An Alkyne-functionalized Arginine for Solid-Phase Synthesis Enabling “Bioorthogonal” Peptide Conjugation

Abstract

Lately, amino-functionalized N ω-carbamoylated arginines were introduced as arginine surrogates enabling peptide labeling. However, this approach is hardly compatible with peptides also containing lysine or cysteine. Here, we present the synthesis of an alkyne-functionalized, N ω-carbamoylated arginine building block (7), which is compatible with Fmoc-strategy solid-phase peptide synthesis. The alkynylated arginine was incorporated into three biologically active linear hexapeptides and into a cyclic pentapeptide. Peptide conjugation to an azido-functionalized fluorescent dye via “click” chemistry was successfully demonstrated. In the case of a peptide also containing lysine besides the alkyne-functionalized arginine, this was feasible in a “bioorthogonal” manner.

Red-Emitting Dibenzodiazepinone Derivatives as Fluorescent Dualsteric Probes for the Muscarinic Acetylcholine M2 Receptor

“…Figure 1B), the antagonistic potencies of 18, 20, 23 and 28 (Table 2) were considerably lower compared to binding data (pK i values) obtained from radioligand competition binding studies (Table 1). Increasing the incubation times in the M 2 R IP1 assay from 30 min to 180 min (preincubation of the cells with antagonist) and from 60 min to 90 min (continued incubation after addition of the agonist CCh), as well as lowering the temperature from 37 °C to 22 °C during preincubation, did not affect (18,28) or led to a moderate increase (20,23) in pIC 50 values (Table 2). Possibly, the type of interaction between 18, 20, 23 or 28 and CCh is different from how the small antagonist 31 inhibits the CCh induced IP1 accumulation, which is supported by a much better correlation of the pIC 50 (pK b ) and pK i value in case of 31 (pIC 50 : 8.09 (Table 2), corresponding pK b : 8.66, pK i : 9.04 (Table 6)).…”

“…For this reason, the pharmacophore (ligand) and the fluorophore are often held apart by a linker or spacer moiety. [6][7][8] There are numerous reports on fluorescent probes for GPCRs, for instance for neuropeptide Y, [9][10][11][12][13][14] histamine, [15][16][17][18][19] opioid, [20][21][22] dopamine, 23,24 neurotensin, [25][26][27][28] and adenosine 29 receptors.…”

“…showing the fastest dissociation of the studied ligands(20,23,28) was chosen to perform dissociation experiments in the presence of the allosteric M 2 R modulators W84 (32),[55][56][57] LY2119620 (33)58,59 or alcuronium (34)60,61 (Structures see Figure S4, Supporting Information). The allosteric ligands, used at a high concentration of 100 µM, did not reduce the observed dissociation rate of 23 from the M 2 R as previously observed for the structurally related radioligands [ 3 H]6 and [ 3 H]7.…”

“…The cell density was adjusted to 0.9-1.1 × 10 6 cells/mL. For saturation(18,20,23,28,30) and competition(20) binding experiments, 1.5 mL reaction vessels were prefilled with 490 μL of the cell suspension.For total binding, DMSO/H 2 O (1:1 v/v) (5 μL) and DMSO/H 2 O (1:1 v/v) (5 μL), containing the fluorescent ligand (100-fold concentrated), were added. To determine unspecific binding and in case of competition binding, DMSO/H 2 O (1:1 v/v) (5 μL), containing atropine (31) (100-fold concentrated) and the compound of interest (100-fold concentrated), respectively, and DMSO/H 2 O (1:1 v/v) (5 μL), containing the fluorescent ligand (100-fold concentrated), were added.…”

N-Terminus to Arginine Side-Chain Cyclization of Linear Peptidic Neuropeptide Y Y4 Receptor Ligands Results in Picomolar Binding Constants

“…This had not been achieved before with small peptides such as hexapeptides. The study demonstrates that the guanidine/carbamoylguanidine bioisosteric approach, reported to be useful for peptide labeling via arginine residues, 19,24,38,39 can be successfully exploited for the preparation of cyclic peptidic receptor ligands with high affinity. The cyclic peptides 18 (UR-AK86c) and 24 (UR-AK95c) were shown to be substrates of angiotensin-converting enzyme (EC 3.4.15.1), which cleaves 18 and 24 at the Leu 4 − Arg 5 bond in their C-terminal exocyclic tripeptide sequence, resulting in moderate in vitro plasma stabilities of 18 and 24 (t 1/2 ca.…”