Metal-Free Click Chemistry

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Figure 1. Post-conjugation schematic of GlycoConnect™ ADC created using BCN and metal-free click chemistry (azide-alkyne cycloaddition).

Synaffix scientists are pioneers in the field of metal-free click chemistry, credited with the discovery of the two world-leading probes, BCN and DBCO, which then led to the creation of Synaffix (Debets et al 2011). Metal-free click chemistry has been widely applied across pharma, biotech and academic groups due to its unique capability for rapid, selective and stable conjugation of complex macromolecules. At Synaffix, we have extensively optimized metal-free click chemistry between cyclooctynes and azides in conjunction with GlycoConnect™ for conjugation of potent cytotoxins site-specifically to mAbs.  The resulting conjugates feature an aromatically stabilized triazole which, by virtue of its high stability, provides a powerful alternative to cysteine-maleimide conjugation chemistry, as applied in the vast majority of ADCs currently in clinical trials (van Geel et al 2015).  It is well known that a cysteine-maleimide bond may display limited (and unpredictable) stability which, in the context of an ADC, translates directly into suboptimal tolerability and reduced efficacy.

At Synaffix, we have explored and extensively evaluated the combination of GlycoConnect™ (with azido-modified substrates) and metal-free click probes BCN and DBCO in terms of (a) conjugation efficiency and (b) aggregation potential of the resulting ADC.  

Studies repeatedly demonstrate that conjugation of BCN-containing constructs is highly favored over DBCO-based constructs.  This is irrespective of length of the PEG-based spacer (x = 4, 8, 12).  Perhaps most surprisingly, it was found that the longest PEG-spacer employed (n=12) in fact worsened the outcome of the conjugation process for DBCO.


Figure 2. Efficiency of conjugation with BCN vs DBCO using increasing sizes of PEG spacer from 4 to 8 and 12 PEG subunits.  Vertical axis is percent conversion after 3 hours.

Finally, it was discovered that the reaction rate constant of BCN and DBCO versus azides is also (besides the nature of the conjugate) highly dependent on the nature of the azide employed.  In deviation from the common perception that DBCO is more reactive than BCN, it was found that reaction of BCN with aromatic azides is accelerated 7-fold with respect to DBCO, which can even be enhanced >30-fold further by decreasing electron-density of the aromatic moiety (Dommerholt et al 2014).