Carbamoylphosphonate-based matrix metalloproteinase inhibitor metal complexes: solution studies and stability constants. Towards a zinc-selective binding group

Overactive matrix metalloproteinases (MMPs) are associated with a variety of disease states. Therefore, their inhibition is a highly desirable goal. Yet, more than a decade of worldwide activity has not produced even one clinically useful inhibitor. Because of the crucial role of zinc in the activity of the enzyme, the design of inhibitors is usually based upon a so-called zinc binding group (ZBG). Yet, many of the hitherto synthesized potent inhibitors failed clinically, presumably because they bind stronger to metals other than zinc. We have developed in vivo potent inhibitors based on the carbamoylphosphonic group as a putative ZBG. In this paper we report stability constants for Ca(II), Mg(II), Zn(II) and Cu(II) complexes of two potent, in vivo active, MMP inhibitors, cyclopentylcarbamoylphosphonic acid (1) and 2-(N,N-dimethylamino)ethylcarbamoylphosphonic acid (2). Precipitation prevented the determination of stability constants for iron(III) complexes of 1 and 2. For comparison with carbamoylphosphonates 1 and 2, we synthesized 2-cyclohexyl-1,1-difluoroethylphosphonic acid (3), which does not inhibit MMP, and determined the stability constants of its complexes with Mg(II), Ca(II) and Zn(II). Comparison with the values obtained from the complexes of 1 and 2 with those from 3 indicates participation of the C=O group in the metal binding of the former compounds. The complex stability orders for both 1 and 2 are Ca(II)<Mg(II)<Zn(II)<Cu(II). In addition, the results indicate that at pH>8 the dimethylamino group of compound 2 can also participate in the binding of the transition metals Cu and Zn. On the other hand, the amino group in carbamoylphosphonic acid 2 lowers the stability of the complexes with metals favoring oxygen ligands (Ca, Mg and Fe) and increases the selectivity towards Zn. These results are helpful for rationalizing the results observed on our MMP inhibitors hitherto examined, and are expected to be useful for the design of new selective inhibitors.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0524-5     

Innate protection conferred by fucosylated oligosaccharides of human milk against diarrhea in breastfed infants

To test the hypothesis that human milk fucosyloligosaccharides are part of an innate immune system, we addressed whether their expression (1) depends on maternal genotype and (2) protects breastfed infants from pathogens. Thus the relationship between maternal Lewis blood group type and milk oligosaccharide expression and between variable oligosaccharide expression and risk of diarrhea in their infants was studied in a cohort of 93 Mexican breastfeeding mother-infant pairs. Milk of the 67 Le(a-b+) mothers contained more LNF-II (Le(a)) and 3-FL (Le(x)) (oligosaccharides whose fucose is exclusively alpha 1,3- or alpha 1,4-linked) than milk from the 24 Le(a-b-) mothers; milk from Le(a-b-) mothers contained more LNF-I (H-1) and 2'-FL (H-2), whose fucose is exclusively alpha 1,2-linked. The pattern of oligosaccharides varied among milk samples; in each milk sample, the pattern was summarized as a ratio of 2-linked to non-2-linked fucosyloligosaccharides. Milks with the highest ratios were produced primarily by Le(a-b-) mothers; those with the lowest ratios were produced exclusively by Le(a-b+) mothers (p<0.001). Thus maternal genetic polymorphisms expressed as Lewis blood group types are expressed in milk as varied fucosyloligosaccharide ratios. The four infants who developed diarrhea associated with stable toxin of Escherichia coli were consuming milk with lower ratios (4.4 +/- 0.8 [SE]) than the remaining infants (8.5 +/- 0.8; p<0.001). Furthermore, the 27 infants who developed moderate to severe diarrhea of any cause were consuming milk with lower ratios (6.1 +/- 0.9) than the 26 who remained healthy (10.5 +/- 1.9; p = 0.042). Thus, milk with higher 2-linked to non-2-linked fucosyloligosaccharide ratios affords greater protection against infant diarrhea. We conclude that specific oligosaccharides constitute a major element of an innate immune system of human milk.     

Prediction of pKa and redox properties in the thioredoxin superfamily

Electrostatic interactions play important roles in diverse biological phenomena controlling the function of many proteins. Polar molecules can be studied with the FDPB method solving the Poisson-Boltzmann equation on a finite difference grid. A method for the prediction of pK(a)s and redox potentials in the thioredoxin superfamily is introduced. The results are compared with experimental pK(a) data where available, and predictions are made for members lacking such data. Studying CxxC motif variation in the context of different background structures permits analysis of contributions to cysteine DeltapK(a)s. The motif itself and the overall framework regulate pK(a) variation. The reported method includes generation of multiple side-chain rotamers for the CxxC motif and is an effective predictive tool for functional pK(a) variation across the superfamily. Redox potential follows the trend in cysteine pK(a) variation, but some residual discrepancy indicates that a pH-independent factor plays a role in determining redox potentials for at least some members of the superfamily. A possible molecular basis for this feature is discussed.     

Monomers for Oligonucleotide Synthesis with Linkers Carrying Reactive Residues: II. The Synthesis of Phosphoamidites on the Basis of Uridine and Cytosine and Containing a Linker with Methoxyoxalamide Groups in Position 2′

A convenient preparative synthesis of 2-amino-2-deoxyuridine was developed. Starting from 2-amino-2-deoxyuridine and 2-amino-2-deoxycytidine, monomers for the phosphoamidite oligonucleotide synthesis were obtained that carry a linker with methoxyoxalamide groups in position 2.     

Zinc triflate-benzoyl bromide: a versatile reagent for the conversion of ether into benzoate protecting groups and ether glycosides into glycosyl bromides

A simple and efficient method is developed for the chemoselective one-pot conversion of ethers (benzyl, TBDMS and acetal) to the corresponding benzoates by zinc triflate-catalyzed deprotection and benzoylation by benzoyl bromide. In the same reaction, methyl or p-methoxyphenyl glycosides are converted into glycosyl bromides that are useful in glycosylation reactions.     

Characterization of an acetylated heteroxylan from Eucalyptus globulus Labill

A heteroxylan was isolated from Eucalyptus globulus wood by extraction of peracetic acid delignified holocellulose with dimethyl sulfoxide. Besides (1-->4)-linked beta-D-xylopyranosyl units of the backbone and short side chains of terminal (1-->2)-linked 4-O-methyl-alpha-D-glucuronosyl residues (MeGlcA) in a 1:10 molar ratio, this hemicellulose contained galactosyl and glucosyl units attached at O-2 of MeGlcA originating from rhamnoarabinogalactan and glucan backbones, respectively. About 30% of MeGlcA units were branched at O-2. The O-acetyl-(4-O-methylglucurono)xylan showed an acetylation degree of 0.61, as determined by 1H NMR spectroscopy, and a weight-average molecular weight (M(w)) of about 36 kDa (P=1.05) as revealed from size-exclusion chromatography (SEC) analysis. About half of the beta-D-xylopyranosyl units of the backbone were found as acetylated moieties at O-3 (34 mol%), O-2 (15 mol%) or O-2,3 (6 mol%). Practically, all beta-D-xylopyranosyl units linked at O-2 with MeGlcA residues were 3-O-acetylated (10 mol%).     

Growth and Respiration in Plants from Various Adaptation Groups as Affected by Mineral Nutrient Deficiency

The effect of the deficiency in mineral nutrients was investigated in plant species representing various adaptation groups (stress-tolerant, competitive, and ruderal plants). Dry and fresh weight, as well as the length of shoots and underground organs, were determined in 20- to 50-day-old seedlings. The ratio between the dry weights of shoot and root (SRR), relative growth rate (RGR), the rate of total dark respiration (R), gross photosynthesis (P _g), and the proportion of the respiratory expenditures to gross photosynthesis (R/P _g) were calculated. When affected by a deficiency in mineral nutrients, the weight of the whole plant decreased. In resistant species of clover (Trifolium pratense L.) and alfalfa (Medicago sativa L.), this reduction was insignificant, whereas, in the ruderal species amaranth (Amaranthus retroflexus L.), it was at its highest. In all the species investigated, the ratio R/P _g was 38–46%. Under stress conditions, this index increased. Given a deficiency in mineral nutrients, the changes in SRR, RGR, and R/P _g were greater in amaranth, suggesting that this plant species is less tolerant to stress. The correlation between RGR and R observed in amaranth under normal conditions indicates that the major energy expenditures are associated with growth. Under stress conditions, such a correlation was not observed. In more resistant species of clover and alfalfa, a weak positive correlation between RGR and R was observed both under normal and stress conditions. In these species, the deficiency in mineral nutrients probably brought about a reduction in the growth component of total dark respiration and a rise in the adaptation component. The complex of indices (R/P _g, RGR, and SRR) and the extent of their variation in the seedlings describe the potential productivity and resistance of particular species to a deficiency in mineral nutrients and may characterize the adaptation type of the plants.     

Preparation of orthogonally protected chitosan oligosaccharides: observation of an anomalous remote substituent effect

Orthogonally protected chitosan tetrasaccharides were synthesized in a convergent fashion by trichloroacetimidate activation. The anomeric substituent at the reducing end of the disaccharide acceptor has a remarkably strong influence on glycosidic coupling; a thiophenyl-substituted disaccharide was observed to be an unusually poor glycosyl acceptor in comparison with the corresponding allyloxy-substituted disaccharide.     

Expression and evolution studies of ets genes in a primitive coelomate,the polychaete annelid,Hediste (Nereis) diversicolor

The Ets family includes numerous proteins with a highly conserved DNA-binding domain of 85 amino acids named the ETS domain. Phylogenetic analyses from ETS domains revealed that this family could be divided into 13 groups, among them are ETS and ERG. The ets genes are present in the Metazoan kingdom and we have previously characterized the Nd ets and Nd erg genes in the polychaete annelid Hediste diversicolor. Here, we isolated a fragment encoding the ETS domain from Nd Ets, by genomic library screening. By Northern blot analysis, we showed that this gene was transcribed as one major mRNA of 2.6 kb and one minor mRNA of 3.2 kb. By in situ hybridization, we observed that Nd ets was expressed in the intestine and oocytes and that Nd erg was expressed in cellular clumps present in the coelomic cavity, in an area of proliferating cells situated between the last metamere and the pygidium. Finally, we showed that Nd erg shared the expression pattern of Nd ets in oocytes. Molecular modeling studies have revealed that the spatial structure of ETS domain of Nd Ets and Nd Erg was conserved, in comparison to the murine Ets-1 and human Fli-1 proteins, respectively.     

Nepotistic vigilance behavior in Siberian jay parents

Many Siberian jay offspring (up to 50%) postpone independenceand stay with their parents for up to 3 years. Parents offersuch nondispersers a benefit in that they increase their vigilancewhile feeding together with retained offspring. In contrast,parents reduce their vigilance while in company of nonrelatedflock members according to the "many eyes" principle. The preferentialtreatment offered by the parents provides an incentive for offspringto forego dispersal. Given evidence for mortality via surpriseattacks by predators (goshawks), such nepotistic vigilance byparents could have a bearing on offspring survival and therebypromote delayed dispersal.