Self‐Liganded Suzuki–Miyaura Coupling for Site‐Selective Protein PEGylation

The discovery and development of palladium-catalyzed carbon–carbon bond formation has revolutionized modern organic chemistry in recent decades. The versatility and robustness of these processes have enabled major progress in total synthesis, materials development, and medicinal chemistry. Currently, a large variety of nucleophiles and electrophiles can be efficiently coupled, and part of the efforts to increase the utility of these reactions are now directed towards the development of simpler, more active, and economic catalytic systems that work under mild conditions. Owing to their broad functional-group tolerance and low associated toxicity, as well as their chemical and biological compatibility, Pd-catalyzed reactions are now attractive approaches for protein modification. These processes are particularly interesting, as a variety of non-natural amino acids can be genetically incorporated into peptides and proteins, thus providing specific chemical handles for a number of post-translational manipulations, including palladium-mediated reactions. Recent contributions from our group and others report robust and highly efficient Suzuki–Miyaura and Sonogashira cross-coupling on protein substrates bearing a halide or alkyne moiety, respectively, under ambient conditions both in vitro and on cell surfaces. As part of our continuing interest in developing chemical tools for the mild and specific post-translational modification of proteins, we investigated the use of the Suzuki–Miyaura coupling to site-selectively attach polyethylene glycol (PEG) chains to proteins. Protein PEGylation is a widespread approach to improve the stability and pharmacokinetics of protein drugs by reducing clearance rates, while providing a steric shield from proteolytic enzymes and immune system recognition. However, reduced biological activity is often observed for PEG-conjugated proteins. This effect is attributed to steric crowding resulting from the low positional control achieved with traditional PEGylation chemistry; commonly used PEG-derivatives react with the side chains of natural amino acids, such as lysines or cysteines, or protein termini and disulfide bonds. Whenmore than one reactive site is present on the protein, these approaches lead to heterogenous mixtures of potentially less-/in-active conjugates that are difficult to separate. The possibility of siteselectively PEGylating proteins could potentially overcome this problem but, to date, only a few examples use the PEGderivatization of genetically-encoded non-natural amino acids. These include Sonogashira coupling at homopropargyl-glycine, and triazole formation or Staudinger phosphite reaction at p-azidophenylalanine. The two former examples involve metal catalysts and required exogenous ligands and consequent optimization. Airand moisture-stable Pd-ligands provide convenient systems that do not require inert atmosphere or degassed solvents. Nonetheless, even more direct “ligandless” (selfliganded or lacking an added ligand) methods are of significant interest. This would provide a more straightforward method that is particularly attractive in biotechnological applications to be conducted on scale such as PEGylation. Pdnanoparticle-based catalysts and metal catalysts stabilized by polymers such as polyethylene glycol (PEG) have been reported. Taking advantage of these potential stabilizing properties of PEG, we considered the possibility of direct palladium-catalyzed PEGylation of halogenated amino acids with PEG–boronic acid derivatives in the absence of additional ligand. Herein, we report PEGylation of amino acids, peptides, and proteins through palladium-catalyzed Suzuki–Miyaura coupling of 2 kDa and 20 kDa monomethoxy PEG (mPEG) phenylboronic acids (mPEG2k-PBA and mPEG20k-PBA, respectively). An evaluation of pyrimidineor guanidinebased palladium complexes and comparison with PEG reagents alone reveals that the latter can act as potent selfliganding agents for the stabilization of metal species, thus allowing complete conversion using water-soluble palladium salts in the absence of external ligands. The resulting reaction system combines the advantages of reaction biocompatibility for protein modification with the possibility of combining a highly simple, single-component, economic, and environmentally friendly catalyst with a self-liganding coupling partner. We believe this shows the first example of ligandless metal-mediated ligation on a protein surface. Our initial study focused on the PEGylation of N-Boc-4iodo-l-phenylalanine (1; Boc-pIPhe) with mPEG20k-PBA (2) by Suzuki–Miyaura cross-coupling in 20 mm phosphate buffer, pH 8.0. The palladium catalyst system first tested used an additional ligand 2-amino-4,6-dihydroxy-pyrimidine (L1), [*] Dr. A. Dumas, C. D. Spicer, Dr. Z. Gao, Y. A. Lin, Prof. B. G. Davis Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford OX13TA (UK) E-mail: ben.davis@chem.ox.ac.uk