Preparation, supramolecular aggregation and immunological activity of the bona fide vaccine adjuvant sulfavant S

Article abstract of DOI:10.3390/md18090451

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), 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 1–3 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.

Full text of DOI:10.3390/md18090451

marine drugs
Article
Preparation, Supramolecular Aggregation and
Immunological Activity of the Bona Fide Vaccine
Adjuvant Sulfavant S
1
, ,†
1,2,†
, Laura Fioretto
, Carmela Gallo 1 , Marcello Ziaco ,
3
Emiliano Manzo *
Genoveffa Nuzzo 1 , Giuliana D’Ippolito , Assunta Borzacchiello , Antonio Fabozzi ,
Raffaele De Palma 1 and Angelo Fontana *
1
4
4
,5
1,
1
Bio-Organic Chemistry Unit, CNR-Institute of Biomolecular Chemistry, Via Campi Flegrei 34,
8
0078 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.)
Consorzio Italbiotec, Via Fantoli, 16/15, 20138 Milano, Italy
BioSearch Srl., Villa Comunale c/o Stazione Zoologica “A.Dohrn”, 80121 Napoli, Italy; m.ziaco@icb.cnr.it
Institute for Polymers, Composites and Biomaterials (IPCB), CNR, 80125 Naples, Italy;
bassunta@unina.it (A.B.); Sirfabozzi@hotmail.it (A.F.)
Medicina Interna, Immunologia Clinica e Medicina Traslazionale,
Università di Genova and IRCCS-Ospedale S. Martino, 16131 Genova, Italy
Correspondence: emanzo@icb.cnr.it (E.M.); afontana@icb.cnr.it (A.F.)
These authors contributed equally.
2
3
4
5
*
†
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
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 ( ) and two epimers, Sulfavant R (
1
2
) and Sulfavant S ( ), differing only
3
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
of mixtures of 2/3 proved that the bizarre in vitro response to
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 (
and Sulfavant S ( ) proves, for the first time, that supramolecular organization of a mixture of active
3
was achieved, and the study of the immunomodulatory behavior
1–3
effectively depends on the
2
)
3
epimers in aqueous solution can bias evaluation of their biological and pharmacological potential.
Keywords: sulfavants; adjuvant; immunomodulatory activity; colloid; aggregates
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 arrangement 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,
Mar. Drugs 2020, 18, 451; doi:10.3390/md18090451
www.mdpi.com/journal/marinedrugs

Mar. Drugs 2020, 18, 451
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even if it can seriously affect 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 affecting the overall quality of a drug candidate in
cellular assays. In physiological media, lipophilic substances produce complex equilibria involving
free molecules and many aggregates differing in size and shape. Therefore, as aggregates are not
involved in the pharmacodynamic interaction, these equilibria determine the effective 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 differentiation
of lymphocytes [4–11]. In particular, the identification of pattern recognition receptors (PRRs)
as primary effectors 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
collectively named Sulfavants. These synthetic molecules were inspired by natural and marine
-sulfoquinovosyl-diacylglycerols ( -SQDGs) occurring as membrane constituents in photosynthetic
organisms [12 15]. Sulfavant A ( ) (1,2-O-distearoyl-3-O-( -sulfoquinovosyl)-R/S-glycerol),
β-sulfoquinovosyl-diacyl glycerols (β-SQDGs) as a novel class of vaccine adjuvants
α
α
–
1
β
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
the pharmacological properties of this new class of molecules, we also synthetized two enantiopure
analogues named Sulfavant R ( ) and S ( ) [12]. Surprisingly, compound showed maturation
of hDCs at nanomolar concentrations with 1000-fold increase of the activity in comparison to the
epimeric mixture . We never had the opportunity to test rigorously the biological response to pure
Sulfavant S ( ) 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 ( ) and 20% Sulfavant R ( ) was also
active on DCs but at concentrations between those of and . All of these compounds gave bell-shaped
1 is a 1.3:1 mixture of R/S epimers at carbon 2 of glycerol moiety. In order to investigate
2
3
2
1
3
(
3
2
1
2
concentration–response curves, and chemo-physical analysis revealed a clear correlation between size
of the microparticles in water and activation of hDC maturation [12].
O
O
-
+
SO -K+
3
SO K
3
O
O
O
O
HO
HO
O
HO
HO
O
O
O
R
S
OH
OH
O
O
Sulfavant R (2)
Sulfavant S (3)

Mar. Drugs 2020, 18, 451
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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.
BF
O
3
S
OAc
O
O
O
H
δ
+
AcO
AcO
O
AcO
O
N
HO
O
H
SO -K+
3
OH
O
OAc
O
1
. Ac
2
O / Pyridine
S-glycerol acetonide
CCl
3
OAc
O
AcO
2S:2R 4:1
OStearoyl
OStearoyl
S
O
HO
HO
AcO
AcO
AcO
AcO
HO
HO
O
OH
O
O
O
2
. BnNH
2
/ THF
AcO
CHNCO
BF
3
-Et
2
O
OH
D-glucose
OH
Cl
3
O
O
R
3
. CCl CN / DBU
3
OAc
O
Sulfavant S
(20% of Sulfavant R)
O
AcO
AcO
H
AcO
O
N H
CCl
3
Scheme 1. Literature synthetic approach for the preparation of Sulfavant S (3) (with 20% of R epimer)
according to Manzo et al., 2019 [12].
2
. Results and Discussion
The use of (R)-(-)-1,2-isopropylideneglycerol as an acceptor in the coupling step with the
trichloroacetamidate sugar gave high stereospecificity for the synthesis of Sulfavant R ( ) [12]. However,
2
a similar reaction with (S)-(+)-1,2-isopropylideneglycerol led to a low diastereoselective yield because
of opening and closure of the acetonide ring under acidic conditions due to the boron trifluoride catalyst.
As described in Scheme 1, formation of a complex between boron trifluoride and the acetonide oxygen
atoms can start a fast and reversible process of racemization at C-2 of glycerol. The rearrangement
rate was comparable to formation of the glycosidic bond, thus resulting in 20% epimerization of the
glycosylated product. In order to overcome this issue, we prepared an alternative acceptor for the
glycosylation reaction (Scheme 2).
Stearic acid
BnBr/NaH
20h-60°C
Dowex-H+
DCC
O
O
OH
OStearoyl
OStearoyl
DMAP-16h
1
h r.t.
O
O
OH
HO
BnO
BnO
BnO
94%
93%
9
2%
4
5
6
(
S)-1,2-isopropylideneglycerol
Pd-C
H -16h
2
35%
OStearoyl
OStearoyl
HO
(S)-1,2-O-distearoyl-glycerol
Scheme 2. Synthesis of the acceptor (S)-1,2-O-distearoylglycerol.
Starting from the usual S-1,2-isopropylideneglycerol, the first step was the protection of the
primary hydroxy group by benzylation with benzyl bromide and sodium hydride. Treatment with
+
Dowex H led to the diol derivative (5) from which the distearoyl-benzyl intermediate (6) was
obtained by acylation with stearic acid and N,N’-dicyclohexylcarbodiimide. Without affecting the
chirality of the stereocenter at C-2 of glycerol, debenzylation by hydrogenolysis on palladium gave
the (S)-1,2-O-distearoylglycerol acceptor with 35% overall yield. The new acceptor was immediately
coupled with the peracetylated glucosyl-trichloroacetimidate donor without loss of stereospecificity.
As shown in Scheme 3, after deacetylation of the sugar moiety by hydrazinolysis, the synthesis of the
sulfolipid followed the same sequence of reactions previously reported to give pure Sulfavant S [12
]
(Figure 1).

Mar. Drugs 2020, 18, 451
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3
CCl CN (10 eq)
DBU (0.5 eq)
2h/0°C
OH
O
HO
OH
OAc
O
OAc
O
OAc
O
Ac
2
O/Pyridine
9%
BnNH2 (1.5eq)/THF
HO
AcO
AcO
AcO
AcO
AcO
AcO
OH
OH
OAc
81%
9
OAc
OAc
75%
AcO
3
CHNCO
sugar trichloroacetimidate donor
Cl
D-glucose
OStearoyl
HO
OStearoyl
TMSOTf / 5h-0°C
(
S)-1,2-O-distearoyl glycerol
4
5%
acceptor
I
2
(1.5 eq) / P(Ph)
2,6-lutidine (20 eq)
3
(1.5 eq)
2 2 2
NH NH -H O (2.2 eq)
EtOH 85%/44°C/8h
I
OH
O
OAc
O
OStearoyl
OStearoyl
OStearoyl
OStearoyl
OStearoyl
O
AcO
AcO
O
OStearoyl
HO
HO
O
AcO
AcO
O
Ac2O/pyridine
51%
OAc
OH
OAc
9
9%
8
7
2
3
-butanone/CH COSK
80°C/2h
99%
2 2
H O
KAc-buffer
16h/40°C
2 2 2
NH NH -H O (2.4 eq)
EtOH 85%/44°C/6h
SO
3
-K+
SAc
O
SO
O
3
-K+
OStearoyl
OStearoyl
OStearoyl
OStearoyl
OStearoyl
OStearoyl
O
AcO
AcO
O
AcO
AcO
O
HO
HO
O
79%
55%
OAc
OAc
OH
Pure Sulfavant S
Scheme 3. Synthesis of the diastereopure Sulfavant S (3).
1
Figure 1. H-NMR of pure 3 in CDCl :CD OD 1:1 at 600MHz.
3
3
Enantiopure Sulfavant S (
monocytes [13]. DCs are the most efficient antigen-presenting cells (APCs) [16
nature’s adjuvant” [23] for their ability to induce activation and specific expansion of CD4 helper
3) was tested for the activation of hDCs derived from blood
–
22], often called
+
“
+
T (Th) and CD8 cytotoxic T (CTL) lymphocytes. The search for substances able to activate and
prepare DCs to face pathogens represents a key tool in the development of new molecular adjuvants
for vaccines against tumors or infections [24
measured by upregulation of CD83, an integral membrane protein belonging to the immunoglobulin
superfamily and selectively expressed on mature DCs [27]. The activity of pure Sulfavant S ( ) was
similar to that of Sulfavant R ( ) [12], with maximum CD83 expression at nanomolar concentration
Figure 2).
–26]. The effect of compound 3 on hDC maturation was
3
2
(

Mar. Drugs 2020, 18, 451
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+
Figure 2. Percentage of CD83 cells stimulated by pure Sulfavant S (
differences from untreated cells; * p < 0.5, **** p < 0.0001.
3); asterisks indicate significant
In addition, as previously observed for compounds
dose activity curve that is common to other lipophilic drugs and reflects the formation of aggregates in
the aqueous media [28 32]. In confirmation, Dynamic Light Scattering (DLS) measurements showed
that pure formed very small colloidal particles with a hydrodynamic radius of about 50 nm. The size
of these particles was very similar to those we have previously observed for the epimer Sulfavant R
) [12], thus proving that pure isomers have very similar biological and chemo-physical properties.
Notably, both compounds were 1000-fold more active than Sulfavant A ( ), which is composed of a
.3:1 mixture of 2 and 3.
In order to test the effect of the aggregation on the immunological response and to test our
1 and 2, Sulfavant S (3) gave a bell-shaped
–
3
(2
1
1
hypothesis on the reduction of the activity due to mixing of the two active epimers, we analyzed the
effect of different combinations of Sulfavant R and Sulfavant S on hDC maturation.
Sulfavant R (
effective in triggering CD83 expression than the pure molecules (Figure 3A). The addition of the S
epimer ( ) to the R epimer ( ) determined a linear decrease of the activity in the range from 100% to 40%
2) was slightly more active than Sulfavant S (3), but their mixtures were always less
3
2
of R. Further additions revert the response (20% and 0% of R) as expected for the formation of mixture
where the S epimer became progressively predominant. Furthermore, in complete agreement with
the response elicited by
by mixture of and with a 1.3:1 ratio, which is identical to the composition of Sulfavant A (
Figure 3B). On the whole, these experiments proved that the mixing ratio can significantly affect the
1, the immunomodulatory activity increased to micromolar concentrations
2
3
1)
(
activity of amphiphilic compounds. In the case of Sulfavants, there is an incredibly large difference
between potency of pure products and their mixture, thus determining erroneous evaluation of the
therapeutic potential.

Mar. Drugs 2020, 18, 451
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Figure 3. Effect of combination of
different combinations of and at an overall concentration of 0.1
.3:1 mixture of and at concentrations from 1 nM to 100 M; asterisks indicate significant differences
from untreated cells; *** p < 0.001, **** p < 0.0001.
2
and
3
on hDC maturation. (
A
) CD83+ DC cells triggered by
) CD83+ DC cells triggered by
2
3
µM; (
B
1
2
3
µ
In order to test the role of aggregation on changes of the biological activity of 1–3, we characterized
the suspension of pure products and their mixture in water by Dynamic Light Scattering (DLS)
(Figure 4).
Figure 4. Effect of combination of
Light Scattering.
2 and 3 on the hydrodynamic radius evaluated by means of Dynamic
DLS measures showed a constant increase of the aggregate size moving from Sulfavant R (
about 50 nm) to the 1:1 mixture of Sulfavant R ( ) and Sulfavant S ( ) (about 170 nm). In analogy with
the effect on the biological activity, further additions of compound shrank the particle up to 50 nm
of the pure Sulfavant S ( ). This surprising behavior was linked to the different way that Sulfavants
2)
(
2
3
3
3
self-organize in aqueous solution [12]. The evidence that almost identical bioactive molecules (like
epimers) could dramatically lose the biological activity as a result of their mixing represents a new
paradigm to be evaluated in the study of bioactive lipophilic substances. We have previously suggested
that the stability of the final aggregates has a relevant role in influencing the effective bioavailability
of free active molecules in equilibrium with self-aggregates [12]. The current results obtained with
a mixture of pure Sulfavant R (2) and Sulfavant S (3) prove, for the first time, that supramolecular
organization of mixture of active epimers in aqueous solutions can bias evaluation of their biological
and pharmacological potential.

Mar. Drugs 2020, 18, 451
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3
. Materials and Methods
NMR spectra were recorded on a Bruker Avance-400 (400.13 MHz) and on a Bruker DRX-600
equipped with a TXI CryoProbe in CDCl and in CDCl :CD OD 1:1 ( values referred to CHCl3
δ
3
3
3
and CH OH at 7.26 and 3.34 ppm respectively). HR-MS spectra were acquired by a Q-Exactive
3
Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA). TLC plates
(Kieselgel 60 F254) and silica gel powder (Kieselgel 60, 0.063–0.200 mm) were from Merck.
All the reagents were purchased from Sigma-Aldrich and used without any further purification.
DLS measurements were performed with a homemade instrument composed with a Photocor compact
goniometer, a SMD 6000 Laser Quantum 50 mW light source operating at 5325 Å, a photomultiplier
(PMT-120-OP/B) and a correlator (Flex02-01D, Correlator.com).
3
.1. Dynamic Light Scattering (DLS)
◦
0.05) C with temperature controlled through the use
Measurements were performed at (25.00
±
(2)
of a thermostat bath. In DLS, the intensity autocorrelation function, g (t), was measured for the
◦
instrument configuration corresponding to the scattering angle of 90 . The intensity autocorrelation
function is related to the electric field autocorrelation function through the Siegert relation. The electric
(
1)
field autocorrelation function, g (t), is defined as
+
∞
Z
ꢀ
ꢁ
t
τ
g(1)(t) =
τA(τ) exp − d ln τ
(1)
−∞
where
index of the solvent,
the relaxation rate
τ
= 1/
Γ
and q is the modulus of the scattering vector q = 4
λ
n /
λ
sin (
θ
/2), n = 1.33 is the refractive
0
0
λ
is the incident wavelength and
θ
represents the scattering angle. Evaluation of
2
Γ
distribution allows calculating the translational diffusion coefficient: D =
Γ
/q :
(J. B. a. R. Pecora, Dynamic Light Scattering: With Applications to Chemistry, Biology, and Physics,
Couvire Dover Publications, 2003.)
Inverse Laplace transforms were performed using a variation of the CONTIN algorithm
incorporated in Precision Deconvolve software. For spheres diffusing in a continuum medium
at infinite dilution, in the approximation of spherical objects, the diffusion coefficient is related to the
hydrodynamic radius, R , through the Stokes–Einstein equation:
h
kT
Rh =
(2)
6
πη D
0
where k is the Boltzmann constant, T is the absolute temperature and
η
0
= 0.89cP is the solvent viscosity.
For not spherical particles, R represents the radius of a spherical aggregate with the same diffusion
h
coefficient measured. In the present system, due to the high dilution, it is possible to consider the
approximation that η ꢀ η0, where
η represents the solution viscosity. In this hypothesis, Equation (2)
can be reasonably used to estimate the averaged hydrodynamic radius of the particles.
3
.2. Synthetic Procedures and Characterization of Intermediates 4–7, (S)-1,2-O-Distearoyl Glycerol and Pure
Sulfavant S (3)
Compound 4: Sodium hydride (0.235 g, 0.01 mol) was portion-wise added to (S)-(+)-1,2-
isopropylideneglycerol (0.6 g, 0.00457 mol) dissolved in THF (7.5 mL), and after 30 min of stirring
◦
benzyl bromide (0.85 g, 0.005 mol) was added; after 20 h at 60 C the mixture was evaporated and
purified by silica gel chromatography using a light petroleum ether/diethyl ether gradient to give
1
4
4
2
(1.0 g, 0.0045 mol, 94%); H-NMR (400 MHz, CDCl ):
δ
7.19–7.08 (5H, overlapped), 4.38 (2H, bs),
3
.12 (1H, m), 3.86 (1H, m), 3.58 (1H, m), 3.38 (1H, m), 3.30 (1H, m), 1.27 (3H, s), 1.20 (3H, s); HRESIMS m/z
45.1140 [M + Na] (calcd for C H O Na , 245.1148).
+
+
13
18
3

Mar. Drugs 2020, 18, 451
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Compound 5: Compound
Dowex H (7.3 g) was added; after stirring for 1.5 h the mixture was filtered and evaporated giving
4
(1.0 g, 0.0045 mol) was dissolved in methanol/water 95/5 (7 mL) and
+
1
compound
5
(0.762 g, 0.0042 mol, 93%); H-NMR (400 MHz, CDCl ):
δ
7.29–7.17 (5H, overlapped, Ph),
3
+
4
.58 (2H, bs), 3.86 (1H, m), 3.60 (1H, m), 3.52 (1H, m), 3.44 (2H, m); HRESIMS m/z 205.0829 [M + Na]
+
(calcd for C H O Na , 205.0835).
10 14 3
Compound 6: Compound
5 (0.762 g, 0.0042 mol) was dissolved in anhydrous dichloromethane (7 mL)
before addition of 1.1 equiv. of stearic acid, N,N’-dicyclohexylcarbodiimide (1.0 g, 0.008 mol) and
4
2
-dimethylaminopyridine (0.51 g, 0.0042 mol) under argon. The reaction mixture was stirred for 16 h at
5 C; after evaporation under reduced pressure, the mixture was purified by silica gel chromatography
◦
using a gradient of petroleum ether/diethyl ether to give compound
6 (2.76 g, 0.0039 mol, 92%) as
1
pale-yellow oil. H-NMR (400 MHz, CDCl ):
δ 7.29–7.17 (5H, overlapped, Ph), 5.28, (1H, m, H-2),
3
4
3
.57 (2H, CH Bn), 4.39 (1H, dd, = 3.76 and 11.8 Hz, H-1a), 4.23 (1H, dd, J = 6.4 and 11.8 Hz, H-1b),
2
.63 (2H, bd, J = 5.2 Hz, H-3), 2.33 (4H, m,
α
-methylene), 1.63 (4H, m,
β
-methylene), 1.28–1,32 (56H, m,
+
acyl chains CH ), 0.83 (6H, overlapped, acyl chains CH ); HRESIMS m/z 737.6062 [M + Na] (calcd for
C H O Na , 737.6054).
2
3
+
46
82
5
(
S)-1,2-O-distearoyl glycerol
:
Compound
6
(2.76 g, 0.0039 mol) was dissolved in THF/MeOH 1/1 (25 mL)
◦
and Pd-C (10%) (0.350 g) was added; after stirring for 16 h at 25 C the mixture was filtered, evaporated
and purified by silica gel chromatography using a light petroleum ether/ethyl acetate gradient to
1
give (S)-1,2-O-distearoyl glycerol (0.875 g, 0.0014 mol, 35%); H-NMR (400 MHz, CDCl ):
δ 5.1 (1H,
3
m, H-2), 4.32 (1H, dd, J = 4.4 and 11.9 Hz, H-1a), 4.23 (1H, dd, J = 5.8 and 11.9 Hz, H-1b), 3.72 (2H,
bd, J = 4.8 Hz, H-3), 2.33 (4H, m, -methylene), 1.63 (4H, m, -methylene), 1.21–1.35 (56H, m, acyl
chains CH ), 0.85 (6H, overlapped, acyl chains CH ); HRESIMS m/z 647.5591 [M + Na] (calcd for
α
β
+
2
3
+
C H O Na , 647.5585).
39
76
5
Compound 7: Peracetylated glucosyl-trichloroacetimidate (0.491 g, 0.001 mol) and (S)-1,2-O- distearoyl
glycerol (0.625 g, 0.001 mol) were dissolved in anhydrous dichloromethane (15 mL), and the solution
◦
was kept at 0 C; then, trimethylsilyl trifluoromethanesulfonate (TMSOTf) (35
µ
L in 1.5 mL of CH Cl )
2
2
was added dropwise. The reaction mixture was stirred on activated 3 Å molecular sieves under
◦
argon for 5 h at 0 C and quenched adding triethylamine (150
µL); after evaporation under reduced
pressure the crude product was purified by silica gel chromatography using a gradient of petroleum
1
ether/diethyl ether to give compound
7
(0.430 g, 0.00045 mol, 45%). H-NMR (300 MHz, CDCl ):
δ
5.19
3
(1H, m), 5.06 (1H, overlapped), 4.99 (1H, t, J = 9.5 Hz), 4.88 (1H, dd, J = 9.5 and 7.9 Hz), 4.52 (1H, d),
4.02–4.32 (4H, overlapped), 3.93 (1H, dd, J = 11.0 and 4.9 Hz), 3.68 (2H, m), 2.3 (4H, m,
α-methylenes)
1
.98–2.02 (12H, bt), 1.60 (4H, m,
β
-methylenes), 1.32–1.22 (56H, acyl chains CH ), 0.89 (6H, overlapped,
2
+
acyl chains CH ); HRESIMS m/z 977.6545 [M + Na] (calcd for C H O Na, 977.6541).
3
53 94 14
1
Sulfavant S (3): white solid; H-NMR (400 MHz, CD OD/CDCl 1/1):
δ values are referred to CHD OD
2
3
3
(
3.34 ppm and 49.0 ppm):
δ
5.28 (1H, m, H-2), 4.40 (1H, dd, J = 2.7, 12.0 Hz, H-1a), 4.31 (1H, d,
0
J = 7.6 H-1 ), 4.24 (1H, dd, J = 6.9, 12.0 Hz, H-1b), 4.05 (1H, dd, J = 5.4, 11.0 Hz, H-3a), 3.79–3.71 (3H,
H-3b, H-3 , H-4 ), 3.41 (1H, bt, J = 8.9 Hz, H-2 ), 3.26 (1H, overlapped, H-6 a), 3.25 (1H, overlapped,
0
0
0
0
0
0
H-5 ), 3.09 (1H, dd, J = 7.2, 15.7 Hz, H-6 b), 2.36–2.27 (4H,
.65–1.56 (4H, -methylenes of stearoyl portions), 1.36–1.20 (60H, aliphatic methylenes), 0.89 (6H, bt,
J = 6.0 Hz, 2CH ); C-NMR (100MHz, CD OD:CDCl 1:1):
α-methylenes of stearoyl portions),
1
β
13
δ
0
174.1, 173.7 (C, acyl esters of stearoyl part),
3
3
3
0
0
0
0
1
03.2 (CH, C1 ), 76.1 (CH, C2 ), 73.8 (CH, C5 ), 72.4 (CH, C3 ), 72.3 (CH, C4 ), 70.2 (CH, C2), 68.2 (CH ,
2
0
C3), 63.2 (CH , C1), 53.6 (CH , C6 ), 34.2 (CH ,
α
-methylene of stearoyl portion), 32.2–29.0 (CH2,
2
2
2
methylenes of stearoyl portion), 24.9 (CH2,
stearoyl portion); HRESIMS m/z 849.5772 [M-K] (calcd for C H O S , 849.5767).
β-methylene of stearoyl portion), 13.8 (CH , methyls of
3
-
-
45
85 12

Mar. Drugs 2020, 18, 451
9 of 11
3
.3. Human Monocyte-Dendritic Cell Differentation
For each assay, human peripheral blood mononuclear cells were isolated from two healthy
donors by standard Ficoll density gradient centrifugation. Monocytes were purified from human
peripheral blood mononuclear cells using MACS CD14 microbeads (Miltenyi Biotech, Auburn, AL,
USA) according to the manufacturer’s recommendation. Purity was checked by staining with a
FITC-conjugated anti-CD14 antibody (Milteny Biotech) and FACS analysis and was routinely found to
6
−1
be greater than 98%. Immature DCs were obtained by incubating monocytes at 1
×
10 cells mL in
an RPMI 1640 medium supplemented with 10% fetal calf serum, 1% L-glutamine 2mM, 1% penicillin
and streptomycin, human IL-4 (5 ng/mL), and human GM-CSF (100 ng/mL) for five days.
3
.4. Cells Staining and Stimulation
After five days in culture, surface staining was performed on monocyte-derived dendritic cells
6
(
moDCs) for flow cytometry analysis. moDCs (0.8
×
10 cell/well) were then incubated with synthetic
−1
compounds in 12 wells at the reported concentrations. Stimulation with PAM2CSK4 1
Invivogen) was used as positive control. After 24 h, expression of all surface markers was estimated
µg mL
(
by using the following conjugated mAbs from Miltenyi Biotec: HLA-DR FITC, CD83 PE and CD86
APC, and analyzed by a flow cytometer (BD ACCURI, BD Bioscience, Milano, Italy) according to
standard protocol.
3
.5. Statistical Analysis
All data were analyzed by one-way ANOVA, followed by the Tukey test for multiple comparisons.
A p-value less than 0.05 was considered statistically significant. All analyses were performed using the
GraphPad Prism 8.00 for Windows software (GraphPad Software, San Diego, CA, USA).
3
.6. Characterization of Colloid Nanoparticles
After purification by HPLC, samples were prepared in 1 mL of Millipore water at 0.1 mM of
◦
each compound
1
–
3
and mixtures
2
and
3
. After sonication for 30 min at 35 C, the solutions were
◦
maintained at room temperature (20 C) for 24 h. The mean diffusion coefficients were obtained as an
◦
average of at least three measurements at 25 C.
4
. Conclusions
Sulfavants are bioactive molecules able to induce DC maturation and trigger an adaptive immune
response in vivo 13 15]. The activity of these products is largely dependent on the formation of colloidal
particles under aqueous conditions [12]. Mixing diastereoisomers and , both active at nanomolar
[
–
2
3
concentrations in pure form, determined a consistent decrease of the biological response in agreement
with formation of supramolecular aggregates of different size and stability. Multiple equilibria between
free active monomers and different aggregates control the concentration of the products able to bind
the cellular targets, thus affecting the results of biological tests and accuracy of the evaluation of the
therapeutic potential. In our opinion, this behavior is common to many other lipophilic or amphiphilic
compounds, such as natural products, lipopeptides and glycolipids, whose activity can be significantly
altered by supramolecular self-assembly in aqueous media. This effect could be more relevant in
in vitro assays than in vivo trials since the presence of proteins and other molecules in the body fluids
can reduce the tendency of these compounds to aggregate. Sulfavant S (3) was synthesized by a
modified protocol involving the preparation of (S)-1,2-O-distearoylglycerol as an alternative acceptor of
the glucosyl-trichloroacetimidate donor. This approach allows to overcome the technical issues arising
by the low stereocontrol of the coupling reaction and represents a starting point for the preparation of
other members of this new family of immunomodulatory agents.
Author Contributions: E.M. and A.F. conceived and coordinated the study. L.F., M.Z., G.N. and G.D. carried
out the experimental works and data analysis. L.F., A.B. and A.F. (Antonio Fabozzi) DLS analysis. R.D.P. and

Mar. Drugs 2020, 18, 451
10 of 11
C.G. performed biological tests. E.M. and A.F. (Angelo Fontana) wrote the manuscript with contributions of all
coauthors. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded in the frame of the project ADViSE Antitumor Drugs and Vaccines from the
SEa approved by Campania with D.D 403 of 12/11/2018 and integration D.D: n.422 of 16/11/2018.
Acknowledgments: E.M. and A.F. thank BioSEArch SRL for the generous support; moreover, the authors wish
to thank European Union (FSE, PON Ricerca e Innovazione 2014–2020, Azione I.1 “Dottorati Innovativi con
caratterizzazione Industriale”) for funding a Ph.D. grant to one of the authors (Laura Fioretto).
Conflicts of Interest: The authors declare no conflict of interest.
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β
β
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(