Linkers and conjugation chemistry

Linker and conjugation technology has attracted increasing interest due to its key role in the development of complex (bio)molecules, such as prodrugs, ADCs, SMDCs, PROTACs, codrugs, diagnostic probes, and drug-delivery systems[1]. Linkers, which can be cleavable or noncleavable, connect a functional (bio)molecule with a molecular tag to form a conjugate. The linker’s physicochemical properties must be carefully chosen to enable the function of the (bio)molecule. (Bio)conjugation needs to be compatible with the nature of the linked molecular tag. Symeres has considerable experience in the synthesis of a range of linkers and (bio)conjugates, and we offer our clients a tailor-made solution.

Cleavable linkers
The chemical triggers controlling the cleavage of a linker are of the utmost importance. For example, in the development of ADCs, the linker that connects the antibody to the cytotoxic payload needs to be highly stable in the systemic circulation and efficiently cleaved in the target tissues. To achieve this, different linkers with various chemical triggers have been developed, such as enzyme- and acid-cleavable linkers.

Cleavable linkers often contain, or are used in combination with, a self-immolative group. Self-immolative linkers can spontaneously degrade in response to a specific stimulus. A well-known example of a self-immolative linker is the para-aminobenzyl alcohol group (PAB), which degrades via an electronic cascade process.

We not only offer our clients support in the synthesis, analysis, purification, and conjugation of known linkers, but we also use our considerable experience in the development of new linkers and conjugation to their molecular constructs. An example of this is the synthesis of a new class of tunable, acid-sensitive linkers for the development of nanoparticulate drug-delivery systems[6]. Symeres designed the synthesis of these linkers, based on the trityl protecting group, which allowed the tunable release of the drug under slightly acidic conditions.

Noncleavable linkers
Noncleavable linkers are characterized by their high stability, and thus, lack a release mechanism. Linkers of various lengths, polarity, stability, and flexibility have been used in different molecular constructs. These can be used in modalities that need to stay intact to fulfill their function, such as PROTACs. There has been increasing interest in these linkers in the ADC area. ADCs containing a noncleavable linker release the payload–linker after ADC internalization and digestion of the antibody by lysosomal enzymes.

Conjugation chemistry
Conjugation chemistry is the synthetic process by which two or more functional (bio)molecules are covalently bound, either via directly attached reactive functional groups or via more remote groups, and bridged by a noncleavable linker. This requires the presence of reactive handles that can react with a specific functional group (amines, sulfhydryls, azides, etc.). The conditions used to perform this conjugation need to be compatible with the nature of the (bio)molecules. A breakthrough development in conjugation is the introduction of bioorthogonal chemistry in chemical biology. Many applications of bioorthogonal conjugation chemistry have been extensively reported in the literature[7],[8] and recently the Nobel prize was awarded to Carolyn Bertozzi, Morten Meldal, and Barry Sharpless for their work on click chemistry.

At Symeres, we have applied all the reported methods in conjugation and click chemistry (see Figure). We also use our synthesis skills to support our clients in the development of new reactive handles for use in (bio)conjugation chemistry. An example is the synthesis of TMTH–sulfoximine (TMTHSI) for copper-free click chemistry published in Chemical Science[9]. With its small size and low hydrophobicity, this reagent can be used in highly aqueous environments. While its reactivity can compete with best-in-class examples in the field, this reagent shows far better stability and can conveniently be functionalized on the sulfoximine to attach a variety of payloads[10].

CliCr® is provided under an intellectual property license from Cristal Therapeutics.

CliCr® is provided under an intellectual property license from Cristal Therapeutics.

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[1] G. Leriche et al., Bioorg. Med. Chem. 2012, 20 , 571–582 DOI:

[2] Z. Su. et al., Acta Pharm. Sin. 2021, 11, 3889-3901 DOI:

[3] T. Nakada et al., Bioorg. Med. Chem. Lett 2016, 26, 1542–1545 DOI:

[4] J. C. Kern et al., J. Am. Chem. Soc 2016, 138, 1430−1445 DOI:

[5] S. C. Jeffrey et al., Bioconjugate Chem 2006, 138, 831−840 DOI:

[6] M. Timmers et al., Bioconjugate Chem. 2022, 33 , 1707–1715 DOI:

[7] G. Hermans et al., WO2022/055351 A1

[8] G. Hermans et al., WO2022/055352 A1

[9] J. Weterings et al., Chem. Sci. 2020, 11 , 9011 DOI:

[10] Synaffix press release

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