Piedad Fernández-Resa

Identification of Insulin-like Growth Factor-binding Protein-1 as a Potential Physiological Substrate for Human Stromelysin-3

To elucidate the physiological role of human stromelysin-3 (hST-3) in tumor progression and/or wound healing, insulin-like growth factor-binding protein-1 (IGFBP-1) was analyzed as a potential physiological substrate. hST-3 proteolysis generates two fragments of 16 and 9 kDa that react with IGFBP-1 monoclonal antibody, although they do not bind insulin-like growth factor-I (IGF-I) in ligand blot. N-terminal sequencing shows that hST-3 cleaves IGFBP-1 at the His140-Val141 bond located in the IGFBP-1 midregion. We show that IGFBP-1 inhibits IGF-I-induced survival and proliferation of BAF/3 cells, as well as IGF-I-mediated activation of phosphatidylinositol 3-kinase (PI 3-K). Co-incubation of the IGF-I·IGFBP-1 complex with hST-3 restores IGF-I-induced proliferation and PI 3-K kinase activity in these cells. BAF/3 proliferation is significantly increased with the hST-3-treated IGF-I·IGFBP-1 complex compared with that obtained using IGF-I alone. To produce this enhanced proliferation, IGF-I must bind to IGFBP-1 before hST-3 proteolysis, demonstrated using an IGF-I variant that does not bind IGFBP. IGFBP-1 also inhibits IGF-I-induced proliferation of the MCF-7 breast adenocarcinoma, and this inhibition was not seen in hST-3-transfected MCF-7 cells. Such proteolysis may thus play a role in in vivo tumor progression. These results indicate that hST-3 may regulate IGF-I bioavailability by proteolyzing IGFBP, thus favoring cell survival and proliferation.

Evaluation of fluorometric and zymographic methods as activity assays for stromelysins and gelatinases.

To measure matrix metalloproteinase (MMP) activity in a large number of samples it is advisable to use easily automated methods. We have evaluated and compared the activity of stromelysin-1 (MMP-3), matrilysin (MMP-7), 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) by zymogram analysis and fluorescent substrate degradation assays. FITC-casein and the fluorogenic peptide Dnp-Pro-β-cyclohexyl-Ala-Gly-Cys(Me)-His-Ala-Lys-(N-Me-Abz)-NH were used as fluorescent substrates. FITC-casein was more efficiently degraded than the fluorogenic peptide by all MMPs tested except MMP-9. MMP-2 was not significantly able to degrade the fluorogenic peptide. Gelatin zymography was the most sensitive method to detect the activity of both gelatinases but quantitation problems compromise its use. The degradation of fluorogenic substrates by MMPs could be inhibited by the chelating agent EDTA and by the tissue inhibitor of metalloproteinases 2 (TIMP-2), an MMP-specific inhibitor. Fluorometric methods represent a good alternative for MMP activity measurement, especially when a large number of samples must be processed.

Synthesis of ribosyl and arabinosyl cyanides by reaction of 1-O-acyl sugars with trimethylsilyl cyanide

A new procedure is described for the synthesis of glycosyl cyanides by reaction of 1-O-acyl sugars with trimethyl-silyl cyanide in a polar aprotic solvent and in the presence of a Lewis acid as catalyst. A variety of ribosyl and arabinosyl cyanides have been made in this way from sugar derivatives having acyl, chloro, or methoxy leaving groups at the anomeric position, furanose or pyranose rings, and acyl or benzyl protecting groups. The 1,2-Trans-glycosyl cyanide was formed when the starting sugar had a participating 2-O-acyl substituent. A mixture of cyanide anomers was obtained when the starting sugar was protected with non-participating benzyl groups.

Cyanosugars—II: Synthesis of hexopyranosyl cyanides by reaction of glycals with trimethylsilyl cyanide

Abstract 3,4,6-Tri-O-acetyl-O-glucal and 2,3,4,6-tetra-O-acetyl-2-hydroxy-D-glucal reacted with Me3SiCN in the presence of a Lewis acid (SnCl4, BF3) as catalyst to give anomeric pairs of 2,3-dideoxy-hex-2-enopyranosyl cyanides and 3-deoxy-hex-2-enopyranosyl cyanides. These 2,3-unsaturated glycopyranosyl cyanides were hydrogenated over Pd C to afford the corresponding 2,3-dideoxy- and 3-deoxy-hexopyranosyl cyanides.

Cyanosugars III : The reaction of trimethylsilyl cyanide with keto, epoxy and acetal derivatives of carbohydrates

Abstract Reaction of methyl 2-acetamido-4,6- O -benzylidene-2-deoxy-α- D - ribo -hexopyranosid-3-ulose with Me3SiCN afforded methyl 2-acetamido-4,6- O -benzylidene-3- C -cyano-2-deoxy-3- O -trimethylsilyl-α- D - allo - Reaction of ethyl 4,6-di- O -acetyl-2,3-anhydro-α- D -mannopyranoside with Me3SiCN gave the corresponding ethyl 4,6-di- O -acetyl-2- C -cyano-2-deoxy-α- D -glucopyranoside. Reaction of methyl 4,6- O -benzylidene-2,3-anhydro-α- D -allopyranoside or methyl 4,6- O -benzylidene-2,3-di- O -tosyl-α- D -glucopyranoside with Me3SiCN at - 75° or - 50° gave the corresponding methyl 6- O -[(R)-cyano phenyl methyl]-α- D -glyco-pyranosides with high or total regio and stereoselectivity.

Uridine-5′-Diphosphate Glucose Analogues. 21. Nucleoside Modified Analogues of Antiviral 5′-O-[[[[(α-D-Glucopyranosyl)Oxy Carbonyl]Amino]Sulfonyl]Uridine Derivatives

Abstract A series of uridine modified analogues of antiviral 5′-O-[[[[(α-D-glucopyranosyl) oxy] carbonyl]amno]sulfonyl]uridine derivatives has been synthesized by reaction of suitably protected glucose and glucosamine derivatives with CISO2-N=C=O and thymidine, 2′-deoxyuridine, 3-methyluridine, 5, 6-dihydrouridine and 1-[(2-hydroxyethoxy) methyl] uracil derivatives. The antiviral activity against HSV-1 has been determined.

Stereoselective synthesis of cis-4-(substituted) monobactams from ethyl acetoacetate

The stereoselective synthesis of cis-3-amino-4-methyl-2-oxoazetidine-1-sulphonic acid (25) from ethyl acetoacetate is described. Nitrosation of this compound and reduction of the resulting oxime gave the corresponding amine, which after treatment with different acyl halides, yielded the acylamino derivatives (6)–(8). Condensation with p-anisidine gave the enamines (12)–(14), which were then reduced to the β-amino acid esters (15)–(17). Stereoselective cyclization with Grignard reagents as base, and appropriate deprotection and sulphonation of the resulting β-lactams (18)–(20), led to the title compounds.

Enantioselective synthesis of (3S)-trans-4-(substituted methyl)monobactams from 2,3-O-isopropylidene-D-glyceraldehyde

The enantioselective synthesis of various (3S)-trans-4-(substituted methyl)-2-oxoazetidine-1-sulphonic acid derivatives from 2,3-O-isopropylidene-D-glyceraldehyde is described. Reaction of this aldehyde with trimethylsilyl cyanide gave a 80:20 ratio of the corresponding threo and erythroα-aminonitriles, which by amino protection and subsequent treatment with basic hydrogen peroxide provided the corresponding α-amino carboxamides. Successive selective protection, activation, sulphonation and, finally, stereospecific cyclization of the threoα-carboxamide yielded (2S,4R)-3-benzyloxycarbonylamino-4-hydroxymethyl-2-oxoazetidine-1-sulphonic acid, an intermediate for the synthesis of the corresponding 4-acyloxymethyl-, 4-carbamoyloxymethyl-, 4-methylsulphonyloxymethyl-, 4-iodomethyl-, and 4-methyl-2-oxozetidines.

Analogues of Uridinediphosphatehexoses. A New Type of Protein Glycosylation Inhibitors That Show Antiviral Activity

Abstract A series of analogues of UDP-Glc and UDP-GlcNAc prepared by reaction of protected hexoses with ClSO2NCO and 2′3′-O-isopropylideneuridine, inhibited glycosylation of proteins in HSV-1 infected HeLa cells and were active against several enveloped viruses.