1,9-Bis(2-pyridyl)-1,2,8,9-tetrathia-5-oxanonane

Disulfide crosslinking of proteins is typically performed by treating proteins bearing cysteine residues with small-molecule disulfide reagents. The process results in the formation of a mixed disulfide intermediate, which then reacts with the cysteine residue of another protein molecule to form the crosslinked product. This second step requires the intimate association of two large reactants. The ensuing steric hindrance can result in poor crosslinking yields. Here, we introduce a bis(disulfide) reagent in which activated disulfides are separated by linkers that can alleviate steric hindrance and thereby potentially increase the efficiency of crosslinking.

dithiopyridine disulfide [11] or 5,5′-dithiobis(2-nitrobenzoic acid) [12]. This reaction results in the formation of a labile mixed disulfide intermediate. In the second step, this intermediate reacts with another protein molecule bearing a reactive cysteine to form the desired crosslinked product. This second step can be problematic because it entails a reaction between two large reactants that can be impeded by steric hindrance, compromising crosslinking yields.
To mitigate steric hindrance, we designed 1,9-bis(2-pyridyl)-1,2,8,9-tetrathia-5-oxanonane (1), a reagent that contains two labile disulfides separated by a linker. Upon treatment with a protein bearing a reactive cysteine, one disulfide of the reagent reacts with the protein to form a stable mixed disulfide. In a second step, the remaining labile disulfide reacts with a cysteine residue of another protein to form the desired crosslinked product. This method is likely to increase crosslinking efficiency compared to the conventional method.
The synthesis of bis(disulfide) 1 was accomplished in a single step with commercial starting materials (Scheme 1). We used the same route to avail a longer congener, 1,12-bis(2pyridyl)-1,2,11,12-tetrathia-5,8-dioxadodecane (2). We chose oligoethylene oxides to serve as the linker within both reagents because oligo-and polyethylene oxides have favorable properties such as high water solubility, chemical inertness, and low toxicity [13]. Molecules containing polyethylene oxide backbones with various chain lengths are available. Hence, this strategy can be employed to generate reagents possessing a wide range of linker lengths, enabling utility in a variety of applications.

Instrumentation
NMR spectra were acquired with a Bruker AC+ 300 spectrometer ( 1 H NMR: 300 MHz, 13 C NMR: 75 MHz) at the Magnetic Resonance Facility in the Department of Chemistry at Madison. 13 C spectra were proton-decoupled. Mass spectra were obtained with electrospray ionization (ESI) techniques.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Scheme 1.
Route for the syntheses of bis(disulfide) 1 and 2.