Conjugation Strategies for Probe Development

Hello Dr.GPCR readers! This is Lucía from the Celtarys Research chemistry team. 

For our very first post in this ecosystem, we wanted to highlight a huge part of our work at Celtarys Research: conjugation strategies. You can check what we do here on our website!  

Conjugation strategies for small molecules are very versatile! In this case, we would like to focus on the synthesis of fluorescent probes. Traditionally, the most reliable and commonly used method is the amide coupling using acid and amine.[1]

This method has several advantages: it is usually very robust, good yields, reagents are found in most chem labs (like HATU, HoBT, EDCI etc.). Still, there are some downsides, such as the byproduct obtained by the O-acylisourea rearranging intramolecularly into the N-acylurea.[2] 

NHS ester amide coupling is the most suited for bioconjugation with proteins, DNA, etc, thanks to its reaction with the free amino groups present in these biomolecules. NHS esters are not very stable even in aqueous environment but they only need a slightly basic medium for the reaction to work, so they have to be used quickly and stored correctly. Not only do they work in aqueous medium, but also in aprotic solvents like DMF, where you will need to add a base such as TEA. [3] 

Maleimide conjugation with thiols present Cys residues. This conjugation is very useful for tagging biomolecules and can also be used to develop fluorescent probes with small molecules. Its biggest advantage is the presence of Cys residues in proteins, although sometimes S-S bridge reduction is needed, and how quickly the reaction takes place. The biggest detractor? It’s reversible under non-reducing conditions. [4] 

Other strategies include click chemistry, more specifically, the CuAAC (Cu(I)- catalyzed azide-alkyne 1,3-dipolar cycloaddition), which is a very robust conjugation strategy to obtain linkers with a rigid moiety (the triazol). But it also presents some issues, such as synthesizing the as the presence of the copper catalyst, which has to be removed completely, otherwise it can quelate biomolecules or induce cell toxicity. [5] 

At Celtarys’ we have our conjugation strategy - our own proprietary technology- which bypasses some of the issues seen before. There’s no need for any catalysts; all reagents will be incorporated in the structure of the final compound. The reaction is convergent, efficient and robust. Thanks to the unique linker structure we obtain, which can be divided into three differentiated parts, we can modify the rigidity of the linker as well as the physicochemical properties of the whole probe. This property comes from the wide chemical space this reaction can access – we can substitute one reagent and make an unprecedented combination, also using commercially available precursor, which improves the performance of the probes.  It also poses some disadvantages – just like acid-amine amide coupling, some byproducts are obtained during the synthesis. However, these are usually easily removable. Besides, it’s an eco-friendlier method, which always helps future-proof our probes! 

References

(1) Brown, D. G.; Boström, J. Analysis of Past and Present Synthetic Methodologies on Medicinal Chemistry: Where Have All the New Reactions Gone?: Miniperspective. J. Med. Chem. 2016, 59 (10), 4443–4458. https://doi.org/10.1021/acs.jmedchem.5b01409. 

(2) Sam, S.; Touahir, L.; Salvador Andresa, J.; Allongue, P.; Chazalviel, J.-N.; Gouget-Laemmel, A. C.; Henry De Villeneuve, C.; Moraillon, A.; Ozanam, F.; Gabouze, N.; Djebbar, S. Semiquantitative Study of the EDC/NHS Activation of Acid Terminal Groups at Modified Porous Silicon Surfaces. Langmuir 2010, 26 (2), 809–814. https://doi.org/10.1021/la902220a. 

(3) Fan, J.; Toth, I.; Stephenson, R. J. Chapter Three - Bioconjugated Materials in the Development of Subunit Vaccines. In Comprehensive Analytical Chemistry; Verma, S. K., Das, A. K., Eds.; Elsevier, 2023; Vol. 103, pp 59–103. https://doi.org/10.1016/bs.coac.2023.02.005. 

(4) Fontaine, S. D.; Reid, R.; Robinson, L.; Ashley, G. W.; Santi, D. V. Long-Term Stabilization of Maleimide–Thiol Conjugates. Bioconjugate Chem. 2015, 26 (1), 145–152. https://doi.org/10.1021/bc5005262. 

(5) Meldal, M.; Tornøe, C. W. Cu-Catalyzed Azide−Alkyne Cycloaddition. Chem. Rev. 2008, 108 (8), 2952–3015. https://doi.org/10.1021/cr0783479.