Nα-Z-Nω-(4-toluenesulfonyl)-L-arginine

1. Relaxation of the methoxamine-precontracted rat small mesenteric artery by endothelium-derived hyperpolarizing factor (EDHF) was compared with relaxation to the cannabinoid, anandamide (arachidonylethanolamide). EDHF was produced in a concentration- and endothelium-dependent fashion in the presence of NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) by either carbachol (pEC50 [negative logarithm of the EC50] = 6.19 +/- 0.01, Rmax [maximum response] = 93.2 +/- 0.4%; n = 14) or calcium ionophore A23187 (pEC50 = 6.46 +/- 0.02, Rmax = 83.6 +/- 3.6%; n = 8). Anandamide responses were independent of the presence of endothelium or L-NAME (control with endothelium: pEC50 = 6.31 +/- 0.06, Rmax = 94.7 +/- 4.6%; n = 10; with L-NAME: pEC50 = 6.33 +/- 0.04, Rmax = 93.4 +/- 6.0%; n = 4). 2. The selective cannabinoid receptor antagonist, SR 141716A (1 microM) caused rightward shifts of the concentration-response curves to both carbachol (2.5 fold) and A23187 (3.3 fold). It also antagonized anandamide relaxations in the presence or absence of endothelium giving a 2 fold shift in each case. SR 141716A (10 microM) greatly reduced the Rmax values for EDHF-mediated relaxations to carbachol (control, 93.2 +/- 0.4%; SR 141716A, 10.7 +/- 2.5%; n = 5; P < 0.001) and A23187 (control, 84.8 +/- 2.1%; SR 141716A, 3.5 +/- 2.3%; n = 6; P < 0.001) but caused a 10 fold parallel shift in the concentration-relaxation curve for anandamide without affecting Rmax.

Synthesis and characterization of a dozen derivatives of ornithine and arginine bearing 2-methyl or 2-carboxymethyl substituents are described. These substituents were introduced by dilithiation of 3-(4-methoxybenzylidineamino)-2-piperidinone with lithium diisopropylamide followed by regiospecific alkylation at C-3 with iodomethane or ethyl bromoacetate. 2-Methyl-D,L-ornithine was obtained in three steps from 3-amino-2-piperidinone in 68% overall yield, and 2-carboxymethyl-D,L-ornithine was isolated in 56% overall yield. 2-Methyl- and 2-carboxymethyl-D,L-arginine were obtained by mild acid hydrolysis to remove the 4-methoxybenzylidine group, N-acylation with 4-toluenesulfonyl chloride, mild alkaline hydrolysis of the lactam ring and the ester group, guanidination of the 5-amino group with O-methylisourea, and strong acid hydrolysis to remove the 4-tolunenesulfonyl group. Several of these compounds are inhibitors of carboxypeptidase B.

The long-term cryopreservation of cells, tissues and organs is limited by both osmotic stress and ice injury during freezing or thawing. Introduction of biocompatible cryoprotective agents is promising for cell cryopreservation based on membrane stabilization of glycopeptides and trehalose. Herein, a series of trehalose-functional (glyco)peptides were synthesized by successively tethering Nω-(4-toluenesulfonyl)-l-arginine and carboxylated trehalose to the side amino groups of ε-polylysine. The specifically synthesized glycopeptide demonstrated enhanced cryosurvival of sheep red blood cells (RBCs) from 49.1 ± 0.9% to 75.0 ± 2.4% at pH 7.4, along with trehalose molecules. Cryopreservation via membrane stabilization was proposed such that the glycopeptide could attach on the cell membrane surface of RBCs to protect the cell membrane from osmotic injury by electrostatic interactions, π-π stacking and hydrogen bonding. Meanwhile, trehalose could prevent RBCs from ice injury via ice recrystallization inhibition activity. Thus, membrane protection for preventing osmotic stress and ice injury could be boosted via synergistic interaction between the glycopeptide and trehalose. The combined usage of biocompatible glycopeptides and trehalose could potentially be applied in the glycerol-free cryopreservation of cells and tissues in biomedicine.