Synthesis of the hexacyclic triterpene core of the jujuboside saponins via tandem Wolff rearrangement-intramolecular ketene hetero-Diels-Alder reaction.

The jujubosides are saponin natural products reported to have immunoadjuvant, anticancer, antibacterial, antifungal, and antisweet activities. The triterpene component, jujubogenin contains a unique tricyclic ketal motif comprising the DEF ring system. Herein, we describe our efforts toward the total synthesis of jujubogenin, using a sterically-demanding intermolecular Diels-Alder reaction to assemble the C-ring and a tandem Wolff rearrangement-intramolecular ketene hetero-Diels-Alder reaction to form the DF-ring system. Acid-catalyzed cyclization of the resulting bicyclic enol ether then closes the E-ring to provide the hexacyclic core of jujubogenin.

Development of Improved Vaccine Adjuvants Based on the Saponin Natural Product QS-21 through Chemical Synthesis.

Vaccines based on molecular subunit antigens are increasingly being investigated due to their improved safety and more precise targeting compared to classical whole-pathogen vaccines. However, subunit vaccines are inherently less immunogenic; thus, coadministration of an adjuvant to increase the immunogenicity of the antigen is often necessary to elicit a potent immune response. QS-21, an immunostimulatory saponin natural product, has been used as an adjuvant in conjunction with various vaccines in numerous clinical trials, but suffers from several inherent liabilities, including scarcity, chemical instability, and dose-limiting toxicity. Moreover, little is known about its mechanism of action. Over a decade-long effort, beginning at the University of Illinois at Urbana-Champaign and continuing at the Memorial Sloan Kettering Cancer Center (MSKCC), the group of Prof. David Y. Gin accomplished the total synthesis of QS-21 and developed a practical semisynthetic approach to novel variants that overcome the liabilities of the natural product. First, semisynthetic QS-21 variants were designed with stable amide linkages in the acyl chain domain that exhibited comparable in vivo adjuvant activity and lower toxicity than the natural product. Further modifications in the acyl chain domain and truncation of the linear tetrasaccharide domain led to identification of a trisaccharide variant with a simple carboxylic acid side chain that retained potent adjuvant activity, albeit with reemergence of toxicity. Conversely, an acyl chain analogue terminating in a free amine was inactive but enabled chemoselective functionalization with radiolabeled and fluorescent tags, yielding adjuvant-active saponin probes that, unlike inactive congeners, accumulated in the lymph nodes in vaccinated mice and internalized into dendritic cells. Subtle variations in length, stereochemistry, and conformational flexibility around the central glycosidic linkage provided QS-21 variants with adjuvant activities that correlated with specific conformations found in molecular dynamics simulations. Notably, deletion of the entire branched trisaccharide domain afforded potent, truncated saponin variants with negligible toxicity and improved synthetic access, facilitating subsequent investigation of the triterpene core. The triterpene C4-aldehyde substituent, previously proposed to be important for QS-21 adjuvant activity, proved to be dispensable in these truncated saponin variants, while the presence of the C16 hydroxyl group enhanced activity. Novel adjuvant conjugates incorporating the small-molecule immunopotentiator tucaresol at the acyl chain terminus afforded adjuvant-active variants but without significant synergistic enhancement of activity. Finally, a new divergent synthetic approach was developed to provide versatile and streamlined access to additional linear oligosaccharide domain variants with modified sugars and regiochemistries, opening the door to the rapid generation of diverse, synthetically accessible analogues. In this Account, we summarize these multidisciplinary studies at the interface of chemistry, immunology, and medicine, which have provided critical information on the structure-activity relationships (SAR) of this Quillaja saponin class; access to novel, potent, nontoxic adjuvants for use in subunit vaccines; and a powerful platform for investigations into the mechanisms of saponin immunopotentiation.

Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORγt activity.

CD4(+) T helper lymphocytes that express interleukin-17 (T(H)17 cells) have critical roles in mouse models of autoimmunity, and there is mounting evidence that they also influence inflammatory processes in humans. Genome-wide association studies in humans have linked genes involved in T(H)17 cell differentiation and function with susceptibility to Crohn's disease, rheumatoid arthritis and psoriasis. Thus, the pathway towards differentiation of T(H)17 cells and, perhaps, of related innate lymphoid cells with similar effector functions, is an attractive target for therapeutic applications. Mouse and human T(H)17 cells are distinguished by expression of the retinoic acid receptor-related orphan nuclear receptor RORγt, which is required for induction of IL-17 transcription and for the manifestation of T(H)17-dependent autoimmune disease in mice. By performing a chemical screen with an insect cell-based reporter system, we identified the cardiac glycoside digoxin as a specific inhibitor of RORγt transcriptional activity. Digoxin inhibited murine T(H)17 cell differentiation without affecting differentiation of other T cell lineages and was effective in delaying the onset and reducing the severity of autoimmune disease in mice. At high concentrations, digoxin is toxic for human cells, but non-toxic synthetic derivatives 20,22-dihydrodigoxin-21,23-diol and digoxin-21-salicylidene specifically inhibited induction of IL-17 in human CD4(+) T cells. Using these small-molecule compounds, we demonstrate that RORγt is important for the maintenance of IL-17 expression in mouse and human effector T cells. These data indicate that derivatives of digoxin can be used as chemical templates for the development of RORγt-targeted therapeutic agents that attenuate inflammatory lymphocyte function and autoimmune disease.

Cell-selective labeling using amino acid precursors for proteomic studies of multicellular environments.

We report a technique to selectively and continuously label the proteomes of individual cell types in coculture, named cell type-specific labeling using amino acid precursors (CTAP). Through transgenic expression of exogenous amino acid biosynthesis enzymes, vertebrate cells overcome their dependence on supplemented essential amino acids and can be selectively labeled through metabolic incorporation of amino acids produced from heavy isotope-labeled precursors. When testing CTAP in several human and mouse cell lines, we could differentially label the proteomes of distinct cell populations in coculture and determine the relative expression of proteins by quantitative mass spectrometry. In addition, using CTAP we identified the cell of origin of extracellular proteins secreted from cells in coculture. We believe that this method, which allows linking of proteins to their cell source, will be useful in studies of cell-cell communication and potentially for discovery of biomarkers.

Design, synthesis, and immunologic evaluation of vaccine adjuvant conjugates based on QS-21 and tucaresol.

Immunoadjuvants are used to potentiate the activity of modern subunit vaccines that are based on molecular antigens. An emerging approach involves the combination of multiple adjuvants in a single formulation to achieve optimal vaccine efficacy. Herein, to investigate such potential synergies, we synthesized novel adjuvant conjugates based on the saponin natural product QS-21 and the aldehyde tucaresol via chemoselective acylation of an amine at the terminus of the acyl chain domain in QS saponin variants. In a preclinical mouse vaccination model, these QS saponin-tucaresol conjugates induced antibody responses similar to or slightly higher than those generated with related QS saponin variants lacking the tucaresol motif. The conjugates retained potent adjuvant activity, low toxicity, and improved activity-toxicity profiles relative to QS-21 itself and induced IgG subclass profiles similar to those of QS-21, indicative of both Th1 cellular and Th2 humoral immune responses. This study opens the door to installation of other substituents at the terminus of the acyl chain domain to develop additional QS saponin conjugates with desirable immunologic properties.

Total synthesis, relay synthesis, and structural confirmation of the C18-norditerpenoid alkaloid neofinaconitine.

The first total synthesis of the C18-norditerpenoid aconitine alkaloid neofinaconitine and relay syntheses of neofinaconitine and 9-deoxylappaconitine from condelphine are reported. A modular, convergent synthetic approach involves initial Diels-Alder cycloaddition between two unstable components, cyclopropene 10 and cyclopentadiene 11. A second Diels-Alder reaction features the first use of an azepinone dienophile (8), with high diastereofacial selectivity achieved via rational design of siloxydiene component 36 with a sterically demanding bromine substituent. Subsequent Mannich-type N-acyliminium and radical cyclizations provide complete hexacyclic skeleton 33 of the aconitine alkaloids. Key endgame transformations include the installation of the C8-hydroxyl group via conjugate addition of water to a putative strained bridghead enone intermediate 45 and one-carbon oxidative truncation of the C4 side chain to afford racemic neofinaconitine. Complete structural confirmation was provided by a concise relay synthesis of (+)-neofinaconitine and (+)-9-deoxylappaconitine from condelphine, with X-ray crystallographic analysis of the former clarifying the NMR spectral discrepancy between neofinaconitine and delphicrispuline, which were previously assigned identical structures.

Chemical glycosylation in the synthesis of glycoconjugate antitumour vaccines.

Therapeutic vaccines derived from carbohydrate antigen-adjuvant combinations are a promising approach for cancer immunotherapy. One of the critical limitations in this area is access to sufficient quantities of tumour-associated carbohydrate antigens and glycoconjugate adjuvants. At present, availability of the complex oligosaccharide constructs that are needed for the systematic design and evaluation of novel vaccine formulations relies on de novo chemical synthesis. The use of both state-of-the-art and emerging glycosylation technologies has led to significant advances in this field, allowing the clinical exploration of carbohydrate-based antigens in the treatment of cancer.

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.

Design and synthesis of potent Quillaja saponin vaccine adjuvants.

The success of antitumor and antiviral vaccines often requires the use of an adjuvant, a substance that significantly enhances the immune response to a coadministered antigen. Only a handful of adjuvants have both sufficient potency and acceptable toxicity for clinical investigation. One promising adjuvant is QS-21, a saponin natural product that is the immunopotentiator of choice in many cancer and infectious disease vaccine clinical trials. However, the therapeutic promise of QS-21 adjuvant is curtailed by several factors, including its scarcity, difficulty in purification to homogeneity, dose-limiting toxicity, and chemical instability. Here, we report the design, synthesis, and evaluation of chemically stable synthetic saponins. These novel, amide-modified, non-natural substances exhibit immunopotentiating effects in vivo that rival or exceed that of QS-21 in evaluations with the GD3-KLH melanoma conjugate vaccine. The highly convergent synthetic preparation of these novel saponins establishes new avenues for discovering improved molecular adjuvants for specifically tailored vaccine therapies.

Annulation of thioimidates and vinyl carbodiimides to prepare 2-aminopyrimidines, competent nucleophiles for intramolecular alkyne hydroamination. Synthesis of (-)-crambidine.

A convergent synthesis of (-)-crambidine is reported. The sequence capitalizes on two novel key transformations, including a [4+2] annulation of thioimidates with vinyl carbodiimides and an alkyne hydroamination employing 2-aminopyrimidine nucleophiles.

Ring-opening of aziridine-2-carboxamides with carbohydrate C1-O-nucleophiles. Stereoselective preparation of alpha- and beta-O-glycosyl serine conjugates.

The stereoselective formation of the alpha-GalNAc-Ser linkage via the ring opening of aziridine-2-carboxamides with pyranose C1-O-nucleophiles is described. The process is tolerant to the native C2-NHAc group, can be modulated to provide either the alpha- or beta-glycoside through judicious choice of solvent and metal counterion, and is amenable to other classes of O-glycosyl-Ser constructs such as the beta-GlcNAc-Ser and alpha-Man-Ser linkages. This coupling reaction also led to the development of the o-allylbenzyl (ABn) moiety as a new C-terminus carboxyl protective group, which allows for the use of novel methods for N- and C-terminus extension of amino acids following carbohydrate conjugation.

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.

Application of an intramolecular dipolar cycloaddition to an asymmetric synthesis of the fully oxygenated tricyclic core of the stemofoline alkaloids.

An intramolecular non-stabilized azomethine ylide dipolar cycloaddition was applied toward the first non-racemic synthesis of the fully-oxygenated bridged pyrrolizidine core (45) of (+)-stemofoline (1) in eleven steps from a commercially available starting material.

Rapid assembly of the doubly-branched pentasaccharide domain of the immunoadjuvant jujuboside A via convergent B(C6F5)3-catalyzed glycosylation of sterically-hindered precursors.

A convergent synthesis of the complex, doubly-branched pentasaccharide domain of the natural-product immunoadjuvant jujuboside A is described. The key step is a sterically-hindered glycosylation reaction between a branched trisaccharide trichloroacetimidate glycosyl donor and a disaccharide glycosyl acceptor. Conventional Lewis acids (TMSOTf, BF3·Et2O) were ineffective in this glycosylation, but B(C6F5)3 catalyzed the reaction successfully. Inherent complete diastereoselectivity for the undesired α-anomer was overcome by rational optimization with a nitrile solvent system (1 : 5 t-BuCN/CF3Ph) to provide flexible, effective access to the ß-linked pentasaccharide.

Semisynthesis of Analogues of the Saponin Immunoadjuvant QS-21.

Saponins are triterpene glycoside natural products that exhibit many different biological properties, including activation and modulation of the immune system, and have therefore attracted significant interest as immunological adjuvants for use in vaccines. QS-21 is the most widely used and promising saponin adjuvant but suffers from several liabilities, such as scarcity, dose-limiting toxicity, and hydrolytic instability. Chemical synthesis has emerged as a powerful approach to obtain homogeneous, pure samples of QS-21 and to improve its properties and therapeutic profile by providing access to optimized, synthetic saponin variants. Herein, we describe a general method for the semisynthesis of these molecules from QS-21, with detailed synthetic protocols for two saponin variants developed in our recent work.

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 QS21 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.

Enantioselective approach to the hetisine alkaloids. Synthesis of the 3-methyl-1-aza-tricyclo[5.2.1.0(3,8)]decane core via intramolecular dipolar cycloaddition.

[structure: see text]. An efficient, enantioselective approach to the hetisine class of the C(20)-diterpenoid alkaloids is described. The strategy involves an intramolecular oxidopyridinium dipolar cycloaddition as the key transformation, in which simultaneous formation of the C5-C6 and C10-C20 bonds in the 3-methyl-1-aza-tricyclo[5.2.1.0(3,8)]decane core of the hetisine alkaloids is effected.

Highly convergent synthesis of C3- or C2-symmetric carbohydrate macrocycles.

[structure: see text] A highly convergent strategy for the synthesis of C3- or C2-symmetric oligosaccharide macrocycles is reported. Molecular modeling indicates these macrocycles possess sterically congested cavities. Weak host-guest interactions are observed that should be beneficial for applications such as functionalized molecular pores.

Correction: Versatile strategy for the divergent synthesis of linear oligosaccharide domain variants of Quillaja saponin vaccine adjuvants.

Correction for 'Versatile strategy for the divergent synthesis of linear oligosaccharide domain variants of Quillaja saponin vaccine adjuvants' by Alberto Fernández-Tejada et al., Chem. Commun., 2015, DOI: 10.1039/c5cc05244k.

Versatile strategy for the divergent synthesis of linear oligosaccharide domain variants of Quillaja saponin vaccine adjuvants.

We describe a new, versatile synthetic approach to Quillaja saponin variants based on the natural product immunoadjuvant QS-21. This modular, divergent strategy provides efficient access to linear oligosaccharide domain variants with modified sugars and regiochemistries. This new synthetic approach opens the door to the rapid generation of diverse analogues to identify novel saponin adjuvants with improved synthetic accessibility.