Synthesis and biological evaluation of chemical tools for the study of Dolichol Linked Oligosaccharide Diphosphatase (DLODP)

Article abstract of DOI:10.1016/j.ejmech.2016.10.013

Citronellyl- and solanesyl-based dolichol linked oligosaccharide (DLO) analogs were synthesized and tested along with undecaprenyl compounds for their ability to inhibit the release of [³H]OSP from [³H]DLO by mammalian liver DLO diphosphatase activity. Solanesyl (C45) and undecaprenyl (C55) compounds were 50–500 fold more potent than their citronellyl (C10)-based counterparts, indicating that the alkyl chain length is important for activity. The relative potency of the compounds within the citronellyl series was different to that of the solanesyl series with citronellyl diphosphate being 2 and 3 fold more potent than citronellyl-PP-GlcNAc₂and citronellyl-PP-GlcNAc, respectively; whereas solanesyl-PP-GlcNAc and solanesyl-PP-GlcNAc₂were 4 and 8 fold more potent, respectively, than solanesyl diphosphate. Undecaprenyl-PP-GlcNAc and bacterial Lipid II were 8 fold more potent than undecaprenyl diphosphate at inhibiting the DLODP assay. Therefore, at least for the more hydrophobic compounds, diphosphodiesters are more potent inhibitors of the DLODP assay than diphosphomonoesters. These results suggest that DLO rather than dolichyl diphosphate might be a preferred substrate for the DLODP activity.

Full text of DOI:10.1016/j.ejmech.2016.10.013

European Journal of Medicinal Chemistry 125 (2017) 952e964
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and biological evaluation of chemical tools for the study of
Dolichol Linked Oligosaccharide Diphosphatase (DLODP)
a
,
, 2
Michael Bosco 1, Ahmad Massarweh b, Soria Iatmanen-Harbi b, Ahmed Bouhss c
,
€
Isabelle Chantret b, Patricia Busca a, Stuart E.H. Moore b, Christine Gravier-Pelletier a
Universite Paris Descartes, CICB-Paris, CNRS UMR 8601, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, 45 rue des Saints-Peres,
,
*
a
ꢀ
ꢁ
75006, Paris, France
b
ꢀ
Universite Paris Diderot, INSERM U1149, 16 rue Henri Huchard, 75018, Paris, France
c
ꢀ
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Universite Paris-Saclay, 91198, Gif-sur-Yvette, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Citronellyl- and solanesyl-based dolichol linked oligosaccharide (DLO) analogs were synthesized and
tested along with undecaprenyl compounds for their ability to inhibit the release of [3H]OSP from [3H]
DLO by mammalian liver DLO diphosphatase activity. Solanesyl (C45) and undecaprenyl (C55) com-
pounds were 50e500 fold more potent than their citronellyl (C10)-based counterparts, indicating that
the alkyl chain length is important for activity. The relative potency of the compounds within the cit-
ronellyl series was different to that of the solanesyl series with citronellyl diphosphate being 2 and 3 fold
more potent than citronellyl-PP-GlcNAc2 and citronellyl-PP-GlcNAc, respectively; whereas solanesyl-PP-
GlcNAc and solanesyl-PP-GlcNAc2 were 4 and 8 fold more potent, respectively, than solanesyl diphos-
phate. Undecaprenyl-PP-GlcNAc and bacterial Lipid II were 8 fold more potent than undecaprenyl
diphosphate at inhibiting the DLODP assay. Therefore, at least for the more hydrophobic compounds,
diphosphodiesters are more potent inhibitors of the DLODP assay than diphosphomonoesters. These
results suggest that DLO rather than dolichyl diphosphate might be a preferred substrate for the DLODP
activity.
Received 27 July 2016
Received in revised form
19 September 2016
Accepted 7 October 2016
Available online 8 October 2016
Keywords:
CDG
Diphosphatase
Phosphosugars
Disubstituted diphosphates
Biological evaluation
Glycochemistry
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
mutations in genes encoding proteins of the dolichol cycle lead to
type I congenital disorders of glycosylation (CDG-I) [2], a group of
N-glycans play crucial roles in cell growth, differentiation and
communication [1]. Protein N-glycosylation occurs by transfer of
Glc3Man9GlcNAc2, from dolichol-linked oligosaccharide (DLO,
Glc3Man9GlcNAc2-PP-dolichol), onto polypeptides containing an
Asn residue in the Asn-X-Ser/Thr glycosylation sequon. Dolichyl-
diphosphate, the by-product of this reaction, is recycled into DLO.
This sequence of reactions constitutes the dolichol cycle, and
because dolichol-P (DolP) is rate limiting for protein glycosylation,
its interruption leads to hypoglycosylation of glycoproteins. In man,
rare inherited diseases, manifesting multisystemic clinical pictures,
whose hallmark is the presence of hypoglycosylated serum glyco-
proteins [3]. Of particular interest for the study of these diseases are
DLO regulation and the fate of truncated DLO intermediates often
seen in CDG-I. In fact, data show that truncated DLO species are
cleaved by a DLO diphosphatase (DLODP), to yield DolP and
oligosaccharyl-phosphates (OSP) in cells derived from CDG-I pa-
tients [4,5] and it has been hypothesized that such a mechanism
may restrict truncated DLO accumulation while at the same time
allowing DolP recycling [6]. In order to understand the role of a
recently described Co2þ-dependent DLODP activity [7,8] (Fig. 1) we
initiated a chemistry program aimed at generating chemical tools
that are required for DLODP characterization.
* Corresponding author.
E-mail address: christine.gravier-pelletier@parisdescartes.fr (C. Gravier-
Pelletier).
In particular, we were interested in defining the structural ele-
ments required for molecules to interact with DLODP. Thus,
the synthesis of simplified DLO analogs (Fig. 2) has been
carried out taking the simplest GlcNAc-diphosphoryl-dolichol
1
Present address: Normandie Univ, UNIROUEN, INSA Rouen, CNRS, COBRA (UMR
6014), 76000 Rouen, France.
2
ꢀ
ꢀ
Present address: Laboratoire Structure-Activite des Biomolecules Normales et
ꢀ
Pathologiques (SABNP), INSERM UMRS1204 and Universite Evry-Val d'Essonne,
Evry, France.
http://dx.doi.org/10.1016/j.ejmech.2016.10.013
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

M. Bosco et al. / European Journal of Medicinal Chemistry 125 (2017) 952e964
953
Additionally, the related monophosphates (R1-O-P or R2-O-P)
were also prepared to assist the identification of the reaction
products. Finally, the synthesis of citronellyl medronate in which a
methylene group replaces the central oxygen atom of the diphos-
phate moiety has also been achieved as a non-cleavable substrate.
Results concerning the biochemical characterization of the Co2þ
-
dependent DLODP have recently been published [7]. Here we report
the full results dealing with the efficient synthesis of these complex
compounds in pure form and the complete comparison of their
inhibition of DLODP activity.
Fig. 1. Role of the DLODP.
2. Results and discussion
(GlcNAc-PP-dolichol, R1O-P-O-P-OR2) as a model. In order to
investigate the relative importance of each part of the DLODP
substrate, the R1 position has been substituted with either a GlcNAc
residue or a di-N-acetylchitobiose moiety, which occur in the nat-
ural substrate, and the dolichol residue at the R2 position has been
replaced by the shorter citronellyl or solanesyl moieties. Such R2
groups were intended to probe the possible requirement of the
diphosphatase activity for long polyprenyl chains. Furthermore, to
evaluate the importance of the sugar chain for recognition by
DLODP, the corresponding citronellyl and solanesyl diphosphates
have also been synthesized (R1 ¼ H).
2.1. Chemistry
The preparation of the targeted disubstituted diphosphates A
has been envisaged through the coupling of the corresponding
phosphosugars B and phosphodolichol mimics C (Fig. 3).
The peracetylated GlcNAc 1 (Scheme 1) was prepared from
commercially available GlcNAc by treatment with acetic anhydride
in excess in pyridine and the pure
yield after flash chromatographic purification. Peracetylated
GlcNAc2 2 was obtained by acetolysis of chitin by H2SO4 and acetic
a anomer was isolated in 89%
Fig. 2. Structure of DLO and of the targeted compounds.

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M. Bosco et al. / European Journal of Medicinal Chemistry 125 (2017) 952e964
mixture of THF and CH3CN was followed by oxidation with
hydrogen peroxide to give 5 [12] and 6 [13] in 83% and 78% yield,
respectively. Then, benzyl esters cleavage by hydrogenolysis in the
presence of 10% Pd/C in EtOH for 5 and in a mixture of EtOH/CH2Cl2
for 6 gave the corresponding phosphates that were quantitatively
isolated as their mono-triethylammonium salts 7 and 8. Meth-
anolysis of the acetates on the saccharidic units by sodium meth-
ylate in MeOH afforded the sodium salts of GlcNAc and GlcNAc2
phosphates 9 and 10 in 94% and 97% yields.
We next turned to the preparation of citronellyl phosphate 11,
diphosphate 12 and medronate 13, starting from (S)-citronellol
(Scheme 2). Phosphorylation of citronellol was carried out ac-
cording to L. L. Danilov et al. [14] by an excess of POCl3 and Et3N in
CH2Cl2 (path a). After dissolution of the crude in a 9/1 acetone/
water mixture at 0 ꢀC, cyclohexylamine addition afforded cit-
ronellyl phosphate as its dicyclohexylammonium salt 11 in 33%
yield after recrystallization in acetone/water (9/1). On another
hand (path b), activation of citronellol by tosyl chloride in the
presence of DMAP in CH2Cl2 furnished the intermediate tosylate
which was reacted with either tris(tetrabutylammonium)hydrogen
diphosphate or medronate in CH3CN to afford, after reverse phase
HPLC purification, the triammonium salts of citronellyl diphos-
phate 12 and medronate 13.
Fig. 3. Retrosynthetic analysis.
anhydride under sonication conditions [9]. Flash chromatographic
purification of the crude permitted us to isolate pure compound 2
in 5% yield and also to recover a small amount of compound 1 (1%
isolated yield). The selective deprotection of the anomeric acetate
of compound 1 and 2 was performed by treatment with ammo-
nium acetate in the presence of diethylamine in DMF [10] leading to
the hemiacetals 3 and 4 in 81% and 47% yield, respectively. The
phosphoramidite-oxidation strategy [11] was chosen for the gly-
cosylphosphates 5 and 6 synthesis. The preparation of the inter-
mediate phosphites by reaction of compounds 3 and 4 with
The solanesol was phosphorylated with smoother conditions
dibenzyl N,N-diethylphosphoramidate and 1H-tetrazole in
a
Scheme 1. a) Ac2O, pyridine, 89% b) Ac2O, H2SO4, 35 ꢀC, 14 h, 1% for 1, 5% for 2. c) CH3COONH4, DIEA, DMF, RT, 3 days, 81% for 3, 47% for 4, d) i. dibenzyl N,N-diethylphosphoramidate,
1H-tetrazole, THF, CH3CN, RT, 14h; ii. H2O2, -78 ꢀC to RT, 1 h, 83% for 5, 78% for 6. e) H2, Pd/C 10%, EtOH, then triethyamine, quant. for 7, H2, Pd/C 10%, EtOH, CH2Cl2, then triethylamine
quant. for 8. f) MeONa, MeOH, RT, 1 h, 94% for 9, 14 h, 97% for 10.

M. Bosco et al. / European Journal of Medicinal Chemistry 125 (2017) 952e964
955
microsomes in the presence of Co2þ at pH 5.5. Enzyme activity is
quantitated by expressing the radioactivity associated with the [3H]
OSP generated as a fraction of the total radioactivity recovered from
the incubations (non-hydrolysed [3H]DLO, [3H]OSP and neutral [3H]
oligosaccharides and [3H]mannose). In order to gain insight into the
physiological susbstrates for this activity, we screened compounds
for their ability to interfere with the above-described assay. First,
commonly occurring water soluble phospho-compounds were
tested (AMP, ADP, ATP, NADP, GDP-Man, UDP-GlcNAc, UDP-Glc),
and it was found that such structures had IC50 concentrations in
the low millimolar range, which is over 1000 fold higher than the
substrate concentration. An example of such a compound is
GlcNAc-1P 9 that corresponds to the simplest OSP that could be
hydrolysed from a DLO (GlcNAc-PP-dolichol) by DLODP, which has
an IC50 of greater than 3 mM in the DLODP assay (Table 1).
Nevertheless, this compound proved useful for examining DLODP
activity in microsomes generated from different sources.
Scheme 2. a) POCl3, Et3N, CH2Cl2, RT, 1 h, then acetone/Et3N/water, RT, 14 h, recrys-
tallization with cyclohexylamine, 33%. b) TsCl, DMAP, CH2Cl2, RT, 14 h, 65%. c) tris(te-
trabutylammonium) hydrogen diphosphate or medronate, CH3CN, RT, 18 h, then
reverse phase HPLC, 12: 38%, 13: 37%.
As shown in the upper panels A of Fig. 4, microsomes generated
from the human hepatocellular carcinoma HepG2 cell line, after 3, 8
and 16 days in culture, generate uniquely OSP from DLO. By contrast
(upper panels B), microsomes derived from 3, 8 and 16 day cultures
of the TC7 clone of the human colon carcinoma Caco-2 cell line
yield both neutral oligosaccharides (nfOS) and OSP as a function of
cell growth. Microsomes derived from early growth cells (day 3)
generate predominantly OSP whereas at day 16, the microsomes
yield predominantly nfOS. At the intermediate growth time (day 8),
cells yield microsomes whose capacity to hydrolyse DLO is almost
two fold higher than those generated from early or late growth
cells.
Because the increase in nfOS is at the expense of OSP between
the 15 and 20 min incubation time points, it would appear that
nfOS are not being liberated directly from DLO, but rather by
dephosphorylation of DLODP-generated OSP. This was underlined
when DLODP assays were conducted in the presence of increasing
concentrations of GlcNAc-1P (9). As shown in the middle panel of
Fig. 4B, at low concentrations this compound inhibits nfOS pro-
duction while increasing OSP production and at higher concen-
trations begins to inhibit OSP production as well. The DLODP
activity found in microsomes from HepG2 cells is also inhibited at
higher GlcNAc-1P (9) concentrations (middle panel of Fig. 4A).
These data are consistent with OSP dephosphorylation during
DLODP assays using Caco-2 cell microsomes. This hypothesis is
underlined by the fact that during Caco-2, but not HepG2, cell
growth (lower panels in Fig. 4A and B), there is a differentiation-
dependent expression of cell surface alkaline phosphatase, which
is known to be able to dephosphorylate sugar 1 phosphates.
(Scheme 3) because allylic phosphates are known to easily undergo
elimination side-reactions. Thus, the treatment of solanesol with
Cl3CCN and tetrabutyl ammonium dihydrogen phosphate in excess
in CH2Cl2 afforded a one pot activation e phosphorylation process
[15] that leads to either the monophosphate 14 or diphosphate 15
depending on the reaction duration (Scheme 3). Accordingly, after
one hour of reaction, the solanesyl phosphate 14 could be obtained
in 33% yield after purification by flash chromatography in
dichloromethane/methanol/14% aqueous ammonia 80/18/2 (v/v/v),
while the diphosphate synthesis was achieved in 14 h of reaction
and its purification required DEAE anion exchange chromatography
to give the expected compound 15 in 27% yield.
The synthesis of diphosphates 16e19 was achieved by the
coupling of phosphosugars 7 and 8 with the phosphorimidazolides
intermediates [16] in situ generated from the phosphodolichol
mimics 11 and 14 (Scheme 4). Typical conditions involved careful
drying the partners separately by co-evaporation with toluene and
triethylamine followed by treatment of phospholipid 11 or 14 with
1,10-carbonyldiimidazole in CH2Cl2 to give the phosphor-
imidazolides [17]. Condensation of the resulting crude phosphor-
imidazolides onto phosphosugar 7 or 8 followed by acetates
methanolysis afforded the corresponding diphosphates that were
purified by flash chromatography on silica gel or by reverse phase
HPLC to afford the pure targeted diphosphates 16e19 in yield
varying between 12 and 30% over three steps from the dolichol
phosphate mimics 11 or 14.
2.2. Biological studies
2.2.1. GlcNAc-1P blocks phosphomonoesterase activity in the
DLODP assay
2.2.2. Biological testing of water-soluble citronellyl compounds and
solanesyl compounds in the DLODP assay
Previously, DLODP has been assayed by incubating detergent
Next, the citronellyl- and solanesyl-based compounds were
tested for their ability to interfere with the standard DLODP
suspended Glc3-0[3H]Man9-5GlcNAc2-PP-dolichol with hepatocyte
Scheme 3. a) Tetrabutylammonium dihydrogen phosphate, CCl3CN, CH2Cl2, 1 h, RT, 33% for 14 or 14 h, RT, 27% for 15.

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M. Bosco et al. / European Journal of Medicinal Chemistry 125 (2017) 952e964
Scheme 4. a) i. Co-evaporation with Et3N and toluene; ii. CDI, THF, 1.5 h from 11 or CDI, CH2Cl2, 2 h from 14. b) RT, THF, 12 h for 16, RT, DMF, 12 h for 17, RT, DMF, 6 days for 18, RT,
pyridine, 7 days for 19. c) MeONa, MeOH, RT then DOWEX 50WX8, Hþ form.
Table 1
3 fold more potent than the diphosphodiesters citronellyl-PP-
GlcNAc2 17 and citronellyl-PP-GlcNAc 16, respectively. It is note-
worthy that citronellyl medronate 13 is 7 fold less active than the
diphosphate 12, suggesting that the observed interference of the
DLODP assay by the latter compound is not entirely related to its
physicochemical properties. The decreased potency of the diphos-
phodiesters 16/17 compared to that of the diphosphomonoester 12,
indicates that the physiological susbstrate of the DLODP activity
may be a diphosphomonoester, like dolichyl-diphosphate rather
than a diphosphodiester like DLO. However, this was not the case
for the solanesyl-based compounds. Here (solanesyl-diphospho-
GlcNAc2 19 > solanesyl-diphospho-GlcNAc 18 > solanesyl-diphos-
phate 15 > solanesyl-phosphate 14), the diphosphodiesters 18/19
were 4 and 8 fold more potent, respectively, than the diphosphate
15. In order to get further information on this point, a series of
undecaprenyl (C55)-based compounds was tested. Results shown
in the lower panel of Fig. 5 and Table 1 demonstrate that the ability
of these compounds to interfere with the DLODP assay is similar to
that of the solanesyl-based compounds. In addition, the relative
potency of these compounds (undecaprenyl-diphospho-MurNAc(-
pentapeptide)GlcNAc/undecaprenyl-diphospho-
Effects of compounds on DLODP activity in HepG2 membranes.
Compound
Number
IC50 (mM)a
Monophosphates
Propyl phosphate
ni at 3.0 mM
nd > 3.0 mM
4.2
0.3
0.0078
GlcNAc-1-phosphate
p-nitrophenyl phosphate
Citronellyl phosphate
Solanesyl phosphate
Undecaprenyl phosphateb
Diphosphates
9
11
14
~0.010
Diphosphate
Medronate
nd > 3.0 mM
ni at 3.0 mM
0.1
0.7
0.0047
Citronellyl diphosphate
Citronellyl medronate
Solanesyl diphosphate
Undecaprenyl diphosphateb
Phosphodiesters
12
13
15
0.0040
Bis(p-nitrophenyl) phosphate
Uridinyl-PP-GlcNAc
1.2
ni at 3.0 mM
0.2
0.3
0.0012
0.0005
0.0005
Citronellyl-PP-GlcNAc
Citronellyl-PP-GlcNAc2
Solanesyl-PP-GlcNAc
Solanesyl-PP-GlcNAc2
Undecaprenyl-PP-GlcNAcc
Undecaprenyl-PP-MurNAc
(pentapeptide)-GlcNAc (Lipid II)d
16
17
18
19
GlcNAc
> undecaprenyl-diphosphate > undecaprenyl-mono-
phosphate), again shows that the diphosphodiesters interfere
better with the assay than the diphosphomonoester.
9
0.0005
a
OSP release from DLO was measured as described in Experimental Procedures.
Where possible, the concentration of inhibitor causing 50% inhibition of OSP gen-
eration was determined (IC50). In some cases, no inhibition was noted at the con-
centration indicated (ni). In other cases, inhibition was observed, but the maximum
concentrations used did not allow determination of IC50 values (nd).
2.2.3. Further examination of the importance of the R1 moiety for
inhibition of the DLODP assay
b
Undecaprenyl phosphate and undecaprenyl diphosphate were provided by the
Inhibition of the DLODP assay by the solanesyl and undecap-
renyl compounds indicates that the R2 moiety has a role to play.
There are several possibilities for this phenomenon. First, a hy-
drophilic headgroup may simply help physically orientate the
diphosphate moiety of these amphiphilic compounds in their lipid/
detergent milieu for access to the enzyme. Second, the mono-
saccharides in close proximity to the diphosphate moiety may
directly interact with the enzyme and therefore promote substrate
binding. The potential role of mannose and GlcNAc residues of DLO
in recognition by DLODP was addressed by evaluating the capacity
of various monosccharides/disaccharides to interfere with the
Institute of Biochemistry and Biophysics of the Polish Academy of Sciences, Warsaw,
Poland.
c
Undecaprenyl-PP-GlcNAc was produced by enzymatic synthesis, using the
WecA enzyme [18,19].
d
Lipid II (Undecaprenyl-PP-MurNAc(-pentapeptide)-GlcNAc), was produced by
enzymatic synthesis, using the purified MraY and MurG enzymes as previously
described [20,21].
assay. Data shown in Fig. 5 and Table 1 demonstrate that the
former compounds were 50e500 fold less potent than their sol-
anesyl counterparts. Furthermore, the relative potency of the
compounds within each series was different. For the citronellyl-
based products (citronellyl diphosphate 12 > citronellyl-diphos-
DLODP assay. As shown in Fig. 6, at 50 mM, mannose, a-methyl
mannoside and N- acetylglucosamine did not inhibit OSP
pho-GlcNAc 16
>
citronellyl-diphospho-GlcNAc2 17/citronellyl
generation.
phosphate 11 > citronellyl medronate 13), the diphosphate is 2 and
Although 50 mM
b-methyl mannoside isopropylate caused a

M. Bosco et al. / European Journal of Medicinal Chemistry 125 (2017) 952e964
957
Fig. 4. DLODP activity as a function of cell growth and differentiation. HepG2 (A) and
Caco-2 (B) cells were cultivated for 3, 8 and 16 days and total cell membranes were
prepared as described in Experimental Procedures. Upper panels. DLODP assays were
performed under standard conditions using the indicated amounts of membrane
protein, and radioactivity associated with OSP and nfOS was quantitated by scintilla-
tion counting. Middle panels. DLODP assays using 20 mg membrane protein derived
from cultivated for 8 days were performed in the standard incubation mixture sup-
plemented with the indicated amounts of GlcNAc-1P (9), and radioactivity associated
with OSP and nfOS was quantitated. Lower panels. Alkaline phosphatase was measured
in the membrane preparations described above.
Fig. 5. Inhibition of DLODP assay by different citronellyl and solanesyl compounds.
Using 40 nM Glc3-0[3H]Man5GlcNAc2-PP-dolichol as substrate, standard DLODP assays
were conducted in the presence of different concentrations of the indicated compounds.
Dose response curves were generated and the IC50 values are reported in Table 1.
Concerning the biological activity of solanesyl compounds (shown in the middle panel),
this research was originally published in the Journal of Lipid Research [7] A. Massarweh,
M. Bosco, S. Iatmanen-Harbi, C. Tessier, N. Auberger, P. Busca, I. Chantret, C. Gravier-
Pelletier, S. E. H. Moore, Demonstration of an oligosaccharide-diphosphodolichol
diphosphatase activity whose subcellular localization is different than those of
dolichyl-phosphate-dependent enzymes of the dolichol cycle, Journal Lipid Res. 57
10% inhibition of OSP generation, the same effect was obtained with
50 mM isopropanol alone, suggesting that
itself has no effect on OSP production at this concentration.
GlcNAc( 1,4)GlcNAc caused < 10% inhibition of the reaction,
whereas at the same concentration Man( 1,4)GlcNAc provoked a
30% inhibition of OSP release from DLO. By contrast, the Gal( 1,4)
b-methyl mannoside
b
b
b
©
(2016) 1029e1042 the American Society for Biochemistry and Molecular Biology.
GlcNAc disaccharide, which does not occur in DLO, did not provoke
a statistically significant reduction in DLDOP action. Accordingly
the ensemble of this data indicates that the Man(b1,4)GlcNAc motif
3. Conclusion
and to a lesser extent the di-N-acetylchitobiose motif interfere with
the DLODP assay. These observations suggest that the sugar moiety
of DLO may have a role to play in substrate recognition by the OSP-
generating activity.
Two series of citronellyl- and solanesyl-based DLO analogs
containing, or not, a mono- or di-saccharidic GlcNAc moiety were

958
M. Bosco et al. / European Journal of Medicinal Chemistry 125 (2017) 952e964
recorded with a TOF mass analyzer under electrospray ionization
(ESI) in positive or negative ionization mode detection, atmo-
spheric pressure chemical ionization. All reactions were carried out
under a argon atmosphere, and were monitored by thin-layer
chromatography with Merck 60F-254 precoated silica (0.2 mm)
on glass. Flash chromatography was performed with Merck Kie-
selgel 60 (200e500
reverse phase purification were done on a STABILITY C18 100 Å
column; 5
mm); the solvent systems were given v/v HPLC
m
m; 250 ꢁ 20 mm at a flow rate 4 mL/min in the indi-
cated solvents.
4.1.1. 2-Deoxy-2-acetamido-4-O-(2-deoxy-2-acetamido-30,40,60-tri-
O-acetyl-
2
b-D-glucopyranosyl)-1,3,6-tri-O-acetyl-a-D-glucopyranose
To a suspension of chitin (15 g) in acetic anhydride (150 mL,
1.59 mol) was added dropwise concentrated sulfuric acid (15 mL,
4.13 mmol). The reaction mixture was sonicated for 3 h. The
resulting suspension was warmed at 35 ꢀC for 14 h then poured in
ice (200 g). The pH of the solution was adjusted at 7 with sodium
acetate (120 g). The precipitate was separated by centrifugation and
the supernatant was extracted four times with dichloromethane
(100 mL). The combined extracts were washed with a saturated
aqueous solution of NaHCO3 (400 mL), water (400 mL), dried over
Na2SO4, filtered and concentrated in vacuo. Purification of the res-
idue by column chromatography (silica gel 300 g, EtOAc) afforded
peracetate 1 (347 mg, 0.89 mmol, 1%) as a white solid and per-
acetate 2 (1.2 g, 1.77 mmol, 5%) as a white solid. Compound 2 [22]:
Fig. 6. Inhibition of DLODP assay by different mono- or disaccharides. Using Glc3-0[3H]
Man5GlcNAc2-PP-dolichol as substrate, standard DLODP assays were conducted in the
presence of 50 mM of the indicated compounds. The abbreviations are: Gal; galactose,
GlcNAc; N-acetylglucosamine, Man; mannose,
aMeMan; a-methyl mannoside, bMe-
Man; -methyl mannoside. For each compound, inhibition values were determined at
b
least in triplicate (n) and the mean and standard error of the mean are indicated. The
dotted line indicates the DLODP activity observed in the absence of inhibitors.
synthesized according to appropriate routes. Glycosyl phosphates
were obtained by the phosphoramidite-oxidation strategy from
peracetylated GlcNAc or GlcNAc2 derivatives, while citronellyl
phosphate was efficiently obtained by treatment of citronellol with
phosphorus oxychloride. The corresponding diphosphate resulted
from displacement of the tosylate with hydrogen diphosphate salt.
The allylic solanesyl mono and diphosphate were advantageously
prepared in milder conditions involving a one pot activation e
phosphorylation process. Finally, the coupling of glycosyl phos-
phates with the phosphorimidazolides derived from citronelly and
solanesyl phosphates afforded the DLO mimics. The resulting
compounds were tested for their ability to inhibit the release of [3H]
OSP from [3H]DLO. The solanesyl compounds were 50e500 fold
more potent than their water-soluble citronellyl-based counter-
parts. These data demonstrate the importance of the alkyl chain
(R2) length of the compounds for interfereing with the DLODP
assay. The role of the glycan (R1) moiety is less clear, but at least for
compounds containing the long chain R2 moieties, the diphos-
phodiesters inhibit the reaction more efficiently than the diphos-
phomonoesters. How the R1 substituents promote better inhibition
of the DLODP assay will have to await further investigation.
Nevertheless these compounds are being used for the character-
ization of OSP-generating systems from various sources.
[
a
]D þ 29.5 (c 1.0, CHCl3); [
a
]D þ 29.2 (c 1.0, CHCl3); 1H NMR
(CDCl3)
d
6.09 (d, J ¼ 3.6 Hz, 1H, H-1), 5.96 (d, J ¼ 9.1 Hz, 1H, NHAc*),
5.65 (d, J ¼ 9.1 Hz,1H, NHAc), 5.22 (dd, J ¼ 11.0, 9.0 Hz,1H, H-3), 5.13
(dd, J ¼ 10, 9.1 Hz, 1H, H-3*), 5.05 (t, J ¼ 10 Hz, 1H, H-4*), 4.48 (d,
J ¼ 8.4 Hz, 1H, H-1*), 4.44 (dd, J ¼ 12.2, 3.3 Hz, 1H, H-6), 4.40e4.32
(m, 2H, H-2, H-6*), 4.18 (dd, J ¼ 12.2, 2.4 Hz, 1H, H-60*), 4.02 (dd,
J ¼ 12.2, 2.2 Hz, 1H, H-60), 3.95 (dt, J ¼ 9.1, 8.4 Hz, 1H, H-2*), 3.89
(ddd, J ¼ 10.0, 3.3, 2.2 Hz, 1H, H-5), 3.74 (broad dd, J ¼ 10.0, 9.0 Hz,
1H, H-4), 3.62 (ddd, J ¼ 10, 4.1, 2.4 Hz, 1H, H-5*), 2.18 (s, 3H, CH3CO),
2.14 (s, 3H, CH3CO), 2.08 (s, 3H, CH3CO), 2.05 (s, 3H, CH3CO), 2.01 (s,
3H, CH3CO), 2.00 (s, 3H, CH3CO), 1.95 (s, 3H, CH3CO), 1.92 (s, 3H,
CH3CO); 13C NMR (CDCl3)
d 171.6, 171.4, 170.9, 170.6, 170.5, 170.2,
169.4, 169.0 (8C, COCH3), 101.9 (C-1*), 90.7 (C-1), 76.1 (C-4), 72.7 (C-
3*), 72.1 (C-5*), 70.9 (C-5), 70.8 (C-3), 68.1 (C-4*), 61.9 (C-6*), 61.6
(C-6), 54.7 (C-2*), 51.3 (C-2), 23.3, 23.2, 21.1, 21.1, 20.8, 20.7, 20.7,
20.7 (8C, CH3CO); ESI-MS: Calcd for C28H41N2O17: 677.2. [MþH]þ
Found: 677.2; ESI-HRMS: Calcd for C28H41N2O17: 677.2400. [MþH]þ
Found: 677.2393.
4.1.2. 2-Deoxy-2-acetamido-3,4,6-tri-O-acetyl-
a-D-glucopyranose
3 and 2-deoxy-2-acetamido-4-O-(2-deoxy-2-acetamido-30,40,6’-tri-
4. Experimental
O-acetyl-
4
b-D-glucopyranosyl)-3,6-di-O-acetyl-a-D-glucopyranose
4.1. Chemical synthesis
A solution of peracetate 1 (5 g, 12.8 mmol) in a mixture of dry
DMF (50 mL) and N,Nediisopropylethylamine (19.7 mL,
119.4 mmol) was stirred with ammonium acetate crystals (3.96 g,
51.4 mmol) under argon atmosphere. After 3 days, the reaction
mixture was decanted from the undissolved crystals of ammonium
acetate, diluted with dichloromethane (700 mL) and washed with a
saturated aqueous solution of sodium bicarbonate (200 mL), then
water (200 mL), then brine (150 mL), dried over sodium sulfate,
filtered, and concentrated in vacuo. Purification of the residue by
column chromatography (silica gel 200 mL, cyclohexane/EtOAc, v/v,
MS and/or analytical data were obtained using chromato-
graphically homogeneous samples.1H NMR (500 MHz), 13C NMR
(126 MHz) and 31P (202 MHz) spectra were recorded a Bruker
Avance or Avance II in the given solvents unless otherwise indi-
cated. Chemical shifts (d) are reported in ppm and coupling con-
stants are given in Hz. To facilitate the understanding of NMR
spectroscopic data, the numbering of atoms for the following
representative compound 19 is as indicated (Fig. 7).
Optical rotations were measured on a Perkin-Elmer 341 polar-
imeter with sodium (589 nm) lamp at 20 ꢀC. Low resolution mass
spectra (LRMS) were recorded with an ion trap mass analyzer un-
der electrospray ionization (ESI) in positive and negative ionization
mode detection. High resolution mass sprectra (HRMS) were
2/8, 1/9, 0/1) afforded a 98/2
10.4 mmol, 81%) as a colourless oil. For the
(CDCl3)
a
/
b
mixture of lactol acetates 3 (3.62 g,
a
anomer [23]: 1H NMR
d
6.00 (d, J ¼ 9.5 Hz, 1H, NHAc), 5.28 (dd, J ¼ 10.7, 9.5 Hz, 1H,
H-3), 5.23 (d, J ¼ 3.5 Hz, 1H, H-1), 5.11 (t, J ¼ 9.5 Hz, 1H, H-4), 4.26
(ddd, J ¼ 10.7, 9.5, 3.5 Hz, 1H, H-2), 4.22e4.17 (m, 2H, H-5, H-6),

M. Bosco et al. / European Journal of Medicinal Chemistry 125 (2017) 952e964
959
Fig. 7. Numbering of compound 19.
4.14e4.07 (m, 1H, H-60), 2.07 (s, 3H, CH3CO), 2.01 (s, 3H, CH3CO),
2.01 (s, 3H, CH3CO), 1.95 (s, 3H, CH3CO); 13C NMR (126 MHz, CDCl3)
5.18e5.01 (m, 6H, H benzylic ꢁ 4, H-3, H-4), 4.37 (ddt, J ¼ 10.7, 9.2,
3.3 Hz, 1H, H-2), 4.13 (dd, J ¼ 12.5, 4.0 Hz, 1H, H-6), 4.00 (ddd,
J ¼ 9.7, 4.0, 2.2 Hz, 1H, H-5), 3.92 (dd, J ¼ 12.5, 2.2 Hz, 1H, H-60), 2.02
(s, 3H, CH3CO), 2.00 (s, 3H, CH3CO), 2.00 (s, 3H, CH3CO), 1.71 (s, 3H,
d
171.5, 171.1, 170.7, 169.6 (4C, CH3CO), 91.6 (C-1), 71.1 (C-3), 68.5 (C-
4), 67.6 (C-5), 62.3 (C-6), 52.5 (C-2), 23.2, 20.9, 20.8, 20.7 (4C,
CH3CO); ESI-MS: Calcd for C14H22NO9: 348.1. [MþH]þ Found: 348.1;
CH3CO); 13C NMR (CDCl3)
d 171.2, 170.6, 170.3, 169.2 (4C, CH3CO),
ESI-HRMS: Calcd for
348.1281.
C
14H22NO9: 348.1289. [MþH]þ Found:
135.5 (d, J ¼ 6.3 Hz, Cq aromatic), 135.3 (d, J ¼ 6.5 Hz, Cq aromatic),
129.1, 128.9, 128.9,128.2,128.2 (10 C, C aromatic), 96.4 (d, J ¼ 6.6 Hz,
C-1), 70.2 (C-3), 70.09 (d, J ¼ 5.8 Hz, C benzylic), 70.04 (d, J ¼ 5.8 Hz,
C benzylic), 69.8 (C-5), 67.5 (C-4), 61.4 (C-6), 51.9 (d, J ¼ 7.7 Hz, C-2),
A solution of peracetate 2 (1.02 g, 1.51 mmol) in a mixture of dry
DMF (15 mL) and N,Nediisopropylethylamine (1 mL, 6.06 mmol)
was stirred with ammonium acetate crystals (1 g, 13.0 mmol) under
argon atmosphere for 3 days. Then, the same treatment as for
compound 3 was carried out. Recrystallization of the residue using
22.8, 20.7, 20.7, 20.6 (4C, CH3CO); 31P NMR (CDCl3)
d
ꢂ2.48; ESI-MS:
Calcd for C28H35NO12P: 608.2. [MþH]þ Found: 608.2; ESI-HRMS:
Calcd for C28H35NO12P: 608.1891. [MþH]þ Found: 608.1883.
a mixture of dichloromethane and cyclohexane afforded a 96/4
mixture of lactol acetates 4 (452 mg, 0.71 mmol, 47%) as a white
solid.: 1H NMR (CDCl3) for the major
anomer [22,24] 7.21 (d,
a
/b
To a solution of lactol 4 (420 mg, 662 mmol) in dry THF (12 mL)
were successively added a solution of 1H-tetrazole in acetonitrile
(5.9 mL, 0.45 M, 1.65 mmol) and dibenzyl N,N-diethylphosphor-
amidite (1.9 mL, 5.76 mmol). The reaction mixture was stirred
14 h at room temperature. Then, the same treatment as for com-
pound 5 was carried out. Purification of the crude by column
chromatography (silica gel 150 mL, cyclohexane/EtOAc with 1% of
a
d
J ¼ 10.0 Hz, 1H, NHAc), 6.20 (d, J ¼ 8.5 Hz, 1H, NHAc*), 5.67 (dd,
J ¼ 10.8, 9.3 Hz, 1H, H-3), 5.21 (d, J ¼ 3.3 Hz, 1H, H-1), 5.08 (t,
J ¼ 9.6 Hz, 1H, H-4*), 4.97 (t, J ¼ 9.6 Hz, 1H, H-3*), 4.42 (dd, J ¼ 12.5,
4.1 Hz, 1H, H-6*), 4.32e4.27 (m, 2H, H-6, H-2), 4.18e4.11 (m, 3H, H-
1*, H-60, H-2*), 4.07 (ddd, J ¼ 10.0, 3.8, 1.8 Hz, 1H, H-5), 4.03 (dd,
J ¼ 12.5, 2.0 Hz, 1H, H-60*), 3.64 (t, J ¼ 9.3 Hz, 1H, H-4), 3.57 (ddd,
J ¼ 9.6, 4.1, 2.0 Hz,1H, H-5*), 2.13 (s, 3H, CH3CO), 2.08 (s, 3H, CH3CO),
2.06 (s, 3H, CH3CO), 2.01 (s, 3H, CH3CO), 2.01 (s, 3H, CH3CO), 2.01 (s,
Et3N, v/v, 2/3, 1/3) afforded the phosphate 6 (464 mg, 518
78%) as a white solid. [ ꢂ15 (c 1.1,
]D þ 21.3 (c 1.0, CHCl3); [
CHCl3) [24]; 1H NMR (CDCl3) in agreement with reported data
[24,27]
7.42e7.30 (m, 10H, H aromatic), 5.85 (d, J ¼ 8.9 Hz, 1H,
mmol,
a
a]
D
d
3H, CH3CO), 1.94 (s, 3H, CH3CO); 13C NMR (CDCl3)
d
172.2, 171.5,
NHAc*), 5.62 (dd, J ¼ 5.8, 3.3 Hz, 1H, H-1), 5.58 (d, J ¼ 9.3 Hz, 1H,
NHAc), 5.20 (dd, J ¼ 10.4, 9.4 Hz, 1H, H-3*), 5.14 (dd, J ¼ 10.9, 9.1 Hz,
1H, H-3), 5.13e5.00 (m, 5H, H benzylic, H-4*), 4.55 (d, J ¼ 8.3 Hz,1H,
H-1*), 4.37 (dd, J ¼ 12.4, 4.2 Hz,1H, H-6*), 4.31e4.22 (m, 2H, H-6, H-
2), 4.06 (dd, J ¼ 12.3, 2.2 Hz, 1H, H-6), 4.00 (dd, J ¼ 12.4, 2.4 Hz, 1H,
H-60*), 3.94 (ddd, J ¼ 10.2, 3.1, 2.2 Hz, 1H, H-5), 3.82 (dt, J ¼ 10.4,
8.3 Hz, 1H, H-2*), 3.71 (dd, J ¼ 10.2, 9.1 Hz, 1H, H-4), 3.61 (ddd,
J ¼ 10.0, 4.2, 2.4 Hz, 1H, H-5*), 2.07 (s, 6H, CH3CO), 2.02 (s, 3H,
CH3CO), 2.01 (s, 3H, CH3CO), 2.00 (s, 3H, CH3CO),1.93 (s, 3H, CH3CO),
1.69 (s, 3H, CH3CO); 13C NMR (CDCl3) in agreement with reported
171.3, 171.0, 170.7, 169.3 (7C, COCH3), 102.5 (C-1*), 91.9 (C-1), 76.7
(C-4), 72.2, 72.1 (2C, C-5*, C-3*), 71.1 (C-3), 68.3 (C-5), 67.9 (C-4*),
62.5 (C-6), 61.8 (C-6*), 54.3 (C-2*), 52.01 (C-2), 23.3, 23.0, 21.1, 20.9,
20.8, 20.7, 20.7 (7C, CH3CO). ESI-MS: Calcd for C26H39N2O16: 635.2.
[MþH]þ Found: 635.3; ESI-HRMS: Calcd for C26H39N2O16
:
635.2294. [MþH]þ Found: 635.2286.
4.1.3. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-
1-dibenzylphosphate 5 and 2-deoxy-2-acetamido-4-O-(2-deoxy-2-
acetamido-30,40,60-tri-O-acetyl-
-D-gluco pyranosyl)-3,6-di-O-
acetyl- -D-glucopyranose 1-dibenzylphosphate 6
To a solution of lactol 3 (1 g, 2.89 mmol) in dry THF (32 mL) were
successively added solution of 1H-tetrazole in acetonitrile
a-D-glucopyranose
data [27]
d 171.2, 171.0, 170.8, 170.7, 170.4, 170.3, 169.5 (7C, COCH3),
b
135.5 (d, J ¼ 6.2 Hz, Cq aromatic), 135.4 (d, J ¼ 6.6 Hz, Cq aromatic),
129.1,129.0,128.9,128.3,128.2 (10C, CH aromatic), 101.3 (C-1*), 96.2
(d, J ¼ 6.2 Hz, C-1), 75.7 (C-4), 72.6 (C-3*), 72.1 (C-5*), 70.8 (C-5),
70.3 (C-3), 70.1 (d, J ¼ 5.6 Hz, CH benzylic), 70.0 (d, J ¼ 5.4 Hz, CH
benzylic), 68.2 (C-4*), 61.9 (C-6*), 61.4 (C-6), 55.1 (C-2*), 52.1 (d,
J ¼ 7.7 Hz, C-2), 23.3, 22.9, 21.0, 20.8, 20.7 (7C, CH3CO); 31P NMR
a
a
(25.6 mL, 0.45 M, 11.5 mmol) and dibenzyl N,N-diethylphosphor-
amidite (1.9 mL, 5.76 mmol). The reaction mixture was stirred
14 h at room temperature. Then the solution was cooled by a dry
ice/acetone bath and an aqueous solution of hydrogen peroxide
(0.88 mL, 30%, 8.64 mmol) was added dropwise. The reaction
mixture was stirred at ꢂ78 ꢀC during 1 h then it was allowed to
warm at room temperature. The solution was diluted with dieth-
ylether (300 mL). The organic layer was washed with an aqueous
solution of NaHSO3 (10% in weight, 100 mL), brine (100 mL), dried
over Na2SO4, filtered and concentrated in vacuo. Purification of the
residue by column chromatography (silica gel 200 mL, cyclo-
hexane/EtOAc with 1% of Et3N, v/v, 2/3, 1/3, 0/1) afforded the
(CDCl3) in agreement with reported data [22]
d
ꢂ2.23; ESI-MS:
Calcd for C40H51N2O19P: 895.3. [MþH]þ Found: 895.2; ESI-HRMS:
Calcd for C40H51N2O19P: 895.2896. [MþH]þ Found: 895.2882.
4.1.4. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-
1-phosphate 7 and 2-deoxy-2-acetamido-4-O-(2-deoxy-2-
acetamido-30,40,60-tri-O-acetyl-
-D-glucopyranosyl)-3,6-di-O-
acetyl- -D-glucopyranose 1-phosphate 8
To a solution of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
glucopyranose 1-dibenzylphosphate 5 (100 mg, 164 mol) in
ethanol (10 mL) under argon atmosphere was added a catalytic
amount of palladium on charcoal (10 mg, 9 mol). Then, the reac-
tion mixture was purged with hydrogen gas and stirred during
a-D-glucopyranose
b
a
a-D-
phosphate 5 (1.46 g, 2.40 mmol, 83%) as a white solid [12,25]. [
D þ 59.7 (c 1.0, CHCl3); [ ]D þ 29.0 (c 0.17, MeOH) [25]; [ ]D þ 4.8 (c
1.0, CHCl3) [26]; 1H NMR (CDCl3)
7.41e7.31 (m, 10H, H aromatic),
5.76 (d, J ¼ 9.2 Hz, 1H, NHAc), 5.66 (dd, J ¼ 6.0, 3.3 Hz, 1H, H-1),
a
]
m
a
a
d
m

960
M. Bosco et al. / European Journal of Medicinal Chemistry 125 (2017) 952e964
30 min under hydrogen atmosphere. The resulting solution was
(D2O)
d
2.22 (s); 31P NMR non decoupled (D2O)
d
2.22 (d, J ¼ 7.5 Hz);
degassed with argon atmosphere and it was filtered through a
ESI-MS: Calcd for C8H15NO9Pꢂ: 300.0. [M]- Found: 300.0; ESI-
celite© patch. To the filtrate was added triethylamine (50
m
L) and
concentration under vacuum furnished the product 7 (86 mg,
162 5.49 (dd,
mol, quant.) as a white solid [25]. 1H NMR (CD3OD)
HRMS: Calcd for C8H15NO9Pꢂ: 300.0479. [M]- Found: 300.0471.
To
acetamido-30,40,60-tri-O-acetyl-
acetyl- -glucopyranose 1-phosphate 8 (43 mg, 60 m
a
solution of 2-deoxy-2-acetamido-4-O-(2-deoxy-2-
-glucopyranosyl)-3,6-di-O-
mol, 1 eq.) in
m
d
b-D
J ¼ 7.0, 3.4 Hz, 1H, H-1), 5.31 (dd, J ¼ 10.6, 9.5 Hz, 1H, H-3), 5.09 (t,
J ¼ 9.8 Hz, 1H, H-4), 4.30 (ddd, J ¼ 9.8, 3.5, 2.1 Hz, 1H, H-5),
4.30e4.25 (m, 1H, H-6), 4.30 (ddd, J ¼ 10.6, 3.4, 2.2 Hz, 1H, H-2),
4.17e4.11 (m, 1H, H-60), 3.18 (q, J ¼ 7.3 Hz, 8H, (CH3CH2)3NHþ), 2.05
(s, 3H, CH3CO), 2.01 (s, 3H, CH3CO), 1.97 (s, 3H, CH3CO), 1.94 (s, 3H,
CH3CO), 1.31 (t, J ¼ 7.3 Hz, 12H, (CH3CH2)3NHþ); 13C NMR (CD3OD)
a
-D
methanol (20 mL) was added dropwise a solution of sodium
methoxide (1.3 mL, 0.325 M, 7 eq.). The reaction mixture was
stirred at room temperature for 14 h. Then the same procedure as
for compound 9 was carried out to furnish the product 10 (32 mg,
58
m
mol, 97%) as a white solid [28]. 1H NMR (D2O)
d
5.37 (dd, J ¼ 7.6,
d
173.5, 172.4, 172.0, 171.3 (4C, CH3CO), 95.06 (d, J ¼ 5.9 Hz, C-1),
2.9 Hz, 1H, H-1), 4.66 (d, J ¼ 8.5 Hz, 1H, H-1*), 4.05e4.01 (m, 1H, H-
5), 4.00e3.92 (m, 3H, H-2, H-5*, H-6), 3.88 (dd, J ¼ 12.1, 1.6 Hz, 1H,
H-6*), 3.83e3.75 (m, 2H, H-2*,H-6), 3.73e3.66 (m, 2H, H-6*, H-4),
3.65e3.60 (m, 1H, H-3*), 3.57e3.49 (m, 2H, H-3, H-4*), 2.11 (s, 3H,
72.8 (C-3), 69.9 (C-4), 69.6 (C-5), 63.0 (C-6), 53.5 (d, J ¼ 7.8 Hz, C-2),
47.5 (4C, (CH3CH2)3NHþ), 22.6, 20.7, 20.6 (4C, CH3CO), 9.2 (4C,
(CH3CH2)3NHþ); 31P NMR (CD3OD)
d
ꢂ1.11; 31P NMR non decoupled
(CD3OD)
d
ꢂ1.11 (d, J ¼ 6.3 Hz); ESI-MS: Calcd for C28H45N2O24Pꢂ2 :
CH3CO), 2.09 (s, 3H, CH3CO); 13C NMR (D2O)
d 174.7 (s, CH3CO),174.6
853.2. [2MþH]- Found: 853.3; ESI-HRMS: Calcd for C14H21NO12Pꢂ:
(s, CH3CO), 101.3 (s, C-1*), 92.4 (d, J ¼ 5.4 Hz, C-1), 79.7 (s, C-4), 76.0
(s, C-3), 73.6 (s, C-3*), 70.5 (s, C-5), 70.1 (s, C-5*), 69.8 (s, C-4*), 60.6
(s, C-6), 60.2 (s, C-6*), 55.7 (s, C-2*), 53.7 (d, J ¼ 7.4 Hz, C-2), 22.2 (s,
426.0807. [M]- Found: 426.0791.
To
acetamido-30,40,60-tri-O-acetyl-
acetyl- -glucopyranose 1-dibenzylphosphate 6 (55 mg, 74
a
solution of 2-deoxy-2-acetamido-4-O-(2-deoxy-2-
b
-D-glucopyranosyl)-3,6-di-O-
CH3CO), 22.2 (s, CH3CO); 31P NMR (D2O) 2.21 (s); 31P NMR non
d
a
-
D
mmol)
decoupled (D2O)
d
2.19 (d, J ¼ 7.2 Hz); ESI-MS: Calcd for
17H31N2O14Pꢂ: 518.2. [M]- Found: 518.4; ESI-HRMS: Calcd for
16H28N2O14Pꢂ: 503.1273. [M]- Found: 503.1275.
in a mixture of ethanol (8 mL) and dichloromethane (5.5 mL) under
argon atmosphere was added a catalytic amount of palladium on
C
C
charcoal (11 mg, 10
pound 5 was carried out to furnish the product 8 (60 mg, 74
quant.) as a white solid [13]. 1H NMR (CD3OD)
5.43 (dd, J ¼ 6.9,
m
mol). Then, the same procedure as for com-
mmol,
4.1.6. Dicyclohexylammonium (S)-citronellyl phosphate 11
To dichloromethane (60 mL) under argon atmosphere at room
temperature were added successively POCl3 (4.2 mL, 45.2 mmol, 3
d
3.4 Hz, 1H, H-1), 5.37 (dd, J ¼ 10.4, 9.3 Hz, 1H, H-3*), 5.25 (dd,
J ¼ 10.6, 9.1 Hz, 1H, H-3), 4.95 (dd, J ¼ 9.9, 9.3 Hz, 1H, H-4*), 4.80 (d,
J ¼ 8.4 Hz, 1H, H-1*), 4.54 (dd, J ¼ 12.1, 1.9 Hz, 1H, H-6), 4.43 (dd,
J ¼ 12.4, 4.1 Hz, 1H, H-6*), 4.21e4.14 (m, 2H, H-2, H-5), 4.06 (dd,
J ¼ 12.1, 4.2 Hz, 1H, H-60), 4.02 (dd, J ¼ 12.4, 2.3 Hz, 1H, H-60*), 3.89
(dd, J ¼ 9.9, 9.1 Hz,1H, H-4), 3.79 (ddd, J ¼ 10.1, 4.1, 2.3 Hz,1H, H-5*),
3.57 (dd, J ¼ 10.4, 8.4 Hz, 1H, H-2*), 3.12 (q, J ¼ 7.3 Hz, 8H,
(CH3CH2)3NHþ), 2.10 (s, 3H, CH3CO), 2.06 (s, 3H, CH3CO), 2.03 (s, 3H,
CH3CO), 1.98 (s, 3H, CH3CO), 1.97 (s, 3H, CH3CO), 1.93 (s, 3H, CH3CO),
1.89 (s, 3H, CH3CO),1.28 (t, J ¼ 7.3 Hz,12H, (CH3CH2)3NHþ); 13C NMR
eq.), triethylamine (6.3 mL, 45.2 mmol, 3eq.) and (S)-(ꢂ)-
b-citro-
nellol (3 mL, 15.1 mmol, 1 eq.). The reaction mixture was kept under
agitation at room temperature then 90 mL of an acetone, triethyl-
amine and water (88/10/2, v/v/v) solution was added. After 14 h
stirring, solvents were removed. The crude product was diluted
with water (15 mL). The aqueous layer was extracted with
dichloromethane (3 ꢁ 75 mL). The combined organic layers were
washed with water (45 mL), brine (50 mL), dried over Na2SO4,
filtered and concentrated in vacuo. The crude product was dissolved
in a mixture of acetone and water (120 mL, 9/1, v/v) and cyclo-
hexylamine (3 mL) was added. The mixture was kept at 0 ꢀC for 1 h.
The formed crystals were collected by filtration and they were
submitted to recrystallization in 45 mL of a mixture of acetone and
water (9/1, v/v) to furnish the product 11 (2.22 g, 4.94 mmol, 33%) as
white crystals. A part of this product was purified by reverse phase
HPLC (0% MeCN 100% aq. NH4HCO3 25 mM t0, 100% MeCN 0% aq.
NH4HCO3 25 mM over 50 min; tR 28 min) to furnish the di-
ammonium salt of (S)-citronellyl phosphate 11. 1H NMR (D2O)
(CD3OD)
d 173.6, 173.5, 172.6, 172.3, 172.2, 171.8, 171.3 (7C, COCH3),
101.6 (C-1*), 95.0 (d, J ¼ 5.9 Hz, C-1), 77.0 (C-4), 73.5 (C-3*), 73.3 (C-
3), 72.8 (C-5*), 70.5 (C-5), 70.0 (C-4*), 63.4 (C-6), 63.1 (C-6*), 56.7
(C-2*), 53.6 (d, J ¼ 8.1 Hz, C-2), 47.7 (4C, (CH3CH2)3NHþ), 22.9, 22.6,
21.1, 20.9, 20.7, 20.6, 20.5 (7C, CH3CO), 9.4 (4C, (CH3CH2)3NHþ); 31
P
NMR (CD3OD)
d
ꢂ1.12; 31P NMR non decoupled (202 MHz, CD3OD)
d
ꢂ1.09 (d, J ¼ 6.1 Hz); ESI-MS: Calcd for C26H38N2O19Pꢂ: 713.2. [M]-
Found: 713.6; ESI-HRMS: Calcd for C26H38N2O19Pꢂ: 713.1812. [M]-
Found: 713.1802.
d
5.29 (tq, J ¼ 7.2, 1.4 Hz, 1H, H-6), 4.02e3.88 (m, 2H, H-1), 2.15e1.99
4.1.5. 2-Acetamido-2-deoxy-
2-deoxy-2-acetamido-4-O-(2-deoxy-2-acetamido-
glucopyranosyl)- -D-glucopyranose 1-phosphate 10
To a solution of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
glucopyranose 1-phosphate 7 (72 mg, 169 mol, 1 eq.) in methanol
a
-D-glucopyranose 1-phosphate 9 and
(m, 2H, H-5), 1.77e1.69 (m, 4H, H-2, CH3-9), 1.68 (s, 3H, CH3-10),
1.67e1.58 (m, 1H, H-3), 1.53e1.45 (m, 1H, H-20), 1.44e1.36 (m, 1H,
H-4), 1.24 (dddd, J ¼ 13.5, 9.2, 7.7, 6.2 Hz, 1H, H-40), 0.95 (d,
b-D-
a
a
-D
-
J ¼ 6.7 Hz, 3H, CH3-8); 13C NMR (D2O)
d 133.2 (s, C-7), 125.3 (s, C-6),
m
64.4 (d, J ¼ 5.5 Hz, C-1), 36.8 (d, J ¼ 6.8 Hz, C-2), 36.3 (s, C-4), 28.6 (s,
(20 mL) was added dropwise a solution of sodium methoxide
(1.6 mL, 0.53 M, 5 eq.). The reaction mixture was stirred at room
temperature for 1 h. The reaction mixture was neutralized with
excess of cation exchange resin (Dowex 50Wx8, Hþ form). The resin
was filtered and washed with methanol. The combined phases
were evaporated. The obtained white solid was dissolved in water
C-3), 24.8 (s, C-5), 24.8 (s, C-9), 18.6 (s, C-8), 16.9 (s, C-10); 31P NMR
non decoupled (D2O)
d
0.53 (t, J ¼ 6.6 Hz); 31P NMR (D2O)
d 0.54 (s);
ESI-MS: Calcd. For C10H20O4Pꢂ: 235.1. [MþH]- Found 235.3; ESI-
HRMS: Calcd. For C10H20O4Pꢂ: 235.1105. [MþH]- Found 235.1094.
4.1.7. (S)-Citronellyl diphosphate 12
and was freeze dried to furnish the product 9 (55 mg, 159
m
mol,
To a solution of (S)-(ꢂ)-
b-citronellol (0.5 mL, 2.51 mmol) in
94%) as a white solid [11]. 1H NMR (D2O)
d
5.38 (dd, J ¼ 7.5, 3.2 Hz,
dichloromethane (10 mL) under argon atmosphere were added
triethylamine (0.38 mL, 2.76 mmol), tosyl chloride (280 mg,
2.76 mmol) and 4-(dimethylamino)pyridine (30 mg, 0.25 mmol).
The reaction mixture was stirred during 14 h at room temperature,
and then diluted in diethyl ether (50 mL). The resulting precipitate
was removed by filtration through a celite pad. The filtrate was
concentrated in vacuo and the crude product was purified by flash
1H, H-1), 4.00 (ddd, J ¼ 9.6, 5.2, 2.1 Hz, 1H, H-5), 3.95 (dd, J ¼ 10.4,
3.2, 1.8 Hz, 1H, H-2), 3.92 (d, J ¼ 12.3, 2.1 Hz, 1H, H-6), 3.83 (broad
dd, J ¼ 10.4, 9.6 Hz,1H, H-3), 3.80 (dd, J ¼ 12.3, 5.2 Hz,1H, H-60), 3.51
(t, J ¼ 9.6 Hz, 1H, H-4), 2.10 (s, 3H, CH3CO); 13C NMR (D2O)
d 174.7 (s,
CH3CO), 92.7 (d, J ¼ 5.5 Hz, C-1), 72.1 (s, C-5), 71.6 (s, C-3), 70.2 (s, C-
4), 60.8 (s, C-6), 54.2 (d, J ¼ 7.3 Hz, C-2), 22.2 (s, CH3CO); 31P NMR

M. Bosco et al. / European Journal of Medicinal Chemistry 125 (2017) 952e964
961
chromatography on silica gel column (50 g, EtOAc/cyclohexane, 1/9,
v/v) to yield the citronellyl tosylate as a colourless oil (532 mg,
1.64 mmol, 65%).
toluene and methanol (3.25 mL, 1/1, v/v) was added. After 30 min,
the precipitate was removed by filtration and the filtrate was
concentrated in vacuo. The crude product was purified by flash
chromatography on silica gel column (5 g, dichloromethane/
methanol/ammonia 14%, 80/18/2, v/v/v) to yield the tetrabuty-
lammonium solanesyl phosphate 14 as a colourless oil (21 mg,
To
a suspension of tris(tetrabutylammonium) hydrogen
diphosphate (668 mg, 0.74 mmol) in distilled acetonitrile (10 mL)
under argon atmosphere was added the previous citronellyl tosy-
late (100 mg, 0.31 mmol). The reaction mixture was stirred during
18 h at room temperature and concentrated in vacuo. It was then
dissolved in water (10 mL) and DOWEX 50WX8 (NHþ4 form) was
added to exchange tetrabutylammonium ions. Resin was removed
by filtration and the filtrate was freeze dried. The lyophilisate was
dissolved in 100 mM aq. NH4HCO3 (3 mL) and a mixture of aceto-
nitrile/isopropanol (1/1, v/v, 3 mL) was added. After centrifugation
of the suspension for 5 min at 2000 rpm, the supernatant was
removed and the process was repeated three times. The combined
supernatants were concentrated in vacuo and freeze dried. The
lyophilisate was purified by reverse phase HPLC (0% MeCN 100% aq.
NH4HCO3 25 mM t0, 0e15% MeCN over 20 min, 15e25% over
30 min; tR 37.1 min) to afford the desired citronellyl diphosphate 12
as a white powder (39 mg, 0.12 mmol, 38%). 1H NMR (D2O)
28
m
mol, 33%). 1H NMR (250 MHz, CDCl3)
d
5.39 (t, J ¼ 5.9 Hz, 1H, H-
2), 5.11 (t, J ¼ 6.2 Hz, 8H, 8 ꢁ H-7), 4.45 (t, J ¼ 5.7 Hz, 2H, 2 ꢁ H-1),
3.42e3.18 (m, 8H, (CH3CH2CH2CH2)4Nþ), 2.17e2.02 (m,16H,14 ꢁ H-
6, 2 ꢁ H-11), 2.01e1.86 (m, 16H, 14 ꢁ H-9, 2 ꢁ H-4), 1.70e1.55 (m,
8H, (CH3CH2CH2CH2)4Nþ), 1.67 (s, 6H, CH3-5, CH3-15), 1.62 (s, 3H,
CH3-14), 1.59 (s, 21H,
7
ꢁ
CH3-10), 1.52e1.36 (m, 8H,
(t, 7.2 Hz, 12H,
1.62 (s); ESI-MS
(CH3CH2CH2CH2)4Nþ),
0.98
J
¼
(CH3CH2CH2CH2)4Nþ); 31P NMR (101 MHz, CDCl3)
d
Calcd. For C45H74O4Pꢂ: 709.6. [MþH]- Found 708.7; ESI-HRMS
Calcd. For C45H74O4Pꢂ: 709.5530. [MþH]- Found 709.5329.
4.1.10. Ammonium solanesyl diphosphate 15
To a solution of solanesol (95 mg, 150
methane (2 mL) was added tetrabutylammonium phosphate
(204 mg, 600 mol, 4 eq.). The reaction mixture was placed in a
dark place. Then trichloroacetonitrile (75 L, 750 mol, 5 eq.) was
mmol, 1 eq.) in dichloro-
d
5.34e5.24 (m, 1H, H-6), 4.09e3.91 (m, 2H, H-1), 2.13e1.97 (m, 2H,
m
H-5), 1.78e1.69 (m, 4H, H-2, CH3-9), 1.67 (s, 3H, CH3-10), 1.65e1.58
(m, 1H, H-3), 1.55e1.46 (m, 1H, H-20), 1.45e1.37 (m, 1H, H-4),
1.29e1.18 (m, 1H, H-40), 0.95 (d, J ¼ 6.6 Hz, 3H, CH3-8); 13C NMR
m
m
added to the mixture. After 14 h stirring at room temperature, the
solvent was evaporated. To the residue was added THF (1.5 mL) and
a concentrated solution of aqueous ammonium hydroxide (28%,
0.3 mL). After 30 min stirring at room temperature, a mixture of
toluene and methanol (8 mL, 1/1, v/v) was added. After 30 min, the
precipitate was removed by filtration and the filtrate was concen-
trated in vacuo. The crude product was purified by DEAE anion
exchange chromatography. The product was eluted with a mixture
of chloroform/methanol/water (10/10/3, v/v/v), then a mixture of
chloroform/methanol/5 mM aqueous ammonium acetate (10/10/3,
v/v/v), then a mixture of chloroform/methanol/100 mM aqueous
ammonium acetate (10/10/3, v/v/v). Fractions containing the
desired product were pooled and lyophilized to furnish the product
(D2O)
d
133.1 (s, C-7), 125.4 (s, C-6), 64.7 (d, J ¼ 5.7 Hz, C-1), 37.0 (d,
J ¼ 7.3 Hz, C-2), 36.5 (s, C-4), 28.7 (s, C-3), 24.9 (s, C-5), 24.8 (s, C-9),
18.7 (s, C-8), 16.9 (s, C-10).
31P NMR non decoupled (202 MHz, D2O)
d
ꢂ6.03 (d, J ¼ 21.7 Hz,
P-A), ꢂ10.08 (dt, J ¼ 21.7, 6.4 Hz, PeB). 31P NMR (202 MHz, D2O)
d
ꢂ6.02 (d, J ¼ 21.7 Hz, P-A), ꢂ10.08 (d, J ¼ 21.7 Hz, PeB); ESI eMS
Calcd. For C10H21O7Pꢂ2 : 315.1. [M þ 2H]- Found 315.3; ESI-HRMS
Calcd. For C10H21O7Pꢂ2 : 315.0768. [M þ 2H]- Found 315.0773.
4.1.8. (S)-Citronellyl medronate 13
The medronate was prepared by the same methodology as the
citronellyl diphosphate 12 starting from tris(tetrabutylammonium)
hydrogen medronate (800 mg, 0.89 mmol) and citronellyl tosylate
(170 mg, 0.55 mmol) in distilled acetonitrile (10 mL) to afford after
reverse phase HPLC purification (0% MeCN 100% aq. NH4HCO3
25 mM t0, 0e20% MeCN over 20 min, 20e50% over 30 min; tR
36.0 min) the desired citronellyl medronate 13 as a white powder
15 (34 mg, 40
CDCl3, 19.5% CD3OD, 2.5% D2O)
m
mol, 27%) as a white solid. 1H NMR (250 MHz, 78%
5.33e5.19 (m, 1H, H-2), 5.11e4.90
d
(m, 8H, 7 ꢁ H-7, H-12), 4.41e4.24 (m, 2H, 2 ꢁ H-1), 2.07e1.90 (m,
16H, 14 ꢁ H-6, 2 ꢁ H-11), 1.90e1.75 (m, 16H, 14 ꢁ H-9, 2 ꢁ H-4),
1.54, 1.53 (2 ꢁ s, 6H, CH3-5, CH3-15), 1.46 (s, 24H, 7 ꢁ CH3-10, CH3-
14); 31P NMR (101 MHz, 78% CDCl3, 19.5% CD3OD, 2.5% D2O)
d
ꢂ5.04
(75 mg, 0.21 mmol, 37%). 1H NMR (D2O)
d
5.33e5.22 (m, 1H, H-6),
(d, J ¼ 17.1 Hz, P-A), -6.05 (d, J ¼ 17.1 Hz, PeB); ESI-HRMS Calcd. For
4.09e3.91 (m, 2H, H-1), 2.30 (t, J ¼ 20.1 Hz, 2H, H-11), 2.14e1.98 (m,
2H, H-5), 1.78e1.69 (m, 4H, H-2, CH3-9), 1.67 (s, 3H, CH3-10),
1.65e1.59 (m,1H, H-3),1.55e1.46 (m,1H, H-20),1.45e1.36 (m,1H, H-
4), 1.29e1.18 (m, 1H, H-40), 0.95 (d, J ¼ 6.6 Hz, 3H, CH3-8); 13C NMR
C
45H74O7Pꢂ2 : 789.4994. [MþH]- Found 789.4999.
4.1.11. P1-(2-Acetamido-2-deoxy-
-(S)-citronellyl diphosphate 16
a-D
-glucopyranosyl) P2-(S)-(ꢂ)-
b
(D2O)
d
133.2 (s, C-7), 125.3 (s, C-6), 64.0 (d, J ¼ 5.9 Hz, C-1), 37.0 (d,
To a solution of (S)-citronellyl phosphate 11 [264 mg, 607
mmol,
J ¼ 6.1 Hz, C-2), 36.3 (s, C-4), 28.6 (s, C-3), 26.7 (t, J ¼ 125 Hz, C-11),
1.3 eq., previously co-evaporated with dry toluene (5 mL) and
24.9 (s, C-5), 24.8 (s, C-9), 18.7 (s, C-8), 16.9 (s, C-10); 31P NMR (D2O)
triethylamine (50 mL) three times] in dry THF (15 mL) was added
d
17.86 (d, J ¼ 9.2 Hz, PeB), 17.22 (d, J ¼ 9.2 Hz, P-A); 31P NMR non
1,10-carbonyldiimidazole CDI (325 mg, 2 mmol, 4.3 eq.) under argon
atmosphere. After 1.5 h stirring at room temperature, dry methanol
decoupled (D2O)
P-A).
d
18.05e17.65 (m, PeB), 17.22 (td, J ¼ 20.1, 9.2 Hz,
(210 mmol) was added and the mixture was stirred for 1 h to destroy
ESI-MS: Calcd. For C11H23O7Pꢂ2 : 313.1. [M þ 2H]- Found 313.3;
ESI-HRMS Calcd. For C11H23O7Pꢂ2 : 313.0975. [M þ 2H]- Found
313.0979.
the excess of CDI. Then, solvents were removed. The resulting crude
activated phosphate was solubilized in dry THF (15 mL) and was
transferred to a flask containing the phosphate 7 [225 mg,
427
m
mol, 1 eq., previously co-evaporated with dry toluene (5 mL)
L) three times]. The reaction mixture was
4.1.9. Tetrabutylammonium solanesyl phosphate 14
and triethylamine (50
m
To a solution of solanesol (50 mg, 79
methane (1 mL) was added tetrabutylammonium phosphate
m
mol, 1 eq.) in dichloro-
stirred overnight at room temperature and then was concentrated
in vacuo. The crude product was quickly purified by column chro-
matography on silica gel (50 g, CH2Cl2/MeOH/H2O/Et3N, 90/10/0/
0.2 to 80/19/1/0.2, v/v/v/v) to furnish a mixture of partially deace-
tylated diphosphate (120 mg).
At room temperature, the previous mixture (120 mg) was dis-
solved in methanol (12 mL) then a solution of sodium methoxide
(1 mL, 0.43 M, 430 mmol) was added to the reaction mixture. After
(87 mg, 256
mmol, 3.2 eq.). The reaction mixture was placed in a
ꢀ
dark place. Then, trichloroacetonitrile (39
m
L, 390
m
mol, 5 eq.) was
added to the mixture. After 1 h stirring at room temperature, the
solvent was evaporated. To the residue was added THF (0.75 mL)
and a concentrated solution of aqueous ammonium hydroxide
(28%, 0.15 mL). After 14 h stirring at room temperature, a mixture of

962
M. Bosco et al. / European Journal of Medicinal Chemistry 125 (2017) 952e964
30 min stirring, the reaction was stopped with addition of a cation
exchange resin (DOWEX 50WX8, Hþ form). After 30 min stirring,
the resin was removed by filtration and the resin was washed with
methanol. The filtrate was concentrated in vacuo. The residue was
purified by column chromatography on silica gel (12 g, CH2Cl2/
MeOH/H2O/Et3N, 90/10/0/0.2 to 75/25/1/0.2, v/v/v/v) to furnish the
11), 1.80e1.70 (m, 1H, H-8), 1.68 (s, 3H, CH3-16), 1.66e1.58 (m, 1H,
H-9), 1.50 (td, J ¼ 13.6, 7.0 Hz, 1H, H-80), 1.45e1.33 (m, 1H, H-10),
1.31e1.19 (m, 1H, H-100), 0.96 (d, J ¼ 6.6 Hz, 3H, CH3-14); 13C NMR
(D2O) d 174.7 (s, CH3CO), 174.5 (s, CH3CO), 133.1 (s, C-13), 125.4 (s, C-
12), 101.3 (s, C-1*), 94.1 (d, J ¼ 6.1 Hz, C-1), 79.2 (s, C-4), 76.0 (s, C-
5*), 73.7 (s, C-3*), 71.5 (s, C-5), 69.9 (s, C-3), 69.8 (s, C-4*), 65.3 (d,
J ¼ 6.1 Hz, C-7), 60.7 (s, C-6*), 59.9 (s, C-6), 55.7 (s, C-2*), 53.2 (d,
J ¼ 8.4 Hz, C-2), 36.8 (d, J ¼ 7.2 Hz, C-8), 36.4 (s, C-10), 28.7 (s, C-9),
24.9 (s, C-11), 24.9 (s, C-15), 22.2 (s, CH3CO), 22.2 (s, CH3CO), 18.7 (s,
product 16 (80 mg, 124
m
mol, 29% over two steps) as
a
monotriethyl-ammonium salt [23]. 1H NMR (D2O)
d
5.53 (dd,
J ¼ 7.2, 3.3 Hz, 1H, H-1), 5.32e5.23 (m, 1H, H-12), 4.09e4.00 (m, 3H,
H-2, 2 ꢁ H-7), 3.98 (ddd, J ¼ 10.2, 4.6, 2.3 Hz, 1H, H-5), 3.92 (dd,
J ¼ 12.4, 2.3 Hz, 1H, H-6), 3.85 (t, J ¼ 9.8 Hz, H-3), 3.85 (dd, J ¼ 12.4,
4.6 Hz, 1H, H-60), 3.58 (dd, J ¼ 10.2, 9.8 Hz, 1H, H-4), 3.24 (q,
J ¼ 7.3 Hz, 6H, (CH3CH2)3NHþ), 2.12 (s, 3H, CH3CONH), 2.14e1.99 (m,
2H, 2 ꢁ H-11), 1.80e1.69 (m, 1H, H-8), 1.74 (s, 3H, CH3-15), 1.67 (s,
3H, CH3-16), 1.63 (td, J ¼ 12.0, 7.1 Hz, 1H, H-9), 1.50 (ddd, J ¼ 16.5,
12.0, 5.0 Hz,1H, H-80),1.46e1.35 (m,1H, H-10),1.32 (t, J ¼ 7.3 Hz, 9H,
(CH3CH2)3NHþ), 1.29e1.17 (m, 1H, H-100), 0.95 (d, J ¼ 6.6 Hz, 3H,
C-14), 17.00 (s, C-16); 31P NMR (D2O)
d
ꢂ10.63 (broad s, PeB), -13.15
(broad s, P-A); ESI-MS Calcd. For C26H47O17Pꢂ2 : 721.2. [MþH]- Found
721.5; ESI-HRMS Calcd. For C26H47O17Pꢂ2 : 721.2355. [MþH]- Found
721.2359.
a-D
4.1.13. P1-(2-Acetamido-2-deoxy- -glucopyranosyl) P2-solanesyl
diphosphate 18
To a solution of solanesyl phosphate 14 [50 mg, 52
previously co-evaporated with dry toluene (5 mL) and triethyl-
amine (50 L) three times] in dry dichloromethane (5 mL) was
added 1,10-carbonyldiimidazole CDI (43 mg, 262
mol, 5 eq.) under
argon atmosphere. After 2 h stirring at room temperature, dry
methanol (100 mol) was added and the mixture was stirred for 1 h
mmol, 1 eq.,
CH3-14); 13C NMR (D2O)
d 174.8 (s, CH3CONH), 133.2 (s, C-13), 125.3
(s, C-12), 94.5 (d, J ¼ 6.3 Hz, C-1), 73.0 (s, C-5), 71.0 (s, C-3), 69.6 (s,
C-4), 65.3 (d, J ¼ 6.1 Hz, C-7), 60.4 (s, C-6), 53.7 (d, J ¼ 8.6 Hz, C-2),
46.7 (s, 3C, (CH3CH2)3NHþ), 36.8 (d, J ¼ 7.3 Hz, C-8), 36.4 (s, C-10),
28.6 (s, C-9), 24.9 (s, C-11), 24.8 (s, C-15), 22.1 (s, CH3CONH), 18.6 (s,
C-14), 16.9 (s, C-16), 8.25 (s, 3C, (CH3CH2)3NHþ); 31P NMR (D2O)
m
m
m
to destroy the excess of CDI. Then solvents were removed. The
resulting crude activated phosphate was solubilized in dry DMF
(5 mL) and was transferred to a flask containing the phosphate 7
d
ꢂ10.70 (d, J ¼ 20.7 Hz, PeB), -13.22 (d, J ¼ 20.7 Hz, P-A); 31P NMR
non decoupled (D2O)
d
ꢂ10.71 (dt, J ¼ 20.7, 7.2 Hz, 1H, PeB), -13.23
(dd, J ¼ 20.7, 7.8 Hz, 1H, P-A); ESI-MS Calcd. For C18H34O12Pꢂ2 : 518.2.
[MþH]- Found 518.4; HRMS (ESI-) Calcd. For C18H34O12Pꢂ2 : 518.1562.
[MþH]- Found 518.1556.
[40 mg, 75
mmol, 1.8 eq., previously co-evaporated with dry toluene
(5 mL) and triethylamine (50
m
L) three times]. The reaction mixture
was stirred 6 days at room temperature. The reaction mixture was
concentrated in vacuo. The crude product was purified by column
chromatography on silica gel (5 g, CH2Cl2/MeOH/NH4OH 14%, 80/
18/2 to 70/27/3, v/v/v) to furnish the tri-O-acetyl diphosphate
4.1.12. P1-[2-Acetamido-2-deoxy-
b-D-glucopyranosyl-(1 / 4)-2-
Acetamido-2-deoxy-
diphosphate 17
a-D
-glucopyranosyl] P2-(S)-(ꢂ)-
b -citronellyl
(26 mg, 22.5
At room temperature, the previous tri-O-acetyl diphosphate
(26 mg, 22.5 mol, 1 eq.) was dissolved in methanol (3 mL) and
dichloromethane (3 mL) then a solution of sodium methoxide in
methanol (250 L, 0.43 M, 107 mol, 4.8 eq.) was added to the re-
mmol, 43%).
To a solution of (S)-citronellyl phosphate 11 [236 mg, 545
mmol,
3.3 eq., previously co-evaporated with dry toluene (5 mL) and
m
triethylamine (50 mL) three times] in dry THF (15 mL) was added
1,10-carbonyldiimidazole CDI (380 mg, 2.34 mmol, 14.2 eq.) under
argon atmosphere. After 1.5 h stirring at room temperature, dry
m
m
action mixture. After 1 h 30 stirring, the reaction was stopped by
addition of a cation exchange resin (DOWEX 50WX8, Hþ form).
After 30 min stirring, the resin was removed by filtration and the
resin was washed with methanol. The filtrate was concentrated in
vacuo. The residue was purified by column chromatography on
silica gel (1 g, CH2Cl2/MeOH/H2O/Et3N, 90/10/0/0.2 to 75/25/1/0.2,
methanol (240 mmol) was added and the mixture was stirred for 1 h
to destroy the excess of CDI. Then, solvents were removed. The
resulting crude activated phosphate was solubilized in dry DMF
(10 mL) and was transferred to a flask containing the phosphate 8
[135 mg,165
mmol,1 eq., previously co-evaporated with dry toluene
(5 mL) and triethylamine (50
m
L) three times]. The reaction mixture
v/v/v/v) to furnish the product 18 (14 mg, 13.6
two steps) as a di-ammonium salt. 1H NMR (250 MHz, 78% CDCl3,
19.5% CD3OD, 2.5% D2O) 5.44e5.32 (m, 1H, H-1), 5.25 (broad t,
mmol, 60%, 26% over
was stirred overnight at room temperature. The reaction mixture
was concentrated in vacuo. The crude product was purified by
column chromatography on silica gel (40 g, CH2Cl2/MeOH/H2O/
Et3N, 90/10/0/0.2 to 75/22/3/0.2, v/v/v/v) to furnish a mixture of
partially deacetylated diphosphate (105 mg).
At room temperature, the previous mixture (105 mg) was dis-
solved in methanol (10 mL) then a solution of sodium methoxide in
methanol (5 mL, 0.43 M, 2.15 mmol) was added to the reaction
mixture. After overnight stirring, the reaction was stopped with
addition of a cation exchange resin (DOWEX 50WX8, Hþ form).
After 1 h stirring, the resin was removed by filtration and the resin
was washed with methanol. The filtrate was concentrated in vacuo.
The residue was purified by reverse phase HPLC (0% MeCN 100% aq.
NH4HCO3 25 mM t0, 0e20% MeCN over 10 min, 20e30% over
d
J ¼ 6.5 Hz, 1H, H-8), 5.00 (broad t, J ¼ 5.9 Hz, 8H, 7 ꢁ H-12, H-16),
4.42e4.25 (m, 2H, 2 ꢁ H-7), 3.88e3.72 (m, 3H, H-5, H-2, H-6),
3.72e3.50 (m, 2H, H-60, H-4), 3.30 (broad t, J ¼ 9.3 Hz, 1H, H-4),
2.05e1.77 (m, 35H, 2 ꢁ H-10, NCOCH3, 14 ꢁ H-11, 14 ꢁ H-14, 2 ꢁ H-
15), 1.56 (s, 6H, CH3-18, CH3-21), 1.48 (s, 24H, 7 ꢁ CH3-19, CH3-20);
31P NMR (101 MHz, 78% CDCl3, 19.5% CD3OD, 2.5% D2O)
d
ꢂ6.16
(broad s, PeB), -8.27 (broad s, P-A); ESI-MS Calcd. For
C
C
53H88O12NPꢂ2 : 992.6. [MþH]- Found 993.1; ESI-HRMS Calcd. For
53H88O12NPꢂ2 : 992.5743. [MþH]- Found 992.5787.
4.1.14. P1-[2-Acetamido-2-deoxy-
b
-
D
-glucopyranosyl-(1 / 4)-2-
acetamido-2-deoxy-
19
a-D
-glucopyranosyl] P2-solanesyl diphosphate
20 min; tR 13.0 min) to furnish the product 17 (32 mg, 50
mmol, 30%
over two steps) as a di-ammonium salt. 1H NMR (D2O)
d
5.52 (dd,
To a solution of a crude mixture containing tetrabutylammo-
J ¼ 7.3, 3.2 Hz, 1H, H-1), 5.34e5.24 (m, 1H, H-12), 4.67 (d, J ¼ 8.5 Hz,
1H, H-1*), 4.09e3.93 (m, 6H, H-2, 2 ꢁ H-7, H-5, H-6*, H-3), 3.89 (dd,
J ¼ 12.3, 1.9 Hz, 1H, H-6), 3.83e3.74 (m, 3H, H-2*, H-6*, H-4), 3.72
(dd, J ¼ 12.3, 4.4 Hz, 1H, H-6), 3.62 (dd, J ¼ 10.4, 8.7 Hz, 1H, H-3*),
3.58e3.52 (ddd, J ¼ 9.7, 5.9, 2.1 Hz, 1H, H-5*), 3.50 (dd, J ¼ 9.7,
8.7 Hz, 1H, H-4*), 2.11 (s, 6H, 2 ꢁ CH3CO), 2.19e1.98 (m, 2H, 2 ꢁ H-
nium solanesyl phosphate 14 and tetrabutylammonium solanesyl
diphosphate 15 [14/15 z 2/1, 100 mg, 94
evaporated with dry toluene (5 mL) and triethylamine (50
three times] in dry dichloromethane (5 mL) was added 1,10-car-
bonyldiimidazole CDI (85 mg, 525 mol) under argon atmosphere.
After stirring 2 h at room temperature, methanol (100 mol) was
mmol, previously co-
mL)
m
m

M. Bosco et al. / European Journal of Medicinal Chemistry 125 (2017) 952e964
963
added and the mixture was stirred 1 h to destroy the excess of CDI.
Then solvents were removed. The resulting crude activated phos-
phate was solubilized in dry pyridine (2 mL) and was transferred to
activity is defined as cpm FA/(cpm DLO þ cpm FA). Percent inhi-
bition of DLODP activity is defined as 100 ꢂ (100 ꢁ DLODP activi-
tyþinhibitor/DLODP activitycontrol). Alkaline phosphatase was assayed
another flask containing the phosphate 8 [60 mg, 74
previously co-evaporated with dry toluene (5 mL) and triethyl-
amine (50 L) three times]. The reaction mixture was stirred for 7
mmol, 1 eq.,
as previously described. GlcNAc(
Gal( 1,4)GlcNAc were obtained from Dextra Laboratories (Reading,
UK), whereas -methyl mannoside and -methyl mannoside were
b1,4)GlcNAc, Man(b1,4)GlcNAc and
b
m
a
b
days at room temperature. The reaction mixture was concentrated
in vacuo. The crude product was purified by column chromatog-
raphy on silica gel (50 g, CH2Cl2/MeOH/NH4OH 14%, 80/18/2 to 70/
purchased from Toronto Research Chemicals (Toronto, CA).
Cell culture e HepG2 cells (ATCC, Rockville, MD) were cultivated
in RPMI 1640 Glutamax™ medium containing 10% fetal calf serum
and 1% penicillin/streptomycin. The TC7 clone derived from the
colonic adenocarcinoma Caco-2 cell line was a kind gift from Dr.
Monique Rousset [29] and was cultivated in RPMI 1640 Glutamax™
medium containing 10% fetal calf serum and 1% penicillin/strep-
tomycin and 1% non essential amino acids. All cells were cultivated
at 37 ꢀC under an atmosphere containing 5% CO2.
27/3, v/v/v) to furnish acetylated diphosphate (25 mg, 15
25%) and acetylated triphosphate (17 mg, 9.6 mol, 30%).
At room temperature, the acetylated diphosphate (25 mg,
mol) was dissolved in a mixture of methanol (3 mL) and
dichloromethane (3 mL) then a solution of sodium methoxide
(160 L, 0.43 M, 69 mol) was added to the reaction mixture. After
mmol,
m
15
m
m
m
1 h 30 stirring, the reaction was stopped by addition of a cation
exchange resin (DOWEX 50WX8, Hþ form). After 30 min stirring,
the resin was removed by filtration and the resin was washed with
methanol. The filtrate was concentrated in vacuo. The residue was
purified by column chromatography on silica gel (900 mg, CH2Cl2/
MeOH/14% aqueous NH4OH, 80/18/2 to 70/27/3, v/v/v then CH2Cl2/
MeOH/H2O/Et3N, 70/27/2/1, v/v/v/v) to furnish the product 19
Acknowledgments
We gratefully acknowledge la Fondation pour la Recherche
Medicale (FRM: DCM20121225751) for the financial support of this
ꢀ
work and for a post-doctoral fellowship granted to M.B. This work is
supported by the European Union FP6-Coordination Action
EUROGLYCANET (LSHM-CT-2005-512131), the E-Rare-2 Joint
Transnational Call 2011 (EURO-CDG) and institutional funding from
(10 mg, 7.1
m
mol, 47%) as a ditriethyl-ammonium salt. 1H NMR
5.40e5.30 (m, 1H,
(250 MHz, 78% CDCl3, 19.5% CD3OD, 2.5% D2O)
d
H-1), 5.28e5.19 (m, 1H, H-8), 5.00 (t, J ¼ 5.9 Hz, 8H, 7 ꢁ H-12, H-16),
4.41 (d, J ¼ 8.3 Hz, 1H, H-1*), 4.38e4.30 (m, 2H, 2 ꢁ H-7), 3.83e3.28
(m, 12H, H-2, H-5, H-2*, H-5*, H-6, H-60, H-6*, H-6’*, H-3, H-3*, H-4,
H-4*), 3.01 (q, J ¼ 7.2 Hz, 12H, 2 ꢁ (CH3CH2)3NHþ), 2.04e1.78 (m,
38H, 2 ꢁ H-10, 2 ꢁ NCOCH3, 14 ꢁ H-11, 14 ꢁ H-14, 2 ꢁ H-15), 1.55 (s,
6H, CH3-18, CH3-21), 1.48 (s, 24H, 7 xCH3-19, CH3-20), 1.18 (t,
J ¼ 7.2 Hz, 18H, (CH3CH2)3NHþ); 31P NMR (101 MHz, 78% CDCl3,
ꢁ
ꢀ
INSERM, CNRS and the Ministere de l’Enseignement Superieur et de
la Recherche. Ahmad Massarweh is supported by French
a
government/An-Najah National University in Palestine joint
doctoral fellowship. Assia Hessani (Universite Paris Decartes) is
gratefully acknowledged for assistance with low resolution and
high resolution mass spectra analyses. The NMR experiments were
performed at the Interdisciplinary Center for Chemistry and
Biology, Paris.
ꢀ
19.5% CD3OD, 2.5% D2O)
d
ꢂ7.00 (broad s, PeB), -9.16 (broad s, P-A);
ESI-MS Calcd. For C61H101O17N2Pꢂ2 : 1195.7. [MþH]- Found 1196.1;
ESI-HRMS Calcd. For C61H101O17N2Pꢂ2 : 1195.6570. [MþH]- Found
1195.6543.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.10.013.
4.2. Source of undecaprenyl compounds
Undecaprenyl phosphate and undecaprenyl diphosphate were
provided by the Institute of Biochemistry and Biophysics of the
Polish Academy of Sciences, Warsaw, Poland. Undecaprenyl-PP-
GlcNAc was produced by enzymatic synthesis, using the WecA
enzyme [18,19]. Lipid II (Undecaprenyl-PP-MurNAc(-pentapep-
tide)-GlcNAc), was produced by enzymatic synthesis, using the
purified MraY and MurG enzymes as previously described [20,21].
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