Anionic glycolipids related to glucuronosyldiacylglycerol inhibit protein kinase Akt.

New glucuronosyldiacylglycerol (GlcADG) analogues based on a 2-O-ß-D-glucopyranosyl-sn-glycerol scaffold and carrying one or two acyl chains of different lengths have been synthesized as phosphatidylinositol 3-phosphate (PI3P) mimics targeting the protein kinase Akt. The Akt inhibitory effect of the prepared compounds was assayed using an in vitro kinase assay. The antiproliferative activity of the compounds was tested in the human ovarian carcinoma IGROV-1 cell line in which we found that two of them could inhibit proliferation, in keeping with the target inhibitory effect.

New 6-amino-6-deoxy-glycoglycerolipids derived from 2-O-ß-D-glucopyranosylglycerol: insights into the structure-activity relationship of glycoglycerolipids as anti-tumor promoters.

As part of a project aimed at obtaining compounds capable of inhibiting tumor promotion, new 6-amino-6-deoxyglycoglycerolipids (AGGLs) derived from 2-O-ß-D-glucopyranosyl-sn-glycerol were synthesized and tested for their anti-tumor-promoting activity using a short-term in vitro assay of the inhibition of Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). The corresponding 6-amino-6-deoxy-ß-D-octylglucosides were also prepared as simplified aminoglycolipid models and tested. Comparison with the activity of a series of previously studied glycoglycerolipids showed that replacing the 6-oxygen of the glucose moiety by a nitrogen atom greatly reduced the in vitro activity of the compounds. A two-stage mouse skin carcinogenesis test of two representative aminoglycoglycerolipids confirmed their reduced activity also in this in vivo model.

A practical route to long-chain non-natural α,ω-diamino acids.

An efficient method for the synthesis of long-chain α,ω-diamino acids, starting from natural α-amino acids, has been developed. The long-chain skeleton has been generated through condensation between a protected aldehyde, derived from L-aspartic acid, and an ylide obtained from an ω-hydroxy-alkyl phosphonium salt. After conversion of the ω-hydroxy group into an amine, catalytic hydrogenation produced the N,N'-protected α,ω-diamino acid. The present route to α,ω-diamino acids allows the modulation of the chain length depending on the length of the ylide used for the Wittig olefination reaction.

Development of a fluorescent probe for the study of the sponge-derived simplexide immunological properties.

Simplexide is a glycolipid of marine origin, endowed with immunological properties, composed of a long chain secondary alcohol glycosylated by an α-d-glucosyl-(1→4)-ß-D-galactosyl disaccharide residue. Herein we describe the preparation of a fluorescent derivative of simplexide, labeled at position 6 of the distal glucose with a dansyl group, as a probe for future studies on the mechanism by which simplexide affects the immune system. Fluorescent simplexide was prepared from a 6″-amino functionalized compound, which in turn was obtained through a highly efficient glycosylation between the preformed activated disaccharide and the long chain secondary alcohol 18-pentatriacontanol.

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.

Synthesis of the sulfonate analogue of seminolipid via Horner-Wadsworth-Emmons olefination.

The first synthesis of the sulfonate analogue of seminolipid, the main sulfoglycolipid in mammalian sperm, is reported. Installation of the sulfonate unit was accomplished by a quite unexplored strategy based on Horner-Wadsworth-Emmons olefination on a 3 '-keto-galactoside, followed by stereoselective double bond reduction.

Synthesis, molecular dynamics simulations, and biology of a carba-analogue of the trisaccharide repeating unit of Streptococcus pneumoniae 19F capsular polysaccharide.

The synthesis of a carba-analogue corresponding to the trisaccharide repeating unit of Streptococcus pneumoniae type 19F capsular polysaccharide, where a residue of carba-L-rhamnose has been inserted into the natural trisaccharide in place of L-rhamnose, is described. The conformational properties of the analogue were investigated with the aid of molecular dynamics simulations and were strictly analogous to those of the natural compound. The biological activity of the carba-analogue was comparable to that of the corresponding natural repeating unit, thus suggesting that this compound, more stable to hydrolysis, is a good mimic of the natural structure.

2-O-beta-d-glucopyranosyl-sn-glycerol based analogues of sulfoquinovosyldiacylglycerols (SQDG) and their role in inhibiting Epstein-Barr virus early antigen activation.

New sulfoquinovosyldiacylglycerols derived from 2-O-beta-d-glucopyranosyl-sn-glycerol, carrying acyl chains of various length on the glycerol moiety, were prepared through a convenient synthetic procedure in which a sulfonate is introduced at the C-6 position of glucose by oxidation of a thioacetate in the presence of the unprotected secondary hydroxyl groups, and tested for their anti-tumor-promoting activity using a short-term in vitro assay for Epstein-Barr virus early antigen (EBV-EA) activation. Our study has allowed to ascertain the role of the 6'-sulfonate group and the need of a free hydroxyl group on the glycerol moiety in inhibiting the EBV activation promoted by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA).

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.

A general and stereoselective route to alpha- or beta-galactosphingolipids via a common four-carbon building block.

A general synthetic strategy toward alpha- or beta-galactosylceramides and their analogues from 3-azido-2-O-benzyl-1-O-(4-methoxybenzyl)butane-1,2,4-triol is described. The key steps for the installation of the main lipid chain are either a diasteroselective alkynylation reaction yielding the 4R stereocenter of phytosphingosine or a Wittig olefination generating the trans double bond of sphingosine. The methodology allows the preparation of different glycolipids with variations in the structure of the sphingoid base. In particular, three alpha-GalCer-related compounds have been synthesized and evaluated for their ability to activate CD1d-restricted T-cells.

Synthesis and evaluation of human T cell stimulating activity of an alpha-sulfatide analogue.

A concise synthesis of alpha-sulfatide 1, an analogue of natural glycolipid antigens with potential anti-tumor activity, was performed. Two different approaches to the alpha-glycosidic bond were explored, resulting in a high yield and excellent stereoselectivity. Compound 1 combines the structural features of sulfated beta-GalCer (sulfatide) and alpha-GalCer, which activate specific T cells. alpha-Sulfatide 1 was stimulatory for CD1d-restricted semi-invariant Natural Killer T (iNKT) cell clones, although less potent than alpha-GalCer, while it was not recognized by CD1a-restricted sulfatide-specific T cells.

The conformational behaviour of the C-glycosyl analogue of sulfatide studied by NMR in SDS micelles.

The conformational behaviour of sulfatide and its C-glycosyl analogue has been studied by using a combination of J and NOE data assisted by molecular mechanics calculations. There is a major exoanomeric conformation around the phi angle of both molecules with two or three conformers contributing to the equilibrium around psi. The MM3* calculations only provide a qualitative description of the actual population distribution. Despite this geometrical similarity, the quantitative analysis of the NOE intensities at a variety of mixing times indicates that the motion around the pseudoglycosidic linkages of the C-glycosyl analogue is faster than that for the natural compound.

Cyclic and branched acyl chain galactoglycerolipids and their effect on anti-tumor-promoting activity.

Fifteen new galactoglycerolipid analogues, in which one or two branched, alicyclic or aromatic acyl chains are linked to 2-O-beta-D-galactosylglycerol (6'-position or 1,6' positions), were prepared and tested for their anti-tumor-promoting activity using a short-term in vitro assay for Epstein-Barr virus early antigen (EBV-EA) activation. All compounds were active in inhibiting the EBV activation promoted by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), the branched compounds resulting in the most active glycoglycerolipid analogues of the series. The branched 2-O-[6-O-(3-methylbutanoyl)-beta-D-galactopyranosyl]-sn-glycerol (1a) and the structurally related alicyclic 2-O-[6-O-(2-cyclohexylethanoyl)-beta-D-galactopyranosyl]-sn-glycerol (1d), when tested in an in vivo two-stage carcinogenesis test, exhibited inhibitory effects on mouse skin tumor promotion.

Stereoselective synthesis and immunogenic activity of the C-analogue of sulfatide.

[Structure: see text] The C-sulfatide 1b was synthesized through a [2,3]-Wittig sigmatropic rearrangement and a Horner-Wadsworth-Emmons olefination as the key steps. The C-analogue 1b is less immunogenic than natural sulfatide 1a, but induces a preferential secretion of the proinflammatory cytokine IFN-gamma.

Synthesis of sulfated galactocerebrosides from an orthogonal beta-D-galactosylceramide scaffold for the study of CD1-antigen interactions.

CD1a protein binds sulfatide (3-O-sulfo-beta-D-galactosylceramide) to form an antigen complex that interacts with T cell receptors and activates T cells. To assess the role of the position of the sulfate in T cell activation, the synthesis of three beta-D-galactosylceramides, variously bearing a sulfate at position 2, 4, or 6 of galactose, has been planned and carried out. The compounds were synthesized by an orthogonal sulfation strategy from a common beta-D-galactosylceramide scaffold, which was in turn obtained through an efficient glycosylation reaction between a fully orthogonally protected galactosyl imidate and 3-O-benzoylazidosphingosine. Immunological evaluation of the three sulfated compounds in CD1a-mediated T cell activation, in comparison with natural sulfatide, provided evidence of the influence of the sulfate position in the recognition event between the antigen, the CD1 protein and the T cell receptor.

Crystal structures of 10alpha-gon-5-enes from the synthetic pathway to desogestrel.

X-ray crystallographic studies performed on the product of the ketalization reaction of 13beta-ethyl-11alpha-hydroxy-gon-5-ene-3,17-dione have lead to the unequivocal assignment of the 10alpha stereochemistry to C10, showing that an inversion of configuration occurred during formation of the 3,17-diketal. From the Swern oxidation of this compound, 11alpha-(methylthio)methoxy-10alpha-gonene was obtained as the major product instead of the desired 11-ketone. Modeling studies showed that the configurational instability at C10 is determined by the presence of the 11alpha-hydroxyl group.

Anti-tumor-promoting activity of simple models of galactoglycerolipids with branched and unsaturated acyl chains.

Six new galactoglycerolipid analogs, in which one or two 4-methylpentanoyl or trans-2-butenoyl groups are linked to the 2-O-beta-D-galactosylglycerol skeleton, were tested for their anti-tumor-promoting activity using a short-term in vitro assay for Epstein-Barr virus early antigen (EBV-EA) activation. All these compounds were more active than their linear or saturated reference compounds in inhibiting the EBV activation promoted by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), the diester 1-O-(4-methylpentanoyl)-2-O-[6-O-(4-methylpentanoyl)-beta-D-galactopyranosyl]-sn-glycerol resulting the most active glycoglycerolipid analog till now tested. Four compounds (three butenoates and one 4-methylpentanoate), when tested in the in vivo two-stage carcinogenesis test, exhibited also inhibitory effects on mouse skin tumor promotion.

A theoretical study of the conformational behavior of analogues of alpha-L-rhamnose-1-phosphate.

The conformational behavior of methyl(2-O-methyl-alpha-L-rhamnopyranosyl)phosphate, together with a group of potentially more stable analogues, was investigated through a DFT approach at the B3LYP/6-31G(d) level; the energy of all the optimized structures was recalculated using a continuum solvent model, C-PCM, choosing water as the solvent. The compounds exhibited several, sometimes tenths of populated conformations so that the overall properties of flexibility and mobility were evaluated. The analogue in which the pyranose oxygen atom is replaced by a methylene group emerges as the best candidate as a mimic of the reference 1-phosphate, in spite of the fact that it lacks the anomeric and exo-anomeric effects. The other analogues result poorer mimics because of a conformational equilibrium at the pyranose ring or of an excessive rigidity of the aglycone moiety.

Crystal structure of steroidal monoenes obtained by reduction of the aromatic A ring of 13-ethyl-3-ethoxy-gona-1,3,5(10)-triene-11alpha,17beta-diol.

The crystal structures of 13-ethyl-gona-1(10)-ene-11alpha,17beta-diacetate (3b) and 13-ethyl-10alpha-gona-4-ene-11alpha,17beta-diacetate (5b), two steroidal monoenes obtained as minor products from the reduction, then acetylation, of the aromatic A ring of 13-ethyl-3-ethoxy-gona-1,3,5(10)-triene-11alpha,17beta-diol (1), were determined by X-ray diffraction. The conformations of the rings A, B, C, and D and the unusual stereochemistry at C-10 of the 10alpha-gona-4-ene (5b) are discussed.

Synthesis and anti-tumor-promoting activity of glycoglycerolipid analogues lacking the glycerol backbone.

Glycoglycerolipid analogues lacking the glycerol backbone were prepared through a lipase catalyzed transesterification of beta-D-galactosylethylene glycol. The inhibitory effect of the resultant isomeric hexanoates at the primary alcoholic positions, beta-D-galactosylethylene glycol itself and nonyl beta-D-galactopyranoside, was tested on Epstein-Barr virus early antigen (EBV-EA) activation in Raji cells promoted by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), as a primary screening test for inhibitors of tumor promotion. All the compounds assayed were found to be less active than the reference 2-O-beta-D-galactopyranosylglycerol derivatives, of which they are simplified models, indicating that the anti-tumor-promoting activity is very closely related to the presence of a free hydroxymethylene group on the glycerol-like aglycone moiety.