Towards Amplified Probing of Weak Intermolecular Interactions on the External Surfaces of Molecular Capsules.

We report a sensitive method for comparing weak interactions between aryl rings located on the external surfaces of equilibrating homo- and heterodimeric capsules. Two identical self-complementary resorcin[4]arene tetrabenzoate molecules and one tetramethylammonium cation form in CDCl3 hydrogen-bonded homodimeric capsules whose exteriors are decorated with four tight pairs of weakly interacting aryl rings. The pair wise mixing of six different homodimers establishes their equilibria with the corresponding heterodimeric species in which two types of aryl rings exert on each other some gentle forces. This equilibrium is significantly shifted either towards homo- or heterodimers depending on the nature and location of the substituents in the weakly interacting aryl rings. The thermodynamic favorability or disadvantage of the heterodimerization is determined by stronger or weaker aryl-aryl attractions in the hetero- or homodimeric capsules, respectively. The four-fold amplification of weak aryl-aryl interactions on the external surfaces of the equilibrating capsules is responsible for high sensitiveness of our approach.

Peptide identification and angiotensin converting enzyme (ACE) inhibitory activity in prolyl endoproteinase digests of bovine α(s)-casein.

Incubation of sodium caseinate (NaCN) and purified α-casein (αs-CN) with an Aspergillus niger derived prolyl endoproteinase (An-PEP) for 1, 2, 3, 4, 8 and 24 h resulted in the generation of potent angiotensin converting enzyme (ACE) inhibitory hydrolysates. An ACE IC50 of 21.1±5.1 µg/ml was obtained on incubation of An-PEP with NaCN for 4 h. Fractionation of the NaCN hydrolysates using 3 kDa centrifugal filters resulted in highly active permeate fractions, the most potent being obtained from the 3 h hydrolysate (ACE IC50=2.9±0.3 µg/ml). The hydrolytic specificity of An-PEP for purified α-CN was assessed using UPLC ESI MS/MS. The analysis confirmed An-PEP's cleavage preference for the C-terminal side of Pro and also confirmed that An-PEP has the ability to cleave at the C-terminal of Ala, Leu, Arg and His residues.

Extraction of antioxidant and ACE inhibitory peptides from Thai traditional fermented shrimp pastes.

Antioxidant and angiotensin converting enzyme (ACE) inhibitory peptides were extracted and isolated from two different types of Thai traditional fermented shrimp pastes, Kapi Ta Dam (Kp-B6) and Kapi Ta Deang (Kp-R6). Compounds with masses less than 500Da were found to be predominantly presented in both extracts. Following fractionation with sequential anion exchange chromatography and solid phase extraction (C18 matrix), three dipeptides were identified. Ser-Val and Ile-Phe were shown to exhibit ACE inhibitory activity with IC50 values of 60.68±1.06 and 70.03±1.45µM, respectively. Trp-Pro was shown to have high 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging activity (EC50 17.52±0.46µM). These results indicate that Thai traditional fermented shrimp pastes are potential sources of bioactive peptides possessing ACE inhibitory and antioxidant activities.

Characterisation of the hydrolytic specificity of Aspergillus niger derived prolyl endoproteinase on bovine ß-casein and determination of ACE inhibitory activity.

The hydrolytic specificity of Aspergillus niger prolyl endoproteinase (An-PEP) on purified ß-casein (ß-CN) was assessed. This analysis confirmed cleavage at the C-terminal side of Pro residues. An-PEP also had the ability to cleave at the C-terminal side of Ala, Glu, Gly, Ser, Lys and Leu. Incubation of purified ß-CN with An-PEP resulted in the generation of highly potent angiotensin converting enzyme (ACE) inhibitory hydrolysates. The most potent hydrolysate was obtained after 24h incubation (ACE IC50=16.41±6.06µg/mL). Fourteen ß-CN derived C-terminal Pro-containing di-, tri, and tetrapeptides which were predicted in silico to be released following An-PEP hydrolysis or which were detected by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) in the 24h hydrolysate were synthesised and characterised for their ACE inhibitory activity. The most potent inhibitory peptides were Ile-Gln-Ala (ß-CN f187-189) and Val-Glu-Pro (ß-CN f116-118) having ACE IC50 values of 32.9±9.2 and 63.7±12.0µM, respectively. The hydrolysates generated appear to have the most potent ACE IC50 values reported for a food derived hydrolysate to date.

Antioxidant activity of bovine casein hydrolysates produced by Ficus carica L.-derived proteinase.

A Ficus carica L. latex proteinase preparation was investigated for its ability to produce antioxidant hydrolysates/peptides from bovine casein (CN). The Oxygen Radical Absorbance Capacity (ORAC) values for NaCN and ß-CN hydrolysates ranged from 0.06 to 0.18, and from 0.51 to 1.19µmol Trolox equivalents/mg freeze-dried sample, respectively. Gel permeation HPLC showed that the ß-CN hydrolysate with a degree of hydrolysis of 21% had 65% of peptide material with a molecular mass <500Da. The RP-UPLC profiles also indicated that ß-CN was substantially hydrolysed during the early stages of hydrolysis. Analysis of the 4h ß-CN hydrolysate by LC-ESI-MS/MS allowed identification of 8 peptide sequences with potential antioxidant properties.

Liporetro-D-peptides - a novel class of highly selective thrombin inhibitors.

INTRODUCTION: Plasma serine protease thrombin plays a key role in coagulation, haemostasis and thromboembolic diseases. Direct thrombin inhibitors could be beneficial for future anticoagulant therapy. We have synthesized and studied liporetro-D-peptides - efficient thrombin inhibitors resistant to enzymatic degradation. MATERIALS AND METHODS: Compounds X-D-Arg-D-Phe-OMe, where X=residue of lauric or myristic acid or 9-fluorenylmethoxycarbonyl, have been synthesized by conventional peptide synthesis in solution and their comparative inhibitory analysis in relation to thrombin, factor X, plasmin and trypsin has been conducted. RESULTS: Modification of the synthetic liporetro-D-peptides with the myristic acid residue was the most successful one. This modification has dramatically increased the inhibition efficacy (Ki=0,17 µM) and selectivity toward the chosen target enzyme, thrombin, in comparison to factor X, plasmin and trypsin (more than 600, 900, and 5000-fold, respectively). CONCLUSIONS: Our findings establish an important role of the fatty moiety in the structure of peptide inhibitors with regards to their potency and selectivity toward thrombin.

Influence of aromatic and aliphatic moieties on thrombin inhibitors potency.

Thrombin is a plasma serine protease that plays a key role in coagulation and hemostasis but also in thromboembolic diseases. Direct thrombin inhibitors could be beneficial for future anticoagulant therapy in the prophylaxis of venous and arterial thrombosis as well as myocardial infarction. To design the efficient thrombin inhibitors we have synthesized and studied peptide-based inhibitors resistant to enzymatic degradation. Compounds with general formula X-DArg-D-Phe-OMe, where X = residue of 3-[6-ethyl-7-hydroxy-3-(4-methyl-thiazol-2-yl)-4-oxo-4H-chromen-2-yl]-propionic acid (chromone) and lauric acid were synthesized by classic methods of peptides synthesis in solution. The comparative inhibitory analysis of prepared compounds in relation to thrombin was conducted. The analysis of the inhibition effect of the peptide with retro-D-sequence modified by residues of natural organic compounds (chromone or fatty acid moiety) has demonstrated that modification with the fatty acid residue appeared to be the most successful one. Introduction of lauric acid residue (Ki = 1,76 muM) maximally increased the inhibition effect. These findings establish an important role of fatty moiety in structure of inhibitors in preferential binding and inhibition of thrombin active side.