Synthesis of 3-O-acyl esters of serine and threonine

The 3-O-acyl derivatives of serine and threonine have been prepared by reacting oleoyl chloride and palmitoyl chloride with N-t-butoxycarbonyl (N-T-BOC) serine and N-t-BOC threonine. The t-BOC group was removed by treatment with 4 N HCl in dioxane. The products were identified by proton magnetic resonance spectroscopy, infrared spectroscopy, elemental analysis and chromatographic properties. The O-acyl serines and O-acyl threonines were converted to their methyl esters by treatment with boron trifluoride in methanol and were converted to their dinitrophyl derivatives by treatment with dinitrofluorobenzen (DNFB). The yield of the dinitrophenyl derivatives was very high but the yield of methyl esters was low due mainly to methanolysis and loss of the fatty acyl group. The O-acyl serines and O-acyld threonines prepared will provide standards for researchers who are interested in identifying fatty acids esterified to serine and threonine hydroxyl groups in membrane proteins.     

The use of 1-O-sulfonyl-d-mannopyranose derivatives in β-d-mannopyranoside synthesis

Several 1-O-sulfonyl derivatives of d-mannopyranose having a nonparticipating benzyl ether group at C-2 and ester functions at C-6 and C-4 were synthesized from the corresponding d-mannopyranosyl chloride derivatives with silver sulfonates in acetonitrile. The reaction of 1-O-sulfonyl-d-mannopyranose compounds with methanol in various solvents at room temperature gave high yields of glycosides with low degrees of stercoselectivity. On the other hand, 1-O-suffonyl-d-mannopyranose derivatives having an acyl participating-group at O-2 and benzyl ethers at C-3, C-4, and C-6 gave high yields and high stereoselectivity of α-d-mannopyranosides with primary and secondary alcohols in several solvents. Model studies were carried out to determine the best combination of 2-O-acyl group, solvent, time, temperature, and 1-O-sufonyl group to give high yields with high stereoselectivity. The method has been used to prepare in good yields more complex glycosides, including perbenzylated methy 2-O-(α-d-mannopyranosyl)-α-d-mannopyranoside.     

O-Aryl α,β-d-ribofuranosides: Synthesis & highly efficient biocatalytic separation of anomers and evaluation of their Src kinase inhibitory activity

A series of peracetylated O-aryl α,β-d-ribofuranosides have been synthesized and an efficient biocatalytic methodology has been developed for the separation of their anomers which was otherwise almost impossible by column chromatographic or other techniques. The incubation of 2,3,5-tri-O-acetyl-1-O-aryl-α,β-d-ribofuranoside with Lipozyme® TL IM immobilized on silica led to the selective deacetylation of only one acetoxy group, viz the C-5′-O-acetoxy group of the α-anomer over the other acetoxy groups derived from the two secondary hydroxyl groups present in the molecule and also over three acetoxy groups (derived from one primary and two secondary hydroxyls of the β-anomer). This methodology led to the easy synthesis of both, α- and β-anomers of O-aryl d-ribofuranosides. All the arylribofuranosides were screened for inhibition of Src kinase. 1-O-(3-Methoxyphenyl)-β-d-ribofuranoside exhibited the highest activity for inhibition of Src kinase (IC₅₀ = 95.0 μM).     

Synthesis and anti-tumor activities of methyl 2-O-aryl-6-O-aryl′-d-glucopyranosides

A synthetic method of introducing bulky aryl groups at the 2-O- and 6-O-positions on glucopyranosides was developed. A total of 37 new compounds of this class were obtained successfully. These compounds were tested on several tumor cell lines by MTT assays, and some of them exhibited encouraging inhibitory activities. The most potent compound, CAB-SHZH-27, exhibited EC₅₀ values of 14, 12, and 10 μmol/L on A549, MDA-MB-231 and HeLa cells, respectively. A preliminary structure–activity relationship analysis indicates that the two free hydroxyl groups on the d-glucose core are indispensable for the biological activities of this class of compounds, and the aryl group at the 6-O-position has a more obvious impact than the one at the 2-O-position. An interesting ‘on–off’ mechanism of this class of compounds was also observed in our MTT assays, which remains to be explored.     

Reactions of chitosan: 2. Preparation and reactivity of N-acyl derivatives of chitosan

A study has been made of the influence of reaction medium on the N-acetylation of chitosan under heterogeneous conditions. The results show that provided a pre-steeping treatment is given a range of reaction media permit rapid N-acetylation. The influence of the nature of the N-acyl group on O-acetylation has also been studied. In general the larger the N-acyl group the greater the ease of O-acetylation, although too bulky a group inhibits reaction through steric hindrance. In all cases the rate of O-acetylation falls to nearly zero when ～ 50% of the hydroxyl groups have reacted, and prolonged reaction times are required if a more highly acetylated product is required.     

Blocksynthese von O-glycopeptiden und anderen T-antigen strukturen

Under the conditions of in situ anomerisation, the 2-azido-4,6-di-O-benzoyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-α-d-galactopyranosyl bromide reacted directly and with high selectivity with the reactive hydroxyl groups of l-serine and l-threonine derivatives to form α-glycosidically-linked products. Thus, the glycopeptides containing l-serine and l-threonine are more accessible. The disaccharide block could also be coupled with other reactive hydroxyl compounds to give compounds that contain the T-receptor.     

Platelet-aggregating effects of platelet-activating factor-like phosphollpids formed by oxidation of phosphatidylcholines containing an sn-2-polyunsaturated fatty acyl group

Previously, we reported the formation of four kinds of pfaosphatidylcholines (PC) with a short-chain monocarboxylate, dicarboxylate, dicarboxylate semialdehyde or w-hydroxymonocarboxylate group by oxidation of PCs containing polyunsaturated fatty acid (PUFA) in an FeSO₄ /ascorbate /EDTA system. In this study, we identified these novel phospholipids by GC-MS as oxidation products of two alkyl ether-linked PCs. 1-O-hexadecyl-2-docosahexaenoyl and 1-O-hexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine (GPC). The sn-2-acyl moieties of oxidatively fragmented PCs derived from PCs containing docosahexaenoate were one methylene unit shorter than those detected as major oxidation products of PCs containing arachidonate. The platelet-aggregations induced by the oxidized PCs were all inhibited by FR-900452, an antagonist of platelet activating factor (PAF). The PAF-like activity of oxidized 1-O-hexadecyl-2-docosahexaenoyl-GPC, which was equivalent of 1372 ± 262 pmol 16: 0-PAF/μmol starting PC, was 5 times that of oxidized 1-O-hexadecyl-2-arachidonoyl-GPC and 150 times that of oxidized 1-palmitoyl-2-docosahexaenoyl-GPC, suggesting that both an sn-1-alkyl ether linkage and an sn-2-acyl group with a short chain length are important structural requirements for induction of platelet aggregation. These possibilities were confirmed by experiments on the platelet-aggregating activities of synthetic PAF-like compounds. Quantitative measurements by GC-MS of PAF-like phospholipids formed by lipid peroxidation and the activities of synthetic PAF-like phospholipids, suggested that the activities of most oxidized PCs containing PUFA were ascribable to those of PCs with an sn-2-short-chain monocarboxylate group.     

Synthesis of nonreducing-sugar subunit analogs of bacterial lipid a carrying an amide-group (3R)-3-acyloxytetradecanoyl group

Two types of optically active, 4-O-phosphono-d-glucosamine derivatives related to the nonreducing-sugar subunit of bacterial lipid A, one being 2-[(3R)-3-acyloxytetradecanamido]-2-deoxy-4-O-phosphono-3-O-tetradecanoyl-d-glucose (GLA-27 type; GLA-57 and GLA-58), and the other 2-[(3R)-3-acyloxytetradecanamido]-2-deoxy-3-O-[(3R)-3-hydroxytetradecanoyl]-4-O-phosphono-d-glucose (GLA-59 type; GLA-61 and GLA-62), have been synthesized. The amino group of benzyl 2-amino-2-deoxy-4,6-O-isopropylidene-β-d-glucopyranoside was first acylated with the (3R)-3-dodecanoyloxytetradecanoyl or (3R)-3-hexadecanoyloxytetradecanoyl group, and then the remaining hydroxyl group was esterified with the tetradecanoyl or (3R)-3-(benzyloxymethoxy)tetradecanoyl group, respectively. The resulting protected intermediates were each converted, by the sequence of O-deisopropylidenation, 6-O-trityltion, and 4-O-phosphorylation, into the desired compounds.     

Epstein-Barr Virus Early Antigen Inducing Activity of 14-O-Derivatives of (*#x2212;)-Indolactam V

New 14-O-derivatives of (?)-indolactam V, which has the basic ring-structure of teleocidins without the monoterpenoid moiety, were prepared and their Epstein–Barr virus early antigen (EBV-EA) inducing activity was tested. A series of 14-O-alkyl derivatives was far less active than (?)-indolactam V, but the 14-O-acyl derivatives showed a high EBV-EA inducing activity. These results suggest that the free hydroxyl group at C-14 plays an important role in inducing the EBV-EA, and that the activity of the acyl derivatives arises through hydrolysis of the ester groups.     

Microbial hydroxylation and glycosylation of pentacyclic triterpenes as inhibitors on tissue factor procoagulant activity

To discover new inhibitors on tissue factor procoagulant activity, 20 pentacyclic triterpenes were semi-synthetized through microbial transformation and assayed on the model of human THP-1 cells stimulated by lipopolysaccharide. In the biotransformation two types of reactions were observed, regio-selective hydroxylation and glycosylation. The bioassay results showed that most of tested compounds were significant effective on this model and two of the biotransformation products 23-hydroxy-28-O-β-d-glucopyranosyl betulinic acid (3d) and 28-O-β-d-glucopyranosyl oleanic acid (1a) exhibited most potential activities with the IC₅₀ values of 0.028, 0.035 nM respectively. The preliminary structure and activity relationship analysis revealed that the aglycones with single free hydroxyl group on the skeleton (1, 1j) were less effective than that with more free hydroxyl groups (1d, 1f, 2), mono-glycosylation can significantly enhance their inhibitory effects. Our findings also provide some potential leading compounds for tissue factor-related diseases, such as cancer and cardiovascular diseases.     

Regioselective synthesis of flavonoid bisglycosides using Escherichia coli harboring two glycosyltransferases

Regioselective glycosylation of flavonoids cannot be easily achieved due to the presence of several hydroxyl groups in flavonoids. This hurdle could be overcome by employing uridine diphosphate-dependent glycosyltransferases (UGTs), which use nucleotide sugars as sugar donors and diverse compounds including flavonoids as sugar acceptors. Quercetin rhamnosides contain antiviral activity. Two quercetin diglycosides, quercetin 3-O-glucoside-7-O-rhamnoside and quercetin 3,7-O-bisrhamnoside, were synthesized using Escherichia coli expressing two UGTs. For the synthesis of quercetin 3-O-glucoside-7-O-rhamnoside, AtUGT78D2, which transfers glucose from UDP-glucose to the 3-hydroxyl group of quercetin, and AtUGT89C1, which transfers rhamnose from UDP-rhamnose to the 7-hydroxyl group of quercetin 3-O-glucoside, were transformed into E. coli. Using this approach, 67 mg/L of quercetin 3-O-glucoside-7-O-rhamnoside was synthesized. For the synthesis of quercetin 3,7-O-bisrhamnoside, AtUGT78D1, which transfers rhamnose to the 3-hydroxy group of quercetin, and AtUGT89C1 were used. The RHM2 gene from Arabidopsis thaliana was coexpressed to supply the sugar donor, UDP-rhamnose. E. coli expressing AtUGT78D1, AtUGT89C1, and RHM2 was used to obtain 67.4 mg/L of quercetin 3,7-O-bisrhamnoside.     

Characterization of the Intestinal Absorption of Seven Flavonoids from the Flowers of Trollius chinensis Using the Caco-2 Cell Monolayer Model

The human Caco-2 cell monolayer model was used to investigate the absorption property, mechanism, and structure-property relationship of seven representative flavonoids, namely, orientin, vitexin, 2”-O-β-L-galactopyranosylorientin, 2”-O-β-L-galactopyranosylvitexin, isoswertisin, isoswertiajaponin, and 2”-O-(2”‘-methylbutanoyl)isoswertisin from the flowers of Trollius chinensis. The results showed that these flavonoids were hardly transported through the Caco-2 cell monolayer. The compounds with 7-OCH₃ including isoswertisin, isoswertiajaponin and 2”-O-(2”‘-methylbutanoyl)isoswertisin were absorbed in a passive diffusion manner, and their absorbability was increased in the same order as their polarity. The absorption of the remaining compounds with 7-OH including orientin, vitexin, 2”-O-β-L-galactopyranosylorientin, and 2”-O-β-L-galactopyranosylvitexin involved transporter mediated efflux in addition to passive diffusion. Among the four compounds with 7-OH, those with a free hydroxyl group at C-2” such as orientin and vitexin were the substrates of P-glycoprotein (P-gp) and that with a free hydroxyl group at C-2’ such as 2”-O-β-L-galactopyranosylorientin was the substrate of multidrug resistance protein 2 (MRP2). The results of this study also implied that the absorbability of the flavonoids should be taken into account when estimating the effective components of T. chinensis.     

Semi-synthetic salinomycin analogs exert cytotoxic activity against human colorectal cancer stem cells

Salinomycin, a polyether antibiotic, is a well-known inhibitor of human cancer stem cells. Chemical modification of the allylic C20 hydroxyl of salinomycin has enabled access to synthetic analogs that display increased cytotoxic activity compared to the native structure. The aim of this study was to investigate the activity of a cohort of C20-O-acyl analogs of salinomycin on human colorectal cancer cell lines in vitro. Two human colorectal cancer cell lines (SW480 and SW620) were exposed to three C20-O-acylated analogs and salinomycin. The impact of salinomycin and its analogs on tumor cell number, migration, cell death, and cancer stem cell specifity was analyzed. Exposure of human colorectal cancer cells to the C20-O-acylated analogs of salinomycin resulted in reduced tumor cell number and impaired tumor cell migration at lower concentrations than salinomycin. When used at higher (micromolar) concentrations, these effects were accompanied by induction of apoptotic cell death. Salinomycin analogs further expose improved activity against cancer stem cells compared to salinomycin.     

Oxazoline synthesis of 1,2-trans-2-acetamido-2-deoxyglycosides. Glycosylation with 2-methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-D-galactopyrano)-[2′,1′:4,5]-2-oxazoline

The glycosylating activity of 2-methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-D-galactopyrano)-[2′,1′:4,5]-2-oxazoline has been tested in reaction with partially protected saccharides having free primary or secondary hydroxyl groups or with hydroxy amino acids. 3-O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranosyl)-N-benzyloxycarbonyl-L-serine benzyl ester (3), 6-O-(2-acetamido-2-deoxy-β-D-galactopyranosyl)-D-galactopyranose (5), p-nitrophenyl 2-acetamido-6-O-(2-acetamido-2-deoxy-β-D-galactopyranosyl)-2-deoxy-β-D-glucopyranoside (7), 6-O-(2-acetamido-2-deoxy-β-D-galactopyranosyl)-D-glucose (9), and 3-O-(2-acetamido-2-deoxy-β-D-galactopyranosyl)-D-glucose (11) were synthesized in high yield.     

ω-O-Acylceramides but not ω-hydroxy ceramides are required for healthy lamellar phase architecture of skin barrier lipids

Epidermal omega-O-acylceramides (ω-O-acylCers) are essential components of a competent skin barrier. These unusual sphingolipids with ultralong N-acyl chains contain linoleic acid esterified to the terminal hydroxyl of the N-acyl, the formation of which requires the transacylase activity of patatin-like phospholipase domain containing 1 (PNPLA1). In ichthyosis with dysfunctional PNPLA1, ω-O-acylCer levels are significantly decreased, and ω-hydroxylated Cers (ω-OHCers) accumulate. Here, we explore the role of the linoleate moiety in ω-O-acylCers in the assembly of the skin lipid barrier. Ultrastructural studies of skin samples from neonatal Pnpla1^+/+ and Pnpla1^-/- mice showed that the linoleate moiety in ω-O-acylCers is essential for lamellar pairing in lamellar bodies, as well as for stratum corneum lipid assembly into the long periodicity lamellar phase. To further study the molecular details of ω-O-acylCer deficiency on skin barrier lipid assembly, we built in vitro lipid models composed of major stratum corneum lipid subclasses containing either ω-O-acylCer (healthy skin model), ω-OHCer (Pnpla1^-/- model), or combination of the two. X-ray diffraction, infrared spectroscopy, and permeability studies indicated that ω-OHCers could not substitute for ω-O-acylCers, although in favorable conditions, they form a medium lamellar phase with a 10.8 nm-repeat distance and permeability barrier properties similar to long periodicity lamellar phase. In the absence of ω-O-acylCers, skin lipids were prone to separation into two phases with diminished barrier properties. The models combining ω-OHCers with ω-O-acylCers indicated that accumulation of ω-OHCers does not prevent ω-O-acylCer-driven lamellar stacking. These data suggest that ω-O-acylCer supplementation may be a viable therapeutic option in patients with PNPLA1 deficiency.     

Conjugates of betulin derivatives with AZT as potent anti-HIV agents

Fourteen novel conjugates of 3,28-di-O-acylbetulins with AZT were prepared as anti-HIV agents, based on our previously reported potent anti-HIV triterpene leads, including 3-O-acyl and 3,28-di-O-acylbetulins. Nine of the conjugates (49–53, 55, 56, 59, and 60) exhibited potent anti-HIV activity at the submicromolar level, with EC₅₀ values ranging from 0.040 to 0.098 μM in HIV-1_NL4-3 infected MT-4 cells. These compounds were equipotent or more potent than 3-O-(3′,3′-dimethylsuccinyl)betulinic acid (2), which is currently in Phase IIb anti-AIDS clinical trial.     

Glucosylation of Isoflavonoids in Engineered Escherichia coli

A glycosyltransferase, YjiC, from Bacillus licheniformis has been used for the modification of the commercially available isoflavonoids genistein, daidzein, biochanin A and formononetin. The in vitro glycosylation reaction, using UDP-α-D-glucose as a donor for the glucose moiety and aforementioned four acceptor molecules, showed the prominent glycosylation at 4′ and 7 hydroxyl groups, but not at the 5^th hydroxyl group of the A-ring, resulting in the production of genistein 4′-O-β-D-glucoside, genistein 7-O-β-D-glucoside (genistin), genistein 4′,7-O-β-D-diglucoside, biochanin A-7-O-β-D-glucoside (sissotrin), daidzein 4′-O-β-D-glucoside, daidzein 7-O-β-D-glucoside (daidzin), daidzein 4′, 7-O-β-D-diglucoside, and formononetin 7-O-β-D-glucoside (ononin). The structures of all the products were elucidated using high performance liquid chromatography-photo diode array and high resolution quadrupole time-of-flight electrospray ionization mass spectrometry (HR QTOFESI/MS) analysis, and were compared with commercially available standard compounds. Significantly higher bioconversion rates of all four isoflavonoids was observed in both in vitro as well as in vivo bioconversion reactions. The in vivo fermentation of the isoflavonoids by applying engineered E. coli BL21(DE3)/ΔpgiΔzwfΔushA overexpressing phosphoglucomutase (pgm) and glucose 1-phosphate uridyltransferase (galU), along with YjiC, found more than 60% average conversion of 200 μM of supplemented isoflavonoids, without any additional UDP-α-D-glucose added in fermentation medium, which could be very beneficial to large scale industrial production of isoflavonoid glucosides.     

Studies of carbohydrate derivatives having the -CH2F function

The following primary sulphonates have been converted into the corresponding deoxyfluoro derivatives by reaction with potassium fluoride in ethylene glycol:1,2:3,4-di-O-isopropylidene-6-O-tosyl α-D-galactopyranose (1), methyl 2,3-O2-isopropyliden-5-O-tosyl-α,β-D-ribofuranoside (2), 1,2:3,4-di-O-methylene-6-O-tosyl-α-D-glucofuranose (3), 3,5-di-O-benzylidene-1,2-O-isopropylidene-6-O-tosyl-α-D-glucofuranose (4), and 1,2:3,5-di-O-isopropylidene-6-O-tosyl-α-D-glucofuranose (5). The yields were generally poor; in the reaction of 1, a major by-product was 6-O-(2-hydroxyethyl)-1,2:3,4-di-O-isopropylidene-α-D-galactopyranose (11). The reaction of the primary hydroxyl precursor of each of the above tosylates with N2-(2-chloro- 1,1,2-trifluoroethyl)-N,N-diethylamine generally yielded the O-chlorofluoroacetyl derivative; however, 1,2:3,5-di-O-methylene-α-D-glucofuranose (12) was converted into the 6-deoxy-6-fluoro derivative (8). The ¹⁹F resonances of compounds containing the CH₂F moiety fall between φ_C +213 and φ_C +235 p.p.m. The differences between the vicinal¹⁹F-¹H couplings of compounds having the D-gluco and D-galacto configurations clearly reflect the influence of the C-4O-4 substitutents on the populations of the C-5C-6 rotamers. A novel type of noise-modulated, heteronuclear decoupling experiment is described.     

Dioxolane and dioxane acetal derivatives of D-allose: Condensation of 3-O-benzyl-D-allose with acetaldehyde

Condensation of 3-O-benzyl-D-allose with acetaldehyde forms a complex mixture from which potentially useful mono- and di-O-ethylidene derivatives were isolated and identified. Compounds isolated and identified after conversion of unsubstituted hydroxyl groups into the corresponding acetates included 1,2-di-O-acetyl-3-O-benzyl-4,6-O-ethylidene-β-D-allopyranose; 5,6-di-O-acetyl-3-O-benzyl-1,2-O-(R)-ethylidene-α-D-allofuranose; and two 3-O-benzyl-1,2:5,6-di-O-ethylidene-α-D-allofuranoses, both having the R configuration in the 1,2-O-ethylidene ring. Furanose and pyranose conformations were determined by n.m.r. analysis, and the location and configuration of each acetal ring was established. The benzyl ether group in the furanose derivatives was removed by catalytic hydrogenation with subsequent formation of 3-O-acetyl analogs.     

Antiprotozoal activity of 8-acyl and 8-alkyl incomptine A analogs

The activities of 11 C-8-O-acyl and alkyl analogs of the antiprotozoal sesquiterpene lactone, incomptine A (1) against Entamoeba histolytica and Giardia lamblia, were determined. Here, the effects of different lengths and amounts of branching of the acyl and alkyl groups on the antiprotozoal activity of the synthesized incomptine A-analogs are reported.