Bioconjugate Chem. 1991, 2, 13-15
13
LETTERS (N-Succinimidyl 4 - pentynoate)hexacarbonyldicobalt: A
Transition-Metal Carbonyl Complex Having Similar Uses to the Bolton-Hunter Reagent Michele Salmain, Anne Vessi&res,Ian S . Butler,+ and G6rard Jaouen* Ecole Nationale Supgrieure de Chimie de Paris, U.A. C.N.R.5' 403, 1 1 rue Pierre et Marie Curie, 75231 Paris Cedex 05, France. Received November 27, 1990
The synthesis of the transition-metal carbonyl complex (N-succinimidyl4-pentynoate)hexacarbonyldicobalt [ [ (C~H~O~N)O(CO)CH~CH~C=CH]CO~(CO)~] is described. This cobalt carbonyl complex is structurally similar to the Bolton-Hunter conjugation reagent and has been successfully employed as a nonradioactiue tracer for labeling the drug carbamazepine. The metal carbonyl tracer can be detected a t extremely low concentrations (ca. 1 pmol) by FT-IR spectroscopy in the v(C0) region (2150-1800 cm-l). The cobalt carbonyl labeled carbamazepine retains good recognition for anti-carbamazepine antibodies. This novel labeling procedure, which can be broadly termed carbonylmetalloimmunoassay (CMIA), has considerable potential for assaying a wide range of biological materials.
The labeling of biomolecules with radionuclides and other conjugates is an extremely important field for clinical diagnosis and there is a constant search for new and more sensitive analytical techniques for the detection of biological tracers ( I ) . The Bolton-Hunter reagent, I25Ilabeled N-succinimidyl 3-(4-hydroxyphenyl)propionate (11, was the first conjugation species to be used extensively
1
to prepare high-activity, high-specificity, radiolaudel tracers for immunoassay ( 2 , 3 ) . However, chiefly because of the attendant legal and technical problems associated with 1251and other radioisotopes (4), considerable effort is being placed nowadays into the development of nonradioactive tracers for clinical diagnosis. In 1977,Cais et al. ( 5 )introduced a nonradioactive metalloimmunoassay procedure in which bioligands labeled with organometallic groups were used for assaying the free bioligands. Several such studies have now been reported in which the metallic species are detected by atomic absorption (6) or electrochemically ( 7 ) . Unfortunately, + Permanent address: Department of Chemistry, McGill University, 801 Sherbrooke St. West, Montreal, Quebec, Canada H3A 2K6.
1043- 180219 112902-00 13$02.50/0
these detection techniques are often hampered by metal contamination of the biologicalmaterials being examined. In our approach, biomolecules such as hormonal steroids (8-1 0) and mycotoxines ( 11 ) are labeled with various metal carbonyl fragments which can then be unambiguously detected by FT-IR spectroscopy in the v(C0) region (21501800 cm-'1. The FT-IR sensitivity can be increased markedly by incorporating a liquid-nitrogen cooled, indium antimonide detector and by employing an ultralowvolume, gold light-pipe, solution cell (12). Our overall objective is to dc !lop a new analytical technique for immunoassay, which we term carbonylmetalloimmunoassay (CMIA). Labeled biomolecules that exhibit good molecularrecognition properties are usually synthesized by direct complexation of a reactive precursor ( 13). Indirect labeling of hormonal steroids with C02(C0),3fragments has been successful in certain cases due to the alkylating properties of carbenium ions (10,14). At the present time, there are no direct or indirect methods of labeling polyfunctional molecules,such as proteins, with metal carbonyl fragments. We report here the synthesis of a cobalt carbonyl complex that is structurally similar to the Bolton-Hunter reagent (3) and describe its use in labeling the antiepileptic drug carbamazepine (4). Compound 3 was prepared by reaction of N-succinimidyl 4-pentynoate (2) with Coz(CO)8 (Scheme I). Carbamazepine (4) was labeled as shown in Scheme 11. This labeling can be done in various solvents, including water. In most direct-labeling experiments, the solvent is THF, in which many biological substances, especially proteins, are insoluble. There are three characteristic v(C0) peaks for Co2(CO)6-labeledcarbamazepine (6) in CCld solution (Figure 1). The best correlation between the quantity of the marker injected into the IR cell (in the range 10-7-10-5 M) and the intensities of the v(C0) peaks is obtained for the 2052 cm-l peak (r = 0.998), supporting the possibility of quantitative work. Approximately 1pmol of 6 can be detected by the CMIA technique and this detection level 0 1991 American Chemical Society
Bioconjugate Chem., Vol. 2, No. 1, 1991
14
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Figure 1. Expansion of the v ( C 0 )region of the FT-IRspectrum of 6 (c = lo-' M; 1.6 pmol injected; CCld solution; 20-mm gold light-pipe cell; 10 scans; 4 cm-1 resolution).
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Scheme I. Synthetic Scheme for the Preparation of 3 0 HC
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Scheme 11. Synthetic Scheme for the Preparation of 6 R 1) COCI,, ioluene
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appears to be the lowest ever reported for a solution-phase FT-IR experiment over such a short time period (10 scans, 4 cm-l resolution, 66 8). In a preliminary CMIA study using rabbit anti-carbamazepine antibodies, Co2(CO)6-labeledcarbamazepine (6) as the tracer, and FT-IR detection of the v(C0) mode at 2052 cm-l, we have established that it is possible toperform a high-sensitivity immunoassay of carbamazepine (15).A typical standard immunoassay curve is obtained in the presence of 10 pmol of 6 and of 0-80 pmol of carbamazepine per tube. Under these conditions, 32 pmol of carbamazepine are required to displace 50 % of the binding
of 6 to the antibodies. For a physiological level of carbamazepine (16), only 2.5 pL of serum is necessary to perform the immunoassay. The affinity constant of the antibodies for 6 is 4.5 X lo7 M-l, while the number of binding sites is estimated to be 2.6 X 10+ M. These results confirm the feasibility of a FT-IR immunoassay of carbamazepine. Finally, we have found that other methods can be used to label peptides with organometallic units, such as the complexation of norleucine acetylenic fragbut the herein described conjugationprocedure ments (13, is particularly straightforward. Experimental Procedures. All solvents were freshly distilled under argon. Infrared spectra were recorded on a Bomem Michelson 100 FT spectrometer equipped with an In/Sb detector and a beam condensor (to produce the required 1.0-mm IR microbeam). The NMR, UV-visible, and mass spectra were measured on a 250-MHz Bruker, a Kontron Uvikon 860, and a Nermag spectrometer, respectively. (N-Succinimidyl 4-pentynoate)hexacarbonyldicobalt (3). 4-Pentynoic acid (200 mg, 2.0 mmol) and N-hydroxysuccinimide (230 mg, 2.0 mmol) were added to a solution of dicyclohexylcarbodiimide (DCC, 500 mg, 2.4 mmol) in THF (5mL) at -10 OC. The mixture was stirred at -10 "C for 2 h and was then allowed to warm up to ambient temperature overnight. Acetic acid (25 pL) was added and the dicyclohexylurea which formed was removed by filtration. The filtrate was stripped down to give a white product, N-succinimidyl4-pentynoate(2) (ca. 400 mg): IR (KBr) u(CH2) 2933 (s), 2853 (s), v(C=C) 2114, v(C0) 1815 (s), 1786 (s), 1728 (s) cm-'. Two-hundred milligrams (1.0 mmol) of 2 was dissolved in THF (7 mL), and Coa(CO)s(350mg, 1.2 mmol) was added. Thereaction mixture was stirred at room temperature for 1 h. The solvent was almost completely evaporated off under reduced pressure, and the residue remaining was chromatographed on a silica gel TLC plate (Kieselgel60 GF 254) using ether/pentane (1/1) as eluent. The crude product (Rf= 0.4) recovered from the TLC plate was recrystallized from the same solvent mixture at -20 "C to yield bright-purple crystals of 3 (150 mg, 31 % ;mp 62 OC): lH NMR (acetone-d6) 6 2.90 (s, 4 H of cycle), 3.05 (t, 2 H, 3J = 7.3 Hz, C(O)CH2),3.39 (t, 2 H, 3J = 7.3 Hz, CCH2), 6.52 ppm (t, 1 H, *J = 1.0 Hz, HCEC); IR (KBr) v(C0) 2099 (s), 2046 (s), 2006 (e), 1996 (sh) cm-l;UV-vis (MeOH) 406 (em= = 640), 347 (emar. = 3070), 207 nm (em= = 23 600); MS (DCI/NH3) base peak m / e = 499 (MNH4+). 5- [N-(2-Aminoethyl)carbamoyl]-5H-dibenz[ b,flazepine (5) was prepared essentially according to the literature method (18),except that a 4-fold excess of ethylenediamine was added. Compound 5 was obtained as a beige powder (1.31 g, 47% yield): 'H NMR (CD30D) d 2.61 (t,2 H, 3J = 5.0 Hz, HZNCH~), 3.10 (t, 2 H, 3J = 5.0 Hz, CHaNH), 6.93 (9, 2 H, vinylic), 7.39 ppm (s, 8 H, aromatic); IR (KBr) v(NH) 3360, v(C0) (ketone) 1660, v[amide(II)]1510, v(CN) 1319,798cml-l; UV-vis (EtOH) 282 (em= = 8910), 238 (emm = 123601, 216 nm (em= = 24 540); MS (DCI/NH3) base peak m / e = 280 (MH+). [5-[ N-[2-(4-Pentynamido)ethyl]carbamoyl]-5Hdibenz[b,fJazepine]hexacarbonyldicobalt(6). Acetonitrile solutions of 3 (48 mg, 0.1 mmol, 1.0 mL) and 5 (30 mg, 0.1 mmol, 4 mL) were stirred together at 4 "C for 3.5 h. The reaction mixture was chromatographed on silica preparative TLC plates with ether/ethyl acetate (2/1) as eluent. The product (Rf= 0.67) was scraped off the TLC plate and was recrystallized from CHzClz/hexane (7/1) as a brick-red powder (22 mg, 50% yield): lH NMR (CD3COCD3) 6 1.28 and 5.46 (s, large, NH), 2.50 (t, 2 H, 3J =
Letters
7.9 Hz, CH2),3.20 (m, 6 H, CH2), 6.98 ( s , 2 H, vinylic), 7.39 ppm (m, 8 H, aromatic); IR (KBr disk) u(NH) 3310, u(C0) 2092 (s), 2050 (vs),2017 (vs),u(C0) (ketone) 1650,u[amide(II)] 1510,u[amide(VI)]518 cm-l; UV-vis (EtOH) 347 (cmax = 2130), 225 (e, = 28 290). ACKNOWLEDGMENT
This research was performed in part under the auspices of a France-Quebec Exchange (Biotechnology). Operating grants in partial support of the research from ANVAR, CNRS, and RBgion Bourgogne are also acknowledged. LITERATURE CITED (1) Schall, R. F., and Tenoso, H. J. (1981) Alternatives to Radioimmunoassay Labels and Methods. Clin. Chem. 27,11571164. (2) Bolton, A. E., and Hunter, W. M. (1973) The Labelling of Proteins to High Specific Activities by Conjugation to a 1251containing Acylating Agent. Biochem. J. 133,529-539. (3) Langone, J. J. (1980) Radioiodination by the Use of the Bolton-Hunter and Related Reagents. Methods Enzymol. 70, 221-243. (4) Gosling, J. P. (1990) A Decade of Development in Immunoassay Methodology. Clin. Chem. 36, 1408-1427. (5) Cais, M., Dani, S., Eden, Y., Gandolfi, O., Horn, M., Isaacs, E. E., Josephy, Y., Saar, Y., Slovin, E., and Snarsky, L. (1977) Metalloimmunoassay. Nature 270, 534-535. (6) (a) Cais, M., Slovin, E., and Snarsky, L. (1978) Iron-Metallohaptens from Estrogen Steroids. J. Organomet. Chem. 160, 223-230. (b) Cais, M. (1983) Metalloimmunoassay: Principles and Practice. Methods Enzymol. 92,445-458. (c) Cheret, P. (1987) Ph.D. Thesis, Universitk de Bourgogne. (7) Kricka, L. J. (1985) Ligand-Binder Assays: Labels and Analytical Strategies, Marcel-Dekker, New York and Basel. (8) Butler, I. S.,Vessiires, A., and Jaouen, G. (1989) Application of Organotransition Metal Carbonyl Complexes as Infrared Markers for Hormonal Steroids in Biological Processes. Comments Inorg. Chem. 8, 269-286. (9) VessiBres, A., Top, S., Ismail, A. A., Butler, I. S., Louer, M., and Jaouen, G. (1988) Organometallic Estrogens: Synthesis,
Bioconjugte Chem., Vol. 2, No. 1, 1991 15
Interaction with Lamb Uterine Estrogen Receptor, and Detection by Infrared Spectroscopy. Biochemistry 27,66596666. (10) Vessiires, A., Jaouen, G., Gruselle, M., Rossignol, J. L., Savignac, M., Top, S., and Greenfield, S. (1988) Synthesis and Receptor Binding of Polynuclear Organometallic Estradiol Derivatives. J. Steroid Biochem. 30, 301-305. (11) Gruselle, M.,Deprez, P., Vessiires, A., Greenfield, S., Jaouen, G., Larue, J.-P., and Thouvenot, D. (1989) Cobalt and Molybdenum Carbonyl Clusters in Immunology. Synthesis and Binding Properties of Mycotoxin Derivatives of Zearalenone. J. Organomet. Chem. 359, C53-C56. (12) VessiBres, A., Jaouen, G., Salmain, M., and Butler, I. S. (1990) An Ultra-low Volume, Gold Light-pipe Cell for the IR Analysis of Dilute Organic Solutions. Appl. Spectrosc. 44, 1091-1094. (13) See, for example: Zalutaky, M. R., Noska, M. A., Colapinto, E. V., Garg, P. K., and Bigner, D. D. (1989) Enhanced Tumor Localization and in Vivo Stability of a Monoclonal Antibody Radioiodinated Using N-Succinimidyl3-(Tri-n-stannyl) benzoate. Cancer Res. 49, 5543-5549. (14) Gruselle, M., Greenfield, S., and Jaouen, G. (1987) The Selective Introduction of Organometallic Markers into Oestrogens. C-16 prop-2-ynylation of Oestrone. J. Chem. SOC. Chem. Commun. 1353-1355. (15) Salmain, M. (1990) Ph.D. Thesis, Universitb de Paris VI. (16) Kutt, K. Clinical Pharmocology of Carbamazepine. In Pippenger, C. E., Penry, J. K., and Kutt, K. (Eds.) (1978) Antiepileptic Drugs: Quantitative Analysis and Interpretation, pp 297-305, Raven Press, New York. (17) (a) Sasaki, N. A., Potier, P., Savignac, M., and Jaouen, G. (1988) Organometallic Derivatives of Peptides: Application to Peptide Receptor Analysis Tetrahedron Lett. 29, 57595762. (b) Le Borgne, F., and Beaucourt, J. P. (1988) Cobalt Carbonyl Complexes of Some Peptides as Infrared Markers. Tetrahedron Lett. 29, 5649-5652. (18) Sidki, A. M., Landon, J., and Rowell, F. (1984) Influence of the Hapten-Fluorophore Bridge on Binding Parameters in a Fluoroimmunoassay for Carbamazepine. Clin. Chem. 30, 1348-1352.
