a b l e s u b s t a n c e s , p a r t i c u l a r l y biological ones. M o r e o v e r t h e 9 - a n t h r y l a c y l h y d r o x y m e t h a n e l u m i n e s c e n t g r o u p f o r m e d as a result of t h e r e a c t i o n has optical p r o p e r t i e s f a c i l i t a t i n g t h e m e a s u r e m e n t s , i.e. a n intense l u m i n e s c e n t b a n d in t h e visible region o f t h e s p e c t r u m , possessing a v e r y v a r i a b l e s t r u c t u r e a n d a h i g h degree o f polarization. CONCLUSIONS
(1) A m e t h o d for t h e p r e p a r a t i o n of c a r b o x y l a t e d p o l y m e r s w i t h c o v a l e n t l y a d d e d l u m i n e s c e n t groups h a s b e e n proposed. T h e m e t h o d is b a s e d on t h e reaction of 9 - d i a z o m e t h y l a n t h r a c e n e w i t h C O O H g r o u p s of t h e m a c r o m o l e c u l e s . (2) P o l y a c r y l i c , p o l y m e t h a c r y l i c a n d p o l y g l u t a m i c acids a n d s o m e of t h e i r esters ( p o l y m e t h y l m e t h a c r y l a t e , p o l y b u t y l m c t h a c r y l a t c ) possessing t h e lumin e s c e n t m a r k e r s h a v e b e e n synthesized. (3) Some optical characteristics ( a b s o r p t i o n a n d fluorescent spectra, polarization of luminescence) of t h e 9 - a n t h r y l a e y l h y d r o x y m e t h a n e g r o u p s a d d e d to the polymers have been investigated. Translated by R. J. A. HENDRY REFERENCES
1. G. WEBER, Biochem. J. 52: 145, 1952 2. G. KEMERER, Polymer Chem. and Technol., No. 3, 26, 1966 3. M. G. KRAKOVYAK, Ye. V. ANUFRIEVA and S. S. SKOROKHODOV, Vysokomol. soyed. 8: 1681, 1966 (Translated in Polymer Sci. U.S.S.R. 8: 10, 1853, 1966) 4. Ye. V. ANUFRIEVA, M. V. VOL'KENSHTEIN, M. G. KRAKOVYAK and T. V. SHEVlWLEVA, Dokl. AN SSSR 182: 361, 1968 5. R. F. STEINER and H. EDELHOCH, Chem. Revs. 62: 457, 1962 6. H. Z0LLINGER, Azo- and diazochemistry of aliphatie and aromatic compounds, New York-London, 1961 7. Yu. N. PANOV, Thesis, 1967 8. A. KATCHALSKY and H. EISENBERG, J. Polymer Sei. 6: 145, 1951 9. M. Ya. PORMALE, Ye. A. PLISKO and S. N. DANILOV, Zh. prikl, khimii 39: 2310, 1966 10. E. KRICK and P. EMMELOT, Bioehim et biophys, aeta 91: 59, 1964 11. S. NEWMAN, W. R. KRIEGBAUM, $. L'AUGIER and P. FLORY, J. Polymer Sci. 14: 451, 1954 12. T. N. NEKRASOVA, O. B. PTITSYN and M. S. SHIKANOVA, Vysokomol. soyed. AI0: 627, 1968 (Translated in Polymer Sei. U.S.S.R. 10: 7, 1771, 1968) 13. Syntheses of Organic Compounds, ed. by Kazanskii, No. 2, p. 174, 1949 14. H. SCHUBERT and L. SELISKO, J. Pract. Chem. 16: 1, 1962 15. P. YATES, B. L. SHAPIRO, N. YODA and L. FUGGER, J. Amer. Chem. Soc. 79: 5756, 1957 16. V. BADDELEY, J. Chem. Soe., 99, 1947 17. T. NAKAYA, T. TOMOMOTO and M. IMOTO, Bull. Chem. Soc. Japan 40: 691, 1967