PotM: Furan-Crosslinking and 5HP2O as Maleimide Alternative

The so-called surfaceome (= plasma membrane proteins exposed to the extracellular space) is involved in a variety of cellular processes. Aberrant structures are associated with severe diseases, e.g., wrong/missing glycosylation pattern on surface proteins is related to Alzheimer’s disease and cancer. Thus, methods for the analysis of the surfaceome and its proteins are highly thought after. However, one major challenge is the low abundance of surface proteins compared to intracellular proteins. A frequently used technique in this context is the biotinylation of surface proteins allowing for their enrichment via avidin/streptavidin affinity and thus facilitated detection and analysis.

In our portfolio, we offer a wide variety of differently functionalized biotinylation reagents that may target specific chemical moieties, e.g., as present in side chains of surface proteins. In this context, the research group around Prof. Annemieke Madder (Uni Ghent) developed a protocol for surface biotinylation based on a furan-biotin.

Synthesis of furan-biotin starting from D-biotin (LS-1070) and furfurylamine.

In vitro, the furan moiety can be oxidized upon singlet oxygen production by visible light irradiation in the presence of a photosensitizer or by adding N-bromosuccinimide (NBS) or endogenously upon the action of reactive oxygen species (ROS). In its oxidized, ring-open form, it may target the side chains of surface-exposed cysteine, lysine, and tyrosine residues of membrane proteins.

The example illustrated below shows the reaction of a peptide containing furyl-alanine with a nearby peptide/protein carrying an exposed lysine side chain. This reaction may be carried out under physiological conditions in aqueous solutions.

Proximity labelling reaction. The furan moiety is activated by oxidation and reacts with the side chain amine of a nearby lysine.

In our portfolio, we offer 2-furyl-L-alanine as Fmoc-protected building block (FAA4250) and as free base (HAA2930).

Besides, furan can also react in a Diels-Alder reaction with maleimide as dienophile, e.g., for the distinct biorthogonal labeling of peptides with fluorochromes or for the preparation of antibody-drug-conjugates (ADCs).

On-column-derivatization of a peptide containing furyl-alanine by a Diels-Alder reaction with a maleimide as dienophile. The modification may be carried out at room temperature by adding the dienophile in a suitable inert solvent. Workup and deprotection, e.g., with TFA/TIS/water (95:2.5:2.5), 2 hours.

Despite various possibilities for the modification of and conjugation to side chain functional groups of histidine, lysine, methionine, tryptophane, and tyrosine, respectively, cysteine remains the most attractive target due to its rare occurrence in natural proteins typically allowing for its site-selective modification upon introduction at a specific position. In terms of cysteine modification, maleimides are the reactive moieties of choice acting via a Michael addition reaction. However, maleimides are susceptible to hydrolysis, retro-Michael reactions and/or thiol exchange reactions leading to protein conjugates with overall poor stability.

In this context, starting from furan, the research group around Prof. Annemieke Madder developed 5-hydroxy-1,5-dihydro-2H-pyrrol-2-ones (5HP2Os) – representatives of the 5-hydroxy-pyrrolone family – as maleimide replacement technology avoiding all the above-mentioned maleimide-drawbacks. The substituents R1 and R2 – as shown in the scheme below – allow for further derivatization and can be chosen depending on the intended subsequent use.

Singlet oxygen-induced formation of 5HP2O-derivatives in one pot starting from a substituted furan and a substituted-amine in the presence of methylene blue, oxygen, and dimethyl sulfide in methanol upon visible light irradiation. The 5HP2O reacts with a peptide containing a sulfhydryl group (typically from a cysteine). This reaction usually completes within 1 hour with 0.3 equivalents Na₄EDTA in MeOH or with aqueous MeCN (3/2) at pH 8.0 as solvent.

→ Register for our online workshop featuring Prof. Annemieke Madder and learn more about furan and the 5HP2O technology! 

→ Stay tuned – various 5HP2O derivatives will soon be added to our portfolio!

References:

5-Hydroxy-pyrrolone based building blocks as maleimide alternatives for protein bioconjugation and single-site multi-functionalization; E. De Geyter, E. Antonatou, D. Kalaitzakis, S. Smolen, A. Iyer, L. Tack, E. Ongenae, G. Vassilikogiannakis, A. Madder; Chem. Sci. 2021; 12: 5246-5252. https://doi.org/10.1039/d0sc05881e

Cell Surface Biotinylation Using Furan Cross-Linking Chemistry; E. Fernandez, L. Miret-Casals, A. Madder, K. Gevaert; Methods Mol. Biol. 2023; 2718: 11-21. https://doi.org/10.1007/978-1-0716-3457-8_2

Equipping Coiled-Coil Peptide Dimers With Furan Warheads Reveals Novel Cross-Link Partners; L. Miret-Casals, S. Van De Putte, D. Aerssens, J. Diharce, P. Bonnet, A. Madder; Front. Chem. 2022; 9: 799706. https://doi.org/10.3389/fchem.2021.799706

Furan-modified PNA probes for covalent targeting and ligation of nucleic acids; L. De Paepe, E. Cadoni, A. Manicardi, A. Madder; Methods 2023; 218: 210-223. https://doi.org/10.1016/j.ymeth.2023.08.010

Exploiting furan's versatile reactivity in reversible and irreversible orthogonal peptide labeling; K. Hoogewijs, D. Buyst, J. M. Winne, J. C. Martins, A. Madder; Chem. Commun. (Camb). 2013; 49(28): 2923-2929. https://doi.org/10.1039/c3cc40588e

Furan oxidation based cross-linking: a new approach for the study and targeting of nucleic acid and protein interactions; L. L. G. Garrette, E. Gyssels, N De Laet, A. Madder; Chem. Commun. (Camb). 2016; 8: 1539-1554. https://doi.org/10.1039/c5cc08766j

Novel furan-oxidation based site-specific conjugation methodology for peptide labeling and antibody drug conjugates; A. Van Den Bulcke, E. Antonatou, W. Vannecke, K. Hoogewijs, A. Madder; 6th World ADC meeting October 19th-22nd 2015; San Diego, California, USA.

Bioconjugation Reagent and Methods. A. Madder, E. De Geyter, E. Antonatou, S. Smolen, D. Kalaitzakis, D. Vassilikogiannakis; 2020. WO2020/174086A2

2-Hydroxypyrrolidin-5-ones for bioconjugation and methods for their production. A. Madder, E. De Geyter, E. Antonatou, S. Smolen, D. Kalaitzakis, D. Vassilikogiannakis; 2020. WO2020/174086A3