**1. Introduction**

Development of new drugs is a long and expensive process, and often many unpredictable problems arise in clinical phases due to the chemical-physical behavior of some molecules in physiological environments. Several pharmacologically active compounds and commercial drugs are in fact amphiphilic substances able to self-aggregate in aqueous solutions. Self-assembly in water is a spontaneous process involving the arrangemen<sup>t</sup> in supramolecular structures that are stabilized by non-covalent interactions and minimizes the direct contact between the hydrophobic part of the molecule and the polar solvent [1]. This behavior is as common as it is poorly considered, even if it can seriously a ffect biological activity and pharmacological developments. Only recently, studies have focused on the relevance of physicochemical properties, such as lipophilicity, in the in vitro selection of drug candidates and likelihood of success in development [2]. In fact, molecular aggregation can be critical in determining in vivo ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties, but also in a ffecting the overall quality of a drug candidate in cellular assays. In physiological media, lipophilic substances produce complex equilibria involving free molecules and many aggregates di ffering in size and shape. Therefore, as aggregates are not involved in the pharmacodynamic interaction, these equilibria determine the e ffective concentration of the bioactive product on the target and in cellular tests.

Adjuvants are chemical components that are combined with antigens to enhance the immune response to vaccines [3]. Traditionally, adjuvants are composed of a suspension of insoluble compounds (e.g., oils, aluminum, particulate materials containing small molecules) in water. In the last few years, a major breakthrough has been the discovery of the link between adjuvants and innate immune response triggered by antigen-presenting cells (APCs) that capture and process antigens for presentation to T-lymphocytes, and to produce signals required for the proliferation and di fferentiation of lymphocytes [4–11]. In particular, the identification of pattern recognition receptors (PRRs) as primary e ffectors of the plastic activation of APCs has rapidly led to the rational design of molecular adjuvants based on single, immunomodulatory molecules. In this context, we recently characterized β-sulfoquinovosyl-diacyl glycerols (β-SQDGs) as a novel class of vaccine adjuvants collectively named Sulfavants. These synthetic molecules were inspired by natural and marine α-sulfoquinovosyl-diacylglycerols ( α-SQDGs) occurring as membrane constituents in photosynthetic organisms [12–15]. Sulfavant A (**1**) (1,2- *O*-distearoyl-3- *O*-(β-sulfoquinovosyl)- *<sup>R</sup>*/*S*-glycerol), the prototype of the family, induces maturation of human dendritic cells (hDCs) at micromolar concentrations with a typical "bell-shaped" dose–response curve that is featured by a maximum around 10 μM. Sulfavant A (**1**) also showed promising adjuvant activity in *in vivo* experiments, as it was able both to boost immune protection in mice and to inhibit tumor growth in an experimental model of cancer vaccine against melanoma [13–15].

Compound **1** is a 1.3:1 mixture of *R*/*S* epimers at carbon 2 of glycerol moiety. In order to investigate the pharmacological properties of this new class of molecules, we also synthetized two enantiopure analogues named Sulfavant R (**2**) and S (**3**) [12]. Surprisingly, compound **2** showed maturation of hDCs at nanomolar concentrations with 1000-fold increase of the activity in comparison to the epimeric mixture **1**. We never had the opportunity to test rigorously the biological response to pure Sulfavant S (**3**) because of a partial loss of stereospecificity of the synthesis for this product due to a fast process of opening and closure of the *S*-glycerol acetonide during the glycosylation step (Scheme 1) [12]. However, a fraction containing 80% Sulfavant S (**3**) and 20% Sulfavant R (**2**) was also active on DCs but at concentrations between those of **1** and **2**. All of these compounds gave bell-shaped concentration–response curves, and chemo-physical analysis revealed a clear correlation between size of the microparticles in water and activation of hDC maturation [12].

In the present work, we implemented a stereospecific synthesis of Sulfavant S (**3**) with the aim to prove that the immunological priming of hDC by the enantiopure isomers is significantly dependent, per se, on the type of aggregation of active products.

**Scheme 1.** Literature synthetic approach for the preparation of Sulfavant S (**3**) (with 20% of *R* epimer) according to Manzo et al., 2019 [12].
