Bioorthogonal Azide–Thioalkyne Cycloaddition Catalyzed by Photoactivatable Ruthenium(II) Complexes

Abstract Tailored ruthenium sandwich complexes bearing photoresponsive arene ligands can efficiently promote azide–thioalkyne cycloaddition (RuAtAC) when irradiated with UV light. The reactions can be performed in a bioorthogonal manner in aqueous mixtures containing biological components. The strategy can also be applied for the selective modification of biopolymers, such as DNA or peptides. Importantly, this ruthenium‐based technology and the standard copper‐catalyzed azide–alkyne cycloaddition (CuAAC) proved to be compatible and mutually orthogonal.

1 H, 13 C , 19 F and 31 P NMR spectra were collected on a 300 MHz (Varian), 400 MHz (Varian) or 500 MHz (Bruker and Varian) in CDCl3, CD2Cl2, CD3OD, DMSO-d6 or DMF-d7. Carbon types and structure assignments were determined from DEPT-NMR. NMR spectra were analyzed using MestreNova© NMR data processing software (www.mestrelab.com). Abbreviations to denote the multiplicity of the signals are s (singlet), d (doublet), t (triplet), q (quartet), quint (quintet), sex (sextet), m (multiplet) and their corresponding combinations. Routine mass spectra were acquired using ITMS Bruker AmaZon SL at CIQUS, High Resolution Mass Spectrawere recordedusing electrospray ionization (ESI) recorded at the CACTUS facility of the University of Santiago de Compostela or at the University of Vigo. HPLC-MS analysis was carried out using Bruker Amazon IT/MS with C18 column using coumarin (2H-chromen-2-one) as internal standard.
Reactions that required the photoactivation of the [Cp*Ru(arene)]X complexes were irradiated at 365 nm with a UV-B LED (Custom apparatus by ThorLabs) for the indicated time, using the following setup.
The resulting solution was refluxed overnight, the solvent was removed under vacuum and the residue was dissolved in a water/Et2O mixture (1:1, 10 mL). The aqueous fraction was retained and washed with Et2O (3 x 5 mL). The aqueous layer was mixed slowly with an aqueous solution of NaBPh4 (5 mL, 0.30 M). The resulting precipitate was filtered and washed with Et2O. If necessary, Ru3 can be further purified through a short column of neutral alumina using acetone as eluent. Yellow-mustard coloured fractions were collected affording [Cp*Ru(naphthalene)]BPh4 (Ru3) as yellow needles (94% yield). The NMR data are in accordance with those previously reported. [  The synthesis of [Cp*Ru(pyrene)]PF6 (Ru4) was carried out according a reported procedure: [12] In an dried Schlenk tube, filled with N2, [Cp*Ru(MeCN)3]PF6 (100 mg, 0.19 mmol, 1.0 eq.) was added to a solution of pyrene (40.6 mg, 0.20 mmol, 1.05 eq.) in degassed 1,2-dichloroethane (5 mL The synthesis of (Ru5) was carried out adapting the previous procedure for the synthesis of Ru4: [12]  Scheme S1. Tentative mechanistic proposal for the formation of Ru2'.

RuAtAC promoted by Ru2 in CH2Cl2
(exemplified for the reaction of 1a and 2a)

Performance of Ru1-Ru4 in the Reaction of 1d and 2a at Micromolar Conditions
Besides the comparison shown in Figure 2

Assessment of the Stability of Ru4 in Biologically Relevant Media
Ruthenium complex Ru4 (2.5 μL from a stock solution 10 mM in DMSO) was added to a vial containing 500 μL of the corresponding reaction media (HeLa Cell lysates or DMEM), and the mixture was stirred for 24 h at rt.

After the indicated time the solution was diluted with MeOH and analyzed by HPLC MS.
In neither case decomposition of the catalyst was observed.