Straightforward synthesis of novel Akt inhibitors based on a glucose scaffold

Article abstract of DOI:10.1016/j.carres.2009.12.013

Glucose-based analogues of phosphatidylinositol 3-phosphate were straightforwardly synthesised from 2,3,4,6-tetra-O-acetyl-D-glucosyl bromide as protein kinase B (PKB/Akt) inhibitors. β-D-Glucuronyl diethyl phosphoramidate was identified as a promising hit through biological screening in two different cellular systems. In addition, RNA interference experiments (siRNA) provide evidence of the ability of the compound to exert biological effects specifically through Akt signalling.

Full text of DOI:10.1016/j.carres.2009.12.013

Carbohydrate Research 345 (2010) 1291–1298
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Straightforward synthesis of novel Akt inhibitors based on a glucose scaffold
*
Laura Cipolla , Cristina Redaelli, Francesca Granucci, Giuseppe Zampella, Antonio Zaza, Riccardo Chisci,
Francesco Nicotra
Department of Biotechnology and Biosciences, University of Milano-Bicocca P.za della Scienza 2, 20126 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 October 2009
Received in revised form 26 November 2009
Accepted 13 December 2009
Available online 16 December 2009
Glucose-based analogues of phosphatidylinositol 3-phosphate were straightforwardly synthesised from
2,3,4,6-tetra-O-acetyl-D-glucosyl bromide as protein kinase B (PKB/Akt) inhibitors. b-D-Glucuronyl
diethyl phosphoramidate was identified as a promising hit through biological screening in two different
cellular systems. In addition, RNA interference experiments (siRNA) provide evidence of the ability of the
compound to exert biological effects specifically through Akt signalling.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Sugar phosphoramidates
Enzyme inhibitors
PKB
Glycolipid analogues
Cyclitol analogues
Glucose
1. Introduction
Due to its central role in these processes, Akt has recently
gained great attention as a promising molecular target in cancer6
Monosaccharides provide an excellent platform to tailor molec-
ular diversity by appending desired substituents at selected posi-
tions around the sugar scaffold. The presence of functionalized
and stereocontrolled centres on the sugar scaffolds affords an effi-
cient tool to custom design molecules to a pharmacophore model
towards bioactive compounds.1 Hence, we envisaged the possibil-
ity of using glucose as a scaffold for a straightforward synthesis of
phosphatidylinositol mimics as protein kinase inhibitors.
Protein kinases comprise a large family of enzymes that cata-
lyse the transfer of the terminal phosphate group from ATP (aden-
osine triphosphate) to protein substrates, specifically to the
hydroxyl group of serine or threonine (Ser/Thr kinases) or tyrosine
(Tyr kinases). The serine/threonine protein kinase B (PKB), also
known as Akt,2 phosphorylates diverse protein substrates to pro-
mote diverse cellular responses. For example, the phosphatidylin-
ositol 3-kinase (PI3K)–PKB pathway is responsible for most of the
metabolic actions of insulin.3 In addition, many lines of evidence
demonstrate that Akt is a critical player in the development,
growth,4 and therapeutic resistance5 of cancers. Up-regulation
and increased Akt activity induce oncogenic transformation of cells
and tumour formation in the breast, prostate, ovary and pancreas.
Blockage of Akt signalling then results in apoptosis and growth
inhibition of tumour cells with elevated Akt activity.
and diabetes7 therapy. To date, three members of the Akt family
(Akt1, Akt2 and Akt3) have been identified.1b Structurally, all Akt
isoforms are composed of an N-terminal pleckstrin homology
(PH) domain, a central kinase catalytic domain and a C-terminal
hydrophobic regulatory motif (HM).
Akt can be activated in a phosphatidylinositol 3-kinase (PI3K)-
dependent manner: PI3K phosphorylates the 3-position of phos-
phatidylinositols giving rise to three signalling phospholipids
(PI(3)P’s). Binding of the PH domain of Akt to membrane PI(3)P’s
is fundamental for Akt activation, causing a conformational change
in the protein and its translocation to the plasma membrane; phos-
phorylation of Thr 308 of the activation loop in the kinase domain
and Ser 473 of the hydrophobic motif (HM), completes Akt activa-
tion. Inappropriate activation of the PI3K/Akt pathway has been
linked to the development of several human pathological
states.2c,3b,7b,8
In light of these considerations, widespread efforts towards Akt
inhibitors have recently emerged.9 Part of these drug-development
efforts have been focused on ATP-binding-site inhibitors, such as
some unnatural canthine alkaloids,10 azepane derivatives,11 ‘aktst-
atins’,12 or other classes of compounds.13 However, since the ATP-
binding site is highly conserved in kinases, the design of selective
inhibitors is a formidable challenge.12,13a,d,e A second valuable ap-
proach to Akt-specific inhibitors is the development of substrate–
competitive inhibitors;14 because the substrate-binding domain
is less conserved than the ATP fold, they should be more specific.
Indeed, a substrate–competitive inhibitor was found to be much
* Corresponding author. Fax: +39 02 64483565.
E-mail address: laura.cipolla@unimib.it (L. Cipolla).
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.12.013

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L. Cipolla et al. / Carbohydrate Research 345 (2010) 1291–1298
superior to an ATP mimic.14b More recently, a bisubstrate analogue
has also been designed.15 Allosteric inhibitors have also been pro-
posed.2c,16 An additional approach targets the pleckstrin homology
(PH) domain, and over the past few years a few inhibitors based on
this approach have been developed.17
CH2OAc
CH2OAc
AcO
AcO
c
AcO
AcO
O
O
b
O
P(OR)2
N
R
H
OAc
OAc
R = a-Br
R = b-N3
In this context, we propose a novel class of phosphatidylinositol
(PI) mimics targeted to the PH domain as Akt inhibitors, based on
10
: R = Et, 98%
8:
9:
a
11
12
: R = Bu, 96%
: R = iDecyl 80%
D-glucose as a scaffold (Fig. 1) mimicking the inositol ring: the D-
glucopyranose structure is a good bioisostere of the myo-inositol
moiety, as already proposed by other research groups.18 The pro-
posed inhibitors can be easily obtained in only four synthetic steps
from the commercially available 2,3,4,6-tetra-O-acetyl-D-glucopyr-
anosyl bromide.
CH2OH
d
COOH
HO
HO
O
O
HO
HO
O
O
P(OR)2
N
H
P(OR)2
N
H
OH
2. Results and discussion
OH
2: R = Et, 98%
3: R = Bu, 90%
4: R = iDecyl 98%
5:
R = Et, 54%
6:
7:
R = Bu, 55%
R = iDecyl 30%
The designed inhibitors (2–7, Fig. 1) have four main features: (i)
the metabolically labile phosphate ester linkage between the ino-
sitol ring and the diacylglycerol moiety is substituted by a phos-
Scheme 1. Reagents: (a) TBAHS, NaHCO3, NaN3, CH2Cl2, 93%; (b) P(OR)3, CH2Cl2; (c)
Na, MeOH; (d) TEMPO, KBr, NaHCO3, NaOCl.
phoramidate group as
a stable phosphate mimic. (ii) The
phosphate group in position 3 of the inositol ring (position 5 of
the sugar) is substituted by a hydroxymethylene or a carboxylic
group; these groups should highlight the relevance of an acidic
moiety for biological activity. (iii) As the degradation of inositol
phospholipids occurs through the enzymatic action of PI-specific
phospholipase C (PIPLC) with the formation of a 1,2-cyclic phos-
phate,19 deletion of the inositol 2-OH group to block the formation
of the cyclic phosphate is a common feature of the synthesized
mimetics and carbon-2 is replaced by the endocyclic oxygen of
the glucopyranose ring. (iv) Finally, lipophilic acyl chains of the
diacyl glycerol moiety have been mimicked by hydrophobic groups
of different sizes, reasoning that the long chains are not needed for
enzyme recognition, but only for membrane anchoring.17b
nium hydrogen sulfate (TBAHS) and sodium azide.20 Alkyl
phosphoramidates 10–12 were synthesized by Staudinger reaction
of azide 9 with the corresponding trialkyl phosphite, as reported by
Kannan and co-workers who proposed the synthesis of several gly-
cosyl phosphoramidates as isosteric analogues of native glycosyl
phosphates.21 All products were obtained with total stereoselec-
tion at the anomeric position as determined by 1H NMR. For all
compounds coupling constants between H-1 and H-2 (around
9.0 Hz) typical for a trans-diaxial arrangement of the substituents,
indicated a b-orientation of the azido group in a 4C1 chair confor-
mation. 31P NMR of derivatives 2–7 confirmed the presence of
the NH–P@O group in which the phosphorous atom resonates at
approximately 8–10 ppm. Deprotection of phosphoramidates 10–
12 under Zèmplen conditions (cat. Na, MeOH) afforded compounds
2–4, which were selectively oxidised at the primary hydroxyl
The synthesis of the potential inhibitors 2–7 is straightforward
from 2,3,4,6-tetra-O-acetyl-D-glucosyl bromide (8), Scheme 1. The
b- -glucopyranosyl azide 9 was prepared by the reaction of gluco-
D
syl bromide 8 under phase transfer catalysis with tetrabutylammo-
O
OH
2-O3PO
O
2
HO
HO
3
O
O
O
P
O-
1
HO
O
1)
PtdIns3P (
O
OEt
P
O
OBu
H
H
N
OEt
N
OBu
2
5
3
: R1 =
: R1 =
P
O
R2= CH2OH
R2= COOH
Oidec
P
O
H
N
Oidec
4: R1 =
R2
5
O
HO
HO
R1
1
OEt
H
N
OBu
P
HO
H
N
6
: R1 =
: R1 =
OEt
OBu
P
glucose scaffold
O
O
OiDec
P
O
H
N
OiDec
7: R1 =
iDec =
Figure 1. Structure of phosphatidylinositol-3-phosphate (1) and its mimics.

L. Cipolla et al. / Carbohydrate Research 345 (2010) 1291–1298
1293
group
by
2,2,6,6-tetramethyl-1-piperidinyloxyl/hypochlorite
With the aim of disclosing key atomic details related to the
binding of the synthesised phosphatidylinositol-3-phosphate ana-
logues, a set of docking simulations was carried out on most active
compounds 2 and 5. The adopted docking protocol (see Section 3),
which was already used for the investigation of ligand–receptor
interactions,23 has been initially validated on the X-ray structure
of the complex between Akt PH domain and PtdIns(3,4,5)P3. The
relative stability data obtained by molecular modelling studies
on compounds 2 and 5 are summarized in Table 1.
According to this computational analysis, binding of the uronic
acid 5 is favoured compared to the corresponding glucose derivative
2 (Fig. 3), in nice agreement with preliminary in vitro assays. Indeed,
both compounds bind to Akt in a similar fashion (Fig. 3) with the glu-
cose OH groups involved in H-bond interactions with Lys14, Arg25
and Arg86 of the pleckstrin homology domain of Akt. Moreover,
the phosphoramidate group interacts, via H-bond, with Glu17. The
higher affinity of 5 can be essentially ascribed to the chelating inter-
action between the carboxylic group and Arg23 (Fig. 3B), which is
weaker and partially lost in the –CH2OH analogues (Fig. 3A). It is also
worth notingthat, on the basis of the docking simulations, the length
of the alkyl chains in the phosphoramidate group has no relevant ef-
fects on binding, as already reported by Kozikowski and co-work-
ers.17b These results are in agreement with molecular modelling
studies of the natural substrates and inositol mimics already
reported.17b
(TEMPO),22 to the corresponding uronates (5–7).
All compounds 2–7 were assayed for in vitro inhibitory activity
against Akt1, using an in vitro Kinase Assay Kit (StressXpressÒ
non-radioactive Akt/PKB Kinase activity),Figure 2A. These prelimin-
ary data show that phosphoramidate 5 is the most active derivative
among the synthesized compounds, followed by glucose diethyl
phosphoramidate 2; it is worth noting that uronic acid 5 is twice
as active as the parent glucose derivative 2. It can also be noted that
the uronic acids are more active than the corresponding glucose
counterparts, suggesting that the carboxylic group may be relevant
for interactions of the putative inhibitors with the binding site.
ELISA tests and molecular modelling studies highlighted com-
pound 5 as a good hit as an Akt inhibitor. Hence, in vitro activity
on dendritic cells (DCs) as a cellular system was also carried out,
since it has been recently reported that Akt1 has an essential role
in DC function.24 DCs are a special class of leucocytes that play a
fundamental role in regulating innate and adaptive immune re-
sponses.25 They can be activated following encounter with micro-
bial cell products that signal through specific receptors called toll-
like receptors (TLRs).26,27 TLR engagement leads to nuclear factor
(NF)-kB activation through the MyD88-dependent pathway28 and
also through the activation of the phosphatidylinositol-3-kinase
(PI3K) that phosphorylates its downstream target Akt. Inhibition
of Akt phosphorylation leads to partial DC activation and reduced
DC functionality.21 To test the inhibitory activity of compound 5
we took advantage of the well-characterized murine DC line
D1,29 which can undergo maturation in vitro upon encountering
of microbial cell products, such as lipopolysaccharides (LPS), lead-
ing to the activation of PI3K/Akt pathway, required to have effi-
cient IL-2 production.30
The ability of compound 5 to inhibit LPS-induced Akt phosphor-
ylation in D1 cells was investigated by both Western blot and cyto-
fluorimetric analysis. In particular, D1 cells were pretreated with
compound 5 for 1 h and then incubated with LPS (1 lg/mL) for an
additional 20 min. Immunoblotting experiments (Fig. 2B) were per-
formed using a Ser473 phospho-specific antibody, while cytofluori-
metric analyses (Supplementary data, Fig. S1) were performed using
a Thr308 phospho-specific antibody. As shown in Figure 2B, com-
pound 5 inhibited Akt phosphorylation in a dose-dependent manner
(IC50 97 lM). To assess whether compound 5 could be cytotoxic, tox-
icity was investigated with the annexin V test. D1 cells did not show
any appreciable percent of apoptotic cells after 24-h incubation with
Figure 2. Biological activity of compound 5. Bar heights indicate the means SEM,
determined from at least three independent experiments. (A) In vitro Akt Kinase
Assay (ELISA) of compounds 2–7, 1L6-hydroxymethyl-chiro-inositol-2-(R)-2-O-
methyl-3-O-octadec-yl-sn-glycerocarbonate17c was used as the positive control
(CTR). Statistical analysis was done using the one-way ANOVA followed by Tukey’s
*
test as post hoc comparison; P <0.05 versus CTR; (B) Western blot analysis showing
the inhibitory activity of compound 5 with D1 cells at the indicated concentrations
(lg/mL); Wortmannin (W) was used as the positive control. The protein levels of
phosphorylated Akt (P-Akt) and Akt were determined using monoclonal antibodies
anti-phospho-Ser473-Akt; (C) IL-2 levels in the supernatant of D1 cells cultured for
Table 1
Relative stability data (kcal/mol), which correspond to (Edocking Ligand ꢀ Edocking
)
5
related to the binding of the inhibitors 2 and 5 to the PH domain
Ligands
Relative stability
24 h in the presence of LPS with the indicated amounts (lg/mL) of compound 5.
Wortmannin (W) was used as the positive control. Statistical analysis was done
using the Kruskal–Wallis ANOVA for non-parametric values followed by Dunns test
2
5
+16.3
0
**
*
as post hoc comparison. P <0.001 versus LPS, P <0.05 versus LPS.

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L. Cipolla et al. / Carbohydrate Research 345 (2010) 1291–1298
Figure 3. 3-D view of the binding mode of 2 (A) and 5 (B) to the PH domain. Relevant interatomic distances are shown in Å.
LPS and compound 5 at the concentration showing maximal inhibi-
tory activity (data not shown).
maturation state was analysed by evaluating the up-regulation
of the co-stimulatory molecule B7.2 and the ability of D1 cells
to produce inflammatory cytokines was tested by evaluating
IL-2 concentration in the supernatant. Wortmannin was always
used as the positive control. Similarly to wortmannin, compound
5 did not block the up-regulation of B7.2 induced by LPS (data
not shown) and inhibited IL-2 production in a dose-dependent
manner (Fig. 2C).
Further biological investigations were carried out to best
characterize the biological activity of compound 5. In particular,
inhibition of cell maturation and cytokine production induced by
LPS was analysed. To this end, D1 cells were pre-treated with
increasing amounts of the inhibitor 5 for 1 h and then cultured
in the presence of LPS (1 lg/mL) for the remaining 24 h. D1 cell

L. Cipolla et al. / Carbohydrate Research 345 (2010) 1291–1298
1295
Figure 4. Effects of compound 5 on twitch amplitude on not-transfected and Akt1-silenced cardiomyocytes. Representative twitch recordings during superfusion of control
and compound solutions on not-transfected cells (A) and myocytes silenced for Akt1 (B). (C) Statistical analysis of twitch amplitude calculated for NT and Tr cells (NT: n = 6;
CTR = 159.8 53.2; Cpd = 187.9 54.6; iRNA: n = 8; CTR = 279.5 23.7; Cpd = 283.3 25.3; *P <0.05 vs CTR).
Recent studies showed that PI3K/Akt pathway is also involved in
cardiomyocytes function.31 To obtain preliminary information on
the cardiac effect of inhibitor 5, we studied its effect on contraction
(unloaded twitch) of adult rat ventricular myocytes during field
stimulation at 2 Hz (Figs. 4 and S2, Supplementary data). This type
of measurement is suitable to detect mechanical abnormalities as
well as arrhythmias ensuing during short-lasting exposure to drugs.
3. Experimental section
3.1. Synthesis, general procedures
Reactions were carried out using the commercially available
starting materials and solvents without further purification. All sol-
vents were dried over molecular sieves, for at least 24 h prior to use.
When dry conditions were required, the reaction was performed un-
der Ar atmosphere. Thin-layer chromatography (TLC) was per-
formed on Silica Gel 60 F254 plates (Merck) charring with a
solution containing concd H2SO4/EtOH/H2O in a ratio of 5:45:45 or
with an oxidant mixture composed of (NH4)Mo7O24 (21 g), Ce(SO4)2
(1 g), concd H2SO4 (31 mL) in water (500 mL). Flash column chroma-
tography was performed on silica gel 230–400 mesh (Merck). NMR
spectra were recorded at 400 MHz (1H) 100.57 MHz (13C) and
162.01 MHz (31P) on a Varian Mercury instrument. Chemical shifts
are reported in ppm downfield from TMS as an internal standard; J
values are given in hertz. For all compounds assignments of the 1H
NMR spectra were based on 2D proton–proton shift-correlation
spectra. The assignments of 13C NMR spectra were based on car-
bon–proton shift-correlation spectra. Carbon signals of the C10
chains of the phosphoramidate moiety have been omitted in the car-
bon spectra descriptions. Mass spectra were recorded on a MALDI2
Kompakt Kratos instrument, with gentisic acid (DHB) as the matrix.
At 50 lM, compound 5 reversibly enhanced twitch amplitude
(Fig. 4A; P <0.05), without inducing unstimulated contractions or
other signs of acute toxicity. To test whether the inotropic effect of
compound 5 depended on PH-domain antagonism, we repeated
the experiment after silencing the expression of Akt1, the main ki-
nase activated through PH-domain interaction, by RNA interference.
As shown inFigure 4B, compound 5 effect was completely abolished
in Akt1-silenced myocytes, thus suggesting that it indeed occurred
through PH-domain antagonism and, at the same time, proving the
involvement of Akt signalling pathway.
The positive effect of compound 5 on cardiomyocyte contractility
was unexpected and suggests the absence of acute cellular toxicity.
Nevertheless, depending on their nature, cellular mechanisms res
ponsible of acute inotropy may lead to cell damage if sustained
chronically (e.g., cell Ca2+ overload). Therefore, further experiments
will be required to assess the mechanism of compound 5 inotropic
effect and rule out that it may result in myocardial damage during
long-term administration.
2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl azide (9) was synthe-
In conclusion, this work shows a novel class of kinase inhibitors,
based on a glucose scaffold straightforwardly obtained from the
commercially available 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl
sized from the corresponding peracetylated glycosyl bromide (8)
under phase transfer catalysis as described by Tropper and co-
workers.20
bromide in only four synthetic steps. Preliminary biological data
on dendritic cell and cardiomyocytes systems identified compound
5 as a promising lead. In addition, the abolition of compound 5 ef-
fects by Akt1 silencing provides further evidence of the ability of
the compound to exert biological effects through the modulation
of PH-domain activated signalling. Further biological studies and
lead optimization are underway and will be reported in due course.
3.2. General Procedure for glycosyl phosphoramidates
synthesis (10–12)
To a solution of 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl azide
(9) (1.0 equiv) in dry dichloromethane (CH2Cl2) trialkyl phosphite
(2.0 equiv) was added dropwise. The reaction mixture was allowed

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L. Cipolla et al. / Carbohydrate Research 345 (2010) 1291–1298
to stir overnight at rt, then concentrated to dryness. The crude was
purified as described below.
19.10, 19.08 (2t, 2 (CH2)2CH2CH3), 14.03, 14.00 (2t, 2 (CH2)3CH3);
31P NMR (162.01 MHz, CDCl3): d 6.47. MALDI-MS m/z [M+Na]+,
562; [M+K]+, 578. Elemental Anal. Calcd (%) for C22H38NO12P: C,
48.98; H, 7.10; N, 2.60; P, 5.74. Found: C, 48.95; H 7.12; N 2.59; P,
5.72.
3.3. General procedure for Zèmplen methanolysis32
To a solution of the per-O-acetylated phosphoramidates (10–
12) (1.0 equiv) in dry methanol (MeOH) catalytic amount of metal-
lic sodium was added. The reaction mixture was allowed to stir till
completion (2–5 h), then Amberlite IR 120 H+ was added till solu-
tion neutrality. The resin was filtered off and the solvent was evap-
orated to dryness affording phosphoramidates (2–4).
3.7. 2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl diisodecyl
phosphoramidate (12)
The crude obtained as described in the general procedure was
first washed with petroleum ether, then purified by flash chroma-
tography (eluent: 4:6 petroleum ether/EtOAc). Yellow oil, yield
80%. 1H NMR (400 MHz, CDCl3) d 5.23 (t, 1H, J = 9.5 Hz, H-3), 5.02
(t, 1H, J = 9.5 Hz, H-4), 4.85 (t, 1H, J = 9.4 Hz, H-2), 4.55 (dd, 1H,
J = 19.4, 8.8 Hz, H-1), 4.24 (dd, 1H, J = 11.8, 3.4 Hz, H-6a), 4.09–3.96
(m, 5H, H-6a, OCH2(CH2)6CH(CH3)2), 3.73 (ddd, 1H, J = 10.0, 4.2,
1.8 Hz, H-5), 3.51 (t, 1H, J = 10.4 Hz, NH), 2.06, 2.04, 2.02, 2.00 (4s,
12H, 4COCH3), 1.73–0.72 (br m, 38H, 2 CH2(CH2)6CH(CH3)2). 13C
NMR (100.57 MHz, CDCl3) d 170,64, 170.61, 170.03, 169.65 (4s,
COCH3), 83.05 (d, C-1), 73.45 (d, C-5), 73.02 (d, C-3), 71.77 (d,
3.4. General procedure for uronates synthesis (5–7)
To a solution of glucosyl phosphoramidate (2–4) (1.0 equiv) in
satd aq NaHCO3, 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO,
0.1 equiv) in CH3CN (0.08 M) and KBr (0.1 equiv) in satd aq NaHCO3
(0.05 M) were added at 0 °C; then a solution of 5% aq NaOCl
(1.25 equiv) was added dropwise. The reaction mixture was allowed
to stir at 15–18 °C. After 2 h additional 5% aq NaOCl (1.25 equiv) was
addeddropwiseandthereactionmixtureallowedtostirovernightat
rt The reaction mixture was made acidic with 5% HCl in EtOH, then
the solvent evaporated to dryness. The residue was dissolved in eth-
anol, the precipitate filtered off, and the filtrate was evaporated un-
der reduced pressure. The crude was finally purified by flash
chromatography with suitable eluents.
3J(C,P) = 9.9 Hz, C-2), 68.63 (d, C-4), 66.70, 66.10 (2t,
2
CH2(CH2)6CH(CH3)2), 62.23 (t, C-6), 21.06 (4q, 4 CH3CO); 31P NMR
(162.01 MHz, CDCl3):
d
10.36. MALDI-MS m/z [M+Na]+, 730
[M+K]+, 746. Elemental Anal. Calcd (%) for C34H62NO12P: C, 57.69;
H, 8.83; N 1.98; P, 4.37. Found: C, 57.74; H, 8.85; N, 1.99; P, 4.36.
3.8. b-D-Glucopyranosyl diethyl phosphoramidate (2)
3.5. 2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl diethyl
phosphoramidate (10)
Amorphous white solid, yield 96%. 1H NMR (400 MHz, D2O) d
4.09 (t, 1H, J = 9.0 Hz, H-1), 3.98–3.91 (br s, 4H, CH2CH3), 3.67
(dd, 1H, J = 12.1, 1.4 Hz, H-6a), 3.52 (dd, 1H, J = 12.1, 4.7 Hz, H-
6b), 3.30 (t, 1H, J = 9.0 Hz, H-3), 3.23–3.15 (m, 2H, H-4,H-5),
3.06 (t, 1H, J = 9.0 Hz, H-2), 1.12 (t, 6H, J = 7.0 Hz, CH2CH3). 13C
NMR (100.57 MHz, D2O) d 86.37 (d, C-1), 79.83 (d, C-5), 79.21
(d, C-3), 76.09 (d, 3J(C,P) = 9.2 Hz, C-2), 72.21 (d, C-4), 67.01,
66.87 (2t, 2 CH2CH3), 63.44 (d, C-6), 18.35, 18.29 (2q, 2 CH2CH3).
31P NMR (162.01 MHz, D2O): d 9.95. MALDI-MS m/z [M+H]+, 316;
[M+Na]+, 338 [M+K]+, 354; [M+K]. Elemental Anal. Calcd (%) for
The crude obtained as described in the general procedure was
first washed with petroleum ether, then purified by recrystalliza-
tion from 4:1 EtOAc–petroleum ether affording the desired per-
O-acetylated phosphoramidate (10). Amorphous white solid, yield
98%. 1H NMR (400 MHz, CDCl3) d 5.24 (t, 1H, J = 9.5 Hz, H-3), 5.01
(t, 1H, J = 9.6 Hz, H-4), 4.85 (t, 1H, J = 9.3 Hz, H-2), 4.54 (dd, 1H,
J = 19.4, 8.5 Hz, H-1), 4.21 (dd, 1H, J = 12.2, 4.7 Hz, H-6a), 4.11–
4.00 (m, 5H, 2 OCH2CH3, H-6b), 3.81–3.68 (m, 1H, H-5), 3.58 (t,
1H, J = 10.6 Hz, NH), 2.06, 2.05, 2.02, 2.00 (4s, 12H, CH3CO), 1.30–
0.90 (m, 6H, OCH2CH3). 13C NMR (100.57 MHz, CDCl3) d 170.63,
170.61, 170.02, 169.66 (4s, 4 COCH3), 82.95 (d, C-1), 73.45 (d, C-
5), 73.02 (d, C-3), 71.79 (d, 3J(C,P) 9.7 Hz, C-2), 68.76 (d, C-4),
63.00, 62.92 (2t, 2 CH2CH3), 62.30 (t, C-6), 21.15, 21.10, 21.04,
21.03 (4q, 4 COCH3), 16.61, 16.53 (2q, 2 CH2CH3). 31P NMR
C10H22NO8P: C, 38.10; H, 7.03; N, 4.44; P, 9.82. Found: C,
38.16; H, 7.05; N, 4.43; P, 9.79.
3.9. b-D-Glucopyranosyl dibutyl phosphoramidate (3)
Yellow oil, yield 90%. 1H NMR (400 MHz, D2O) d 4.10 (t, 1H,
J = 8.7 Hz, H-1), 3.96–3.89 (m, 4H, CH2(CH2)2CH3), 3.69 (dd, 1H,
J = 12.1, 1.5 Hz, H-6a), 3.55 (dd, 1H, J = 12.2, 4.6 Hz, H-6b), 3.32 (t,
1H, J = 8.9 Hz, H-4), 3.23–3.19 (m, 2H, H-3, H-5), 3.08 (t, 1H,
J = 8.9 Hz, H-2), 1.53–1.46 (m, 4H, CH2CH2CH2CH3), 1.27–1.18 (m,
4H, (CH2)2CH2CH3), 0.75 (t, 6H, J = 7.4 Hz, (CH2)3CH3). 13C NMR
(100.57 MHz, D2O) d 88.43 (d, C-1), 81.99 (d, C-5), 81.74 (d, C-3),
78.13 (d, 3J(C,P) = 9.7 Hz, C-2), 74.24 (d, C-4), 70.55, 70.38 (2t, 2
CH2(CH2)2CH3), 65.56 (t, C-6), 36.41, 36.34 (2t, 2 CH2CH2CH2CH3),
22.79 (2t, 2 (CH2)2CH2CH3), 16.97 (2q, 2 (CH2)3CH3). 31P NMR
(162.01 MHz, D2O): d 10.05. MALDI-MS m/z [M+H]+, 372; [M+Na]+,
394; [M+K]+, 410. Elemental Anal. Calcd (%) for C14H30NO8P: C,
45.28; H, 8.14; N, 3.77; P, 8.34. Found: C, 45.32; H, 8.16; N, 3.78; P,
8.36.
(162.01 MHz, CDCl3):
d
6.35. MALDI-MS m/z [M+Na]+, 506;
[M+K]+, 522. Elemental Anal. Calcd (%) for C18H30NO12P: C, 44.72;
H, 6.25; N, 2.90; P, 6.41. Found: C, 44.75; H, 6.24; N, 2.89; P, 6.43.
3.6. 2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl dibutyl
phosphoramidate (11)
The crude obtained as described in the general procedure was
first washed with petroleum ether, then purified by recrystallization
from 4:1 EtOAc–petroleum ether. White amorphous solid, yield 96%.
1H NMR (400 MHz, CDCl3) d 5.19 (t, 1H, J = 9.5 Hz, H-3), 4.98 (t, 1H,
J = 9.7 Hz, H-4) 4.82 (t, 1H, J = 9.4 Hz, H-2), 4.50 (ddd, 1H, J = 11.2,
9.1, 7.8 Hz, H-1), 4.18 (dd, 1H, J = 12.3, 4.7 Hz, H-6a), 4.04 (dd, 1H,
J = 12.3, 2.2 Hz, H6b), 3.99–3.86 (m, 4H, OCH2(CH2)2CH3), 3.70
(ddd, 1H, J = 10.1, 4.7, 2.2 Hz, H-5), 3.61 (t, 1H, J = 10.6 Hz, NH),
2.01,2.00, 1.98, 1.96 (4s, 12H, COCH3), 1.63–1.54 (m, 4H,
OCH2CH2CH2CH3), 1.38–1.28 (m, 4H, O(CH2)2CH2CH3), 0.95, 0.88
(2t, 6H, J = 7.3, Hz, O(CH2)3CH3). 13C NMR (100.57 MHz, CDCl3) d
170.57, 170.57, 169.99, 169.56 (4s, 4 COCH3), 82.96 (d, C-1), 73.37
(d, C-5), 73.01 (d, C-3), 71.72 (d, 3J(C,P) = 9.5 Hz, C-2), 68.65 (d, C-
4), 66.83, 66.67 (2t, 2 CH2(CH2)2CH3), 62.27 (t, C-6), 32.53, 32.53
(2t, 2 CH2CH2CH2CH3), 21.06, 21.06, 21.00, 21.00 (4t, 4 CH3CO),
3.10. b-D-Glucopyranosyl diisodecyl phosphoramidate (4)
Yellow oil, yield 96%. 1H NMR (400 MHz, DMSO) d 5.59 (t, 1H,
J = 11.3 Hz, NH), 3.97–3.85 (m, 5H, H-1, OCH2 (CH2)6CH(CH3)2),
3.60 (dd, 1H, J = 11.3, 4.8 Hz, H-6a), 3.41 (dd, 1H, J = 11.6, 5.6 Hz,
H-6b), 3.10–2.95 (m, 4H, H-3, H-4, H-5, H-2), 1.68–1.02 (br m, 26H,
2 CH2(CH2)6CH(CH3)2), 0.88–0.58 (br m, 6H, 2 CH2(CH2)6CH(CH3)2).
13C NMR (100.57 MHz, CDCl3) d 89.82 (d, C-1), 83.39 (d, C-5), 83.07

L. Cipolla et al. / Carbohydrate Research 345 (2010) 1291–1298
1297
d, (C-3), 79.15 (d, 3J(C,P) = 9.9 Hz, C-2), 75.92 (d, C-4), 70.81, 69.28
(2t, 2 CH2(CH2)6CH(CH3)2), 66.90 (t, C-6). 31P NMR (162.01 MHz,
CDCl3): d 8.82. MALDI-MS m/z [M+Na]+, 562; [M+K]+, 578. Elemental
Anal. Calcd (%) for C26H54NO8P: C, 57.86; H, 10.08; N, 2.59; P, 5.74.
Found: C, 57.89; H, 10.10; N, 2.61; P, 5.72.
force field. The MMFF94s force field has been already shown to
be well suited to study protein–ligand interactions when ligands
are organic derivatives of carbohydrates.35 (iii) An extensive
systematic search on the torsion phase space of the ligands has
been carried out to locate the global energy minimum for each li-
gand. (iv) The potential energy surface describing protein–ligand
interaction has been sampled using Autodock, according to the
previous studies.36 The best 40 poses of the ligand in the active site
have been considered for the refinement steps. (v) Each of the 40
protein–ligand complexes obtained at point 4 has been used as
an input of a MM optimization using the MMFF94s forcefield.
The ligand and all protein atoms within 10 A from the ligand has
been allowed to change their position during optimization. The
remainder of protein atoms has been held fixed. (vi) The protein–
ligand configurations obtained after MM optimization have been
used as an input of simulated annealing (SA) simulations, carried
out using two subsequent temperature ramps, in order to avoid
freezing of the system in local metastable minima. In the first tem-
perature ramp the molecular system has been gradually (50 ps)
warmed up to 800 K. Then, the system was subjected to 1000 ps
of classical Molecular Dynamics according to the NVT ensemble. Fi-
nally, the system has been slowly brought down to 5 K (500 ps)
and an MM energy minimization has been started from the last tra-
jectory frame. Further SA cycles (up to 5) were carried out on each
system to confirm that the adopted procedure led to exhaustive
potential energy function sampling. Edocking related to the forma-
tion of the protein–ligand complex has been calculated according
to the following equation:
3.11. b-D-Glucuronyl diethyl phosphoramidate (5)
Uronate 5 obtained as described in the general procedure was
purified by flash chromatography (eluent: 1:1 CHCl3/MeOH + 0.1%
AcOH). Hygroscopic amorphous white solid, yield 54%. 1H NMR
(400 MHz, D2O) d 4.24 (t, 1H, J = 9.0 Hz, H-1), 4.12–4.05 (m, 4H,
CH2CH3), 3.67 (d, 1H, J = 9.4 Hz, H-5), 3.52–3.43 (m, 2H, H-3, H-
4), 3.27 (t, 1H, J = 8.6 Hz, H-2), 1.27–1.20 (m, 6H, CH2CH3). 13C
NMR (100.57 MHz, D2O) d 178.90 (s, C-6), 86.20 (d, C-1), 80.66
(d, C-5), 78.94 (d, C-3), 75.92 (d, 3J(C,P) = 9.9 Hz, C-2), 74.65 (d, C-
4), 67.19, 67.10 (2t, 2 CH2CH3), 18.26, 18.23 (2t, 2 CH2CH3). 31P
NMR (162.01 MHz, D2O): d 9.92. MALDI-MS m/z [M+H]+, 330;
[M+Na]+, 352; [M+K]+, 368. Elemental Anal. Calcd (%) for
C10H20NO9P: C, 36.48; H, 6.12; N, 4.25; P, 9.41. Found: C, 36.45;
H, 6.10; N, 4.26; P, 9.38.
3.12. b-D-Glucuronyl dibutyl phosphoramidate (6)
Uronate 6 obtained as described previously was purified by
flash chromatography (eluent: CHCl3/MeOH/H2O 1:1:0.1). Amor-
phous white solid, yield 55%. 1H NMR (400 MHz, D2O) d 4.08 (t,
1H, J = 8.7 Hz, H-1), 3.96–3.88 (m, 4H, CH2(CH2)2CH3), 3.52 (d,
1H, J = 9.3 Hz, H-5) 3.37–3.31 (m, 2H, H-3, H-4), 3.13 (t, 1H,
J = 8.8 Hz, H-2), 1.51–1.46 (m, 4H, CH2CH2CH2CH3), 1.26–1.18 (m,
4H, (CH2)2CH2CH3), 0.77–0.72 (t, 6H, J = 7.4 Hz, (CH2)3CH3). 13C
NMR (100.57 MHz, D2O) d 178.40 (s, C-6), 86.18 (d, C-1), 80.85
(d, C-5), 78.96 (d, C-3), 75.95 (d, 3J(C,P) = 10.0 Hz, C-2), 74.69 (d,
C-4), 70.53, 70.43 (2t, 2 CH2(CH2)2CH3), 34.43, 34.36 (2t, 2
CH2CH2CH2CH3), 21.09, 21.07 (2t, 2 (CH2)2CH2CH3), 15.79, 15.79
(2q, 2 (CH2)3CH3). 31P NMR (162.01 MHz, D2O): d 10.01. MALDI-
MS m/z [M+Na]+, 408; [M+K]+, 424. Elemental Anal. Calcd (%) for
Edocking ¼ EPL ꢀ EP ꢀ EL
being the terms on the right-hand side representative of minima on
the potential energy surface (PES) of the protein–ligand system (PL),
the sole protein (P) and the ligand (L).
Acknowledgement
We gratefully acknowledge C.I.N.M.P.I.S. for financial support.
C
14H28NO9P: C, 43.64; H, 7.32; N, 3.63; P, 8.04. Found: C, 43.67;
Supplementary data
H, 7.34; N, 3.62; P, 8.02.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2009.12.013.
3.13. b-D-Glucuronyl diisodecyl phosphoramidate (7)
Uronate 7 obtained as described previously was purified by
flash chromatography (eluent: CHCl3/MeOH 1:0.2?CHCl3/MeOH
1:1). Yellow oil, yield 54%. 1H NMR (400 MHz, DMSO) d 5.59 (t,
1H, J = 11.3 Hz, NH), 4.00–3.92 (m, 5H, H-1, OCH2(CH2)6CH(CH3)2),
3.23 (d, 1H, J = 9.6 Hz, H-5), 3.21–3.12 (m, 2H, H-3, H-4),3.00 (t, 1H,
J = 9.5 Hz, H-2), 1.57–1.04 (br m, 26H, 2 CH2(CH2)6CH(CH3)2), 0.84–
0.58 (br, 6H, 2 CH2(CH2)6CH(CH3)2). 13C NMR (100.57 MHz, DMSO)
d 178.43 (s, C-6), 89.33 (d, C-1), 82.52 (d, C-3), 80.23 (d, C-5), 78.65
(d, 3J(C,P) = 10.7 Hz, C-2), 77.36 (d, C-4), 67.53, 65.43 (2t, 2
CH2(CH2)6CH(CH3)2). 31P NMR (162.01 MHz, DMSO): d 9.42. MAL-
DI-MS m/z [M+Na]+, 576; [M+K]+, 592. Elemental Anal. Calcd (%)
for C26H52NO9P: C, 56.40; H, 9.47; N, 2.53; P, 5.59. Found: C,
56.45; H, 9.49; N, 2.54; P, 5.60.
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