Molecular conjugation using non-covalent click chemistry


Molecular conjugation refers to methods used in biomedicine, advanced materials and nanotechnology to link two partners — from small molecules to large and sometimes functionally complex biopolymers. The methods ideally have a broad structural scope, proceed under very mild conditions (including in H2O), occur at a rapid rate and in quantitative yield with no by-products, enable bioorthogonal reactivity and have zero toxicity. Over the past two decades, the field of click chemistry has emerged to afford us new and efficient methods of molecular conjugation. These methods are based on chemical reactions that produce permanently linked conjugates, and we refer to this field here as covalent click chemistry. Alternatively, if molecular conjugation is undertaken using a pair of complementary molecular recognition partners that associate strongly and selectively to form a thermodynamically stable non-covalent complex, then we refer to this strategy as non-covalent click chemistry. This Perspective is concerned with this latter approach and highlights two distinct applications of non-covalent click chemistry in molecular conjugation: the pre-assembly of molecular conjugates or surface-coated nanoparticles and the in situ capture of tagged biomolecular targets for imaging or analysis.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Non-covalent click chemistry.
Fig. 2: Pre-assembly of molecular conjugates using non-covalent click chemistry.
Fig. 3: In situ capture using non-covalent click chemistry.


  1. 1.

    Janaratne, T. K., Okach, L., Brock, A. & Lesley, S. A. Solubilization of native integral membrane proteins in aqueous buffer by noncovalent chelation with monomethoxy polyethylene glycol (mPEG) polymers. Bioconjug. Chem. 22, 1513–1518 (2011).

    CAS  Article  Google Scholar 

  2. 2.

    Kolb, H. C., Finn, M. G. & Sharpless, K. B. Click chemistry: diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. 40, 2004–2021 (2001).

    CAS  Article  Google Scholar 

  3. 3.

    Xu, Z., Wang, L., Fang, F., Fu, Y. & Yin, Z. A review on colloidal self-assembly and their applications. Curr. Nanosci. 12, 725–746 (2016).

    CAS  Article  Google Scholar 

  4. 4.

    Plutschack, M. B., Pieber, B., Gilmore, K. & Seeberger, P. H. The hitchhiker’s guide to flow chemistry. Chem. Rev. 117, 11796–11893 (2017).

    CAS  Article  Google Scholar 

  5. 5.

    Liu, K. et al. Molecular imaging probe development using microfluidics. Curr. Org. Synth. 8, 473–487 (2011).

    CAS  Article  Google Scholar 

  6. 6.

    Cheng, Z., Al Zaki, A., Hui, J. Z., Muzykantov, V. R. & Tsourkas, A. Multifunctional nanoparticles: cost versus benefit of adding targeting and imaging capabilities. Science 338, 903–910 (2012).

    CAS  Article  Google Scholar 

  7. 7.

    Marqués-Gallego, P. & de Kroon, A. I. P. M. Ligation strategies for targeting liposomal nanocarriers. Biomed Res. Int. 2014, 129458 (2014).

    Article  Google Scholar 

  8. 8.

    Abd Ellah, N. H. & Abouelmagd, S. A. Surface functionalization of polymeric nanoparticles for tumor drug delivery: approaches and challenges. Expert Opin. Drug Deliv. 14, 201–214 (2017).

    CAS  Article  Google Scholar 

  9. 9.

    Stéen, E. J. L. et al. Pretargeting in nuclear imaging and radionuclide therapy: improving efficacy of theranostics and nanomedicines. Biomaterials 179, 209–245 (2018).

    Article  Google Scholar 

  10. 10.

    Yoon, H. Y., Koo, H., Kim, K. & Kwon, I. C. Molecular imaging based on metabolic glycoengineering and bioorthogonal click chemistry. Biomaterials 132, 28–36 (2017).

    CAS  Article  Google Scholar 

  11. 11.

    Rossin, R. & Robillard, M. S. Pretargeted imaging using bioorthogonal chemistry in mice. Curr. Opin. Chem. Biol. 21, 161–169 (2014).

    CAS  Article  Google Scholar 

  12. 12.

    Patterson, D. M., Nazarova, L. A. & Prescher, J. A. Finding the right (bioorthogonal) chemistry. ACS Chem. Biol. 9, 592–605 (2014).

    CAS  Article  Google Scholar 

  13. 13.

    Freidel, C., Kaloyanova, S. & Peneva, K. Chemical tags for site-specific fluorescent labeling of biomolecules. Amino Acids 48, 1357–1372 (2016).

    CAS  Article  Google Scholar 

  14. 14.

    Rashidian, M., Dozier, J. K. & Distefano, M. D. Enzymatic labeling of proteins: techniques and approaches. Bioconjug. Chem. 24, 1277–1294 (2013).

    CAS  Article  Google Scholar 

  15. 15.

    Dundas, C. M., Demonte, D. & Park, S. Streptavidin–biotin technology: improvements and innovations in chemical and biological applications. Appl. Microbiol. Biotechnol. 97, 9343–9353 (2013).

    CAS  Article  Google Scholar 

  16. 16.

    Jain, A. & Cheng, K. The principles and applications of avidin-based nanoparticles in drug delivery and diagnosis. J. Control. Release 245, 27–40 (2017).

    CAS  Article  Google Scholar 

  17. 17.

    Miller, L. W., Cai, Y., Sheetz, M. P. & Cornish, V. W. In vivo protein labeling with trimethoprim conjugates: a flexible chemical tag. Nat. Methods 2, 255–257 (2005).

    CAS  Article  Google Scholar 

  18. 18.

    Xu, S. & Hu, H.-Y. Fluorogen-activating proteins: beyond classical fluorescent proteins. Acta Pharm. Sin. B 8, 339–348 (2018).

    Article  Google Scholar 

  19. 19.

    Uchinomiya, S., Ojida, A. & Hamachi, I. Peptide tag/probe pairs based on the coordination chemistry for protein labeling. Inorg. Chem. 53, 1816–1823 (2014).

    CAS  Article  Google Scholar 

  20. 20.

    Gatterdam, K., Joest, E. F., Gatterdam, V. & Tampé, R. Scaffold design of trivalent chelator heads dictates high-affinity and stable His-tagged protein labeling in vitro and in cellulo. Angew. Chem. Int. Ed. 57, 12395–12399 (2018).

    CAS  Article  Google Scholar 

  21. 21.

    You, C. & Piehler, J. Multivalent chelators for spatially and temporally controlled protein functionalization. Anal. Bioanal. Chem. 406, 3345–3357 (2014).

    CAS  Article  Google Scholar 

  22. 22.

    Bouhedda, F., Autour, A. & Ryckelynck, M. Light-up RNA aptamers and their cognate fluorogens: from their development to their applications. Int. J. Mol. Sci. 19, 44 (2018).

    Article  Google Scholar 

  23. 23.

    Zhang, H. et al. Assembling DNA through affinity binding to achieve ultrasensitive protein detection. Angew. Chem. Int. Ed. 52, 10698–10705 (2013).

    CAS  Article  Google Scholar 

  24. 24.

    Rodell, C. B., Mealy, J. E. & Burdick, J. A. Supramolecular guest−host interactions for the preparation of biomedical materials. Bioconjug. Chem. 26, 2279–2289 (2015).

    CAS  Article  Google Scholar 

  25. 25.

    Wang, L., Li, L.-L., Fan, Y.-s & Wang, H. Host–guest supramolecular nanosystems for cancer diagnostics and therapeutics. Adv. Mater. 25, (3888–3898 (2013).

    Google Scholar 

  26. 26.

    Liu, W., Samanta, S. K., Smith, B. D. & Isaacs, L. Synthetic mimics of biotin/(strept)avidin. Chem. Soc. Rev. 46, 2391–2403 (2017).

    CAS  Article  Google Scholar 

  27. 27.

    Peck, E. M. et al. Pre-assembly of near-infrared fluorescent multivalent molecular probes for biological imaging. Bioconjug. Chem. 27, 1400–1410 (2016).

    CAS  Article  Google Scholar 

  28. 28.

    Shaw, S. et al. Non-covalently pre-assembled high-performance near-infrared fluorescent molecular probes for cancer imaging. Chem. Eur. J. 24, 13821–13829 (2018).

    CAS  Article  Google Scholar 

  29. 29.

    van Dun, S., Ottmann, C., Milroy, L.-G. & Brunsveld, L. Supramolecular chemistry targeting proteins. J. Am. Chem. Soc. 139, 13960–13968 (2017).

    Article  Google Scholar 

  30. 30.

    Webber, M. J. et al. Supramolecular PEGylation of biopharmaceuticals. Proc. Natl Acad. Sci. USA 113, 14189–14194 (2016).

    CAS  Article  Google Scholar 

  31. 31.

    Patra, M., Zarschler, K., Pietzsch, H.-J., Stephan, H. & Gasser, G. New insights into the pretargeting approach to image and treat tumours. Chem. Soc. Rev. 45, 6415–6431 (2016).

    CAS  Article  Google Scholar 

  32. 32.

    Kim, T. H. et al. Mix to validate: a facile, reversible pegylation for fast screening of potential therapeutic proteins in vivo. Agnew. Chem. Int. Ed. 52, 6880–6884 (2013).

    CAS  Article  Google Scholar 

  33. 33.

    Liu, X., Sun, J. & Gao, W. Site-selective protein modification with polymers for advanced biomedical applications. Biomaterials 178, 413–434 (2018).

    CAS  Article  Google Scholar 

  34. 34.

    Mantooth, S. M., Munoz-Robles, B. G. & Webber, M. J. Dynamic hydrogels from host–guest supramolecular interactions. Macromol. Biosci. 19, 1800281 (2019).

    Article  Google Scholar 

  35. 35.

    Liu, Y. & Hsu, S.-h. Synthesis and biomedical applications of self-healing hydrogels. Front. Chem. 6, 449 (2018).

    CAS  Article  Google Scholar 

  36. 36.

    Diba, M. et al. Self-healing biomaterials: from molecular concepts to clinical applications. Adv. Mater. Interfaces 5, 1800118 (2018).

    Article  Google Scholar 

  37. 37.

    Sun, C. et al. Polymeric nanomedicine with “Lego” surface allowing modular functionalization and drug encapsulation. ACS Appl. Mater. Interfaces 10, 25090–25098 (2018).

    CAS  Article  Google Scholar 

  38. 38.

    Kim, S. et al. Cucurbit[6]uril-based polymer nanocapsules as a non-covalent and modular bioimaging platform for multimodal: in vivo imaging. Mater. Horiz. 4, 450–455 (2017).

    CAS  Article  Google Scholar 

  39. 39.

    Zhang, S. et al. Precise supramolecular control of surface coverage densities on polymer micro- and nanoparticles. Chem. Sci. 9, 8575–8581 (2018).

    CAS  Article  Google Scholar 

  40. 40.

    Zhou, Z., Han, Z. & Lu, Z. R. A targeted nanoglobular contrast agent from host-guest self-assembly for MR cancer molecular imaging. Biomaterials 85, 168–179 (2016).

    CAS  Article  Google Scholar 

  41. 41.

    Li, Q.-L. et al. Supramolecular nanosystem based on pillararene-capped CuS nanoparticles for targeted chemo-photothermal therapy. ACS Appl. Mater. Interfaces 10, 29314–29324 (2018).

    CAS  Article  Google Scholar 

  42. 42.

    Wang, Y.-X., Zhang, Y.-M., Wang, Y.-L. & Liu, Y. Multifunctional vehicle of amphiphilic calix[4]arene mediated by liposome. Chem. Mater. 27, 2848–2854 (2015).

    CAS  Article  Google Scholar 

  43. 43.

    Hou, C., Huang, Z., Fang, Y. & Liu, J. Construction of protein assemblies by host–guest interactions with cucurbiturils. Org. Biomol. Chem. 15, 4272–4281 (2017).

    CAS  Article  Google Scholar 

  44. 44.

    Liu, Y., Yang, H., Wang, Z. & Zhang, X. Cucurbit[8]uril-based supramolecular polymers. Chem. Asian J. 8, 1626–1632 (2013).

    CAS  Article  Google Scholar 

  45. 45.

    Samanta, S. K., Moncelet, D., Briken, V. & Isaacs, L. Metal–organic polyhedron capped with cucurbit[8]uril delivers doxorubicin to cancer cells. J. Am. Chem. Soc. 138, 14488–14496 (2016).

    CAS  Article  Google Scholar 

  46. 46.

    Park, K. M., Murray, J. & Kim, K. Ultrastable artificial binding pairs as a supramolecular latching system: a next generation chemical tool for proteomics. Acc. Chem. Res. 50, 644–646 (2017).

    CAS  Article  Google Scholar 

  47. 47.

    Zhu, H. et al. Pillararene-based host–guest recognition facilitated magnetic separation and enrichment of cell membrane proteins. Mater. Chem. Front. 2, 1475–1480 (2018).

    CAS  Article  Google Scholar 

  48. 48.

    Finbloom, J. A. & Francis, M. B. Supramolecular strategies for protein immobilization and modification. Curr. Opin. Chem. Biol. 46, 91–98 (2018).

    CAS  Article  Google Scholar 

  49. 49.

    Li, G.-P., Zhang, H., Zhu, C.-M., Zhang, J. & Jiang, X.-F. Avidin–biotin system pretargeting radioimmunoimaging and radioimmunotherapy and its application in mouse model of human colon carcinoma. World J. Gastroenterol. 11, 6288–6294 (2005).

    CAS  Article  Google Scholar 

  50. 50.

    Schubert, M. et al. Novel tumor pretargeting system based on complementary l-configured oligonucleotides. Bioconjug. Chem. 28, 1176–1188 (2017).

    CAS  Article  Google Scholar 

  51. 51.

    Strebl, M. G., Yang, J., Isaacs, L. & Hooker, J. M. Adamantane/cucurbituril: a potential pretargeted imaging strategy in immuno-PET. Mol. Imaging 17, 1536012118799838 (2018).

    Article  Google Scholar 

  52. 52.

    Spa, S. J. et al. A supramolecular approach for liver radioembolization. Theranostics 8, 2377–2386 (2018).

    CAS  Article  Google Scholar 

  53. 53.

    Kim, K. L. et al. Supramolecular latching system based on ultrastable synthetic binding pairs as versatile tools for protein imaging. Nat. Commun. 9, 1712 (2018).

    Article  Google Scholar 

  54. 54.

    Sasmal, R. et al. Synthetic host–guest assembly in cells and tissues: fast, stable, and selective bioorthogonal imaging via molecular recognition. Anal. Chem. 90, 11305–11314 (2018).

    CAS  Article  Google Scholar 

  55. 55.

    Rood, M. T. M. et al. Obtaining control of cell surface functionalizations via pre-targeting and supramolecular host guest interactions. Sci. Rep. 7, 39908 (2017).

    CAS  Article  Google Scholar 

  56. 56.

    Liu, S. et al. The cucurbit[n]uril family: prime components for self-sorting systems. J. Am. Chem. Soc. 127, 15959–15967 (2005).

    CAS  Article  Google Scholar 

  57. 57.

    Welling, M. M. et al. In vivo stability of supramolecular host–guest complexes monitored by dual-isotope multiplexing in a pre-targeting model of experimental liver radioembolization. J. Control. Release 293, 126–134 (2019).

    CAS  Article  Google Scholar 

  58. 58.

    Samanta, S. K., Moncelet, D., Vinciguerra, B., Briken, V. & Isaacs, L. Metal organic polyhedra: a click-and-clack approach toward targeted delivery. Helv. Chim. Acta 101, e1800057 (2018).

    Article  Google Scholar 

  59. 59.

    Bak, M., Jølck, R. I., Eliasen, R. & Andresen, T. L. Affinity induced surface functionalization of liposomes using Cu-free click chemistry. Bioconjug. Chem. 27, 1673–1680 (2016).

    CAS  Article  Google Scholar 

  60. 60.

    Robinson, P. V., de Almeida-Escobedo, G., de Groot, A. E., McKechnie, J. L. & Bertozzi, C. R. Live-cell labeling of specific protein glycoforms by proximity-enhanced bioorthogonal ligation. J. Am. Chem. Soc. 137, 10452–10455 (2015).

    CAS  Article  Google Scholar 

  61. 61.

    Long, M. J. C., Poganik, J. R. & Aye, Y. On-demand targeting: investigating biology with proximity-directed chemistry. J. Am. Chem. Soc. 138, 3610–3622 (2016).

    CAS  Article  Google Scholar 

  62. 62.

    Cañeque, T., Müller, S. & Rodriguez, R. Visualizing biologically active small molecules in cells using click chemistry. Nat. Rev. Chem. 2, 202–215 (2018).

    Article  Google Scholar 

  63. 63.

    Xi, W., Scott, T. F., Kloxin, C. J. & Bowman, C. N. Click chemistry in materials science. Adv. Funct. Mater. 24, 2572–2590 (2014).

    CAS  Article  Google Scholar 

  64. 64.

    Leppiniemi, J. et al. Bifunctional avidin with covalently modifiable ligand binding site. PLOS ONE 6, e16576 (2011).

    CAS  Article  Google Scholar 

  65. 65.

    Saunders, M. J. et al. Fluorogen activating proteins in flow cytometry for the study of surface molecules and receptors. Methods 57, 308–317 (2012).

    CAS  Article  Google Scholar 

  66. 66.

    Ouellet, J. RNA fluorescence with light-up aptamers. Front. Chem. 4, 29 (2016).

    Article  Google Scholar 

  67. 67.

    Zhang, J. X. et al. Predicting DNA hybridization kinetics from sequence. Nat. Chem. 10, 91–98 (2018).

    CAS  Article  Google Scholar 

  68. 68.

    Assaf, K. I. & Nau, W. M. Cucurbiturils: from synthesis to high-affinity binding and catalysis. Chem. Soc. Rev. 44, 394–418 (2015).

    CAS  Article  Google Scholar 

  69. 69.

    Murray, J., Kim, K., Ogoshi, T., Yao, W. & Gibb, B. C. The aqueous supramolecular chemistry of cucurbit[n]urils, pillar[n]arenes and deep-cavity cavitands. Chem. Soc. Rev. 46, 2479–2496 (2017).

    CAS  Article  Google Scholar 

  70. 70.

    Liu, W., Peck, E. M., Hendzel, K. D. & Smith, B. D. Sensitive structural control of macrocycle threading by a fluorescent squaraine dye flanked by polymer chains. Org. Lett. 17, 5268–5271 (2015).

    CAS  Article  Google Scholar 

  71. 71.

    Gómez-Durán, C. F. A., Liu, W., Betancourt-Mendiola, M. L. & Smith, B. D. Structural control of kinetics for macrocycle threading by fluorescent squaraine dye in water. J. Org. Chem. 82, 8334–8341 (2017).

    Article  Google Scholar 

  72. 72.

    Ogoshi, T., Yamagishi, T.-a. & Nakamoto, Y. Pillar-shaped macrocyclic hosts pillar[n]arenes: new key players for supramolecular chemistry. Chem. Rev. 116, 7937–8002 (2016).

    CAS  Article  Google Scholar 

  73. 73.

    Sadrerafi, K., Moore, E. E. & Lee, M. W. Association constant of β-cyclodextrin with carboranes, adamantane, and their derivatives using displacement binding technique. J. Incl. Phenom. Macrocycl. Chem. 83, 159–166 (2015).

    CAS  Article  Google Scholar 

Download references


The authors are grateful for a grant from the US National Institutes of Health (GM059078) and AD&T Berry Family Foundation fellowship from the University of Notre Dame.

Author information




Both authors contributed equally to the preparation of this manuscript.

Corresponding author

Correspondence to Bradley D. Smith.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Schreiber, C.L., Smith, B.D. Molecular conjugation using non-covalent click chemistry. Nat Rev Chem 3, 393–400 (2019).

Download citation

Further reading


Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing