Synthesis and conformational analysis of a novel carbohydrate-fused bis-crown ether: crown-CyPLOS

Article abstract of DOI:10.1016/j.tet.2009.09.100

A novel sugar-based macrocycle consisting of a phosphate-linked 12-membered disaccharide ring (cyclic phosphate-linked oligosaccharide, CyPLOS), fused to two 18-crown-6 ether residues, is here described. The synthesis of the target compound has been accom

Full text of DOI:10.1016/j.tet.2009.09.100

Tetrahedron 65 (2009) 9694–9701
Contents lists available at ScienceDirect
Tetrahedron
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Synthesis and conformational analysis of a novel carbohydrate-fused bis-crown
ether: crown-CyPLOS
,
Cinzia Coppola a, Ada Virno b, Lorenzo De Napoli a, Antonio Randazzo b, Daniela Montesarchio a
*
a
`
Dipartimento di Chimica Organica e Biochimica, Universita di Napoli ‘Federico II’, Via Cintia, 4, I-80126 Napoli, Italy
b
`
Dipartimento di Chimica delle Sostanze Naturali, Universita di Napoli ‘Federico II’, Via D. Montesano 48, I-80131 Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 June 2009
Received in revised form
10 September 2009
Accepted 24 September 2009
Available online 30 September 2009
A novel sugar-based macrocycle consisting of a phosphate-linked 12-membered disaccharide ring (cyclic
phosphate-linked oligosaccharide, CyPLOS), fused to two 18-crown-6 ether residues, is here described.
The synthesis of the target compound has been accomplished in 23% overall yield for 11 reaction steps,
exploiting phosphoramidite chemistry for the dimerization and a classical phosphotriester methodology
for the cyclization reaction. NMR-based conformational analysis studies have been carried out on the
fully deprotected macrocycle, showing a characteristic arched-structure with C2-symmetry.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Chemical synthesis
Macrocycles
Carbohydrates
Crown ether derivatives
NMR
1. Introduction
compounds we named CyPLOS (Cyclic Phosphate-Linked OligoSac-
charides), preferentially adopts a concave conformation, potentially
In order to achieve efficient recognition of a specific guest, most
host agents in supramolecular chemistry exhibit pre-organized con-
formations, ensured by a well-defined three-dimensional pre-
sentation of the interacting moieties.1 In the design of effective
artificial hosts, the presence of chiral, amphiphilic and cyclic struc-
tural motifs is highly desirable, and cyclodextrins, calixarenes, cyclic
peptides are typical examples in this frame. Among the most efficient
and selective receptors for cations are crown ethers, which, in con-
trast, exhibit an achiral, rather simple backbone. Since the pioneering
work of Pedersen,2 crown ethers have attracted a great deal of at-
tention over the last three decades, and a large number of diverse
derivatives have been designed and synthesized for a variety of su-
pramolecular applications. Bis- or poly-crown ether compounds have
proven to be much more effective extraction agents - and more active
transmembrane ion channels or mobile carriers - than their mono-
meric counterparts.3 Relevant examples are, among others, crown
ether-modified cyclodextrins,4 calixarenes5 and resorcinarenes,6 as
well as the synthetic ionophores developed by Gokel and co-
workers.7
able to bind metal ions.
HO
O
OH
PhO
O
O
P
O-
O
O
P
O
O
O
OPh
-O
HO
OH
1
Figure 1. Chemical structure of macrocycle 1.
It was originally envisaged that 1 would provide a useful syn-
thetic platform for the construction of new artificial receptors for
transmembrane ion transport: the hydroxy groups at C-2 and C-3 of
each saccharide residue can in fact be exploited as synthetic han-
dles for the attachment of suitable lipophilic appendages assuring
the insertion into the hydrophobic core of lipid systems. Following
this design, by covalently linking alkyl or polyether linear tentacles
onto the disaccharide macrocycle, a series of amphiphilic, jellyfish-
shaped CyPLOS derivatives endowed with good ion transport ac-
tivity have been prepared (2a–c, Fig. 2). It was also demonstrated
that the presence of the disaccharide macrocycle was essential for
the ionophore activity, probably furnishing a portal entry for ions at
the water-membrane interface.9
In a previous paper,8 we have showed that cyclic phosphodiester-
linked disaccharide 1 (Fig. 1), as the first member of a family of
* Corresponding author. Tel.: þ39 081 674126; fax: þ39 081 674393.
E-mail address: daniela.montesarchio@unina.it (D. Montesarchio).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.09.100

C. Coppola et al. / Tetrahedron 65 (2009) 9694–9701
9695
the formation of the desired 18-crown-6 containing derivative. In
fact, assuming the alkylation on the 2-OH group as the first event, the
close proximity of the 3-OH associated with the template effect of the
metal ion (Naþ or Kþ) should strongly favour the cyclization over
undesired dimerization events.11 Indeed, by reacting 4 with NaH in
anhydrous THF for 30 min, then adding pentaethylene glycol dito-
sylate and, after additional 30 min, diluting the reaction system, the
sole target compound 5 was obtained in very satisfactory yields (92%
after chromatography). This building block was then elaborated to
give the desired 4-phosphorylated and 4-phosphoramidite de-
rivatives 8 and 9, respectively, following the reactions described in
Scheme 1.
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
-
O
-
O
PhO
O
PhO
-
O
O
O
O
PhO
P
P
O
O
P
O
O
P
O
P
O
O
O
P
O
O
O
O
O
O
O
O
O
O
O
OPh
O
OPh
O
-
O
-
O
OPh
O
O
-
O
O
O
Ph
O
Ph
O
O
O
O
O
a
O
O
O
OPh
OPh
O
HO
O
OH
O
O
O
O
O
(4)
O
O
O
O
O
O
O
(5)
b
O
O
O
2c
2b
O
2a
ODMT
Figure 2. Chemical structure of macrocycles 2a–c.
HO
HO
c
O
O
HO
O
OPh
OPh
O
O
O
These promising results prompted us to explore other variants
O
O
of functionalized CyPLOS, exhibiting diverse structural motifs use-
ful for ion recognition. We here describe novel polycyclic derivative
3 (Fig. 3), carrying a cyclic phosphate-linked disaccharide skeleton
fused to two 18-crown-6 units, which potentially offer additional
complexing sites, thus contributing to the ionophore properties of
the macrocycle.10 Attachment of these moieties to the vicinal OH
groups of a glucoside scaffold was considered to introduce only
marginal rigidification into the crown ether backbone, thus leaving
the six oxygen atoms in the cavity of each 18-crown-6 unit with
optimal orientation to host cations.
O
O
O
O
O
O
(7)
(6)
e
d
Cl
-
OH
ODMT
O
P
N
P
O
O
O
O
O
O
CEO
O
OPh
OPh
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
(8)
(9)
O
O
Scheme 1. a) Pentaethylene glycol ditosylate, NaH, THF, reflux, 12 h, 92% yield; b) 10%
TFA in CH2Cl2, 0 ꢀC, 4 h, 99% yield; c) DMTCl, pyridine, rt, 12 h, 98% yield; d) 2-chloro-
phenyldichlorophosphate, 1,2,4-triazole, TEA, pyridine, rt, 3 h, followed by chro-
matographic purification on a column eluted with 0.1% TFA in a CH2Cl2/MeOH mixture,
92% yield; e) 2-O-cyanoethyl-N,N-diisopropylaminochlorophosphoramidite, DIPEA,
CH2Cl2, r. t., 2 h, 96% yield.
-
O
PhO
O
O
P
O
O
P
O
O
-
O
O
OPh
O
O
O
O
O
This synthetic strategy essentially follows the route adopted for
the preparation of CyPLOS derivatives 1 and 2a–c.8,9 Treatment of 5
with 10% TFA in CH2Cl2 at 0 ꢀC for 4 h allowed the complete removal
of the benzylidene protecting group, leading to 6 isolated in almost
quantitative yields. Successive reaction of diol 6 with DMTCl in
pyridine gave compound 7, recovered in 98% yield after column
chromatography. Phosphorylation of 7, by treatment with 2-chloro-
phenyl-dichlorophosphate in the presence of 1,2,4-triazole and TEA in
pyridine, followed by acidic work-up of the reaction mixture, gave,
in a one-pot reaction, detritylated compound 8 in 92% yields for two
steps. 4-Phosphoramidite derivative 9 was obtained in 96% yield,
essentially following protocols well established in the elaboration of
building blocks for oligonucleotide synthesis.12 Linear dimer 10 was
obtained through a phosphoramidite procedure, as described in
Scheme 2, by coupling 8 and 9 in the presence of 0.25 M DCI in
CH3CN, followed by oxidation with 5.5 M tert-butylhydroperoxide
(t-BuOOH) in n-decane. Upon DMT removal, linear dimer 11 was
isolated as a stable compound, after column chromatography, in 50%
overall yield for three steps.
O
O
Figure 3. Chemical structure of macrocycle 3.
Under the assumption that the binding abilities of the crown
ether residues in derivative 3 are preserved, if two monopositive
ions are bound, this compound results in an electrically neutral,
internal salt, which could be spatially organized into a peculiar
molecular architecture.
2. Results and discussions
2.1. Synthesis
We reasoned that treatment of 4,6-benzylidene protected gluco-
side 4 8 with a small excess of penta(ethylene glycol) ditosylate in the
presence of a strong base as sodium or potassium hydride, i.e. under
classical Williamson reaction conditions, would preferentially lead to

9696
C. Coppola et al. / Tetrahedron 65 (2009) 9694–9701
column eluted with H2O/EtOH 4:1 (v/v), furnishing the pure target
compound in 70% yield for the two deprotection steps. Following the
described procedures, 3 was prepared in 11 steps and 23% overall
yield from 4,6-protected phenyl-b-D-glucopyranoside 4.
All the synthesized compounds were purified by column chro-
matography, in all cases allowing to isolate homogeneous com-
pounds, and characterized by 1H, 13C (and 31P, where present) NMR
spectroscopy and mass analysis.
Cl
O-
P
OH
ODMT
N
O
O
P
O
O
O
NCCH2CH2O
O
OPh
OPh
O
O
O
O
+
O
O
O
O
O
O
O
O
(8)
(9)
a
2.2. NMR analysis and structural studies
R1O
O-
O
R3O
O
OPh
O
P
The final cyclic dimer compound proved to be a very hydrophilic
tool, highly soluble in water and, to a lesser extent, in methanol. In
contrast to our original expectations, 3 could not be investigated in
transmembrane ion transport assays, since, differently from related
analogue 2b, it did not permeate lipid systems at all. Due to this
marked hydrophilicity, the binding properties of host 3 towards
cations could not be investigated through classical alkali metal
picrate extraction experiments from water to chloroform. There-
fore, a detailed NMR spectroscopic analysis was undertaken, in
order to elucidate the conformational features of the synthesized
compound, also in comparison with parent macrocycle 1, and to
determine its binding properties through 1H NMR-based titrations.
1H NMR spectra of 3 were recorded in water in the concentra-
tion range 0.1–2.0 mM. Upon varying the concentration, no signif-
icant change was observed in the shape or distribution of the 1H
NMR resonances, thus allowing to exclude aggregation phenomena
under these conditions. As a completely deprotected macrocycle, it
shows a high degree of internal symmetry, as expected for a flexible
C2-symmetric dimer. The 1H NMR spectra show the two glucoside
monomers as magnetically equivalent, with a unique signal present
for each type of nucleus (Fig. 4). In fact, the 1D proton spectrum of 3
(700 MHz, D2O, T¼298 K) shows the presence of only one anomeric
signal at dH 5.21 ppm. Additional four non overlapped signals are
clearly discernible in the region between 3.7 and 4.3 ppm, succes-
sively assigned to H6a (dH 4.18), H4 (dH 4.03), H5 (dH 3.82), H3 (dH
3.70) of the sugar rings.
O
R2O
P
O
O
O
O
O
O
O
O
O
O
O
O
OPh
O
O
O
(10) R1 = DMT; R2 = 2-cyanoethyl; R3 = 2-cholophenyl
(11) R1 = H; R2 = 2-cyanoethyl; R3 = 2-chlorophenyl
b
c
PhO
OR3
P
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
P
O
O
O
OR2
O
OPh
(12) R2 = 2-cyanoethyl; R3 = 2-chlorophenyl
(3) R2 = H; R3 = H
d
Scheme 2. a) 0.25 M DCI in CH3CN, rt, 2 h., then 5.5 M t-BuOOH in decane, rt, 30 min,
50% yield; b) 1% TCA in CH2Cl2 (v/v), rt, 30 min, 99% yield; c) MSNT, DMAP, pyridine, rt,
12 h, 83% yield; d) piperidine, 60 ꢀC, 12 h, then aq ammonia, 60 ꢀC, 12 h, 70% yield.
Cyclization was then achieved by exploiting a phosphotriester
methodology,13 well optimized both in solution and in the solid
phase for the synthesis of cyclic oligonucleotides.14 Using 1-mesity-
lensulfonyl-3-nitro-1,2,4-triazole (MSNT) as the condensing agent in
pyridine in association with DMAP, under high dilution conditions
(10ꢁ3 M), fully protected cyclic compound 12 was obtained in 83%
yield. Final deprotection of the macrocycle was achieved in two steps,
involving, first, a basic treatment with anhydrous piperidine (60 ꢀC,
The signals in the aromatic region at dH 7.29, 7.05 and 7.03 ppm
account for the phenyl protons. A severe overlapping of signals was
observed at dH 3.6 ppm, due to the methylene groups of the crown
ether moieties. In addition, a unique signal is apparent for the two
phosphorus nuclei in the 31P NMR spectra. Proton (700 MHz,
T¼298 K) and carbon (175 MHz, T¼298 K) assignments were
obtained through an in-depth analysis of two-dimensional PE-COSY,
TOCSY, HSQC, and HMBC NMR experiments. Furthermore, 2D ROESY
experiment allowed confirmation of the assignments and gave us
12 h) for the removal of the 2-cyanoethyl protecting groupthrough b-
elimination, followed by a basic hydrolytic treatment with aq am-
monia at 60 ꢀC to cleave the 2-chlorophenyl group. Final purification
was carried out by gel filtration chromatography on a G25 Sephadex
Figure 4. Selected regions of the 1H NMR (700 MHz, D2O) and–in the inset–of the 31P NMR (161.98 MHz, D2O) spectrum of 3.

C. Coppola et al. / Tetrahedron 65 (2009) 9694–9701
9697
structural information as well. Essentially all ROEs are between 1,3-
diaxial protons within the sugar, thus suggesting that the -gluco-
groups forming, with the crown ethers themselves, a pocket capable
to comfortably host cations.
b-D
pyranoside moieties adopt a classical 4C1 chair conformation and are
not distorted. This observation is also confirmed by the analysis of the
proton coupling constants (9 Hz) measured for the sugar ring protons
(H1, H2, H3, H4, and H5). Relevant structural information relative to
the central 12-membered ring could be derived from the analysis of
the multiplicity and coupling constants of the H4 and H6a (for their
couplings with P) and H5 (for its couplings with H6a and H6b) reso-
nances. H6a resonates as a double-double-doublet, characterized by
coupling constants of 22, 12 and 5 Hz. The first coupling constant
(22 Hz) is due to the coupling with the phosphorus, the coupling
constant of 12 Hz is indeed generated by geminal coupling with H6b,
and the smallest coupling constant is relative to the coupling with H5.
The signal attributed to H5 appears as a double triplet and exhibits
a coupling constant of 5 Hz (due to the coupling with H6a), and
a coupling constant of 9 Hz (generating the triplet), due to the
coupling with H4 and H6b. This suggests that both the dihedral angles
H5-C5-C6-H6b and H5-C5-C4-H4 are close to either 0ꢀ or 180ꢀ.
Analogously, the dihedral angle H6a-C6-O6-P is very close to 180ꢀ
(3JHP¼22 Hz), whilenoinformationcouldberetrievedforthedihedral
angle H6b-C6-O6-P. Differently from what observed for 1, H4 is
characterized by a triplet multiplicity, due to the scalar couplings
(9 Hz) with H3 and H5. This means that no coupling is present with
phosphorus and that the dihedral angle H4-C4-O-P is close to 90ꢀ (or
270ꢀ). Three-dimensional structures of 3, satisfying all the ROEs and
In order to test the complexing abilities of compound 3, a series
of 1H NMR-based titrations were performed. However, when alkali
metal cations–i.e., sodium, potassium, cesium or rubidium, given in
the form of chlorides or perchlorates–were progressively added to
the host compound solution up to a large excess, only marginal
changes in the 1H NMR signals of the substrate were observed in all
cases, both at the level of the chemical shifts and J values. Very
small downfield or upfield shifts should only account for the effects
due to the increased ionic strength, ruling out the possibility of
effective binding, as would be explained in the case of prevailing
competing solvation effects. Alternatively, these results can also be
interpreted assuming that sodium, incorporated during the NaH-
mediated crown ether formation in compound 5, is never lost in the
successive synthetic elaborations leading to the final macrocycle,
nor successively displaced by other cations. The close proximity of
two negatively charged phosphate groups could indeed dramati-
cally increase the affinity of the 18-crown-6 ether derivatives for
sodium cations, which may be assumed to be tightly bound in the
crown ether pockets in 3 independently from the successive ad-
dition of external cations.15 In order to establish whether Naþ can
actually be hosted in the crown ethers and simultaneously interact
with the phosphate groups present in 3, a novel set of molecular
dynamic and mechanic calculations was performed in the presence
of sodium cations. These calculations were carried out by using the
same constraints and procedures used for those performed in the
absence of cations. In order to keep the Naþ ions close to the crown
ethers, we have introduced additional constraints into the calcu-
lations. In particular, we have used a range of distances between the
oxygens of the crown ethers and the sodium ions of 2.4–5.0 Å. The
width of this range, if necessary, guarantees the Naþ ions to freely
move towards the negatively charged phosphate groups, and, at the
same time, to remain in proximity of the crown ethers. The calcu-
lations show structures which are almost superimposable to the
ones obtained in the absence of ions. Interestingly, the ions are well
accommodated within the two crown ether residues and, as
a matter of fact, they are very close (2.7 Å) to the phosphates (see
Fig. 6).
3
dihedral angle constraints (3JHH and JHP), were built by restrained
simulated annealing calculations. An initial structure of 3 was gen-
erated and minimized to eliminate any possible conformational bias.
Restrained simulations were carried out in vacuo for 230 ps. The dy-
namics started at 1000 K using the consistent valence force field, as
implemented in the Discover software (Accelrys, San Diego, USA).
Thereafter, the temperature was decreased stepwise to 250 K. The
final step was to energy-minimize and refine the structures obtained
by using the steepest descent and the quasi-Newton-Raphson
(VA09A) algorithms. Out of 500 structures generated, 50 structures
with the lowest energies were selected. An excellent superimposition
ofthe50structures withrootmeansquaredeviation(RMSD)values of
0.18ꢂ0.08 forall heavyatoms could be obtained. The lack of violations
and the low RMSD value suggest that the minimized conformation of
3, reported in Figure 5, is consistent with the experimentally de-
termined restraints. As expected, the three-dimensional structure
obtained for 3 is very different from the one previously derived for 1
(see Fig. 5). In fact, macrocycle 3 adopts an arched structure, in which
the two phosphate functionalities point out towards the crown ether
These findings suggest that the studied molecule is indeed well
pre-organized for hosting cations and, particularly, can be re-
alistically complexed with Naþ. Definitive confirmation of the
presence of bound cations in 3 was expected from X-ray crystal-
lography studies, but all the attempts carried out so far to obtain
good quality crystals have been unsuccessful.
Figure 5. Minimized conformations for compounds 1 (Ref. 8) and 3.

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C. Coppola et al. / Tetrahedron 65 (2009) 9694–9701
MALDI TOF mass spectrometric analyses were performed on a Per-
Septive Biosystems Voyager-De Pro MALDI mass spectrometer in the
Linear mode using 2,5-dihydroxybenzoic acid as the matrix. NMR
spectra for the characterizationofcompounds5–12 were recorded on
Bruker WM-400 or Varian Inova 500 spectrometers. All the chemical
shifts areexpressedinppmwithrespecttotheresidualsolventsignal;
J values are in Hz. The following abbreviations were used to explain
the multiplicities: s¼singlet; d¼doublet; t¼triplet; q¼quartet;
m¼multiplet; dd¼double doublet; ddd¼double double doublet. 31P
NMR spectra have been registered using 85% H3PO4 as the external
reference. UV measurements were carried out on a Jasco V-530 UV
spectrophotometer equipped with a Jasco ETC-505 T temperature
controller unit. Circular dichroism (CD) experiments were registered
on a JASCO J-715 spectrophotometer equipped with a thermostati-
cally controlled cuvette holder (JASCO PTC-348), in a 0.1 cm path
length cuvette. CD spectra were collected from 200 to 300 nm, at
20 nm/min, with a response time of 4 s and at 1 nm bandwidth.
The following abbreviations were used throughout the text:
AcOEt¼ethyl acetate; t-BuOOH¼tert-butylhydroperoxide; CE¼2-
cyanoethyl; DCI¼4,5-dicyanoimidazole; DIPEA¼N,N-diisopropy-
lethylamine; DMAP¼N,N-dimethyl-aminopyridine; DMT¼4,40-
dimethoxytriphenylmethyl; EtOH¼ethanol; Glu¼glucose residue;
MeOH¼methanol; MSNT¼1-mesitylensulfonyl-3-nitro-1,2,4-tri-
azole; TCA¼trichloroacetic acid; TEA¼triethylamine; TFA¼tri-
fluoroacetic acid; THF¼tetrahydrofuran.
Figure 6. Superimposition of the structures obtained for 3 with (yellow) and without
(magenta) sodium ions.
Circular dichroism analysis is consistent with the NMR data for
3; CD spectra of substrate 3 dissolved in water in the absence and in
the presence of 2 equiv. NaCl were found to be absolutely super-
imposable, showing no evident conformational difference due to
a prolonged contact of 3 with added sodium cations (see Supple-
mentary data).
Experiments are in progress to investigate the antimicrobial,
antifungal and antitumoral profile of this novel macrocycle.
4.2. Nuclear magnetic resonance
NMR spectra for compound 3 were determined in D2O (Aldrich,
Milwaukee, USA). The NMR samples were prepared at a concentra-
tion range of 0.1–2.0 mM. NMR spectra were recorded with a Varian
UnityINOVA 700 MHz and 500 MHz spectrometers. Phase sensitive
ROESY spectra16 were recorded with mixing times in the range 250–
500 ms (T¼298 K). TOCSY spectrum17 with mixing time of 100 ms
was recorded. ROESY and TOCSY were recorded using TPPI18 pro-
cedure for quadrature detection. {1H-13C}-HSQC19 and {1H-13C}-
3. Conclusions
A novel C2-symmetric basket-shaped macrocycle with a central
phosphate-linked disaccharide skeleton, decorated with two 18-
crown-6 ether moieties (crown-CyPLOS), has been prepared in 11
steps with 23% yields. Conformational preferences in solution of the
bis-crown ether-fused compound have been investigated by a de-
tailed NMR analysis, suggesting that the supramolecular capacities
of the crown ethers into this macrocycle can be dramatically en-
hanced by the close interaction with negatively charged phosphate
groups, presumably leading to a stable, internal salt, tightly binding
monopositive ions. Such a circumstance is indeed detrimental for
ionophore activity, where loose binding is required for effective ion
exchange in different media. Even though the synthesized macro-
cycle did not prove to be a useful candidate for transmembrane ion
transport experiments, these data are useful to guide future studies,
addressed to the synthesis of related polycyclic analogues, finely
tuning the saccharide decorations so to obtain more effective iono-
phores and biologically active compounds.
1
2,3
HMBC20 were optimized for JC–H¼135 Hz and
J
¼10 Hz,
C–H
respectively. In all the 2D experiments, time domain data consisted
of 2048 complex points in t2 and 400–512 fids in t1 dimension. The
relaxation delay was kept at 1.2 s for all the experiments. The spectra
were calibrated relative to HDO (4.75 ppm) as the internal standard.
The NMR data were processed on a SGI Octane workstation using
FELIX 98 software (Accelrys, San Diego, USA) and on a iMAC using
the software iNMR (www.inmr.net).
4.3. Structure calculations
Crosspeakvolume integrations wereperformed with the program
FELIX 98, using the ROESY experiment collected at mixing time of
250 ms andwith arelaxationdelayof 3 s. TheNOEvolumeswerethen
converted to distance restraints after they were calibrated using
known fixed distances of H6a and H6b of the glucose subunit. Then an
NOE restraint file was generated with three distance classifications
as follows: strong NOEs (1.5 Åꢄrijꢄ2.5 Å), medium NOEs
(2.0 Åꢄrijꢄ3.5 Å) and weak NOEs (3.0 Åꢄrijꢄ5.0 Å). A total of 20 NOE-
(10 foreach symmetric moiety)deriveddistance restraintswere used.
Proton/proton (3JHH) and proton/phosphorus (3JHP) coupling con-
stants were measured from 1D proton spectra and from PE-COSY
spectrum. Dihedral angles were calculated using the J-values extrac-
ted by solving the Karplus equation21 with coefficients A¼6.98,
4. Experimental section
4.1. Materials and methods
TLC analyses were carried out on silica gel plates from Merck (60,
F254). Reaction products on TLC plates were visualized by UV light
and then by treatment with a 10% Ce(SO4)2/H2SO4 aqueous solution.
For column chromatography, silica gel from Merck (Kieselgel 40,
0.063–0.200 mm) was used. HPLC analyses were performed on
a Beckman System Gold instrument equipped with a UV detector
module 166 and a Shimadzu Chromatopac C-R6A integrator. By HPLC
analysis on
a
Nucleosil 100–5C18 Supelco analytical column
m), eluted with a linear gradient from 0 to 100% in
B¼ꢁ1.38, C¼1.72 for 3JHH
,
22 and A¼15.3, B¼ꢁ6.2, C¼1.5 for 3JHP
.
23 Only
(250ꢃ4.6 mm, 5
m
those angles yielding two real roots when solving the Karplus equa-
tion were used during structural calculation. Three-dimensional
structures, which satisfy NOE and dihedral angle constraints were
constructed by simulated annealing calculations. All the calculations
used a distance dependant macroscopic dielectric constant of 80*r
30 min of CH3CN in H2O, flow¼0.8 mL/min, detection at ¼264 nm,
l
all the synthesized compounds were more than 98% pure. For the ESI
MS analyses, a Waters Micromass ZQ instrument–equipped with an
Electrospray source–was used in the positive and/or negative mode.

C. Coppola et al. / Tetrahedron 65 (2009) 9694–9701
9699
and an infinite cut-off for nonbonded interactions to partially com-
pensate for the lack of the solvent.24 Initial structures of 3din the
absence and in the presence of sodium ionsdwere built using
a completely random array of atoms. Using the steepest descent fol-
lowed by quasi-Newton-Raphson method (VA09A) the conforma-
tional energy was minimized. Restrained simulations were carried
out in vacuo for 230 ps at 1000 K using the CVFF force field as
implemented in Discover software (Accelrys, San Diego, USA). Then,
the temperature was decreased stepwise until 250 K. The final step
was again to energy-minimize so to refine the structures obtained,
using successively the steepest descent and the quasi-Newton-
Raphson (VA09A) algorithms. Both dynamic and mechanic calcula-
tionswere carriedoutbyusing a 10 (kcal/mol)/Å2 flatwellrestraints. A
total of 50 structures were generated. Illustrations of structures were
generated using the INSIGHT II program, version ’98 (Accelrys, San
Diego, USA). All the calculations were performed on a SGI Octane
workstation.
aromatic protons), 4.99 (d, J¼10.0 Hz, 1H, H-1), 4.29–4.25 [m, 1H,
(CH2CHaO-Glu-C-2)], 4.06–3.83 [overlapped signals, 4H, H-6a,
(CH2CH2O-Glu-C-3) and (CH2CHbO-Glu-C-2)], 3.79–3.60 [over-
lapped signals, 18H, 4x(OCH2CH2O), H-4 and H-6b], 3.48 [over-
lapped signals, 2H, H-2 and H-5], 3.40 (m, 1H, H-3); dC (125 MHz,
CDCl3): 157.0 129.5, 122.5 and 116.3 (aromatic carbons), 101.2 (C-1),
84.6 (C-3), 82.1 (C-2), 75.3 (C-5), 72.8 (CH2CH2O-Glu-C-2), 71.7, 71.5
and 71.4 [(CH2CH2O-Glu-C-3), 2x(CH2CH2O-Glu)], 70.6, 70.5, 70.3,
70.1 and 70.0 [3x(OCH2CH2O) and, overlapped, C-4], 62.6 (C-6); ESI-
MS (positive ions): calcd for C22H34O10, 458.21; m/z, found 480.59
(MþNaþ), 496.56 (MþKþ); HRMS (MALDI-TOF, positive ions): m/z:
calcd for C22H34O10Na: 481.2050; found 481.2060 (MþNaþ).
4.3.3. Synthesis of derivative 7. Compound 6 (295 mg, 0.645 mmol),
dissolved in anhydrous pyridine (3.0 mL), was treated with 4,40-
dimethoxytriphenylmethylchloride (DMTCl) (283 mg, 0.839 mmol)
for 12 h at room temperature. The reaction mixture was then con-
centrated under reduced pressure and purified on a silica gel col-
umn. Elution with CH2Cl2, added with 1% pyridine (v/v), containing
increasing amounts of MeOH (up to 5%) allowed to recover pure 7
(480 mg, 0.632 mmol) in 98% yield.
4.3.1. Synthesis of derivative 5. Derivative 48 (250 mg, 0.725 mmol)
was dissolved into anhydrous THF (20 mL) and treated with NaH
(104 mg, 4.35 mmol, 60% mineral oil dispersion); after 10 min,
pentaethyleneglycol ditosylate (540 mg, 1.08 mmol) was added and
the resulting mixture left under vigorous stirring at room temper-
ature. After 20 min, an additional volume of anhydrous THF (12 mL)
was added and the reaction left at reflux for 12 h. The reaction was
quenched by addition of a few drops of MeOH, the resulting mixture
was concentrated under reduced pressure, diluted with CHCl3,
transferred into a separatory funnel and washed twice with a satd
aq NaCl solution. The organic layer was collected, taken on anhy-
drous sodium sulfate, filtered, concentrated under reduced pressure
and then purified on a silica gel column. Elution of the column with
CHCl3 containing increasing amounts of MeOH (from 0% to 5%)
allowed to recover pure 5 (364 mg, 0.666 mmol), in 92% yield.
4.3.3.1. Compound 7. Yellow oil, Rf¼0.8 (CH2Cl2/CH3OH, 95:5,
v/v); nmax (liquid film) 3390 (br), 1056, 1037, 1006 cmꢁ1
; dH (500 MHz,
CDCl3): 7.45–6.75 (complex signals, 18H, aromatic protons), 4.95 (d,
J¼8.0 Hz,1H, H-1), 4.27–4.22 [m,1H, (CH2CHaO-Glu-C-2)], 4.12–4.08
[m,1H, (CH2CHaO-Glu-C-3)], 4.02–3.98 [m,1H, (CHaCH2O-Glu-C-3)],
3.93–3.88 [m, 1H, (CHaCH2O-Glu-C-2)], 3.77 (s, 6H, 2ꢃOCH3 of DMT
group), 3.73–3.62 [overlapped signals, 16H, 4x(OCH2CH2O)], 3.58
(apparent t, J¼10.0 and 8.5 Hz, 1H, H-4), 3.53–3.48 [overlapped
signals, 2H, H-2 and H-5], 3.45–3.42 (m, 1H, H-6a), 3.37–3.31
(overlapped signals, 2H, H-3 and H-6b); dC (125 MHz, CDCl3): 158.3,
157.2, 144.8, 135.9, 130.0, 129.3, 128.1, 127.7, 126.6, 122.4, 116.8 and
113.0 (aromatic carbons), 101.3 (C-1), 86.1 (quaternary C of DMT
group), 84.9 (C-3), 82.2 (C-2), 74.5 (C-5), 72.9 (CH2CH2O-Glu-C-2),
71.8, 71.3 and 71.2 [(CH2CH2O-Glu-C-3) and 2x(CH2CH2O-Glu)], 70.9,
70.8, 70.6, 70.5, 70.4 and 70.2 [2x(OCH2CH2O) and, overlapped, C-4],
63.6 (C-6), 55.1 [2x(OCH3 of DMT group)]; ESI-MS (positive ions):
calcd for C43H52O12, 760.35; m/z, found 782.16 (MþNaþ); HRMS
(MALDI-TOF, positive ions): m/z: calcd for C43H52O12Na: 783.3356;
found 783.3370 (MþNaþ).
4.3.1.1. Compound 5. oil, Rf¼0.6 (CHCl3/CH3OH, 98:2, v/v); nmax
(liquid film) 1490, 1452, 1354, 1234, 1059, 1028, 1012 cmꢁ1
; dH
(500 MHz, CDCl3): 7.49–7.03 (complex signals, 10H, aromatic pro-
tons), 5.55 (s, 1H, Ph-CH), 5.06 (d, J¼7.5 Hz, 1H, H-1), 4.35 (dd, J¼5.0
and 10.5 Hz, 1H, H-4), 4.09–4.00 [overlapped signals, 5H,
2x(CH2CH2O-Glu) and H-6a], 3.79 (t, J¼10.0 and 10.0 Hz, 1H, H-3),
3.73–3.58 [overlapped signals,17H, 4x(OCH2CH2O) and H-6b], 3.56–
3.47 (overlapped signals, 2H, H-2 and H-5); dC (125 MHz, CDCl3):
157.0, 137.1, 129.6, 129.4, 128.8, 128.1, 127.8, 125.9, 122.8 and 116.8
(aromatic carbons), 101.7 (C-1), 101.0 (Ph-CH), 82.3 (C-2), 81.5 and
80.8 [2x(CH2CH2O-Glu)], 72.7 and 72.6 [2x(CH2CH2O-Glu)], 70.8,
70.7, 70.6, 70.5 and 70.4 [3x(OCH2CH2O) and, overlapped, C-6], 68.5
(overlapped signals, C-4 and C-3), 66.0 (C-5); ESI-MS (positive
ions): calcd for C29H38O10, 546.25; m/z, found 568.59 (MþNaþ),
584.57 (MþKþ); HRMS (MALDI-TOF, positive ions): m/z: calcd for
C29H38O10Na: 569.2363; found 569.2390 (MþNaþ).
4.3.4. Synthesis of derivative 8. To compound 7 (152 mg, 0.201 mmol),
dissolved in anhydrous pyridine (2.0 mL), 1,2,4-triazole (111 mg,
1.61 mmol), previously dried in vacuo, was added. To the solution
taken at 0 ꢀC, TEA (225
chlorophoshate (130
m
L, 1.61 mmol) and 2-chlorophenyldi-
m
L, 0.804 mmol) were sequentially added. The
reaction mixture was left under stirring at room temperature for 3 h,
then the solution was concentrated under reduced pressure, the
resulting mixture redissolved in CHCl3, transferred into a separatory
funnel and washed twice with a satd aq NaCl solution. The organic
layer was collected, taken on anhydrous sodium sulfate, filtered,
concentrated under reduced pressure and the residue purified on
a silica gel column. Elution of the column with CH2Cl2, added with
0.1% of TFA (v/v), containing increasing amounts of MeOH (up to 10%
in volume) allowed to isolate pure 8 (120 mg, 0.185 mmol) in 92%
yield.
4.3.2. Synthesis of derivative 6. Compound 5 (352 mg, 0.645 mmol)
was treated with a 10% TFA solution in CH2Cl2 (v/v, 10 mL total
volume) under stirring at 0 ꢀC for 4 h. The reaction was then
quenched by addition of a satd aq NaHCO3 solution, the resulting
mixture diluted with CHCl3, transferred into a separatory funnel
and washed twice with a satd aq NaCl solution. The organic layer
was collected, taken on anhydrous sodium sulfate, filtered, con-
centrated under reduced pressure and the residue purified on
a silica gel column. Elution of the column with CHCl3 containing
increasing amounts of MeOH (from 0% to 10%) allowed to obtain
pure 6 (295 mg, 0.645 mmol) in an almost quantitative yield.
4.3.4.1. Compound 8. Yellow oil, Rf¼0.3 (CH2Cl2/CH3OH, 95:5,
v/v); nmax (liquid film) 3322 (br), 1642, 1480, 1227, 1059, 1028,
1012 cmꢁ1
; dH (500 MHz, CDCl3): 7.71–6.85 (complex signals, 9H,
aromatic protons), 4.96 (broad signal, 1H, H-1), 4.64 (m, 1H, H-4),
4.10–3.36 [overlapped signals, 25H, H2-6, H-2, H-5, 5x(OCH2CH2O)
and H-3]; dC (125 MHz, CDCl3): 162.3, 157.2, 149.3, 129.3, 127.6,
124.9, 123.2, 122.3 and 116.5 (aromatic carbons), 101.4 (C-1), 83.1
(C-3), 79.6 (C-2), 75.9 (C-5), 70.9, 70.7, 70.5, 69.8, 69.3, 68.9 and 68.8
4.3.2.1. Compound 6. Oil, Rf¼0.3 (CHCl3/CH3OH, 95:5, v/v); nmax
(liquid film) 3398 (br), 1591, 1493, 1452, 1348, 1230, 1066,
1028 cmꢁ1
; dH (500 MHz, CDCl3): 7.29–6.99 (complex signals, 5H,

9700
C. Coppola et al. / Tetrahedron 65 (2009) 9694–9701
[5x(OCH2CH2O) and C-4], 60.3 (C-6); dP (161.98 MHz, CDCl3): ꢁ3.1;
ESI-MS (negative ions): calcd for C28H38ClO13P, 648.17; m/z, found
646.85 (MꢁH)ꢁ. HRMS (MALDI-TOF, negative ions): m/z: calcd for
C28H37ClO13P: 647.1660; found 647.1690.
protons), 4.85 [broad signal, 2H, 2x(H-1)], 4.65–3.32 [overlapped
signals, 48H, 2x(CH2CH2O-Glu-C-2) and 2x(CH2CH2O-Glu-C-3),
2x(H-4); 2x(H2-6), 8x(OCH2CH2O) and (OCH2CH2CN)], 3.07–2.50
[overlapped signals, 8H, 2x(H-2), 2x(H-5), 2x(H-3) and
(OCH2CH2CN)]; dC (125 MHz, CDCl3): 156.9,156.8,149.8,135.7, 129.5,
127.4,123.6,122.8 and 116.9 (aromatic carbons),117.3 (CN),101.8 and
101.6 (C-1), broad signal centered at 81.8 [2x(C-3) and 2x(C-2)],
broad signal centered at 70.4 [10x(OCH2CH2O), 2x(C-5) and 2x(C-4)],
65.3 (C-6 adjacent to the phosphate), 62.0 (C-6 adjacent to OH), 60.8
(OCH2CH2CN), 25.6 (OCH2CH2CN); dP (161.98 MHz, CDCl3): over-
lapped, broad signals, extended from þ7.6 to ꢁ4.4; MALDI-MS
(positive ions): calcd for C53H73ClNO25P2, 1220.36; m/z, found
1243.67 (MþNaþ); HRMS (MALDI-TOF, positive ions): m/z: calcd for
C53H73ClNO25P2Na: 1243.3533; found 1243.3587 (MþNaþ).
4.3.5. Synthesis of derivative 9. Compound 7 (175 mg, 0.231 mmol)
was dissolved in anhydrous CH2Cl2 (2.0 mL) and left in contact with
activated 3 Å molecular sieves. DIPEA (320
cyanoethyl-N,N-diisopropylaminochlorophosphoramidite (205
m
L, 1.85 mmol) and 2-O-
L,
m
0.93 mmol) were sequentially added dropwise under anhydrous
nitrogen atmosphere. After 2 h at room temperature the reaction
mixture was concentrated under reduced pressure and the result-
ing residue purified on a silica gel column. Elution of the column
with AcOEt/n-hexane 7:3 (v/v), added with 1% TEA, containing in-
creasing volumes of AcOEt (up to 100%) allowed to isolate pure 9
(213 mg, 0.221 mmol) in 96% yield.
4.3.7. Synthesis of derivative 12. Compound 11 (30 mg, 0.024 mmol)
was dissolved in anhydrous pyridine (25 mL) and then reacted with
DMAP (3.0 mg, 0.020 mmol) and MSNT (218 mg, 0.74 mmol). The
reaction mixture was left at room temperature under stirring for
12 h, then concentrated under reduced pressure and the resulting
residue redissolved in CH2Cl2, transferred into a separatory funnel
and washed twice with a satd aq NaCl solution. The organic layer
was collected, taken up in anhydrous sodium sulfate, filtered,
concentrated under reduced pressure and the residue purified on
a silica gel column. Elution of the column with CH2Cl2 containing
increasing volumes of MeOH (from 0 to 15%) allowed to isolate pure
12 (24 mg, 0.020 mmol) in 83% yield.
4.3.5.1. Compound 9. Oil, mixture of diastereomers, Rf¼0.8
(CH2Cl2/CH3OH, 95:5, v/v); nmax (liquid film) 1066,1037,1012 cmꢁ1
;
dH (400 MHz, CDCl3): 7.45–6.70 (complex signals, 36H, aromatic
protons), 4.99 [m, 2H, 2x(H-1)], 4.20–3.87 [overlapped signals, 8H,
2x(CH2CH2O-Glu-C-2) and 2x(CH2CH2O-Glu-C-3)], 3.75 [s, 12H,
4x(OCH3 of DMTgroup)], 3.80–3.46 [overlapped signals, 42H, 2x(H-
4), 8x(OCH2CH2O), 2x(OCH2CH2CN) and 2x(H2-6)], 3.43–3.20
{overlapped signals, 10H, 2x(H-3), 2x(H-2), 2x(H-5) and
2ꢃN[CH(CH3)2]2}, 2.75–2.55 [m, 4H, 2x(OCH2CH2CN)], 1.32–0.84
{overlapped signals, 24H, 2ꢃN[CH(CH3)2]2}; dC (100 MHz, CDCl3):
158.2,157.2,157.1,145.0,144.9,136.2,136.1,136.0,135.9,130.1,130.0,
129.3, 128.2, 128.1, 127.5, 126.5, 122.3, 116.7, 116.6 and 112.8 (aro-
matic carbons), 117.6 and 117.2 (CN), 101.0 and 100.9 (C-1), 85.8 and
85.7 (quaternary C of DMTgroup), 84.8 and 84.1 (C-3), 82.5 and 82.4
(C-2), 75.3 and 75.2 (C-5), 72.6, 72.5, 72.3 and 72.2 [2x(CH2CH2O-
Glu-C-2) and 2x(CH2CH2O-Glu-C-3)], 70.8, 70.7, 70.6, 70.5, 70.5 and
70.4 [8x(OCH2CH2O)], 70.2 [2x(C-4)], 63.6 and 63.2 (C-6), 58.4 and
58.2 (OCH2CH2CN), 55.0 [4x(OCH3 of DMT group)], 42.9 and 42.7
{N[CH(CH3)2]2}, 24.3 and 24.2 {N[CH(CH3)2]2}, 22.8 and 22.7
(CH2CH2CN); dP (161.98 MHz, CDCl3): 151.1 and 150.4; ESI-MS
(positive ions): calcd for C52H69N2O13P, 960.45; m/z, found 983.19
(MþNaþ). HRMS (MALDI-TOF, positive ions): m/z: calcd for
C52H69N2O13PNa: 983.4435; found 983.4398 (MþNaþ).
4.3.7.1. Compound 12. Yellow oil, mixture of diastereomers,
Rf¼0.5 (CH2Cl2/CH3OH, 95:5, v/v); nmax (liquid film) 1563, 1518, 1471,
1420, 1379, 1310, 1221, 1177, 1094, 1012 cmꢁ1
; dH (400 MHz, CD3OD):
7.49–6.87 (complex signals, 28H, aromatic protons), 5.23–5.08
[overlapped signals, 4H, 4x(H-1)], 4.59–4.34 [overlapped signals,12H,
4x(CH2CH2O-Glu-C-2) and 2x(OCH2CH2CN)], 4.18–3.40 [overlapped
signals, 96H, 4x(CH2CH2O-Glu-C-3), 4x(H-3), 4x(H-4), 16x(OCH2
-
CH2O), 4x(H2-6), 4x(H-2) and 4x(H-5), 2.78 [m, 4H, 2x(OCH2CH2CN)];
dC (100 MHz, CD3OD): 165.1, 158.7, 147.2, 141.1, 140.8, 138.7, 133.3,
132.2, 131.5, 131.1, 129.7, 128.4, 123.8, 123.2, 124.5, 118.4, 118.2 and
116.9 (aromatic carbons), 117.1 (CN), 101.9 and 101.4 (C-1), 83.0, 81.8,
80.0, 79.7 and 79.3 (C-3 and C-2), 76.9 and 76.7 (C-5), 74.0, 73.5, 71.9
[20x(OCH2CH2O) and C-4], 69.9, 68.4, 68.0 and 67.5 (C-6), 64.9
[2x(OCH2CH2CN)], 31.1 [2x(OCH2CH2CN)]; dP (161.98 MHz, CDCl3):
-1.25, -3.10, -3.59 and ꢁ5.70; MALDI-MS (positive ions): calcd for
C53H72ClNO24P2, 1203.36; m/z, found 1226.21 (MþNaþ), 1242.37
(MþKþ); HRMS (MALDI-TOF, positive ions): m/z: calcd for
C53H72ClNO24P2Na: 1226.3506; found 1226.3459 (MþNaþ).
4.3.6. Synthesis of linear dimer 11. Compound 8 (120 mg, 0.185 mmol)
and compound 9 (213 mg, 0.222 mmol), coevaporated several times
with anhydrous CH3CN, were reacted with a 0.25 M DCI solution
(6.0 mL) in CH3CN, previously left in contact with activated 3 Å mo-
lecular sieves. The resulting mixture was left under stirring at room
temperature for 2 h, then a 5.5 M t-BuOOH solution (425 mL) in n-
decane was added. After 30 min the reaction mixture was concentrated
under reduced pressure and the resulting residue redissolved in CH2Cl2,
transferred into a separatory funnel and washed twice with a satd aq
NaCl solution. The organic layer was collected, taken on anhydrous
sodium sulfate, filtered, concentrated under reduced pressure and the
residue purified on a silica gel column. Elution of the column with
CH2Cl2, added with 1% pyridine (v/v), containing increasing amounts of
MeOH (from 0% to 5%) allowed to obtain the pure DMT-protected linear
dimer 10 (140 mg, 0.092 mmol) in 50% yield for the two steps. This was
then treated with a 1% TCA solution (4 mL) in CH2Cl2 (v/v) for 30 min at
room temperature. The reaction mixture was concentrated under re-
duced pressure and the resulting residue purified on a silica gel column
eluted with CH2Cl2 containing increasing volumes of MeOH (from 2%up
to 10%). Pure compound 11 (113 mg, 0.092 mmol) could be isolated in
an almost quantitative yield.
4.3.8. Synthesis of 3. Derivative 12 (24 mg, 0.020 mmol) was dis-
solved in anhydrous piperidine (500 mL) and left under stirring for
12 h at 60 ꢀC. After TLC monitoring confirmed the total disappearance
of the starting compound, the reaction mixture was concentrated
under reduced pressure, redissolved in aq ammonium hydroxide
solution (500 m
L) and left under stirring for 12 h at 60 ꢀC. The reaction
mixture was concentrated under reduced pressure, redissolved in
H2O and purified on a Sephadex G25 column, eluted with H2O/EtOH,
4:1 (v/v). From UV spectrophotometric measurements, fractions ab-
sorbing at
l
¼264 nm were collected and taken to dryness, yielding
pure 3 (15 mg, 0.014 mmol) with 70% yield for the two steps.
4.3.8.1. Compound 3. Colourless oil. Rf¼0.3 (CH2Cl2/CH3OH,
85:15, v/v); nmax (liquid film) 1553, 1492, 1454, 1356, 1097, 1068,
1015 cmꢁ1
; dH (700 MHz, D2O): 7.32–7.02 (complex signals, 10H,
aromatic protons), 5.21 [d, J¼9.0 Hz, 2H, 2x(H-1)], 4.18 [ddd, 2H,
2x(H-6a)], 4.03 [t, J¼9.0 and 9.0 Hz, 2H, 2x(H-4)], 3.98–3.85 [over-
lapped signals, 10H, 2x(CH2CH2O-Glu-C-2), 2x(CH2CH2O-Glu-C-3
and 2x(H-6b)), 3.82 [m, 2H, 2x(H-5)], 3.70 [t, J¼9.0 and 9.0 Hz, 2H,
4.3.6.1. Compound 11. Yellow oil, Rf¼0.3 (CH2Cl2/CH3OH, 95:5,
v/v); nmax (liquid film) 3402 (br),1582,1474,1351,1237,1053 cmꢁ1
; dH
(500 MHz, CDCl3): 7.77–6.98 (overlapped signals, 14H, aromatic

C. Coppola et al. / Tetrahedron 65 (2009) 9694–9701
9701
8. Di Fabio, G.; Randazzo, A.; D’Onofrio, J.; Ausı`n, C.; Grandas, A.; Pedroso, E.; De
Napoli, L.; Montesarchio, D. J. Org. Chem. 2006, 71, 3395–3408.
9. (a) Coppola, C.; Saggiomo, V.; Di Fabio, G.; De Napoli, L.; Montesarchio, D. J. Org.
Chem. 2007, 72, 9679–9689; (b) Licen, S.; Coppola, C.; D’Onofrio, J.; Mon-
tesarchio, D.; Tecilla, P. Org. Biomol. Chem. 2009, 7, 1060–1063.
2x(H-3)], 3.67–3.55 [overlapped signals, 32H, 8x(OCH2CH2O)], 3.51
[m, 2H, 2x(H-2)]; dC (175 MHz, D2O): 158.8, 132.7, 126.4 and 119.3
(aromatic carbons),103.3 (C-1), 83.5 (C-3), 83.4 (C-2), 80.4 (C-4), 74.5,
74.4 and 72.8 [10x(OCH2CH2O)], 69.9 (C-5), 69.0 (C-6); dP
(161.98 MHz, D2O): 0.08; ESI-MS (negative ions): calcd for
C44H66O24P2, 1040.34; m/z, found 1038.79 (MꢁH)ꢁ; 517.82
(Mꢁ2H)2ꢁ; HRMS (MALDI-TOF, negative ions): m/z: calcd for
C44H65O24P2: 1039.3341; found 1039.3293.
10. For recent examples of crown ethers playing key roles in functionalized cal-
ixarene-based chemosensors, see, among others: (a) Chang, K.-C.; Su, I.-H.;
Senthilvelan, A.; Chung, W.-S. Org. Lett. 2007, 9, 3363–3366; (b) Zhang, D.; Cao,
X.; Purkiss, D. W.; Bartsch, R. A. Org. Biomol. Chem. 2007, 5, 1251–1259.
11. See, for example: (a) Gibson, H. W.; Wang, H.; Bonrad, K.; Jones, J. W.;
Slebodnick, C.; Zackharov, L. N.; Rheingold, A. L.; Habenicht, B.; Lobue, P.; Ratliff,
A. Org. Biomol. Chem. 2005, 3, 2114–2121; (b) Macrocyclic Chemistry; Dietrich, B.,
Viout, P., Lehn, J. M., Eds.; VCH: New York, NY, 1993; (c) Antonini Vitali, C.;
Masci, B. Tetrahedron 1989, 45, 2201–2212; (d) Ercolani, G.; Mandolini, L.; Masci,
B. J. Am. Chem. Soc. 1981, 103, 2780–2782; (e) For a very recent example, see:
Illuminati, G.; Mandolini, L. Acc. Chem. Res. 1981, 14, 95–102; Porwanski, S.;
Marsura, A. Eur. J. Org. Chem. 2009, 13, 2047–2050 and references cited therein.
12. (a) Adinolfi, M.; De Napoli, L.; Di Fabio, G.; Iadonisi, A.; Montesarchio, D.;
Piccialli, G. Tetrahedron 2002, 58, 6697–6704; (b) Adinolfi, M.; De Napoli, L.; Di
Fabio, G.; Iadonisi, A.; Montesarchio, D. Org. Biomol. Chem. 2004, 2, 1879–1886.
13. For a recent review, see for example: Reese, C. B. Org. Biomol. Chem. 2005, 3,
3851–3868.
Acknowledgements
We thank MIUR (PRIN) for grants in support of this investigation
`
and Centro di Metodologie Chimico-Fisiche (CIMCF), Universita di
Napoli ‘Federico II’, for the MS and NMR facilities. We also thank
`
prof. Paolo Tecilla, from Universita di Trieste, Italy, for the trans-
membrane ion transport assays.
14. (a) Moggio, L.; De Napoli, L.; Di Blasio, B.; Di Fabio, G.; D’Onofrio, J.;
Montesarchio, D.; Messere, A. Org. Lett. 2006, 8, 2015–2018; (b) Alazzouzi, E. M.;
Escaja, N.; Grandas, A.; Pedroso, E. Angew. Chem., Int. Ed. 1997, 36, 1506–1508
and references cited therein.
15. To further corroborate this hypothesis, which would well explain the irre-
sponsiveness of 3 towards the addition of metal cations, two kinds of experi-
Supplementary data
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.tet.2009.09.100.
ments were carried out. The first one involved the treatment of
3 with
competing binders as EDTA, which was left in contact with the bis-crown
derivative for 72 h; however no apparent change was observed in the 1H NMR
resonances in 3 as such, nor when successively left in contact with metal
cations. Similar results were obtained treating 3 with strong cation exchange
resins, as Chelex-100. In a second set of experiments, the apolar, fully protected
macrocycle 12 was filtered through basic alumina, reported also by other au-
thors (cf.r. ref. 6b and references cited therein) as a standard chromatographic
procedure in crown ether chemistry for cations removal. Indeed 12, originally
only soluble in methanol, after filtration on alumina was converted into
a chloroform soluble compound, with a higher Rf, still having the same MS
features. However, if it could be reasonably concluded that this treatment was
effective in removing bound cations from the crown ether pockets in 12, no
different behaviour was then observed when analyzing the resulting 3. It is to
be noted, however, that 3 is derived from 12 through two deprotection steps,
with the latter one carried out in aq ammonia, requiring for the purification
a prolonged contact with aq solutions. So, even if efficiently removed from 12,
Naþ or Kþ ions could be successively extracted from aq solutions and/or from
glassware, so to give a stable complex of 3, which is then obviously unreactive
towards additional metal ions.
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