Novel octavalent cross-linker displays efficient trapping of protein– protein interactions{

Transient protein–protein interactions regulate a diversity of cellular responses. Currently, it is difficult to predict such interactions a priori from sequence information. Thus, methods to characterise protein–protein interactions are of significant interest for cell biology as well as the development of small molecule modulators of such interfaces. A comparison of wide-scope studies of the Saccharomyces cerevisiae protein interactions, including yeast two-hybrid and tandem affinity purification, has highlighted poor overlap of data-sets and the need for an intersection of data derived from diverse techniques. Chemical cross-linking provides covalent capture of transient protein–protein interactions and can facilitate topological analysis using mass spectrometry. A limitation of cross-linking reagents is that tether length and reactivity must be optimized for individual protein–protein complexes. Furthermore, it is difficult to discriminate between intervs. intra-molecular links or nonproductive modifications arising from protein modification at one terminus of the cross-linker and, hydrolysis (for example) at the other. Recently an isotope coding strategy has been used to facilitate discrimination between some of these outcomes in the mass spectrum. A modular synthetic route that offers rapid access to a versatile arsenal of cross-linkers has also been reported, but the optimum reagent for each protein system must be selected by evaluation of each individual reagent. There remains an unmet need to augment the ratio of crosslinked compared to surface-labeled protein species. A reagent that does not require optimisation for individual protein systems would be of general utility. Increasing the efficiency of crosslinking reagents is therefore a key design criterion. This communication describes a novel cross-linker architecture that is a more efficient cross-linker than a representative panel of commercial reagents in preliminary comparative experiments. We envisaged that a resorcinarene scaffold could be elaborated to display multiple functional groups over a large surface area. In turn, this should bias the reaction toward inter-protein links by offering a greater number and span of reactive functionality than contemporary cross-linkers, thus yielding greater efficiency. The well defined geometry available from the resorcinarene architecture offers potentially useful features for future developments, such as analysis of its ‘footprint’ at the protein surface by mass spectrometry. Furthermore, various aldehydes incorporated during resorcinarene synthesis could be easily varied to allow modular enhancement of the design, for example by attaching functionality that may assist in affinity purification. N-Hydroxysuccinimidylester octavalent cross-linker, SOXL 1, was rapidly synthesised over four linear steps in y40% overall yield on a gram scale. Reaction of 4-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)benzaldehyde with resorcinol in acidic absolute ethanol at 80 uC furnished the resorcinarene precursor in 78% yield after recrystallisation from hot ethanol (Scheme 1). Subsequent alkylation of all eight resorcin[4]arene phenols proceeded in good overall yield of the octaethyl ester 2 using a two molar excess of ethyl 2-bromoacetate for each phenolate. Saponification of the resulting ethyl esters provided the octa-acid 3, which was treated with oxalyl chloride, then N-hydroxysuccinimide (NHS) to yield the prototypical resorcin[4]arene derived succinimidyl octaester cross-linker SOXL 1. We found that employing the polymersupported base piperidinomethyl polystyrene in this transformation provided high yields of essentially pure product 1. Comparison of NMR data with closely related polyether derivatives initially suggested a flattened cone structure, but we subsequently obtained crystals of the ethyl ester 2 in which the asymmetric unit contains two independent half molecules each lying about an inversion centre. This structure confirmed our assignment of the C2h isomer in which adjacent pairs of aldehyde derived groups sit on opposite faces of the central macrocycle. The terminal esters occupy a distorted rhomboid geometry, with C...C distances for the carbonyls ranging between 4.5 and 13.5 Å (Fig. 1). We then set out to compare the octaester 1 with a commercial homobivalent cross-linker disuccinimidyl suberate, DSS 4, for their efficiency to cross-link a known protein dimer. Glutathione S-transferases (GST) [E.C.2.5.1.18] exist as homodimers, although higher order oligomers have also been reported. Experiments were performed on the Schistosoma japonicum form of GST purified from E. coli transformed with the pGEX2T expression vector. Aliquots of between 1 and 8 molar equivalents of SOXL 1 were added to GST solution (2.7 mM), quenched after 1 h and separated through 10% poly-acrylamide SDS gel, with immunoblotting providing the principal means for visual inspection of the results. In the absence of cross-linker, only monomeric GST is apparent (Fig. 2(a), lane 13). At the same stoichiometry DSS was School of Chemistry, University of Nottingham, University Park, Nottingham, UK NG7 2RD. E-mail: [email protected]; Fax: 0115 95135654; Tel: 0115 9513566 Department of Pharmacy and Pharmacology, University of Bath, Calverton Down, Bath, UK BA2 7AY { Electronic supplementary information (ESI) available: General experimental details and characterisation data for compounds 1–3. See DOI: 10.1039/b701542a COMMUNICATION www.rsc.org/chemcomm | ChemComm