The side-chains of lysine (-NH₂) and cysteine (-SH) as well as the N-terminal amino group of peptides are well-known points of conjugation for the modification of peptides, e.g., labeling or cyclization. However, if modification at these sites might affect a peptide’s bioactivity, if they are not accessible or simply not present within the sequence, alternatives are in demand.

Here, our amino-functionalized N^ω-carbamoylated arginines come into play. These derivatives are obtained by replacing the native guanidino group with a suitable carbamoyl guanidine moiety. The carbamoyl guanidino group is a chemically stable structure. Its basicity is lower compared to the unsubstituted guanidine group in arginine, but basic enough (pKa approx. 8) to be protonated at physiological pH. Thus, it serves as bioisosteric arginine substitute enabling further conjugation, e.g. with a fluorescent label.

Chemical structure of our available N^ω-carbamoylated arginine building blocks for SPPS.

Our building blocks FAA7210 and FAA7220 can easily be incorporated by standard solid-phase peptide synthesis (SPPS). In both arginine derivatives, their side-chain amino groups are orthogonally protected with Boc, and may be derivatized with amino reactive molecules like, e.g., activated esters after deprotection.

Such modified arginines have been used to, e.g., create cyclic peptides, as well as to attach fluorescent dyes, isotope labels and bioorthogonally reactive groups for Click Chemistry.

Schematic illustration of the synthesis of a cyclic model peptide, made with the N^ω-carbamoylated arginine building block FAA7210 (R^carb): First, the peptide ETLR^carbTAF is synthesized by Fmoc-SPPS on Ramage Resin (RAM, which will yield an amidated C-terminus). After the cleavage from the resin with TFA (which also removes the Boc and OtBu protecting groups), the side chain carboxylic group of the N-terminal glutamate reside is activated with diisopropyl-carbodiimide (DIC) in the presence of OxymaPure and reacts with the amino group of the diaminobutyl spacer which has been introduced with the carbamoylated arginine. Finally, the N-terminal Fmoc protection is removed with piperidine to yield the desired product.

The incorporation of these building blocks into peptides has been carried out by SPPS using standard coupling reagents (OxymaPure/DIC, HBTU/HOBt or PyBOP/HOBt, DIPEA as base, with DMF/NMP 80:20 as solvent). To ensure a high coupling efficiency, anhydrous solvents and slightly elevated temperature (35-40 °C) should be used. Under these conditions, three equivalents of the building blocks and a prolonged reaction time (16 h) provided the products in high yield.

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