“…Although it has been described that the pH of the duodenum may vary within the range 5-7 [27][28][29], the most accurate range seems to be 6-6.5 [30][31][32][33][34]. Therefore, the pH of the digests was adjusted to 6.5 and the following were added (a) a bile salt mixture containing equimolar quantities (0.125 m) of sodium taurocholate (Sigma) and glycodeoxycholic acid (Calbiochem, La Jolla, CA, USA), …”
Section: In Vitro Duodenal Digestion (Phase 2)mentioning
confidence: 99%
“…In vitro duodenal digestion was performed by using 120‐min gastric digests as the starting material. Although it has been described that the pH of the duodenum may vary within the range 5–7 [27–29], the most accurate range seems to be 6–6.5 [30–34]. Therefore, the pH of the digests was adjusted to 6.5 and the following were added (a) a bile salt mixture containing equimolar quantities (0.125 m ) of sodium taurocholate (Sigma) and glycodeoxycholic acid (Calbiochem, La Jolla, CA, USA), (b) 1 m CaCl 2 (BDH), (c) 0.25 m Bistris, pH 6.5 (Sigma), (d) porcine pancreatic lipase (EC 3.1.1.3; 20 µL per 10 mL of total volume) (0.1% w/v; Sigma product no.…”
Section: In Vitro Duodenal Digestion (Phase 2)mentioning
2S storage albumins occur in a diverse range of plant seeds, are members of the prolamin superfamily [1] and constitute one of the most important major plant food allergens that sensitize via the gastrointestinal (GI) tract [2]. Among the tree nuts, Brazil nut is frequently associated with immunoglobulin E (IgE)-mediated food allergy [3], the 2S albumin, known as Ber e 1, being the major allergen [4]. 2S albumins are considered to be structurally homologous, typically heterodimeric (small and large subunits of 4000 and 9000 M r , respectively) globular proteins. They have a conserved skeleton of cysteine residues (typical of members of the prolamin superfamily), which form four intermolecular disulphide bonds that hold the two subunits together and contribute to their stability and compactness [5]. This rigid The major 2S albumin allergen from Brazil nuts, Ber e 1, was subjected to gastrointestinal digestion using a physiologically relevant in vitro model system either before or after heating (100°C for 20 min). Whilst the albumin was cleaved into peptides, these were held together in a much larger structure even when digested by using a simulated phase 1 (gastric) followed by a phase 2 (duodenal) digestion system. Neither prior heating of Ber e 1 nor the presence of the physiological surfactant phosphatidylcholine affected the pattern of proteolysis. After 2 h of gastric digestion, 25% of the allergen remained intact, 50% corresponded to a large fragment of M r 6400, and the remainder comprised smaller peptides. During duodenal digestion, residual intact 2S albumin disappeared quickly, but a modified form of the 'large fragment' remained, even after 2 h of digestion, with a mass of 5000 Da. The 'large fragment' comprised several smaller peptides that were identified, by using different MS techniques, as deriving from the large subunit. In particular, sequences corresponding to the hypervariable region (Q37-M47) and to another peptide (P42-P69), spanning the main immunoglobulin E epitope region of 2S albumin allergens, were found to be largely intact following phase 1 (gastric) digestion. They also contained previously identified putative T-cell epitopes. These findings indicate that the characteristic conserved skeleton of cysteine residues of 2S albumin family and, particularly, the intrachain disulphide bond pattern of the large subunit, play a critical role in holding the core protein structure together even after extensive proteolysis, and the resulting structures still contain potentially active B-and T-cell epitopes.Abbreviations GI, gastrointestinal; IgE, immunoglobulin E; SGF, simulated gastric fluid; PtdCho, egg l-phosphatidylcholine.
“…Although it has been described that the pH of the duodenum may vary within the range 5-7 [27][28][29], the most accurate range seems to be 6-6.5 [30][31][32][33][34]. Therefore, the pH of the digests was adjusted to 6.5 and the following were added (a) a bile salt mixture containing equimolar quantities (0.125 m) of sodium taurocholate (Sigma) and glycodeoxycholic acid (Calbiochem, La Jolla, CA, USA), …”
Section: In Vitro Duodenal Digestion (Phase 2)mentioning
confidence: 99%
“…In vitro duodenal digestion was performed by using 120‐min gastric digests as the starting material. Although it has been described that the pH of the duodenum may vary within the range 5–7 [27–29], the most accurate range seems to be 6–6.5 [30–34]. Therefore, the pH of the digests was adjusted to 6.5 and the following were added (a) a bile salt mixture containing equimolar quantities (0.125 m ) of sodium taurocholate (Sigma) and glycodeoxycholic acid (Calbiochem, La Jolla, CA, USA), (b) 1 m CaCl 2 (BDH), (c) 0.25 m Bistris, pH 6.5 (Sigma), (d) porcine pancreatic lipase (EC 3.1.1.3; 20 µL per 10 mL of total volume) (0.1% w/v; Sigma product no.…”
Section: In Vitro Duodenal Digestion (Phase 2)mentioning
2S storage albumins occur in a diverse range of plant seeds, are members of the prolamin superfamily [1] and constitute one of the most important major plant food allergens that sensitize via the gastrointestinal (GI) tract [2]. Among the tree nuts, Brazil nut is frequently associated with immunoglobulin E (IgE)-mediated food allergy [3], the 2S albumin, known as Ber e 1, being the major allergen [4]. 2S albumins are considered to be structurally homologous, typically heterodimeric (small and large subunits of 4000 and 9000 M r , respectively) globular proteins. They have a conserved skeleton of cysteine residues (typical of members of the prolamin superfamily), which form four intermolecular disulphide bonds that hold the two subunits together and contribute to their stability and compactness [5]. This rigid The major 2S albumin allergen from Brazil nuts, Ber e 1, was subjected to gastrointestinal digestion using a physiologically relevant in vitro model system either before or after heating (100°C for 20 min). Whilst the albumin was cleaved into peptides, these were held together in a much larger structure even when digested by using a simulated phase 1 (gastric) followed by a phase 2 (duodenal) digestion system. Neither prior heating of Ber e 1 nor the presence of the physiological surfactant phosphatidylcholine affected the pattern of proteolysis. After 2 h of gastric digestion, 25% of the allergen remained intact, 50% corresponded to a large fragment of M r 6400, and the remainder comprised smaller peptides. During duodenal digestion, residual intact 2S albumin disappeared quickly, but a modified form of the 'large fragment' remained, even after 2 h of digestion, with a mass of 5000 Da. The 'large fragment' comprised several smaller peptides that were identified, by using different MS techniques, as deriving from the large subunit. In particular, sequences corresponding to the hypervariable region (Q37-M47) and to another peptide (P42-P69), spanning the main immunoglobulin E epitope region of 2S albumin allergens, were found to be largely intact following phase 1 (gastric) digestion. They also contained previously identified putative T-cell epitopes. These findings indicate that the characteristic conserved skeleton of cysteine residues of 2S albumin family and, particularly, the intrachain disulphide bond pattern of the large subunit, play a critical role in holding the core protein structure together even after extensive proteolysis, and the resulting structures still contain potentially active B-and T-cell epitopes.Abbreviations GI, gastrointestinal; IgE, immunoglobulin E; SGF, simulated gastric fluid; PtdCho, egg l-phosphatidylcholine.
“…These problems seem to be circumvented to some degree by the recent report of a large number of weakly basic inhibitors, which do not contain the above-mentioned S1 anchoring group but incorporate isosters of these moieties, as well as other structural modifications that reduce the highly basic p K a of the parent inhibitors (in the range of 11−13) (). Among the most promising new S1 anchoring groups recently reported are the following moieties: oxoguanidine (A) (p K a 7) (); hydroxyguanidine (B) (p K a 9) (); acylguanidine (C) (p K a 7.6) (); aminohydrazone (D) (p K a 8.7) (); benzamidrazone (E) (p K a 8.9) (); sulfonylguanidine (F) (p K a 8.3) ( , ); sulfonylaminoguanidine (G) (p K a 8.4) ( , ); imidazole (H) (p K a around 7) (); 1-aminoisoquinoline (I) (p K a 7.5) (); 2-aminopyridine (J) (p K a around 7) (); benzylamine (K) (p K a 9.4) (); aniline (L) (p K a not provided but stated as “neutral”) ( , ); benzonitrile (M) (); and arylsulfonyldicyandiamide (N) (p K a of 7.9−8.2) () among others (Figure ). Compounds incorporating some of these groups, such as LB-30057 ( 1 ), CGH 1668 ( 2 ), L-375378 ( 3 ), or the Organon derivative 4 (Figure ), are currently under clinical investigation as antithrombotic drugs ( , ).…”
mentioning
confidence: 99%
“…Compounds incorporating some of these groups, such as LB-30057 ( 1 ), CGH 1668 ( 2 ), L-375378 ( 3 ), or the Organon derivative 4 (Figure ), are currently under clinical investigation as antithrombotic drugs ( , ). 1 Examples of S1 anchoring groups with reduced basicity incorporated in thrombin inhibitors: A, oxoguanidine (); B, hydroxyguanidine (); C, acylguanidine (); D, aminohydrazone (); E, benzamidrazone (); F, sulfonylguanidine ( , ); G, sulfonylaminoguanidine ( , ); H, imidazole (); I, 1-aminoisoquinoline (); J, 2-aminopyridine (); K, benzylamine (); L, aniline ( , ), M, benzonitrile (); N, arylsulfonyldicyandiamide (). 2 Examples of thrombin inhibitors: 1 , LB-30057; 2 , CGH 1668; 3 , L-375378; 4 , 2-(7-methoxynaphthalene-2-sulfonylamido)-3-(4-amino-5-azabenzothiophen-2-yl)propanoic acid 4-methylpiperidide; 5 , 4-[α-(4-toluenesulfonylureido)phenylalanylamido]benzenesulfonylguanidine; 6 , 4-[(2-ethyl-4,6-diphenylpyridinium)- N -propylcarboxamido]benzenesulfonyl- N -1-aminoguanidine; 7 , 4-aminobenzenesulfonylguanidine; 8 , 4-aminobenzenesulfonyl- N -1-aminoguanidine.…”
mentioning
confidence: 99%
“…Examples of S1 anchoring groups with reduced basicity incorporated in thrombin inhibitors: A, oxoguanidine (); B, hydroxyguanidine (); C, acylguanidine (); D, aminohydrazone (); E, benzamidrazone (); F, sulfonylguanidine ( , ); G, sulfonylaminoguanidine ( , ); H, imidazole (); I, 1-aminoisoquinoline (); J, 2-aminopyridine (); K, benzylamine (); L, aniline ( , ), M, benzonitrile (); N, arylsulfonyldicyandiamide ().…”
To prepare weakly basic thrombin inhibitors with modified S1 anchoring groups, two series of compounds were synthesized by reaction of guanidine or aminoguanidine with acyl halides and N,N-disubstituted carbamoyl chlorides. pK(a) measurements of these acylated guanidines/aminoguanidines showed a reduced basicity, with pK(a) values in the range of 8.4-8.7. These molecules typically showed inhibition constants in the range of 150-425 nM against thrombin and 360-965 nM against trypsin, even though some bulky derivatives, such as N,N-diphenylcarbamoylguanidine/aminoguanidine and their congeners, showed much stronger thrombin inhibitory activity, with inhibition constants in the range of 24-42 nM. Unexpectedly, very long incubation times with both proteases revealed that aminoguanidine derivatives behaved as irreversible inhibitors. To assess the molecular basis responsible for the high affinity observed for these molecules toward thrombin, the crystal structure of the thrombin-hirugen-N,N-diphenylcarbamoylaminoguanidine complex has been solved at 1.90 A resolution. The structural analysis of the complex revealed an unexpected interaction mode with the protease, resulting in an N,N-diphenylcarbamoyl intermediate covalently bound to the catalytic serine as a consequence of its hydrolysis together with the release of the aminoguanidine moiety. Surprisingly, in this covalent adduct a phenyl group was found in the S1 specificity pocket, which usually recognizes positively charged residues. These findings provide new insights in the design of low basicity serine protease inhibitors.
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