Bioisosteric Argininine Analogs

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.

References:

Illuminating Neuropeptide Y Y4 Receptor Binding: Fluorescent Cyclic Peptides with Subnanomolar Binding Affinity as Novel Molecular Tools; J. Gleixner, S. Kopanchuk, L. Grätz, M.-J. Tahk, T. Laasfeld, S. Veikšina, C. Höring, A. O. Gattor, L. J. Humphrys, C. Müller, N. Archipowa, J. Köckenberger, M. R. Heinrich, R. J. Kutta, A. Rinken, M. Keller; ACS Pharmacol. Transl. Sci. 2024; 7(4): 1142-1168. https://doi.org/10.1021/acsptsci.4c00013

Mimicking of Arginine by Functionalized N(omega)-carbamoylated Arginine As a New Broadly Applicable Approach to Labeled Bioactive Peptides: High Affinity Angiotensin, Neuropeptide Y, Neuropeptide FF, and Neurotensin Receptor Ligands As Examples; M. Keller, K. K. Kuhn, J. Einsiedel, H. Hubner, S. Biselli, C. Mollereau, D. Wifling, J. Svobodova, G. Bernhardt, C. Cabrele, P. M. Vanderheyden, P. Gmeiner, A. Buschauer; J. Med. Chem. 2016; 59(5): 1925-45. https://doi.org/10.1021/acs.jmedchem.5b01495

Fluorescence Labeling of Neurotensin(8-13) via Arginine Residues Gives Molecular Tools with High Receptor Affinity; M. Keller, S. A. Mahuroof, V. Hong Yee, J. Carpenter, L. Schindler, T. Littmann, A. Pegoli, H. Hubner, G. Bernhardt, P. Gmeiner, N. D. Holliday; ACS Med. Chem. Lett. 2020; 11(1): 16-22. https://doi.org/10.1021/acsmedchemlett.9b00462

[³H]UR-JG102–A Radiolabeled Cyclic Peptide with High Affinity and Excellent Selectivity for the Neuropeptide Y Y4 Receptor; J. Gleixner, A. O. Gattor, L. J. Humphrys, T. Brunner, M. Keller; J. Med. Chem. 2023; 66(19): 13788-13808. https://doi.org/10.1021/acs.jmedchem.3c01224

Development of a Neurotensin-Derived ⁶⁸Ga-Labeled PET Ligand with High In Vivo Stability for Imaging of NTS₁ Receptor-Expressing Tumors; L. Schindler, J. Moosbauer, D. Schmidt, T. Spruss, L. Gratz, S. Lüdeke, F. Hofheinz, S. Meister, B. Echtenacher, G. Bernhardt, J. Pietzsch, D. Hellwig, M. Keller; Cancers 2022; 14(19): 4922-51. https://doi.org/10.3390/cancers14194922

An Alkyne-functionalized Arginine for Solid-Phase Synthesis Enabling “Bioorthogonal” Peptide Conjugation; K. Spinnler, L. von Krüchten, A. Konieczny, L. Schindler, G. Bernhardt, M. Keller; ACS Med. Chem. Lett. 2020; 11(3): 334-339. https://doi.org/10.1021/acsmedchemlett.9b00388

N‑Terminus to Arginine Side-Chain Cyclization of Linear Peptidic Neuropeptide Y Y4 Receptor Ligands Results in Picomolar Binding Constants; A. Konieczny, M. Conrad, F. J. Ertl, J. Gleixner, A. O. Gattor, L. Grätz, M. F. Schmidt, E. Neu, A. H. C. Horn, D. Wifling, P. Gmeiner, T. Clark, H. Sticht, M. Keller; J Med. Chem. 2021; 64(22): 16746-16769. https://doi.org/10.1021/acs.jmedchem.1c01574

Neurotensin analogs by fluoroglycosylation at N^ω‑carbamoylated arginines for PET imaging of NTS1‑positive tumors; L. Schindler, K. Wohlfahrt, L. Gluhacevic von Krüchten, O. Prante, M. Keller, S. Maschauer; Sci Rep. 2022; 12: 15028. https://doi.org/10.1038/s41598-022-19296-0

High Affinity Agonists of the Neuropeptide Y (NPY) Y4 Receptor Derived from the C‑Terminal Pentapeptide of Human Pancreatic Polypeptide (hPP): Synthesis, Stereochemical Discrimination, and Radiolabeling; K. K. Kuhn, T. Ertl, S. Dukorn, M. Keller, G. Bernhardt, O. Reiser, A. Buschauer; J. Med. Chem. 2016; 59(13): 6045-6058. https://doi.org/10.1021/acs.jmedchem.6b00309

Structure-based design of high-affinity fluorescent probes for the neuropeptide Y Y1 receptor; C. Müller, J. Gleixner, M.-J. Tahk, S. Kopanchuk, T. Laasfeld, M. Weinhart, D. Schollmeyer, M.U. Betschart, S. Lüdeke, P. Koch, A. Rinken, M. Keller; J. Med. Chem. 2022; 65(6): 4832−4853. https://doi.org/10.1021/acs.jmedchem.1c02033

Insertion of Nanoluc into the Extracellular Loops as a Complementary Method To Establish BRET-Based Binding Assays for GPCRs; L. Grätz, C. Müller, A. Pegoli, L. Schindler, G. Bernhardt, T. Littmann; ACS Pharmaco.l Transl. Sci. 2022 ; 31(5): 1142-1155. https://doi.org/10.1021/acsptsci.2c00162