Quillaja saponin variants with central glycosidic linkage modifications exhibit distinct conformations and adjuvant activities

Immunological adjuvants such as the saponin natural product QS-21 help stimulate the immune response to co-administered antigens and have become increasingly important in the development of prophylactic and therapeutic vaccines. However, clinical use of QS-21 is encumbered by chemical instability, dose-limiting toxicity, and low-yielding purification from the natural source. Previous studies of structure–activity relationships in the four structural domains of QS-21 have led to simplified, chemically stable variants that retain potent adjuvant activity and low toxicity in mouse vaccination models. However, modification of the central glycosyl ester linkage has not yet been explored. Herein, we describe the design, synthesis, immunologic evaluation, and molecular dynamics analysis of a series of novel QS-21 variants with different linker lengths, stereochemistry, and flexibility to investigate the role of this linkage in saponin adjuvant activity and conformation. Despite relatively conservative structural modifications, these variants exhibit striking differences in in vivo adjuvant activity that correlate with specific conformational preferences. These results highlight the junction of the triterpene and linear oligosaccharide domains as playing a critical role in the immunoadjuvant activity of the Quillaja saponins and also suggest a mechanism of action involving interaction with a discrete macromolecular target, in contrast to the non-specific mechanisms of emulsion-based adjuvants.     

Synthesis and Preclinical Evaluation of QS-21 Variants Leading to Simplified Vaccine Adjuvants and Mechanistic Probes

QS-21 is a potent immunostimulatory saponin that is currently under clinical investigation as an adjuvant in various vaccines to treat infectious diseases, cancers, and cognitive disorders. Herein, we report the design, synthesis, and preclinical evaluation of simplified QS-21 congeners to define key structural features that are critical for adjuvant activity. Truncation of the linear tetrasaccharide domain revealed that a trisaccharide variant is equipotent to QS-21, while the corresponding disaccharide and monosaccharide congeners are more toxic and less potent, respectively. Modification of the acyl chain domain in the trisaccharide series revealed that a terminal carboxylic acid is well-tolerated while a terminal amine results in reduced adjuvant activity. Acylation of the terminal amine can, in some cases, restore adjuvant activity and enables the synthesis of fluorescently labeled QS-21 variants. Cellular studies with these probes revealed that, contrary to conventional wisdom, the most highly adjuvant active of these fluorescently labeled saponins does not simply associate with the plasma membrane, but rather is internalized by dendritic cells.     

Synthetic studies of complex immunostimulants from Quillaja saponaria: synthesis of the potent clinical immunoadjuvant QS-21Aapi

QS-21 is one of the most promising new adjuvants for immune response potentiation and dose-sparing in vaccine therapy given its exceedingly high level of potency and its favorable toxicity profile. Melanoma, breast cancer, small cell lung cancer, prostate cancer, HIV-1, and malaria are among the numerous maladies targeted in more than 80 recent and ongoing vaccine therapy clinical trials involving QS-21 as a critical adjuvant component for immune response augmentation. QS-21 is a natural product immunostimulatory adjuvant, eliciting both T-cell- and antibody-mediated immune responses with microgram doses. Herein is reported the synthesis of QS-21A(api) in a highly modular strategy, applying novel glycosylation methodologies to a convergent construction of the potent saponin immunostimulant. The chemical synthesis of QS-21 offers unique opportunities to probe its mode of biological action through the preparation of otherwise unattainable nonnatural saponin analogues.     

Synthesis and structure verification of the vaccine adjuvant QS-7-Api. Synthetic access to homogeneous Quillaja saponaria immunostimulants

QS-7-Api is an exceedingly potent immuno-adjuvant isolated from the bark of Quillaja saponaria. It is significantly less toxic than QS-21, a related saponin that is currently the favored adjuvant in anticancer and antiviral vaccine clinical trials. Tedious isolation/purification protocols and uncertainty in its structural constitution have hindered the clinical development of QS-7. A chemical synthesis of QS-7-Api is described, providing structural verification of the adjuvant. A novel semisynthetic sequence to QS-7-Api has also been established, greatly facilitating access to QS-7 for preclinical and clinical evaluation.     

Synthesis of the trisaccharide portion of the immunologic adjuvant QS-21A via sulfonium-mediated oxidative and dehydrative glycosylation

[see structure]. The first synthesis of the trisaccharide fragment of the potent immunologic adjuvant QS-21A is reported. The key steps involve the application of sulfonium-mediated oxidative and dehydrative glycosidic couplings to construct the anomeric linkages in a short and convergent assembly of the branched trisaccharide.     

Synthesis of the potent immunostimulatory adjuvant QS-21A

QS-21A is one of the most promising new adjuvants for immune response potentiation and dose-sparing in vaccine therapy, given its exceedingly high level of potency and its favorable toxicity profile. Melanoma, breast cancer, small cell lung cancer, prostate cancer, HIV-1, and malaria are among the numerous maladies targeted in more than 80 recent and ongoing clinical trials involving QS-21A as a critical component for immune response augmentation in microgram doses. Herein is reported the first synthesis and structure verification of QS-21Aapi, applying novel glycosylation methodologies in the convergent modular construction of this rare and potent natural product immunostimulant.     

Structure of triterpene saponins from the roots ofGlycyrrhiza echinata

Conclusions On the basis of a physicochemical study of the saponins ofGlycyrrhiza echinata L., their molecular weights have been determined. It has been found that the two saponins are biosides of macedonic acid (saponin A) and echinatic acid (saponin B), the carbohydrate moieties of which contain L-rhamnose and D-glucuronic acid.In both saponins, the L-rhamnose is bound directly to the aglycones.Khimiya Prirodnykh Soedinenii, Vol. 5, No. 1, pp. 17–19, 1969     

Chemical synthesis and immunological evaluation of new generation multivalent anticancer vaccines based on a Tn antigen analogue

Tumor associated carbohydrate antigens (TACAs), such as the Tn antigen, have emerged as key targets for the development of synthetic anticancer vaccines. However, the induction of potent and functional immune responses has been challenging and, in most cases, unsuccessful. Herein, we report the design, synthesis and immunological evaluation in mice of Tn-based vaccine candidates with multivalent presentation of the Tn antigen (up to 16 copies), both in its native serine-linked display (Tn-Ser) and as an oxime-linked Tn analogue (Tn-oxime). The high valent vaccine prototypes were synthesized through a late-stage convergent assembly (Tn-Ser construct) and a versatile divergent strategy (Tn-oxime analogue), using chemoselective click-type chemistry. The hexadecavalent Tn-oxime construct induced robust, Tn-specific humoral and CD4⁺/CD8⁺ cellular responses, with antibodies able to bind the Tn antigen on the MCF7 cancer cell surface. The superior synthetic accessibility and immunological properties of this fully-synthetic vaccine prototype makes it a compelling candidate for further advancement towards safe and effective synthetic anticancer vaccines.

A fully-synthetic anticancer vaccine candidate incorporating an hexadecavalent Tn antigen analogue display via oxime linkages induced tumor-specific IgG antibodies and cellular immune responses in mice coadministered with QS-21 as an adjuvant.     

Kinetic peculiarities of <Emphasis Type="Italic">Quillaja Saponaria Molina</Emphasis> saponin sorption by chitosan

A relationship between adsorption layer structure at different stages of sorbent filling, hydrophilicity, and spatial orientation of saponin in chitosan phase is revealed by joint analysis of the kinetic curves of saponin sorption, IR spectra of chitosan and saponin samples, and computer-simulation data. The sorbent–sorbate complex is formed due to electrostatic interactions between protonated amino groups in chitosan and carboanions of glucuronic acid in saponin, as well as hydrogen bonding between NH₂ and OH groups in chitosan molecules and OH groups in the carbohydrate moiety of saponin molecules.     

Comparison of Silica-, C18−, and NH2−HPLC Columns for the Separation of Neutral Steroid Saponins from Dioscorea Plants

Abstract

Six neutral steroid saponins including dioscin and gracillin from the tubers of Dioscorea plants and their peracetates were chromatographed by silica-, C₁₈?, and NH₂?column high-performance liquid chromatography (HPLC). The eluent containing water was used in the silica-column HPLC for the separation of these saponins. These HPLC systems complement each other and allow separation of the saponins. The number of carbohydrate units in the saponin molecule plays the most important role for the separations. The more carbohydrate units in the molecule, the more polar is the saponin or its peracetate. Separation of saponins containing the same number of carbohydrate units can be improved by using their peracetates.     

Antimicrobial activity of simplified mimics of celogentin C

Linear and bicyclic analogues of the peptide natural product, celogentin C, have been prepared in which the sidechain–sidechain crosslinks in celogentin are omitted or replaced with a mesitylenyl moiety. The simplified bicyclic peptides display moderate antibacterial activity, potentially through inhibition of bacterial protomicrotubule formation, while the linear analogs show higher antibacterial activity through a possible membrane disruption mechanism.     

Separation by hydrophobic interaction chromatography and structural determination by mass spectrometry of mannosylated glycoforms of a recombinant transferrin-exendin-4 fusion protein from yeast

Obtaining sufficient amounts of pure glycoprotein variants to characterize their structures is an important goal in both functional biology and the biotechnology industry. We have developed preparative HIC conditions that resolve glycoform variants on the basis of overall carbohydrate content for a recombinant transferrin-exendin-4 fusion protein. The fusion protein was expressed from the yeast Saccharomyces cerevisiae from high density fermentation and is post-translationally modified with mannose sugars through O-glycosidic linkages. Overall hydrophobic behavior appeared to be dominated by the N-terminal 39 amino acids from the exendin-4 and linker peptide sequences as compared to the less hydrophobic behavior of human transferrin alone. In addition, using LC techniques that measure total glycans released from the pure protein combined with new high resolution technologies using mass spectrometry, we have determined the locations and chain lengths of mannose residues on specific peptides derived from tryptic maps of the transferrin-exendin-4 protein. Though the protein is large (80,488 kDa) and contains 78 possible serine and threonine residues as potential sites for sugar addition, mannosylation was observed on only two tryptic peptides located within the first 55 amino acids of the N-terminus. These glycopeptides were highly heterogeneous and contained between 1 and 10 mannose residues scattered among the various serine and threonine sites which were identified by electron transfer dissociation mass spectrometry. Glycan sequences from 1 to 6 linear mannose residues were detected, but mannose chain lengths of 3 or 4 were more common and formed 80% of the total oligosaccharides. This work introduces new technological capabilities for the purification and characterization of glycosylated variants of therapeutic recombinant proteins.     

Saponin Stabilization via Progressive Freeze Concentration and Sterilization Treatment

Saponin is a biopesticide used to suppress the growth of the golden apple snail population. This study aims to determine the stabilized conditions for saponin storage. The maceration process was used for saponin extraction, and for saponin concentration, progressive freeze concentration (PFC) was used. Afterwards, stability analysis was performed by storing the sample for 21 days in two conditions: Room temperature (26 °C) and cold room (10 °C). The samples kept in a cold room were sterilized samples that undergo thermal treatment by placing the sample in the water bath. The non-sterilized samples were kept in room temperature condition for 21 days. The results showed that saponin stored in the cold room (sterilized sample) has low degradation with higher concentration than those stored at room temperature in stability analysis with the highest saponin concentration (0.730 mg/mL) at a concentration temperature of −6 °C and concentration time of 15 min. The lowest saponin concentration obtained by saponin stored at room temperature (non-sterilized sample) is 0.025 mg/mL at a concentration temperature of −6 °C and concentration time of 10 min. Thus, the finding concluded that saponin is sensitive to temperature. Hence, the best storage condition to store saponin after thermal treatment is to keep it in a cold room at 10 °C.     

Thermal Stabilization of an Endoglucanase by Cyclization

An intein-driven protein splicing approach allowed for the covalent linkage between the N- and C-termini of a polypeptide chain to create circular variants of the endo-??-1,3-1,4-glucanase, LicA, from Bacillus licheniformis. Two circular variants, LicA-C1 and LicA-C2, which have connecting loops of 20 and 14 amino acids, respectively, showed catalytic activities that are approximately two and three times higher, respectively, compared to that of the linear LicA (LicA-L1). The thermal stability of the circular variants was significantly increased compared to the linear form. Whereas the linear glucanase lost half of its activity after 3?min at 65?°C, the two circular variants have 6-fold (LicA-C1) and 16-fold (LicA-C2) increased half-life time of inactivation. In agreement with this, fluorescence spectroscopy and differential scanning calorimetry studies revealed that circular enzymes undergo structural changes at higher temperatures compared to that of the linear form. The effect of calcium on the conformational stability and function of the circular LicAs was also investigated, and we observed that the presence of calcium ions results in increased thermal stability. The impact of the length of the designed loops on thermal stability of the circular proteins is discussed, and it is suggested that cyclization may be an efficient strategy for the increased stability of proteins.     

Analysis of soybean sapogenins by high-performance liquid chromatography

A method for the analysis of soybean sapogenins is described. The method is based on the extraction of soybean saponins from a defatted sample. The triterpene glycosides are then hydrolysed with subsequent analysis of the liberated sapogenins by high-performance liquid chromatography using gradient elution and mass detection. By use of a sapogenin/carbohydrate ratio, an estimate of the total saponin content can be made.     

Determination of genetic transferrin variants in human serum by high‐resolution capillary zone electrophoresis†

High‐resolution capillary zone electrophoresis in the routine arena with stringent quality assurance is employed for the determination of carbohydrate‐deficient transferrin in human serum. The assay comprises mixing of human serum with a Fe^III‐containing solution prior to analysis of the iron‐saturated mixture in a dynamically double‐coated capillary using a commercial buffer at alkaline pH. In contrast to other assays, it provides sufficient resolution for proper recognition of genetic transferrin variants. Analysis of 7290 patient sera revealed 166 isoform patterns that could be assigned to genetic variants, namely, 109 BC, 53 CD, one BD and three CC variants. Several subtypes of transferrin D can be distinguished as they have large enough differences in pI values. Subtypes of transferrin C and B cannot be resolved. However, analysis of the detection time ratios of tetrasialo isoforms of transferrin BC and transferrin CD variants revealed multimodal frequency histograms, indicating the presence of subtypes of transferrin C, B and D. The data gathered over 11 years demonstrate the robustness of the high‐resolution capillary zone electrophoresis assay. This is the first account of a capillary zone electrophoresis based carbohydrate‐deficient transferrin assay with a broad overview on transferrin isoform patterns associated with genetic transferrin variants.     

茶皂素―金属复合抗菌剂的制备及抗菌性能研究

通过抑菌环法探讨了茶皂素、金属离子、茶皂素―金属复合抗菌剂对大肠杆菌和金黄色葡萄球菌的抗菌活性,考察了茶皂素纯度、浓度、金属离子种类等因素对抗菌剂抗菌活性的影响。结果表明,茶皂素的抗菌活性与单一的金属离子的抗菌活性相当,茶皂素对大肠杆菌的最低抑制浓度为5 mg/mL,对金黄色葡萄球菌的最低抑制浓度是10 mg/mL;茶皂素与金属离子复配抗菌活性具有协同效应,尤其茶皂素―锌复合抗菌剂对大肠杆菌的抑制效果大大加强。     

The substrate specificity of a glucoamylase with steroidal saponin-rhamnosidase activity from Curvularia lunata

In previous work, we studied and reported that an enzyme from Curvularia lunata 3.4381 had the novel specificity to hydrolyze the terminal rhamnosyl at C-3 position of steroidal saponin and obtained four transformed products; the enzyme was purified and ascertained as glucoamylase (EC 3.2.1.3 GA). In this work, the enzyme exhibiting steroidal saponin-rhamnosidase activity was systematically studied on 21 steroidal saponins and 6 ginsenosides. The results showed that the α-1,2-linked end-rhamnosyl residues at C-3 position of steroidal saponins could be hydrolyzed to corresponding secondary steroidal saponins, among which 18 compounds were isolated and identified, including 3 new secondary compounds. For the furostanosides having glucosyl residues at the C-26 position, hydrolysis occurred first at end-rhamnosyl at C-3 position to produce secondary furostanosides. The reaction of hydrolyzing glucosyl at C-26 position depended considerably on longer reaction times yielding the corresponding secondary spirostanosides (without rhamnosyl and glucosyl residues). The enzyme had the strict specificity for the terminal α-1,2-linked rhamnosyl residues of linear chain, or the terminal α-1,2-linked rhamnosyl residues with branched chain of 1,4-linked glycosyl residues of sugar chain at C-3 position of steroidal saponins, it was not specific for different aglycones, different glycons, and the number of glycon of sugar chain of steroidal saponin. The end-rhamnosyl of ginsenosides and p-nitrophenyl-α-l-rhamnopyranoside (pNPR) could not be hydrolyzed by the enzyme from C. lunata.     

Sequence requirements and an optimization strategy for short antimicrobial peptides

Short antimicrobial host-defense peptides represent a possible alternative as lead structures to fight antibiotic resistant bacterial infections. Bac2A is a 12-mer linear variant of the naturally occurring bovine host defense peptide, bactenecin, and demonstrates moderate, broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria as well as against the yeast Candida albicans. With the assistance of a method involving peptide synthesis on a cellulose support, the primary sequence requirements for antimicrobial activity against the human pathogen Pseudomonas aeruginosa of 277 Bac2A variants were investigated by using a luciferase-based assay. Sequence scrambling of Bac2A led to activities ranging from superior or equivalent to Bac2A to inactive, indicating that good activity was not solely dependent on the composition of amino acids or the overall charge or hydrophobicity, but rather required particular linear sequence patterns. A QSAR computational analysis was applied to analyze the data resulting in a model that supported this sequence pattern hypothesis. The activity of selected peptides was confirmed by conventional minimal inhibitory concentration (MIC) analyses with a panel of human pathogen bacteria and fungi. Circular-dichroism (CD) spectroscopy with selected peptides in liposomes and membrane depolarization assays were consistent with a relationship between structure and activity. An additional optimization process was performed involving systematic amino acid substitutions of one of the optimal scrambled peptide variants, resulting in superior active peptide variants. This process provides a cost and time effective enrichment of new candidates for drug development, increasing the chances of finding pharmacologically relevant peptides.     

A new saponin lactone from Astragalus corniculatus

A new oleanane-type saponin lactone was isolated from the ethanolic extract of the aerial parts of Astragalus corniculatus Bieb. The structure of the saponin was elucidated as 19-hydroxyolean-12-ene-28, 21beta-olide 3beta-D-xylopyranoside (1) by chemical and spectral methods.