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Despite of the enormous efforts put in the research of new vaccines and vaccine adjuvants, approved formulations are far to be efficient against challenging diseases. In this frame, the development of new molecular vaccine adjuvants represents the most innovative technology in the identification of new vaccines. Inspired by a natural immunogenic α-sulfoquinovosyldiacylglycerols (α-SQDGs), this PhD project was aimed to the identification of glycolipids with immunomodulant activity and their development as vaccine adjuvants. The first synthetized sulfolipid was a β-SQDGs (1,2-O-distearoyl-3-O-β-D-sulfoquinovosylglycerol) named Sulfavant A. Sulfavant A presented the sulfonic group typical of natural α-SQDGs and the β anomeric configuration usual for LPS-based glycolipids. Sulfavant A activated dendritic cells (DC) with up-regulation of the CD83 as well as the gene expression of proinflammatory cytokines suggesting an interesting immunomodulant activity TLR-independent. Sulfavant A was also tested in in vivo: in a first experiment the antibodies response was evaluated, and the results were comparable to commercial adjuvants; in a second experiment the adjuvant properties of Sulfavant A were evaluated in a model of vaccination against melanoma in mice obtaining promising results. Starting from these results we explored the effect of structural changing on sulfolipids bioactivity introducing different acyl chains and modifying the stereochemistry at glyceridic carbon 2. In particular, two palmitoyl and two nonadecanoyl chains were introduced and for each molecule the epimeric mixture at glyceridic carbon 2 and the diasteropure analogues were synthetized. The biological assays suggested that even slight modifications in the hydrophobic part induced partial loss of activity, independently from the stereochemistry of the glyceridic carbon 2. The synthetic strategy was improved and employed for the preparation of the two diasteropure analogues of Sulfavant A, 1,2-O-distearoyl-3-O-β-D-sulfoquinovosyl-(R)-glycerol (named Sulfavant R) and 1,2-O-distearoyl-3-O-β-D-sulfoquinovosyl-(S)-glycerol (named Sulfavant S), with defined stereochemistry at carbon 2. The biological evaluation of Sulfavant R and Sulfavant S bioactivity led to different response correlated with stereochemical aspects; in fact, efficacy of single epimers (Sulfavant S and R) on DCs maturation was maximum at concentration 1000-fold lower than that occurred for the Sulfavant A while all the activity curves were featured by a peculiar bell-shaped dose response. A hypothesis of this different behaviour was that Sulfavants could self-assemble in aqueous environment influencing the bioavailability of the active molecule to the cellular target. The self-assembling properties of Sulfavants was investigated in this PhD thesis. In particular, Dynamic Light Scattering (DLS) gave the first evidence that Sulfavants aggregate in water forming supramolecular structure of different size. Despite some difficulties to analyse Sulfavants solutions at very low concentrations, the Critical Micellar Concentration (CMC) was evaluated by surface tension measurements and fluorescence spectroscopy. The assessment of the effect of a detergent on Sulfavant A showed that aggregation negatively affected the immunomodulant activity, since with the detergent the activation of DCs underwent shift at lower concentrations. Cryo-TEM images showed direct evidences of Sulfavant A and Sulfavant R aggregation at 10 nM and 10 µM concentrations, underlining the differences between the supramolecular structures at different concentrations at which they were able to stimulate DCs maturation. Furthermore, a fluorescent analogue of Sulfavants was synthesised for bioimaging studies in order to get information about the mechanisms responsible of immunogenic reaction. In particular, a derivative of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dye (BODIPY) was introduced on the hydrophobic moiety of Sulfavant R by new synthetic strategy. Moreover, higher amount of Sulfavant A was prepared by an improved and scalable synthesis. Finally, according to my PhD project, during an internship at the University of British Columbia, a synthetic strategy for the preparation of 1-O-alkyl-2-O-(2’-O-β-D-glucopyranosil-β-D-xylopyranosil)-glycerol has been designed; the above glycolipid was the main component of a natural bioactive mixture isolated by Professor Andersen and coworkers, from the UBC (Vancouver-Canada).

Sulfavant A (1) and Sulfavant R (2) are amphiphilic sulfoquinovoside-glycerol lipids under study as molecular adjuvants of vaccines. Their structures differ only for the configuration of the oxymethine carbon of glycerol that is R/S in 1 and R in 2. The in vitro activity of both products follows a bell-shaped dose-response curve but 1 gave the best response between 10 and 30 µM, while 2 was active at 10 nM. Characterization of aqueous self-assembly of these molecules by a multi-technique approach based on surface tensiometry, dynamic light scattering (DLS), nuclear magnetic resonance (NMR), fluorescence light phase contrast, and cryo-electron microscopy has finally clarified this divergent and controversial biological outcome. The study proved the occurrence of supramolecular structures at concentrations much lower than the critical aggregation concentration (CAC) calculated by surface tensiometry for both products. The shape and size of these aggregates varied as a function of the concentrations differently for 1 and 2. At nanomolar concentration, Sulfavant A (1) formed cohesive vesicular structures, while Sulfavant R (2) arranged in small spherical micellar particles whose reduced stability was responsible for an increase of the monomer concentration that well agreed with the immunomodulatory activity. On the other side, micromolar concentrations led to an increase of particle sizes of both compounds and a transition from micellar to the vesicular state of Sulfavant R (2). The thermodynamic stability of these aggregates correlated with the activity of 1 above 10 µM and the complete loss of efficacy of 2 at micromolar concentrations. The study of Sulfavants provides clear evidence of how self-aggregation and thus the equilibria between the monomers and the supramolecular forms of lipophilic products in aqueous media determine the overall response of these substances in cell-based assays.

Immunotherapy takes advantage of the immune system to prevent, control, and eliminate neoplastic cells. The research in the field has already led to major breakthroughs to treat cancer. In this work, we describe a platform that integrates in vitro bioassays to test the immune response and direct antitumor effects for the preclinical discovery of anticancer candidates. The platform relies on the use of dendritic cells that are professional antigen-presenting cells (APC) able to activate T cells and trigger a primary adaptive immune response. The experimental procedure is based on two phenotypic assays for the selection of chemical leads by both a panel of nine tumor cell lines and growth factor-dependent immature mouse dendritic cells (D1). The positive hits are then validated by a secondary test on human monocyte-derived dendritic cells (MoDCs). The aim of this approach is the selection of potential immunotherapeutic small molecules from natural extracts or chemical libraries.

In aqueous conditions, amphiphilic bioactive molecules are able to form self-assembled colloidal structures modifying their biological activity. This behavior is generally neglected in preclinical studies, despite its impact on pharmacological development. In this regard, a significative example is represented by a new class of amphiphilic marine-inspired vaccine adjuvants, collectively named Sulfavants, based on the &beta, sulfoquinovosyl-diacylglyceride skeleton. The family includes the lead product Sulfavant A (1) and two epimers, Sulfavant R (2) and Sulfavant S (3), differing only for the stereochemistry at C-2 of glycerol. The three compounds showed a significant difference in immunological potency, presumably correlated with change of the aggregates in water. Here, a new synthesis of diastereopure 3 was achieved, and the study of the immunomodulatory behavior of mixtures of 2/3 proved that the bizarre in vitro response to 1&ndash, 3 effectively depends on the supramolecular aggregation states, likely affecting the bioavailability of agonists that can effectively interact with the cellular targets. The evidence obtained with the mixture of pure Sulfavant R (2) and Sulfavant S (3) proves, for the first time, that supramolecular organization of a mixture of active epimers in aqueous solution can bias evaluation of their biological and pharmacological potential.

Recently, we reported the promising activity of a novel class of sulfoquinovosyl-diacylglycerols named Sulfavants as molecular vaccine adjuvants. Herein, we describe a modified and improved chemical synthesis of the lead product Sulfavant A (1), with the aim to produce from milligrams to 10 g of the pure compound that is necessary for the preclinical development. Starting from the versatile synthesis based on the trichloroacetimidate methodology, up-scaled preparation of Sulfavant A (1) was achieved in 11 steps by elimination and modification of those reactions that negatively affected the overall yield by the previous procedure. The novel strategy gave 17% overall yield of the target compound 1 and also paved the way for the gram-scale preparation of a wide range of other charged and neutral glycoglycerolipids.

A fast and sensitive method that is based on Ultra High Performance Liquid Chromatography coupled with High Resolution Mass Spectrometry (UHPLC-HRMS) for the measurement of Sulfavant A, a molecular adjuvant with a sulfolipid skeleton, is described. The method has been validated over the linearity range of 2.5–2000 ngmL–1 using a deuterated derivative (d70-Sulfavant A) as internal standard. Chromatographic separation is based on a UHPLC Kinetex® 2.6 µm PS C18 column and a gradient of methanol in 0.32 mM ammonium hydroxide solution buffered at pH 8. The lowest limit of quantification of Sulfavant A was 6.5 ngmL–1. The analytical procedure was tested on an extract of mice lung spiked with 30, 300, and 1500 ng of Sulfavant A. The analysis revealed a precision and accuracy value (as a mean value of all the quality control samples analyzed) of 4.7% and 96% in MeOH and 6.4% and 93.4% in the lung extracts, respectively.

Vaccines play a primary role in the protection of human health by preventing infectious and chronic diseases. Recently we have reported 1,2-O-distearoyl-3-O-beta-d-sulfoquinovosylglycerol (beta-SQDG18), here named Sulfavant A (1), which shows promising properties as a new molecular adjuvant in in vitro and in vivo tests. In the present manuscript, we provide full details about a synthetic strategy for the preparation of 1, including a discussion of chemical determinants of the activity and the major technical hurdles we faced during the study. Synthesis of Sulfavant A (1) is achieved by a versatile procedure based on a trichloroacetimidate methodology and peracetate sugar precursors. The final design opens possibilities for the preparation of a series of interesting analogs for further pharmacological optimization and development, including derivatives containing different saturated and polyunsaturated fatty acids (e.g., 17 and 22).