Laura Franchini

CD1a-binding glycosphingolipids stimulating human autoreactive T-cells: synthesis of a family of sulfatides differing in the acyl chain moiety

Abstract Native sulfatide (a mixture of 3-sulfated β- d -galactopyranosylceramides with different fatty acids at the ceramide moiety) is an antigen presented by CD1a proteins. Herein the preparation of four sulfatides, which are constituents of the natural mixture and bear palmitic, stearic, behenic or nervonic fatty acid chains, is described. Azidosphingosine was stereoselectively synthesized through a CuCN-catalyzed allylic alkylation of a hexenitol dimesylate derived from d -xylose; β-glycosylation of azidosphingosine with a suitable d -galactosyl trichloroacetimidate led, after reduction of the azido group, to the galactosylsphingosine skeleton, which was derivatized with the different fatty acids. Final regioselective 3-sulfation gave the desired sulfatides, which were tested for activation of sulfatide-specific and CD1a-restricted T-cell clones.

A formal synthesis of 3-O-(4-methoxybenzyl)-azidosphingosine by a modified Julia olefination

Abstract The stereoselective construction of the d - erythro -azidosphingosine characteristic trans double bond was accomplished by condensation between tetradecanal and a heterocyclic sulfone derived from diethyl- d -tartrate, following the Kocienski modification of the Julia olefination.

Antimicrobial host defence peptide, LL-37, as a potential vaginal contraceptive

STUDY QUESTION Does antimicrobial peptide, LL-37, inhibit sperm fertilizing ability? SUMMARY ANSWER Our results indicate that LL-37 inhibits mouse and human sperm fertilizing ability. WHAT IS KNOWN ALREADY LL-37, a cationic antimicrobial peptide, exerts its microbicidal effects through the disruption of microbial cytoplasmic membranes following its interaction with microbial surface anionic phospholipids. ALL-38 (an LL-37 close analogue: LL-37 + Ala at the N-terminus) is produced in the vagina 2-6 h post-intercourse from its precursor hCAP-18, a seminal plasma component. At this time, motile sperm have already swum into the uterine cavity, thus unexposed to ALL-38. Since sperm contain a substantial amount of acidic sulfogalactosylglycerolipid (SGG) on their surface, treatment of sperm with LL-37 may cause their membrane disruption in an analogous manner to that occurring on microbial membranes. STUDY DESIGN, SIZE AND DURATION Mouse/human sperm treated (2-30 min) with LL-37 in a physiological concentration range (up to 10.8 µM) were assessed for SGG-dependent LL-37 binding, and parameters relevant to fertilizing ability, namely motility and intactness of the sperm acrosome and plasma membrane. Ability of mouse sperm to fertilize eggs in vitro was also evaluated. Each study was performed with greater than or equal to three different sperm samples. The efficacy of LL-37 to inhibit sperm fertilizing ability in vivo was determined in female mice (n = 26 each for LL-37 treatment and no treatment), using sperm retrieved from 26 males. PARTICIPANTS/MATERIALS, SETTING, METHODS Human sperm samples were donated by fertile men. LL-37 was chemically synthesized and was biotinylated for sperm binding studies. Sperm motility was assessed by videomicroscopy and the acrosomal status by Coomassie blue staining of acrosome-intact mouse sperm or the exposure of CD46, an inner acrosomal membrane protein, of acrosome reacted human sperm. Sperm membrane permeabilization/disruption was assessed by the loss of hypo-osmotic swelling response, an incorporation of Sytox Green (a membrane impermeable fluorescent DNA dye), and electron microscopy. Mouse IVF was scored by the presence of two pronuclei in eggs 6 h post-insemination. Ability of mouse sperm to fertilize eggs in vivo was determined by the pregnancy outcome of female mice injected transcervically with sperm with or without LL-37. MAIN RESULTS AND THE ROLE OF CHANCE Biotinylated LL-37 bound to both mouse and human sperm and the binding was partially dependent on sperm surface SGG. Mouse and human sperm became immotile and underwent a premature acrosome reaction upon treatment with LL-37 at 3.6 and 10.8 µM, respectively. The initial action of LL-37 on both mouse and human sperm appeared to be through permeabilization/disruption of sperm surface membranes evidenced by the loss of hypo-osmotic swelling response, Sytox Green staining and electron microscopy revealing ultrastructural damage. Mouse sperm treated with 3.6 µM LL-37 lost the ability to fertilize eggs both in vitro and in vivo. All 26 female mice inseminated with sperm and LL-37 did not become pregnant. No apparent damage to the reproductive tract was observed as revealed by histological characterization in LL-37-inseminated mice and these females resumed fecundity following mating with fertile males. LIMITATIONS, REASONS FOR CAUTION Direct demonstration that LL-37 treated human sperm fail to fertilize eggs was limited by legal restrictions on obtaining human eggs for such use. WIDER IMPLICATIONS OF THE FINDINGS Our results reveal selective inhibitory effects of LL-37 on sperm fertilizing ability in mice without apparent impairment to the female reproductive tract. LL-37 is therefore a promising candidate to be developed into a vaginal contraceptive with microbicidal activity. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by Grand Challenges Explorations grant from the Bill & Melinda Gates Foundation (OPP1024509), Canadian Institutes of Health Research (MOP119438 & CCI82413) and International Collaboration and Exchanges NSFC of China (No.30611120525). There are no competing interests to declare.

Glycoglycerolipid analogues inhibit PKC translocation to the plasma membrane and downstream signaling pathways in PMA-treated fibroblasts and human glioblastoma cells, U87MG.

Glycoglycerolipid analogues, derived from 2-O-β-D-galactosylglycerol, have been synthesized on the base of the structure of natural glycoglycerolipids showing anti-tumor and anti-inflammatory efficacy. These compounds have been previously demonstrated to inhibit phorbol 12-myristate-13-acetate (PMA) induced tumor promotion in mouse skin, but their mechanism of action has never been elucidated. In this work, we studied the effects of glycoglycerolipid analogues on PKC activation induced by PMA and its downstream target molecules, in human fibroblasts. Our results proved that: a) the tested compounds were able to block PKC translocation to the plasma membrane, promoted by PMA, in a dose-dependent manner (IC50: 0.48 μM for the most active compound 2); b) the efficacy of these compounds was strongly connected to their acyl chain linked to galactose; in particular, the addition of hexanoyl and branched chains enhanced PKC inhibition, the presence of a cyclohexane ring and an excessive length of the acyl chain, or its lack, exerted a negative effect; c) the inhibition of PKC translocation blocked enzyme activation and downstream signaling pathways, MAPK and FAK, involved in proliferation and adhesion/migration control. In addition, the branched glycoglycerolipid (compound 2) was able to inhibit PKC translocation and activation in naturally highly PKC activating glioblastoma cells, U87MG. As consequence, U87MG cell proliferation and, especially, migration potential resulted to be markedly reduced (-30% and -84%, respectively). Thus, these results reveal the role of a PKC-dependent mechanism in glycoglycerolipid analogues mediated protective effects and highlight their possible employment in the field of prevention/treatment of cancer.

Chemoenzymatic stereoconvergent synthesis of 3-O-benzoyl azidosphingosine

Abstract The synthesis of 3- O -benzoyl azidosphingosine 1 through a stereoconvergent approach is described. Nucleophilic addition of the Grignard reagent of 1-pentadecyne to cyclohexylidene- d -glyceraldehyde results in a mixture of diastereoisomeric propargylic alcohols. Subsequent enzymatic separation of these diastereoisomers, mediated by lipase from Candida antarctica , Mitsunobu inversion on the wrong diastereoisomer and extremely efficient introduction of azide using a chloromesylate leaving group affords the title compound in 30% overall yield.

Quantification of seminolipid by LC-ESI-MS/MS-multiple reaction monitoring: compensatory levels in Cgt mice

Seminolipid, also known as sulfogalactosylglycerolipid (SGG), plays important roles in male reproduction. Therefore, an accurate and sensitive method for SGG quantification in testes and sperm is needed. Here we compare SGG quantitation by the traditional colorimetric Azure A assay with LC-ESI-MS/MS using multiple reaction monitoring (MRM). Inclusion of deuterated SGG as the internal standard endowed accuracy to the MRM method. The results showed reasonable agreement between the two procedures for purified samples, but for crude lipid extracts, the colorimetric assay significantly overestimated the SGG content. Using ESI-MS/MS MRM, C16:0-alkyl/C16:0-acyl SGG of Cgt+/− mice was quantified to be 406.06 ± 23.63 μg/g testis and 0.13 ± 0.02 μg/million sperm, corresponding to 78% and 87% of the wild-type values, respectively. CGT (ceramide galactosyltransferase) is a critical enzyme in the SGG biosynthesis pathway. Cgt−/− males depleted of SGG are infertile due to spermatogenesis arrest. However, Cgt+/− males sire offspring. The higher than 50% expression level of SGG in Cgt+/− animals, compared with the wild-type expression, might be partly due to compensatory translation of the active CGT enzyme. The results also indicated that 78% of SGG levels in Cgt+/− mice were sufficient for normal spermatogenesis.

An efficient and convenient synthesis of deuterium-labelled seminolipid isotopomers and their ESI-MS characterization

Seminolipids 1a and 1b and galactosylalkylacylglycerols 2a and 2b, labelled with deuterium on the alkyl or acyl chain, respectively, were obtained isotopically and chemically pure through a straightforward synthesis from protected glycidyl galactoside 3 in an overall 22% yield. The identity and purity of compounds was ascertained by NMR spectroscopy and ESI mass spectrometry analysis. These labelled compounds are important as internal standards for quantification of these lipids by mass spectrometry, and they could also be used in metabolic studies in in vitro and even in vivo systems. Extension of the procedure could provide a route for the preparation of isotopomers of other compounds of the same general class.

Synthetic Potential of Fucosyltransferase III for the Synthesis of Fluorescent‐labeled Milk Oligosaccharides

Various fundamental biologic roles of milk oligosaccharides have been recognized; however, their structure‐affinity relationship is still not fully revealed. Herein, we describe the synthesis of the fluorescent‐labeled milk oligosaccharides 3‐(5‐dimethylaminonaphthalene‐1‐sulfonylamino)propyl β‐D‐galactopyranosyl‐(1→3)‐[α‐L‐fucopyranosyl‐(1→4)]‐2‐acetamido‐2‐deoxy‐β‐D‐glucopyranoside (1) and 3‐(5‐dimethylamino‐naphthalene‐1‐sulfonylamino)propyl β‐D‐galactopyranosyl‐(1→3)‐[α‐L‐fucopyranosyl‐(1→4)]‐β‐D-glucopyranoside (2) as useful tools for synthetic, analytic, and biologic applications. For the fucosylation of lactose and lacto‐N‐biose, the chemical and the enzymatic syntheses using fucosyltransferase III were compared.

Anti-Tumor-Promoting Activity of Tibolone and its Metabolites

The aim of this study was to evaluate the cancer chemopreventive potential of the widely prescribed drug tibolone (17alpha-ethynyl-7alpha-methyl-5(10)-estren-3-one, CAS 5630-53-5) and its main metabolites, 17alpha-ethynyl-7alpha-methyl-4-estren-3-one (CAS 1162-60-3), 17alpha-ethynyl-7alpha-methyl-5(10)-estrene-3alpha,17beta-diol (CAS 100239-44-9) and 17alpha-ethynyl-7alpha-methyl-5(10)-estrene-3beta,17beta-diol (CAS 100239-45-0), by studying their anti-tumor-promoting activity. To this aim the test compounds were submitted to the short term in vitro assay for the inhibition of Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) as a primary screening for anti-tumor promoters. All the compounds showed high inhibitory activity and low cytotoxicity as compared to literature data. To extend the study to an animal model, tibolone and its 3alpha-hydroxy metabolite (CAS 100239-44-9) were also assayed in the in vivo two-stage on mouse skin carcinogenesis test, exhibiting significant inhibitory effects on TPA promoted mouse skin papillomas formation. A comparison with literature data indicated them as more potent compounds than other steroids previously studied such as digitoxigenin, cortisone, hydrocortisone, and prednisolone.