A straightforward synthetic access to symmetrical glycosyl disulfides and biological evaluation thereof.

Symmetrical glycosyl disulfides can be prepared within a few hours from per-O-acetylated precursors via a sequential approach entailing short reactions and no purification of any intermediate. The final thiolate-to-disulfide oxidation step is noticeably accelerated by low amounts of phenyl diselenide under air. Applicability of the strategy to non-saccharidic symmetrical alkyl disulfides has also been examined. A preliminary assay of the cytotoxic activity of symmetrical 1,1'- disulfides was performed on two human tumor cell lines, and a noteworthy activity was recorded for a range of these synthetic compounds.

An easy and versatile approach for the regioselective de-O-benzylation of protected sugars based on the I2/Et3 SiH combined system.

The use of cheap and easy to handle reagents, such as I(2) and Et(3) SiH, at low temperature allows the regioselective removal of benzyl protecting groups from highly O-benzylated carbohydrates. The observed regioselectivity is dependent on the nature of the precursor, the least accessible carbinol often being liberated. A mechanistic investigation reveals that in situ generated HI is the promoter of the process, whereas the regioselectivity appears to be mainly controlled by steric effects. However, the presence of an electron withdrawing acyl protecting group can switch the regioselectivity to favour deprotection of the carbinol position farthest from the ester group. The protocol is experimentally simple and provides straightforward access in useful yields to a wide range of partially protected mono- and disaccharide building blocks that are valuable for the synthesis of either biologically useful oligosaccharides or highly functionalised chiral compounds. Partially protected sugars thus obtained can also be coupled in situ with a glycosyl donor, as illustrated by the one-pot synthesis of a Lewis X mimic from fully protected precursors.

Rapid assembly of gp120 oligosaccharide moieties via one-pot glycosidation-deprotection sequences.

Mannosyl trihaloacetimidate donors equipped with a 2-O-Fmoc group can be effectively activated by catalytic Bi(OTf)(3) in glycosidations. Despite the expected participating effect of the Fmoc group, the reaction solvent was found to be decisive for obtaining highly selective alpha-mannosylations. The Fmoc 2-O-protecting group can be then simply removed from the obtained di-oligosaccharide in the same vessel where the glycosidation is conducted. The resulting oligosaccharide can thus be directly employed as a glycosyl acceptor for further elongation. The preparation of biologically important linear and branched oligomannoses incorporated into HIV gp120 demonstrates that iteration of this one-pot sequence leads to very straightforward oligosaccharide assembly. As an additional result, a rapid approach has been disclosed for accessing a 3,6-OH mannose building-block to be incorporated in branched structures. This relies on a double reductive opening of a di-O-benzylidene mannose intermediate whose regioselectivity appears to be independent of the configuration of the five-membered benzylidene.

Disentangling eumelanin "black chromophore": visible absorption changes as signatures of oxidation state- and aggregation-dependent dynamic interactions in a model water-soluble 5,6-dihydroxyindole polymer.

A fundamental unsettled issue concerning eumelanins, the functional biopolymers of human skin and hair, is why they are black. The experimental difficulty lies in the virtual insolubility of these pigments, causing marked scattering effects and hindering characterization of the intrinsic absorption properties of the heterogeneous species produced by oxidative polymerization of 5,6-dihydroxyindole (DHI) and related monomer precursors. The synthesis of spectrally robust, water-soluble DHI polymers is therefore an important goal in the prospects of disentangling intrinsic absorption properties of eumelanin components by circumventing scattering effects. Reported herein is the first water-soluble DHI polymer produced by oxidation of ad hoc designed 5,6-dihydroxy-3-indolyl-1-thio-beta-D-galactopyranoside (1). The dark brown polymer exhibited a distinct band at 314 nm and a broad visible absorption, resembling that of natural eumelanins. Main isolable oligomer intermediates including 2,7'- and 2,4'-biindolyls 2 and 3, attest the close resemblance to the mode of coupling of the parent DHI. Sodium borohydride reduction caused decoloration and a marked absorbance decrease in the visible region around 550 nm, but did not affect the UV band at 314 nm. Measurements of absorbance variations with dilution indicated a linear response at 314 nm, but a significant deviation from linearity in the visible region, with the largest decrease around 500 nm. It is argued that eumelanin black color is not only intrinsically defined by the overlap of pi-electron conjugated chromophores within the individual polymer components, as commonly believed, but also by oxidation state- and aggregation-dependent interchromophoric interactions causing perturbations of the heterogeneous ensemble of pi-electron systems and overall spectral broadening.

Rapid prenatal diagnosis of common chromosome aneuploidies by QF-PCR, results of 9 years of clinical experience.

BACKGROUND: Despite being deliberately targeted to common chromosome aneuploidies, the rapid quantitative fluorescent polymerase chain reaction (QF-PCR) tests can detect the majority of chromosome abnormalities in prenatal diagnosis. The main advantages of this assay are low cost, speed and automation allowing large-scale application. METHODS: We developed a QF-PCR test that was applied on 43 000 clinical samples reporting results in 24 h. Most common indications were biochemical risk (32%) and advanced maternal age (30%). Samples were also tested by cytogenetic analysis and the results compared. RESULTS: Aneuploidies involving chromosomes 21, 18, 13, X and Y were detected with 100% specificity. Several cases of partial trisomies and mosaicism were also identified. Overall 95% of clinically relevant abnormalities were readily detected and termination of affected pregnancies could be performed without waiting for the cytogenetic results. CONCLUSIONS: Our study supports the possibility of reducing the load of prenatal cytogenetic tests if the pregnancies are carefully monitored by non-invasive screening. In case of abnormal QF-PCR results, medical action can be taken within few hours from sampling. In cases of negative QF-PCR results, cytogenetic analyses might only be performed for fetuses with ultrasound abnormalities. In countries where large-scale cytogenetic tests are not available, QF-PCR may be used as the only prenatal diagnostic procedure.

Sequential one-pot glycosidations catalytically promoted: unprecedented strategy in oligosaccharide synthesis for the straightforward assemblage of the antitumor PI-88 pentasaccharide.

The pentasaccharide sequence of the most active components of the antitumor drug PI-88, currently in phase II clinical trial, has been rapidly assembled in high overall yield and in only three steps starting from three monosaccharide building blocks. The procedure takes advantage of the first reported strategy of sequential one-pot glycosidations conducted exclusively under catalytic activation. In addition, the procedure relies only on shelf-stable and mild promoters such as Yb(OTf)(3) and Bi(OTf)(3).

Novel approaches for the synthesis and activation of thio- and selenoglycoside donors.

Alkyl thio-, phenyl seleno-, and phenyl thioglycosides can be prepared through short synthetic sequences based on the generation of glycosyl iodides as versatile intermediates. In addition, a novel cheap combined system (stoichiometric NBS and catalytic Bi(OTf)3) has been developed for rapid and efficient activation of a wide variety of thio- and selenoglycoside donors.

Rapid prenatal diagnosis by QF-PCR: evaluation of 30,000 consecutive clinical samples and future applications.

Rapid prenatal diagnoses of major chromosome abnormalities can be performed on a large scale using highly polymorphic short tandem repeats (STRs) amplified by the quantitative fluorescent polymerase chain reaction (QF-PCR). The assay was introduced as a preliminary investigation to remove the anxiety of the parents waiting for the results by conventional cytogenetic analysis using amniotic fluid or chorionic cells. However, recent studies, on the basis of the analyses of several thousand samples, have shown that this rapid approach has a very high rate of success and could reduce the need for cytogenetic investigations. Its high efficiency, for example, allows early interruption of affected fetuses without the need of waiting for completion of fetal karyotype. The main advantages of the QF-PCR are its accuracy, speed, automation, and low cost that allows very large number of samples to be analyzed by few operators. Here, we report the results of using QF-PCR in a large series of consecutive clinical cases and discuss the possibility that, in a near future, it may even replace conventional cytogenetic analyses on selected samples.

Versatile use of ytterbium(III) triflate and acid washed molecular sieves in the activation of glycosyl trifluoroacetimidate donors. Assemblage of a biologically relevant tetrasaccharide sequence of Globo H.

The nonreducing tetrasaccharide terminus of Globo H has been assembled in good yield and excellent stereocontrol exclusively by using mild and moisture stable agents such as Yb(OTf)(3) and acid washed molecular sieves for the activation of glycosyltrifluoroacetimidate donors in the glycosylation steps.

Non-invasive screening and rapid QF-PCR assay can greatly reduce the need for conventional cytogenetic analyses in prenatal diagnosis.

In 2004, the UK National Screening Committee suggested that rapid screening tests, such as fluorescence in-situ hybridization (FISH) and/or quantitative fluorescence PCR (QF-PCR), should replace prenatal diagnosis of Down syndrome performed by conventional karyotyping. However, doubts have been expressed that replacement of conventional cytogenetic investigations would result in a substantial number of infants affected by preventable handicaps. Based on a brief analysis of 28,000 prenatal tests performed in genetic units, this paper discusses the advantages of using QF-PCR. All normal fetuses were correctly diagnosed without false positive results and approximately 93% major chromosome disorders were detected by the molecular approach. The need for cytogenetic tests was thus greatly reduced, since pregnancy can be terminated, if necessary, without the need to confirm the results. A careful combination of accurately performed non-invasive ultrasound and maternal blood tests, eventually followed by QF-PCR, should reduce the need for conventional chromosome analyses.

Modulating the activity of oligonucleotides by carbohydrate conjugation: solid phase synthesis of sucrose-oligonucleotide hybrids.

In order to expand the repertoire of available oligosaccharide-oligonucleotide hybrids, the on-line solid phase synthesis of oligonucleotides conjugated at the 3'-and/or 5'-end with a preformed disaccharide unit has been performed. The key compound in the synthetic scheme described here is an appropriate phosphoramidite derivative of fully protected sucrose, used in association with a solid support functionalized with DMT-protected sucrose. The sucrose units at both ends of selected oligonucleotide sequences were shown to increase their chemical and enzymatic stability, while not interfering with duplex formation and with the ability of G-rich sequences to adopt a quadruplex structure.

Comparison between fluorescence in situ hybridization (FISH) and quantitative-fluorescent polymerase chain reaction (QF-PCR) for the detection of aneuploidies in single blastomeres.

OBJECTIVES: The aim of our investigation was to compare the efficiencies of the fluorescence in situ hybridization (FISH) and the quantitative-fluorescent PCR (QF-PCR) methods for the detection of sexing and numerical chromosome disorders in single blastomeres collected from the same preimplantation human embryos. METHODS: FISH analysis was carried out on 145 blastomeres from the 79 surplus embryos with probes specific for chromosomes 13, 18, 21, X, and Y. QF-PCR was performed with each one or two of the primers specific for the same chromosomes on 151 blastomeres from the same embryos obtained from patients undergoing IVF treatment. RESULTS: Analyses were possible on 135 blastomeres (93%) by FISH and on 117 blastomeres (77%) by QF-PCR. Of 65 embryos, which could be analyzed by both methods, 20 embryos (31%) were diagnosed as abnormal. CONCLUSION: The present study shows that FISH tests are more accurate than QF-PCR assays for the detection of numerical chromosome disorders when performed on single blastomeres.

Very early prenatal diagnosis on coelomic cells using quantitative fluorescent polymerase chain reaction.

Quantitative fluorescent PCR (QF-PCR) has been shown to be an accurate assay for the rapid prenatal diagnosis of chromosome disorders. The extra-embryonic coelom develops during week 4 of gestation and it can be aspirated from the following week, making coelocentesis the earliest possible method of prenatal diagnosis after implantation. The possibility of using the QF-PCR assay performed on DNA extracted from cells present in the extra-embryonic coelom has been evaluated for the detection of aneuploidies. QF-PCR amplification using several markers for chromosomes X, Y, 21, 18 and 13 was successfully achieved on all 17 serial samples of exo-coelomic fluid (ECF), placental tissue and maternal blood. Multiplex analyses of maternal blood samples and chorionic tissues allowed the distinction of fetal from maternal patterns and, eventually, the identification of maternal contamination of the ECF samples. Prenatal detection of fetal gender was successful in all cases. When tested with autosomal primers, seven samples were found to contain exclusively fetal DNA. Eight samples contained small amounts of maternal DNA that did not interfere with the QF-PCR analysis. One fetus showed trisomy 13. QF-PCR requires very small volumes of sample compared with cell culture, suggesting that coelocentesis may prove useful for very early prenatal diagnosis.

Activation of glycoyl trihaloacetimidates with acid-washed molecular sieves in the glycosidation reaction.

[reaction: see text] Commercially available 4 A acid washed molecular sieves (4 A AW 300 MS) are efficient activators of glycosyl trichloro- and N-phenyltrifluoroacetimidates. These promoters allow glycosidation of primary and secondary saccharidic acceptors to be performed in high yield, under very mild conditions and by an experimentally simple procedure. In addition, the recyclability of such promoters also has been demonstrated.

HLA-G positive trophoblastic cells in transcervical samples and their isolation and analysis by laser microdissection and QF-PCR.

OBJECTIVE: To assess the frequency of cytotrophoblastic cells in endocervical samples collected by lavage at early stages of gestation using a specific anti-HLA-G McAb (G233). From a set of four selected samples, cells identified by immunostaining were collected by laser microdissection and then tested by quantitative fluorescent polymerase chain reaction (QF-PCR) for the presence of paternally derived DNA markers, in order to establish their fetal origin. METHODS: Syncytial fragments and cytotrophoblastic cells from 23 transcervical samples were identified by immunostaining with McAb G233 reacting against HLA-G antigen and with antibodies against cytokeratin. Slides from the same samples were also tested by fluorescent in situ hybridization (FISH), while selected samples were analysed by QF-PCR. Slides from four samples retrieved from mothers with male fetuses were immunolabelled and then cytotrophoblastic cells, syncytial fragments and maternal epithelial cells were collected by laser microdissection and tested by QF-PCR. RESULTS: All endocervical samples retrieved from mothers with male fetuses were found to contain some cells with chromosome Y-specific signals when tested by FISH. Using McAb anti- HLA-G, cytotrophoblastic cellular elements were detected in about 50% of the samples. From four samples, cellular elements identified by immunostaining as cytotrophoblast or syncytial fragments were collected by laser microdissection and shown to be of fetal origin when tested by QF-PCR for the presence of fetal DNA markers. CONCLUSIONS: These results confirm that, during an early phase of gestation, fetal cells are released in the lower uterine cavity and that they can be isolated and analysed for prenatal diagnosis of single gene defects and aneuploidies.

X chromosome dosage by quantitative fluorescent PCR and rapid prenatal diagnosis of sex chromosome aneuploidies.

During the past few years, rapid prenatal diagnosis of chromosome aneuploidies has been successfully achieved by quantitative fluorescent PCR (QF-PCR) amplification of chromosome-specific small tandem repeats (STR). This approach has proven to be very useful in clinical settings, since it allows the detection of major numerical disorders in a few hours after sampling. For the detection of Turner's syndrome (45,X), several highly polymorphic STR on the X chromosome are needed in order to reduce the likelihood that a normal female might be homozygous for all sequences and, consequently, that the test could fail to discriminate between samples retrieved from a Turner's and a normal female fetus. Here we report a new method for rapid and accurate detection of X chromosome copy number in prenatal samples that does not depend on STR heterozygosity. The test is based on QF-PCR amplification of the X-linked HPRT together with the autosomal D21S1411 used as internal control for quantification. In the course of this study, this assay allowed the prenatal diagnosis of a rare case of a normal female homozygous for four selected highly polymorphic X chromosome STR, as well as the assessment of the normal chromosome complement of a fetus homozygous for five chromosome 21 markers.