Sulfhydryl-Selective, Covalent Labeling of Biomolecules with

Sep 1, 2005 - Synthesis of metallocarbonyl substituted 1,2,3-triazole complexes via copper(I)-catalyzed azide–alkyne cycloaddition. Bogna Rudolf. Ca...
0 downloads 0 Views 145KB Size
Download PDF
Bioconjugate Chem. 2005, 16, 1218−1224

1218

Sulfhydryl-Selective, Covalent Labeling of Biomolecules with Transition Metallocarbonyl Complexes. Synthesis of (η5-C5H5)M(CO)3(η1-N-Maleimidato) (M ) Mo, W), X-ray Structure, and Reactivity Studies Bogna Rudolf,† Marcin Palusiak,‡ Janusz Zakrzewski,*,† Miche`le Salmain,*,§ and Ge´rard Jaouen§ Department of Organic Chemistry, University of Lodz, 90-136 Lodz, Narutowicza 68, Poland, Department of Crystallography, University of Lodz, 90-236 Lodz, Pomorska 149/153, Poland, and Ecole Nationale Supe´rieure de Chimie de Paris, Laboratoire de Chimie et Biochimie des Complexes Mole´culaires (UMR CNRS 7576), 11 rue Pierre et Marie Curie 75231 Paris Cedex 05, France. Received March 11, 2005; Revised Manuscript Received July 25, 2005

The photochemical reaction of (η5-C5H5)Mo(CO)3I with maleimide in the presence of diisopropylamine yielded complex (η5-C5H5)Mo(CO)3(η1-N-maleimidato) 4 in 52% yield. The single-crystal X-ray structure of this complex was determined and shows unusual interactions between oxygen atoms of the maleimidato ligand and carbon atoms of the cis-CtO ligands. The tungsten analogue of 4, (η5-C5H5)W(CO)3(η1-N-maleimidato) 5, was synthesized in 37% yield by the reaction of (η5-C5H5)W(CO)3I with the thallium(I) salt of maleimide. Complexes 4 and 5 reacted with cysteine ethyl ester and glutathione to afford products of the addition of the sulfhydryl group to the ethylenic bond of the maleimidato ligand. The reaction of 4 and 5 with glutathione proceeded faster than the reaction of the analogous complex (η5-C5H5)Fe(CO)2(η1-N-maleimidato) (3). However, all these complexes react with glutathione more slowly than N-ethylmaleimide. Complexes 4 and 5 were used for labeling of bovine serum albumin (BSA), enriched in thiol groups by reaction with Traut’s reagent. Reaction of thiolated BSA containing 7.4 SH groups with 4 and 5 gave bioconjugates bearing 6.9 and 6.4 metallocarbonyl moieties, respectively. Under the same conditions, reaction with 3 afforded a BSA conjugate containing 7.6 metallocarbonyl moieties. Labeling was presumed to be site-specific, as the number of metallocarbonyl entities matched very well with the initial number of SH groups measured for the thiolated BSA sample. IR spectra of BSA labeled with 4 and 5 show intense ν(CtO) bands (2042 and 1948 cm-1 in the latter case), enabling sensitive detection of the bioconjugates in biological samples. Complexes 4 and 5 (especially the latter) should be of interest as heavy atom phasing reagents for protein X-ray crystallography.

INTRODUCTION

Side-chain selective, covalent labeling of biomolecules such as proteins with transition organometallic complexes endows the resulting bioconjugates with unusual redox properties (1), IR absorption (2) or luminescence features (3) enabling their sensitive detection in biological samples. In the particular case where the transition metal belongs to the second or third row, thus exhibiting high electron density, such a labeling may also be helpful in overcoming “the phase problem”, one of the bottlenecks of X-ray protein crystallography (4). The thiol group of free cysteine residues side-chain is the most reactive protein function because of its high nucleophilicity and as such an interesting target to introduce transition organometallic complexes into proteins. Maleimido compounds are often used to selectively alkylate thiols (5) but are seldom encountered in the * To whom correspondence should be addressed. J.Z.: Telephone: +4842 635 5750. fax:+ 4842 678 e-mail: janzak@ uni.lodz.pl. M.S.: Telephone: +33 1 44 27 67 32. fax: +33 1 43 26 00 61. e-mail: [email protected]. † Department of Organic Chemistry, University of Lodz. ‡ Department of Crystallography, University of Lodz. § Ecole Nationale Supe ´ rieure de Chimie de Paris.

transition organometallic series. For instance, a tetrairidium cluster carrying a single maleimido group was synthesized (6) and used to introduce heavy atoms into ribososomal particles for X-ray crystallographic studies (7). A related tetrairidium cluster was used for cryoelectron microscopy studies of the hepatitis B virus capsid (8). The ferrocenyl maleimides 1a and 1b were prepared and successfully used to attach a redox active ferrocenyl entity to genetically engineered cytochrome P450cam (9) and β-lactamase (10). The cationic rhenium(I) polypyridine tricarbonyl maleimide complex 2 was used to attach strongly luminescent groups to several polypeptides and oligonucleotides (2c). A few years ago we described the synthesis of the iron(II) dicarbonyl maleimide complex 3 (11). This compound was initially designed to introduce IR-detectable (η5C5H5)Fe(CO)2 (Fp) moieties into thiol-containing biomolecules, such as the protein bovine serum albumin (BSA) (12) and the tripeptide glutathione (13) More recently, we were also able to label PAMAM generation 4 dendrimer by alkylation of its terminal free amino groups by complex 3 (14). The Fp moiety, as all the metallocarbonyl complexes, gives rise to very strong IR absorption bands, νCtO, appearing in the 1850-2100 cm-1 spectral

10.1021/bc050073d CCC: $30.25 © 2005 American Chemical Society Published on Web 09/01/2005

Sulfhydryl-Selective, Covalent Labeling of Biomolecules

region which is devoid of any absorption due to proteins or biological matrixes.

Herein we describe the synthesis of the molybdenum(II) and tungsten(II) cyclopentadienyl tricarbonyl complexes 4 and 5, respectively, both containing the η1-Nmaleimidato ligand, along with reactivity studies toward cysteine ethyl ester, the cysteine-containing tripeptide glutathione, and a model protein, thiolated BSA. These two new metallocarbonyl maleimides are of interest as protein labeling reagents since they combine two features: an easy and sensitive IR detection of the organometallic probe with a possibility of application in protein X-ray crystallography (especially complex 5). EXPERIMENTAL SECTION

All syntheses were carried out under argon. Solvents were dried by using standard procedures. Compounds (η5C5H5)M(CO)3I (M ) Mo, W) were prepared by reaction of [(η5-C5H5) M(CO)3]2 with iodine (15). Compound 3 was prepared according to an earlier published procedure (11). All other reagents are commercially available and were used as received. Chromatographic separations were carried out on silica gel 60 (Merck, 230-400 mesh ASTM). Phosphate-buffered saline (PBS) pH 7.4 was prepared by dissolving 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO4‚2H2O, and 0.34 g KH2PO4 in 1 L of double-distilled grade water. Borate buffer pH 8.0 was prepared by dissolving 0.87 g B(OH)3 and 0.57 g Na2B4O7‚10 H2O in 0.1 L of double-distilled grade water. 1H NMR spectra were recorded on a Varian Gemini 200 BB (200 MHz) and a Bruker DRX 500 (500 MHz) spectrometers and were referenced to internal TMS. Reverse-phase HPLC analysis of glutathione conjugates was performed on a System Gold (Beckman Coulter) equipped with a 4.6 × 250 mm, 5 µm Si-C8 column (Kromasil, Eka Nobel). Compounds were eluted by applying 5 min after the injection a linear gradient from 10 to 100% of MeCN0.1% TFA in water - 0.1% TFA in 20 min. (η5-C5H5)Mo(CO)3 (η1-N-Maleimidato) (4). A stirred, water-ice cooled, and argon-saturated solution of (η5C5H5)Mo(CO)3I (244 mg, 0.67 mmol) and maleimide (58 mg, 0.6 mmol) in toluene (20 mL) containing diisopropylamine (1 mL) was illuminated with visible light (4 × 100 W tungsten lamps) for 2 h. The initial dark red color

Bioconjugate Chem., Vol. 16, No. 5, 2005 1219

of the solution turned orange-yellow upon illumination. The grayish solid formed was filtered off, and the filtrate was evaporated to dryness under reduced pressure. The residue was dissolved in chloroform and purified by column chromatography. A deep red band containing a small amount of the starting molybdenum complex was eluted with chloroform, followed by an orange band of 4 eluted with chloroform-methanol 3:1. The product was crystallized from chloroform-heptane. X-ray grade crystals were obtained from layered dichloromethane-hexane. Yield 106 mg (52%). 1H NMR (δ, ppm): 6.70 (s, 2H, olefinic H’s), 5.53 (s, 5H, Cp). IR (cm-1): 2054, 1977 (CtO), 1659 (CO imide). Anal. (C12H7MoNO5) C, H, N. (η5-C5H5)W(CO)3 (η1-N-Maleimidato) (5). The thallium(I) salt of maleimide was prepared by treatment of a solution of maleimide (152 mg, 1.56 mmol) in ethanol (3.5 mL) with thallium(I) ethoxide (120 µL, 1.56 mmol). After 20 min stirring, the yellow solid formed was filtered off, washed with ethanol, and dried under vacuum. Then it was dissolved in DMF (6 mL), (η5-C5H5)W(CO)3I (162 mg, 0.35 mmol) was added, and the resulting solution was heated, with stirring, to 50 °C for 2 h. The solid formed was filtered off and the filtrate evaporated to dryness. The residue was dissolved in chloroform, washed five times with water, and dried (Na2SO4). Evaporation to dryness and flash chromatography recovered a small amount of (η5-C5H5)W(CO)3I (red band eluted with dichloromethane), followed by 5 (orange band eluted with dichloromethane-methanol 3:1). An analytical sample of 5 was prepared by recrystallization from dichloromethane-hexane. Yield 55 mg (37%). 1H NMR (δ, ppm): 6.72 (s, 2H, olefinic H’s), 5.64 (s, 5H, Cp). IR (cm-1): 2046, 1957 (CtO), 1664 (CO imide). Anal. (C12H7NO5W) C, H, N. Reaction of 4 and 5 with L-Cysteine Ethyl Ester. A solution of L-cysteine ethyl ester hydrochloride (28 mg, 0.15 mmol) and 4 or 5 (0.13 mmol) in methanol (5 mL) containing triethylamine (0.3 mL) was stirred at. r.t. for 2 h and evaporated to dryness. The residue, dissolved in chloroform, was chromatographed on SiO2. First, small amounts of unreacted 4 or 5 were eluted with chloroform and then the products 6 or 7 were eluted with chloroformmethanol (95:5). 6: (orange oil). Yield 55 mg (87%). 1H NMR (δ, ppm): 5.52 (s, 5H), 4.20 (q, J ) 7.2 Hz, 2H), 3.80 (m, 2H), 2.83.4 (m, 3H), 2.47 (dd, J ) 18.1 Hz, 3.8 Hz, 1H), 1.81 (bs, 2H) 1.288 and 1.284 (two triplets, J ) 7.2 Hz, 3H). IR: (cm-1) 2054, 1977, 1728, 1649. Anal. (C17H18 MoN2O7S) C, H, N. 7: (orange oil). Yield 68 mg (91%). 1H NMR (δ, ppm): 5.63 (s, 5H), 4.20 (q, J ) 7.2 Hz, 2H), 3.65 (m, 2H), 2.83.4 (m, 3H), 2.49 (dd, J ) 18.0 Hz, 3.8 Hz, 1H), 1.81 (bs, 2H) 1.29 (t, J ) 7.2 Hz, 3H). IR: (cm-1) 2046, 1959, 1732, 1655. Anal. (C17H18 N2O7SW) C, H, N. Kinetics Studies. Solutions (1 mL) containing compounds 4 or 5 (1 × 10-4 M) and glutathione (1 × 10-4 M) were prepared in PBS, and the reactions were monitored at 285 nm at room temperature. Second-order rate constants k and half-lives were calculated from the equation (A∞ - Ao)/(A∞ - A) ) kct + 1 where Ao is the absorbance at t ) 0, A∞ is the final absorbance, and c the initial concentration of reagents (i.e. 1 × 10-4 M). Thiolation of BSA with Traut’s Reagent. A mixture of BSA (5 × 10-5 M) and Traut’s reagent (1 × 10-3 M) in borate buffer containing 10 mM EDTA was incubated for 1 h at 26 °C (sample volume ) 3 mL). The solution was applied to a prepacked 10 mL gel desalting column

1220 Bioconjugate Chem., Vol. 16, No. 5, 2005

(Pierce Chemicals), and species were eluted with PBS containing 1 mM EDTA. The fractions containing the protein were pooled and the concentration of sulfhydryl groups was immediately determined by the Ellman’s assay (16) whereas the BSA concentration was determined by optical density measurement at 280 nm taking an extinction coefficient  of 43 600 M-1 cm-1 (17). Labeling of Thiolated BSA with 3, 4, or 5. Mixtures of freshly prepared thiolated BSA (2.1 × 10-5 M) and 3, 4, or 5 (2.86 × 10-4 M) were incubated in PBS containing 1 mM EDTA for 24 h at 26 °C (sample volume ) 1 mL). Solutions were applied to a 5 mL prepacked gel desalting column (Pierce Chemicals) and species eluted with PBS. Fifteen 0.5 mL fractions were collected, and those containing the protein were pooled and submitted to UVvisible analysis. The concentration of molybdenum and tungsten tricarbonyl succinimidato moieties was calculated from the optical density of the corresponding sample at 438 nm, taking  equal to 460 M-1‚cm-1 for Mo or 710 M-1‚cm-1 for W. The concentration of iron dicarbonyl succinimidato moieties was calculated from the optical density of the corresponding sample at 365 nm, taking  equal to 600 M-1‚cm-1. The concentration of BSA was calculated from the optical density of the solutions at 280 nm after subtraction of the contribution of the metallocarbonyl succinimidato entities at this wavelength ((Mosuccinimidato) ) 2960 M-1‚cm-1; (W-succinimidato) ) 3500 M-1‚cm-1; (Fe-succinimidato) ) 2400 M-1‚cm-1). RESULTS AND DISCUSSION

Synthesis of 4 and 5. The iron complex 3 had been prepared by the photochemical reaction of (η5-C5H5) Fe(CO)2I with maleimide in the presence of diisopropylamine (dipa) (11). Considering the close analogy in the photochemical behavior of (η5-C5H5) Fe(CO)2I and (η5C5H5)M(CO)3I (M ) Mo, W) (18), we thought at first that it would be possible to apply the same approach for the synthesis of 4 and 5. We found unexpectedly that only (η5-C5H5)Mo(CO)3I underwent photochemical substitution of iodide by the anion of maleimide upon illumination with visible light with maleimide and dipa in benzene (eq 1). Product 4 was isolated in 52% yield and characterized by spectroscopic methods, elemental analysis, and X-ray diffraction (vide infra).

Attempts to prepare 5 in the same way starting from (η5-C5H5)W(CO)3I failed. This complex proved completely inert when irradiated with visible light in benzene containing maleimide and dipa. A significantly lower photochemical reactivity of (η5-C5H5)W(CO)3I in comparison to its molybdenum analogue had been previously reported and attributed to shorter lifetimes of the chemically reactive excited states, due to enhanced spin-orbit coupling facilitating physical deactivation pathways (18c). We thus prepared 5 by reaction of (η5-C5H5)W(CO)3I with the thallium salt of maleimide (eq 2). (A similar method was used earlier to prepare the Mn(CO)5 complex of the succinimide anion (19)).

Rudolf et al.

The thallium(I) salt of maleimide was prepared by reaction of maleimide with thallium(I) ethoxide in anhydrous ethanol. The product precipitating from the reaction mixture (assumed to be the expected thallium salt) was filtered off, dried, and used immediately in the reaction with (η5-C5H5)W(CO)3I. The reaction proceeded smoothly in DMF at 50 °C and was accompanied by precipitation of TlI. Complex 5 was isolated in 37% yield and characterized by spectral methods and elemental analysis. The IR and 1H NMR spectral data of complexes 3-5 are collected in Table 1. Similarly as in 3, the η1-Nmaleimidato ligand in 4 and 5 displays its ν(CdO) imide band shifted toward lower wavenumbers in comparison to that of maleimide (1713 cm-1). The NMR signals of the olefinic protons in 3-5 were slightly shifted upfield relative to the signal of the olefinic protons in maleimide (6.74 ppm). The above data are consistent with a significant increase of the electron density at the η1-N-maleimidato ligand in 3-5 in comparison to maleimide, due to a high polarity of the metal-nitrogen bond and a dπ-pπ repulsion. Therefore, a reduced reactivity of 3-5 toward thiols can be expected, which was confirmed earlier for 3. Hopefully, this complex still proved sufficiently reactive to enable the labeling of BSA (12). Complexes 4 and 5 are also characterized by the presence of two ν(C≡O) bands in agreement with the local C3v symmetry of the metallocarbonyl units. Solid-State Structure of 4. The structure of 4 in the solid state, as determined by X-ray diffraction, is shown in Figure 1. Crystal and refinement data are collected in Table 2, and selected bond distances and angles in Table 3. Compound 4 crystallizes in the monoclinic P21/c space group with one molecule in the asymmetric unit cell. The molybdenum atom is five-coordinated, with the Cg of the cyclopentadienyl ring at the apex and three carbonyls and the nitrogen atom of the η1-N-coordinated maleimidato ligand in the basal positions of the square pyramid (a ”four-legged piano stool structure”). Such a structure is typical for CpM(CO)3X molecules, where X denotes a 2e anionic ligand (20). The distance between the molybdenum atom and a Cg of the cyclopentadienyl ring is 2.001(2) Å. The Mo-C bond for the trans-CtO ligand (1.991(3) Å) is substantially shorter than those for for the cis-CtO ligands (2.029(3) Å and 2.028(3) Å). This means that the trans-CtO ligand is stronger backbonded than its cis-counterparts, presumably because of σ-donor and π-donor properties of the imidato ligand (19). An interesting feature observed in the crystal structure of 4 are short contacts between oxygen atoms of the maleimidato ligand and carbon atoms of the cis-CtO ligands (the distances O(14)‚‚‚C(20) and O(11)‚‚‚C(40) are Table 1. IR and 1H NMR Data of Complexes 3-5 (in CHCl3 or CDCl3) complex

ν(CtO), cm-1

ν(CdO), cm-1

δ(Cp), ppm

δ (maleimide), ppm

3 4 5

2056, 2008 2054, 1977 2046, 1957

1651 1659 1664

5.06 5.53 5.64

6.62 6.70 6.72

Bioconjugate Chem., Vol. 16, No. 5, 2005 1221

Sulfhydryl-Selective, Covalent Labeling of Biomolecules

Table 3. Structure of 4a Mo-N10 Mo-C20 Mo-C30 Mo-C40 Mo-C1 Mo-C2 Mo-C3 Mo-C4 Mo-C5 N10-C11 N10-C14 C12-C13 O11-C11 O14-C14 Mo-C20-O20 Mo-C30-O30 Mo-C40-O40 C11-N10-C14 Mo-N10-C11-C12 C11-C12-C13-C14 C12-C13-C14-O14 O11-C11-C12-C13

Figure 1. Molecular view of compound 4 with atom labeling scheme. Ellipsoids are drawn at 30% probability. Table 2. Crystal and Refinement Data for 4 empiric formula formula weight crystal description crystal size (mm3) space group unit cell dimensions a (Å) b (Å) c (Å) β (deg) V (Å3) dx (g cm-3)

Crystal Data C12 H7 Mo N O5 341.13 Orange plate 0.4 × 0.4 × 0.05 P 21/c 9.8718(10) 8.740(3) 15.8384(14) 116.062(6) 1227.6(5) 1.846

Data Collection Rigaku AFC5S Mo KR 0.71069 1.083 293(2) -12 e h e 12 -10 e k e 10 -19 e l e 19 numbers of reflections measured 9286 numbers of independent reflections 2405 Rint 0.0547 numbers of reflections with I > 2σ (I) 1621

diffractometer radiation type λ (Å) µ (mm-1) temperature (K) data collected (h, k, l)

Solution and Refinement Patterson method full-matrix least-squares on F2 number of parameters 172 R(F)a (all data) 0.0537 wR(F2)b (all data) 0.0479c R(F)a 0.0231 for 1622 reflections wR(F2)b 0.0459c for 1622 reflections (∆/σ)max 0.000 different peak/hole (eÅ-3) 0.487/-0.389 solution refinement method

c

a R(F) ) Σ(|F - F |)/|F |. b wR(F2) ) [Σw(|F - F |)2/Σ|F |2]1/2. o c o o c o w ) 1/[σ2 (Fo2) + (0.0182P)2] where P ) (Fo2 + 2Fc2)/3.

2.673(4)Å and 2.796(4)Å, respectively). These interactions, which may have electrostatic nature, bring about tilting of C(20) and C(40) toward O(14) and O (11), respectively, and change angles O20-C20-Mo and O40C40-Mo (172.7(3)° and 173.3(2)°, respectively, to compare with the angle O30-C30-Mo that equals 178.3(3)°). Similar interactions between organic carbonyl groups are frequently observed in X-ray structures (21). Since these interactions are intramolecular, they may exist not only in the crystal state, but also in solution, and may influence the reactivity of the maleimidato ligand. Reaction of 4 and 5 with L-Cysteine Ethyl Ester and Glutathione. Having complexes 4 and 5 in hand, we first checked their reactivity toward the thiol group

a

2.180(2) 2.029(3) 1.991(3) 2.028(3) 2.288(4) 2.354(3) 2.376(4) 2.325(4) 2.289(4) 1.389(4) 1.394(4) 1.315(5) 1.217(4) 1.213(5) 172.7(3) 178.3(3) 173.3(2) 106.3(2) -172.4(2) -0.2(4) 178.1(3) -177.0(3)

Selected bond lengths (Å) and angles (deg).

of L-cysteine ethyl ester. We found that this compound (generated in situ by reaction of L-cysteine ethyl ester hydrochloride with triethylamine) was readily S-alkylated by either 4 or 5 in methanol to afford the η1-Nsuccinimidato complexes 6 and 7 in high yields (Scheme 1). The addition of the sulfhydryl group of L-cysteine ethyl ester to the CdC bond of the maleimidato ligand was confirmed by the lack of the characteristic ν(S-H) absorption in the IR spectra of 6 and 7 (this absorption appears at 2474 cm-1 in the IR spectrum of L-cysteine ethyl ester). The complexity of the 1H NMR spectra of 6 and 7 suggests the formation of two diastereomers as a result of the creation of the stereogenic center at the succinimide ring. To compare the reactivity of 4 and 5 with that of 3, the rate of reaction of these complexes with the cysteinecontaining tripeptide glutathione was monitored from the change of absorbance at 285 nm in the UV-vis spectra accompanying the addition of glutathione to the maleimide CdC bond. Formation of the S-alkylation adducts was also monitored by reverse phase HPLC. In all cases, the reaction rate followed a second-order kinetics. Measured rate constants and reaction half-lives are gathered in Table 4. As seen from Table 4, the addition of glutathione to 4 and 5 is faster than its addition to 3. This difference may arise from a weaker dπ-pπ repulsion between the formally anionic maleimidato ligand and the d4 metal (Mo(II) or W(II)) in 4 and 5 than the d6 Fe(II) center in 3. This repulsion is expected to increase the electron density at the CdC bond, making it less reactive toward nucleophilic attack. Similarly, a decrease of dπ-pπ repulsion is expected on going from Mo to W (the latter participates more in the back-bonding toward CO ligands), which may explain the slightly larger reaction rate of 5 in comparison with 4. The aforementioned trends in back-bonding toward maleimidato ligand are reflected in the IR spectra of 3-5 (Table 1). The ν(CdO) of imide carbonyls increases in the order 3 < 4 < 5. Consequently, the extent of the back-bonding to the maleimidato ligand should follow the order 3 > 4 > 5. Note finally that the rate of S-alkylation of glutathione with 4 or 5 was still slower than that of N-ethylmaleimide. Both glutathione conjugates, analyzed by reverse phase HPLC under conditions reported in Experimental Sec-

1222 Bioconjugate Chem., Vol. 16, No. 5, 2005

Rudolf et al.

Scheme 1. S-Alkylation of l-Cysteine Ethyl Ester by 4 or 5

Table 4. Rate Constants and Half-Lives of Reaction of 3-5 and N-Ethylmaleimide with Glutathione in Water-Methanol (9:1) at pH ) 7.4 and 24 °C

Table 5. Labeling of Thiolated BSA (containing 7.4 SH groups) with 3-5 (1.8 molar equiv per SH group) at pH 7.4 and 26 °C for 24 h

complex

k [M-1‚min-1]

t1/2 [min]

reference

labeling complex

n (yield)

3 4 5 N-ethylmaleimide

382 1111 1732 9532

26.0 9.0 5.8 1.0

12 this work this work 12

4 5 3

6.9 (93%) 6.4 (86%) 7.6 (103%)

Scheme 2

tion, were eluted in the form of two very close peaks at retention time of 15.7 min, in agreement with the formation of two diastereoisomers. The almost equal intensity of both peaks suggests that addition of glutathione to 4 or 5 is not stereoselective. Reactivity of 4 and 5 toward Thiolated BSA. Having demonstrated the reactivity of 4 and 5 toward the sulfhydryl group in two cysteine derivatives, we became interested in their reactivity toward a protein containing this group. Bovine serum albumin (BSA) is a well-characterized, low cost protein, which is widely used in labeling experiments. Unfortunately, BSA contains only one free cysteine (cysteine 34), and in the commercially available BSA samples, this residue is partially blocked by conjugation with other sulfhydryl compounds such as glutathione (17). Therefore, spectroscopic evidence of BSA labeling with 4 or 5 could be difficult to determine because of the expected very low small number of organometallic units bound to the protein. To overcome this problem, we decided to introduce onto BSA additional sulfhydryl groups before reaction with 4 or 5. This was achieved by treatment of BSA with Traut’s reagent, which enables conversion of some of the protein free amino groups (BSA possesses 60 such groups) into sulfhydryl groups (5) (Scheme 2). Under the conditions described in Experimental section, we obtained a thiolated BSA sample containing an average of 7.4 SH groups per protein (including the existing free cysteine residue). This modified protein was then incubated with a 2-fold molar excess of 4 or 5 over

the starting concentration of SH groups at pH 7.4 and 26 °C for 24 h. For comparison, an experiment using 3 in place of 4 or 5 was also run under the same conditions. These reaction conditions had been previously shown to enable selective labeling of BSA’s thiol (12). The metalloconjugates were purified by gel filtration chromatography. The numbers n of metallocarbonyl entities introduced to thiolated BSA, as determined from UV-visible measurements, are reported in Table 5. It appears from Table 5 that complexes 4 and 5 (as well as complex 3) were very efficient labeling agents of the protein thiols, with labeling yields all above 85%. The

Figure 2. UV-visible spectra of complex 4 (2 × 10-4 M in MeOH, thin line) and its thiolated BSA conjugate (1.97 × 10-5 M in PBS, thick line).

Figure 3. UV-visible spectra of complex 5 (2 × 10-4 M in MeOH, thin line) and its thiolated BSA conjugate (2.07 × 10-5 M in PBS, thick line).

Sulfhydryl-Selective, Covalent Labeling of Biomolecules

Figure 4. IR spectrum of the conjugate resulting from the reaction of 5 with thiolated BSA (10 µL of a 2.07 × 10-5 M deposited on a 6 mm diameter nitrocellulose membrane).

reaction also appeared to be site-specific in the chosen conditions of reaction, as the number of metallocarbonyl entities matched very well with the initial number of SH groups measured for the thiolated BSA sample. The UVvisible and IR spectra of the metalloconjugates depicted in Figures 2-4 confirmed the covalent binding of both metallocarbonyl complexes to BSA. CONCLUSION

We have synthesized metallocarbonyl complexes of Mo and W containing the (η1-N-maleimidato) ligand, (η5-C5H5)M(CO)3(η1-N-maleimidato). These complexes react with sulfhydryl groups of cysteine in cysteine ethyl ester and glutathione as well as with sulfhydryl groups introduced to BSA in reaction with Traut’s reagent to afford the corresponding S-alkylated IR-detectable bioconjugates. The potential advantage of the synthesized complexes is the presence of heavy metal atoms, which may be helpful for solving proteins structure by X-ray crystallography. ACKNOWLEDGMENT

The Centre National de la Recherche Scientifique is gratefully acknowledged for financial support of B.R.’s stay in Paris. Supporting Information Available: Tables of crystallographic data. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Van Staveren, D. R., and Metzler-Nolte, N. (2004) Bioorganometallic chemistry of ferrocene. Chem. Rev. 104, 59315985. (2) Salmain, M., and Jaouen, G. (2003) Side-chain selective and covalent labelling of proteins with transition organometallic complexes. C. R. Chimie 6, 249-258. (3) (a) Guo, X. Q., Castellano, F. N., Li, L., and Lakowicz, J. R. (1998) Use of a long-lifetime Re(I)complex in fluorescence polarization immunoassays of high-molecular-weight analytes. Anal. Chem. 70, 632-637.(b) Dattelbaum, J. D., Abugo, O. O., and Lakowicz, J. R. (2000) Synthesis and characterization of a sulfhydryl-reactive rhenium metal-ligand complex. Bioconjugate Chem. 11, 533-536. (c) Lo, K. K., Hui, W. K., Ng, D. C., and Cheung, K. K.(2002) Synthesis, characterization, photophysical properties, and biological labeling studies of a series of luminescent rhenium(I) polypyridine maleimide complexes. Inorg. Chem. 41, 40-46. (4) Blundell, T. L., and Johnson, L. N. (1976) Protein Crystallography, Academic Press, London.

Bioconjugate Chem., Vol. 16, No. 5, 2005 1223 (5) Hermanson, G. T. (1996) Bioconjugate Techniques, Academic Press, San Diego CA. (6) Jahn, W. (1989) Synthesis of water soluble tetrairidium clusteres suitable for heavy atom labelling of proteins. Z. Naturforsch. 44b, 79-82. (7) Weinstein, S., Jahn, W., Hansen, H., Wittmann, H. G. and Yonath, A. (1989) Novel procedures for derivatization of ribosomes for crystallographic studies. J. Biol. Chem. 264, 19138-19142. (8) Cheng, N. Conway, J. F., Watts, N. R., Hainfeld, J. F., Joshi, V., Powell, R. D., Stahl, S. J., Wingfield, P. E., and Steven, A. C. (1999) Tetrairidium, a four-atom cluster as a density label in three-dimensional cryo-EM maps of proteins at 1025 Å resolution. J. Struct. Biol. 127, 169-176. (9) (a) Lo, K. K.-W, Wong, L.-L., and Hill, H. A. O. (1999) Surface-modified mutants of cytochrome P450cam: enzymatic properties and electrochemistry. FEBS Lett. 451, 342-346. (b) Di Gleria, K., Nickerson, D. P., Hill, H. A. O., Wong, L.L., and Fu¨lo¨p, V. (1998) Covalent attachment of an electroactive sulfhydryl reagent in the active site of cytochrome P450cam as revealed by the crystal structure of the modified protein. J. Am. Chem. Soc. 120, 46-52. (c) Di Gleria, K., Hill, H. A. O., and Wong, L.-L. (1996) N-(2-Ferrocene-ethyl)maleimide: a new electroactive sulfhydryl-specific reagent for cysteine-containing peptides and proteins. FEBS Lett. 390, 142-144. (10) Di Gleria, K., Halliwell, C. M., Jacob, C., and Hill, H. A. (1997) Site-specific introduction of an electroactive label into a non-electroactive enzyme (β-lactamase I). FEBS Lett. 400, 155-157. (11) Rudolf, B., and Zakrzewski,J. (1994) (η5-Cyclopentadienyl) Fe(CO)2-complex of maleimide anion: An organometallic carbonyl probe for biomolecules containing HS groups. Tetrahedron Lett. 35, 9611-9612. (12) Rudolf, B., Zakrzewski, J., Salmain, M., and Jaouen, G. (1998) Metallo-carbonyl complexes based on the CpFe(CO)2(η1-N- imidato) system as protein labelling reagents: reactivity and selectivity studies using bovine serum albumin as a model protein. New J. Chem. 813-818. (13) Salmain, M., Jaouen G., Rudolf, B., and Zakrzewski, J. (1999) Inhibition and photo-deinhibition of glutathione Stransferase activity by an organometallic complex: S-[3CpFe(CO)2(η1-N-succinimidato]glutathione. J. Organomet. Chem. 589, 98-102. (14) (a) Salmain, M., Fischer-Durand, N., Cavalier, L., Rudolf, B., Zakrzewski, J., and Jaouen, G. (2002) Transition metalcarbonyl labeling of biotin and avidin for use in solid-phase Carbonyl Metallo Immunoassay (CMIA). Bioconjugate Chem. 18, 693-698. (b) Rudolf, B., Zakrzewski, J., Celichowski, G., Salmain, M., Vessie`res, A., and Jaouen, G. (2004) (b) Specific binding of a biotinylated, metallocarbonyl-labelled dendrimer to immobilised avidin detected by Diffuse Reflectance Infrared Fourier Transform (DRIFT) spectroscopy. Appl. Organomet. Chem. 18, 105-110. (c) Fischer-Durand, N., Salmain, M., Rudolf, B., Vessie`res, A., Zakrzewski, J., and Jaouen. G. (2004) Synthesis of metal carbonyl-dendrimer-antibody immunoconjugates: towards a new format for Carbonyl Metallo ImmunoAssay (CMIA). ChemBioChem. 5, 519-525. (15) (a) Abel, A. E., Singh, W., and Wilkinson, G. (1960) Some π-cyclopentadienyl- molybdenum and -tungsten halides. J. Chem. Soc. 1321-1324. (b) Burckett- St. Laurent, J. C. T. R., Field, J. S., Haines, R. J., and McMahon, M. (1978) Halogenation of bis (η-cyclopentadienyltricarbonylmolybdenum and -tungsten) via bridged intermediates- formation of some monocyclopentadienyl derivatives of molybdenum (IV). J. Organomet. Chem. 153, C19-C22. (16) Ellman, G. L. (1958) A colorimetric method for determining low concentrations of mercaptans. Arch. Biochem. Biophys. 74, 443-450. (17) Peters, P. J. (1985) Serum albumin. Adv. Protein Chem. 37, 161-245. (18) (a) Alway, D. G., and Barnett, K. W. (1978) Photochemical processes in cyclopentadienylmetal carbonyl complexes. Adv. Chem. Ser. 168, 115-131. (b) Alway, D. G., Barnett, K. W. (1975) Photochemical isomerization of cyclopentadienylmetal complexes. J. Organomet. Chem. 99, C52-C54. (c) Alway, D.

1224 Bioconjugate Chem., Vol. 16, No. 5, 2005 G., and Barnett, K. W. (1980) Photochemistry of cyclopentadienylmolybdenum and cyclopentadienyltungsten carbonyl halide complexes. Inorg. Chem. 19, 1533-1543. (d) Pan, X., Philbin, C. E., Castellani, M. P., and Tyler, D. R. (1988) Photochemistry of organometallic halide complexes. Mechanism for the formation of ionic products. Inorg. Chem. 27, 671-676. (19) Adams, H., Bailey, N. A., Briggs, T. N., McCleverty, J. A., Colquhoun, H. M., and Williams, D. J. (1986) Transitionmetal complexes with terminal and bridging imidato (1-) ligands. X-ray crystal structures of trans-[Ir(CO)(NCOC2F4CO)(PPh3)2] and [{Pd(o-C5H4CHNPh)(m-NCOC2H4CO)}2]‚ CH2Cl2, spectroscopic studies on [Mn(CO)5(NCOC2H4CO)] and

Rudolf et al. the nature of the metal-nitrogen bond. J. Chem. Soc., Dalton Trans. 813-821. (20) Bueno, C., and Churchill, M. R. (1981) Structural studies on some (η5-C5H5)M(CO)3X molecules: (η5-C5H5)W(CO)3Cl, (η5-C5H5)Mo(CO)3Cl and (η5-C5H5)Mo(CO)3HgCl. Inorg. Chem. 20, 2197-2202. (21) Allen, F. H., Baalham, C. A., Lommerse, J. P. M., and Raithby, P. R. (1998) Carbonyl-carbonyl interaction can be competitive with hydrogen bond. Acta Crystallogr. B54, 320-329.

BC050073D