Synthetic Partial Structures Research Articles

Structure Determination, Chemical Synthesis, and Evaluation of Biological Activity of Super Carbon Chain Natural Products

Abstract Marine microorganisms are known to produce natural products, so called super carbon chain compounds whose molecular weights (MWs) exceed one thousand, such as amphidinol 3 (AM3) and maitotoxin (MTX). AM3 (MW 1328) is a long linear compound containing polyol, bis-tetrahydopyran, and polyene moieties, and elicits potent antifungal activities. During the course of our studies on AM3, originally proposed absolute configuration was revised by the chemical synthesis of partial structures in combination with degradation of the natural product. Furthermore, the first total synthesis of AM3 has been achieved based on the convergent method via three components coupling. In addition, we have succeeded in design and synthesis of an artificial truncated analog of AM3 which elicits comparable antifungal activity with those of the natural product. On the other hand, MTX (MW 3422) is a ladder-shaped polyether comprised of thirty-two cyclic ethers, and it elicits potent Ca2+ influx activity. During the course of our structure-activity relationship studies of MTX, we have developed three methods for synthesizing ladder-shaped polyethers via two-ring construction through the coupling of two fragments. The synthetic partial structures corresponding to the hydrophobic parts of MTX inhibited the Ca2+ influx induced by MTX.

Synthesis and Biological Activity of the QRS Ring System of Maitotoxin

Abstract Maitotoxin (MTX) is a ladder-shaped polyether produced by an epiphytic dinoflagellate. As a part of our structure–activity relationship studies using synthetic partial structures of MTX, the QRS ring comprising a 6/6/7 tricyclic system was synthesized through stereoselective construction of five contiguous stereogenic centers on the Q ring via i) coupling of a Weinreb amide and a furyllithium, followed by ii) Noyori asymmetric transfer hydrogenation, iii) the Achmatowicz reaction, iv) chemoselective methylation of a methyl acetal in the presence of a carbonyl group by treatment with Me2Zn/BF3·OEt2, v) highly diastereoselective dihydroxylation, and vi) ring expansion of a six-membered to a seven-membered ring ketone. The synthetic specimen inhibited MTX-induced Ca2+ influx with an IC50 value of 44 µM.

Assembling different antennas of the gp120 high mannose-type glycans on gold nanoparticles provides superior binding to the anti-HIV antibody 2G12 than the individual antennas

In order to re-build Man9GlcNAc2 clusters of the HIV gp120 glycoprotein, ∼2nm gold glyconanoparticles (GNPs) were coated with the synthetic partial structures of Man9, the tetramannoside Manα1–2Manα1–2Manα1–3Manα1– and the pentamannoside Manα1–2Manα1–3[Manα1–2Manα1–6]Manα1–. Their interactions with the anti-HIV broadly neutralizing antibody 2G12 were studied by surface plasmon resonance (SPR)-based biosensors and saturation transfer difference (STD)-NMR spectroscopy. A synergistic effect of the tetra- and pentamannosides multimerized on a same GNP was observed. The assembly of these antennas of the gp120 high-mannose type glycan on GNPs provided superior binding to the anti-HIV antibody 2G12 with respect to GNPs carrying only the individual oligomannosides. The results presented in this work provide new molecular information on the interactions between clusters of oligomannosides and 2G12 that could help in the design of a carbohydrate-based vaccine against HIV.

Synthetic (glyco-)peptides of the homophilic recognition domain of E-cadherin lead to increased E-cadherin mRNA synthesis and are inductors of cell differentiation in primary lung cancer cell lines

E-cadherin is one of the critical molecules involved in the metastatic process in many types of cancer. Once combined, E-cadherin exceeds the amount of membranous E-cadherin on the cellular surface by activation of intracellular signaling cascades. Studies on transformed keratinocytes of the HaCat cell line showed induction of differentiation by synthetical partial structures of the homophilic binding region of E-cadherin. The knowledge of effects in lung cancer cells is sparse. Therefore, the effects in primary lung cancer cell lines were investigated. Four primary lung cancer cell lines were incubated for 3, 6, 12, 15, 18, and 24 h with synthetic partial structures (peptide and glycopeptide). The control substance was sodium butyrate. mRNA was isolated, and relative quantification of E-cadherin was performed using the Real-Time PCR. During the stimulation period, morphologic pictures were taken, and immunohistochemical staining of membranous E-cadherin was performed. Life/dead assays were used to display cell vitality. The intracellular E-cadherin mRNA amount was increased after incubation with the synthetic partial structures. Life/dead assays showed improved survival and integrated cell/cell bindings after stimulation with the partial structures. Increased cell mortality was revealed after sodium butyrate incubation. An effect mediated via E-cadherin on the cellular surface is proposed. The two synthetic partial structures of the homophilic binding region of E-cadherin increased the intracellular E-cadherin mRNA amount, cell–cell bindings, and survival of the tumor cells. Extracellular binding by synthetic partial structures to the binding region may have a beneficial influence on tumor progression in the metastatic process.

Synthetic and model computational studies of molar rotation additivity for interacting chiral centers: a reinvestigation of van't Hoff's principle.

When plane-polarized light impinges on a solution of optically active molecules, the polarization of the light that emerges is rotated. This simple phenomenon arises from the interaction of light with matter and is well understood, in principle, van't Hoff's rule of optical superposition correlates the molar rotation with the individual contributions to optical activity of isolated centers of asymmetry. This straightforward empirical additivity rule is rarely used for structure elucidation nowadays because of its limitations in the assessment of conformationally restricted or interacting chiral centers. However, additivity can be used successfully to assign the configuration of complex natural products such as hennoxazole A if appropriate synthetic partial structures are available. Therefore, van't Hoff's principle is a powerful stereochemical complement to natural products' total synthesis. The quest for reliable quantitative methods to calculate the angle of rotation a priori has been underway for a long time. Both classical and quantum methods for calculating molar rotation have been developed. Of particular practical importance for determining the absolute structure of molecules by calculation is the manner in which interactions between multiple chiral centers in a single molecule are included, leading to additive or non-additive optical rotation angles. This problem is addressed here using semi-empirical electronic structure models and the Rosenfeld equation.

Characterization of monoclonal antibodies recognizing three distinct, phosphorylated carbohydrate epitopes in the lipopolysaccharide of the deep rough mutant I‐69 Rd−/b+ of Haemophilus influenzae

Monoclonal antibodies against the lipopolysaccharide (LPS) of the deep rough mutant I-69 Rd-/b+ of Haemophilus influenzae were obtained after immunization of mice with sheep erythrocytes which had been coated with de-O-acylated LPS. Characterization of antibodies was performed by enzyme immuno assay (EIA) using LPS or neoglycoconjugates containing partial structures of LPS as solid-phase antigens and by haemagglutination with sheep erythrocytes coated with de-O-acylated LPS. Binding data were confirmed by EIA inhibition experiments using deacylated LPS or synthetic partial structures thereof. Three antibodies were specific for 3-deoxy-D-manno-octulopyranosonic acid- (Kdo) 5-phosphate, one for Kdo-4-phosphate, and one required, in addition to a Kdo-phosphate, parts of the phosphorylated glucosamine backbone of lipid A. All antibodies also bound in (i) Western blots to bacterial whole-cell lysates or isolated LPS separated by SDS-PAGE, (ii) bacterial colony blots, and (iii) immunofluorescence with live bacteria. The latter result indicated that Kdo-4- and Kdo-5-phosphate are synthesized by the bacteria and are not the result of phosphate migration.

Specificity of monoclonal antibodies against Escherichia coli K-12 lipopolysaccharide

Three monoclonal antibodies against the lipopolysaccharide (LPS) of Escherichia coli strain K-12 were obtained after immunization of BALB/c mice with heat-killed bacteria and were serologically characterized by hemagglutination, enzyme-immunoassay, Western, dot, and colony blot using as antigens bacteria, LPS, de-O-acylated LPS, dephosphorylated LPS, the core oligosaccharide and synthetic partial structures representing a di-, tri-and tetrasaccharide of the K-12 outer core region. In addition, deacylated LPS and synthetic oligosaccharides were covalently linked to bovine serum albumin resulting in artificial glycoconjugate antigens. In all assays used, the antibodies were specific for K-12 LPS. These antibodies detect E. coli K-12 LPS in its isolated form as well as on the surface of bacteria and are, thus, useful for all working with this microorganism.

Monoclonal antibodies against the heptose region of enterobacterial lipopolysaccharides

Murine monoclonal antibodies (MAbs) against the rough mutant lipopolysaccharides (LPS) of Salmonella minnesota strain R4 and R7 (chemotypes Rd2P - and Rd1P-, respectively) were obtained after immunization of BALB/c and NZB mice, respectively, with heat-killed bacteria. A total of 5 MAbs was obtained which were serologically characterized by hemagglutination, EIA and Western blot using LPS, de-O-acylated LPS and dephosphorylated LPS. In addition, the completely deacylated carbohydrate backbone, the deacylated, dephosphorylated and reduced carbohydrate backbone, and synthetic partial structures were covalently linked to bovine serum albumin resulting in artificial glycoconjugate antigens. Both groups of MAbs (anti-R4 and anti-R7) were highly specific for the Rd2P- and Rd1P- chemotypes, respectively, since they did not react with LPS of the Ra, Rb, Rc or Re chemotype. The R4 antibodies required for binding the core region [composed of 3-deoxy-α-D- manno-octulosonic acid (Kdo) and L-glycero-α-D- manno-heptopyranose (Hep)] and the phosphorylated glucosamine backbone of the lipid A moiety. The R7 antibodies bound to some extent to the core oligosaccharide Hep(1→3)Hep(1→5)Kdo(2→6), however, better binding was observed when the glucosamine backbone of lipid A was also present. In this case however, phosphoryl groups were not required. These antibodies can detect enterobacteria with an LPS of the Rd-chemotype, and may be useful to characterize also other naturally occurring rough mutants or genetically manipulated strains used to clone genes of the rfa locus.

Specificity of rabbit antisera against the rough lipopolysaccharide ofSalmonella minnesotastrain R7 (chemotype Rd1P−)

Rabbit polyclonal antibodies against the lipopolysaccharide (LPS) of the Rd1P- mutant strain R7 of Salmonella minnesota were serologically characterized using R7 LPS, dephosphorylated LPS, deacylated LPS, deacylated, dephosphorylated and reduced LPS, and synthetic partial structures. The latter comprised partial structures of the core region of Rd1P- LPS bound to the beta 1-->6-linked glucosamine dissacharide with two amide-linked 3-hydroxytetradecanoic acid residues or artificial glycoconjugates comprised of the synthetic oligosaccharides coupled to bovine serum albumin. Using a passive hemolysis and an enzyme immunoassay, absorption and inhibition experiments, the antibody specificites present could be determined. One group of antibodies required components of the core region and the phosphorylated glucosamine disaccharide of the lipid A moiety for binding. The majority of phosphate-independent antibodies was directed against the trisaccharide L-glycero-alpha-D-manno-heptopyranose(1-->3)- L-glycero-alpha-D-manno-heptopyranose(1-->5)3-deoxy-D-manno-oct ulo sonic acid. Antibodies against the 1-->3- and 1-->7-linked heptose disaccharides and against a single heptose were also detected, however, with low titers. No antibodies were found which required the presence of fatty acids.

Specificity of rabbit antisera against the rough lipopolysaccharide of Salmonella minnesota R4 (chemotype Rd2P-)

Rabbit polyclonal antibodies against the rough mutant lipopolysaccharide (LPS) of Salmonella minnesota R4 (chemotype Rd2P-) were serologically characterized by using R4 LPS, deacylated LPS, dephosphorylated LPS, and synthetic partial structures, including compounds comprising the core region of Rd2P- LPS bound to the beta 1-->6-linked glucosamine disaccharide with two amide-linked 3-hydroxytetradecanoic acid residues or coupled to bovine serum albumin. By using a passive hemolysis assay and an enzyme immunoassay and absorption and inhibition experiments, the antibody specificities present could be determined. One group of antibodies required components of the core oligosaccharide (with or without the side chain 3-deoxy-D-manno-octulosonic acid [Kdo]) and the phosphorylated glucosamine disaccharide of the lipid A moiety for binding. The phosphate-independent antibodies were directed against the core oligosaccharide, recognizing an epitope consisting of one terminal heptose linked to Kdo or to the reducing moiety of the alpha 2-->4-linked Kdo disaccharide. Antibodies requiring the presence of acyl residues and those reacting with a single heptose or Kdo residue were not detected.

Characterization of murine monoclonal and murine, rabbit, and human polyclonal antibodies against chlamydial lipopolysaccharide

Murine monoclonal and rabbit, murine, and human polyclonal antibodies against chlamydial lipopolysaccharide (LPS) were characterized by the passive hemolysis and passive hemolysis inhibition assays and by absorption experiments with LPSs of Chlamydia psittaci, Chlamydia trachomatis, and a recombinant strain of Salmonella minnesota Re (r595-207) expressing the chlamydia-specific LPS epitope, as well as natural and synthetic partial structures of chlamydial LPS. Eleven monoclonal antibodies of the immunoglobulin M and G classes were characterized as chlamydia-specific by their failure to react with Re-type LPS, binding to a similar epitope for which the trisaccharide alpha-3-deoxy-D-manno-2-octulosonic acid (KDO)-(2-8)-alpha-KDO-(2-4)-alpha-KDO was an absolute prerequisite. For optimal binding, parts of the lipid A moiety were also involved; however, phosphoryl and ester-linked acyl groups and the reducing glucosamine residue of lipid A were dispensable. A similar antibody specificity was detected in lapine and murine hyperimmune sera after immunization with chlamydia, in addition to those recognizing more complex (e.g., those requiring the presence of phosphoryl residues) and less complex epitopes. Among the latter were those cross-reacting with Re-type LPS, which could be removed by absorption. The titers of different antibody specificities, in particular the ratio of chlamydia-specific to cross-reactive antibodies, present in murine polyclonal antisera depended on the immunization protocol. The preferential formation of chlamydia-specific antibodies was observed after immunization with liposome-incorporated immunogens. Human sera from patients with suspected genital chlamydial infections were also found to contain chlamydia-specific and cross-reactive antibodies, the latter of which could be removed by absorption with Re-type LPS.

Induction of Tumor Necrosis Factor-Alpha Release by Lipopolysaccharide and Defined Lipopolysaccharide Partial Structures

We have investigated the release of tumor necrosis factor-alpha (TNF-α) by human mononuclear cells (MNC) and isolated human monocytes/macrophages stimulated with S- and R-form lipopolysaccharide (LPS), natural lipid A, and natural and synthetic partial structures thereof. We found that LPS of Salmonella minnesota (S.min.) Rb 2, which represents a partial structure of wildtype LPS of Salmonella abortus equi (S.a.e.) lacking the 0-chain and parts of the outer core region, was the most active inducer of all substances tested, even more active than the wildtype LPS. Lipid A also induced the production of TNF-α by monocytes/macrophages but was less active than wildtype LPS.The natural Escherichia coli (E.coli) type hexaacyl lipid A (compound 506) was more active than the natural S.min. type heptaacyl lipid A (compound 516).The 1- and 4′-monodephospho partial structures (compounds 505 and 504) of E.coli lipid A were less active and represented the smallest structures tested that were able to induce TNF-α release by monocytes/macrophages. Synthetic tetraacyl lipid A precursor la of E.coli lipid A, lacking non-hydroxylated fatty acids (compound 406), and the monosaccharide precursor lipid X did not induce the release of TNF-α in MNC or isolated monocytes/macrophages.This might indicate that the ability of a lipid A structure to induce the release of TNF-α is closely connected with the conditions to be at least hexaacylated and/or to contain hydroxylated fatty acids. These results demonstrate a structure-dependent hierarchy of LPS and natural or synthetic partial structures in their capacity of inducing TNF-α release by mono cytes/macrophages.

Epitope specificities of murine monoclonal and rabbit polyclonal antibodies against enterobacterial lipopolysaccharides of the Re chemotype

Murine monoclonal and rabbit polyclonal antibodies raised against the lipopolysaccharides (LPS) of Re mutants of Salmonella minnesota, Proteus mirabilis, and Escherichia coli were serologically characterized. Using natural Re LPS and natural and synthetic partial structures thereof, representing the 3-deoxy-D-manno-2-octulosonic acid (KDO) or lipid A region or both, the epitope specificities of four monoclonal antibodies were defined. Clones 20 (immunoglobulin M [IgM]) and 25 (IgG3) recognize a terminal alpha-pyranosidically linked KDO monosaccharide residue and the alpha-2,4-linked KDO disaccharide, respectively, as the immunodominant group. Therefore, these two antibodies are core antibodies which do not require the presence of lipid A constituents for binding. The minimal structure enabling binding of clone 17 (IgG2b) is a pseudotetrasaccharide of the sequence alpha-KDO-(2----4)-alpha-KDO-(2----6)-beta-glucosamine-(1----6)- glucosaminitol with two amide-linked 3-hydroxytetradecanoic acid residues. The smallest structure with which clone 22 (IgG3) reacted was de-O-acylated Re LPS. Therefore, clones 17 and 22 are LPS antibodies requiring both the lipid A and the KDO region for binding. Phosphoryl residues of the lipid A moiety in Re LPS are dispensable for the reaction with clone 17, whereas they are necessary for that with clone 22. These four different antibody types were also detected in polyclonal rabbit antisera and could be distinguished from each other by absorption experiments. It was found that type 20 and 25 antibodies either were not present or were present only in small amounts and that the majority of the antibodies were of types 17 and 22. From these data, we conclude that the immunodominant structures of Re LPS comprise both the KDO and lipid A domains.

IL-1 induction-capacity of defined lipopolysaccharide partial structures.

Natural and synthetic lipid A as well as natural and synthetic oligosaccharide partial structures of LPS were examined in dose-response experiments to define the minimal structure necessary for IL-1 induction and release in cultures of human mononuclear cells. Wild type LPS (S. abortus equi) and rough mutant LPS was active in minimal-doses of 1 to 100 pg/ml, whereas synthetic heptaacyl and hexaacyl lipid A (Salmonella minnesota and Escherichia coli lipid A, respectively) induced IL-1 in minimal-doses of 100 to 1,000 pg/ml and 10 to 1,000 pg/ml, respectively. Nanogram amounts (0.1 to 10 ng/ml) of synthetic monodephospho partial structures of E. coli lipid A were necessary for IL-1 induction. Synthetic pentaacyl partial structures induced IL-1 very weakly. Synthetic tetraacyl and bisacyl partial structures lacking non-hydroxylated fatty acids were not active. Compared to LPS million-fold higher doses of natural and synthetic 3-deoxy-D-manno-octulosonic acid containing core oligosaccharides were necessary for IL-1 induction. Dose-response investigations with LPS and natural or synthetic partial structures established the following hierarchy in IL-1 induction-capacity: LPS greater than lipid A much greater than lipid A partial structures greater than core oligosaccharides greater than oligoacyl lipid A. Lipid A was shown here to be the portion of LPS mainly responsible for induction of IL-1 activity. The high potency of lipid A in inducing IL-1 release and the failure of the precursor Ia of lipid A to induce IL-1 production and release was also observed measuring intracellular IL-1 activity after freeze-thawing the cells. Levels of IL-1 beta mRNA in extracts of mononuclear cells correlated with biologic activity. In co-incubation experiments, precursor Ia of lipid A produced dose-dependent inhibition of production and release of IL-1 activity induced by lipid A or LPS, but not by Staphylococcus epidermidis or PHA. Incubation of cells with precursor Ia for 1h, followed by a medium change and further incubation of stimulus without precursor Ia of lipid A also resulted in inhibition. We conclude that lipid A is the main portion of LPS responsible for induction of IL-1, and that specific activation- and/or binding-mechanisms are involved in stimulation of cells with LPS and/or lipid A.

An Unfortunate Precedent

Natural and synthetic lipid A as well as natural and synthetic oligosaccharide partial structures of LPS were examined in dose-response experiments to define the minimal structure necessary for IL-1 induction and release in cultures of human mononuclear cells. Wild type LPS (S. abortus equi) and rough mutant LPS was active in minimal-doses of 1 to 100 pg/ml, whereas synthetic heptaacyl and hexaacyl lipid A (Salmonella minnesota and Escherichia coli lipid A, respectively) induced IL-1 in minimal-doses of 100 to 1,000 pg/ml and 10 to 1,000 pg/ml, respectively. Nanogram amounts (0.1 to 10 ng/ml) of synthetic monodephospho partial structures of E. coli lipid A were necessary for IL-1 induction. Synthetic pentaacyl partial structures induced IL-1 very weakly. Synthetic tetraacyl and bisacyl partial structures lacking non-hydroxylated fatty acids were not active. Compared to LPS million-fold higher doses of natural and synthetic 3-deoxy-D-manno-octulosonic acid containing core oligosaccharides were necessary for IL-1 induction. Dose-response investigations with LPS and natural or synthetic partial structures established the following hierarchy in IL-1 induction-capacity: LPS greater than lipid A much greater than lipid A partial structures greater than core oligosaccharides greater than oligoacyl lipid A. Lipid A was shown here to be the portion of LPS mainly responsible for induction of IL-1 activity. The high potency of lipid A in inducing IL-1 release and the failure of the precursor Ia of lipid A to induce IL-1 production and release was also observed measuring intracellular IL-1 activity after freeze-thawing the cells. Levels of IL-1 beta mRNA in extracts of mononuclear cells correlated with biologic activity. In co-incubation experiments, precursor Ia of lipid A produced dose-dependent inhibition of production and release of IL-1 activity induced by lipid A or LPS, but not by Staphylococcus epidermidis or PHA. Incubation of cells with precursor Ia for 1h, followed by a medium change and further incubation of stimulus without precursor Ia of lipid A also resulted in inhibition. We conclude that lipid A is the main portion of LPS responsible for induction of IL-1, and that specific activation- and/or binding-mechanisms are involved in stimulation of cells with LPS and/or lipid A.