Exploring the Chemoselectivity towards Cysteine Arylation by Cyclometallated AuIII Compounds: New Mechanistic Insights

Abstract To gain more insight into the factors controlling efficient cysteine arylation by cyclometallated AuIII complexes, the reaction between selected gold compounds and different peptides was investigated by high‐resolution liquid chromatography electrospray ionization mass spectrometry (HR‐LC‐ESI‐MS). The deduced mechanisms of C−S cross‐coupling, also supported by density functional theory (DFT) and quantum mechanics/molecular mechanics (QM/MM) calculations, evidenced the key role of secondary peptidic gold binding sites in favouring the process of reductive elimination.

The peptide ANGELACASINI (Ala-Asn-Gly-Glu-Leu-Ala-Cys-Ala-Se-Ile-Asn-Ile, AC, Figure S1) was prepared following the solid phase peptide synthesis Fmoc protocol, using the CEM Liberty blue peptide synthesizer. The peptide was prepared on Rink amide MBHA resin (0.49 mmol·g -1 ). Fmoc deprotection was carried out with 10% w/v piperazine in NMP:EtOH Excess TFA was removed and diethyl ether (-20 °C) added to precipitate the product. The resin was filtered off and the solution was transferred into an Eppendorf tube, centrifuged at 4000 rpm for 4 min and the solution decanted to yield the crude peptide. The crude product was dissolved in distilled water up to 1 mL and purified in a semi-preparative HPLC in water (0.1% TFA) with a gradient of increasing ACN (0.1% TFA) from 10 to 40% over 32 min (Yield = 5%, MW:1174.31 g/mol) ( Figure S4).  Figure S2. Structure of CASINI.

Synthesis of CASINI (C)
The synthesis of the peptide CASINI (Cys-Ala-Ser-Ile-Asn-Ile , C, Figure S2) was prepared following the method of solid phase peptide synthesis. In detail, 204 mg of rink amide HMBA resin (0.49 mmol·g -1 ) was added to a peptide synthesis vessel. Following swelling (5 mL of 1:1 DMF: DCM for 30 min), the resin was treated with 5 mL deprotection solution (20% v/v piperidine/DMF) twice (5 min each). The resin was then washed with DMF (15 x 5 mL). Two equivalents Fmoc-amino acid was dissolved in a solution of HBTU (0.5 M in DMF, 0.57 mL, 0.4 eq) and HOBt (0.5 M in DMF, 0.55 mL, 3.8 eq.) that was activated by the addition of DIPEA (140 µL, 8 eq.) for 30 s. The solution was added into the resin and stirred for 40 min. The coupling procedure was repeated from the C to N terminus. Final deprotection was achieved by treating the resin with 5 mL deprotection solution twice (5 min each). The resin was washed with DMF (15 x 5 mL) and finally diethyl ether (5 x 5 mL). The resin was dried under nitrogen and subsequently added with the cleavage cocktail (10 mL 92.5:2.5:2.5:2.5 TFA:TIPS:DODT:H2O). The mixture was left to stir magnetically for 2 h. Excess TFA was removed and diethyl ether (-20 °C) was added to precipitate the product. The resin was filtered off and the solution was transferred into an Eppendorf tube, centrifuged at 4000 rpm for 4 min and the solution decanted to yield the crude peptide.
The crude product was dissolved in 1 mL distilled water and purified by HPLC in water (0.1% TFA) with a gradient of increasing ACN (0.1% TFA) from 10 to 40% over 46 min, (Yield = 7%, MW: 618.72 g/mol) ( Figure S5). The synthesis of the peptide LFRANALK (Leu-Phe-Arg-Ala-Asn-Ala-Leu-Lys, L, Figure S3) is following the method of solid phase peptide synthesis. 204 mg Rink amide HMBA resin (0.49 mmol g -1 ) was weighed in a peptide synthesis vessel. Following swelling (5 mL 1:1 DMF:DCM, 30 min), the resin was filtered and treated with 5 mL 20% v/v piperidine/DMF solution twice (5 min each). The resin was then washed with DMF (15 x 5 mL). Two equivalents of Fmoc amino acid were dissolved in a solution of HBTU (0.5 M in DMF, 0.57 mL, 0.4 eq.) and HOBt (0.5 M in DMF, 0.55 mL, 3.8 eq.) and activated by DIPEA (140 µL, 8 eq.) for 30 s. The solution was then added to the resin and stirred for 40 min. The coupling procedure was repeated from C to N terminus. Final deprotection was achieved by adding 5 mL 20% v/v piperidine/DMF (2 x 5 min). The resin was washed with DMF (15x5 mL) and diethyl ether (5x5 mL). The resin was dried under nitrogen and added with the cleavage cocktail (10 mL 92.5:2.5:2.5:2.5 TFA:TIPS: DODT: H2O). After stirring for 2 hours, TFA was removed and diethyl ether (-20 °C) was added to precipitate the product. The resin was centrifuged at 4000 rpm for 4 mins and the solution decanted to yield the crude peptide. The crude product was dissolved in 1 mL distilled water and purified by HPLC in water (0.1% TFA) with a gradient of ACN (0.1% TFA) increasing from 10-40% over 30 min, (Yield = 51.5%, MW: 931.12 g/mol) ( Figure S6). °C, respectively. Mass range was set at 100-3000 m/z. The crude peptide was analyzed using a method 5-70% ACN (0.1% HCO2H) in water (0.1% HCO2H) over 38 min. The pure product after the purification was analyzed by LCMS using a method 5-35% ACN (0.1% HCO2H) in water (0.1% HCO2H) over 38 min. The pure product was analyzed by UPLC using a method water (0.1% HCO2H) with increasing gradient of ACN (0.1% HCO2H) from 1 to 98% over 13 min.
High-resolution mass spectra were recorded on a Waters Synapt G2-Si quadrupole time of flight mass spectrometer operated in electrospray positive ionisation mode.

HPLC-ESI-MS studies.
High Resolution HPLC-ESI-MS spectra of gold compounds/peptide adducts were recorded on Synapt  A competition experiment, in which gold complex 1 competed for ZF vs LE, was carried out at a molar ratio of 3:1:1 (Au:ZF:LE), following the same preparation method described above, using an Acquity UPLC protein BEH C4 column (300 Å, 1.7 μm, 2.1 mm × 100 mm).

Tandem mass spectrometry.
Tandem mass spectra (ESI-MS/MS or ESI-MS 2 ) were acquired in a HPLC-MS setup by selecting the appropriate mass signal and fragmenting the parent ions at 18-34 eV.

Computational studies.
DFT calculations, with full geometry optimization, were performed on the structures of the adducts of complex 1 [Au(C CO N)Cl2] with different amino acids following the substitution of a chlorido ligand ( Figure   S28-S29) and of the cross-coupling products obtained by the reaction of compounds 1 and 2 with GSH ( Figures S30 and S31), by following computational approaches and models recently reported. [2b, 3] In detail, the M06-L DFT functional, [4] the Lanl2tz(f) [5] basis set for Au and the 6-311G(d,p) [6] basis set for the other atoms, were used. Water solvent effects were implicitly evaluated by the polarizable continuum model (PCM). [7] Transition state structures were located by the synchronous transit guided quasi-Newton method. [8] Vibration frequency analysis, within the harmonic approximation, was performed on each optimized geometry, to check whether it matched with an energy minimum or to a first-order saddle point (for transition state structures) in the potential energy surface, and to evaluate their standard Gibbs free energy values, at 298.15 K.
The standard formation Gibbs free energy values of the adducts of 1 [Au(C CO N)Cl2], reported in Table   S8, were obtained by hypothesizing the occurrence of the following reaction:  Figure 3), composed by the Asn2, Cys7 and Asn11 residues of the peptide and by the gold compound. The UFF force field [9] was used in the MM layer (atoms in wires). Full geometry optimization was followed by a frequency analysis, to confirm that the obtained structure corresponded to an energy minimum in the potential energy surface. All calculations were performed by the Gaussian 09 program package. [10] All pictures of the molecular models were produced by the UCSF Chimera software. [11] Tables ZF Cys2His2      Au-based species Figure S8 -HPLC-ESI-MS analysis of the reaction of compound 2 with the ZF Cys2His2 model peptide