On the compatibility of azides in phosphoramidite-based couplings: Synthesis of a novel, convertible azido-functionalized CyPLOS analogue

Article abstract of DOI:10.1002/ejoc.201001057

With the aim of preparing a library of ion transporters based on a CyPLOS (cyclic phosphate-linked oligosaccharide) backbone, we describe the synthesis of a novel azido-derivatized CyPLOS analogue that is tailored for practical post-synthetic functionaliz

Full text of DOI:10.1002/ejoc.201001057

FULL PAPER
DOI: 10.1002/ejoc.201001057
On the Compatibility of Azides in Phosphoramidite-Based Couplings: Synthesis
of a Novel, Convertible Azido-Functionalized CyPLOS Analogue
Cinzia Coppola,[a] Luca Simeone,[a] Lorenzo De Napoli,[a] and Daniela Montesarchio*[a]
Keywords: Azides / Carbohydrates / Macrocycles / Phosphorylation
With the aim of preparing a library of ion transporters based
on CyPLOS (cyclic phosphate-linked oligosaccharide)
backbone, we describe the synthesis of a novel azido-deriva-
tized CyPLOS analogue that is tailored for practical post-syn-
thetic functionalization with a variety of labels. As a prereq-
uisite for the effective preparation of this phosphate-linked
macrocycle, the compatibility of azido alcohols as coupling
agents in phosphoramidite synthetic protocols was prelimi-
narily investigated in solution by using 31P NMR spec-
troscopy to monitor the standard coupling of a nucleoside 3Ј-
phosphoramidite with suitable model alcohols and azides.
a
CyPLOS produced the fluorescent analogue 5; the insertion
of dansyl units at the extremities of the TEG tentacles re-
sulted in a more active ion transporter, and also provided
deeper insights into its mechanism of action.[4]
Introduction
In the search for selective artificial receptors, we recently
described the synthesis and conformational properties of
CyPLOS (cyclic phosphate-linked oligosaccharides), which
is a novel class of phosphodiester-linked oligosaccharide
analogues (1a–c, Figure 1).[1]
Figure 1. Chemical structures of CyPLOS 1a–c (Ph = phenyl).
Amphiphilic macrocycles with a remarkable propensity
toward aggregation were then obtained by attaching long
tentacles with different lipophilicity, particularly n-undecyl
or tetraethylene glycol (TEG) chains, to the CyPLOS back-
bone (2–4, Figure 2).[2] Initial studies on the ionophoric ac-
tivity of amphiphilic CyPLOS[3] showed 4 to be the most
effective compound in the series 2–4; this compound was
able to completely discharge a pH gradient across liposom-
ial membranes in less than 20 min at 2% ionophore concen-
tration. This property was strictly correlated to the presence
of TEG chains, with 4 being much more active than 3, and
tetra-alkylated derivative 2 being almost completely inac-
tive. Subsequent optimization of the design of amphiphilic
Figure 2. Chemical structures of amphiphilic CyPLOS 2–5.
The dansyl moieties in 5 were introduced at the level of
the fully protected monosaccharide, which was synthesized
from the TEG-azide, after reduction to the amine and cou-
pling with dansyl chloride (II, III and IV, respectively, Fig-
ure 3). With the aim of introducing different reporter
groups to the CyPLOS tentacles, which play a major role
in self-aggregation and ion transport activity, we realized
that the set of accessible CyPLOS analogues following this
strategy was limited. Once inserted in the monosaccharide
building blocks, most labels, in fact, do not survive the reac-
tion conditions necessary to convert them into the target
macrocycle. Therefore, a general approach to the synthesis
of diversely end-functionalized analogues – starting from a
[a] Department of Organic Chemistry and Biochemistry,
University “Federico II” of Napoli,
Via Cintia 4, 80126 Napoli, Italy
Fax: +39-081-674393
E-mail: daniela.montesarchio@unina.it
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201001057.
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unique precursor, in a flexible post-synthetic functionaliza- (4-carboxy TEMPO) residue,[23] were designed (Figure 4).
tion protocol – would be highly desirable. In this perspec- However, before undertaking the synthesis of CyPLOS A,
tive, terminal azido moieties, if kept intact until the final it was necessary to first ascertain whether phosphoramidite-
step of the synthesis, can be used as useful, convertible based coupling protocols, which are required to build the
groups for post-synthetic decoration of CyPLOS.
backbone of the target molecule, could be applied without
significantly interfering with the azido functions. Here we
describe coupling experiments, based on standard phos-
phoramidite protocols, between a nucleoside 3Ј-phos-
phoramidite and TEG alcohols or TEG azides, monitored
by 31P NMR spectroscopy. On the basis of the obtained
results, the convergent synthesis of CyPLOS derivatives A
and B has been successfully realized.
Figure 3. Synthetic scheme for the preparation of dansyl-labeled
CyPLOS 5 developed previously.[4] Ms = mesyl; DMT = 4,4Ј-di-
methoxytrityl group.
Many recent works have emphasized the variety of appli-
cations of azido moieties in organic chemistry[5] as chemi-
cally stable groups that are easily amenable to useful trans-
formations, such as the Staudinger reaction[6–8] and copper-
catalyzed 1,3-dipolar cycloaddition, known as “click chem-
istry”.[9–11] Both reactions are extensively exploited in the
synthesis of bioconjugates because they involve mild reac-
tion conditions and simple work-up procedures, and gen-
erally give high yields.
Figure 4. Conversion of azido-functionalized CyPLOS A into spin-
labeled B – obtained as a mixture of regioisomers – through azide
reduction followed by coupling with 4-carboxy TEMPO. Reagents
and conditions: (i) (a) Ph3P, THF, room temp., 12 h; (b) H2O, room
temp., 48 h (quant.); (ii) 4-carboxy-TEMPO, DIPEA, DCC, room
temp.,12 h (67%).
Results and Discussion
Encouraged by the increasing use of azido groups as ver-
satile chemical handles for easy synthetic access to various
The problem of compatibility of azido groups with stan-
conjugates of multifunctional biopolymers, such as pep- dard phosphoramidite chemistry clearly emerged in several
tides[12] and oligonucleotides,[13–22] we revisited our original studies on oligonucleotide conjugates.[15–22] This aspect was
design so that this chemical entity could be kept on the first studied by Sekine et al., who concluded that nucleo-
CyPLOS backbone. Azido groups – if inserted at the ex- sides carrying azido groups cannot be adopted in phos-
tremities of the tentacles of these macrocycles – would allow phoramidite-based oligonucleotide synthesis because of the
post-synthetic condensation with labels that would enable interfering Staudinger reaction that takes place between az-
the properties of these artificial ionophores to be studied. ides and PIII derivatives.[24] In their experiment, 2-azido-2Ј-
We envisioned that the incorporation of a spin label into the deoxyadenosine was treated with 1.5 equiv. of diethyl, N,N-
CyPLOS tentacle should provide a useful tool that could be diisopropylphosphoramidite in N,N-dimethylformamide
used to investigate interactions within phospholipid bilayers (DMF)/CD3CN (9:1, v/v). The progress of the reaction,
by means of ESR spectroscopy. Therefore, starting from which was monitored by 31P NMR spectroscopic analysis
azido-functionalized CyPLOS A, derivatives B, which in- of the reaction mixture, showed initial formation of the
corporate one 4-carboxy-2,2,6,6-tetramethyl-1-piperidinoxy phosphazide intermediate of the Staudinger reaction, which
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Synthesis of an Azido-Functionalized CyPLOS Analogue
then converted into the iminophosphorane within approxi- benzyl N,N-diisopropyl-phosphoramidite, coupling with the
mately two hours. These results indicate that azido alcohols sugar hydroxyl group gave a stable derivative that did not
cannot be transformed into the corresponding azido phos- undergo further conversion, probably due to the bulkiness
phoramidites by treatment with phosphitylating reagents, of the substituents at the PIII atom.
because of the significant nucleophilicity of PIII towards az-
For a clear elucidation of the effective potential of azido-
ides. This limitation was circumvented by using H-phos- functionalized building blocks in phosphoramidite chemis-
phonate chemistry for the oligonucleotide chain as- try protocols, here required for the synthesis of A-type com-
sembly.[15,16,24]
pounds, we first studied a simple model system. For this
In a more recent paper, van Delft et al. prepared a 2Ј-O- purpose, commercially available 5Ј-O-DMT-N4-benzoyl-2Ј-
(3-azidopropyl) adenosine derivative that was subsequently deoxycytidine-3Ј-phosphoramidite (6) was reacted under
converted into the corresponding phosphoramidite nucleo- the standard phosphoramidite coupling procedure, by using
side.[19] This building block was isolated, albeit in extremely 0.25 m 4,5-dicyanoimidazole (DCI) in anhydrous CH3CN
low yields, but could not be incorporated at the 5Ј-end of as the activator, with azido alcohol N3-TEG-OH (7a;
an oligonucleotide still anchored to the solid support and, Table 1). This substrate, which was designed for insertion
when dissolved in acetonitrile, was found to decompose into CyPLOS A, was prepared from tetraethylene glycol as
within two weeks. This degradation was not investigated in described previously.[4]
detail, but the Staudinger-type condensation of the PIII
-
Table 1. Chemical structures of the TEG-derivatives tested in the
containing moiety with the azido function can reasonably
account for the increased lability of the phosphoramidite
nucleoside in solution. To avoid this detrimental side reac-
tion, the azido function was introduced by a time-consum-
ing post-elongation synthetic protocol in solution.[16,19] Al-
ternatively, 5Ј-azido-oligonucleotides were prepared
through 5Ј-iodination,[13] or by use of bromoalkyl building
blocks. The latter were inserted into an oligonucleotide se-
quence[21] or at the 5Ј-end,[15,17] by using an automated so-
lid-phase synthesis protocol. Both synthetic ways were fol-
lowed by treatment with sodium azide. Interestingly,
Lonnberg et al. introduced the azido function into an oligo-
nucleotide sequence by exploiting a 4Ј-(azidomethyl)thym-
idine-3Ј-H-phosphonate building block; chain elongation
was then realized by using either H-phosphonate or phos-
phoramidite chemistry without observing the Staudinger
reaction.[18] Indeed, the experiments carried out in the
group of van Delft can be interpreted by considering that
the competition for a preformed phosphoramidite, which is
dissolved in acetonitrile in the presence of an acidic cata-
lyst, by an azido function in solution and a hydroxyl group
in the solid phase, completely favors the homogeneous reac-
tion over the heterogeneous condensation.[19] In line with
this interpretation, parallel experiments show that if both
the azido and the hydroxyl functions are in the solid phase,
the support-bound azides do not disturb the phosphor-
amidite coupling, and phosphite triester bonds are realized
smoothly.[18,22] To the best of our knowledge, the concomi-
tant presence in solution of an alcohol and an azide, both
competing for the condensation with an activated phos-
phoramidite, was not investigated in detail. This issue was
briefly reported in a recent paper, in which the conversion
of a 4-azido-4-deoxy-d-galactoside into 4-deoxy-d-erythro-
hexos-3-ulose by reaction with a phosphoramidite reagent
and tetrazole as a catalyst, was described.[25] In all cases,
the ready formation of the phosphite derivative of the 4-
azido sugar was observed to take place in less than 5 min
and – only in the presence of unhindered phosphoramid-
ites – an intramolecular Staudinger reaction occurred as a
subsequent step, requiring 5–12 h to go to completion. On
coupling with 2Ј-deoxycytidine-3Ј-phosphoramidite 6.
Entry
Chemical structure
7a
7b
7c
HO(CH2CH2O)3CH2CH2N3
HO(CH2CH2O)3CH2CH2ODMT
N3(CH2CH2O)3CH2CH2N3
To obtain suitable reference compounds, the nucleoside
phosphoramidite 6 was also reacted under the same condi-
tions with related TEG derivatives carrying only one pri-
mary alcohol (DMTO-TEG-OH, 7b) or azido group (N3-
TEG-N3, 7c). In parallel, the same experiment with 7c was
repeated in the absence of activators. The coupling, which
was carried out in an NMR tube with CDCl3 as a co-sol-
vent in the presence of activated molecular sieves (4 Å), was
monitored by recording a set of 31P NMR spectra at dif-
Scheme 1. General scheme for the coupling of 6 with alcohols 7a
(N3-TEG-OH) or 7b (DMTO-TEG-OH). Reagents and conditions:
(i) 7a or 7b (1.2 equiv.), DCI (600 μL, 0.25 m in CH3CN/CDCl3,
5:1); (ii) tBuOOH (50 μL, 5.5 m in decane). Bz = benzoyl; CE = 2-
cyanoethyl; DMT = 4,4Ј-dimethoxytrityl; R for 10a, 11a =
–(CH2CH2O)3CH2CH2N3;
the other hand, it was also noted that, in the case of di- CH2CH2ODMT.
R
for 10b, 11b
=
–(CH2CH2O)3-
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ferent times. Subsequently, a large excess of the oxidizing
agent tert-butyl hydroperoxide (tBuOOH) was added to the
reaction mixtures. The general reaction for coupling phos-
phoramidite 6 with alcohols is depicted in Scheme 1.
As expected, in the reaction with 7b (Scheme 2), the
ready formation (less than 10 min) of the phosphite triester
10b was observed (31P NMR: δ = 145 ppm); this was ob-
tained through the transient activated phosphoramidite 8
(31P NMR: δ = 135 ppm) along with traces of H-phos-
phonate diester 9 (due to advantageous moisture in the
NMR tube) accounting for the two signals at δ = 15 and
11 ppm (identified as a doublet with JHP = 729 Hz). This
reaction mixture, monitored over a period of 6 h, did not
show further spontaneous transformations. As expected,
upon oxidation the phosphite triester 10b was rapidly
(within less than 5 min) converted into phosphate triester
11b, which gave rise to a single 1H NMR signal at δ =
2.8 ppm, whereas the H-phosphonate derivative was essen-
tially unaltered.[26]
Mixing of 6 and 7c (Scheme 3) gave, as expected, dif-
ferent results. In the absence of an acidic activator, the 31P
NMR spectrum showed a unique signal due to phos-
phoramidite 6, and only after 2 h were two new, small sig-
nals (δ = 31 and 16 ppm) observed; after 4 h, the signal
at δ = 31 ppm disappeared, and no further changes were
observed, even not after 12 h. In analogy with the assign-
ments given by the group of Sekine, the signal at δ =
31 ppm was attributed to the Staudinger phosphazide inter-
mediate 10c. The signal at δ = 16 ppm may be due to the
iminophosphorane-type adduct 11c, which was obtained as
a stable derivative upon loss of a nitrogen molecule.[27]
After oxidation, the signal at δ = 153 ppm disappeared
and a new signal at δ = 8 ppm emerged that was attributable
to phosphoramidate 12, obtained from 6, and the signal
at approximately δ = 16 ppm (due to 11c) increased. The
Staudinger adduct 11c was also found in the coupling be-
tween 6 and 7c in the presence of DCI (Scheme 4), but this
reaction proved to be much slower than the corresponding
reaction carried out without DCI. In fact, after 4 h, the
only detectable event was the conversion of 6, through the
reactive intermediate 8, into H-phosphonate 9, with the sig-
nals at δ = 153 ppm (due to 6) and at δ = 135 ppm (due to
8) still prevailing. After a further 16 h reaction time, 6 al-
most completely degraded to 9. Only two new, small signals
emerged: the first (δ = 13 ppm) was attributed to 11c,
whereas the second (broad resonance at δ = 0 ppm), could
be due to the phosphate diester, generated by hydrolysis of
11c.[28]
The reaction route observed in the coupling of 6 with
azido alcohol 7a (Scheme 5) was indeed very similar to that
observed in the coupling with 7b. Within less than 10 min,
the signal attributable to 6 (δ = 153 ppm) disappeared, and
new signals emerged at δ = 144 (prevailing, trialkyl phos-
phite adduct 10a), 15, and 10 ppm (minor signals, H-phos-
phonate derivative 9). Changes in the 31P NMR spectrum
Scheme 2. Reaction scheme and 31P NMR spectra recorded during
the coupling of nucleoside 6 with alcohol 7b: Reagents and condi-
tions: (i) 7b (1.2 equiv.), DCI (0.25 m in CH3CN/CDCl3, 5:1); (ii)
tBuOOH (5.5 m in decane).
were observed only after 4 h, when a new, very small signal natively to the cyclic derivative generated by intramolecular
was apparent at δ = 13 ppm due either to adduct 13, gener- Staudinger reaction of azido-funcionalized phosphite tri-
ated by nucleophilic attack of 10a on residual 7a, or alter- ester 10a; after oxidation, the signal at δ = 144 ppm disap-
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Synthesis of an Azido-Functionalized CyPLOS Analogue
Scheme 3. Reaction scheme and 31P NMR spectra recorded during
the coupling of nucleoside 6 with TEG-azide 7c in the absence of
activator. Reagents and conditions: (i) 7c (1.2 equiv.), CH3CN/
CDCl3, 5:1; (ii) tBuOOH (5.5 m in decane).
Scheme 4. Reaction scheme and 31P NMR spectra recorded during
the coupling of 6 with TEG azide 7c in the presence of activator.
Reagents and conditions: (i) 7c (1.2 equiv.), DCI (0.25 m in
CH3CN/CDCl3, 5:1); (ii) tBuOOH (5.5 m in decane).
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peared, a large new signal emerged at δ = 1.9 ppm, attribut-
able to phosphate triester 11a, while the resonances at 15,
13 and 10 ppm remained unaltered. The identities of the
stable compounds, before and after oxidation, were con-
firmed by comparison of the 31P NMR spectroscopic data
with literature data[24] and by MS and IR experiments. No-
tably, the band at 2212 cm–1 in the IR spectrum of the reac-
tion mixture of 6 and 7a, which is diagnostic of the azide
moiety, did not change either in frequency or relative inten-
sity with time, or upon oxidative treatment. For this reac-
tion, compound 11a was the only product isolated after col-
umn chromatography; it was obtained in 75% yield for the
1
two steps and was characterized by H, 13C, 31P NMR and
ESI-MS analyses.
The results described above are consistent with the fol-
lowing interpretation: the coupling of an azido alcohol with
an activated phosphoramidite compound under the stan-
dard conditions used in oligonucleotide synthesis protocols
(fast reaction times, typically requiring 5–30 min) leads to
an azido phosphite triester derivative, which is then rapidly
converted into a stable azido phosphate triester by oxi-
dation in situ. Only the prolonged contact of a PIII com-
pound with the azido alcohol gives also, as minor reaction
products, classical Staudinger adducts, which finally col-
lapse into iminophosphorane-type adducts.
These data are in agreement with the results of Sekine
and co-workers, and can be easily rationalized by taking
into account that the PIII atom in a phosphoramidite rea-
gent behaves differently if it is in the presence of an acidic
promoter. Indeed, two different reaction pathways have to
be taken into account due to the peculiar behavior of the
P–N linkage: when the phosphoramidite reagent is em-
ployed under acidic conditions, fast protonation of the ni-
trogen atom occurs, with the adjacent PIII center becoming
dramatically more electrophilic. Rapid attack by available
nucleophiles then follows, with the most reactive nucleo-
philes being preferred.[29] On the other hand, under neutral
conditions, the nucleophilic character of PIII prevails and
the only effective mechanism is the nucleophilic attack of
phosphoramidites to azides, giving stable iminophos-
phoranes. Therefore, it can be concluded that a phos-
phoramidite derivative can be efficiently reacted with an
azido alcohol to finally give an azido phosphotriester com-
pound, provided that the coupling reaction is fast (less than
30 min) and that the PIII adduct is rapidly oxidized to the
corresponding PV derivative.
Encouraged by these results, we then focused on our
original target; the synthesis of an A-type compound as a
readily convertible CyPLOS derivative. In particular, the
synthesis of linear dimer 19 was first realized by coupling
two differently substituted monosaccharides: the known
18,[2] carrying benzyl end-capped TEG tentacles, and the
novel TEG-azido-derivatized glucoside 17. This compound
was synthesized in four steps starting from 14,[4] involving
first benzylidene removal leading to 15, installation of the
DMT group on the 6-hydroxyl position to give 16, followed
by a two-step, one-pot reaction for the insertion of a phos-
phorylated moiety at the 4-hydroxyl group and successive
Scheme 5. Reaction scheme and 31P NMR spectra for the coupling
of nucleoside 6 with azido alcohol 7a: Reagents and conditions: (i)
7a (1.2 equiv.), DCI (0.25 m in CH3CN/CDCl3, 5:1); (ii) tBuOOH
(5.5 m in decane).
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Synthesis of an Azido-Functionalized CyPLOS Analogue
DMT removal, affording 17 (Scheme 6). Standard phos-
phoramidite coupling of monosaccharides 17 and 18 gave
the desired dimer 19 as a unique compound, as determined
by TLC monitoring of the reaction mixture; the compound
was isolated after column chromatography in good yields
(75% isolated yield for three steps). Cyclization of linear
dimer 19 was then carried out by applying a phosphotri-
ester methodology; using 1-mesitylenesulfonyl-3-nitro-
1,2,4-triazole (MSNT) as the condensing agent in associa-
tion with 4-(dimethylamino)pyridine (DMAP) as a nucleo-
philic catalyst, under high dilution conditions (ca. 10–3 m),
fully protected 20 was recovered with 75% yield.
Scheme 7. Synthetic scheme for the preparation of cyclic dimer A:
Reagents and conditions: (i) (a) DCI (0.25 m in CH3CN), room
temp., 2 h; (b) tButOOH (5.5 m in decane), room temp., 30 min; (c)
on-column detritylation (74% for the three steps); (ii) DMAP,
MSNT, Py, room temp., 12 h (75%); (iii) piperidine, 70 °C, 12 h
(quant.); (iv) satd. aq. LiOH/dioxane (5:1), 50 °C, 12 h (96%).
Scheme 6. Preparation of phosphorylated building block 17: Rea-
gents and conditions: (i) TFA/CH2Cl2/H2O (1:10:0.5), 4 h, 0 °C
(quant.); (ii) DMTCl, Py, 12 h, room temp. (97%); (iii) (a) 2-chloro-
phenyldichlorophosphate, triethylamine (TEA), triazole, Py, 3 h,
room temp.; (b) on-column DMT removal (92% for two steps).
gested the presence of strongly self-aggregated systems in
CDCl3, with typical chemical shift anisotropy and line
Subsequent phosphate deprotection reactions, involving broadening. This was not observed, however, when the sam-
first 2-cyanoethyl removal by treatment with piperidine, fol- ple was dissolved in CD3OD, in which a unique, sharp sig-
lowed by reaction of 21 with a LiOH solution in dioxane/ nal was observed for the two phosphorus atoms, which is
water (1:5, v/v) at 50 °C to cleave the 2-chlorophenyl group, consistent with a fully symmetric, well-solvated system.
gave target compound A in 94% yield for the two steps Analysis of the 31P NMR signals of A upon varying its
(Scheme 7). Following the described procedure, A was pre- concentration in CDCl3 gave deeper insights into the behav-
pared in four steps and 53% yield on average from building ior of this macrocycle in solution. In detail, when A was
blocks 17 and 18, the former compound was obtained in analyzed as 10 mm samples in CDCl3 (161.98 MHz, 298 K),
four steps with 88% yield from glucoside 14. For its conver- a very broad signal, dispersed over a 10 ppm region, was
sion into spin-labeled derivatives B (Figure 4), the azido observed in the 31P NMR spectrum. This signal, which was
groups in A were first reduced to amines by treatment with unaltered in the temperature interval 288–338 K, was signif-
triphenylphosphane and water. The post-synthetic conden- icantly sharpened only at a concentration of 0.7 mm, to fi-
sation with 0.5 equiv. of 4-carboxy-TEMPO,[23] realized nally give a sharp signal at 0.2 mm, thus allowing a critical
using N,N-dicyclohexylcarbodiimide (DCC) as the con- aggregation concentration (CAC) for this molecule of ap-
densing agent, yielded B, which was obtained as a mixture proximately 0.5 mm to be determined (see the Supporting
of regioisomers. These compounds were isolated in 67% Information). Compared to the CAC value of 6–8 mm
yield after chromatography on a Sephadex G25 column and evaluated for its congener 4,[2] the prepared macrocycle A
1
characterized by MS analysis. As expected, the H and 13C shows a much higher tendency to self-aggregation in
NMR spectra of B showed very large and unresolved sig- CDCl3. These observations could be attributed to different
nals that did not allow a definitive characterization of the end-effects: the tentacles carrying the azides are probably
molecular structure but did confirm its radical nature.
better able to interdigitate with adjacent molecules with re-
All the intermediates were purified by column spect to the more hindered, fully benzyl-capped tentacles in
chromatography and characterized by 1H, 13C (and 31P, 4, thus producing larger aggregates (in this respect, com-
where present) NMR spectrometry and by MS analysis. For pare also the differing behavior of 4 vs. 3, with 3 having
cyclic dimer A, in analogy with the previously synthesized lower, 0.3–0.4 mm, CAC values than 4). This hypothesis
1
congeners, inspection of the H and 31P NMR spectra sug- may be corroborated by a detailed microstructural charac-
Eur. J. Org. Chem. 2011, 1155–1165
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analyses were performed with a PerSeptive Biosystems Voyager–
De Pro MALDI mass spectrometer operating in the Linear mode
using 2,5-dihydroxybenzoic acid as the matrix. NMR spectra were
recorded with Bruker WM-400 or Varian Inova 500 spectrometers.
All the chemical shifts are expressed in ppm with respect to the
residual solvent signal; J values are in Hz. The following abbrevi-
ations are used for the multiplicities: s = singlet; d = doublet; t =
triplet; q = quartet; m = multiplet; dd = double doublet. 31P NMR
spectra were recorded with 85% H3PO4 as the external reference.
terization study on this novel macrocycle; the results will be
compared to those obtained in previous investigations with
2–5.[4,30]
Further experiments, involving an ESR-based study of
the mobility and mechanism of action of macrocycles B
within phospholipids bilayers, as well as the preparation of
a set of differently end-modified CyPLOS analogues start-
ing from convertible compound A, are in progress in our
laboratories and will be reported in due course.
General Procedure for the Coupling of 5Ј-O-DMT-N4-benzoyl-2Ј-de-
oxycytidine-3Ј-phosphoramidite 6 with 7a, 7b, or 7c in the Presence
of Activator: Nucleoside 6 (40 mg, 0.05 mmol) and compound 7a,
7b, or 7c (1.2 equiv.), were treated with a 0.25 m DCI solution in
anhydrous CH3CN (500 μL). The coupling was performed in a
standard NMR tube with the addition of 100 μL CDCl3 and acti-
vated molecular sieves (4 Å). 31P NMR spectra were recorded at
fixed times; after 4 h, a 5.5 m tBuOOH solution in decane (50 μL)
was added to the reaction mixtures.
Conclusions
In this work we describe the synthesis of novel CyPLOS
derivative A, which is functionalized with terminal azido
moieties that can be used for post-synthetic derivatization
with various labels by exploiting either “click chemistry” or
the Staudinger reaction. As a valuable example, its conver-
sion into a spin-labeled derivative, which is of interest for
ESR-based investigations into the mechanism of action and
ionophoric activity of CyPLOS analogues, has been de-
scribed. The linear precursor 19, obtained by phosphoram-
idite-based coupling between monosaccharides 17 and 18,
was obtained in good yields (75% isolated yield for three
steps). The finding that azides did not detectably interfere
in the coupling steps under standard conditions thus dem-
onstrates the compatibility of azido alcohols in the phos-
phoramidite chemistry approach. Before undertaking this
synthesis, the feasibility of the design of A was preliminarily
proven in dedicated coupling studies. The obtained results
demonstrate that a reactive phosphoramidite derivative can
be profitably condensed with an azido alcohol in a standard
phosphoramidite protocol in solution to give the desired,
stable phosphodiester adduct, provided that the reaction is
fast and that the phosphite triester is immediately oxidized
in situ to the more stable phosphate triester. These data
corroborate the analogous results obtained by Morvan et
al.,[22] for reactions carried out in the solid phase, thus ex-
panding the repertoire of reactions available to obtain func-
tionalized phosphodiester-linked scaffolds.
Procedure for the Coupling of 5Ј-O-DMT-N4-benzoyl-2Ј-deoxycytid-
ine-3Ј-phosphoramidite (6) with 7c in the Absence of an Activator:
Nucleoside 6 (40 mg, 0.05 mmol) and compound 7c (1.2 equiv.)
were both dissolved in anhydrous CH3CN (500 μL) in an NMR
tube with the addition of CDCl3 (100 μL) and activated molecular
sieves (4 Å). 31P NMR spectra were recorded at fixed times; after
4 h, a 5.5 m tBuOOH solution in decane (50 μL) was added to the
reaction mixture.
5Ј-O-(4,4Ј-Dimethoxytriphenylmethyl)-N4-benzoyl-2Ј-deoxycytidinyl
3Ј-O-{2-Cyanoethyl2-[2-(2-azidoethoxy)diethoxy]ethyl} Phosphate
(11a): The coupling mixture of nucleoside 6 and 7a described above
was evaporated to dryness and then purified by column chromatog-
raphy (CHCl3/CH3OH, 1 to 5% with a few drops of TEA) to give
pure 11a (35 mg, 0.037 mmol) in 75% yield as the only product.
Oil, Rf = 0.40 [CHCl3/CH3OH, 95:5 (v/v)]. 1H NMR (CDCl3,
500 MHz, mixture of two diastereomers): δ = 8.31 (br., 1 H, 6-H),
7.67–6.82 (m, 19 H, ArH and 5-H), 6.27 (br., 1 H, 1Ј-H), 5.18 (br.,
1 H, 3Ј-H), 4.41–4.20 (m, 4 H, TEG-CH2-O-P and O-CH2CH2CN),
3.98 (br., 1 H, 4Ј-H), 3.80 (s, 6 H, 2ϫ OCH3 of DMT), 3.73–3.63
(m, 12 H, 3ϫ O-CH2CH2-O), 3.54 (m, 1 H, 5Ј-Ha), 3.41–3.37 (m,
3 H, CH2-N3 and 5Ј-Hb), 2.89–2.84 (m, 1 H, 2Ј-Ha), 2.81 (t, J =
6.0, 5.5 Hz, 2 H, O-CH2CH2CN), 2.57–2.51 (m, 1 H, 2Ј-Hb) ppm.
13C NMR (CDCl3, 100 MHz): δ = 168.9 (CO), 162.0, 158.7, 143.8,
134.9, 133.1, 130.0, 128.9, 128.0, 127.4, 113.3 (10ϫ ArC), 117.5
(CN), 88.1 (C-1Ј), 87.0 (quaternary C of DMT), 81.3 (C-4Ј), 70.5,
70.3, 69.9, 69.7 (O-CH2-TEG and C-3Ј), 62.5, 62.0 (C-5Ј and
OCH2CH2CN), 55.1 (2ϫ OCH3 of DMT), 50.6 (2ϫ TEG-
CH2N3), 40.3 (C-2Ј), 19.5 (OCH2CH2CN) ppm. 31P NMR (CDCl3,
161.98 MHz): δ = –2.03 ppm.
Experimental Section
General Methods: TLC analyses were carried out on silica gel plates
from Merck (60, F254). Reaction products on TLC plates were
visualized under UV light and then by treatment with a 10%
Ce(SO4)2/H2SO4 aqueous solution. For column chromatography,
Preparation of the Monosaccharide Building Block 17. Synthesis of
Phenyl 2,3-Di-O-{2-[2-(2-azidoethoxy)diethoxy]ethyl}-β-D-glucopyr-
anoside (15): Phenyl 2,3-di-O-[(2-azidoethoxy)diethoxyethyl]-4,6-O-
benzylidene-β-d-glucopyranoside (14; 665 mg, 0.90 mmol, 1 equiv.)
silica gel from Merck (Kieselgel 40, 0.063–0.200 mm) was used. was treated with a TFA/CH2Cl2/H2O (1:10:0.5, v/v/v, 5 mL) solu-
HPLC analyses were performed with a Beckman System Gold in-
strument equipped with a UV detector module 166 and a Shim-
adzu Chromatopac C-R6A integrator. All the synthesized com-
pounds were found to be more than 98% pure, as assessed by
HPLC analysis on a Nucleosil 100–5 C18 Supelco analytical col-
umn (250ϫ4.6 mm, 5 μm), eluted with a linear gradient from 0 to
100% in 30 min of CH3CN in H2O, flow = 0.8 mL/min, detection
at λ = 264 nm. For the ESI MS analyses, a Waters Micromass ZQ
instrument equipped with an electrospray source was used in the
positive and/or negative mode. MALDI TOF mass spectrometric
tion at 0 °C. After 4 h, the reaction mixture was diluted with
CH2Cl2, the resulting solution was washed twice with water and
then concentrated under reduced pressure. The crude material was
purified by column chromatography (CHCl3/CH3OH, 5 to 15%) to
give pure 15 (592 mg, 0.90 mmol) in almost quantitative yield. Oil,
1
Rf = 0.50 (CHCl3/CH3OH, 9:1). H NMR (CDCl3, 500 MHz): δ =
7.30–7.00 (m, 5 H, ArH), 4.96 (d, J = 6.5 Hz, 1 H, 1-H), 4.16–4.05
[m, 2 H, (-CHa-O-C-2) and (-CHa-O-C-3)], 3.93–3.82 [m, 4 H,
(-CHb-O-C-2), (-CHb-O-C-3), 4-H and 6-Ha], 3.75 (m, 1 H, 6-Hb),
3.68–3.54 (m, 25 H, 12ϫ O-CH2-TEG and 3-H), 3.45 (m, 1 H, 5-
1162
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© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 1155–1165

Synthesis of an Azido-Functionalized CyPLOS Analogue
H), 3.39–3.35 [m, 5 H, 2-H and 2ϫ (-CH2-N3)] ppm. 13C NMR
(CDCl3, 50 MHz): δ = 157.0, 129.5, 122.6, 116.4 (ArH), 101.1 (C-
1), 85.8 (C-2), 82.0 (C-5), 75.3 (C-3), 72.3, 71.9 [(CH2-O-C-2) and
(CH2-O-C-3)], 70.8, 70.7, 70.5, 70.3, 70.2, 69.9 [12ϫ (O-CH2-TEG)
and C-4], 62.8 (C-6), 50.5 [2ϫ (-CH2-N3)] ppm. MALDI (+ve):
calcd. for C28H46N6O12: 658.32; found: 681.58 [M + Na+]. HRMS
was stirred at room temperature and monitored by TLC (CH2Cl2/
CH3OH, 95:5). After 2.0 h, 5.5 m tBuOOH solution in n-decane
(300 μL) was added to the mixture, which was stirred at room tem-
perature. After 30 min the reaction mixture was diluted with
CH2Cl2 and purified by column chromatography (CH2Cl2/CH3OH,
1 to 10% with a few drops of TFA) to afford pure 19 (91 mg,
(MALDI-TOF): calcd. for C28H46N6O12Na: 681.3071; found: 0.052 mmol) in 74% yield as an oil. Rf = 0.4 (CH2Cl2/CH3OH,
1
681.3109.
9:1). H NMR (CDCl3, 500 MHz): δ = 7.45–6.89 (m, 24 H, ArH),
4.85 and 4.77 (m, 2ϫ 1 H, 1-H and 1Ј-H), 4.47 [br. s, 4 H, 2ϫ
(CH2Bn)], 3.97–3.03 [m, 78 H, 16ϫ (CH2CH2OTEG), 2ϫ (4-H),
2ϫ (6-H2), 2ϫ (5-H), 2ϫ (3-H), 2ϫ (2-H) and (O-CH2CH2CN)],
Phenyl 2,3-Di-O-{2-[2-(2-azidoethoxy)diethoxy]ethyl}-6-O-(4,4Ј-di-
methoxytriphenylmethyl)-β-D-glucopyranoside (16): Compound 15
(592 mg, 0.90 mmol, 1 equiv.), dissolved in anhydrous pyridine
(2.2 mL), was treated with DMTCl (365 mg, 1.1 mmol, 1.2 equiv.).
The reaction mixture was stirred at room temperature overnight
then diluted with CH3OH and concentrated under reduced pres-
sure. The crude material was purified on a silica gel column
(CH2Cl2/CH3OH, 1 to 5% with a few drops of pyridine) to give
2.60 [t,
2
H, (O-CH2CH2CN)] ppm. 31P NMR (CDCl3,
161.98 MHz): δ = –1.5, –2.5, –6.87 ppm. ESI-MS (+ve): calcd. for
C79H113ClN7O31P2: 1751.65; found: 1774.56 [M + Na+], 1791.69
[M + K+], 1854.68 [M + Et3N+]. HRMS (MALDI-TOF): calcd.
for C79H112ClN7O31P2Na: 1774.6464; found: 1774.6509.
pure 16 (580 mg, 0.88 mmol) in 97% yield. Glassy compound; Rf
= 0.7 (CHCl3/CH3OH, 98:2, v/v). H NMR (CDCl3, 500 MHz): δ 1 equiv.), which was previously dried by repeated coevaporation
Synthesis of Cyclic Dimer 20: Derivative 19 (35 mg, 0.020 mmol,
1
= 7.48–6.70 (m, 18 H, ArH), 4.97 (d, J = 7.4 Hz, 1 H, 1-H), 4.17–
3.84 [m, 5 H, (-CH2-O-C-2), (-CH2-O-C-3) and 4-H], 3.75 [s, 6 H, MSNT (177 mg, 0.6 mmol, 30 equiv.) were dissolved in anhydrous
2ϫ (OCH3 of DMT)], 3.73–3.50 [m, 31 H, 14ϫ (O-CH2-TEG), 6- pyridine (20 mL) and stirred overnight at room temperature. The
Ha, 5-H, 3-H], 3.45 (m, 1 H, 2-H), 3.37 (m, 1 H, 6-Hb) ppm. 13C reaction mixture was concentrated under reduced pressure, dis-
with anhydrous pyridine, DMAP (3 mg, 0.020 mmol, 1 equiv.), and
NMR (CDCl3, 50 MHz): δ = 158.2, 157.3, 145.0, 136.1, 132.7,
130.1, 129.4, 128.2, 127.6, 126.5, 123.6, 122.4, 116.8, 112.9 (ArC),
solved in ethyl acetate, transferred into a separatory funnel and
washed three times with water. The organic phase was concentrated
101.2 (C-1), 86.3 (quaternary C of DMT), 85.8 (C-2), 82.1 (C-5), under reduced pressure and purified by column chromatography
75.3 (C-3), 72.4, 71.9 [(CH2-O-C-2) and (CH2-O-C-3)], 70.5, 70.4, (CH2Cl2/CH3OH, 1 to 5%) to afford pure 20 (25 mg, 0.014 mmol)
1
70.1, 69.9 [12ϫ (O-CH2-TEG) and (C-4)], 63.4 (C-6), 55.1 [2ϫ
(OCH3 of DMT)], 50.6 [2ϫ (TEG-CH2N3)] ppm. MALDI (+ve):
calcd. for C49H64N6O14: 960.45; found: 983.91 [M + Na+], 682.25
[M – DMT + Na+], 698.31 [M – DMT + K+]. HRMS (MALDI-
TOF): calcd. for C49H64N6O14Na: 983.4378; found: 983.4407.
in 75% yield as an oil. Rf = 0.5 (CH2Cl2/CH3OH, 9:1). H NMR
(CDCl3, 500 MHz; a mixture of two diastereomers in a 1:0.6 ratio):
δ = 7.48–6.80 (m, 24 H, ArH), 5.05, 4.90 (2ϫd, J = 8.0 and 5.5 Hz,
2 H, 1-H and 1Ј-H, minor stereoisomer), 5.00 and 4.94 (2ϫ d, J =
7.5 and 7.5 Hz, 2 H, 1-H and 1Ј-H, major stereoisomer), 4.53 [br.
s, 4 H, 2ϫ (CH2Bn)], 4.41–3.27 [m, 76 H, 16ϫ (CH2CH2OTEG),
2ϫ (4-H), 2ϫ (6-H2), 2ϫ (5-H), 2ϫ (3-H) and 2ϫ (2-H)], 2.78–
Phenyl 2,3-Di-O-{2-[2-(2-azidoethoxy)diethoxy]ethyl}-6-O-(4,4Ј-di-
methoxytriphenylmethyl)-β-D-glucopyranoside-4-O-(2-chlorophenyl)-
2.69 [m,
4
H, (CH2CH2CN)] ppm. 31P NMR (CDCl3,
phosphate Triethylammonium Salt (17): (2-Chlorophenyl) dichlo-
rophosphate (710 μL, 4.4 mmol, 5 equiv.) was added dropwise to a
stirred solution of 16 (580 mg, 0.88 mmol, 1 equiv.), 1,2,4-triazole
(300 mg, 7.0 mmol, 8 equiv.), and triethylamine (1.0 mL, 7.0 mmol,
8 equiv.) in anhydrous pyridine (12 mL) at 0 °C. The mixture was
warmed to room temperature and, after 3 h, the reaction mixture
was concentrated under reduced pressure. The crude material was
diluted with CHCl3, transferred into a separatory funnel and
washed three times with water, then concentrated under reduced
pressure and purified by column chromatography (CH2Cl2/
161.98 MHz): δ = –3.0 and –10.7 (major stereoisomer), –5.3 and
–9.7 (minor stereoisomer) ppm. ESI-MS (+ve): calcd. for
C79H111ClN7O30P2: 1734.65; found: 1758.62 [M + Na+], 1772.55
[M + K+]. HRMS (MALDI-TOF): calcd. for C79H110ClN7-
O30P2Na: 1756.6359; found: 1756.6397.
Synthesis of 21: Compound 20 (25 mg, 0.014 mmol, 1 equiv.),
which was coevaporated several times with anhydrous pyridine and
then dried under reduced pressure, was treated with piperidine
(500 μL) and the resulting mixture was stirred overnight at 70 °C.
CH3OH, 1 to 10% with a few drops of TFA), to afford pure 17 The reaction was stopped by removal of the solvent in vacuo and
(688 mg, 0.81 mmol) in 92% yield as an oil. Rf = 0.3 (CH2Cl2/ the crude material was then purified by column chromatography
CH3OH, 95:5, v/v). 1H NMR (CDCl3, 500 MHz): δ = 7.40–6.98
(CH2Cl2/CH3OH, 1 to 10%) to afford pure 21 (23 mg, 0.014 mmol)
(m, 9 H, ArH), 4.89 (br. s, 1 H, 1-H), 4.04–3.43 [br. m, 20 H, 14ϫ in an almost quantitative yield as an oil. Rf = 0.2 (CH2Cl2/CH3OH,
(O-CH2-TEG), 4-H, 6-H2, 5-H, 3-H, 2-H], 3.34 [m, 4 H, 2ϫ 95:5). 1H NMR (CDCl3, 500 MHz, piperidinium salt): δ = 7.37–
(-CH2-N3)] ppm. 13C NMR (CDCl3, 50 MHz): δ = 157.2, 130.0, 6.85 (m, 24 H, ArH), 4.99–4.84 (m, 2 H, 1-H and 1Ј-H), 4.54 [br.
129.4, 127.5, 124.4, 122.6, 121.9, 116.8, 109.9 (ArC), 101.5 (C-1),
81.8 (C-5), 79.2 (C-3), 72.3, 71.8 [(CH2-O-C-2) and (CH2-O-C-3)], 2ϫ (4-H), 2ϫ (6-H2), 2ϫ (5-H), 2ϫ (3-H) and 2ϫ (2-H)] ppm.
70.4, 70.1, 69.9 [12ϫ (O-CH2-TEG) and (C-4)], 60.3 (C-6), 50.7
13C NMR (CDCl3, 100 MHz): δ = 159.9, 152.8, 152.0, 144.6, 129.5,
[2ϫ (TEG-CH2N3)] ppm. 31P NMR (CDCl3, 161.98 MHz): δ = 128.3, 127.6, 116.8 (ArC), 101.1 (C-1), 99.5 (C-1Ј), 81.1 (C-5 and
s, 4 H, 2ϫ (CH2Bn)], 4.17–2.99 [m, 76 H, 16ϫ (CH2CH2OTEG),
–7.29 (br.) ppm. ESI-MS (+ve): calcd. for C34H50ClN6O15P:
848.27; found: 872.59 [M + Na+], 888.72 [M + K+]. HRMS
(MALDI-TOF): calcd. for C34H50ClN6O15PNa: 871.2658; found:
871.2701.
C-5Ј), 80.3 (C-3 and C-3Ј), 75.0, 73.1, 72.0, 69.9, 68.5 [16ϫ
(CH2CH2OTEG), 2ϫ (CH2Ph), and 2ϫ (C-4)], 65.5 (C-6 and C-
6Ј), 50.9 [2ϫ (TEG-CH2N3)] ppm. 31P NMR (CDCl3,
161.98 MHz): δ = –2.9 and –6.9 ppm. ESI-MS (+ve): calcd. for
C76H107ClN6O30P2: 1681.62; found: 1704.53 [M + Na+]. HRMS
(MALDI-TOF): calcd. for C76H107ClN6O30P2Na: 1705.6093;
found: 1705.6126.
Synthesis of Linear Dimer 19: Derivative 17 (60 mg, 0.070 mmol,
1 equiv.) and known[2] 18 (108 mg, 0.084 mmol, 1.2 equiv.), which
were previously dried by repeated coevaporation with anhydrous
CH3CN and kept under reduced pressure, were treated with a
0.25 m DCI solution in anhydrous CH3CN (2.0 mL). The reaction
Synthesis of A: Compound 21 (23 mg, 0.014 mmol, 1 equiv.), dis-
solved in dioxane (200 μL), was treated with satd. aq. LiOH
Eur. J. Org. Chem. 2011, 1155–1165
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1163

D. Montesarchio et al.
FULL PAPER
(1.0 mL) and the resulting mixture was stirred overnight at 70 °C.
The reaction mixture was concentrated under reduced pressure, dis-
solved in CH2Cl2, transferred into a separatory funnel and washed
three times with water. The organic phase was concentrated under
reduced pressure and purified by column chromatography
(CH2Cl2/CH3OH, 0 to 15%) to give pure cyclic dimer A (21 mg,
0.014 mmol) in 96% yield as an oil. Rf = 0.5 (CH2Cl2/CH3OH,
9:1). 1H NMR (CDCl3, 500 MHz, lithium salt, broadened signals):
δ = 7.33–6.87 (m, 20 H, ArH), 4.97–4.85 (m, 2 H, 1-H and 1Ј-H),
4.55 [br. s, 4 H, 2ϫ (CH2Bn)], 4.10–3.05 [m, 76 H, 16ϫ
(CH2CH2OTEG), 2ϫ (4-H), 2ϫ (6-H2), 2ϫ (5-H), 2ϫ (3-H) and
Supporting Information (see footnote on the first page of this arti-
cle): 31P NMR spectra (CDCl3, 161.98 MHz, 298 K) of macrocycle
A recorded at different concentrations for the determination of the
CAC value.
Acknowledgments
We thank the Italian Ministero dell’Istruzione, dell’Università e
della Ricerca (MIUR) for grants in support of this investigation
(PRIN2008) and Centro di Metodologie Chimico-Fisiche
(CIMCF), Università di Napoli “Federico II” for providing MS
and NMR facilities.
1
2ϫ (2-H)] ppm. H NMR (CD3OD, 500 MHz, lithium salt, sharp
signals): δ = 7.32–6.98 (m, 20 H, ArH), 5.09 (d, J = 7.5 Hz, 2 H,
1-H and 1Ј-H), 4.57–4.50 [m, 4 H, 2ϫ (CH2-Ph)], 4.12–3.91 [m, 14
H, 2ϫ (6-H2), 2ϫ (4-H) and 4ϫ (CH2-CH2-O-sugar)], 3.71–3.46
[m, 56 H, 2ϫ (CH2-O-CH2Ph), (O-CH2-CH2-OTEG), 2ϫ (3-H)
and 2ϫ (5-H)], 3.42–3.33 [m, 6 H, 2ϫ (2-H) and 2ϫ (CH2-
N3)] ppm. 13C NMR (CD3OD, 125 MHz): δ = 156.8, 131.0, 130.1,
129.9, 129.5, 129.4, 124.2, 118.6, 118.3 (ArC), 102.0 (C-1 and C-
1Ј), 84.1, 83.7 (C-5 and C-5Ј), 74.6 [2ϫ (O-CH2-Ph)], 74.0, 73.9
(C-4 and C-4Ј), 72.3, 72.1, 71.6, 71.3, 71.1 (C-2 and C-2Ј, C-3 and
C-3Ј, O-CH2-CH2-OTEG), 69.4 [4ϫ (CH2-CH2-O-sugar)], 68.1 (C-
6 and C-6Ј), 52.3 [2ϫ (O-CH2-N3)] ppm. 31P NMR (CDCl3,
161.98 MHz, 10 mM): δ = ca. –3 (br.) ppm. 31P NMR (CDCl3,
161.98 MHz, 0.2 mM): δ = 0.23 (sharp) ppm. 31P NMR (CD3OD,
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for C70H104N6O30P2: 1570.62; found: 1594.46 [M + Na+]. HRMS
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1593.6280.
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Synthesis of B (Mixture of Regioisomers): CyPLOS A (10 mg,
0.006 mmol, 1 equiv.) was dissolved in anhydrous THF (50 μL).
Triphenylphosphane (4 mg, 0.015 mmol, 2.5 equiv.) was added to
the solution and the reaction was stirred overnight at room tem-
perature. Water (1.0 mL) was added and the resulting system was
stirred for 48 h. The reaction mixture was then treated with dilute
aq. HCl solution and exhaustively washed with diethyl ether to
remove the impurities. The aqueous phase was neutralized and then
extracted with CHCl3/CH3OH (9:1) to furnish the desired, pure
diamino-functionalized CyPLOS (9 mg, 0.006 mmol) as an oil [Rf
= 0.4 (n-butanol/acetic acid/water, 60:15:25)]. This compound
(9 mg, 0.006 mmol, 1 equiv.), previously dried by repeated coevapo-
rations with anhydrous benzene and kept several hours under high
vacuum, was dissolved in anhydrous CH2Cl2 (100 μL). DIPEA
(5 μL, 0.03 mmol, 5 equiv.), DCC (3 mg, 0.015 mmol, 2.5 equiv.),
and TEMPO-carboxylic acid (0.5 mg, 0.003 mmol, 0.5 equiv.) were
sequentially added and the resulting mixture was stirred overnight
at room temperature. The reaction mixture was concentrated under
reduced pressure, diluted with diethyl ether and washed twice with
distilled water. The aqueous phase was concentrated under reduced
pressure, redissolved in H2O and purified on a Sephadex G25 col-
umn (H2O/EtOH, 1:1). From UV measurements, the fractions ab-
sorbing at λ = 264 nm were collected and evaporated to dryness,
yielding target compounds B (7 mg, 0.004 mmol) with 67% yield
as an oil. Rf = 0.4 [n-butanol/acetic acid/water, 60:15:25]. 1H NMR
(CD3OD, 500 MHz, broad, unresolved signals): δ = 7.40–6.90 (m,
20 H, ArH), 5.07 (d, under the HDO signal, 1-H and 1Ј-H), 4.58
[br., 4 H, 2ϫ (CH2-Ph)], 4.06 [m, 8 H, 4ϫ (CH2-CH2-O-sugar)],
3.66–3.16 [m, 62 H, 2ϫ (CH2-O-CH2Ph), 12ϫ (O-CH2-CH2-
OTEG), 2ϫ (3-H), 2ϫ (4-H), 2ϫ (6-H2) and 2ϫ (5-H)], 3.33–3.24
[m, 4 H, 2ϫ (2-H)], 3.02 [br., 4 H, TEG-CH2NH2], 1.98–1.60 [m,
7 H, (CH2 and CH protons of TEMPO)], 1.38–1.05 [m, 12 H, (CH3
of TEMPO)] ppm. ESI-MS (–ve): calcd. for C80H126N3O32P2:
1702.76; found: 850.15 [M – 2H+]2–.
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© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 1155–1165

Synthesis of an Azido-Functionalized CyPLOS Analogue
[26]
[28] As an alternative, following the interpretation given in ref.[27]
,
M. A. Maier, A. P. Guzaev, M. Manoharan, Org. Lett. 2000,
the phosphate diester could be formed through acid-promoted
cleavage of the phosphoramidate diester derivative, see: M.
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7319–7322.
2, 1819–1822.
[27] As an alternative interpretation to the one given by Sekine and
co-workers, this signal could be due to the related phosporam-
idate diester, obtained in a Michaelis–Arbuzov-type rearrange-
ment of adduct 11c. This hypothesis is supported by more re-
cent works (see Serwa et al.[8] and also: I. Wilkening, G. del Si-
gnore, C. P. R. Hackenberger, Chem. Commun. 2008, 2932–
2934, and literature cited therein). Particularly, the presence of
molecular sieves (4 Å) in the NMR tube – added to partially
preserve phosphoramidites from moisture – is described by
Hackenberger and co-workers as an efficient catalyst in this
rearrangement. Neither MS nor IR analysis of this mixture
clarified the results, and purification by column chromatog-
raphy did not allow stable compounds to be isolated. In view
of the fact that this was also not found in the coupling of 6 with
7a, the nature of this adduct was thus not further investigated.
[29] In this respect, a theoretical study by the group of Sekine has
recently contributed to the understanding of O-selectivity in
phosphorylations realized with N-unprotected phosphoramid-
ite nucleosides. This was explained in terms of frontier molecu-
lar orbital interactions between the reactive intermediates and
nucleophiles, such as amino and hydroxyl groups of nucleo-
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Received: July 26, 2010
Published Online: January 11, 2011
Eur. J. Org. Chem. 2011, 1155–1165
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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