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Sep 16, 2016 - Herein, we report a robust and reproducible synthetic strategy for the synthesis of ruthenocenecarboxylic acid, giving the acid in 53% ...
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Efficient Reagent-Saving Method for the N‑Terminal Labeling of Bioactive Peptides with Organometallic Carboxylic Acids by SolidPhase Synthesis Jack C. Slootweg,†,§ H. Bauke Albada,‡,§ Daniel Siegmund,§ and Nils Metzler-Nolte*,§ †

Mercachem, Kerkenbos 1013, 6546 BB Nijmegen, The Netherlands Laboratory of Organic Chemistry, Wageningen University & Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands § Lehrstuhl für Anorganische Chemie I−Bioanorganische Chemie, Fakultät für Chemie und Biochemie, Ruhr-Universität Bochum, D-44801 Bochum, Germany ‡

S Supporting Information *

ABSTRACT: Labeling of biomolecules with organometallic moieties holds great promise as a tool for chemical biology and for the investigation of biochemical signaling pathways. Herein, we report a robust and reproducible synthetic strategy for the synthesis of ruthenocenecarboxylic acid, giving the acid in 53% overall yield. This organometallic label was conjugated via solidphase peptide synthesis in near-quantitative yield to a number of different biologically active peptides, using only 1 equiv of the acid and coupling reagents, thereby avoiding wasting the precious organometallic acid. This optimized method of stoichiometric N-terminal acylation was then also successfully applied to conjugating ferrocenecarboxylic acid and a novel organometallic ReI(CO)3 complex, showing the generality of the synthetic procedure.

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Scheme 1. Synthesis of Ruthenocene Carboxylic Acid 2 via Friedel−Crafts Acylation and Subsequent Hydrolysisa

rganometallic moieties can be used as biocompatible labels to dissect the biological behavior of molecules and to enhance or tune the activity of a bioactive molecular entity.1 If the widely applied ferrocene label is only the tip of the iceberg for potential applications of organometallic moieties in chemical biology, medicinal chemistry, and bioelectronics,2 to name a few, much more can be expected from the field of organometallic chemical biology as a whole. For example, ferrocene’s heavier, larger, but isoelectronic congener ruthenocene has aided in the elucidation of the mode of action of antibacterial peptides.3 Unfortunately, most organometallic fragments have to be prepared manually, using sometimes laborious synthetic techniques. In light of this, it is quite unfortunate that, for the derivatization of resin-bound biomolecule precursors such as peptides, peptide nucleic acid (PNA), and the oligonucleotides DNA and RNA, an excess (typically 4-fold) of the required organometallic building block is used, thereby wasting at least 75% of the material. Although the wasted material can in principle be recovered by collecting and purifying the waste, this is cumbersome and unpopular. Having an improved method for the conjugation of organometallic building blocks to peptides will allow the scientific community to explore many more biomedical properties of organometallic conjugates with biomolecules. Thus, improved synthetic strategies are desired to obtain bioactive organometallic conjugates. Our recent application of ruthenocenecarboxylic acid (2, see Scheme 1) to modulate the antibacterial activity of antimicro© XXXX American Chemical Society

a Reagents and conditions: (a) AlCl3, 2,6-dichlorobenzoyl chloride, CH2Cl2, 0 → 22 °C, 53%; (b) KOtBu, dimethoxyethane, H2O, 105 °C, quantitative.

bial peptides4 has prompted us to optimize the synthetic procedure by which this valuable compound can be prepared.5 Compound 2 was particularly interesting to us in view of its enhanced antibacterial activity and the fact that the presence of the ruthenium metal allows localization of the conjugate using techniques such as AAS6 and TEM. Using the same procedure as for the successful synthesis of ferrocenecarboxylic acid, which typically gives the targeted acid derivative from ferrocene starting material in ca. 65% overall yield, the ruthenocene derivative could only be obtained in 26% yield at most, which is due to its lower intrinsic reactivity.7 Although this was sufficient for the aforementioned initial studies of the effect of this moiety Received: July 8, 2016

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DOI: 10.1021/acs.organomet.6b00544 Organometallics XXXX, XXX, XXX−XXX

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Organometallics on the biological properties of some conjugates, an improved procedure was required for more in-depth studies. The literature procedure requires acylation of commercially available ruthenocene with 2-chlorobenzoyl chloride, followed by alkaline hydrolysis. Another method to synthesize ruthenocenecarboxylic acid is via monolithiation of ruthenocene with tert-butyl lithium and subsequent trapping with CO2.8 Several groups9 have synthesized ruthenocenecarboxylic acid via the acylation/hydrolysis method, and the reported yields vary between 47 and 65%; however, in our hands yields indeed vary broadly and numbers in this range were not achieved (up to half of the reported yields were obtained). Therefore, we proposed to use a more reactive acyl chloride in order to establish a more robust and higher-yielding route by improving the yields of both the Friedel−Crafts acylation reaction and the subsequent hydrolysis of the ketone,7 similar to what has been performed successfully for its ferrocene analogue.10 Thus, changing the acylating agent from 2chlorobenzoyl chloride to 2,6-dichlorobenzoyl chloride not only raised the yield of the Friedel−Crafts acylation from 41% to 53% but also the hydrolysis of the ketone proceeded more smoothly and its conversion to the carboxylic acid increased from 64% to 100%. Thereby the overall yield for the conversion of ruthenocene to ruthenocenecarboxylic acid is raised from 26% to 53%. Almost as important, purification is greatly facilitated by the quantitative yield of the hydrolysis step (Scheme 1b). Next, we set out to reduce the waste of this costly organometallic label in peptide labeling. We reasoned that the application of stoichiometric ratios of the reagents would be possible, even though solid-phase acylation reactions are usually performed with an excess of acylating agent (3−4 equiv) and coupling reagents (3−4 equiv of dehydrating agent and 6−10 equiv of base). Since coupling of the organometallic label is the last step in the conjugation reaction, applying 1 equiv of acylating agent leads to major amounts of labeled peptide and minor amounts of nonlabeled peptide after its cleavage from the solid support. In view of the difference in hydrophobicity of the two products, they would be easily separable by preparative HPLC purification, which often is mandatory anyway due to prior imperfections during the synthesis of the peptide. To test this hypothesis, we set out to acylate the N-terminal amino group of several bioactive peptides using stoichiometric amounts of ruthenocenecarboxylic acid 2 only. In order to screen a small set of reaction conditions, the Nterminus of Leu5-enkephalin was ruthenocenoylated using various reaction times (45 min to 16 h) and varying amounts of acylating/coupling reagent (4, 2, and 1 equiv). In all cases, freshly distilled DIPEA11 was used as base in a 2:1 ratio with respect to 2 and the coupling reagent PyBOP.12 The cleaved peptides were analyzed using HPLC (Figure 1). For comparison, nonacylated peptide was also analyzed. The HPLC trace shows that the cleaved peptide possesses a high purity. Specifically, whereas standard acylation using 4 equiv reagents for 45 min showed complete conversion, and using 2 equiv for the same time period is still acceptable; using only 1 equiv results in a poor conversion to approximately 35% labeled peptide (Figure 1, blue traces). However, increasing the coupling time to 16 h significantly improved the yield of the acylation reaction to near-quantitative yield even when only 1 equiv of acylation reagent was applied (Figure 1, red traces). Even though there is about 10% unreacted peptide left as observable in the HPLC trace, using only 1 equiv of the costly

Figure 1. HPLC traces showing the efficiency of N-terminal acylation on Leu-enkephalin in the solid phase using various amounts of the coupling reagents. The N-terminally unlabeled crude peptide (black trace) is observed at tR = 16.1 min. Coupling reactions were performed over 45 min (blue traces; 4, 2, and 1 equiv from bottom to top, respectively) and 16 h (red traces; 4, 2, and 1 equiv from bottom to top, respectively) showing the ruthenocenoylated product at tR = 19.8 min.

ruthenocenoyl label results in ruthenocene−peptide bioconjugates in very acceptable purified yields. To demonstrate the general applicability of stoichiometric couplings for bioconjugation, we synthesized a variety of ruthenocene−peptide bioconjugates. As peptides, we chose a range of different bioactive peptides, having sequences of intermediate length (6−11 amino acid residues), different Nterminal amino acid residues, and different linkers (i.e., Rink and Wang linkers) on polystyrene (PS) resin (Table 1). These peptides are known to target various cellular organelles, and attaching an organometallic label would allow specific targeting of these organelles. In every case, ruthenocene acid (RcC(O)) was successfully coupled to the N-terminus. Although the peptides contained different N-terminal residues, ranging from easily accessible (glycine (G), entry 5, Table 1) to more sterically demanding (valine (V), entry 6, Table 1) amino groups, in all cases the ruthenocene bioconjugates could be obtained in good quantities after preparative HPLC. The yields (13−33%) are in the range that is often seen for organometallic peptide conjugates, and more importantly, sufficient amounts of peptide, i.e. 10−30 mg, were obtained in all cases to perform in vitro biomedical studies. Encouraged by these results, we determined if the application of stoichiometric amounts of acylation reagent can be used as a general approach to efficiently obtain organometallic peptide bioconjugates. For this, we decided to use two other bioactive organometallic carboxylic acids: (i) the commercially available ferrocenecarboxylic acid and (ii) a Re(CO)3 complex with a tridentate bis(quinolylmethyl)amine ligand (5). Whereas ferrocenoyl (FcC(O)) is usually introduced as a redox-active moiety, the Re(CO)3 complex 5 is reported as a luminescent label and is at the same time a good model for isostructural radioactive technetium imaging agents.13 The Re(CO)3 complex 5 was prepared according to an improved procedure (Scheme 2).13 The synthesis started by preparing the tridentate ligand, via alkylation of 4-aminomethylbenzoic acid methyl ester under reflux conditions, to give bis-quinoline ligand methyl ester 3 in 84% yield. Subsequently, saponification of the methyl ester using sodium hydroxide quantitatively afforded the bis-quinoline ligand carboxylic acid 4. Finally, the Re(CO)3 complex 5 was synthesized by reacting ligand 4 with rhenium pentacarbonyl chloride in MeOH under microwave irradiation. The product 5 was obtained after precipitation in 80% yield. B

DOI: 10.1021/acs.organomet.6b00544 Organometallics XXXX, XXX, XXX−XXX

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Table 1. Yields and Characterization of RcC(O)-Biopeptide Conjugates as Obtained after Purification by Preparative HPLC peptide entry

peptide sequence

resin loading (mmol/g)

m (mg)

n (μmol)a

yield (%)b

HPLC tR (min)c

ESI-MS m/z (found/calcd)

1 2 3 4 5 6 7

RcC(O)-YGGFL-OH RcC(O)-FFKDEL-OH RcC(O)-RRPYIL-OH RcC(O)-KGGAKL-OH RcC(O)-GASDYQRL-OH RcC(O)-VVVKKKRKV-OH RcC(O)-RRRRRRRRRFF-NH2

0.78 0.78 0.78 0.78 0.78 0.53 0.48

20.6 24.6 22.3 21.3 14.6 12.8 27.5

25.4 21.0 17.1 20.1 11.4 6.7 9.2

33 27 22 26 15 13 19

20.85 20.55 18.67 16.83 17.90 17.20 16.33

813.90/814.24d 1056.05/1056.37d 1075.08/1075.47d 831.03/831.33d 1167.02/1167.40d 1341.19/1341.73d 658.80/659.02e

a For the calculation of the molecular weight (FW) one TFA counterion is included for each basic amino acid residue. bCalculations are based on 100 mg of dry peptide-containing resin on which RcC(O)OH was coupled. cDetermined on an analytical C18 column. dm/z values are for [M + H]+ ions. em/z values are for [M + 3H]3+ ions.

Scheme 2. Synthesis of Re(CO)3 Complex 5a

of Leu5-enkephalin via the procedure described above. Much to our delight, these conjugates were also successfully prepared and isolated with >95% purity via preparative HPLC in acceptable yields: i.e. 29% and 18% for FcC(O)-Enk and Re(CO)3-complex-Enk, respectively. Importantly, HPLC analysis of the crude peptides revealed that the amount of the nonlabeled peptide was less than 10%, again showing that the labeling reaction proceeded nearly quantitatively.

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CONCLUSIONS In this work an improved and robust route for the synthesis of ruthenocenecarboxylic acid (2) was developed using a different acylating reagent in the Friedel−Crafts reaction. A series of ruthenocenoylated conjugates of various bioactive peptides was successfully synthesized using stoichiometric acylation at the final coupling step during solid-phase peptide synthesis. Moreover, we demonstrated that the method is applicable for other organometallic complexes. The method described here allows a facile and efficient labeling of peptides with various organometallic carboxylic acid derivatives without unnecessarily wasting costly custom-made organometallic building blocks.

a Reagents and conditions: (a) 2-(chloromethyl)quinoline, K2CO3, MeCN reflux, 84%; (b) NaOH, H2O, dioxane, room temperature, quantitative; (c) [Re(CO)5]Cl, MeOH, 110 °C, 80%.

Crystals suitable for X-ray structural analysis were grown by slow diffusion of diethyl ether into a methanolic solution of 5. The Re complex crystallized in the triclinic space group P1̅ and adopted a distorted-octahedral geometry (Figure 2). The Re− CO bond lengths vary between 1.929 Å (Re−C3) and 1.943 Å (Re−C1). Those, as well as the Re−N bond lengths between 2.221 Å (Re−N2) and 2.238 Å (Re−N1), are in good agreement with comparable complexes in the literature.13,14 With the two complexes in hand, we synthesized the corresponding N-terminal organometallic−peptide conjugates

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EXPERIMENTAL SECTION

General Considerations. All reagents were obtained from commercial sources and used without purification. Solvents were dried according to standard protocols, distilled, and stored over molecular sieves (4 Å). TLC analysis was performed on silica gel 60 F254 aluminum sheets, and spots were visualized by UV light. Elemental analysis was performed in CHN mode. 1H NMR spectroscopy was performed in deuterated solvents at 30 °C on Bruker DPX-200 (1H) or DPX-250 MHz (13C{1H}) instruments. Chemical shifts (δ) are given in parts per million (ppm) relative to TMS, and the solvent residual signal is used as a reference. Abbreviations for peak multiplicities are s (singlet), d (doublet), t (triplet), and m (multiplet). Only absolute values of coupling constants are given in Hz. Mass spectra were measured using electrospray ionization (ESI). Analytical HPLC was performed on an automated HPLC system using a C18-AQ RP column (250 × 4.6 mm) at a flow rate of 1 mL/min. A linear gradient of 5% buffer B per min starting at 5 min of buffer A (A, H2O/MeCN/TFA, 95/5/0.1 v/ v/v; B, MeCN/H2O/TFA, 95/5/0.1 v/v/v) was used. Purification of the peptides was performed on an HPLC machine equipped with PDA detector that was coupled to an RP-18e reversed-phase column (250 × 25 mm), using a gradient similar to that for the analytical HPLC but with a flow rate of 20 mL/min. Crystallographic data for complex 5 have been deposited with the Cambridge Crystallographic Data Centre (CCDC No. 1478615). Copies can be obtained free of charge on application to the CCDC (12 Union Rd., Cambridge CB2 1EZ, U.K. (fax, + 44(1223)336-033; e-mail, [email protected]).

Figure 2. ORTEP plot of Re complex 5 (30% probability ellipsoids). Hydrogen atoms, the chloride counterion, and a cocrystallized molecule of methanol are omitted for clarity. Selected bond lengths (Å): Re−C1 1.943(5), Re−C2 1.930(4), Re−C3 1.929(5), Re−N1 2.238(3), Re−N2 2.221(3), Re−N3 2.227(3). Selected angles (deg): C1−Re−C2 91.1(2), C1−Re−C3 88.1(2), C2−Re−C3 83.0(2), N1− Re−N2 78.1(1), N1−Re−N3 75.1(1), N2−Re−N3 83.5(1), N1−Re− C3 173.8(2), N3−Re−C1 171.9(1), N2−Re−C2 171.6(1). C

DOI: 10.1021/acs.organomet.6b00544 Organometallics XXXX, XXX, XXX−XXX

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0.43 (CH2Cl2/MeOH, 9/1). 1H NMR (200 MHz, CDCl3): δ 8.12 (d, 3 JHH = 8.5 Hz, Ar-H, 2H), 8.03−7.86 (m, Ar-H, 4H), 7.81−7.58 (m, Ar-H, 6H), 7.52−7.34 (m, Ar-H, 4H), 3.92 (s, CH2-quinolyl, 4H), 3.72 (s, CH2-benzyl, 2H). 13C NMR: δ 162.0, 160.0, 147.1, 144.0, 137.1, 130.0, 129.8, 128.9, 128.3, 127.6, 127.5, 126.5, 121.0, 60.7, 58.7. MS (ESI+): calcd C28H23N3O2 433.2; m/z 433.9 [M + H]+, 455.8 [M + Na]+. [Re(CO)3((bis(2-quinolylmethyl)amino)benzoic acid)] Chloride (5). Method A. (Bis(2-quinolylmethyl)amino)benzoic acid (4) (87.0 mg; 0.20 mmol) and [Re(CO)5]Cl (72.4 mg; 0.20 mmol) were dissolved in MeOH (5.0 mL) in a microwave vessel. Then the mixture was allowed to react in a CEM discovery microwave reactor at 110 °C for 30 min. The resulting dark yellow solution was concentrated in vacuo followed by product precipitation via the addition of Et2O (45 mL). The mixture was centrifuged (5000 rpm, 5 min), the supernatant was decanted, and the precipitate was washed with Et2O (45 mL), and this centrifugation/decantation process was repeated. The precipitate was dried under a N2 atmosphere, and the product was obtained as an off-white solid (yield: 118 mg, 1.60 mmol, 80%). Method B. Following method A, (bis(2-quinolylmethyl)amino)benzoic acid (4) (40.0 mg; 92 μmol) and [Re(CO)5]Cl (33.3 mg; 92 μmol) were dissolved in MeOH (4.0 mL) in a microwave vessel and reacted in the microwave reactor at 110 °C for 15 min. The resulting yellow solution was concentrated to about 2 mL and carefully layered with cold diethyl ether. Upon standing at ambient temperature overnight, complex 5 crystallized in the form of colorless needles suitable for X-ray structural analysis. The crystals were collected via filtration and dried in vacuo (yield: 52 mg, 70 μmol, 76%). 1 H NMR (200 MHz, DMSO-d6): δ 8.64 (d, 3JHH = 8.4 Hz, Ar-H, 2H), 8.37 (d, 3JHH = 8.8 Hz, Ar-H, 2H), 8.22−7.85 (m, Ar-H, 8H), 7.80−7.57 (m, Ar-H, 4H), 5.49 (d, 2JHH = 17.7 Hz, CH2-quinolyl, 2H), 5.19 (s, CH2-benzyl, 2H), 4.83 (d, 2JHH = 17.6 Hz, CH2-quinolyl, 2H). 13 C NMR (62.5 MHz, DMSO-d6): δ 196.2, 194.4, 167.1, 165.6, 145.9, 141.4, 136.4, 132.9, 132.1, 129.8, 129.6, 128.1, 127.9, 127.5, 120.5, 67.7. MS (ESI+): calcd C31H23N3O5Re+ 704.1; m/z 703.7 [M]+. Anal. Calcd for (M + MeOH + H2O): C, 48.70; H, 3.70; N, 5.32. Found: C, 48.13; H, 3.40; N, 5.33. Solvent molecules were also found in the Xray crystal structure and detected by 1H NMR in solution, respectively.

2,6-Dichlorobenzoylruthenocene (1). Ruthenocene (500 mg; 2.16 mmol; 1 equiv) was suspended in freshly distilled dry CH2Cl2 (20 mL), and 2,6-dichlorobenzoyl chloride (279 μL; 2.16 mmol; 1 equiv) was added. The mixture was cooled in an ice−salt bath (−20 °C), and to the reaction mixture was then added anhydrous AlCl3 (288 mg; 2.16 mmol; 1 equiv) in one portion. The mixture was warmed to room temperature over the course of 30 min, and stirring was continued overnight. After this, the mixture was cooled to 0 °C, water (3.5 mL) was added, and the layers were separated in a separatory funnel. The organic layer was concentrated under reduced pressure, and the residue was dissolved in EtOAc (20 mL) and subsequently washed with water (1 × 10 mL) and brine (1 × 10 mL). The organic layer was dried over Na2SO4, the crude product was purified using column chromatography (silica; eluent n-hexane/EtOAc 5/1 (v/v)), and the desired ketone was obtained as a yellow solid (yield: 461.9 mg; 1.14 mmol; 53%). Rf: 0.26 (hexane/EtOAc 9/1 v/v). 1H NMR (CDCl3): δ 7.34−7.16 (m, 3H, aromatic H), 4.99−4.86 (m, 2H, Cp-R), 4.82−4.70 (m, 2H, Cp-R), 4.61−4.57 (s, 5H, C5H5). 13C NMR (CDCl3): δ 194.7, 138.4, 132.3, 130.5, 128.4, 84.1, 73.8, 72.9, 71.9. Anal. Found (calcd): C, 50.7 (50.51); H, 3.03 (2.99); Cl, 17.2 (17.54). Mp: 169.7− 171.1 °C. IR (cm−1): 3087 (w), 1646 (s), 1452 (m), 1429 (s), 1372 (m), 1285 (s), 1094 (m), 849 (s), 818 (s), 794 (s), 773 (s), 757 (s), 725 (m), 681 (s). Ruthenocenecarboxylic Acid (2). Bisaryl ketone 1 (461.9 mg; 1.14 mmol) was dissolved in 1,2-dimethoxyethane (8 mL), and KOtBu (1.279 g; 11.4 mmol; 10 equiv) and water (74 μL; 4.1 mmol; 3.6 mmol) were added. The mixture was stirred overnight at under reflux conditions, after which the oil bath was removed and the mixture was diluted with water (10 mL). Then, the mixture was cooled to room temperature, Et2O (20 mL) was added, and the ruthenocene carboxylate was extracted into the aqueous layer using 1 N NaOH (2 × 10 mL). The combined aqueous layers were acidified with 1 N HCl, and the desired acid was extracted into Et2O. After drying of the organic phase over Na2SO4, the filtrate was concentrated and the desired compound was obtained as a yellow solid (312 mg; 1.13 mmol; 100%). 1H NMR (DMSO): δ 12.06 (s, 1H, COOH), 5.02 (t, 3 JHH = 1.8 Hz, 2H, Cp-R), 4.74 (t, 3JHH = 1.8 Hz, Cp-R, 2H), 4.62− 4.58 (s, 5H, C5H5). 13C NMR (DMSO): δ 170.7, 76.3, 72.8, 71.6. (Bis(2-quinolylmethyl)aminomethyl)benzoic Acid Methyl Ester (3). A white suspension consisting of 2-(chloromethyl)quinoline hydrochloride (448 mg; 2.05 mmol; 2.05 equiv), 4-aminomethylbenzoic acid methyl ester hydrochloride (201 mg; 1.00 mmol; 1 equiv), and K2CO3 (690 mg; 5.00 mmol; 5 equiv) in MeCN (15 mL) was heated under reflux for 16 h. After the mixture was cooled to room temperature, the orange suspension was evaporated to dryness. The orange residue was diluted with aqueous saturated NaHCO3 (15 mL) and CH2Cl2 (20 mL). The product was extracted, the organic layer was washed with aqueous saturated NaHCO3 (15 mL, 2×) and dried (Na2SO4), and the solution was concentrated in vacuo. The red brownish, oily residue was purified by silica column chromatography (ethyl acetate/hexane 1/1 → 100% ethyl acetate), and the product was obtained as a yellow oil (yield: 376 mg, 0.84 mmol, 84%). Rf: 0.28 (EtOAc/hexane, 8/2). 1H NMR (200 MHz, CDCl3): δ 8.20−7.89 (m, Ar-H, 6H), 7.85−7.62 (m, Ar-H, 6H), 7.58−7.43 (m, Ar−H, 4H), 4.00 (s, CH2-quinolyl, 4H), 3.89 (s, COOCH3, 3H), 3.80 (s, CH2-benzyl, 2H). 13C NMR (50 MHz, CDCl3): δ 167.1, 160.1, 147.7, 144.6 136.6, 129.8, 129.6, 129.2, 129.1, 127.6, 127.5, 126.4, 121.1, 61.1, 58.6, 52.2. MS (ESI+): calcd C29H25N3O2 447.2; m/z 447.9 [M + H]+, 469.9 [M + Na]+. (Bis(2-quinolylmethyl)aminomethyl)benzoic Acid (4). (Bis(2quinolylmethyl)aminomethyl)benzoic acid methyl ester (3) (348 mg, 0.78 mmol; 1 equiv) was dissolved in dioxane (2.0 mL), a solution of NaOH in H2O (1.95 mL; 2 M; 5 equiv) was added, resulting in a slightly milky solution, and the reaction mixture was stirred at room temperature for 2 h. After dioxane was evaporated in vacuo, the solution was diluted with H2O (15 mL) and neutralized using aqueous (1 M) HCl until the product precipitated. The product was extracted with CHCl3 (3 × 60 mL), the combined organic layers were dried (Na2SO3), and after concentration in vacuo the product was obtained as a light brown solid (yield: 337 mg, 0.78 mmol, quantitative). Rf:

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.organomet.6b00544. Crystallographic data of 5 (CIF) 1 H and 13C NMR spectra of 1−5, chemical structures and HPLC traces of the purified ruthenocenecarboxylic acid, ferrocenecarboxylic acid, and rhenium tridentate carboxylic acid peptide bioconjugates, and crystallographic data of 5 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail for N.M-N.: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was financially supported by a grant from the German Federal Ministry of Science and Technology BMBF (KATMETHAN) to N.M.-N.

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REFERENCES

(1) (a) Joshi, T.; Patra, M.; Gasser, G. Inorganic Chemical Biology: Principles, Techniques and Applications; Wiley: Chichester, U.K., 2014; pp 373−401. (b) Patra, M.; Gasser, G. ChemBioChem 2012, 13, 1232− D

DOI: 10.1021/acs.organomet.6b00544 Organometallics XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.organomet.6b00544 Organometallics XXXX, XXX, XXX−XXX