*Article* **Preparation, Supramolecular Aggregation and Immunological Activity of the Bona Fide Vaccine Adjuvant Sulfavant S**

**Emiliano Manzo 1,\*,**†**, Laura Fioretto 1,2,**†**, Carmela Gallo 1, Marcello Ziaco 3, Genove** ff**a Nuzzo 1, Giuliana D'Ippolito 1, Assunta Borzacchiello 4, Antonio Fabozzi 4, Ra** ff**aele De Palma 1,5 and Angelo Fontana 1,\***

1 Bio-Organic Chemistry Unit, CNR-Institute of Biomolecular Chemistry, Via Campi Flegrei 34, 80078 Pozzuoli, Italy; l.fioretto@icb.cnr.it (L.F.); carmen.gallo@icb.cnr.it (C.G.); nuzzo.genoveffa@icb.cnr.it(G.N.);gdippolito@icb.cnr.it(G.D.);raffaele.depalma@unige.it(R.D.P.)

 2 ConsorzioItalbiotec,ViaFantoli,16/15,20138Milano,Italy

3 BioSearch Srl., Villa Comunale c/o Stazione Zoologica "A.Dohrn", 80121 Napoli, Italy; m.ziaco@icb.cnr.it

4 Institute for Polymers, Composites and Biomaterials (IPCB), CNR, 80125 Naples, Italy;

bassunta@unina.it (A.B.); Sirfabozzi@hotmail.it (A.F.) 5 Medicina Interna, Immunologia Clinica e Medicina Traslazionale,


Received: 20 July 2020; Accepted: 26 August 2020; Published: 29 August 2020

**Abstract:** 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 β-sulfoquinovosyl-diacylglyceride skeleton. The family includes the lead product Sulfavant A (**1**) and two epimers, Sulfavant R (**2**) and Sulfavant S (**3**), di ffering only for the stereochemistry at C-2 of glycerol. The three compounds showed a significant di fference 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**–**3** effectively depends on the supramolecular aggregation states, likely a ffecting the bioavailability of agonists that can e ffectively 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.

**Keywords:** sulfavants; adjuvant; immunomodulatory activity; colloid; aggregates
