ARTICLE pubs.acs.org/Organometallics
Acetal-Functionalized RAPTA Complexes for Conjugation and Labeling Yu Qian Tan,† Paul J. Dyson,‡ and Wee Han Ang*,† † ‡
Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543 Institut des Sciences et Ingenierie Chimiques, Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
bS Supporting Information ABSTRACT:
Ruthenium arene complexes bearing the 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane ligand, namely, RAPTA compounds, are widely investigated as potential antimetastatic agents for cancer therapy. Although much evidence points toward covalent binding with essential protein biomolecules as the key determinant of their activity, the exact biological targets of RAPTA remain elusive. To address this current gap in understanding, RAPTA compounds derivatized with acetal groups via a pendant chain to the arene ligand were developed as a functional probe for selective postlabeling and tagging of covalent RAPTA adducts in cancer cells.
’ INTRODUCTION In recent years, ruthenium complexes have emerged as potential drug candidates for cancer therapy due largely to their low toxicity and high selectivity toward diseased cells in vitro.1 Interest in the field has been fueled by recent successes of two ruthenium-based complexes, namely, NAMI-A and KP1019, in clinical trials (Figure 1).2 Organometallic ruthenium(II) arene complexes have also been extensively investigated as potential anticancer agents.3 In particular, the RAPTA class of complexes has demonstrated antimetastatic activity that is comparable to that of NAMI-A.4 Several structural analogues of the RAPTA scaffold have been investigated, including RAPTA-C and RAPTA-T, with cymene and toluene, respectively, as their arene ligands being the leading candidates (Figure 1).5 Recent studies have pointed toward the covalent binding of RAPTA with essential protein biomolecules as determinants of their antimetastatic action.6 In particular, in vitro studies revealed that RAPTA-T interfered with the ability of cancer cells to detach and re-adhere.7 Detachment and re-adhesion processes are essential for cancer cell proliferation and are mediated by vital proteolytic enzymes, and the protease cathepsin B was found to be inhibited by RAPTA compounds.8 However, the exact biological target of RAPTA compounds that underpins their antimetastatic activity remains elusive. We were, therefore, interested in developing a functional analogue to act as a chemical probe for these interactions. Such a probe should contain a suitable handle for labeling or tagging for pull-down assays so that specific protein biomolecules that interacted with RAPTA complexes could be isolated and identified. With this goal in mind, we prepared an r 2011 American Chemical Society
arene-functionalized RAPTA probe containing an acetal moiety that can be selectively activated for orthogonal conjugation and labeling.9
’ EXPERIMENTAL SECTION All solvents were received and used as-is unless stated otherwise. RuCl3 3 xH2O (Precious Metals Online), 3-(1,4-cyclopentadien-1-yl)propanol (Alfa Aesar), oxalyl chloride (Alfa Aesar), PBS (phosphate buffered saline, Vivantis), sodium oxalate (TCI Chemicals), silver nitrate (Alfa Aesar), sodium chloride (TCI Chemicals), glutathione (GSH, Alfa Aesar), and AlexaFluor488-C5 aminooxyacetamide bis(triethylammonium) salt (Invitrogen) were used without further purification. Procedures were performed under an atmosphere of nitrogen using Schlenk techniques. 1H, 13C{1H}, and 31P{1H} NMR spectra were recorded on a Bruker AC300 spectrometer. ESI mass spectra were obtained on a Thermo Finnigan MAT LCQ spectrometer. UV vis spectroscopy was carried out on a Shimadzu UV-1800 UV vis spectrophotometer.
Preparation of 3-(1,4,-Cyclohexadien-1-yl)-propanal (2). Dimethylsulfoxide (DMSO, 0.55 mL, 7.71 mmol) in dichloromethane (DCM, 3 mL) was added dropwise to a solution of oxalyl chloride (0.4 mL, 4.63 mmol) in DCM (7 mL) at 78 °C and left to stir for 15 min. A solution of 3-(1,4-cyclohexadien-1-yl)-propanol (355.3 mg, 2.57 mmol) in DCM (3 mL) was then added dropwise to the mixture at 78 °C under N2. After 50 min of stirring, triethylamine (2.1 mL, 15.2 mmol) was added to the reaction flask and the crude mixture was warmed to rt. Water (10 mL) was added to the mixture, and the aqueous Received: August 20, 2011 Published: October 08, 2011 5965
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Figure 1. Examples of ruthenium-based anticancer complexes. layer was extracted with DCM (3 15 mL). The combined organic extracts were dried over anhydrous MgSO4 and filtered, and the solvent was removed in vacuo. The product was purified by flash column chromatography with an eluent of ethyl acetate/hexane (1:10). Yield: 284.1 mg (81.2%). 1H NMR (CDCl3) δ/ppm: 2.17 (t, 2H, CH2CH2CHO), 2.41 2.50 (m, 4H, dCHCH2, CH2CHO), 2.53 2.56 (m, 2H, d CH-CH2), 5.31 5.32 (m, 1H, dCH-Cquat), 5.53 5.61 (m, 2H, CHd CH ), 9.64 (t, 1H, CHO), 3J = 1.73 Hz. 13C{1H} NMR (CDCl3) δ/ppm: 26.3 ( CH2CH2CHO), 28.6, 29.05 (dienyl-CH2 ), 41.0 ( CH2CH2CHO), 118.7, 123.5, 123.7 (dienyl-CdCH), 132.9 (dienylCquat), 201.8 ( CHO).
Preparation of [η6-(3,3-Diethoxypropylbenzene)RuCl2]2 (3b). RuCl3 3 xH2O (58.3 mg, 0.21 mmol) and 2 (143 mg, 1.05 mmol)
in ethanol (12 mL) was refluxed at 100 °C under a N2 atmosphere for 4 h. The reaction mixture was concentrated in vacuo, and diethyl ether was added to yield an orange precipitate. The precipitate was washed with diethyl ether (2) and dried in vacuo. Yield: 73.0 mg (90.3%). 1H NMR (CDCl3) δ/ppm: 1.15 (t, 6H, OCH2CH3) 3J = 6.99 Hz, 1.86 1.93 (m, 2H, PhCH2CH2CH ), 2.62 (t, 2H, PhCH2CH2CH ), 3.39 3.65 (m, 4H, OCH2CH3), 4.47 (t, 1H, PhCH2CH2CH ), 3J = 5.43 Hz, 5.39 (d, 2H, Ph-Hortho), 5.56 5.60 (m, 1H, Ph-Hpara), 5.62 5.66 (m, 2H, Ph-Hmeta); single crystals from diethyl ether into DCM solution.
Preparation of [η6-(3,3-Dimethoxypropylbenzene)RuCl2]2 (3a). The same procedure as complex 3a was carried out using 2 (141.1
g, 1.04 mmol) and RuCl3 3 xH2O (41.5 mg, 0.15 mmol) in methanol, yielding an orange powder. Yield: 28.1 mg (52.1%). 1H NMR (CDCl3) δ/ppm: 1.84 1.91 (m, 2H, PhCH2CH2CH ), 2.60 (t, 2H, PhCH2CH2CH ), 3.27 (s, 6H, OCH3), 4.34 (t, 1H, PhCH2CH2CH ), 5.38 (d, 2H, Ph-Hortho), 5.56 5.60 (m, 1H, Ph-Hpara), 5.63 5.64 (m, 2H, Ph-Hmeta); single crystals from diethyl ether into DCM solution.
Preparation of [η6-(3,3-Diethoxypropylbenzene)RuCl2(pta)] (4). pta (16.5 mg, 0.01 mmol) was added to a solution of 3b
(40 mg, 0.05 mmol) in DCM/MeOH (1:1) and refluxed at 60 °C for 2 h. The crude reaction mixture was concentrated, and the product was precipitated with diethyl ether. The precipitate was washed with diethyl ether (3) and dried in vacuo. Yield: 53.4 mg (94.5%). 1H NMR (CDCl3) δ/ppm: 1.18 (t, 6H, OCH2CH3), 1.91 1.96 (m, 2H, PhCH 2CH 2CH ), 2.58 (t, 2H, PhCH2 CH2 CH), 3.44 3.69 (m, 2H, OCH2CH3), 4.35 (s, 6H, PCH2N), 4.51 4.55 (m, 7H, NCH2N, PhCH2CH2CH ), 5.21 5.55 (m, 5H, Ph-H). 31P{1H} NMR (CDCl3) δ/ppm: 33.0. ESI-MS (m/z): 559 [M + Na]+, 502 [M Cl]+; single crystals from diethyl ether into chloroform solution.
Preparation of [η6-(3,3-Diethoxypropylbenzene)Ru(C2O4)(pta)] (5). A 1 M silver nitrate solution (0.24 mL, 0.24 mmol) was
added dropwise to an aqueous solution of sodium oxalate (17 mg, 0.12 mmol) and left to stir for 10 min. The white precipitate was isolated by centrifugation and washed with water (3 1 mL). The isolated silver oxalate was used as-is without drying. (Caution! Dry silver oxalate is
known to be explosive under heating.) 3b (30 mg, 0.04 mmol) was stirred with a suspension of silver oxalate (0.01 mmol) in water (12 mL) at rt for 24 h. The crude solution was filtered through Celite and dried by rotary evaporation. The residue was redissolved in methanol (12 mL) and stirred with pta (13 mg, 0.08 mmol) for 2 h at rt. The reaction mixture was concentrated, and diethyl ether was added to precipitate a yellow solid. The precipitate was washed with diethyl ether (2) and dried in vacuo. Yield: 35.7 mg (80.4%). 1H NMR (CD3OD) δ/ppm: 1.10 (t, 6H, OCH2CH3), 3J = 7.07 Hz, 1.91 1.97 (m, 2H, PhCH2CH2CH ), 2.45 (t, 2H, PhCH2 CH2 CH), 3.48 3.72 (m, 4H, OCH2CH3), 4.13 (s, 6H, PCH2N ), 4.52 (m, 6H, NCH2N ), 4.72 (masked by solvent, 1H, PhCH2CH2CH ), 5.53 (m, 1H, Ph-Hpara), 5.83 5.90 (m, 4H, Ph-H). 31P{1H} NMR (CD3OD) δ/ppm: 31.1. ESI-MS (m/z): 578 [M + Na]+, 1132 [2M + Na]+. X-ray Diffraction Studies. X-ray data were collected with a Bruker AXS SMART APEX diffractometer using Mo Kα radiation at 223(2) K with the SMART suite of Programs.10 Data were processed and corrected for Lorentz and polarization effects with SAINT,11 and for absorption effects with SADABS.12 Structural solution and refinement were carried out with the SHELXTL suite of programs.13 The structure was solved by direct methods to locate the heavy atoms, followed by difference maps for the light, non-hydrogen atoms. All non-hydrogen atoms were generally given anisotropic displacement parameters in the final model. All H atoms were put at calculated positions. A summary of the crystallographic data is given in Table 2. Aquation Studies by UV vis Absorption Spectroscopy. A solution of 4 (0.2 mM, 1 mL) was added to a quartz cuvette, and its UV absorbance was measured at 1 min intervals from 280 to 580 nm. The reaction was repeated using 150 mM NaCl and 1 mM GSH, and 5 with 1 or 10 mM GSH. pH-Dependent Stability Studies. Complex 5 (100 mM) was dissolved in 0.2 M phosphate buffered solutions of pH 5.8, 6.0, 7.0, and 8.0 (0.5 mL) and diluted with D2O (500 μL). The solutions were left at rt for 48 h and analyzed by 1H NMR. Conjugation Studies with o-Benzylhydroxylamine. Complexes 4 (1.0 mg, 1.9 μmol) and 5 (1.0 mg, 1.8 μmol) in water (1 mL) were treated with HCl (10 mM, 1 mL) for 1 h. The solution was adjusted to pH 6.0 using a 100 mM K2CO3 solution, and o-benzylhydroxylamine in DMF (0.33 mg, 2.7 μmol) was added to the reaction mixture. The reaction was stirred at rt for 2 h and analyzed by ESI-MS. Cell-Based Binding Assays. HEK cells were plated on a 96-well microplate (Nunc) at a density of 10 000 cells per well in complete DMEM media. After 24 h, the cells were incubated with a solution of 4 in DMEM (1 mM, 100 μL). After incubating for 6 h, the solution was removed and the cells washed with PBS (2 100 μL). Cold ethanol (100 μL) was added to each well, and the plate was incubated at 20 °C for 5 min. The cells were washed with PBS (2 100 μL), and HCl solution (10 mM, 100 μL) was added to each well. The plate was incubated at rt for 1 h. After washing with PBS (2 100 μL), AlexaFluor488 5966
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Table 1. Selected Bond Distances (Å) and Angles (°) of 3a, 3b, and 4 bond distances and anglesa
3a
3b
4
Ru C
2.149 2.190
2.148 2.189
Ru Clμ
2.4434(8), 2.4439(8)
2.4470(18), 2.4597(18)
2.163 2.274
Ru Clσ
2.4101(9)
2.4193(19)
2.4138(9), 2.4122(9)
Ru P
2.2869(9)
(arene)C C
1.392 1.422
1.395 1.439
1.392 1.440
C O
1.406 1.434
1.411 1.442
1.394 1.439
Clμ Ru Clμ Clμ Ru Clσ
80.59(3) 87.31(3), 87.88(3)
80.73(6) 87.32(6), 88.59(6)
99.41(3)
99.27(6)
Clσ Ru Clσ Ru Clμ Ru
88.13(3)
Clσ Ru P a
84.25(3), 85.51(3)
The esd is given in brackets when applicable.
Table 2. Crystallographic Data for 3a, 3b, and 4 complex
3a
3b
4
chem formula
C22H32Cl4O4Ru2
C26H40Cl4O4Ru2
C19H32Cl2N3O2PRu
fw crystal system
704.42 monoclinic
760.52 monoclinic
537.42 orthorhombic
space group
P21/c
P21/c
Pbca
A (Å)
13.0435(9)
14.922(4)
13.800(2)
B (Å)
10.3684(7)
10.535(3)
11.815(2)
C (Å)
9.7799(7)
9.700(3)
26.418(4)
α (°)
90
90
90
β (°)
100.889(2)
104.268(6)
90
γ (°) volume (Å3)
90 1298.8(16)
90 1477.8(7)
90 4307.5(13)
Z
2
2
8
Dcalc (g cm 3)
1.801
1.709
1.657
F(000)
704
768
2208
μ (mm 1)
1.601
1.414
1.071
temp (K)
223(2)
100(2)
100(2)
wavelength (Å)
0.71073
0.71073
0.71073
measured reflns unique reflns
8938 2978
7274 3358
28163 4946
no. of data/restraints/parameters
2978/0/147
3358/0/165
4946/0/255
Ra [I > 2σ(I)]
0.0352
0.0572
0.0423
wR2a (all data)
0.0898
0.1957
0.0920
GOFb
1.038
1.009
1.218
a R = ∑||Fo| |Fc||/Σ|Fo|, wR2 = {∑[w(Fo2 the number of parameters refined.
Fc2)2]/∑[w(Fo2)2]}1/2. b GOF = {∑[w(Fo2
hydroxylamine (20 μM, 50 μL) was added and incubated for 2 h. The cells were washed with PBS (2 100 μL), and the fluorescence readings were obtained (excitation, 488 nm; emission, 560 nm) using a microplate reader (BioTek Synergy 4). All readings were performed in quadruplicate.
’ RESULTS AND DISCUSSION RAPTA complexes comprise a monodentate 1,3,5-triaza-7phosphatricyclo-[3.3.1.1]decane (pta) ligand, an η6-arene ligand bound to the ruthenium(II) center, and leaving groups in the form of two halides or a chelating bis-carboxylate ligand. 5
Fc2)2]/(n
p)}1/2, where n is the number of data and p is
Modification on the pta ligand had been previously demonstrated to alter the biological activity of the complex while the leaving groups readily undergo aquation to release coordination sites at the metal center.4 It was also shown that RAPTA-C binding to proteins occurs with retention of the arene and pta ligands.6 We, therefore, examined the use of the aldehyde functionality, positioned on the arene ligand, as a possible handle for the probe since it can be readily conjugated via hydrazone and oxime coupling reactions under mild conditions.14 A RAPTA complex bearing a benzaldehyde group on the arene ligand was previously prepared as a carrier drug for direct hydrazone conjugation with recombinant human serum albumin as macromolecular 5967
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Organometallics drug delivery agents (Figure 2).15 In the context of biological investigations, the benzaldehyde group was deemed to be undesirable because the bulky and hydrophobic functionality would drastically alter the physical and chemical properties of the RAPTA pharmacophore and hence would not be a good model. Notably, RAPTA-benzaldehyde is insoluble in water, whereas RAPTA-C and RAPTA-T exhibited good solubility. In addition, the aldehyde group could itself react with abundant amine groups on protein surfaces via imine condensation. With these key considerations, we conceived of a RAPTA probe containing an arene-functionalized acetal moiety, which can be selectively converted to an aldehyde group under mild conditions. The target complex was prepared in two key steps (Scheme 1). First, the facially bound functionalized arene ligand was formed via dehydrogenation of the cyclic diene 2 using hydrated RuCl3 in alcohol solvent to yield dimer 3. Diene 2 was prepared by carrying out Swern oxidation on commercially available 1 in high yield. Treatment of 2 with hydrated RuCl3 in either methanol or ethanol afforded in one step the desired ruthenium dimer containing either the dimethyl acetal 3a or the diethyl acetal 3b functional groups, respectively. 1H NMR spectroscopy revealed formation of the facially bound arene ligand with the aromatic CH resonances lying downfield at 5 6 ppm. The acetal moieties were also readily observed using the 1H NMR spectrum with resonances at 3 5 ppm. In particular, the characteristic CHO resonance in the upfield region was absent. The ability of RuCl3 to catalytically convert aldehyde to acetal groups was reported previously.16 Single crystals of 3a and 3b were obtained by vapor diffusion of diethyl ether into dichloromethane solutions. Their structures are illustrated in Figure 3, and key bond
Figure 2. Functionalized RAPTA complexes for conjugation and tagging.
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lengths and angles are given in Table 1. The bond lengths are essentially of typical values. In keeping with other [(η6-arene)RuCl2]2 complexes, both 3a and 3b are organized around the rhombus-shaped [Ru(μ-Cl)]2 core with the remaining arene and chloride ligands arranged in anti geometry.17 Second, 3b was treated with pta under refluxing conditions to afford the target complex 4 in high yield. Besides the resonance attributable to H atoms on the arene ligand and the acetal moiety, the 1H NMR spectrum of 4 also revealed two singlets with integral values of 6 each at 4 5 ppm corresponding to the P CH2 N and N CH2 N on the pta ligand. 31P{1H} NMR showed a singlet resonance at 33.0 ppm, which is indicative of [(η6-arene)RuCl2(pta)]-type complexes. ESI-MS spectra further showed molecular ions corresponding to [M + Na]+ and [M Cl]+ at m/z 560 and 502, respectively, which was confirmed by isotope pattern and MS2 fragmentation analysis, indicating that the diethyl acetal moiety was preserved. Under the same reaction conditions, precursor 3a reacts with pta to form the putative RAPTA complex, but with some degradation of the dimethyl acetal moiety. In the 1H NMR spectrum, the presence of the upfield CHO resonance was clearly observed after pta treatment, constituting a ca. 10% conversion. The lower stability of the dimethyl acetal complex in comparison to the diethyl acetal derivative was not unexpected due to the higher acidity of the dimethyl acetal group, and it was not pursued further.18 Single crystals of 4 were obtained by vapor diffusion of diethyl ether into chloroform solution. X-ray diffraction analysis confirmed the “piano-stool” structure with the pta and the chloride ligands as the “piano-legs” (Figure 4). In comparison with other RAPTA complexes, the bond lengths and angles were similar,15,19 indicating that the presence of the diethyl acetal handle did not significantly distort the coordination sphere at the Ru center (Table 1). Dimer 3b was further treated with silver oxalate and pta successively to yield 5, the oxalo-RAPTA derivative containing an oxalate ligand in place of chloride ligands. Replacement of the chloride ligands with a chelating oxalate ligand stabilized the RAPTA structure by retarding aquation and suppressing reactivity at the metal center.20 The 1H NMR spectra in D2O revealed resonances of the diethyl acetal moiety, indicating that the functional group was stable in an aqueous environment. It was further
Scheme 1. Synthesis Route for Acetal-Functionalized RAPTA Probes
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Figure 3. Molecular structures of ruthenium arene dimers 3a (left) and 3b (right).
Figure 4. Molecular structure of acetal-functionalized RAPTA probe 4.
confirmed using ESI-MS, which detected parent molecular ions with the diethyl acetal group intact, identified by isotope pattern and MS2 fragmentation analysis. 31P{1H} NMR spectroscopy in D2O also showed only one single resonance, indicating that 5 was stable in water, consistent with earlier investigations using nonfunctionalized [(η6-cymene)Ru(oxalate)(pta)]. Before applying the probe in a cellular environment, the aquatic stability of 4 was examined using time-course UV vis spectroscopy. RAPTA complexes, for example, RAPTA-C and RAPTA-T, are prone to aquation due to the displacement of its labile chloride ligands by water, forming highly reactive aqua species. Upon dissolution in water, 4 was rapidly aquated with the UV vis absorption spectra showing an isosbestic point at ca. 340 nm (Figure S1a, Supporting Information). Aquation was effectively suppressed when 4 was dissolved in an aqueous solution of 150 mM NaCl with no significant shift in the UV vis spectra (Figure S1b, Supporting Information). The suppression of aquation can be rationalized by the fact that the aquation of the first chloride ligand is highly facile compared to that of the second and can be arrested in a high chloride environment.21 As a consequence of aquation, 4 exhibited high reactivity in water. Treatment of 4 with 1 mM glutathione (GSH) at 25 °C resulted in rapid reaction, which was completed within 30 min (Figure S2a,
Supporting Information). In the presence of 150 mM NaCl, reaction with 1 mM GSH was significantly retarded with no visible change in the UV vis absorption spectra within 30 min (Figure S2b, Supporting Information). Indeed, reaction was only completed after 1 day of incubation. Oxalo-RAPTA 5, which was inert to aquation by virtue of its chelating oxalate ligands, did not react with GSH at 1 and 10 mM concentrations, providing further validation that aqua species were important for reactivity and binding (Figure S3, Supporting Information). Since the acetal functionality can be converted to aldehyde under weakly acidic conditions,22 we investigated the stability of the diethyl acetal moiety of 4 under different pH conditions using 1 H NMR spectroscopy. Mild conversion conditions were essential in order to avoid decomposition of the RAPTA core structure. At a physiological pH of 5.8 8.0 in D2O buffer, the diethyl acetal moiety was observed unchanged with characteristic resonances between 1 and 3.5 ppm (Figure S4, Supporting Information). Notably, the aldehyde resonance at the upfield region was absent. However, in the presence of 10 mM HCl, conversion from acetal to aldehyde occurred within 2 h, accompanied by the appearance of the CHO resonance at 10 ppm. Other resonances, attributable to the arene and pta ligands, remained largely unchanged, indicating that the complex remained intact under these conditions. The conversion of diethyl acetal to aldehyde was also observed for 5 under the same conditions. This mild reaction conditions provided an easy way of selectively activating the complexes at the acetal functional group. Next, the conjugation conditions via oxime coupling were investigated using 4 and 5 with o-benzylhydroxylamine by ESIMS (Scheme 2). Complexes 4 and 5 were treated with 10 mM HCl for 2 h, after which the pH was adjusted to 6 and 1.5 equivalents of the o-benzylhydroxylamine substrate were added. 1 H NMR spectroscopy was carried out after 10 mM HCl treatment to ensure conversion of the diethyl acetal species to aldehyde. After a further 2 h incubation, ESI-MS analysis on the reaction mixture of 4 revealed molecular ions at m/z = 533 corresponding to the formation of the oxime-conjugated product with loss of one chloride ligand [M Cl]+ (Figure S5, Supporting Information). Fragmentation analysis on the molecular ion produced fragments at m/z = 376 with the loss of the pta ligand [M Cl pta]+, indicating that the ion was not an aggregate between 4 and o-benzylhydroxylamine. Under the same reaction 5969
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Scheme 2. Conjugation of o-Benzylhydroxylamine to the RAPTA Probe
In cells treated with 4 and labeled with AlexaFluor488, a positive fluorescence signal was detected, corresponding to a ca. 23-fold increase in fluorescence against background (Figure 5, entry 3). Without the fluorophore, only baseline fluorescence was observed (entry 1). With the fluorophore in the absence of 4, a ca. 3-fold increase in fluorescence was detected (entry 2), indicating some nonspecific interactions between the fluorophore with the cell components, presumably endogenous carbonyl products, for example, damaged nucleotides. The positive response over the controls indicated the successful uptake of complex 4 into cells, which can be postlabeled with a functional group-selective fluorophore.
Figure 5. Postlabeling of HEK cells treated with 4 using Alexa488 hydroxylamine dye: (1) cells treated with 4 only, (2) cells treated with dye only, (3) cells treated with both 4 and dye.
conditions, 5 also conjugated specifically at the aldehyde functional group, but with the loss of the oxalate ligand. Notably, coordination of the nucleophilic o-benzylhydroxylamine substrate to the Ru(II) center was not observed. Furthermore, in the absence of 10 mM HCl required for aldehyde conversion, both 4 and 5 did not conjugate with o-benzylhydroxylamine, indicating that the simple diethyl acetal deprotection could be used to selectively activate the RAPTA probe prior to postlabeling. As a proof-of-concept, 4 was used on live mammalian cells to investigate the feasibility of postlabeling covalent RAPTA adducts after treatment. In the proposed workflow, the cells were treated with 4, fixed, activated using aqueous HCl solution, and labeled with a hydroxylamine-functionalized fluorophore. Therefore, HEK cells were treated with 4 for 6 h, fixed with cold ethanol, activated, and labeled with AlexaFluor488 hydroxylamine fluorophore in a 96-well microplate (Figure 5). Fluorescence levels were read using a microplate reader, and controls were carried out by treating the cells with either 4 or the fluorophore.
’ CONCLUSIONS A method for selective postlabeling of covalent RAPTA adducts was developed with the ultimate goal of isolating and identifying cellular biological targets of RAPTA compounds using an acetal-functionalized probe. The method exploits the conversion of diethyl acetal to aldehyde groups under weakly acidic conditions and its facile conjugation to hydroxylamine binding partners. One possible limitation is the presence of endogenous carbonyl moieties that may interfere with hydroxylamine coupling. In addition, the possibility that the acetal moiety may modulate the binding efficacy of the metal to protein biomolecules cannot be ruled out. However, this feature is mitigated by the fact that the RAPTA probe is water-soluble and exhibits similar aquation properties to RAPTA-C and RAPTA-T, indicating that the probe is functionally similar. The positive validation of the approach augurs well for its application as a probe for the biological target elucidation of RAPTA complexes. ’ ASSOCIATED CONTENT
bS
Supporting Information. Crystallographic data (CIF) for the complexes reported in this paper, their NMR and ESIMS spectra, and UV vis spectra. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. 5970
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Organometallics
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’ ACKNOWLEDGMENT The authors thank Lip Lin Koh, Geok Kheng Tan, and Yimian Hong for performing the X-ray crystallographic analysis. Start-up funding from the National University of Singapore (W.H.A.) is gratefully acknowledged. ’ REFERENCES (1) (a) Ang, W. H.; Dyson, P. J. Eur. J. Inorg. Chem. 2006, 4003–4018. (b) Hartinger, C. G.; Dyson, P. J. Chem. Soc. Rev. 2009, 38, 391–401. (2) (a) Hartinger, C. G.; Zorbas-Seifried, S.; Jakupec, M. A.; Kynast, B.; Zorbas, H.; Keppler, B. K. J. Inorg. Biochem. 2006, 100, 891–904. (b) Bergamo, A.; Sava, G. Dalton Trans. 2007, 1267–1272. (3) (a) S€uss-Fink, G. Dalton Trans. 2010, 39, 1673–1688. (b) Bruijnincx, P. C.; Sadler, P. J. Adv. Inorg. Chem. 2009, 61, 1–62. (4) Scolaro, C.; Bergamo, A.; Brescacin, L.; Delfino, R.; Cocchietto, M.; Laurenczy, G.; Geldbach, T. J.; Sava, G.; Dyson, P. J. J. Med. Chem. 2005, 48, 4161–4171. (5) Ang, W. H.; Casini, A.; Sava, G.; Dyson, P. J. J. Organomet. Chem. 2011, 696, 989–998. (6) Wu, B.; Ong, M. S.; Groessl, M.; Adhireksan, Z.; Hartinger, C. G.; Dyson, P. J.; Davey, C. A. Chem.—Eur. J. 2011, 17, 3562–3566. (7) Bergamo, A.; Masi, A.; Dyson, P. J.; Sava, G. Int. J. Oncol. 2008, 33, 1281–1289. (8) (a) Casini, A.; Gabbiani, C.; Sorrentino, F.; Rigobello, M. P.; Bindoli, A.; Geldbach, T. J.; Marrone, A.; Re, N.; Hartinger, C. G.; Dyson, P. J.; Messori, L. J. Med. Chem. 2008, 51, 6773–6781. (b) Fidler, I. J. Nat. Rev. Cancer 2003, 3, 453–458. (9) Dirksen, A.; Dawson, P. E. Bioconjugate Chem. 2008, 19, 2543– 2548. (10) SMART, version 5.628; Bruker AXS Inc.: Madison, WI, 2001. (11) SAINT+, version 6.22a; Bruker AXS Inc.: Madison, WI, 2001. (12) Sheldrick, G. W. SADABS, version 2.10; University of Gottingen: Gottingen, Germany, 2001. (13) SHELXTL, version 6.14; Bruker AXS Inc.: Madison, WI, 2000. (14) Kalia, J.; Raines, R. T. Angew. Chem., Int. Ed. 2008, 47, 7523– 7526. (15) Ang, W. H.; Daldini, E.; Juillerat-Jeanneret, L.; Dyson, P. J. Inorg. Chem. 2007, 46, 9048–9050. (16) De, S. K.; Gibbs, R. A. Tetrahedron Lett. 2004, 45, 8141–8144. (17) (a) Bown, M.; Bennett, M. A. Acta Crystallogr., Sect. C 1999, ubrilo, J.; Hartenbach, I.; Schleid, T.; Winter, R. F. Z. 55, 852–854. (b) C Anorg. Allg. Chem. 2006, 632, 400–408. (c) Vieille-Petit, L.; Therrien, B.; S€uss-Fink, G. Acta Crystallogr., Sect. E 2002, 58, m656–m657. (18) Cordes, E. H.; Bull, H. G. Chem. Rev. 1974, 74, 581–603. (19) (a) Allardyce, C. S.; Dyson, P. J.; Ellis, D. J.; Heath, S. L. Chem. Commun. 2001, 1396–1397. (b) Ang, W. H.; Parker, L. J.; De Luca, A.; Juillerat-Jeanneret, L.; Morton, C. J.; Lo Bello, M.; Parker, M. W.; Dyson, P. J. Angew. Chem., Int. Ed. 2009, 48, 3854–3857. (c) Renfrew, A. K.; Phillips, A. D.; Tapavicza, E.; Scopelliti, R.; Rothlisberger, U.; Dyson, P. J. Organometallics 2009, 28, 5061–5071. (20) Ang, W. H.; Daldini, E.; Scolaro, C.; Scopelliti, R.; JuilleratJeannerat, L.; Dyson, P. J. Inorg. Chem. 2006, 45, 9006–9013. (21) Scolaro, C.; Hartinger, C. G.; Allardyce, C. S.; Keppler, B. K.; Dyson, P. J. J. Inorg. Biochem. 2008, 102, 1743–1748. (22) Wuts, P. G. M.; Greene, T. W. Protection for the Carbonyl Group. Greene’s Protective Groups in Organic Synthesis; John Wiley & Sons, Inc.: Hoboken, NJ, 2006; pp 431 532.
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dx.doi.org/10.1021/om200783r |Organometallics 2011, 30, 5965–5971
