Cyclic phosphate-linked oligosaccharides: Synthesis and conformational behavior of novel cyclic oligosaccharide analogues

Article abstract of DOI:10.1021/jo0600757

CyPLOS (cyclic phosphate-linked oligosaccharides), that is, novel cyclic oligosaccharide surrogates, consisting of two, three, and four phenyl-β-D-glucopyranoside units, 4,6-linked through stable phosphodiester bonds, were prepared by a straightforward an

Full text of DOI:10.1021/jo0600757

Cyclic Phosphate-Linked Oligosaccharides: Synthesis and
Conformational Behavior of Novel Cyclic Oligosaccharide Analogues
Giovanni Di Fabio,† Antonio Randazzo,‡ Jennifer D’Onofrio,† Cristina Aus´ın,§,|
Enrique Pedroso,§ Anna Grandas,§ Lorenzo De Napoli,† and Daniela Montesarchio*,†
Dipartimento di Chimica Organica e Biochimica, UniVersita` degli Studi di Napoli “Federico II”,
Complesso UniVersitario di Monte S. Angelo, Via Cynthia, 4, I-80126 Napoli, Italy, Dipartimento di
Chimica delle Sostanze Naturali, UniVersita` degli Studi di Napoli “Federico II”, Via Domenico
Montesano, 49, I-80131 Napoli, Italy, and Departament de Qu´ımica Orga`nica, Facultat de Qu´ımica,
UniVersitat de Barcelona, Mart´ı i Franque`s 1-11, E-08028 Barcelona, Spain
montesar@unina.it
ReceiVed January 13, 2006
CyPLOS (cyclic phosphate-linked oligosaccharides), that is, novel cyclic oligosaccharide surrogates,
consisting of two, three, and four phenyl-â-D-glucopyranoside units, 4,6-linked through stable phos-
phodiester bonds, were prepared by a straightforward and efficient solid-phase protocol. The assembly
of the linear precursors was achieved by standard phosphoramidite chemistry on an automated DNA
synthesizer, using a suitably protected 4-phosphoramidite derivative of D-glucose as the building block.
For the crucial cyclization step a phosphotriester methodology was exploited, followed by a mild basic
treatment releasing the desired cyclic molecules in solution in a highly pure form. The cyclic dimer and
trimer were also independently prepared by classical solution synthesis, basically following the same
approach. The solution structural preferences of the cyclic dimer and trimer, obtained by detailed NMR
analysis, are also reported.
Introduction
with structurally diverse backbones, including peptide and
oligosaccharide cores, and are also useful synthetic scaffolds
in the design of artificial hosts for selective recognition and
catalysis. Due to their unique ability to bind hydrophobic
molecules within their cavity while dissolved in polar solvents,
cyclodextrins, that is, cyclic oligosaccharides with a toroidal
shape composed of 6, 7, or 8 R-(1,4)-linked glucopyranose
residues, have attracted widespread interest in supramolecular
chemistry.2 In addition to being ideal hosts for a plethora of
guest molecules or ions, cyclodextrins are readily available at
low cost, are not toxic, are nonimmunogenic, and exhibit an
excellent pharmacokinetic profile.3 For these reasons, such
oligosaccharides have found important applications in many
The synthesis of macrocyclic compounds is one of the most
fascinating problems in organic chemistry.1 Large cyclic motifs
are found in a wide number of naturally occurring compounds
* Corresponding author. Phone: +39-081-674126/27/41. Fax: +39-081-
674393.
† Dipartimento di Chimica Organica e Biochimica.
‡ Dipartimento di Chimica delle Sostanze Naturali.
§ Universitat de Barcelona.
| Present address: Laboratory of Chemistry, Center for Drug Evaluation and
Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD
20892.
(1) For recent reviews on macrocycles, see: (a) Roxburgh, C. J.
Tetrahedron 1995, 51, 9767-9822. (b) Blankenstein, J.; Zhu, J. Eur. J.
Org. Chem. 2005, 10, 1949-1964. (c) Gonthier, E.; Breinbauer, R. Mol.
DiVersity 2005, 9, 51-62. (d) Wessjohann, L. A.; Ruijter, E. Top. Curr.
Chem. 2005, 243, 137-184.
(2) For a comprehensive review on cyclodextrins, see: Szejtli, J. Chem.
ReV. 1998, 98, 1743-1753 and references therein.
10.1021/jo0600757 CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/01/2006
J. Org. Chem. 2006, 71, 3395-3408
3395

Di Fabio et al.
SCHEME 1. General Procedure for the Solid-Phase Synthesis of Cyclic Oligomers 1, 2, and 3a
a a: Chain elongation and final DMT removal. b: Cyclization. c: Detachment from the support and deprotection.
different fields as molecular reactors,4 drug delivery systems,5
enzyme mimics,6 electrode surface modifiers,7 and chromato-
graphic materials,8 just to mention a few. Indeed, cyclodextrins
are exploited as pharmaceutical tools mainly to improve drug
bioavailability, but also in cosmetics, in tissues, and in food
and environmental chemistry.9
nected through an amide in lieu of glycosidic bonds.13b-d These
molecules can be useful scaffolds to elicit predictable secondary
structures when inserted in peptide backbones or, in the form
of cyclic homo-oligomers, to produce new analogues of natural
biopolymers, of interest as both peptidomimetics and cyclo-
dextrin mimics.13e-h Other interesting examples of modified
cyclic oligosaccharides have been described by Vasella and co-
workers, who prepared analogues of R-, â- and γ-cyclodextrins
including a buta-1,3-diyne-1,4-diyl or a 1,2,3-triazole moiety
connecting the saccharidic monomers.13i,j Cyclic oligomers
formed by butadiyne-linked carbohydrate residues were also
shown to easily accommodate a nucleoside.13k
Aiming at cyclic oligosaccharide analogues containing glu-
cose units connected through chemically and enzymatically
stable bonds, alternative to the natural 1,4-O-glycosidic linkages,
we here report the synthesis of new cyclic structures we have
named CyPLOS (cyclic phosphate-linked oligosaccharides),
containing two, three, and four phenyl-â-D-glucopyranoside
residues, 4,6-linked through phosphodiester bonds (1, 2, and 3,
Scheme 1), as well as the investigation of their conformational
behavior by NMR. Phosphate bridges, chosen as linkers between
two or more saccharidic moieties in a cyclic arrangement, may
Various chemically modified cyclodextrin derivatives, cy-
clodextrin polymers, and cyclodextrin conjugates with drugs,
peptides, or differently functionalized molecules have been
designed and evaluated.10 A fine-tuning of the properties and
complexation capabilities of cyclodextrins can be achieved by
selectively modifying their oligosaccharide backbone.11 Organic
synthesis offers easy access to the construction of modified
cyclodextrins displaying the desired functional groups in specific
positions on the cyclic molecule.12 A de novo chemical synthesis
approach is particularly useful when incorporation in the cycle
of unnatural monosaccharides or of alternative inter-residue
linkages is desired.13 In the search for cyclic oligosaccharides
more stable to acidic and enzymatic hydrolysis, â-1,6-linked
cycloglucopyranosides, with sulfur replacing oxygen atoms in
the glycosidic bridges, were recently synthesized.13a Sugar amino
acids, that is, carbohydrates bearing both an amino and a
carboxyl group, have been also designed as suitable monomers
for the construction of modified cyclic oligosaccharides con-
(13) (a) Fan, L.; Hindsgaul, O. Org. Lett. 2002, 4, 4503-4506. (b)
Locardi, E.; Stockle, M.; Gruner, S.; Kessler, H. J. Am. Chem. Soc. 2001,
123, 8189-8196. (c) van Well, R. M.; Marinelli, L.; Erkelens, K.; van der
Marel, G. A.; Lavecchia, A.; Overkleeft, H. S.; van Boom, J. H.; Kessler,
H.; Overhand, M. Eur. J. Org. Chem. 2003, 2303-2313. (d) Bornaghi, L.
F.; Wilkinson, B. L.; Kiefel, M. J.; Poulsen, S. A. Tetrahedron Lett. 2004,
45, 9281-9284. (e) von Roedern, E. G.; Lohof, E.; Hessler, G.; Hoffmann,
M.; Kessler, H. J. Am. Chem. Soc. 1996, 118, 10156-10167. (f) van Well,
R. M.; Overkleeft, H. S.; Overhand, M.; Carstenen, E. V.; van der Marel,
G. A.; van Boom, J. H. Tetrahedron Lett. 2000, 41, 9331-9335. (g) van
Well, R. M.; Marinelli, L.; Altona, C.; Erkelens, K.; Siegal, G.; van Raaij,
M.; Llamas-Saiz, A. L.; Kessler, H.; Novellino, E.; Lavecchia, A.; van
Boom, J. H.; Overhand, M. J. Am. Chem. Soc. 2003, 125, 10822-10829.
(h) Mayes, B. A.; Stetz, R. J. E.; Ansell, C. W. G.; Fleet, G. W. J.
Tetrahedron Lett. 2004, 45, 153-156. (i) Stichler-Bonaparte, J.; Vasella,
A. HelV. Chim. Acta 2001, 84, 2355-2367. (j) Hoffmann, B.; Bernet, B.;
Vasella, A. HelV. Chim. Acta 2002, 85, 265-287. (k) Bu¨rli, R.; Vasella,
A. Angew. Chem., Int. Ed. Engl. 1997, 36, 1852-1853. (l) Morales, J. C.;
Zurita, D.; Penade´s, S. J. Org. Chem. 1998, 63, 9212-9222. (m) Belghiti,
T.; Joly, J.-P.; Alem, H.; Chapleur, Y. C. R. Chim. 2003, 6, 553-564. (n)
Bodine, K. D.; Gin, D. Y.; Gin, M. S. J. Am. Chem. Soc. 2004, 126, 1638-
1639. (o) Bodine, K. D.; Gin, D. Y.; Gin, M. S. Org. Lett. 2005, 7, 4479-
4482. (p) Velasco-Torrijos, T.; Murphy, P. V. Tetrahedron: Asymmetry
2005, 16, 261-272.
(3) (a) Davis, M. E.; Brewster, M. E. Nat. ReV. Drug DiscoVery 2004,
3, 1023-1035. (b) Nalluri, B. N.; Chowdary, K. P. R.; Murthy, K. V. R.
Int. J. Pharm. Excipients 2002, 79-91.
(4) Takahashi, K. Chem. ReV. 1998, 98, 2013-2034.
(5) Rajewski, R. A.; Stella, V. J. J. Pharm. Sci. 1996, 85, 1142-1169.
(6) Breslow, R.; Dong, S. D. Chem. ReV. 1998, 98, 1997-2011.
(7) Willner, I.; Willner, B. J. Mater. Chem. 1998, 8, 2543-2556.
(8) Mitchell, C. R.; Armstrong, D. W. Methods Mol. Biol. 2004, 243,
61-112.
(9) Hedged, A. Chem. ReV. 1998, 98, 2035-2044.
(10) Easton, C. J.; Lincoln, S. F. Modified Cyclodextrins: Scaffolds and
Templates for Supramolecular Chemistry; Imperial College Press: London,
1999.
(11) (a) Khan, A. R.; Forgo, P.; Stine, K. J.; D′Souza, V. T. Chem. ReV.
1998, 98, 1977-1996. (b) Engeldinger, E.; Armspach, D.; Matt, D. Chem.
ReV. 2003, 103, 4147-4173.
(12) (a) Gattuso, G.; Nepogodiev, S. A.; Stoddart, J. F. Chem. ReV. 1998,
98, 1919-1958. (b) Yuan, D. Q.; Tahara, T.; Chen, W. H.; Okabe, Y.;
Yang, C.; Yagi, Y.; Nogami, Y.; Fukudome, M.; Fujita, K. J. Org. Chem.
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M. Chem. Commun. 2003, 3020-3021.
3396 J. Org. Chem., Vol. 71, No. 9, 2006

Cyclic Phosphate-Linked Oligosaccharides
SCHEME 2. Synthesis of Building Block 9a
FIGURE 1. Structure of the methyl-R-D-glucopyranoside building
block previously used for the conjugation of oligonucleotides with
glucose-phosphate tails.18
N,N-diisopropylphosphoramidite moiety on a secondary hy-
droxyl group) typically installed on nucleotide monomers used
in standard automated oligonucleotide synthesis. Glucopyrano-
side 9 was obtained in five steps and with 80% overall yields.
Commercially available phenyl-â-D-glucopyranoside 4 was first
converted into 4,6-O-benzylidene derivative 516 by a reaction
with benzaldehyde dimethylacetal in the presence of catalytic
amounts of p-toluenesulfonic acid (PTSA) and then treated with
an excess of benzoyl chloride in pyridine to give 6.16 The
successive addition of I2 in CH3OH in the presence of
triethylsilane (Et3SiH) allowed the selective removal of the
benzylidene protecting group, and the primary hydroxyl group
of diol 717 was then protected by a reaction with 4,4’-
dimethoxytriphenylmethylchloride (DMTCl) in pyridine. Com-
pound 8 was subsequently phosphitylated by addition of
2-cyanoethyl-N,N-diisopropyl-chlorophosphoramidite and di-
isopropylethylamine (DIEA), leading to the desired 9. The
synthetic route to phosphoramidite 9 is essentially similar to
the one previously adopted for the preparation of the 4-phos-
phoramidite derivative of methyl-R-D-glucopyranoside 10 (Fig-
ure 1), recently described for the synthesis of glycoconjugate
oligonucleotides, tethering sugar-phosphate residues.18
The modifications introduced in building block 9 with respect
to 10 led to significant improvements. On one hand, benzoyl
was chosen as the semipermanent protecting group for the
secondary hydroxyl functions because acetyl groups were found
to be not completely stable to the Et3N treatments, required to
remove the 2-cyanoethyl phosphate protecting groups (Monte-
sarchio et al., unpublished results). On the other hand, the UV-
visible phenyl group inserted at the anomeric position proved
to be useful for the final purification of the target cyclic
molecules.
Solid-Phase Synthesis of CyPLOS 1, 2, and 3. With
monomer 9 in hand, we first carried out on the solid phase both
the elongation of the saccharidic chains and the cyclization,
essentially following a procedure already described and opti-
mized for the preparation of cyclic oligonucleotides.15 The major
advantage of this strategy resides in the possibility of obtaining
the desired compounds with a high purity, which is intrinsically
a nontrivial problem when the synthetic targets are macrocycles.
The basic idea is to attach the first monomeric unit to the solid
support through a 2-chlorophenylphosphodiester bridge, which,
only if converted upon cyclization into a 2-chlorophenylphos-
photriester linkage, can be cleaved by the final oximate
treatment, selectively releasing the cyclic phosphodiester-linked
molecule in solution. Following this strategy, unreacted linear
chains or polymers, presumably formed in the solid phase as a
a a: Benzaldehyde dimethylacetal, PTSA cat., DMF, 12 h, 50 °C (97%).
b: BzCl, pyridine, 12 h, room temperature (quantitative yields). c: I2, Et3SiH
cat., CH3OH, 12 h, room temperature (91%). d: DMTCl, pyridine, 12 h,
room temperature (95%). e: 2-Cyanoethyl-N,N-diisopropylaminochloro-
phosphoramidite, DIEA, CH2Cl2, 1 h, room temperature (96%).
provide interesting features: (i) these bonds are chemically
stable in a large pH range; (ii) they are naturally negatively
charged, which could result in efficient cation binding; (iii)
phosphate diesters can be easily and efficiently obtained through
straightforward and high-fidelity reactions, well-optimized in
oligonucleotide synthesis. These advantages have been clearly
recognized by Nicolaou and co-workers, who first proposed
phosphate-linked carbohydrate analogues, termed “carbonucleo-
toids”, describing the synthesis of a linear tetramer by classical
solution methods starting from glucose pentaacetate.14
The here reported synthetic scheme exploits a versatile
approach, based on standard phosphoramidite chemistry for the
assembly of the sugar-phosphate backbone (Scheme 1). The
key intermediate in our strategy is the stable 4-phosphoramidite
derivative of D-glucose 9 (Scheme 2). The 4,4′-dimethoxytriph-
enylmethyl (DMT) group is used for the transient protection of
the 6-OH moiety; the phenyl group blocks the anomeric position
and allows UV monitoring during the final purification step.
Phosphoramidite 9 is exploited for the elongation of the
oligosaccharide chain on the solid matrix in an automated DNA
synthesizer. After cyclization, performed following phosphot-
riester procedures,15 the cyclic molecule is detached from the
solid support by a simple oximate treatment and obtained in a
very pure form. The solution synthesis of the target cyclic
molecules has been achieved following essentially the same
synthetic scheme.
Results and Discussion
Synthesis of Building Block 9. Phosphoramidite 9 was
prepared as summarized in Scheme 2. This building block
contains all the functional groups (DMT as the transient
protecting group for the primary hydroxyl and the 2-cyanoethyl-
(16) Rivaille, R.; Szabo´, L. Bull. Soc. Chim. Fr. 1963, 716-721.
(17) Ziegler, T.; Eckhardt, E.; Herold, G. Liebigs Ann. Chem. 1992, 5,
441-451.
(18) Adinolfi, M.; De Napoli, L.; Di Fabio, G.; Iadonisi, A.; Montesar-
chio, D.; Piccialli, G. Tetrahedron 2002, 58, 6697-6704.
(14) Nicolaou, K. C.; Florke, H.; Egan, H.; Egan, M. G.; Barth, T.;
Estevez, V. A. Tetrahedron Lett. 1995, 36, 1775-1778.
(15) Alazzouzi, E. M.; Escaja, N.; Grandas, A.; Pedroso, E. Angew.
Chem., Int. Ed. Engl. 1997, 36, 1506-1508.
J. Org. Chem, Vol. 71, No. 9, 2006 3397

Di Fabio et al.
SCHEME 3. Synthesis of Functionalized Solid Support 16a
a a: Tetrazole 0.45 M in CH3CN, 4 Å molecular sieves, 1 h, room temperature. b: t-BuOOH in decane, 10 min, room temperature. c: DCC, HOBt in
CH2Cl2/DMF, 12 h, room temperature. d: Piperidine 20% in DMF, 3 × 10 min, room temperature. e: Pyridine/Et3N (1:1, v/v), 3 × 2 h, room temperature.
result of interstrand condensations, are not affected by oximate
and, therefore, are not detached from the support. As the solid
support, a Tentagel amino resin (Tentagel-NH2) was exploited,
first derivatized with the spacer 6-aminohexanoic acid, in the
form of a N-Fmoc (fluorenylmethoxycarbonyl)-protected de-
rivative, to give 13, which was then deprotected at the primary
amino function, yielding 14 (Scheme 3). The subsequent step
was the insertion on the solid support of the bifunctional linker
3-chloro-4-hydroxyphenylacetic acid, bearing the o-chlorophenol
moiety that generates the desired o-chlorophenylphosphodiester
linkage when reacted with phosphoramidite 9. To obtain a
homogeneous functionalization of the solid support with the first
monomer, the linker, in the form of activated 2,4,5-trichlo-
rophenyl ester 1115 was coupled in solution with building block
9 in the presence of tetrazole. The successive oxidation of the
phosphite to phosphate triester function gave adduct 12, which
was then reacted with support 14 in the presence of N,N-
dicyclohexylcarbodiimide (DCC) and N-hydroxybenzotriazole
(HOBt). The loading of the resulting support 15 was always in
the range of 0.13-0.17 mmol/g, as evaluated by spectroscopic
measurements of the DMT cation released upon acidic treatment
from weighed amounts of the dry resin.
ester linkage, as monitored by recording 31P NMR spectra of
the resin suspended in CDCl3. Typically, upon 2-cyanoethyl
removal from the phosphate triester function on resin 15, the
two signals at about -5 ppm were replaced, in resin 16, by a
single signal at -2.4 ppm. The assembly of the linear oligomers,
including final DMT removal, was carried out by a standard
phosphoramidite protocol on an automated DNA synthesizer,
using phosphoramidite 9 as the building block (Scheme 4).
In a typical experiment, 100 mg of the functionalized support
16 were used in a 5-µmol scale synthesis, including, for each
synthetic cycle, the following steps: detritylation, coupling,
oxidation, and capping. Incorporation of one, two, or three
glucopyranose-phosphate units afforded functionalized resins
17, 18, and 19, respectively, from which the DMT group was
then removed. The desired circularization was achieved by the
addition of 1-mesitylensulfonyl-3-nitro-1,2,4-triazole (MSNT)
in pyridine to give solid support 20, 21, or 22, respectively,
following a classical phosphotriester protocol. After treatment
with Et3N/pyridine (1:1, v/v), the selective detachment from the
support of the cyclic molecule was then realized by a reaction
with 1,1,3,3-tetramethylguanidinium 2-nitrobenzaldoximate (0.2
M solution in dioxane/H2O, 1:1, v/v). The eluate was succes-
sively treated with concentrated aq ammonia for complete
hydroxyls deprotection. The concentrated mixture was then
Support 15 was then treated with Et3N/pyridine, until
complete conversion of the phosphotriester into the phosphodi-
3398 J. Org. Chem., Vol. 71, No. 9, 2006

Cyclic Phosphate-Linked Oligosaccharides
SCHEME 4. Solid-Phase Synthesis of Cyclic Oligomers 1, 2,
and 3a
FIGURE 2. HPLC profile of crude cyclic tetramer 3, as released from
the solid support (conditions as reported in Supporting Information).
The peak at 12.5 min, collected and analyzed by ESI-MS, was identified
as the target macrocyclic compound.
in phosphoramidite solution synthesis protocols. Building block
23 was obtained in a one-pot reaction in 90% yields starting
from derivative 8, which was phosphorylated by treatment with
2-chlorophenylphosphoryl pyridinium chloride. This intermedi-
ate was also designed as the model compound to verify the
stability of the linkage connecting the 4-phosphoester group of
the first monomer to the 3-chloro-4-hydroxyphenylacetic linker
on solid-support 16 when exposed to the reaction conditions
required for the phosphoramidite-based chain elongation pro-
tocol. Therefore, compound 23 was subjected to prolonged
treatments (12 h, room temperature) with the deblock [2%
dichloroacetic acid (DCA) in CH2Cl2], the oxidation [0.02 M
I2 in pyridine/H2O/tetrahydrofuran (THF), 7:2:1, v/v/v], and the
capping solution [acetic anhydride (Ac2O)/pyridine, 1:1, v/v)]
routinely used in oligonucleotide assembly protocols. In no case,
degradation products, even in traces, were found, as evaluated
by TLC, HPLC, and NMR analysis of the mixtures.
a a: DCA 2% in CH2Cl2, 10 min, room temperature. b: (i) Coupling
with 9; (ii) oxidation; (iii) capping. c: MSNT, pyridine, 12 h, room
temperature. d: Et3N/pyridine (1:1, v/v), 3 h, room temperature. e: 1,1,3,3-
tetramethylguanidinium 2-nitrobenzaldoximate 0.2 M in H2O/dioxane (1:
1, v/v), 12 h, room temperature. f: NH4OH (17 M), 6 h, 55 °C.
applied to a Sephadex G25 column, which yielded the desired
compound in pure form, not requiring further purification, as
evaluated by HPLC and NMR analysis (see, for example, the
HPLC profile for crude cyclic tetramer 3, as detached from the
solid support, in Figure 2). The dimer 1 in the amount of 4 mg,
2 mg of trimer 2, and 1 mg of tetramer 3 could be on average
isolated for each preparation, with overall yields ranging from
about 40% for the dimer to 5% for the tetramer. As expected,
a marked decrease in cyclization yields was observed upon
increasing the length of the oligomer, in agreement with previous
findings on cyclic oligonucleotides.15,19 Purified cyclic molecules
1, 2, and 3 were fully characterized by ESI-MS and NMR
spectroscopy.
Solution Synthesis of CyPLOS 1 and 2. To further confirm
their structures, 1 and 2 were also independently synthesized
by solution methods that may facilitate the preparation of large
amounts of the desired products. To this purpose, 4-O-(2-
chlorophenyl)phosphate glucopyranoside 23 (Scheme 5) was
prepared and used as the starting material in connection with 9
The synthesis of the linear oligomers was accomplished as
described in Scheme 6. Solution coupling of glucopyranoside
23 with phosphoramidite 9, promoted by standard activator
solution (0.45 M tetrazole in anhydrous CH3CN), followed by
the addition of 5.5 M anhydrous t-ButOOH in decane, for the
phosphite to phosphate oxidation, led to a mixture of DMT-
protected dimer 24 and deprotected linear dimer 25. After
complete DMT removal, obtained compound 25 was subjected
to the cyclization procedure, carried out at 1 × 10-3
M
concentration in anhydrous pyridine by the addition of MSNT.
Protected cyclic dimer 26 could be isolated in 71% yields.
Selective deprotection of the phosphodiester linkages was
achieved by treatment with Et3N/pyridine (1:1, v/v), followed
by the reaction with a 0.2 M solution of 1,1,3,3-tetrameth-
ylguanidinium 2-nitrobenzaldoximate in dioxane/H2O (1:1, v/v),
(19) De Napoli, L.; Galeone, A.; Mayol, L.; Messere, A.; Montesarchio,
D.; Piccialli, G. Bioorg. Med. Chem. 1995, 3, 1325-1329 and references
therein.
J. Org. Chem, Vol. 71, No. 9, 2006 3399

Di Fabio et al.
SCHEME 5. Synthesis of Derivative 23
allowing, respectively, the 2-cyanoethyl and 2-chlorophenyl
group removal.
A final aq ammonia treatment ensured the complete hydroly-
sis of the benzoic ester functions, thus affording target com-
pound 1, which was isolated in 73% yields after a Sephadex
G25 column.
the same multiplicity and coupling constants, indicating that 1
adopts the same conformation in both solvents. The 1D proton
spectrum of 1 (700 MHz, D2O, T ) 298 K) showed the presence
of only seven signals attributable to nonexchangeable protons
in the region between 3.2 and 5.5 ppm, at δH 4.93 (d, H1), 3.22
(t, H2), 3.43 (t, H3), 3.93 (q, H4), 3.50 (br d, H5), 3.86 (br d,
H6a), and 3.70 (br d, H6b).
A mixture of DMT-protected linear trimer 27 and DMT-free
linear trimer 28 was achieved by coupling linear dimer 25 with
phosphoramidite 9, following standard procedures. Trimer 28
(1 × 10-3 M concentration in anhydrous pyridine) was then
cyclized by the addition of MSNT. Analogously to 26, fully
protected cyclic trimer 29, obtained in 56% yields, was next
subjected to the successive Et3N/pyridine, benzaldoximate, and
aq ammonia treatments to remove the phosphate and hydroxyl
protecting groups, respectively, yielding 2 in 75% yields after
a Sephadex G25 column.
All the intermediates and the final compounds were fully
characterized by ESI-MS and NMR spectroscopy. The depro-
tected cyclic molecules obtained by solution synthesis were
found to be identical to the ones previously prepared following
the solid-phase strategy, as confirmed by HPLC coelution
experiments and NMR data comparison.
DMSO-d6 generally allows the observation of exchangeable
protons and is useful to get insight into the hydrogen-bonding
network and, therefore, into the conformational preferences of
the molecule.20 Particularly, we analyzed three parameters of
the two OH signals reported in Table 1: (i) the chemical shift
values; (ii) the multiplicity and vicinal coupling constants
(3JCH,OH); and (iii) the temperature coefficients (∆δ/∆T, in ppb
°C-1). A first inspection of the spectrum clearly showed a
notable difference in the chemical environment experienced by
OH-3 and OH-2. In fact, the signal attributed to OH-3,
resonating at 7.90 ppm (298 K), showed a relevant downfield
shift (ca. 2 ppm) with respect to OH-2 and appeared as a broad
singlet, as if in the presence of slow exchanges on the NMR
time scale. In addition, it exhibited a low-temperature coefficient
(-1.4), indicating a very limited exposure to the solvent. On
the contrary, the signal attributed to OH-2 resonated at 5.27
ppm (298 K) as a well-resolved doublet with a vicinal coupling
constant of 5.1 Hz, which according to a simplified Karplus
equation accounts for a free rotation of the hydroxyl group
around the C-O bond. A large temperature coefficient was
found for this proton (-8.9), which is consistent with intense
solvation effects. Being well-accepted that hydrogen-bonded
protons are significantly deshielded, it can be concluded that
OH-3 is tightly involved in such interactions, whereas OH-2 is
not affected.
NMR Analysis and Structural Studies. Macrocycle 1, 2,
and 3 were converted into the corresponding triethylammonium
salts by cation exchange on a DOWEX (Et3NH+ form) resin to
1
have homogeneous samples. H NMR spectra of all the three
compounds were recorded in DMSO-d6 and D2O.
As far as 1 is concerned, the 1H, 13C, and 31P NMR spectra,
both in D2O and DMSO-d6, show a unique signal for each kind
of nucleus, as if in the presence of a single glucopyranoside
unit. This can be attributed to the high internal symmetry of 1
(2-fold symmetry) and further confirms the cyclic structure of
the synthesized molecule. 1H NMR spectra of 1 were recorded
in the concentration range 0.1-2.0 mM. No significant changes
Altogether, these data agreed with the absence of mutual
H-bonding interactions between OH-3 and OH-2, typically
observed in cyclodextrins.20a,b On the other hand, taking into
account the spatial proximity between OH-3 and the negatively
charged oxygen atom of the 4-phosphate group, it can be
hypothesized that strong intramolecular H-bonding interactions
exist between these two groups.
1
were observed in the shape or distribution of the H NMR
resonances, thus allowing to exclude aggregation phenomena
under these conditions. Proton (700 MHz, T ) 298 K), carbon
(175 MHz, T ) 298 K), and phosphorus (202 MHz, T ) 298
K) assignments were obtained through an in-depth analysis of
one-dimensional (1D) 1H and 31P NMR spectra and two-
dimensional (2D) PE-COSY, TOCSY, HSQC, and HMBC
NMR spectra.
(20) (a) Bernet, B.; Vasella, A. HelV. Chim. Acta 2000, 83, 995-1021.
(b) Bernet, B.; Vasella, A. HelV. Chim. Acta 2000, 83, 2055-2071. (c)
Ivarsson, I.; Sandstro¨m, C.; Sandstro¨m, A.; Kenne, L. J. Chem. Soc., Perkin
Trans. 2 2000, 2, 2147-2152.
The 1H NMR spectra in DMSO-d6 and D2O were very similar
(Figure 3). Particularly, all the signals were characterized by
3400 J. Org. Chem., Vol. 71, No. 9, 2006

Cyclic Phosphate-Linked Oligosaccharides
SCHEME 6. Solution Synthesis of Cyclic Oligomers 1 and 2a
a a: Coupling with 9. b: Oxidation and capping. c: DCA 2% in CH2Cl2, 10 min, room temperature. d: MSNT, pyridine, 12 h, room temperature. e:
Et3N/pyridine (1:1, v/v), 3 h, room temperature. f: 1,1,3,3-tetramethylguanidinium 2-nitrobenzaldoximate 0.2 M in H2O/dioxane (1:1, v/v), 12 h, room
temperature. g: NH4OH (17 M), 6 h, 55 °C.
2D NOESY experiments allowed confirmation of the assign-
ments and the ability to gain structural information. Essentially
all NOEs were between 1,3-diaxial protons within the sugar
(Figure 4), thus suggesting that the â-D-glucopyranoside moieties
assume a classical 4C1 chair conformation and are not distorted.
This observation was 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 can be derived from the 1D 1H NMR spectrum
and particularly from the analysis of the multiplicity and
coupling constants of the H4, H6a, and H6b (for their couplings
with P) and H5 (for its couplings with H6a and H6b) resonances.
All the signals attributed to the H5, H6a, and H6b protons appear
as broad doublets. In particular, H5 exhibits a coupling constant
of 9 Hz (due to the coupling with H4), and H6a and H6b show
a geminal coupling constant of 12 Hz. This suggests that both
the dihedral angles H5-C5-C6-H6a and H5-C5-C6-H6b
are close to 90°. Analogously, the dihedral angles H6a-C6-
O6-P and H6b-C6-O6-P do not sensibly deviate from 90°
as well. On the other hand, the quartet multiplicity of H4 is
justified by scalar couplings exhibiting the same J values (9
3
Hz) with H3, H5, and P. Unfortunately, for JHP ) 9 Hz, four
real roots are obtained when solving the Karplus equation, not
allowing the retrieval of any certain structural information.
Three-dimensional structures of 1, satisfying all the found
NOEs and dihedral angle constraints (3JHH and 3JHP), were built
by restrained simulated annealing calculations. An initial
structure of 1 was constructed and minimized to eliminate any
possible source of initial bias in the conformation. Restrained
simulations were carried out in vacuo for 150 ps 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
J. Org. Chem, Vol. 71, No. 9, 2006 3401

Di Fabio et al.
FIGURE 4. Expanded region of 2D NOESY (700 MHz, T ) 298 K,
mt ) 500 ms) of dimer 1 in DMSO-d6.
behavior of the molecule. In fact, dissolved in D2O at 25 °C,
the 1H NMR spectrum of 2 is very similar to the one of dimer
1, with three magnetically equivalent glucopyranoside residues
(3-fold symmetry). On the contrary, when dissolved in DMSO-
d6 for the analysis of the hydroxyl protons, all the resonances
were not resolved and appeared very broadened, as if in the
presence of slow equilibriums on the NMR time scale, and no
real improvement was obtained by heating the system up to
100 °C. However, it was possible to analyze the signals
attributed to OH-2 and OH-3, which at 298 K resonated at 5.32
and 7.90 ppm and showed temperature coefficients of -6.8 and
-3.0 ppb °C-1, respectively (see Table 1), basically respecting
the same trend observed in cyclic dimer 1, which suggests again
the presence of intramolecular H bonds exclusively involving
the OH-3 groups.
1
FIGURE 3. Selected region of H NMR spectrum (700 MHz, T )
298 K) of dimer 1 in DMSO-d6 (panel A) and in D2O (panel B).
TABLE 1. Chemical Shifts, Vicinal Coupling Constants, and
Temperature Coefficients for the Hydroxyl Protons in Cyclic
Compounds 1 and 2a
HO-C (2)
HO-C (3)
δ (OH) multiplicity, ∆δ/∆T δ (OH)
compounds ppm J (H, OH), Hz ppb, °C ppm
∆δ/∆T
multiplicity ppb, °C
1
2
5.32
5.36
doublet, 5.1 -8.90
doublet, 5.1 -6.80
7.90 broad singlet -1.4
7.90 broad singlet -3.0
a Dissolved in DMSO-d6 (400 MHz, 298 K).
Because the spectrum of 2, when acquired in D2O, is
characterized by well-resolved and sharp resonances, this solvent
was used for the structural characterization. A selected region
of 1D proton NMR spectrum of 2 (700 MHz, T ) 298 K) is
reported in Figure 6.
was to energy-minimize and refine the structures obtained by
using the steepest descent and the quasi-Newton-Raphson
(VA09A) algorithms. Out of 50 structures generated, 20
structures with the lowest energies were selected. It was possible
to obtain an excellent superimposition of the 20 structures with
root mean square distance (RMSD) values of 0.65 ( 0.38 for
heavy atoms. The lack of violations and the low RMSD value
suggest that the minimized conformation of 1, reported in Figure
5, is consistent with the experimentally determined restraints.
Even if no H bond was a priori imposed during the structural
calculations, the obtained structure for 1 is characterized by
hydrogen bonds between the OH-3 and the adjacent negatively
charged oxygen atom of the 4-phosphate group of each subunit,
in perfect agreement with the results of the temperature-
dependence study carried out on the hydroxyl protons.
The overall structure of 1 appears to be quite planar, with
the two sugar moieties, as expected, in the 4C1 chair conforma-
tion and the phosphate groups pointing outward. The central
12-membered ring appears to be too small to host cations in
the cavity, thus forming inclusion complexes. However, the two
phosphate groups pointing toward the same side of the molecule
can create a binding site for positively charged groups. In this
conformation, the distances between the nonbridged oxygen
atoms of the two phosphate groups are in the range 7.1-7.7 Å.
When examining cyclic trimer 2, on the other hand, the nature
of the solvent was found to sensibly affect the conformational
3
3
Analyzing both the JHH and JHP coupling constants, we
found that, analogously to 1, all the â-D-glucopyranoside
4
moieties in 2 are in a C1 chair conformation, because all the
sugar ring H1, H2, H3, H4, and H5 protons show 3JHH ) 9 Hz.
This observation was further inferred by the analysis of the 2D
NOESY spectrum, in which essentially only the NOEs among
the 1,3 diaxial protons were observed (Figure 7).
The multiplicity and coupling constants of H4, H6a, and H6b
(for their couplings with P) and H5 (for its couplings with H6a
and H6b) were analyzed as well. As in 1, H4 is a quartet due to
3
the coupling with H3, H5, and P (J ) 9 Hz). Again, JHP ) 9
Hz yields four real roots when solving the Karplus equation,21
not allowing to retrieve any certain structural information.
3
Similarly, both H6a and H6b show JHP ) 6 Hz, which does
not allow any restraint in the structural calculation. H5 is
characterized, on the other hand, by coupling constants of 6
and 2 Hz, respectively, with H6a and H6b. Also in this case,
3JHH ) 6 Hz could not be used to guide structural calculation.
Even if the number of restraints available to characterize the
central 18-membered ring was very limited, we attempted to
(21) Lankhorst, P. P.; Haasnoot, C. A. G.; Erkelens, C.; Altona, C. J.
Biomol. Struct. Dyn. 1984, 1, 1387-1405.
3402 J. Org. Chem., Vol. 71, No. 9, 2006

Cyclic Phosphate-Linked Oligosaccharides
FIGURE 5. Top (A) and side (B) stereoview representations of the best NMR structure of dimer 1. The molecule is depicted in colored “stick”
form (carbons, green; oxygens, red; hydrogens, white; phosphorus, magenta). Hydrogen bonds are depicted with yellow dashed lines.
1H NMR analysis could not be carried out on cyclic tetramer
3, which, in all the investigated solvents (D2O, DMSO-d6, and
CF3CD2OD), exhibited very complex spectra. These were
characterized by severely overlapping and broadened signals,
whose relative intensities were temperature-dependent, suggest-
ing that 3 exists as a number of different families of conformers
in slow equilibrium on the NMR time scale. No benefit in terms
of resolution could be obtained by either heating the system
(up to 373 K in DMSO-d6) or cooling it (down to 263 K in
CF3CD2OD).
1
FIGURE 6. Selected region of H NMR spectrum (700 MHz, T )
298 K) of trimer 2 in D2O.
Conclusions
A straightforward solid-phase protocol for the synthesis of
CyPLOS, a novel class of cyclic oligosaccharide mimics,
containing two, three, and four phenyl-â-D-glucopyranoside
units, 4,6-linked through stable phosphodiester bonds, has been
here described. A suitable building block (9) has been devised,
which was obtained in five easy and high-yielding steps from
commercially available phenyl-â-D-glucopyranoside. Phosphora-
midite derivative 9 was first derivatized to allow incorporation
into the solid support through a 2-chlorophenylphosphodiester
bridge, essentially following the synthetic strategy previously
developed for the preparation of cyclic oligonucleotides.15 Chain
elongation was carried out on an automated DNA synthesizer
by standard and high-fidelity phosphoramidite chemistry, with
9 as the addition monomer, and a classical phosphotriester
protocol was exploited for the cyclization procedure. Selective
release in solution of the target cyclic molecules could be
achieved using mild basic conditions, under which linear,
unreacted oligomers or even polymeric species, formed on the
solid phase as side-products during the cyclization procedure,
were not detached. After a simple gel filtration chromatography,
cyclic dimer 1, trimer 2, and tetramer 3 were isolated in overall
yields ranging from 40% in the case of the dimer to 5% for the
tetramer. Solution synthesis of 1 and 2 provided independently
prepared samples, which proved to be identical to those
assembled on a solid support, as ascertained by HPLC, NMR,
and ESI-MS analysis. Both solution and solid-phase synthetic
strategies, based on the same well-known and optimized
chemistry, proved to be simple and reliable. In addition, the
FIGURE 7. Expanded region of 2D NOESY (700 MHz, T ) 298 K,
mt ) 500 ms) of trimer 2 in D2O.
carry out a restrained structural calculation study for 2. As
expected, this led to an inextricable mixture of isoenergetic
conformers, which could not be clustered into any well-defined
family. This indicates that, even if strong H-bonding interactions
between the OH-3 and the adjacent phosphate group are
expected, this cyclic molecule is not sufficiently rigidified to
adopt a single, preferential conformation in solution under the
studied experimental conditions.
J. Org. Chem, Vol. 71, No. 9, 2006 3403

Di Fabio et al.
) 6.6 and 10 Hz, 1H, H-3), 3.96-3.54 (overlapped signals, 5H,
H-2, H-4, H-5, and H2-6). 13C NMR (CDCl3, 50 MHz): δ 156.7,
129.5, 129.3, 128.3, 126.9, 126.2, 123.2, and 116.8 (aromatic
carbons); 101.9 (Ph-CH); 101.0 (C-1); 80.2 (C-5); 74.2 (C-3); 73.1
(C-2); 68.5 (C-4); 66.4 (C-6). ESI-MS (positive ions): m/z calcd
for C19H20O6, 344.126; found, 367.19 (M + Na+), 383.25 (M +
K+). HRMS (MALDI-TOF): m/z calcd for C19H20O6Na, 367.1158;
found, 367.1209 (M + Na+).
reported protocols can be in principle suitably and easily
modified so as to obtain all the specific backbone modifications
typically introduced in the ribose-phosphate chain of oligo-
nucleotides, as phosphorothioate, methylphosphonate, or phos-
phoramidate linkages, just to mention a few. Furthermore, the
repertoire of the accessible cyclic phosphodiester-linked oli-
gosaccharides (CyPLOS) can be significantly expanded by ad
hoc modulating the nature, ring size, stereochemistry, and
derivatizations of the saccharidic building block chosen. In this
frame, we are currently studying further functionalizations of
the hydroxyl groups to obtain oligomers with a higher diversity
in terms of structure and function.
A detailed structural study has been carried out by NMR
analysis. Using 1H-1H and 1H-31P vicinal scalar couplings and
NOE data, as well as mechanic and dynamic calculations, we
concluded that cyclic dimer 1, having a 2-fold symmetry, adopts
a cradle-like preferential conformation in solution, identical both
in D2O and in DMSO, stabilized by strong hydrogen-bonding
interactions between the negatively charged phosphate groups
and the adjacent OH-3. Cyclic trimer 2 showed a solvent-
dependent conformational behavior. Particularly, in D2O it
exhibited a very flexible structure, while in DMSO a number
of different families of conformers, in slow equilibrium on the
NMR time scale, were present in a large temperature range. In
the case of cyclic tetramer 3, in all the investigated solvents,
many different conformers in slow equilibrium could be
observed, not allowing a detailed structural analysis.
It could be envisaged, for the largest macrocycles, a cation-
dependent conformational behavior, with the metal coordinated
to the internal phosphate diester groups placed at definite
distances by the pyranoside rings. The potential of these cyclic
oligosaccharide analogues as novel artificial receptors, naturally
carrying one negative charge per residue in a large pH range,
will be investigated in their ability to selectively bind different
metal cations.
The here-reported synthetic strategy is highly versatile,
allowing in principle to prepare a variety of cyclic oligosac-
charide analogues with a high degree of intrinsic chemical
diversity, in which the single monomers are connected through
stable phosphate bonds or related derivatives. This issue is of
the utmost importance in the search for new synthetic molecular
receptors, potentially able to specifically bind metal cations or,
more generally, positively charged molecules, in the frame of
bio-organic and supramolecular chemistry-related applications.
Synthesis of 6. Compound 5 (2.0 g, 5.8 mmol, 1 equiv),
dissolved in anhydrous pyridine (10 mL), was reacted with benzoyl
chloride (3.4 mL, 29 mmol, 5 equiv). The mixture was left under
stirring at room temperature overnight and then concentrated under
reduced pressure. The crude was then diluted with CHCl3,
transferred into a separatory funnel, and washed three times with
a saturated NaHCO3 aqueous solution and then twice with water.
The organic phase, dried over anhydrous Na2SO4 and filtered, was
then concentrated under reduced pressure, furnishing pure 6 (3.2
g, 5.8 mmol) in an almost quantitative yield.
6: mp (from AcOEt/diethyl ether) 199-200 °C (lit.16 203 °C).
1
Rf ) 0.6 (benzene). H NMR (CDCl3, 200 MHz): δ 8.25-7.03
(complex signals, 20H, aromatic protons), 5.97 (t, J ) 9.2 and 9.2
Hz, 1H, H-3), 5.85 (apparent t, J ) 7.8 and 9.2 Hz, 1H, H-2), 5.61
(s, 1H, Ph-CH), 5.48 (d, J ) 7.5 Hz, 1H, H-1), 4.50 (m, 1H, H-5),
4.11 (t, J ) 9.2 and 9.0 Hz, 1H, H-4), 3.93 (m, 2H, H2-6). 13C
NMR (CDCl3, 50 MHz): δ 165.4 and 165.0 (2 CO); 156.7, 136.6,
134.3, 133.1, 133.0, 131.2, 130.3, 129.6, 129.4, 128.9, 128.7, 128.2,
128.0, 126.0, 123.2, and 117.0 (aromatic carbons); 101.3 (Ph-CH);
99.9 (C-1); 78.3 (C-5); 72.1 (C-3); 71.9 (C-2); 68.4 (C-4); 66.5
(C-6). ESI-MS (positive ions): m/z calcd for C33H28O8, 552.178;
found, 575.17 (M + Na+), 591.15 (M + K+). HRMS (MALDI-
TOF): m/z calcd for C33H28O8Na, 575.1682; found, 575.1721 (M
+ Na+).
Synthesis of 7. Compound 6 (3.2 g, 5.8 mmol, 1 equiv),
dissolved in anhydrous CH3OH (40 mL), was reacted with I2 (640
mg, 2.6 mmol, 0.45 equiv) and Et3SiH (45 µL, 0.29 mmol, 0.05
equiv). The reaction mixture, left at room temperature overnight
under stirring, was then diluted with AcOEt, and the organic phase
was washed twice with a saturated NaHCO3 aqueous solution and
then twice with sodium thiosulfate. The organic phase, dried over
anhydrous Na2SO4, was filtered and concentrated under reduced
pressure and then purified by column chromatography. Eluting the
column with CHCl3, containing growing amounts of CH3OH (from
0 to 10%), pure 7 (2.5 g, 5.3 mmol) was recovered in 91% yield.
7: mp (from acetone/n-hexane) 84-86 °C (lit.17 87 °C). Rf )
0.3 [CHCl3/CH3OH, 97/3, (v/v)]. 1H NMR (CDCl3, 200 MHz): δ
7.99-6.93 (complex signals, 15H, aromatic protons), 5.70 (dd, J
) 8.0 and 9.6 Hz, 1H, H-2), 5.49 (apparent t, J ) 9.2 and 9.6 Hz,
1H, H-3), 5.34 (d, J ) 7.8 Hz, 1H, H-1), 4.11-3.73 (overlapped
signals, 4H, H-4, H-5 and H2-6). 13C NMR (CDCl3, 50 MHz): δ
167.6 and 165.2 (2 CO); 156.9, 133.7, 133.3, 130.0, 129.7, 129.6,
129.1, 128.5, 128.4, 123.2, and 116.8 (aromatic carbons); 99.2 (C-
1); 77.2 (C-5); 76.1 (C-3); 71.1 (C-2); 69.9 (C-4); 62.2 (C-6). ESI-
MS (positive ions): m/z calcd for C26H24O8, 464.147; found, 487.14
(M + Na+), 503.15 (M + K+). HRMS (MALDI-TOF): m/z calcd
for C26H24O8Na, 487.1369; found, 487.1407 (M + Na+).
Synthesis of 8. Compound 7 (2.0 g, 4.3 mmol, 1 equiv),
dissolved in anhydrous pyridine (15 mL), was reacted with DMTCl
(1.9 g, 5.6 mmol, 1.3 equiv). The reaction mixture, left at room
temperature overnight under stirring, was then diluted with CH3-
OH and concentrated under reduced pressure. The crude was next
purified on a silica gel column, eluted with cyclohexane containing
growing amounts of AcOEt (from 15 to 30%) in the presence of a
few drops of pyridine, affording pure 8 (3.1 g, 4.1 mmol) in 95%
yield.
Experimental Section
Synthesis of Phenyl-6-O-(4,4′-dimethoxytriphenylmethyl)-2,3-
di-O-benzoyl-â-D-glucopyranoside-4-O-(2-cyanoethyl-N,N-diiso-
propyl)phosphoramidite, 9. Synthesis of 5. Phenyl-â-D-glucopy-
ranoside 4 (3.0 g, 12 mmol, 1 equiv) was dissolved in 25 mL of
anhydrous N,N-dimethylformamide (DMF). PTSA (110 mg, 0.6
mmol, 0.05 equiv) and, dropwise, benzaldehyde dimethylacetal (4
mL, 26 mmol, 2.2 equiv) were sequentially added to the stirred
mixture, which was left overnight at 50 °C. The crude was then
diluted with ethyl acetate (AcOEt), transferred into a separatory
funnel, washed three times with water, concentrated under reduced
pressure, and purified by column chromatography. Eluting the
column with CHCl3, containing growing amounts of CH3OH (from
1 to 5%) gave pure 5 (4.0 g, 11.6 mmol) with 97% yield.
8: glassy solid; mp >90 °C dec. Rf ) 0.4 [cyclohexane/AcOEt,
1
5: mp [from ethanol (EtOH)/acetone] 191-193 °C (lit.16 194-
7/3 (v/v)]. H NMR (CDCl3, 300 MHz): δ 8.03-6.82 (complex
1
195 °C). Rf ) 0.4 (CHCl3/CH3OH, 98/2, v/v). H NMR (CDCl3,
signals, 28H, aromatic protons), 5.74 (dd, J ) 8.1 and 9.0 Hz, 1H,
H-2), 5.52 (apparent t, J ) 9.3 and 9.3 Hz, 1H, H-3), 5.29 (d, J )
7.8 Hz, 1H, H-1), 4.02 (m, 1H, H-4), 3.81 (s, 6H, 2 OCH3 of DMT
200 MHz): δ 7.52-7.00 (complex signals, 10H, aromatic protons),
5.54 (s, 1H, Ph-CH), 5.02 (d, J ) 7.5 Hz, 1H, H-1), 3.36 (dd, J
3404 J. Org. Chem., Vol. 71, No. 9, 2006

Cyclic Phosphate-Linked Oligosaccharides
group), 3.76 (m, 1H, H-5), 3.56 (m, 2H, H2-6). 13C NMR (CDCl3,
75 MHz): δ 167.1 and 165.2 (2 CO); 158.4, 157.0, 144.5, 135.6,
133.4, 133.1, 129.9, 129.9, 129.7, 129.4, 129.1, 129.0, 128.8, 128.3,
128.2, 128.0, 127.8, 126.8, 123.0, 117.1, and 113.1 (aromatic
carbons); 99.3 (C-1); 86.5 (quaternary carbon of DMT group); 77.1
(C-5); 74.9 (C-3); 71.2 (C-2); 70.8 (C-4); 63.4 (C-6); 55.1 (OCH3
of DMT group). ESI-MS (positive ions): m/z calcd for C47H42O10,
766.278; found, 767.39 (M + H+), 789.25 (M + Na+), 805.49 (M
+ K+). HRMS (MALDI-TOF): m/z calcd for C47H42O10Na,
789.2676; found, 789.2711 (M + Na+).
Synthesis of 9. To a solution of compound 8 (500 mg, 0.65
mmol, 1 equiv), dissolved in anhydrous CH2Cl2 (4 mL), DIEA (335
µL, 1.95 mmol, 3 equiv) and 2-cyanoethyl-N,N-diisopropyl-
chlorophosphoramidite (230 µL, 1.04 mmol, 1.6 equiv) were
sequentially added under stirring at room temperature. After 30
min, the reaction mixture was diluted with AcOEt, transferred into
a separatory funnel, and washed three times with a saturated aqueous
NaCl solution and then twice with water. The organic phase was
dried over anhydrous Na2SO4, filtered, and then concentrated under
reduced pressure. The crude was then chromatographed on a silica
gel column, eluted with cyclohexane containing growing amounts
of AcOEt (from 25 to 30%) in the presence of a few drops of
triethylamine, furnishing the desired compound 9 (610 mg, 0.63
mmol) in 96% yield.
9: oil; Rf ) 0.5 [cyclohexane/AcOEt, 7/3 (v/v)]. 1H NMR
(CDCl3, 400 MHz; as a diastereomeric mixture): δ 7.99-6.75
(complex signals, 56H, aromatic protons), 5.71 (overlapped signals,
4H, 2 H-2 and 2 H-3), 5.34 (complex signal, 2H, 2 H-1), 4.10
(complex signal, 2H, 2 H-4), 4.00-3.85 (complex signals, 4H, 2
OCH2CH2CN), 3.78 and 3.77 (2 s, 12H, 2 OCH3 of DMT groups),
3.66 (complex signals, 2H, 2 H-5), 3.45-3.17 (complex signals,
8H, 2 H2-6 and 2 OCH2CH2CN), 2.24 and 2.01 (complex signals,
2H each, N[CH(CH3)2]2), 0.92, 0.90, 0.85, and 0.83 (complex
signals, 24H, N[CH(CH3)2]2). 13C NMR (CDCl3, 100 MHz): δ
165.6 and 165.2 (2 CO); 158.3, 157.1, 144.8, 136.0, 135.8, 133.0,
130.1, 130.0, 129.7, 129.5, 129.3, 128.2, 128.1, 127.6, 126.6, 122.8,
and 112.9 (aromatic carbons); 117.1 (CN); 99.4 and 99.2 (2 C-1);
86.1 and 86.0 (2 quaternary carbons of 2 DMT groups); 75.8, 75.2,
74.6, 71.9, 71.8, and 71.1 (C-2, C-3, C-4, and C-5); 63.4 and 62.7
(2 C-6); 57.8 and 57.6 (OCH2CH2CN); 55.1 (OCH3 of DMT group);
43.0 and 42.8 (N[CH(CH3)2]2); 24.2 (N[CH(CH3)2]2); 19.7 and 19.2
(OCH2CH2CN). 31P NMR (CDCl3, 161.98 MHz): δ 154.2 and
153.0. ESI-MS (positive ions): m/z calcd for C56H59N2O11P,
966.385; found, 967.19 (M + H+), 989.31 (M + Na+), 1005.42
(M + K+). HRMS (MALDI-TOF): m/z calcd for C56H60N2O11P,
967.3935; found, 967.3948 (M + H+).
Synthesis of Support 13. Resin TentaGel-NH2 (300 mg, 0.29
mmol/g, 0.087 mmol, 1 equiv) was treated with N-Fmoc-6-amino-
hexanoic acid (61 mg, 0.17 mmol, 2 equiv), HOBt (23 mg, 0.17
mmol, 2 equiv), previously coevaporated several times with
anhydrous pyridine, and DCC (35 mg, 0.17 mmol, 2 equiv) in a
CH2Cl2/DMF, 7/1 (v/v), solution. The reaction mixture was left at
room temperature overnight under stirring. Solid support 13 was
then exhaustively washed with DMF, CH2Cl2, CH3OH, and CH3-
CN and then dried under reduced pressure. The incorporation of
the 6-aminohexanoic acid spacer was monitored by spectrophoto-
metric Fmoc test (λ ) 301 nm, ꢀ ) 7800 cm-1M-1) after a basic
treatment (20% piperidine in DMF) on a dried and weighed amount
of the resin. The functionalization was 0.20 mmol/g on average,
corresponding to 69% yield. Unreacted amino groups were masked
by a standard capping procedure (Ac2O/pyridine, 1/1, v/v, rt, 2 h).
Synthesis of support 16. For Fmoc removal from support 13
(300 mg), a standard 20% piperidine treatment in DMF was carried
out (3 × 2 mL, 10 min each). Resulting support 14 was left in
contact overnight with derivative 12 (190 mg, 0.15 mmol, 2.5
equiv), DCC (31 mg, 0.15 mmol, 2.5 equiv), and HOBt (23 mg,
0.15 mmol, 2.5 equiv), previously dried by repeated coevaporations
with anhydrous pyridine, in 2.5 mL of CH2Cl2/DMF, 4/1 (v/v),
under stirring at room temperature. Successively, the support was
exhaustively washed with DMF, CH2Cl2, CH3OH, and CH3CN and
then dried under reduced pressure.
The functionalization of support 15 was calculated by spectro-
scopic measurements at λ ) 498 nm (ꢀ ) 71 700 cm-1M-1) of the
DMT cation, released by acidic treatment (HClO4/EtOH, 3/2, v/v)
from dried and weighed amounts of the resin. An average
functionalization of 0.15 mmol/g was obtained, corresponding to a
75% incorporation yield of derivative 12. Resin 15 was then treated
with Et3N/pyridine (1/1, v/v, 3 treatments of 2 h each) to remove
the 2-cyanoethyl group. After exhaustive washings, the resulting
support 16 was dried, then suspended in CDCl3 and analyzed by
31P NMR, which confirmed the total conversion of the phosphot-
riester into phosphodiester groups.
Solid support 16. 31P NMR (CDCl3, 161.98 MHz): δ -2.4.
Solid-Phase Synthesis of CyPLOS 1, 2, and 3. Support 16 (100
mg, 0.015 mequiv), previously washed with anhydrous CH2Cl2,
was treated at room temperature under stirring with 2% DCA in
CH2Cl2 (4 × 1 mL, 3 min each). Successively, the support, after
exhaustive washings with CH2Cl2 and anhydrous CH3CN, was
reacted with phosphoramidite 9 (50 mg), previously dried by
repeated coevaporations with CH3CN, in anhydrous CH3CN (300
µL) and a 0.45 M tetrazole solution in anhydrous CH3CN (600
µL). After 45 min, under stirring at room temperature, the support
was washed several times with CH3CN and then treated with a
0.02 M I2 solution (1 mL) in pyridine/H2O/THF (1/2/7, v/v) for 30
min under stirring. The solid support, repeatedly washed with CH3-
CN, was then dried under reduced pressure. The efficiency of the
incorporation of the second glycosidic residue onto solid support
17 was determined by measuring the absorbance at λ ) 498 nm of
the DMT cation (ꢀ ) 71 700 cm-1M-1) released upon acidic
treatment [HClO4/EtOH, 3/2 (v/v)] of a dried and weighed amount
of the resin. The functionalization was in all cases 0.15 mmol/g,
accounting for an almost quantitative incorporation of the second
monomer onto the solid support. After a standard capping treatment
with Ac2O/pyridine (1/1, v/v, 30 min), support 17 was subjected
to the final detritylation, followed by repeated washings with CH2-
Cl2 and CH3CN. Resulting support 20 was then reacted at rt under
stirring with MSNT (50 mg, 0.17 mequiv), dissolved in anhydrous
pyridine (500 µL, 2 × 3 h, followed by an overnight treatment).
The obtained support was then repeatedly washed with pyridine
and CH3CN and successively treated with Et3N/pyridine (2 mL,
1/1, v/v) for 3 h under stirring, specifically to remove the
2-cyanoethyl protecting group. The cyclic dimer was detached from
the solid support by an overnight treatment with a 0.2 M 1,1,3,3-
tetramethylguanidinium 2-nitrobenzaldoximate solution (1 mL) in
H2O/dioxane (1/1, v/v). The filtered eluate and two washings with
Functionalization of the Solid Support with Phosphoramidite
9. Synthesis of Derivative 12. Compound 9 (192 mg, 0.2 mmol,
1.2 equiv), previously coevaporated several times with anhydrous
CH3CN, was dissolved in a 0.45 M tetrazole solution in CH3CN
(1.2 mL) and reacted with 3-chloro-4-hydroxy-phenylacetic acid
2,4,5-trichloro-phenylester (11, 60 mg, 0.17 mmol, 1 equiv),
dissolved in anhydrous CH2Cl2 (0.5 mL), in the presence of
activated 4 Å molecular sieves. The reaction mixture was left at
room temperature under stirring for 1 h and monitored by TLC,
using CHCl3 as the eluent. Successively, a 5.5 M tert-butylhydro-
peroxide (t-BuOOH) solution in decane (200 µL, 1.7 mmol, 10
equiv) was added to the reaction mixture, which was left for an
additional 10 min under stirring. This was then diluted with CH2-
Cl2, transferred into a separatory funnel, and washed twice with a
saturated NaCl solution. The organic phase, dried over anhydrous
Na2SO4, was filtered, concentrated under reduced pressure, and
crystallized from n-hexane. Isolated compound 12 (168 mg, 0.13
mmol, corresponding to 76% yield) was used as such, without
further purification.
12: 31P NMR (CDCl3, 161.98 MHz; mixture of diastereomers):
δ -5.2 and -5.6. ESI-MS (positive ions): m/z calcd for C64H52-
Cl4NO15P, 1245.183; found, 1247.132 (M + H+), 1269.373 (M +
Na+).
J. Org. Chem, Vol. 71, No. 9, 2006 3405

Di Fabio et al.
H2O (1 mL each) of the support, once combined and concentrated
under reduced pressure, were treated with a concentrated aq
ammonia solution (5 mL) at 55 °C for 6 h. The obtained crude
was finally purified by gel filtration chromatography on a G25
Sephadex column eluted with H2O/EtOH, 4/1 (v/v). The obtained
fractions were monitored by spectrophotometric measurements at
λ ) 264 nm. Pure cyclic dimer 1 (4 mg, 0.0062 mmol, 42% overall
yields) was isolated, characterized by spectroscopic means, and
found to be identical to the compound synthesized in solution as a
control.
H2-6). 1H NMR (DMSO-d6, 373 K, 400 MHz): 7.32-7.01
(complex signals, 5H, aromatic protons), 4.88 (d, J ) 6.9 Hz, 1H,
H-1), 3.82 (m, 1H, H-4), 3.68 (m, 2H, H2-6), 3.54 (overlapped
signals, 2H, H-5 and H-3), 3.36 (m, 1H, H-2). At 295 K, another
set of signals (the major ones were centered at 5.44 and 5.35 ppm,
respectively) were present, attributed to the OH-2 protons, which
further broadened at higher temperatures and completely disap-
peared at 343 K. 31P (D2O, 161.98 MHz): δ 2.6 ppm. ESI-MS
(negative ions): m/z calcd for C48H60O32P4, 1272.202; found, from
combination of multiple charged ions, 1272.80.
Starting from functionalized support 17 (100 mg, 0.015 mmol),
for the synthesis of 2 and 3, the above-described synthetic cycle
was reiterated, once and twice, respectively, including the following
steps: (i) detritylation; (ii) coupling with phosphoramidite 9 (50
mg), dissolved in anhydrous CH3CN (300 µL) and a 0.45 M
tetrazole solution in anhydrous CH3CN (600 µL); (iii) oxidation;
and (iv) capping. Obtained supports 18 and 19 were then respec-
tively reacted with 2% DCA in CH2Cl2 (4 × 1 mL, 3 min each),
giving detritylated supports 21 and 22. Solid-phase cyclization was
then realized by treating 21 or 22 with MSNT (50 mg, 0.17 mmol)
dissolved in anhydrous pyridine (500 µL, 2 × 3 h, followed by an
overnight treatment). All the successive steps for the final depro-
tection and detachment from the solid support of the cyclic
oligomers were carried out as reported above for cyclic dimer 1.
Gel filtration chromatography of the final mixtures on a G25
Sephadex column eluted with H2O/EtOH, 4/1 (v/v), yielded 2 mg
(14% overall yield) and 1 mg (5% overall yield), respectively, of
the pure compounds, which were identified as the desired cyclic
molecules 2 and 3.
Solution Synthesis of CyPLOS 1 and 2. Synthesis of derivative
23. 2-Chlorophenyl-dichlorophosphate (530 µL, 3.3 mmol, d ) 1.52
g/mL, 5 equiv) was dissolved in anhydrous pyridine (2.5 mL) and
left under stirring at room temperature for 10 min. Successively,
H2O (54 µL, 3 mmol) was added to the mixture and left under
stirring for an additional 10 min. Finally, derivative 8 (500 mg,
0.66 mmol, 1 equiv), previously dried by repeated coevaporations
with anhydrous CH3CN and dried under reduced pressure, was
dissolved in anhydrous pyridine (2 mL) and then added to the
mixture containing 2-chlorophenylphosphoryl pyridinium chloride.
The reaction was left under stirring at room temperature overnight
and monitored by TLC using the eluent systems CHCl3/CH3OH,
9/1 (v/v), and CHCl3/CH3OH, 98/2 (v/v). The crude was then
purified by silica gel column, eluted first with AcOEt to recover
the unreacted starting material and then with a mixture AcOEt/
CH3OH, 8/2 (v/v). The fractions containing pure 23, collected and
concentrated under reduced pressure, gave 385 mg (0.59 mmol,
90% yield) of the desired compound.
23: white amorphous solid. Rf ) 0.3 [CHCl3/CH3OH 9/1, (v/
1
1: white amorphous solid. Retention time (conditions as specified
in the Supporting Information) ) 10.2 min; Rf ) 0.15 [CHCl3/
v)]. H NMR (CD3OD, 400 MHz; as pyridinium salt): δ 7.87-
6.71 (complex signals, 19H, aromatic protons), 5.89 (dd, J ) 8.9
and 9.2 Hz, 1H, H-2), 5.52 (overlapped signals, 2H, H-1 and H-3),
4.10-3.93 (overlapped signals, 3H, H-5 and H2-6). H-4 resonance
is submerged under the residual HDO solvent signal. 13C NMR
(CD3OD, 50 MHz): δ 167.3 and 166.8 (2 CO); 158.5, 134.4, 133.9,
130.6, 129.4, 128.9, 128.3, 124.8, 124.0, 122.1, and 117.8 (aromatic
C); 100.1 (C-1); 77.0 (C-2); 75.3 (C-3); 73.9 (C-5); 72.8 (C-4);
61.2 (C-6). 31P NMR (CD3OD, 161.98 MHz): δ -2.5. ESI-MS
(negative ions): m/z calcd for C32H28O11PCl, 654.106; found, 653.20
(M - H)-. HRMS (MALDI-TOF): m/z calcd for C32H27O11PCl,
653.0978; found, 653.0962 (M - H)-.
CH3OH, 9/1 (v/v)]; [R]25 ) -17.8°; λmax ) 264 nm (ꢀ ) 780
D
1
cm-1M-1). H NMR (D2O, 500 MHz; as triethylammonium salt):
δ 7.45-7.19 (complex signals, 5H, aromatic protons), 5.22 (d, J
) 8.0 Hz, 1H, H-1), 4.32 (m, 1H, H-4), 4.22 (br d, J ) 11.6 Hz,
1H, H-6a), 4.15 (br d, J ) 11.6 Hz, 1H, H-6b), 3.90 (overlapped
signals, 2H, H-5 and H-3), 3.70 (t, J ) 8.0 and 8.0 Hz, 1H, H-2),
3.22 (q, 6H, CH2 triethylammonium ion), 1.30 (t, 9H, CH3
1
triethylammonium ion). H NMR (DMSO-d6, 400 MHz, 298 K):
relevant signals at δ 7.90 (br s, 1H, OH-3); 5.32 (d, J ) 5.1 Hz,
1H, OH-2). 13C NMR (D2O, 125 MHz): δ 159.9, 133.1, 126.2,
119.3 (aromatic carbons); 102.7 (C-1); 78.2 (C-3); 75.7 (C-2); 75.5
(C-5); 75.0 (C-4); 66.2 (C-6). 31P NMR (D2O, 161.98 MHz): δ
3.1. ESI-MS (negative ions): m/z calcd for C24H30O16P2, 636.101;
found, 635.25 (M - H)-. HRMS (MALDI-TOF): m/z calcd for
C24H29O16P2, 635.0929; found, 635.0885 (M - H)-.
Synthesis of Linear Precursor 25. Derivative 23 (100 mg, 0.15
mmol, 1 equiv) and compound 9 (177 mg, 0.18 mmol, 1.2 eq),
previously dried by repeated coevaporations with anhydrous CH3-
CN and kept under reduced pressure, were reacted with a 0.45 M
tetrazole solution in anhydrous CH3CN (4 mL, 15 mmol, 10 equiv).
The reaction was left under stirring at room temperature and
monitored by TLC in the eluent system CHCl3/CH3OH, 9/1 (v/v).
After 2 h, a 5.5 M t-BuOOH solution in decane (1 mL) was added
to the mixture and left under stirring at room temperature. After
30 min, TLC monitoring of the reaction in CHCl3/CH3OH, 9/1 (v/
v), showed the formation of two products, one at Rf ) 0.55
(identified as 24) and the other one at Rf ) 0.45 (identified as 25).
The concentrated crude was then purified by silica gel column
chromatography eluted with CHCl3/CH3OH, 98/2 (v/v), containing
a few drops of pyridine, giving DMT-protected linear dimer 24
(46 mg, 0.03 mmol, 20% yield) and detritylated linear dimer 25
(110 mg, 0.09 mmol, 60% yield).
2: white amorphous solid. Retention time (conditions as specified
in the Supporting Information) ) 11.8 min; Rf ) 0.10 [CHCl3/
CH3OH, 8/2 (v/v)]; [R]25 ) -22.3°; λmax ) 264 nm (ꢀ ) 1030
D
1
cm-1M-1). H NMR (D2O, 500 MHz; as triethylammonium salt):
δ 7.45-7.18 (complex signals, 5H, aromatic protons), 5.18 (d, J
) 8.0 Hz, 1H, H-1), 4.35 (m, 1H, H-6a), 4.13 (overlapped signals,
2H, H-6b and H-4), 3.98 (m, 1H, H-5), 3.88 (apparent t, J ) 9.5
and 8.5 Hz, 1H, H-3), 3.55 (apparent t, J ) 8.5 and 8.0 Hz, 1H,
H-2), 3.22 (q, 6H, CH2 triethylammonium ion), 1.30 (t, 9H, CH3
1
triethylammonium ion). H NMR (DMSO-d6, 400 MHz, 298 K):
relevant signals at δ 7.90 (br s, 1H, OH-3), 5.36 (d, J ) 5.1 Hz,
1H, OH-2). 13C NMR (D2O, 100 MHz): δ 157.6, 130.2, 123.4,
and 116.7 (aromatic carbons); 102.3 (C-1); 77.6 (C-3); 76.3 (C-4);
75.9 (C-5); 74.9 (C-2); 67.2 (C-6). 31P NMR (D2O, 161.98 MHz):
δ 2.9. ESI-MS (negative ions): m/z calcd for C36H45O24P3, 954.151;
found, 953.34 (M - H)-, 476.21 (M - 2H)2-. HRMS (MALDI-
TOF): m/z calcd for C36H44O24P3, 953.1434; found, 953.1397 (M
- H)-.
3: white amorphous solid. Retention time (conditions as specified
in the Supporting Information) ) 12.5 min; λmax ) 264 nm (ꢀ )
1430 cm-1M-1). 1H NMR (D2O, 400 MHz): 7.51-7.22 (complex
signals, 5H, aromatic protons), 5.31 (d, J ) 7.5 Hz, 1H, H-1), 4.55-
3.40 (complex region, broadened signals, H-2, H-3, H-4, H-5, and
24: white amorphous solid. Rf ) 0.55 [CHCl3/CH3OH, 9/1 (v/
1
v)]. H NMR (CDCl3, 400 MHz; as pyridinium salt): δ 8.04-
6.55 (complex signals, 47H, aromatic protons); 5.88 (dd, 1H, H-3
A residue); 5.64 (overlapped signals, 2H, H-2 A residue and H-3
B residue); 5.41 (dd, 1H, H-2 B residue); 5.35 (d, J ) 7.6 Hz, 1H,
H-1 A residue); 5.03 (d, J ) 8.0 Hz, 1H, H-1 B residue); 4.72 (m,
1H, H-4 B residue); 4.41 (m, 2H, OCH2CH2CN); 3.93-3.42
(overlapped signals, 7H, H-4 A residue, 2 H-5 and 2 H2-6); 3.74
(s, 6H, 2 OCH3 of the DMT group); 2.59 (t, 2H, OCH2CH2CN).
31P NMR (CDCl3, 161.98 MHz): δ -0.5 and -3.7. ESI-MS
(negative ions): m/z calcd for C82H72NO23P2Cl, 1535.366; found,
3406 J. Org. Chem., Vol. 71, No. 9, 2006

Cyclic Phosphate-Linked Oligosaccharides
1534.61 (M - H)-. HRMS (MALDI-TOF): m/z calcd for C82H71-
NO23P2Cl, 1534.3579; found, 1534.3544 (M - H)-.
128.0, 127.6, 127.2, 123.9, 123.8, 122.1, 117.9, and 117.1 (aromatic
carbons); 116.1 (OCH2CH2CN); 99.9 and 99.8 (2 C-1); 73.8, 73.7,
73.5, 73.4, 72.7, 72.5, 71.4, and 71.0 (2 C-2, 2 C-3, 2 C-4 and 2
C-5); 66.1 and 65.6 (2 C-6); 62.0 and 61.0 (OCH2CH2CN); 18.8
and 18.7 (OCH2CH2CN). 31P NMR (CDCl3, 161.98 MHz): δ -2.3
and -7.5. ESI-MS (positive ions): m/z calcd for C61H52NO20P2Cl,
1215.225; found, 1216.71 (M + H+), 1238.75 (M + Na+), 1254.88
(M + K+). HRMS (MALDI-TOF): m/z calcd for C61H52NO20P2-
ClNa, 1238.2144; found, 1238.2178 (M + Na+).
Solution Synthesis of Cyclic Trimer 2. Synthesis of linear
precursor 28. Derivative 25 (50 mg, 0.04 mmol, 1 equiv) and
derivative 9 (47 mg, 0.05 mmol, 1.2 equiv), dried by coevaporations
with anhydrous CH3CN, were dissolved in 2 mL (10 equiv) of a
0.45 M tetrazole solution in anhydrous CH3CN and left under
stirring at room temperature. After 2 h, 500 µL of a 5.5 M t-BuOOH
solution in decane were added to the reaction mixture and left under
stirring at room temperature. TLC analysis in CHCl3/CH3OH (9/1,
v/v) showed the formation of two new products, respectively, at Rf
) 0.4 and Rf ) 0.3, identified as linear trimer 27 and the
corresponding detritylated derivative 28. The mixture was then
purified by TLC plates, developed using the solvent system CHCl3/
CH3OH, 9/1 (v/v), containing a few drops of pyridine, giving
detritylated linear trimer 28 (25 mg, 0.014 mmol, 35% yields) and
linear trimer 27, retaining the DMT group (6 mg, 0.003 mmol, 8%
yields).
25: white amorphous solid. Rf ) 0.45 [CHCl3/CH3OH, 9/1 (v/
1
v)]. H NMR (CD3OD, 400 MHz; as pyridinium salt): δ 8.02-
6.79 (complex signals, 34H, aromatic protons); 5.92 (dd, J ) 9.6
and 9.2 Hz, 1H, H-3 A residue); 5.74 (dd, J ) 9.6 and 9.2 Hz, 1H,
H-3 B residue); 5.59 (dd, J ) 8.4 and 9.2 Hz, 1H, H-2 A residue);
5.51 (d, J ) 8.0 Hz, 1H, H-1 A residue); 5.43 (dd, J ) 8.0 and 9.6
Hz, 1H, H-2 B residue); 5.26 (d, J ) 8.0 Hz, 1H, H-1 B residue);
4.70 (m, 1H, H-6a signal of B residue); 4.60 (m, 1H, H-4 B residue);
4.27 (m, 1H, H-6b signal of B residue); 4.15 (m, 2H, OCH2CH2-
CN); 4.02-3.90 (overlapped signals, 3H, H-5 and H2-6 of A
residue); 3.79 (m, 1H, H-5 B residue); 2.74 (m, 2H, OCH2CH2-
CN). H-4 of A residue is submerged by the residual HDO signal
in CD3OD. 13C NMR (CD3OD, 100 MHz): δ 167.8, 167.7, 167.2,
and 167.1 (CO); 159.1, 158.8, 150.3, 145.8, 139.2, 135.2, 135.1,
135.0, 134.4, 131.6, 131.3, 131.0, 131.1, 130.9, 130.4, 130.1, 130.0,
129.4, 128.9, 126.1, 125.2, 124.9, 124.6, 122.9, 118.9, and 118.5
(aromatic C); 117.1 (OCH2CH2CN); 100.7 (C-1 A residue and B
residue); 76.8 (C-5 A residue); 76.1 (C-5 B residue); 75.7, 75.6,
and 75.3 (C-3 A residue, C-3 B residue, C-4 A residue); 74.2 (C-2
B residue); 73.9 (C-2 A residue and C-4 B residue); 69.6 (C-6 B
residue); 64.9 (OCH2CH2CN); 61.8 (C-6 OH A residue); 19.8 and
18.9 (OCH2CH2CN). 31P NMR (CD3OD, 161.98 MHz): δ -0.7
and -3.7. ESI-MS (negative ions): m/z calcd for C61H54NO21P2-
Cl, 1233.235; found, 1232.53 (M - H)-. HRMS (MALDI-TOF):
m/z calcd for C61H53NO21P2Cl, 1232.2272; found, 1232.2240 (M
- H)-.
27: white amorphous solid. Rf ) 0.7 [CHCl3/CH3OH, 9/1 (v/
1
v)]. H NMR (CD3OD, 400 MHz; as pyridinium salt): δ 8.12-
6.75 (complex signals, 62H, aromatic protons), 6.13 (dd, J ) 9.0
and 10.0 Hz, 1H, H-3 A residue), 5.87 (dd, J ) 9.5 and 10.0 Hz,
1H, H-3 B residue), 5.71 (overlapped signals, 2H, H-3 and H-1 C
residue), 5.64 (dd, J ) 9.5 and 10.0 Hz, 1H, H-2 A residue), 5.59
(dd, J ) 9.5 and 8.0 Hz, 1H, H-2 B residue), 5.48 (dd, J ) 9.5 and
8.0 Hz, 1H, H-2 C residue), 5.40 (d, J ) 8.0 Hz, 1H, H-1 B residue),
5.16 (d, J ) 7.5 Hz, 1H, H-1 A residue), 4.74 (m, 1H, H-4 A
residue), 4.65 (m, 1H, H-4 B residue), 4.57 (m, 1H, H-4 C residue),
4.29 (m, 1H, H-5 B residue), 4.21 (complex signals, 2H, H-5 A
residue and H-5 C residue), 4.12-4.01 (complex signals, 4H, 2
OCH2CH2CN), 3.99-3.43 (overlapped signals, 6H, 3 × H2-6), 3.79
(s, 6H, OCH3 protons DMT group), 2.78-2.60 (complex signals,
4H, 2 OCH2CH2CN). 13C NMR (CD3OD, 100 MHz): δ 165.8,
165.1, and 165.0 (CO); 158.3, 149.6, 133.5, 133.1, 133.0, 131.2,
130.2, 129.7, 129.5, 129.0, 128.6, 128.3, 127.7, 123.7, 123.4, 123.1,
118.1, and 113.1 (aromatic C); 117.2 (OCH2CH2CN); 99.1, 99.0,
and 98.9 (C-1); 74.6, 74.0, 73.7, 73.6, 73.5, 73.1, 72.7, 71.8, 71.5,
and 71.2 (C-2, C-3, C-4, and C-5); 63.0, 62.9, 62.8, and 62.4 (C-6
and OCH2CH2CN); 55.1 (OCH3); 18.9 and 18.9 (OCH2CH2CN).
31P NMR (CD3OD, 161.98 MHz): δ -1.0, -1.2, -3.6. MALDI-
TOF MS (negative ions): m/z calcd for C111H98N2O33P3Cl, 2114.495;
found, 2114.1 (M - H)-. HRMS (MALDI-TOF): m/z calcd for
Synthesis of Cyclic Dimer 1. Derivative 25 (38 mg, 0.031 mmol,
1 equiv), previously dried by coevaporations with anhydrous
pyridine, and MSNT (183 mg, 0.62 mmol, 20 equiv) were dissolved
in anhydrous pyridine (30 mL) and left under stirring overnight.
The reaction, monitored by TLC in CHCl3/CH3OH, 9/1 (v/v),
showed the complete conversion of derivative 25 into a compound
with a higher Rf. The reaction mixture, taken to dryness under
reduced pressure, was then purified by silica TLC plates, developed
in the eluent system CHCl3/CH3OH, 9/1 (v/v), which afforded pure
26 (27 mg, 0.022 mmol) with 71% yield. This compound,
coevaporated several times with anhydrous pyridine and dried under
reduced pressure, was then treated with Et3N/pyridine (4 mL, 1/1,
v/v, 3 h, rt) to selectively remove the 2-cyanoethyl group. The
reaction, monitored by TLC in CHCl3/CH3OH, 9/1 (v/v), was
quenched by in vacuo removal of the solvent. The crude, after
several coevaporations with anhydrous pyridine, was then treated
with a 0.2 M 1,1,3,3-tetramethylguanidinium 2-nitrobenzaldoximate
solution (1 mL) in H2O/dioxane (1/1, v/v). The reaction mixture
was then left overnight under stirring at room temperature. Taken
to dryness, the crude was then treated with concentrated NH4OH
(1 mL, 6 h, 55 °C). The concentrated mixture was then purified by
gel filtration chromatography on a Sephadex G25 column, eluted
with H2O/EtOH, 4/1 (v/v). Fractions containing the cyclic dimer,
monitored by spectrophotometric measurements at λ ) 264 nm,
were collected and concentrated, affording 10 mg (0.016 mmol,
73% yield) of pure 1.
26: white amorphous solid. Rf ) 0.8 [CHCl3/CH3OH, 9/1 (v/
v)]. 1H NMR (CDCl3, 500 MHz): δ 8.14-6.62 (complex signals,
34H, aromatic protons), 6.03 (dd, J ) 9.2 and 9.2 Hz, 1H, H-3 A
residue), 5.95 (dd, J ) 7.6 and 9.2 Hz, 1H, H-2 A residue), 5.83
(dd, J ) 9.2 and 9.2 Hz, 1H, H-3 B residue), 5.70 (dd, J ) 8.0 and
9.2 Hz, 1H, H-2 B residue), 5.46 (d, J ) 7.6 Hz, 1H, H-1 A residue),
5.41 (m, 1H, H-4 A residue), 5.07 (m, 1H, H-4 B residue), 5.01
(d, J ) 8.0 Hz, 1H, H-1 B residue), 4.75 (m, 1H, H-6a A residue),
4.50 (m, 1H, H-6a B residue), 4.37 (d, J ) 11.6 Hz, 1H, H-6b A
residue), 4.27 (d, J ) 11.2 Hz, 1H, H-6b B residue), 4.04 (m, 1H,
H-5 A residue), 3.96 and 3.36 (2 m, 1H each, OCH2CH2CN), 3.76
(m, 1H, H-5 B residue), 2.30 (m, 2H, OCH2CH2CN). 13C NMR
(CDCl3, 100 MHz): δ 165.8, 165.5, 165.4, and 165.3 (CO); 156.9,
156.8, 133.9, 133.7, 133.6, 133.0, 131.1, 130.3, 130.2, 130.1, 130.0,
129.8, 129.6, 129.5, 129.2 129.1, 129.0, 128.9, 128.7, 128.6, 128.1,
C
111H97N2O33P3Cl, 2113.4873; found, 2113.4844 (M - H)-.
28: white amorphous solid. Rf ) 0.6 [CHCl3/CH3OH, 9/1 (v/
1
v)]. H NMR (CD3OD, 400 MHz; as pyridinium salt): δ 8.07-
6.70 (complex signals, 49H, aromatic protons), 6.12 (dd, J ) 9.0
and 10.0 Hz, 1H, H-3 A residue), 5.91 (dd, J ) 9.5 and 9.0 Hz,
1H, H-3 B residue), 5.81 (dd, J ) 9.5 and 9.0 Hz Hz, 1H, H-3 C
residue), 5.69 (d, J ) 8.0 Hz, 1H, H-1 C residue), 5.61 (overlapped
signals, 2H, H-2 A residue and H-2 C residue), 5.51 (dd, J ) 8.5
and 9.0 Hz, 1H, H-2 B residue), 5.46 (d, J ) 8.0 Hz, 1H, H-1 B
residue), 5.35 (d, J ) 7.5 Hz, 1H, H-1 A residue), 4.94 (m, 1H,
H-4 A residue), 4.77 (m, 1H, H-4 B residue), 4.65 (m, 1H, H-4 C
residue), 4.35 (overlapped signals, 2H, H-5 and H-6a B residue),
4.22-4.14 (complex signals, 7H, H-6b B residue, H2-6 C residue
and 2 OCH2CH2CN), 4.09 (m, 1H, H-5 A residue), 4.04 (m, 1H,
H-6a A residue), 3.98 (m, 1H, H-5 C residue), 3.89 (m, 1H, H-6b
A residue), 2.80-2.67 (complex signals, 4H, 2 OCH2CH2CN). 13
C
NMR (CD3OD, 100 MHz): δ 168.0, 167.9, 167.6, 167.5, 167.2,
and 167.1 (CO); 159.1, 159.0, 158.7, 158.6, 155.0, 151.5, 150.6,
150.7, 135.3, 135.2, 135.1, 135.0, 134.9, 134.3, 131.7, 131.6, 131.3,
131.2, 131.1, 131.0, 130.9, 130.4, 130.1, 130.0, 129.9, 129.4, 128.8,
J. Org. Chem, Vol. 71, No. 9, 2006 3407

Di Fabio et al.
125.1, 124.9, 124.5, 122.9, 119.2, 119.0, 118.8, 118.6, 118.5, and
118.2 (aromatic C); 117.2 and 116.9 (OCH2CH2CN); 101.0 (C-1
B residue); 100.7 (C-1 A residue); 100.4 (C-1 C residue); 76.7 (C-5
B residue); 76.3 (C-5 A residue); 73.9 (C-5 C residue); 75.9, 75.8,
75.7, 75.2, 75.1, 74.4, 74.2, 73.6, and 70.1 (C-2, C-3, and C-4 for
A, B, and C residues); 68.1 (C-6 B residue); 64.9 (overlapped C-6
C residue and OCH2CH2CN); 61.8 (C-6 OH A residue); 20.4 and
20.4 (OCH2CH2CN). 31P NMR (CD3OD, 161.98 MHz): δ -1.6,
-3.2, and -5.6. ESI-MS (negative ions): m/z calcd for C90H80N2O31-
P3Cl, 1812.365; found, 1812.42 (M - H)-. HRMS (MALDI-
TOF): m/z calcd for C90H79N2O31P3Cl, 1811.3566; found, 1811.3523
(M - H)-.
Synthesis of Cyclic Trimer 2. Derivative 28 (18 mg, 0.010
mmol, 1 equiv), previously dried by coevaporations with anhydrous
pyridine, and MSNT (60 mg, 0.21 mmol, 20 equiv) were dissolved
in anhydrous pyridine (10 mL) and left under stirring at room
temperature overnight. The reaction mixture was next concentrated
under reduced pressure and then purified by TLC plates, developed
in CHCl3/CH3OH, 93/7 (v/v). The band at Rf ) 0.9 furnished fully
protected cyclic trimer 29 (10 mg, 0.0056 mmol, 56% yields) as a
pure compound. Successively, treatment with a 1/1 (v/v) Et3N/
pyridine solution (4 mL) for 3 h at room temperature allowed the
removal of the 2-cyanoethyl group from the phosphodiester
linkages. The reaction was quenched by concentrating the mixture
under reduced pressure. The crude, dried by coevaporations with
anhydrous pyridine, was then treated with a 0.2 M 1,1,3,3-
tetramethylguanidinium 2-nitrobenzaldoximate solution (1 mL) in
H2O/dioxane (1/1, v/v). The reaction mixture was left at room
temperature overnight and, after removal of the solvent in vacuo,
treated with concentrated NH4OH (1 mL, 6 h, 55 °C). The reaction
mixture, taken to dryness and redissolved in H2O, was then purified
by gel filtration chromatography on a Sephadex G25 column eluted
with H2O/EtOH, 4/1 (v/v). Fractions containing the cyclic trimer,
monitored by spectrophotometric measurements at λ ) 264 nm,
were collected and concentrated, affording 4 mg (0.0042 mmol,
75% yield) of pure 2.
29: white amorphous solid. Rf ) 0.9 [CHCl3/CH3OH, 93/7 (v/
v)]. 31P NMR (CDCl3, 161.98 MHz): δ 0.4, -0.9, -1.3, -2.3,
-5.8, -6.7. ESI-MS (positive ions): m/z calcd for C90H78N2O30P3-
Cl, 1794.354; found, 1817.72 (M + Na+), 1834.68 (M + K+).
HRMS (MALDI-TOF): m/z calcd for C90H78N2O30P3ClNa,
1817.3439; found, 1817.3471 (M + Na+).
Acknowledgment. We thank MIUR (PRIN) and Regione
Campania (L5) 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. The
authors are also grateful to “Centro di Servizio Interdiparti-
mentale di Analisi Strumentale”, C.S.I.A.S., Universita` di Napoli
“Federico II”, for the high field NMR facilities. The authors
acknowledge Azione Integrata Italia-Spagna (IT324-2001/
HI2000-0031), allowing exchanges between the group of Napoli
and the group of Barcelona.
Supporting Information Available: Materials and methods,
nuclear magnetic resonance methods, and structure calculation
methods. Full scale 1H NMR in D2O and in DMSO-d6 for
compounds 1 and 2, 31P NMR of compounds 1, 2, and 3, and NMR
data for compounds 8, 9, 12, 16, and 23-29. This material is
available free of charge via the Internet at http://pubs.acs.org.
JO0600757
3408 J. Org. Chem., Vol. 71, No. 9, 2006