Bioconjugate Chem. 1997, 8, 489−494
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Cyclopentadienyl Iron Dicarbonyl (η1-N-Phthalimidato) Complexes Containing an Isothiocyanate Function: Synthesis and Application to Protein Side-Chain Selective Labeling Anna Kazimierczak,† Janusz Zakrzewski,*,† Miche`le Salmain,‡ and Ge´rard Jaouen*,‡ Department of Organic Chemistry, University of Lodz, Narutowicza 68, 90-136 Lodz, Poland, and Ecole Nationale Supe´rieure de Chimie, Laboratoire de Chimie Organome´tallique, URA CNRS 403, 11 rue Pierre et Marie Curie, F-75231 Paris Cedex 05, France. Received February 25, 1997X
The two first transition metal carbonyl isothiocyanates were prepared in high yield within two steps from photolysis of CpFe(CO)2I and 3- or 4-aminophthalimide in the presence of diisopropylamine followed by reaction with thiophosgene/triethylamine. Their reaction with a model amino acid, i.e. β-alanine, was performed and led to the expected thioureas. When reacted with bovine serum albumin in aqueous medium, conjugates bearing 6-10 iron-carbonyl fragments were obtained and characterized by Fourier transform infrared spectroscopy, thus demonstrating the usefulness of these reagents for the selective and covalent labeling of proteins.
INTRODUCTION
Organotransition metal complexes having substituents able to react selectively at functional groups present in biomolecules currently attract a great deal of attention as labeling reagents (Furuya et al., 1988; Carver et al., 1993; Wang et al., 1993; Anson et al., 1994; Dalla Riva Toma et al., 1994; Kra¨mer, 1996). During the past few years, we have focused our interest on the use of transition metal carbonyl complexes as reporter groups for the labeling of biologically active molecules (Jaouen et al., 1993). These complexes can be detected in a sensitive and univoque manner by mid-IR1 spectroscopy thanks to their νCO vibration modes that appear in the 1800-2150 cm-1 spectral region. Owing to the fact that quantities of complex in the order of magnitude of the picomole (10-12 mol) can be routinely detected and quantified by current FT spectrometers (Salmain et al., 1991), a new competitive immunological method, coined carbonyl metallo immunoassay (CMIA) has been elaborated for hapten assay (Salmain et al., 1992; Philomin et al., 1994; Varenne et al., 1995). The labeling of high molecular weight antigens or antibodies, which is a prerequisite to the extension of CMIA to proteic antigens, poses new problems as compared to the labeling of haptens, owing to the polyfunctional character of proteins and their tendency to denaturation. These problems have been solved by preparing a series of metal carbonyl complexes containing an N-hydroxysuccinimide (NHS) ester function, which reacts with amino groups at the N-terminal position and the lysine residues of proteins (Varenne et al., 1993; Salmain et al., 1994; Gorfti et al., 1996). Other studies dealt with organometallic reagents containing a pyrylium group (Salmain et al., 1994; Malisza et al., 1995), reactive * Author to whom correspondence should be addressed. † University of Lodz. ‡ Ecole Nationale Supe ´ rieure de Chimie. X Abstract published in Advance ACS Abstracts, June 15, 1997. 1 Abbreviations: BSA, bovine serum albumin; Cp, η5-cyclopentadienyl; CR, coupling ratio; DMF, dimethylformamide; ESIMS, electrospray ionization spectroscopy; FT-IR, Fourier transform infrared; mid-IR, mid-infrared; NHS, N-hydroxysuccinimide; TLC, thin-layer chromatography; TMS, tetramethylsilane; TNBS, trinitrobenzenesulfonic acid.
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toward amino groups. Recently, an organometallic complex containing an N-maleimide function, CpFe(CO)2(η1N-maleimidato), has been synthesized (Rudolf and Zakrzewski, 1994). Although it is believed that this function reacts selectively with sulfhydryl groups (cysteines), it has been demonstrated that this complex can also react with amino groups or imidazole rings (histidines) (Rudolf and Zakrzewski, 1996). However, the coupling selectivity can be tuned by changing the reaction conditions (pH) (unpublished work). Isothiocyanates have found numerous applications in peptide and protein chemistry to date (Wong, 1991). The best known examples include the Edman method of protein amino acid sequence determination by means of phenyl isothiocyanate (Cohen and Strydom, 1988) and the use of fluoresceine isothiocyanate (FITC) for fluorescent labeling of proteins [see, for example, Oshita and Katunuma (1993) and references cited therein]. Ferrocene-methyl isothiocyanate was also used to prepare immunogens so as to eventually produce anti-ferrocene polyclonal antibodies (Gill and Mann, 1966). In this paper, we report the synthesis of two iron carbonyl complexes containing an isothiocyanate function. Their reactivity toward amino groups was checked using β-alanine and a model protein, bovine serum albumin (BSA), which we had previously chosen for NHS esters and pyrylium salt reactivity studies. Their preparation took advantage of the photochemical approach to the synthesis of CpFe(CO)2(η1-N-imidato) complexes (Cp ) η5-cyclopentadienyl) (Zakrzewski, 1989). The idea was to synthesize complexes containing a phthalimidato ligand substituted by an amino function at the aromatic ring and then convert this amino group into an isothiocyanate function. EXPERIMENTAL PROCEDURES
Materials. All chemical reactions were carried out under argon. Column chromatographies were performed with silica gel 60 (230-400 mesh ASTM, Merck). Benzene was distilled over sodium/benzophenone, and dichloromethane and chloroform were distilled over calcium hydride. Diisopropylamine and triethylamine (Aldrich) were stored over KOH. Other analytical grade solvents were used without purification. CpFe(CO)2I was synthesized according to a published procedure (King, 1965). © 1997 American Chemical Society
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Aminophthalimides (Kodak), thiophosgene, and β-alanine (Aldrich) were used as received. Photolyses were carried out by illuminating the solutions with visible light produced by four 150 W domestic tungsten lamps and simultaneous external cooling with a mixture of ice and water. Borate and phosphate buffers were prepared from demineralized water. Protein conjugations were performed with undistilled solvents at room temperature. 1H NMR spectra were recorded on a Gemini 200BB apparatus (200 MHz for 1H, Varian) and were referenced to the internal standard TMS (0.00 ppm). Mass spectra were recorded in the electronic impact mode at 15 eV with a 2091 GCMS spectrometer (LKB). Qualitative IR characterizations were performed on a 75IR spectrometer (Specord). Quantitative IR analyses were performed by depositing 10 µL samples on 9 mm diameter nitrocellulose membranes (Alltech), and spectra were recorded on a MB-100 FT spectrometer (Bomem) equipped with a liquid nitrogen-cooled InSb detector as described (Salmain et al., 1993). UV-vis spectra were recorded on a UV-mc2 spectrometer (Safas). Methods. CpFe(CO)2[η1-(N - 1)(3-aminophthalimidato] (1a). A mixture of CpFe(CO)2I (1.2g, 3.9 mmol), 3-aminophthalimide (0.52 g, 3.2 mmol), and diisopropylamine (4 mL) in benzene (50 mL) was photolyzed for 12 h. The black color turned yellow during the photolysis. The reaction course was followed by TLC using chloroform/ methanol 95:5 as eluent. The solid was filtered off, and the filtrate was concentrated to ca. one-fourth of its original volume. After addition of pentane (50 mL) and cooling to -5 °C, the solid formed was filtered off and dried, and 1a was purified by column chromatography, eluted by chloroform, and crystallized from dichloromethane/pentane: yield 0.99 g (91%); 1H NMR (CDCl3) δ 7.20 (t, J ) 7 Hz, partially obscured by the solvent signal, 1H, H-5), 6.95 (d, J ) 7.0 Hz, 1H, H-4), 6.67 (d, J ) 7.0 Hz, 1H, H-6), 5.11 (s, 5H, Cp) , 5.06 (bs, 2H, NH2); IR (CHCl3) ν (in cm-1) 3360, 3270 (NH2), 2055, 1990 (FeCO), 1640 (imide CO); MS, m/e (assignment) (% intensity) 338 (M) (7), 310 (M - CO) (4), 282 (M - 2CO) (9), 186 (ferrocene?) (100). Anal. Calcd for C15H10N2O4Fe: C, 53.25; H, 2.96; N, 8.28. Found: C, 52.98; H, 2.82; N, 8.43. CpFe(CO)2(η1-(N - 1)(4-aminophthalimidato) (1b). The procedure was similar to the synthesis of 1a. After the photolysis, pentane (50 mL) was added to the photolyte, and after cooling to -5 °C, the solid was filtered off, washed with pentane, triturated with 10% aqueous Na2CO3 (5 mL) and then with water, and dried. The crude product was purified by column chromatography, and 1b was eluted by chloroform and crystallized from dichloromethane/ether: yield 0.71 g (71%); 1H NMR (CDCl3) δ 7.38 (d, J ) 6.9 Hz, 1H, H-6), 6.87 (d, J ) 1.2 Hz , 1H, H-3) and 6.64 (d,d, J ) 6.9 Hz, J ) 1.2 Hz, 1H, H-5), 5.09 (s, 5H, Cp), 4.16 (bs, 2H, NH2); IR (CHCl3) ν (in cm-1) 3410, 3360 (NH2), 2055, 2000 (Fe-CO), 1665, 1635 (imide CO); MS, m/e (assignment) (% intensity) 338 (M) (1), 310 (M - CO) (4), 282 (M - 2CO) (9), 186 (ferrocene?) (100). Anal. Calcd for C15H10N2O4Fe: C, 53.25; H, 2.96; N, 8.28. Found: C, 53.29; H, 2.98; N, 8.17. CpFe(CO)2(η1-(N - 1)(3-isothiocyanatophthalimidato) (2a). A magnetically stirred solution of 1a (700 mg, 2.07 mmol) and triethylamine (0.4 mL) in dichloromethane (33 mL) was treated with thiophosgene (0.207 mL, 2.71 mmol) at room temperature. After 1.5 h, the solvent was evaporated and the residue chromatographed on a short column. 2a was eluted by chloroform and crystallized from dichloromethane/pentane: yield 630 mg (80%); dec 130-140 °C; 1H NMR (CDCl3) δ 7.4-7.55 (m, 3H, aromatic ring protons), 5.12 (s, 5H, Cp); IR (CHCl3) ν (in cm-1) 2100 (br) (NCS), 2050, 2005 (Fe-CO), 1655 (imide
Kazimierczak et al.
CO); UV-vis (ethanol) λmax (in nm) () 275 (15070), 320 (4170); MS, m/e (assignment) (% intensity) 380 (M) (4), 352 (M - CO) (5), 324 (M - 2CO) (100). Anal. Calcd for C16H8N2O4SFe: C, 50.53; H, 2.11; N, 7.37; S, 8.42. Found: C, 50.22; H, 2.03; N, 7.36; S, 8.47. CpFe(CO)2(η1-(N - 1)(4-isothiocyanatophthalimidato) (2b). A magnetically stirred solution of 1b (850 mg, 2.51 mmol) and triethylamine (0.5 mL) in dichloromethane (40 mL) was treated with thiophosgene (0.25 mL, 3.28 mmol) at room temperature. After 1.5 h, the solvent was evaporated and the residue chromatographed on a short column. 2b was eluted by chloroform and crystallized from dichloromethane/ether: yield 880 mg (92%); dec 150-160 °C; 1H NMR (CDCl3) δ 7.42 (d, J ) 1.6 Hz, 1H, H-3), 7.57 (d, J ) 6.5 Hz, 1H, H-6), 7.29 (dd, J ) 6.5 Hz, J ) 1.6 Hz, 1H, H-5), 5.12 (s, 5H, Cp); IR (CHCl3) ν (in cm-1) 2120 (br) (NCS), 2055, 2005 (Fe-CO), 1660 (imide CO); UV-vis (ethanol) λmax (in nm) () 264 (18160), 290 (18960); MS, m/e (assignment) (% intensity) 380 (M) (5), 352 (M - CO) (4), 324 (M - 2CO) (100). Anal. Calcd for C16H8N2O4SFe: C, 50.53; H, 2.11; N, 7.37; S, 8.42. Found: C, 50.50; H, 2.10; N, 7.29; S, 8.11. Reaction of 2a with β-Alanine. 2a (60 mg, 0.16 mmol) and β-alanine (60 mg, 0.67 mmol) were dissolved in pyridine (2 mL). To this solutionn were added 10% aqueous Na2CO3 (5 mL) and some water to obtain a homogeneous solution and pH 9-10. This solution was allowed to stand at room temperature in the dark for 24 h. The solvents were evaporated to dryness, and the residue was dissolved in water (5 mL) and extracted three times with dichloromethane. Then the water layer was acidified with HCl to pH 3-4. The precipitated yellow solid 3a was filtered off, washed with water, and dried under vacuum: yield 64.5 mg (86% calculated from 2a); dec 148 °C; 1H NMR (CDCl3) δ 10.14 (bs, 1H, COOH), 9.32 (d, J ) 8.3 Hz, 1H, NH), 8.61 (bs, 1H, NH), 7.457.30 (m, 3H, benzene ring H’s), 5.10 (s, 5H, Cp), 4.03 (m, 2H, CH2), and 3.00 (m, 2H, CH2); IR (CDCl3) ν (in cm-1) 2055, 1990 (Fe-CO), 1720 (COOH), 1640 (imide CO); UV-vis (ethanol) λmax (in nm) () 255 (23020), 275 (18900), 355 (5250). Anal. Calcd for C19H15N3O6SFe: C, 48.63; H, 3.23; N, 8.95; S, 6.83. Found: C, 48.34; H, 3.45; N, 9.15; S, 7.08. Reaction of 2b with β-Alanine. The same procedure as in the preceeding reaction was applied. However, after acidification, the product 3b did not crystallize. Instead, it was extracted with dichloromethane: yield 33.7 mg (45%); dec 165-170 °C; 1H NMR (CDCl3) δ 10.31 (bs, 1H, COOH), 8.6 (bs, 1H, NH), 7.7-7.3 (m, 3H benzene ring H’s), 7.2 (bs, 1H, NH), 5.11 (s,5H, Cp), 3.95 (m, 2H, CH2), and 2.78 (m, 2H, CH2); IR (CDCl3) ν (in cm-1) 2050, 1990 (Fe-CO), 1720 (COOH), 1635 (imide CO); UV-vis (methanol) λmax (in nm) () 275 (15070), 320 (4170). Anal. Calcd for C19H15N3O6SFe: C, 48.63; H, 3.23; N, 8.95; S, 6.83. Found: C, 48.60; H, 3.50; N, 8.82; S, 6.88. Reaction of 2a,b with BSA. To 2.7 mL of a 50 µM BSA solution in borate buffer pH 9.5 was added 0.3 mL of a 27 mM 2a,b solution in DMF (i.e. 60 molar equiv). Partial precipitation of the organometallic reagent was noticed as soon as both solutions were mixed. The suspension was stirred for one night at room temperature. After centrifugation at 4000 rpm for 15 min, the supernatant was chromatographed on a prepacked gel filtration column (Econopac 10DG, Bio-Rad). Ten 1-mL fractions were collected and spectrophotometrically analyzed. The first four fractions were pooled. The concentration of protein [P] was measured according to the Coomassie blue method (Bradford, 1978) and by UV spectrophotometry at 280 nm (280 ) 35700), after subtraction of the contribution of the label to the total
Iron−Carbonyl Isothiocyanates for Protein Labeling
Figure 1. Synthetic scheme of CpFe(CO)2-(η1-N-phthalimidato) complexes. B ) diisopropylamine.
Figure 2. Synthetic route to compounds 1a,b, 2a,b, and 3a,b: (a) 3-isomer, (b) 4-isomer; (i) diisopropylamine/hν; (ii) CSCl2/ triethylamine; (iii) β-alanine/pH 9-10.
absorbance. The concentration of CpFe(CO)2 groups [M] was measured by UV spectroscopy at 350 nm [350 (3a) ) 5250; 350 (3b) ) 3780] and by IR spectroscopy on nitrocellulose membranes (k ) 18.2) as described (Salmain et al., 1993). The concentration of free protein amino groups was measured according to the TNBS method (Snyder and Sobocinski, 1975). RESULTS
We had earlier reported that irradiation with visible light of CpFe(CO)2I (Cp ) η5-C5H5) in the presence of phthalimide and diisopropylamine in benzene led to the efficient substitution of iodide by the phthalimide anion (Figure 1) (Zakrzewski, 1989; Bukowska-Strzyzewska et al., 1994). Other “N-H acidic” compounds such as pyrrole, indole (Zakrzewski, 1987; Zakrzewski and Gianotti, 1990), and uracils (Zakrzewski et al., 1995) react similarly. In this paper, we used this photochemical reaction for synthesis of 3- and 4-amino derivatives of CpFe(CO)2(η1N-phthalimidato). We found that CpFe(CO)2I reacted with 3- or 4-aminophthalimide and diisopropylamine in benzene, under irradiation with visible light, and yielded the yellow, air-stable complexes 1a,b (Figure 2). However, due to rather poor solubility of the starting aminophthalimides in the reaction medium (and their anticipated lower N-H acidity in comparison to the parent, unsubstituted phthalimide), these reactions required much longer photolysis times than the reaction with phthalimide (12 h vs 2 h). Nevertheless, the yields of isolated compounds 1a,b were relatively high (91 and 71% for 1a and 1b, respectively), and no other reaction
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products were detected in the photolytes by TLC. The presence of the amino substituents on the aromatic ring of phthalimide did not impair the photochemical reaction with the CpFe(CO)2I/diisopropylamine/hν system, which invariably involves coordination of the CpFe(CO)2 moiety to the deprotonated imide nitrogen. The IR spectra of 1a,b displayed two bands in the region of the stretching N-H vibrations, which confirmed the presence of the primary amino function (NH2 symmetric and antisymmetric vibrations). The imide carbonyl stretching frequencies were shifted toward lower wavenumbers (ca. 70-80 cm-1) in comparison to the parent N-H imides. The same shift was observed on going from phthalimide to CpFe(CO)2(η1-N-phthalimidato), suggesting the same coordination mode of the deprotonated phthalimide ligand to the CpFe(CO)2 moiety. Aromatic amines are versatile starting materials for the synthesis of aromatic isothiocyanates (Rifai and Wong, 1989). We found that 1a,b react with thiophosgene in the presence of triethylamine at room temperature to afford the corresponding isothiocyanates 2a,b (Figure 2). These complexes were isolated in 80% (2a) and 92% (2b) yields as yellow, air-stable crystalline solids, readily obtained in the pure form by chromatography and crystallization from CH2Cl2/pentane or CH2Cl2/diethyl ether mixtures. Their structure was confirmed by IR spectroscopy. No absorption in the region of the N-H stretching vibrations was noticed, whereas a relatively strong broad band at ca. 2100 cm-1 (NCS stretching vibration mode) was detected for both compounds. The reactivity of 2a,b toward an amine was checked using β-alanine. Coupling reactions were performed at room temperature for 24 h, in the pyridine/H2O/K2CO3 system. We obtained the expected thioureas 3a,b in 86 and 45% yields, respectively (Figure 2). Interestingly, their formation was ascertained by the disappearing of the νNCS band at ca. 2100 cm-1. Having demonstrated the reactivity of both isothiocyanates with a model amino acid, we tried to label a model protein, i.e., bovine serum albumin (BSA), which is a well-characterized, low-cost protein. The primary sequence of BSA contains 60 amino groups, i.e., 60 potential sites of conjugation for isothiocyanates. Conjugation of BSA was carried out in the presence of 60 molar equiv of isothiocyanates 2a,b, in pH 9.5 borate buffer containing 10% of DMF so as to improve solubility of the iron-carbonyl reagents in the reaction medium. After one night at room temperature, the excess reagent was separated from the protein by centrifugation and gel filtration chromatography. Figure 3 shows the chromatograms obtained by plotting the absorbance of each fraction at 350 nm vs the elution volume. Each chromatogram presents two peaks at ca. 2-3 and 6-7 mL. The first eluted peak was readily assigned to the labeled protein, whereas the second was assigned to the unbound label. The whole-range IR spectrum of one of the protein conjugates was recorded and compared to the IR spectrum of native BSA (Figure 4). While the main features are conserved for the conjugate, two new bands are clearly detected in the νCO region (at 2048 and 2002 cm-1) and assigned to the stretching vibration modes of the metal-carbonyl graft. As for the β-alanine conjugate, effective conjugation can be ascertained by the absence of the νNCS band at 2100 cm-1. To characterize both labeled protein samples, we measured the number of coupled CpFe(CO)2 entities per protein molecule, CR, with CR ) [M]/[P], where [M] is the concentration of CpFe(CO)2 entities and [P] the
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Figure 3. Gel filtration chromatography of BSA-[CpFe(CO)2]n conjugates: (9) reagent ) 2a; (2) reagent ) 2b.
Figure 5. Comparison of the UV-vis spectra of BSA-[CpFe(CO)2]n conjugates (dotted line) with thioureas 3a and 3b (solid line): (A) 3-isomer; (B) 4-isomer.
Figure 4. (A) IR spectrum of BSA; 13-mm KBr pellet of lyophilized protein. (B) IR spectrum of BSA-3b conjugate; 13mm KBr pellet of lyophilized protein.
concentration of protein. Numerous methods have been described to date. The oldest one takes advantage of the UV-vis absorption properties of the introduced chromophore (Erlanger, 1980). More recently, the TNBS assay was used for titration of unreacted protein amino functions (Sashidhar et al., 1994). The most recent and widely applicable method exploits the change of molecular mass resulting from the conjugation of several haptens, which can be accurately detected by electrospray ionization mass spectroscopy (ESI-MS) (Adamczyk et al., 1996). For simplicity reasons, we selected three classical methods further described. The first one is based on the indirect titration of free amino groups according to the TNBS assay. The two others take advantage of the
spectroscopic properties of the metal-carbonyl graft. Indeed, compounds 3a,b absorb above 300 nm (Figure 5). It was then expected that protein conjugates would also absorb in the same range. This allowed us to quantify the concentration of metal carbonyl haptens [M] by direct absorbance measurement at 350 nm. Protein concentration [P] was then deduced by differential spectroscopy at 280 nm. On the other hand, it was also possible to measure [M] by IR spectroscopy according to a previously described method (Salmain et al., 1993). Concentration [P] was this time measured according to a classical colorimetric method (Bradford, 1978). Results are gathered in Table 1. It appears that the coupling ratios calculated from IR spectroscopic measurements are very close to the number of modified amino groups assayed by TNBS titration. Both are in the range of 6-10. This indicates in turn that [M] and [P] are overestimated by UV spectrophotometry. DISCUSSION
Isothiocyanates are useful reagents for the selective modification of protein amino groups. When reacted at basic pH, thiourea bonds are formed, which are stable under normal physiological conditions. Examples of isothiocyanates among organometallic compounds are
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Iron−Carbonyl Isothiocyanates for Protein Labeling Table 1. Analysis of Conjugated BSA Samples labeling reagent 2a Differential UV Spectroscopy [M] (µM) 377a [P] (µM) 31c CR 12.2 IR Spectroscopy [M]e (µM) 242 [P]f (µM) 25 CR 9.7 TNBS Assay no. of free amino groups per protein 49.3 CRg 10.7
2b 542b 43d 12.6 176 27 6.5 52.1 7.9
a [M] ) A b c 350/5250. [M] ) A350/3780. [P] ) (A280 - [M] × 16900)/35700. d [P] ) (A280 - [M] × 16350)/35700. e [M] ) A2052/ 18.2. f Measured according to the Bradford assay. g CR ) 60 (number of free amino groups).
rare and nonexistent in the transition metal-carbonyl series. Two examples of cyclopentadienyl iron dicarbonyl isothiocyanates have been presented herein. Their synthesis took advantage of the easy photolytically induced substitution of iodide and addition of 3- or 4-aminophthalimide anion, then transformation of the amine into an isothiocyanate. Both isomers reacted with an amino acid to form the expected isothioureas. Similarly, they reacted with a model protein to give conjugates bearing several iron-carbonyl moieties per protein molecule. Quantitation of the number of conjugated entities was performed according to several spectroscopic methods. IR spectroscopy and titration of the free amino groups by TNBS assay gave corroborating results. However, UV spectrophotometry gave overestimated measurements even if 3a,b were chosen as standards for [M] determination as they were supposed to best mimic the organometallic label structure bound to the protein. Indeed, a careful comparison between the UV spectra of compounds 3a,b and their protein counterparts in the 250-400 nm region is necessary at this point (Figure 5). In the 3-substituted derivative series, both 3a and the protein conjugate present a maximum at 355 nm but the protein conjugate does not show any maximum at 255 nm. In the 4-substituted derivative series, the protein conjugate presents a shoulder at 320 nm, while 3b shows a shoulder at 290 nm. We then conclude that iron carbonyl entities and protein concentrations are inaccurately measured by UV spectrometry because 3a,b are not appropriate standards. Such a discrepancy had been previously reported in the literature for studies dealing with the characterization of hapten-protein conjugates (i.e. immunogens) (Sashidhar et al., 1994). Very recently, ESI-MS appeared as a reference method, obviating the need of choosing standards, to perform accurate measurements of the degree of conjugation of hapten-protein conjugates (Adamczyk et al., 1996). Thanks to the particular nature of the reporter group, IR spectroscopy of νCO bands also appears as an accurate analytical tool for quantitation of conjugated metal-carbonyl species as νCO band intensities are relatively insensitive to chemical structural changes (Salmain et al., 1991). Under identical reaction conditions, metal-carbonyl NHS esters give slightly higher coupling ratios, ranging from 13 to 30 (Varenne et al., 1992; Salmain et al., 1993; Gorfti et al., 1996). The slightly lower reactivity of isothiocyanates could be explained by their much lower solubility in aqueous medium. Isothiocyanates would eventually require a higher percentage of organic cosolvent (here 10% DMF) to be fully reactive, but one should keep in mind that this could lead to protein denaturation.
Finally, it bears noticing that while assay sensitivities using such reporter groups are theoretically proportional to the number of conjugated species, too high incorporation could be detrimental to the conservation of a high affinity of the conjugates toward their biological target (for example, in antibody-antigen reactions). Thus, by giving less conjugated proteins, metal carbonyl isothiocyanates could be an interesting alternative to NHS esters. ACKNOWLEDGMENT
This work was financially supported by the Polish State Committee for Scientific Research (KBN), Grant 9T09A 008 08, and a Centre National de la Recherche Scientifique (CNRS) research grant. LITERATURE CITED Adamczyk, M., Gebler, J. C., and Mattingly, P. G. (1996) Characterization of protein-hapten conjugates. 2. Electrospray mass spectrometry of bovine serum albumin-hapten conjugates. Bioconjugate Chem. 7, 475-481. Anson, C. E., Creaser, C. S., Egyed, O., Fey, M. A., and Stephenson, G. R. (1994) FTIR detection of an enzyme-bound organometallic carbonyl probe in the presence of the unbound probe molecule. J. Chem. Soc., Chem. Commun., 39-40. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Bukowska-Strzyzewska, M., Tosik, A., Wodka, D., and Zakrzewski, J. (1994) The X-ray crystal structure, 13C and 15N NMR spectra of (η5-C5H5)Fe(CO)2(η1-N-phthalimidato). Polyhedron 13, 1689-1694. Carver, J. A., Fates, B., and Kane-Maguire, L. A. P. (1993) Selective labelling of peptides using (dienyl)iron tricarbonyl cations. J. Chem. Soc., Chem. Commun., 928-929. Cohen, S. A., and Strydom, D. J. (1988) Amino acid analysis utilizing phenyisothiocyanate derivatives. Anal. Biochem. 174, 1-16. Dalla Riva Toma, J. M., and Bergstrom, D. E. (1994) Transition metal labeling of oligodeoxyribonucleotides: synthesis and characterization of (pentacarbonyl)tungsten(0) nucleoside phosphites. J. Org. Chem. 59, 2418-2422. Erlanger, B. F. (1980) The preparation of antigenic haptencarrier conjugates: a survey. Methods Enzymol. 70, 85-97. Furuya, F. R., Miller, L. L., Hainfeld, J. F., Christopfel, W. C., and Kenny, P. W. (1988) Use of Ir4(CO)11 to measure the lengths of organic molecules with a scanning transmission electron microscope. J. Am. Chem. Soc. 110, 641-643. Gill, T. J., III, and Mann, L. T. (1966) Studies on synthetic poylypeptide antigens XV. The immunochemical properties of ferrocenyl-poly Glu58Lys36Tyr6 (No2) conjugates. J. Immunol. 96, 906-912. Gorfti, A., Salmain, M., Jaouen, G., McGlinchey, M. J., Bennouna, A., and Mousser, A. (1996) Covalent and selective labeling of proteins with heavy metals. Synthesis, X-ray structure and reactivity studies of N-succinimidyl and Nsulfosuccinimidyl ester organotungsten complexes. Organometallics 15, 142-151. Jaouen, G., Vessie`res A., and Butler, I. S. (1993) Bioorganometallic chemistry: a future direction for transition metal organometallic chemistry? Acc. Chem. Res. 26, 361-369. King, R. B. (1965) Cyclopentadienyl iron dicarbonyl iodide. In Organometallic Syntheses, Academic Press, New York. Kra¨mer, R. (1996) Application of π-arene-ruthenium complexes in peptide labeling and peptide synthesis. Angew. Chem., Int. Ed. Engl. 35, 1197-1199. Malisza, K. L., Top, S., Vaissermann, J., Caro, B., Se´ne´chalTocquer, M-C., Se´ne´chal, D., Saillard, J-Y., Triki, S., Kahlal, S., Britten, J. F., McGlinchey, M. J., and Jaouen, G. (1995) Organometallics as potential protein labels: Pyrylium and pyridinium salts bearing (C6H5)Cr(CO)3, (C5H4)Mn(CO)3 and ferrocenyl substituents. Organometallics 14, 5273-5280. Oshita, T., and Katunuma, N. (1993) Analysis of lysosomal degradation of fluorescein isothiocyanate-labelled proteins by
494 Bioconjugate Chem., Vol. 8, No. 4, 1997 Toyapearl HW-40 affinity chromatography. J. Chromatogr. 633, 28-286. Philomin, V., Vessie`res, A., and Jaouen, G. (1994) New aplications of carbonylmetalloimmunoassay (CMIA): a non-radioisotopic approach to cortisol assay. J. Immunol. Methods 171, 201-210. Rifai, A., and Wong, S. S. (1986) Preparation of phosphorylcholine-conjugated antigens. J. Immunol. Methods 94, 25-30. Rudolf, B., and Zakrzewski, J. (1994) (η5-cyclopentadienyl)Fe(CO)2 - complex of maleimide anion: An organometallic carbonyl probe for biomolecules containing SH groups. Tetrahedron Lett. 35, 9611-9612. Rudolf, B., and Zakrzewski, J. (1996) Addition of imidazoles and aminoacids to the ethylenic bond in (η5-C5H5)Fe(CO)2(η1-Nimidato). J. Organomet. Chem. 522, 313-315. Salmain, M., Vessie`res, A., Jaouen, G., and Butler, I. S. (1991) Fourier transform infrared spectroscopic technique for the quantitative analysis of transition metal carbonyl-labeled bioligands. Anal. Chem. 63, 2323-2329. Salmain, M., Vessie`res, A., Brossier, P., Butler, I. S., and Jaouen, G. (1992) Carbonylmetalloimmunoassay (CMIA), a new type of non-radioisotopic immunoassay. I. Principles and application to phenobarbital assay. J. Immunol. Methods 148, 65-75. Salmain, M., Gunn, M., Gorfti, A., Top, S., and Jaouen, G. (1993) Labeling of proteins by organometallic complexes of rhenium (I). Synthesis and biological activity of the conjugates. Bioconjugate Chem. 4, 425-433. Salmain, M., Malisza, K. L., Top, S., Jaouen, G., Se´ne´chalTocquer, M-C., Se´ne´chal, D., and Caro, B. (1994) [η5-cyclopentadienyl] metal tricarbonyl pyrylium salts: novel reagents for the specific conjugation of proteins with transition organometallic labels. Bioconjugate Chem. 5, 655-659. Sashidhar, R. B., Capoor, A. K., and Ramana, D. (1994) Quantitation of -amino group using amino acids as reference standards by trinitrobenzene sulfonic acid. J. Immunol. Methods 167, 121-127.
Kazimierczak et al. Snyder, S. L., and Sobocinski, P. Z. (1975) An improved 2,4,6trinitrobenzene sulfonic acid method for the determination of amines. Anal. Biochem. 64, 284-288. Varenne, A., Salmain, M., Brisson, C., and Jaouen, G. (1992) Transition metal carbonyl labeling of proteins. A novel approach to a solid phase two-site immunoassay using Fourier transform infrared spectroscopy. Bioconjugate Chem. 3, 471-476. Varenne, A., Vessie`res, A., Salmain, M., Brossier, P., and Jaouen, G. (1995) Production of specific antibodies and development of a non-isotopic immunoassay for carbamazepine by the Carbonyl metallo immunoassay (CMIA) method. J. Immunol. Methods 186, 195-204. Wang, Z., Roe, B. A., Nicholas, K. M., and White, R. L. (1993) Metal carbonyl labels for oligonucleotide analysis by Fourier transform infrared spectroscopy. J. Am. Chem. Soc. 115, 4399-4400. Wong, S. S. (1991) Chemistry of Protein Conjugation and CrossLinking, CRC Press, Boca Raton, FL. Zakrzewski, J. (1987) The photochemical reaction of (η5-C5H5)Fe(CO)2-diisopropylamine system with pyrrole and indole. J. Organomet. Chem. 327, C41-C42. Zakrzewski, J. (1989) The photosubstitution of iodide in (η5C5H5)Fe(CO)2I. Synthesis of cyclopentadienyliron dicarbonyland carbonyl-phosphine complexes containing η1-N-imidato (1-) ligands. J. Organomet. Chem. 359, 215-219. Zakrzewski, J., and Giannotti, C. (1990) An improved photochemical synthesis of azaferrocene. J. Organomet. Chem. 388, 175-179. Zakrzewski, J., Tosik, A., and Bukowska-Strzyzewska, M. (1995) Photochemical reactions of (η5-cyclopentadienyl) dicarbonyliron iodide with 1-substituted uracils in the presence of diisopropylamine: Crystal structure of the (η5-C5H5)Fe(CO)2 complex of deprotonated 5-fluoro-1-(tetrahydro-2-furyl)uracil (Ftorafur). J. Organomet. Chem. 495, 83-90.
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