Bioconjugate Chem. 1992, 3, 32-36
32
Preparation of Novel Cyclosporin A Derivatives P. A. Paprica,+A. Margaritis,$ and N. 0. Petersen'tt Department of Chemistry and Department of Chemical and Biochemical Engineering, University of Western Ontario, London, Ontario, Canada N6A 5B7. Received June 24, 1991
The hydroxyl group on the 2-N-methyl-(R)-((E)-2-butenyl)-4-methyl-~-threonine residue of cyclosporin A was protected by acetylation, then the double bond on the same amino acid residue was oxidatively cleaved using a periodatetpermanganate reagent. The resultant derivative of cyclosporin A contained a carboxylic acid group which was subsequently reacted with the nucleophiles 5-(aminoacetamid0)fluorescein and poly(L-lysine), in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, to furnish novel cyclosporin A conjugates.
INTRODUCTION Cyclosporin A (CsA, 1) is a secondary metabolite produced by the fungus Beauuaria niuea (I). CsA is an undecapeptide (Chart I, Schemes I and 11) which is prescribed as an immunosuppressant to prevent rejection of transplanted organs in human patients (2,3). Because of its wide prescription throughout North America and Europe, cyclosporin A research is widespread; however, few synthetic schemes for modification of cyclosporin A are known (4-8). Previously published schemes for modification of cyclosporin A involve in vivo amino acid substitution (41, total synthesis of CsA starting from tartaric acid (5-7), or a low-yield oxidation of CsA to a derivative which contains a reactive aldehyde moiety (8). In our laboratory we have prepared a novel cyclosporin A derivative in good yield and used this derivative to prepare conjugates for fluorescence and immunological studies. This report presents the syntheses of four new molecules synthesized from cyclosporin A (3, 8, l l b , llc, Schemes 11-IV). EXPERIMENTAL PROCEDURES Cyclosporin A (1) was obtained as a generous gift from Dr. J. Bore1 of Sandoz. 2- [N-Methyl-N-(7-nitrobenz-2oxa-l,3-diazo-4-yl)aminolethanoic acid (10) was prepared by methods described elsewhere(9). 5-(Aminoacetamid0)fluorescein (7) was purchased from Molecular Probes Inc. (Eugene,OR). Poly-(L-1ysine)hydrobromide(averageMW = 26 500) (9) was purchased from Sigma Chemical Co. (St.Louis, MO). All other reagents were purchased from Aldrich Chemical Co. (Milwaukee, WI) or British Drug House (BDH) (Toronto, ON, Canada); KMn04, NaI04, and K&03 were recrystallized prior to use as described elsewhere (10). IH NMR spectra and 13C NMR APT spectra were recorded on a Varian Gemini 200-MHz spectrometer. Chemical shifts are reported in ppm relative to TMS as an internal standard unless otherwise stated. Mass spectroscopy analyses were performed on a Finnigan MAT 8320 by the chemical ionization (CI) or fast atom bombardment (FAB) technique. Fourier transform infrared spectra were recorded using a Bruker IRS 32 source and an IBM system 9000 processing system. FTIR samples were prepared as thin films from CHCL solutions on NaCl disks. Reactions and purification procedures were monitored by thin-layer chromatography (TLC) using plasticbacked silica gel 60 UV/254 plates (Merck) as the stationary + Department of
Chemistry. Department of Chemical and Biochemical Engineering.
Chart I
1
C
AOH-
S
-
~
To
CsA
2
Scheme I1 C
S
3
-
A
~
1
A
0 C
s
A
4
V
-
A
0 C
S
A
W
5
phase and 60140 (vtv) ethyl acetatelacetone with 1?6 trifluoroacetic acid as the mobile phase. UV spectra were obtained using a Shimadzu UV- 160 UV-visible recording spectrophotometer. Automatic pH-stat work was performed with a radiometer system (Copenhagen) consisting of a PHM82 @ 1992 American Chemical Society
Preparation of Novel Cyclosporin A Derivatives
Bioconjugate Chem., Vol. 3, No. 1, 1992
Scheme I11
33
Scheme IV yo2
O
W
O
H
10
QCozH
6
9
6
lla
(q+r+s), 8
standard pH meter, a TTT80 titrator, and an ABU80 autoburette. CsA Lactone 3. The title compound was prepared by a modification of the periodatelpermanganese oxidation used to cleave unsaturated fatty acids (10). In a typical preparation, 490 pL of 0.15 M aqueous KzC03 (73.5 pmol) and 490 pL of 0.20 M aqueous NaI03 (98.0 pmol) were added to a solution of 14.7 mg of CsA (1) (12.2 pmol) in 1.48 mL of tert-butyl alcohol. Deionized distilled water was added dropwise until all NaI04 had dissolved, then 98.0pL of 0.025 M aqueous KMn04 (2.45 pmol) was added. The solution was stirred at room temperature under N2(g) for 14 h, and then an additional 98 pL of 0.025 M aqueous KMn04 (2.45 pmol) was added to the reaction mixture. Stirring a t room temperature under N2(g) was continued for an additional 6 h (total reaction time 20 h), at which point the solution was pale brownish pink. The reaction was stopped by the addition of 0.147 mL of freshly prepared 40 % (w/v)aqueous NazSz05 solution (309pmol1, 0.250 mL of 1.0 M HzS04 (250 pmol), and 2.5 mL of deionized distilled water. The mixture was stirred for 10 min, and then the aqueous solution was extracted with 3 X 40 mL of diethyl ether. The solvent was removed from the ether extracts under reduced pressure, and then the dry product was dissolved in 2 mL of methanol and loaded on 20 g of Sephadex LH20 gel filtration media (Pharmacia) which had been previously swollen in methanol. The fractions were analyzed by TLC and the fractions containing the first compound to elute from the gel filtration column were collected. Removal of solvent under reduced pressure furnished 12.2 mg (84%) of white solid product 3. 'H NMR (CDCl3): NH groups, 6 8.42, 7.92, 7.50, 7.46 (all d, each 1 H); NCH3 groups, 3.46, 3.39, 3.17, 3.05, 2.66, 2.64 (alls, each 3 H). 13CNMR (CDC13): CH2 carbons, 6 49.92, 40.57,36.51,39.05,36.51,35.90,35.20,25.14,CHOR,82.77; CH=CH carbons, 126.72, 130.14. IR: carbonyl stretch (lactone), 1787 cm-'. MS: mle expected 1188, found 1188 (M+). O-Acetyl-CsA 4. O-Acetyl-CsAwas prepared according to the method of Traber et al. (11). 'H NMR (CDC13): NH groups, 6 8.48, 7.97,7.43,7.38 (alld,each 2 H);NCH3
9
= 125
6, 10, 5
l l a , lib, llc
(3
A
0
groups, 3.38, 3.18, 3.16, 3.13, 3.01, 2.60, 2.58 (all 8, each 3 H), OC(O)CH3 1.93 ( 8 , 3 H) (the complete 'H NMR spectrum is available as supplementary material). 13C NMR (CDC13): CHZcarbons, 6 49.82,40.79,37.95, 36.79, 35.61, 33.48, 24.74; HCOC(O)CH3, 72.31; HCOC(O)CH3, 168.21; CH=CH carbons 129.21, 126.35. All NMR assignments are in accord with those expected on the basis of previous assignments of the 'H and 13C NMR spectra of CsA (12).IR: carbonyl stretch, 1746 cm-l (in addition to a strong amide stretch at 1628 cm-1). MS: m/e (FAB) expected 1245, found 1245 (M)+. O-Acetyl-CsA Acid 5. The title compound was prepared from O-acetyl-CsA 4 by the same method used to prepare CsA lactone 3. Yield: 100%. 'H NMR (CDCl3) (dominant conformer): NH groups, 6 8.38,8.02,7.72,7.45 (d, each 1H); NCH3 groups, 3.49,3.24,3.09,2.69,2.67,(all s, each 3 HI, 3.25 (8, 6 HI; OC(O)CH3, 2.01 (s 3 H)(the complete 'H NMRspectrum is available as supplementary material). 13CNMR (CDCM: CH2 carbons, 6 40.50,38.79, 34.40, 24.92 (multiple conformations of the peptide in solution complicated the 13Cspectrum and precluded the assignment of all expected methylene 13Csignals); HCOC-
34
Bioconiugate Chem., Vol. 3, No. 1, 1992
Paprlca et al.
at 5.50. Products were purified as per 1 la. The first band (0)CH3, 72.31; HCOC(O)CH3, 167.34. IR: carbonyl to elute from the column presumably contained comstretch, 1745cm-l. MS: mle (FAB) expected 1249, found pounds 1 la-c and unreacted polyb-lysine). Unreacted 1249 (M)+. O-acetyl-CsA acid 5 and low molecular weight products 5-(Aminoacetamido)fluorescein-O-Acetyl-CsA from side reactions of 10 were collected in later fractions Amide 8. Compound 8 was prepared using a modification and identified by lH NMR. The lH NMR of the products of the procedure routinely used to react carboxylic acid 1 la-c and 9 contained signals corresponding to both polygroups on proteins (13). In a typical preparation, 33.1 mg (173pmol) of l-ethyl-3-[3-(dimethylamino)propy1lcarbo- (L-lysine) and O-acetyl-CsA acid, but lH NMR was not a sensitive enough technique to detect peaks corresponding diimide (EDC, 6) and 2.5 mg (5.24 pmol) of 54aminoacto protons on the fluorescent moiety. 'H NMR (DzO): etamid0)fluorescein (7) were dissolved in 12 mL of polyb-lysine) peaks, 6 4.05,2.74,1.50,1.22; cyclosporin A deionized distilled water which had been made basic by related peaks, 2.92, 2.62, 2.60, 1.90, 0.73. the addition of 2 drops of 10% NaOH solution. A solution of 10 mg (8 pmol) of O-acetyl-CsA acid 5 in 200 p L of RESULTS AND DISCUSSION tert-butyl alcohol was added to the aqueous solution and the solution was transferred to an autotitrator set on pHCyclosporin A (CsA, 1) is an undecapeptide which stat, where the pH of the solution was adjusted to 5.50 by contains only two chemically reactive sites that can be titration with 1.0 M HC1. The reaction was allowed to modified without destruction of the amide bonds within proceed in the dark for 5 h, during which time the pH of the peptide (see Chart I). Both of these functionalgroups, the solution was maintained at 5.50 by automatic titrathe hydroxyl group and the double bond, are located on tion with 1.0 M HCl. After 5 h the reaction was stopped the 2-N-methyl-(R)-((E)-2-butenyl)-4-methyl-~-threonine by the addition of 1mL of pH 4.75 acetate buffer and the (MeBmt) residue of cyclosporin A, and the chemical solvent was removed by freeze-drying. reactivity of both groups is diminished by steric factors The methanol-soluble dried products were dissolved in arising from the three-dimensional conformation of CsA 2 mL of methanol and loaded on 20 g of Sephadex LH-20 in solution (13). gel filtration media (Pharmacia)which had been previously It has been shown elsewhere (11)that the alcohol moiety swollen in methanol. The fastest running colored comof CsA is sufficiently reactive to produce O-acetyl-CsA pound was collected and the methanol was removed under when combined with acetic anhydride. Consequently, in reduced pressure. CHCl3 (25 mL) was added to the flask our initial experiments we aimed to exploit the reactivity containing the dried products, and then the solution of of the alcohol group and incorporate a new reactive the colored product in CHC13 was washed with 3 X 20 mL fundamental group on CsA by allowingthe alcohol moiety of pH 7.40 phosphate buffer to remove side products. to undergo esterification with small carboxylic acid Removal of the CHCl3 under vacuum furnished 12.0 mg chlorides containing terminal primary bromide groups (i.e. (87%) of orange solid product 8. 'H NMR (CDCld: NCH3 4-bromobutyric acid chloride, 3-bromopropionic acid groups (major conformer in solution) 6 3.48, 3.11, 3.06, chloride). 2.74, 2.71, (all s, each 3 H), 3.25 (s,6 H); aromatic region When 2-octanol is allowed to react with 4-bromobumultiplets, 8.62, 8.34, 7.77, 7.66, 6.78. tyric acid chloride in deuterochloroform in an NMR tube, Compound 1 la. The title compound was prepared by it is possible to follow the reaction by monitoring the lH the same method used to prepare compound 8. In a typical NMR spectrum, since the lH signal corresponding to the reaction 80 mg of 2- [N-methyl-N-(7-nitrobenz-2-oxa-1,3- methylene group adjacent to the carbonyl in 4-bromobudiazo-4-yl)amino]ethanoicacid (10)(317pmol) was allowed tyric acid chloride is 0.58 ppm downfield from the to react with 18.3 mg of poly(L-lysine) (9) r0.691 pmol of analogoussignal of 4-bromobutyric acid 2'-octyl ester (3.02 poly@-lysine),87.7 pmol lysine residues] in the presence vs 2.43 ppm). Using this information we hoped to follow of 161.1 mg of EDC (6) (840 pmol) in 13 mL of deionized the reaction of the alcohol group on CsA with 4-bromobudistilled water at pH 5.50. After 5 h the reaction was tyric acid chloride; however the lH NMR spectra indicated stopped by the addition of 2 mL of pH 4.75 acetate buffer that no new signal at -2.43 ppm was evident after 5 h, and the total volume of the solution was reduced to 2 mL i.e. no reaction had occurred. Additional experiments by freeze-drying. Purification of products was performed where cyclosporin A was combined with 4-bromobutyric on a column of 20 g of Bio-Gel P-2 gel filtration media acid chloride and 3-bromopropionic acid chloride in the (Bio-Rad) which had been previously swollen in pH 7.4 presence of a pyridine catalyst also showed that no ester phosphate buffer solution. The desired product was easily product was formed, even after 24 h. The lack of success identified as the first colored compound to elute from the with the above strategy is perplexing in view of the column. 'H NMR (D20) indicated the presence of polyreactivity of CsA with acetic anhydride (1O), and can most (L-lysine) but was not sensitive enough to detect peaks likely be attributed to the fact that the alcohol moiety of corresponding to protons on the fluorescent moiety. 'H CsA does not readily react intermolecularly with acid NMR (DzO): C(O)NHCH, 6 4.11 (t, 1 H); CH2NHz,2.80 chlorides because of steric hindrance of the alcohol moiety (t, 2 H); CH2CH2NH2,1449(quint, 2 H); CHCH~CH~CHT due to the three-dimensional conformation of the peptide NH2,1.24 (m, 2 H). UV: X = 480 nm. (13). Compound 11b. The title compound was prepared by Since attempts to react the alcohol group of CsA were unsuccessful, a new synthetic strategy was developedbased the same method used to prepare compound 8. In a typical reaction 6.23 mg of poly(L-lysine) [0.235 pmol of poly(Lon oxidative cleavage of the double bond of the MeBmt residue, by analogy with a previous preparation of a CsA lysine), 29.9 pmol of lysine residues] was combined with 1.5 mg of 2- [N-methyl-N-(7-nitrobenz-2-oxa-l,3-diazo-4- derivative containing an aldehyde function ( 4 ) . Using a y1)aminolethanoic acid (10)(5.95 pmol) and 80 mg of EDC modification of a mild oxidation procedure used to cleave the double bonds of unsaturated fatty acids, cyclosporin (6) (417 pmol) in 12 mL of deionized distilled water which A was converted to compound 2 (Scheme I). As expected had been made basic by the addition of 2 drops of 10% NaOH solution. A solution of 10 mg of O-acetyl-CsAacid (1.9, 'H and 13C NMR indicated that the equilibrium 5 (8.01 pmol) in 1mL of tert-butyl alcohol was added and between 2 and the lactone 3 strongly favored lactone the solution was stirred for 5 h while the pH was maintained formation to the exclusion of 2. Additional evidence for
Preparation of Novel Cyclosporin A Derivatives
Bioconjugate Chem., Vol. 3, No. 1, 1992 35
moiety. The UV spectrum of adduct lla confirmed that lactone formation was obtained from the FTIR spectrum, the fluorophore was present, and that the overall coupling which exhibited a characteristic strong stretch at 1787 efficiency for the reaction was low [lo% of the poly(Lcm-'. lysine) in solution reacted to produce conjugate lla]. From the spectral data of 3 it was clear that the reaction Once the reaction of poly(L-lysine) (9) with fluorescent had proceeded cleanly and without any oxidation of amide carboxylic acid 10 had been ascertained, polyb-lysine) (9) bonds in the peptide. However, the fact that lactone 3 and O-acetyl-CsA acid 5 were allowed to react in the was the dominant species of the equilibrium with carpresence of EDC (6) and a small amount of 10 (Scheme boxylic acid 2 suggested that secondary reactions involving IV). The colored high molecular weight products lla, intramolecular nucleophilic attack of the alcohol on a and presumably llc, eluted from a Bio-Gel P-2 column modified terminal carboxylic acid group might occur at a with unreacted poly(L-lysine) (91, and presumably polylater stage in any synthetic scheme. Accordingly, O-acetyl(L-lysine)-O-acetyl-CsA acid conjugate, 1 lb. The presence CsA acid 5 which is incapable of lactonization, was prepared of colored products 1 la and 1 IC allowed for facile isolation by oxidation of O-acetyl-CsA (4) (Scheme 11). of the mixture of poly@-lysine)adducts by gel filtration O-Acetyl-CsA acid 5 was found to be reactive with chromatography, and served to indicate that reaction primary amines in aqueous solution when combined with between poly(L-lysine) and carboxylic acids in solution l-ethyl-3-[3-(dimethylamino)propyllcarbodiimide (EDC, had occurred. Fractions containing unreacted O-acetyl6), a well-known reagent for modification of carboxylic CsA acid 5 and products from side reactions of 10 eluted acid groups in aqueous media (10).By reacting O-acetylafter the fractions containing products lla-c and were CsA acid 5 with 5-(aminoacetamido)fluorescein (7), a characterized by lH NMR. commercially available fluorescent compound, in the We felt it was necessary to include the tracer compound presence of EDC (6), the fluorescent derivative of cyclos10 in our reaction scheme to ensure that conjugation was porin A (8) was obtained (Scheme 111). Fluorescent occurring; however, the presence of 10 inevitably gave rise product 8 was easily identified and isolated as the first to the mixture of products lla-c which could not be colored compound to elute from a gel filtration column separated. Consequently, it was necessary to inject the which fractionates compounds over the molecular weight mixture of products lla-c into the live animal, in the range 100-1800 g/mol. expectation that antibodies will be raised against all One of the main purposes of the cyclosporin A manipantigens. Though it is always preferable to inject a single ulations undertaken in this laboratory was to generate an antigen (in our case llb would be the desired antigen) immunogen with CsA as a hapten which could be used to when generating antibodies, fortunately it is possible to obtain monoclonal antibodies directed against CsA. It select only those antibodies directed against O-acetyl-CsA was necessary to prepare an immunogen because cyclosacid by competitive ELISA screening techniques, or by a porin A is an immunosuppressant and is also liable to be competitive binding assay using fluorescent derivative 8. too small to illicit an immune response in a live animal. Thus we anticipate that screening techniques will furnish The initial synthetic strategy for preparation of an imantibodies directed against the O-acetyl-CsAacid moiety munogen derived from CsA involved the reaction of 0of llb,though compound llb could not be isolated itself. acetyl-CsA acid 5 with bovine serum albumin (BSA) in the presence of EDC (6); however, verification of the CONCLUSIONS couplingof 5 to BSA by amino acid analysis proved difficult for several reasons. Amino acid analysis is based on the The synthesis of compound 5 is an important step toward hydrolysis of amide bonds, therefore the amide bond the synthesis of novel cyclosporinA derivatives. The novel linking O-acetyl-CsAacid 5 and BSA is necessarily cleaved compounds 8, llb, and llc represent a few examples of by this technique. Consequently, using amino acid the numerous cyclosporin A derivatives which can be analysis, one cannot verify whether 5 is chemically linked prepared by reacting 5 with primary amines or other nuto BSA, or whether the two species are simply present in cleophiles. the same solution. Moreover, the coupling efficiency of the reaction of 5 with BSA in the presence of EDC (6) is ACKNOWLEDGMENT very low, and evidence of CsA was not detectable in the Funding for the work described in this paper was hydrolysis mixture. provided by an NSERC postgraduate fellowship (P.A.P.), To address these general problems involved with by NSERC operating grant #3272 (N.O.P.), and by an detection of the immunogen, we decided to increase the NSERC Biotechnology Strategic Grant #STROO40839 coupling probability of the reaction by using poly(L-lysine) (A.M., principal investigator). We thank Dr. J. Borel of rather than BSA and to verify the coupling by introducing Sandoz for providing a cyclosporin A sample. a small amount of fluorescent acid as a tracer. The assumption made was that if we can identify a fluorescent Supplementary Material Available: lH NMR spectra of adduct of polyb-lysine), there is a high probability that 1,4, and 5 (6pages). Ordering informationis given on any current the poly(L-lysine)-0-acetyl-CsA acid conjugate will also masthead page. be present. The compound 2- [N-methyl-N-(7-nitrobenz-2-oxa-l,3LITERATURE CITED diazo-4-y1)aminolethanoic acid (10)was prepared because it is a fluorescent water-soluble compound containing a (1) Margaritis, A., and Chahal, P. S. (1989) Development of a carboxylicacid group, which should coupleto poly(L-lysine) Fructose Medium for Biosynthesis of CyclosporinA by Beaucompetitively with O-acetyl-CsA acid. In our first exvaria nivea. Biotechnology Lett. 11, 765-768. periment, poly(L-lysine) (9) was allowed to react with 10 ( 2 ) Borel, J. F. (1983) Cyclosporine: Historical Perspectives. in the presence of EDC (6) (Scheme IV). Fluorescent Transplant Proc. 15, Suppl. 1, 2230-2241. product lla was isolated as the first colored compound to (3) Stiller, C. R., and Keown, P. A. (1984) Cyclosporin Therapy elute from a gel filtration column and lH NMR confirmed in Perspective. Progress in Transplantation (P. J. Morris, the presence of poly(L-lysine),but was not sensitive enough and N. L. Tilney, Eds.) pp 11-45, Churchill Livingston to detect peaks correspondingto protons on the fluorescent Publishers, London.
36 Bioconjugate Chem., Vol. 3,No. 1, 1992
(4) Wenger,R.,Traber,R. P., Kobel, H., andHofmann, H. (1985) Cyclosporin Derivatives and Their Use. French Patent # 561 651 A l . (5) Wenger, R. M. (1986)Synthesis of Ciclosporin and Analogs:
Structural and Conformational Requirements for Immunosuppressive Activity. Prog. Allergy 38 (Ciclosporin), 46-64. (6) Wenger, R. (1983)Synthesis of Cyclosporineand Analogues: Structure Activity Relationships of New Cyclosporine Derivatives. Transplant. Proc. 15, Suppl. l, 2230-2241. (7) Wenger, R. (1982)Chemistry of Cyclosporin. Cyclosporin A, Proceedings of the International Conference (D. White, Ed.) pp 19-34,Elsevier BiomedicalPress, Amsterdam, Netherlands. (8) Abbot Laboratories (1988)FluorescencePolarization Assay for Cyclosporin A and Metabolites and Related Immunogens and Antibodies. European Patent # EP 0283 801 A2. (9) Petersen, N. 0.(1985)Intramolecular Fluorescence Energy Transfer in Nitrobenzoxadiazole Derivatives of Polyene Antibiotics. Can. J. Chem. 63, 1, 77-85. (10)Longmuir, K. J., Rossi, M. E., and Resele-Tiden, C. (1987) Determination of MonoenoicFatty Acid Double Bond Position by Permanganate-Periodate Oxidation Followed by High
Paprlca et al.
Performance Liquid Chromatography of CarboxylicPhenacyl Esters. Anal. Biochem. 167, 213-221. (11) Traber, R., Loosli, H., Hoffman, H., Kuhn, M., and Von Wartburg,A. (1982)Isolation and Structure Determination of New Cyclosporings E, F, G, H and I. Helv. Chim. Acta 65, F ~ S5,C 1655-1677. (12) Kessler, H., Loosli,H., and Oschkinat,H. (1985)Assignment of the lH-, W-, and 14N-NMRSpectra of Cyclosporin A in CDCls and CsDa by a Combination of Homo- and Heteronuclear Two-DimensionalTechniques. Helv. Chim. Acta 68,661681. (13) Carraway,K.L., and Koshland, D. E. (1972)Carbodiimide Modification of Proteins. Methods Enzymol. 25B, 616-623. (14) Loosli, H.; Kessler, Oschknat, Weber, H., Petcher, T. J., and Windmer, A. (1985)The Conformationof Cyclosporin A in the Crystal and in Solution. Helv. Chim. Acta 68,682-704. (15) Streitwieser, A., Jr., and Heathcock, C. H. (1985)Chapter 27 Hydroxy Acids. Introduction to Organic Chemistry, 3rd ed., pp 859-861, Macmillan Publishing Co., New York. Registry No. 1,59865-13-3; 3,137718-40-2; 4,83602-41-9; 5, 137718-41-3;7, 137718-42-4; 8, 137718-43-5; 9 (homopolymer), 25104-18-1; 9 (SRU),38000-06-5.
