Preparation of Photo-Cross-Linked Small Molecule Affinity Matrices for Affinity Selection of Protein Targets for Biologically Active Small Molecules

  • Hiroshi Takayama
  • Takashi Moriya
  • Naoki KanohEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 800)


Small molecule-immobilized affinity matrices are indispensable tools in chemical genomics to screen and purify protein targets for biologically active small molecules. Usually, prior to immobilization, small ­molecules have to be derivatized at a position that does not significantly abrogate the intrinsic biological activity, or chemically synthesized to have an appropriate functional group for the immobilization chemistry. Here, we describe a photo-cross-linking technique to immobilize biologically active small molecules for protein target screening, without the need for chemical derivatization.

Key words

Photo-cross-linking Affinity matrices Biologically active small molecule Immobilization Diazirine Photo-generated carbine 



This work is supported by a Grant-in-Aid for Young Scientists (A) (No. 19681023; to NK), a Grant-in-Aid for Scientific Research (B) (No. 22310132; to NK), and a Grant-in-Aid for JAPS fellows (No. 2255053; to HT). We also thank Prof. Takayuki Doi and Dr. Masahito Yoshida for their assistance with the LC/MS analysis.


  1. 1.
    Leslie BJ, Hergenrother PJ (2008) Identification of the cellular targets of bioactive small organic molecules using affinity reagents. Chem Soc Rev 37: 1347–1360PubMedCrossRefGoogle Scholar
  2. 2.
    Harding MW, Galat A, Uehling DE et al (1989) A receptor for the immunosuppressant FK506 is a cis-trans peptidyl-prolyl isomerase. Nature 341: 758–760PubMedCrossRefGoogle Scholar
  3. 3.
    Taunton J, Hassig CA, Schreiber SL (1996) A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p. Science 272: 408–411PubMedCrossRefGoogle Scholar
  4. 4.
    Kanoh N, Kumashiro S, Simizu S et al (2003) Immobilization of natural products on glass slides by using a photoaffinity reaction and the detection of protein-small-molecule interactions. Angew Chem Int Edit 42: 5584–5587CrossRefGoogle Scholar
  5. 5.
    Kanoh N, Honda K, Simizu S et al (2005) Photo-cross-linked small-molecule affinity matrix for facilitating forward and reverse chemical genetics. Angew Chem Int Edit 44: 3559–3562CrossRefGoogle Scholar
  6. 6.
    Kanoh N, Nakamura T, Honda K et al (2008) Distribution of photo-cross-linked products from 3-aryl-3-trifluoromethyldiazirines and alcohols. Tetrahedron 64: 5692–5698CrossRefGoogle Scholar
  7. 7.
    Kanoh N, Takayama H, Honda K et al (2010) Cleavable linker for photo-cross-linked small-molecule affinity matrix. Bioconjugate Chemistry 21: 182–186PubMedCrossRefGoogle Scholar
  8. 8.
    Drahl C, Cravatt BF, Sorensen EJ (2005) Protein-reactive natural products. Angew Chem Int Edit 44: 5788–5809CrossRefGoogle Scholar
  9. 9.
    Hatanaka Y, Nakayama H, Kanaoka Y (1993) An improved synthesis of 4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoic acid for photoaffinity-labeling. Heterocycles 35: 997–1004CrossRefGoogle Scholar
  10. 10.
    Girgenti E, Ricoux R, Mahy JP (2004) Design and synthesis of a Mn(III)-porphyrin steroid conjugate used as a new cleavable affinity label: on the road to semi-synthetic catalytic antibodies. Tetrahedron 60: 10049–10058CrossRefGoogle Scholar
  11. 11.
    Gaur RK, Gupta KC (1989) A spectrophotometric method for the estimation of amino-groups on polymer supports. Anal Biochem 180: 253–258PubMedCrossRefGoogle Scholar
  12. 12.
    Kawatani M, Okumura H, Honda K et al (2008) The identification of an osteoclastogenesis inhibitor through the inhibition of glyoxalase I. P Natl Acad Sci USA 105: 11691–11696CrossRefGoogle Scholar
  13. 13.
    Miyazaki I, Simizu S, Okumura H et al (2010) A small-molecule inhibitor shows that pirin regulates migration of melanoma cells. Nat Chem Biol 6: 667–673PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Graduate School of Pharmaceutical SciencesTohoku UniversitySendaiJapan

Personalised recommendations