Palladium-Mediated Incorporation of Carboranes into Small Molecules, Peptides, and Proteins

Abstract

Carboranes represent a class of compounds with increasing therapeutic potential. However, few general approaches to readily embed carboranes into small molecules, peptides, and proteins are available. We report a strategy based on palladium-mediated C-X (X = C, S, and N) bond formation for the installation of carborane-containing moieties onto small molecules and peptides. We demonstrate the ability of Pd-based reagents with appropriate ligands to overcome the high hydrophobicity of the carborane group and enable chemoselective conjugation of cysteine residues at room temperature in aqueous buffer. Accordingly, carboranes can be efficiently installed on proteins by employing a combination of a bis-sulfonated biarylphosphine-ligated Pd reagent in an aqueous histidine buffer. This method is successfully employed on nanobodies, a fully synthetic affibody, and the antibody therapeutics trastuzumab and cetuximab. The conjugates of the affibody Z_HER2 and the trastuzumab antibody retained binding to their target antigens. Conjugated proteins maintain their activity in cell-based functional assays in HER2-positive BT-474 cell lines. This approach enables the rapid incorporation of carborane moieties into small molecules, peptides, and proteins for further exploration in boron neutron capture therapy, which requires the targeted delivery of boron-dense groups.

Figure 1.

Palladium oxidative addition complexes (Pd-OACs) mediate incorporation of carborane groups into therapeutically relevant molecules. (A) Boron delivery agents used in the clinic for BNCT. (B) Versatile access to a variety of carborane-bearing molecules (peptides, proteins, small molecules, etc.) utilizing two Pd-mediated cross-coupling strategies. (C) Carborane-containing small molecules and proteins prepared in this work.

Figure 2.

The incorporation of a (hetero)aryl linker permitted successful formation of a variety of Pd-OACs. (A) Utilizing structures based on scaffold III, we avoided the equilibrium mixture of I and II which strongly favors I. (B) Pd-OACs containing carboranes linked via phenyl, pyridine, pyrimidine, or benzamide groups.

Figure 3.

Two complementary approaches featuring carboranes with embedded electrophilic groups (strategy 1) or nucleophilic groups (strategy 2) provided carborane-containing small molecules, peptides, and proteins. (A) Strategy 1: a range of nucleophiles are compatible with Pd cross-coupling chemistry including thiols, amines, and boronic acids. Amino acid derivatives of cysteine were prepared which combine the properties of BSH and BPA. (B) Strategy 2: Pd-bearing molecules (gefitinib and ribaroxaban) are treated with a meta-carborane-1-thiol to provide thioether-linked carborane derivatives. Yields are given as an average of two runs. Conditions for C–S bond formation, ^aPd = t-BuXPhos Pd G6 TES or 3; b 2-MeTHF, 50 °C; ^cTFA/DCM, 2:3 (v/v), RT; ^dTHF/H₂O 2:1 (v/v) as the solvent, 9 as the OAC reagent. ^e[%] indicates yield of reactions carried out with stoichiometric Pd OAC III. ^fDMSO as the solvent, 4 as the OAC reagent. THF = tetrahydrofuran; DMF = N,N-dimethylformamide; DBU = 1,8-diazabicyclo(5.4.0)undec-7-ene; RT = room temperature.

Figure 4.

Utilizing bsSPhos-based Pd-OACs, near-quantitative carborane conjugation was achieved on mPA (mutant protective antigen K563C protein) in ≥95% aqueous buffer when treated with a variety of biarylphosphine-supported Pd carborane-containing reagents.

Figure 5.

Utilizing a bsSPhos-supported Pd-aryl-carborane complex in histidine buffer afforded >95% phenyl-carboranylation (–Ph-oCB) of therapeutically relevant proteins, avoided protein precipitation, and modulated reaction kinetics. (A) VHH-H11 nanobody that targets cytotoxic T-cell lymphocyte-associated protein 4 (CTLA-4) was conjugated with 97% conversion and isolated in 51% yield (VHH-H11_Cys, calcd. 13,259 Da, obsd. 13,259 Da; VHH-H11_Cys–Ph-oCB, calcd. 13,478 Da, obsd. 13,478 Da). (B) Histidine as an additive gave near-quantitative yield of the VHH-H11 nanobody conjugate within 6 h (blue) with the corresponding consumption of starting material (black). (C) The minimized antibody, the affibody Z_HER2, and trastuzumab (Tmab) both converted into the conjugated proteins in high yields. Loading of carboranes (¹⁰B) on the antibody can be controlled by the equivalents of Pd-OACs. Up to 73 ¹⁰B atoms were attached to Tmab in His buffer with >80% recovery of the conjugated antibody (Z_HER2‑Cys, calcd. 7470 Da, obsd. 7470 Da. Z_HER2‑Cys–Ph-oCB, calcd. 7680 Da, obsd. 7680 Da). (D) The affibody, once conjugated with Ph–o-CB, gives picomolar binding affinity by bio-layer interferometry, in line with reported literature values. (E) Tmab conjugate gives binding data comparable to those of wild-type Tmab with picomolar binding affinity (see Supporting Information Figure S28).

Figure 6.

Carborane-conjugated proteins demonstrated comparable activity in BT-474 cells relative to the wild-type variants. (A) Cell viability assay of the protective antigen-carborane conjugate (PA–Ph-oCB) compared to that of the wild-type protective antigen (PA) (both at 100 nM) in the anthrax-based delivery system with addition of the lethal factor N-terminus (LF_N) conjugated to the diphtheria toxin A chain (DTA). (B) Cell proliferation inhibition by Tmab and the corresponding carborane conjugate Tmab-(Ph-oCB)₄. Cetuximab (Cmab) and PA + LF_N-DTA were used as negative and positive controls, respectively. BT474 cell lines were incubated with proteins at a 100 nM concentration. Cell viability was determined by the relative luminescence from a CellTiter-Glo assay after 72 h incubation with the indicated compounds. Experiments were carried out in triplicate with error bars depicted.