Sequential click reactions for synthesizing and patterning three-dimensional cell microenvironments

Click chemistry provides extremely selective and orthogonal reactions that proceed with high efficiency and under a variety of mild conditions, the most common example being the copper(I)-catalysed reaction of azides with alkynes1,2. While the versatility of click reactions has been broadly exploited3–5, a major limitation is the intrinsic toxicity of the synthetic schemes and the inability to translate these approaches into biological applications. This manuscript introduces a robust synthetic strategy where macromolecular precursors react through a copper-free click chemistry6, allowing for the direct encapsulation of cells within click hydrogels for the first time. Subsequently, an orthogonal thiol–ene photocoupling chemistry is introduced that enables patterning of biological functionalities within the gel in real time and with micrometre-scale resolution. This material system enables us to tailor independently the biophysical and biochemical properties of the cell culture microenvironments in situ. This synthetic approach uniquely allows for the direct fabrication of biologically functionalized gels with ideal structures that can be photopatterned, and all in the presence of cells. An emerging paradigm in organic synthesis is a focus on highly selective and orthogonal reactions that proceed with high efficiency and under a variety of mild conditions. A growing number of these reactions are grouped under the term ‘click chemistry’, and have been used to produce a catalogue of functional synthetic molecules and subsequent materials1,4. Characteristics of modular click reactions include (1) high yields with fast kinetics, (2) regiospecificity and stereospecificity, (3) insensitivity to oxygen orwater and (4)mild reaction conditions, solventless or inwater. While the versatility of click reactions has been broadly exploited in many fields including drug discovery7,8, material science9–11, and bioconjugation3,12,13, a major limitation is the intrinsic toxicity of the synthetic schemes and the inability to translate these approaches to biological applications. Though the 1,3-dipolar Huisgen cycloaddition between azides and alkynes2 is often seen as the quintessential click reaction, the crucial copper catalyst precludes its use with biological systems14,15. This drawback, however, was recently circumvented through the development of novel cyclooctyne moieties whose ring strain and electron-withdrawing fluorine substituents give rise to an activated alkyne. This molecule has been shown to react quickly with azides in the absence of a metal catalyst, enabling the use of traditional click chemistry in living systems6,16. Specifically, azide-labelled cell-surface glycans were reacted with fluorescently labelled cyclooctynes in vivo to enable the visualization of dynamic subcellular development within zebra-fish embryos17. Though this