CellMosaic’s Phenyldiazirine sxLink™ (T2A14) is a proprietary photo-crosslinking reagent developed at CellMosaic for studying biomolecular interactions. This reagent combines the highly efficient carbene-generating phenyldiazirine group with a cleavable disulfide crosslinking group via a super hydrophilic AqueaTether™ (AqT™) based linker. These structural features make it highly advantageous to use this reagent over traditional photo-crosslinking reagents.

1. Trifluoromethyl phenyldiazirine (TFMP) group: TFMP photolyzes around 360 nm, at which photodamage to biomolecules is minimized. The generated carbene inserts C–H bonds into the neighboring biomolecular partner within picoseconds. Because the electron-withdrawing trifluoromethyl group confers stability on the intermediate diazo-isomer, no side products are detected under normal labeling conditions.
2. Reversible disulfide for chemical crosslinking of any biomolecule containing a free thiol. Figure 1 shows a workflow for how a reversible sxLink™ can be used to crosslink the interacting biomolecule partner and identify the crosslinking site or detect the interaction partner.
3. Hydrophilic AqT™ linkers: TFMP group is highly hydrophobic, biomolecules labeled with a TFMP using a traditional ethylene and ethylene glycol-type linker tend to aggregate and destabilize the labeled protein. T2A14 AqT™ linker is made by linking two threitols together, greatly increasing the hydrophilicity of sxLink™ (Figure 2) and its water solubility (2.2 mg/mL saturated solution). The molecule is also more biocompatible with decreased non-specific hydrophobic interactions with other biomolecules, allowing high loading of phenyldiazirine groups.

The product is sold as 1 vial of 250 µg (Cat# CM81401-250UG) or 4 vials of 250 µg (Cat# CM81401-4x250UG) lyophilized powder. One vial of dissolution buffer is included

Chemical Information

• Chemical name: Phenyldiazirine sxLink™ (T2A14)
• Chemical formula: N/A
• Molecular weight: 633.66 Da           CAS number: N/A

Specification

• Physical appearance: white lyophilized powder in a 1.5 mL centrifuge tube
• Storage temp.: -20 °C
• Purity: ≥ 95 % by HPLC

Application of the Product

• Labeling biomolecules containing free thiol groups
• Studying dynamic biomolecule interactions via photo-crosslinking (e.g., studying biological complexes and networks, protein-protein interactions, protein-DNA interactions, small ligand-protein interactions)

Key Features of the Product

• More hydrophilic than traditional photo-crosslinking reagent, soluble, and biocompatible
• High loading with minimized aggregation
• Reversible linkage
• Contains an efficient carbene-generating photo-crosslinking group

Figure 1. sxLink™ workflow using water-soluble Phenyldiazirine sxLink™ (T2A14)

Figure 2. C18 HPLC analysis of 4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoic acid (TFDB, red, in DMSO) and sxLink™ (blue, in water).

References

Protein crosslinking reviews: a) Brunner, J. (1993) Annu. Rev. Biochem. 62, 485−514. b) Freedman, R. B. (1979) Trends Biochem. Sci. 193–197. c) Herrmann, J. M., Westermann, B., Neupert, W. (2001) Methods Cell Biol. 65, 217–230. d) Fancy, D. A. (2000) Curr. Opin. Chem. Biol. 4, 28–32. e) Fasold, H., Klappenberger, J., Meyer, C., Remold, H. (1971) Angew. Chem. Internat. Edit. 10, 795–801. f) Bayley, H. Photogenerated Reagents in Biochemistry and Molecular Biology, Vol. 12. Elsevier, Amsterdam, Neth, 1983.

3-Trifluoromethyl-3-phenyldiazirine reference: Brunner, J., Senn, H. & Richards, F. M. (1980) J. Bio. Chem. 255, 3313−3318.

 Thiol cleavable photo-crosslinking reagents for studying rhodopsin and transducing interactions: a) Resek, J. F., Bhattacharya, S. & Khorana, H. G. (1993) J. Org. Chem. 58, 7598−7601. b) Resek, J. F., Farrens, D. & Khorana, H. G. (1994) Proc. Natl. Acad. Sci. USA 91, 7643−7647. c) Cai, K, Itoh, Y. & Khorana, H. G. (2001) Proc. Natl. Acad. Sci. USA 98, 4877−4882. d) Huang Y, Khorana HG. (2003) Mapping of Contact Sites in Interaction between Transducin and Light-Activated Rhodopsin. Presented at 17th Symposium of the Protein Society, July 26–30, Boston, Massachusetts.