New lupane bidesmosides exhibiting strong cytotoxic activities in vitro.

Triterpene bidesmosides are considered as highly cytotoxic saponins, usually less toxic against normal cells than monodesmosides, and less haemolytic. Biological activity of the betulin-type bidesmosides, rarely found in Nature, and seldom prepared due to serious synthetic problems, is poorly recognized. We report herein a protocol for the preparation of disubstituted lupane saponins (betulin bidesmosides) by treatment of their benzoates with potassium carbonate in dichloromethane / methanol solution. Cytotoxicity of all compounds was tested in vitro for a series of cancer cell lines, as well as normal human skin BJ fibroblasts. Presence of l-rhamnose moiety is crucial for cytotoxicity of betulin bidesmosides. On the other hand, l-arabinose fragment connected to lupane C-3 carbon atom significantly decreases activity. Presented results clearly show that betulin bidesmosides have significant clinical potential as anticancer agents.

Stereoselective synthesis of ß-rhamnopyranosides via gold(I)-catalyzed glycosylation with 2-alkynyl-4-nitro-benzoate donors.

Stereoselective ß-rhamnopyranosylation remains a challenge, due to the unfavorable anomeric effect and steric hindrance of the C2-substituent; herein, this challenge is addressed with a gold(I)-catalyzed SN2-like glycosylation protocol employing α-rhamnopyranosyl 2-alkynyl-4-nitro-benzoates as donors.

Synthesis and biological evaluation of acylated oligorhamnoside derivatives structurally related to mezzettiaside-6 with cytotoxic activity.

Two partially acylated oligorhamnoside derivatives 1 and 2 structurally related to the natural product mezzettiaside-6 were synthesized via a '2 + 1 + 1' convergent strategy. The bioassay results showed that the introduction of the acetyl groups to the 2-position of the terminal l-rhamnose was helpful to improve in vitro cytotoxicity. Compound 1 showed both extensive in vitro cytotoxicity in tumor cell lines and potential antimultidrug resistance capability. Preliminary mechanistic studies demonstrated that compound 1 could inhibit cell growth by inducing apoptosis, arresting cell cycle progression at the S phase in K562 cells.

Synthesis and immunological evaluation of a MUC1 glycopeptide incorporated into l-rhamnose displaying liposomes.

MUC1 variable number tandem repeats (VNTRs) conjugated to tumor-associated carbohydrate antigens (TACAs) have been shown to break self-tolerance in humanized MUC1 transgenic mice. Therefore, we hypothesize that a MUC1 VNTR TACA-conjugate can be successfully formulated into a liposome-based anticancer vaccine. The immunogenicity of the vaccine should be further augmented by incorporating surface-displayed l-rhamnose (Rha) epitopes onto the liposomes to take advantage of a natural antibody-dependent antigen uptake mechanism. To validate our hypothesis, we synthesized a 20-amino-acid MUC1 glycopeptide containing a GalNAc-O-Thr (Tn) TACA by SPPS and conjugated it to a functionalized Toll-like receptor ligand (TLRL). An l-Rha-cholesterol conjugate was prepared using tetra(ethylene glycol) (TEG) as a linker. The liposome-based anticancer vaccine was formulated by the extrusion method using TLRL-MUC1-Tn conjugate, Rha-TEG-cholesterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a total lipid concentration of 30 mM. The stability, homogeneity, and size characterization of the liposomes was evaluated by SEM and DLS measurements. The formulated liposomes demonstrated positive binding with both anti-Rha and mouse anti-human MUC1 antibodies. Groups of female BALB/c mice were immunized and boosted with a rhamnose-Ficoll (Rha-Ficoll) conjugate formulated with alum as adjuvant to generate the appropriate concentration of anti-Rha antibodies in the mice. Anti-Rha antibody titers were >25-fold higher in the groups of mice immunized with the Rha-Ficoll conjugate than the nonimmunized control groups. The mice were then immunized with the TLRL-MUC1-Tn liposomal vaccine formulated either with or without the surface displaying Rha epitopes. Sera collected from the groups of mice initially immunized with Rha-Ficoll and later vaccinated with the Rha-displaying TLRL-MUC1-Tn liposomes showed a >8-fold increase in both anti-MUC1-Tn and anti-Tn antibody titers in comparison to the groups of mice that did not receive Rha-Ficoll. T-cells from BALB/c mice primed with a MUC1-Tn peptide demonstrated increased proliferation to the Rha-liposomal vaccine in the presence of antibodies isolated from Rha-Ficoll immunized mice compared to nonimmune mice, supporting the proposed effect on antigen presentation. The anti-MUC1-Tn antibodies in the vaccinated mice serum recognized MUC1 on human leukemia U266 cells. Because this vaccine uses separate rhamnose and antigenic epitope components, the vaccine can easily be targeted to different antigens or epitopes by changing the peptide without having to change the other components.

Synthesis and evaluation of l-rhamnose 1C-phosphonates as nucleotidylyltransferase inhibitors.

We report the synthesis of a series of phosphonates and ketosephosphonates possessing an L-rhamnose scaffold with varying degrees of fluorination. These compounds were evaluated as potential inhibitors of α-D-glucose 1-phosphate thymidylyltransferase (Cps2L), the first enzyme in Streptococcus pneumoniae L-rhamnose biosynthesis, and a novel antibiotic target. Enzyme-substrate and enzyme-inhibitor binding experiments were performed using water-ligand observed binding via gradient spectroscopy (WaterLOGSY) NMR for known sugar nucleotide substrates and selected phosphonate analogues. IC50 values were measured and Ki values were calculated for inhibitors. New insights were gained into the binding promiscuity of enzymes within the prokaryotic L-rhamnose biosynthetic pathway (Cps2L, RmlB-D) and into the mechanism of inhibition for the most potent inhibitor in the series, L-rhamnose 1C-phosphonate.

Rhamnosylation: diastereoselectivity of conformationally armed donors.

The α/ß-selectivity of super-armed rhamnosyl donors have been investigated in glycosylation reactions. The solvent was found to have a minor influence, whereas temperature was crucial for the diastereoselectivity. At very low temperature, a modest ß-selectivity could be obtained, and increasing temperature gave excellent α-selectivity. The donors were highly reactive, and activation was observed at temperatures as low as -107 °C. Different promoter systems and leaving groups were investigated, and only activation with a heterogeneous catalyst increased the amount of the ß-anomer significantly. By introducing an electron-withdrawing nonparticipating group, benzyl sulfonyl, on 2-O, an increase in ß-product was observed.

The cephalostatins. 21. Synthesis of bis-steroidal pyrazine rhamnosides (1).

The synthesis of bis-steroidal pyrazines derived from 3-oxo-11,21-dihydroxypregna-4,17(20)-diene (4) and glycosylation of a D-ring side chain with α-L-rhamnose have been summarized. Rearrangement of steroidal pyrazine 10 to 14 was found to occur with boron triflouride etherate. Glycosylation of pyrazine 10 using 2,3,4-tri-O-acetyl-α-L-rhamnose iodide led to 1,2-orthoester-α-L-rhamnose pyrazine 17b. By use of a persilylated α-L-rhamnose iodide as donor, formation of the orthoester was avoided. Bis-steroidal pyrazine 10 and rhamnosides 17b and 21c were found to significantly inhibit cancer cell growth in a murine and human cancer cell line panel. Pyrazine 9 inhibited growth of the nosocomial pathogen Enterococcus faecalis.

Synthesis of the beta-rhamnopyranosides and the 6-deoxy-beta-mannoheptopyranosides.

A review is presented of the development of methods for the synthesis of beta-rhamnopyranosides and the related 6-deoxy-beta-mannoheptopyranosides based on the 4,6-O-alkylidene directed synthesis of mannopyranosides and their 6-thia derivatives followed by selective reduction at the 6-position. Applications to total synthesis of complex bacterial oligosaccharides containing the title moieties are presented.

Stereocontrolled synthesis of D- and L-beta-rhamnopyranosides with 4-O-6-S-alpha-cyanobenzylidene-protected 6-thiorhamnopyranosyl thioglycosides.

The synthesis of both enantiomers of a 4-O-6-S-alpha-cyanobenzylidene-protected 6-thiorhamnopyranosyl thioglycoside is described starting from D-mannnose and L-arabinose derivatives for the D- and L-series, respectively. This donor is effective in the preparation of the corresponding beta-glycosides using the 1-benzenesulfinyl piperidine/trifluoromethanesulfonic anhydride protocol. Following desulfurization and concomitant debenzylation with Raney nickel, the so-formed 6-thio-beta-mannosides are converted in high yield to the beta-rhamnopyranosides.

The spirocyclopropyl moiety as a methyl surrogate in the structure of l-fucosidase and l-rhamnosidase inhibitors.

Nitrogen-in-the-ring analogues of l-fucose and l-rhamnose were prepared, which feature a spirocyclopropyl moiety in place of the methyl group of the natural sugar. The synthetic route involved a titanium-mediated aminocyclopropanation of a glycononitrile as the key step. Four new spirocyclopropyl iminosugar analogues were generated, which displayed some activity towards l-fucosidase and l-rhamnosidase.

Stereoselective synthesis of beta-L-rhamnopyranosides.

Stereoselective construction of 1,2-cis-beta-L-rhamnopyranoside was achieved by our effective methodology using naphthylmethyl (NAP) ether-mediated intramolecular aglycon delivery (IAD). The complete stereoselective synthesis of the bacterial extracellular polysaccharide, alpha-L-Rhap-(1-->3)-beta-L-Rhap-(1-->4)Glcp from Sphaerotilus natans, was successfully accomplished, clearly demonstrating that the NAP-IAD methodology is highly versatile.

Biomarker-Based Metabolic Labeling for Redirected and Enhanced Immune Response.

Installation of an antibody-recruiting moiety on the surface of disease-relevant cells can lead to the selective destruction of targets by the immune system. Such an approach can be an alternative strategy to traditional chemotherapeutics in cancer therapy and possibly other diseases. Herein we describe the development of a new strategy to selectively label targets with an antibody-recruiting moiety through its covalent and stable installation, complementing existing methods of employing reversible binding. This is achieved through selective delivery of 1,3,4- O-acetyl- N-azidoacetylmannosamine (Ac3ManNAz) to folate receptor-overexpressing cells using an Ac3ManNAz-folate conjugate via a cleavable linker. As such, Ac3ManNAz is converted to cell surface glycan bearing an azido group, which serves as an anchor to introduce l-rhamnose (Rha), a hapten, via a click reaction with aza-dibenzocyclooctyne (DBCO)-Rha. We tested this method in several cell lines including KB, HEK-293, and MCF7 and were able to demonstrate the following: 1) Rha can be selectively installed to the folate receptor overexpressing cell surface and 2) the Rha installed on the target surface can recruit anti-rhamnose (anti-Rha) antibodies, leading to the destruction of target cells via complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP).

Total synthesis and stereochemical reassignment of (+)-dolastatin 19.

[reaction: see text] A revised configurational assignment for the cytotoxic marine macrolide dolastatin 19 is proposed and validated by total synthesis. Key features of the route include an asymmetric vinylogous aldol reaction to install the isolated C13 stereocenter and (E)-trisubstituted alkene, two sequential 1,4-syn boron-mediated aldol reactions, and a Mukaiyama glycosylation to append the l-rhamnose-derived pyranoside.

4-O-Acetyl-3-O-tert-butyldimethylsilyl-L-rhamnal: a building block in the stereoselective synthesis of 2-deoxy-alpha-L-rhamnopyranosides.

The stereoselective synthesis of 2-deoxy-alpha-L-glycosides by addition of various acceptors to 4-O-acetyl-3-O-tert-butyldimethylsilyl-L-rhamnal promoted by triphenylphosphine-hydrogen bromide is developed.

Alternative synthesis and antibacterial evaluation of 1,5-dideoxy-1,5-imino-L-rhamnitol.

A convenient synthesis is described of 5-azido-5-deoxy-2,3-O-isopropylidene-L-rhamnofuranose from L-rhamnose in seven steps and 17% overall yield. A key feature of the synthesis is the selective oxidation of the secondary alcohol in 2,3-O-isopropylidene-L-rhamnofuranose in the presence of the hemiacetal to give the corresponding ketone in good yield using the Parikh-Doering reagent. 5-Azido-5-deoxy-2,3-O-isopropylidene-l-rhamnofuranose is then converted by a literature protocol to 1,5-dideoxy-1,5-imino-L-rhamnitol, which was found to have no significant antimicrobial activity against Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and Escherichia coli.

Synthesis and structure-activity relationships of 17beta-substituted 14beta-hydroxysteroid 3-(alpha-L-rhamnopyranoside)s: steroids that bind to the digitalis receptor.

The preparation of 17beta-substituted 14beta-hydroxysteroid C-3 alpha-L-rhamnopyranosides is described. These derivatives have a 14beta,20-ether, 14beta,20-lactone, or 17beta-CH2CH2OH, -CH2CH2NH2, -CH=CHNO2(E), -CH=CHCOOH(E), -CH(OH)CH2NO2(R), -CH(OMe)CH2NO2(R), -CH2-CH2COOH, or -CH(OH)CH2NH2(R) group. Derivatives were assayed in a radioligand binding assay for [3H]ouabain in membranes from canine heart muscle. The digitalis "receptor" comprises isoenzymes of the ion-pumping enzyme, Na+,K+-ATPase. The 17beta-CH=CHNO2(E), 17beta-CH=CHCOOH(E), and 17beta-CH(OMe)CH2NO2(R) derivatives were the most potent and equivalent to ouabain with low-nanomolar IC50 values. The very potent binding affinity of the disubstituted compound 17beta-CH(OMe)CH2NO2(R) further demonstrates that 17beta-unsaturated substitution is not required for potent binding affinity. This observation may be of value in the separation of cardiotonic and cardiotoxic effects. Tosylation of the 17beta-CH2OH, prepared from the 17beta-CHO by lithium aluminum hydride reduction, yielded the 14beta,17beta-ether. Synthesis of the 17beta-CH2COOH gave the epimeric 14alpha,17alpha- and 14beta,17beta-lactones. Structures have been established by NMR analysis.

Enantioselective synthesis of N-Cbz-protected 6-amino-6-deoxymannose, -talose, and -gulose.

The enantioselective synthesis of three 6-amino-6-deoxy sugars has been achieved in six to eight steps from furfural. A sequence of diastereoselective oxidation and reduction reactions produced Cbz-protected 6-aminomannose from furfuryl alcohol 3. The incorporation of a Mitsunobu reaction into the reaction sequence allows for the selective synthesis of both N-Cbz-protected 6-aminotalose and 6-aminogulose. The overall procedure allows for the synthesis of either enantiomer of these three aminosugars. [reaction: see text]

The 4,6-O-[alpha-(2-(2- iodophenyl)ethylthiocarbonyl)benzylidene] protecting group: stereoselective glycosylation, reductive radical fragmentation, and synthesis of beta-D-rhamnopyranosides and other deoxy sugars.

[reaction: see text] In the thioglycoside/BSP/Tf(2)O glycosylation method, the 4,6-O-[alpha-(2-(2-iodophenyl)ethylthiocarbonyl)benzylidene] group enforces beta-selectivity in mannopyranosylations. Following glycosylation, treatment with Bu(3)SnH in toluene at reflux affords regioselective, reductive fragmentation to the 6-deoxy-beta-mannosides (beta-rhamnosides). Applied to glucosides, the radical fragmentation provides 6-deoxyglucosides, whereas 4-deoxygalactosides are the preferred products in the galactose series. The radical fragmentation is fully compatible with the presence of benzyl and p-methoxybenzyl ethers and with acetate esters

Direct synthesis of the beta-l-rhamnopyranosides.

The direct formation of beta-l-rhamnopyranosides by means of thioglycoside donors protected with a 2-O-sulfonate ester and, ideally, a 4-O-benzoyl ester, is reported. Activation is achieved with the combination of 1-benzenesulfinyl piperidine and triflic anhydride in the presence of 2,4,6-tri-tert-butylpyrimidine. Selectivities vary from moderate to good, and the sulfonyl group is easily removed post-glycosylation with sodium amalgam in 2-propanol.

A simple method for preparation of D-rhamnose.

A rapid procedure for the preparation of D-rhamnose from bacterial lipopolysaccharide (LPS) has been developed. It involves purification of LPS from Pseudomonas syringae pv. phaseolicola by phenol extraction and hydrophobic interaction chromatography (HIC), followed by mild hydrolysis and cleavage of the O-antigen into D-fucose and D-rhamnose. The monosaccharides were separated by column chromatography, and D-rhamnose recovered after filtration over Sephadex-LH 20.