Bioconjugation Methods for Coupling Targeting Ligands with Fluorescent Dyes

  • Xiaoxi LingEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1444)


Targeted molecular imaging probes are essential tools for visualization of specific molecular processes in cells and living systems. Among these, targeted fluorescent probes are widely used due to the high sensitivity and resolution of fluorescence imaging. The conventional strategy for developing targeted fluorescent probes is to couple targeting ligands with fluorescent dyes by covalent bond via bioconjugation. Here, we describe several commonly used bioconjugation methods, from traditional amide and thiol coupling, to metal-catalyzed coupling reaction and catalyst free cycloaddition.

Key words

Bioconjugation Coupling reaction Amide coupling Thiol coupling Targeted Huisgen cycloaddition Click chemistry Michael addition Suzuki coupling Fluorescent probe Molecular imaging Inverse electron demand Diels–Alder cycloaddition 


  1. 1.
    Zhang S, Shao P, Bai M (2013) In vivo type 2 cannabinoid receptor-targeted tumor optical imaging using a near infrared fluorescent probe. Bioconjug Chem 24(11):1907–1916. doi: 10.1021/bc400328m CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Lee MH, Han JH, Lee J-H, Choi HG, Kang C, Kim JS (2012) Mitochondrial thioredoxin-responding off–on fluorescent probe. J Am Chem Soc 134(41):17314–17319. doi: 10.1021/ja308446y CrossRefPubMedGoogle Scholar
  3. 3.
    Bai M, Rone MB, Papadopoulos V, Bornhop DJ (2007) A novel functional translocator protein ligand for cancer imaging. Bioconjug Chem 18(6):2018–2023. doi: 10.1021/bc700251e CrossRefPubMedGoogle Scholar
  4. 4.
    Nomura W, Ohashi N, Okuda Y, Narumi T, Ikura T, Ito N, Tamamura H (2011) Fluorescence-quenching screening of protein kinase c ligands with an environmentally sensitive fluorophore. Bioconjug Chem 22(5):923–930. doi: 10.1021/bc100567k CrossRefPubMedGoogle Scholar
  5. 5.
    Uddin MJ, Crews BC, Ghebreselasie K, Marnett LJ (2013) Design, synthesis, and structure–activity relationship studies of fluorescent inhibitors of cycloxygenase-2 as targeted optical imaging agents. Bioconjug Chem 24(4):712–723. doi: 10.1021/bc300693w CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Tafreshi NK, Huang X, Moberg VE, Barkey NM, Sondak VK, Tian H, Morse DL, Vagner J (2012) Synthesis and characterization of a melanoma-targeted fluorescence imaging probe by conjugation of a melanocortin 1 receptor (MC1R) specific ligand. Bioconjug Chem 23(12):2451–2459. doi: 10.1021/bc300549s CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Yang Z, Lee JH, Jeon HM, Han JH, Park N, He Y, Lee H, Hong KS, Kang C, Kim JS (2013) Folate-based near-infrared fluorescent theranostic gemcitabine delivery. J Am Chem Soc 135(31):11657–11662. doi: 10.1021/ja405372k CrossRefPubMedGoogle Scholar
  8. 8.
    Ranyuk E, Cauchon N, Klarskov K, Guérin B, van Lier JE (2013) Phthalocyanine–peptide conjugates: receptor-targeting bifunctional agents for imaging and photodynamic therapy. J Med Chem 56(4):1520–1534. doi: 10.1021/jm301311c CrossRefPubMedGoogle Scholar
  9. 9.
    Budin G, Yang KS, Reiner T, Weissleder R (2011) Bioorthogonal probes for polo-like kinase 1 imaging and quantification. Angew Chem Int Ed 50(40):9378–9381. doi: 10.1002/anie.201103273 CrossRefGoogle Scholar
  10. 10.
    Goodreid JD, Duspara PA, Bosch C, Batey RA (2013) Amidation reactions from the direct coupling of metal carboxylate salts with amines. J Org Chem 79(3):943–954. doi: 10.1021/jo402374c CrossRefGoogle Scholar
  11. 11.
    Carpino LA (1993) 1-Hydroxy-7-azabenzotriazole. An efficient peptide coupling additive. J Am Chem Soc 115(10):4397–4398. doi: 10.1021/ja00063a082 CrossRefGoogle Scholar
  12. 12.
    Montalbetti CAGN, Falque V (2005) Amide bond formation and peptide coupling. Tetrahedron 61(46):10827–10852. doi: 10.1016/j.tet.2005.08.031 CrossRefGoogle Scholar
  13. 13.
    Valeur E, Bradley M (2009) Amide bond formation: beyond the myth of coupling reagents. Chem Soc Rev 38(2):606–631. doi: 10.1039/b701677h CrossRefPubMedGoogle Scholar
  14. 14.
    El-Faham A, Albericio F (2011) Peptide coupling reagents, more than a letter soup. Chem Rev 111(11):6557–6602. doi: 10.1021/cr100048w CrossRefPubMedGoogle Scholar
  15. 15.
    Gruber HJ, Kada G, Pragl B, Riener C, Hahn CD, Harms GS, Ahrer W, Dax TG, Hohenthanner K, Knaus H-G (2000) Preparation of thiol-reactive Cy5 derivatives from commercial Cy5 succinimidyl ester. Bioconjug Chem 11(2):161–166. doi: 10.1021/bc990107f CrossRefPubMedGoogle Scholar
  16. 16.
    Song HY, Ngai MH, Song ZY, MacAry PA, Hobley J, Lear MJ (2009) Practical synthesis of maleimides and coumarin-linked probes for protein and antibody labelling via reduction of native disulfides. Org Biomol Chem 7(17):3400–3406. doi: 10.1039/b904060a CrossRefPubMedGoogle Scholar
  17. 17.
    Walker MA (1995) A high yielding synthesis of N-alkyl maleimides using a novel modification of the Mitsunobu reaction. J Org Chem 60(16):5352–5355. doi: 10.1021/jo00121a070 CrossRefGoogle Scholar
  18. 18.
    Hoyle CE, Lowe AB, Bowman CN (2010) Thiol-click chemistry: a multifaceted toolbox for small molecule and polymer synthesis. Chem Soc Rev 39(4):1355–1387. doi: 10.1039/b901979k CrossRefPubMedGoogle Scholar
  19. 19.
    Chinchilla R, Najera C (2011) Recent advances in Sonogashira reactions. Chem Soc Rev 40(10):5084–5121. doi: 10.1039/c1cs15071e CrossRefPubMedGoogle Scholar
  20. 20.
    Bräse S, Gil C, Knepper K, Zimmermann V (2005) Organic azides: an exploding diversity of a unique class of compounds. Angew Chem Int Ed 44(33):5188–5240. doi: 10.1002/anie.200400657 CrossRefGoogle Scholar
  21. 21.
    Ali H, Ait-Mohand S, Gosselin S, van Lier JE, Guérin B (2011) Phthalocyanine- peptide conjugates via palladium-catalyzed cross- coupling reactions. J Org Chem 76(6):1887–1890. doi: 10.1021/jo102083g CrossRefPubMedGoogle Scholar
  22. 22.
    Blackman ML, Royzen M, Fox JM (2008) Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels−Alder reactivity. J Am Chem Soc 130(41):13518–13519. doi: 10.1021/ja8053805 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Taylor MT, Blackman ML, Dmitrenko O, Fox JM (2011) Design and synthesis of highly reactive dienophiles for the tetrazine–trans- cyclooctene ligation. J Am Chem Soc 133(25):9646–9649. doi: 10.1021/ja201844c CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of RadiologyUniversity of PittsburghPittsburghUSA

Personalised recommendations