A straightforward preparation of aminoglycoside-dinucleotide and -diPNA conjugates via click ligation assisted by microwaves

Article abstract of DOI:10.1002/ejoc.201000182

An alternative and straightforward method to prepare aminoglycoside- dinucleotide and -diPNA conjugates is reported, which is based, on copper-catalyzed Huisgen azidealkyne cycloaddition (click chemistry ligation) assisted by microwave irradiation. This method permitted conjugations to be performed in aqueous solution, in very short times and with readily prepared, precursors.

Full text of DOI:10.1002/ejoc.201000182

FULL PAPER
DOI: 10.1002/ejoc.201000182
A Straightforward Preparation of Aminoglycoside–Dinucleotide and –diPNA
Conjugates via Click Ligation Assisted by Microwaves
Javier Alguacil,[a] Sira Defaus,[a] Ana Claudio,[a] Alejandro Trapote,[a] Marta Masides,[a]
and Jordi Robles*[a]
Keywords: Nucleic acids / Carbohydrates / Aminoglycosides / Antibiotics / Cycloaddition / Microwave chemistry
An alternative and straightforward method to prepare ami-
microwave irradiation. This method permitted conjugations
noglycoside–dinucleotide and –diPNA conjugates is re- to be performed in aqueous solution, in very short times and
ported, which is based on copper-catalyzed Huisgen azide-
alkyne cycloaddition (“click chemistry” ligation) assisted by
with readily prepared precursors.
cleic acids (PNAs), the conjugates could behave as selective
artificial ribonucleases.[10–11] Based on these reports, we
were interested in further studying the properties of amino-
glycoside–oligonucleotide conjugates as specific RNA li-
gands. Here, we report on a procedure for obtaining neomy-
cin– or paromomycin–dinucleotide and –diPNA conjugates
(Scheme 1) which combines copper-catalyzed Huisgen
Introduction
Abbreviations: Asc: sodium ascorbate, Bhoc: benzhydryl-
oxycarbonyl, Boc: tert-butoxycarbonyl, CPG: controlled pore glass
CuAAC: copper-catalyzed Huisgen azide-alkyne cycloaddition,
DIEA: N,N-diisopropylethylamine, DMF: N,N-dimethylform-
amide, DMSO: dimethyl sulfoxide, ESI: electrospray ionization
mass spectrometry, Fmoc: 9-fluorenylmethoxycarbonyl, HOBt:
N-hydroxybenzotriazole, HPLC: high performance liquid azide-alkyne cycloaddition (CuAAC) with microwave irra-
chromatography, MALDI-TOF: matrix assisted laser desorption
ionization-time of flight mass spectrometry, MW: microwave irradi-
ation, NMP: N-methylpyrrolidone, PNA: peptide nucleic acid,
TBTA: tris(benzyltriazolylmethyl)amine, TFA: trifluoroacetic acid,
THAP: 2,4,6-trihydroxyacetophenone, tR: retention time.
diation (MW).
Results and Discussion
Aminoglycoside antibiotics are known bactericidal
agents. However, toxicity, target promiscuity and the ap-
pearance of resistance mechanisms have depreciated their
clinical use.[1] The alarming decrease in the activity of the
current antibiotic repertoire has renewed the interest for
chemical analogues of aminoglycosides.[2] Efforts have re-
cently been fuelled by the vast knowledge acquired on the
structure of ribosomal RNA[3] (the main biological target
of aminoglycosides) but also by potential therapies based
on RNA ligands.[4] Among many other aminoglycoside de-
rivatives, aminoglycoside–oligonucleotide conjugates[5–7]
have recently been considered as specific ligands of bacterial
and viral RNA due to the additional chemical recognition
properties conferred by oligonucleotide strands. Further-
more, free aminoglycosides[8] and aminoglycosides as CuII
complexes[9] are also known to produce the degradation of
nucleic acids. Recent results suggested that if aminoglyco-
sides are conjugated with oligonucleotides or peptide nu-
Aminoglycoside–oligonucleotide conjugates were known
by forming thiourea,[6,12] amide,[7,13–14] and phosphate[11]
linkages, or by nucleobase derivatization.[15] We reasoned
that CuAAC, the best known “click chemistry“ transforma-
tion,[16] could also be a useful method for producing the
ligation of aminoglycosides and oligonucleotides. CuAAC,
specifically a 1,3-dipolar cycloaddition of azides and alk-
ynes which produces a triazole ring,[17] has become an out-
standing method in medicinal chemistry[18] and means of
building new materials.[19] Moreover, more recently it has
been used for biomolecule ligation and bioconjug-
ation.[20–21] Consequently, CuAAC has been extensively em-
ployed to synthesize both oligonucleotide[22–26] and amino-
glycoside derivatives.[27–29] In connection with our objec-
tives, during this work, the synthesis of aminoglycoside–oli-
gonucleotide conjugates by CuAAC was reported for the
first time by ligation of a 4Ј-C-(azidomethyl)thymidine-con-
taining oligonucleotide and an alkynyl neamine deriva-
tive.[30]
[a] Departament de Química Orgànica, Facultat de Química, and
Institut de Biomedicina, Universitat de Barcelona,
Martí i Franquès 1-11, 08028 Barcelona, Spain
Fax: +34-933-397-878
Here, we describe a straightforward alternative approach
to obtain aminoglycoside–nucleotide conjugates via
CuAAC, by coupling azido-aminoglycosides with alkynyl
dinucleotides in aqueous solution (Scheme 2). The reasons
for this choice were that alkynyl oligonucleotides can be
E-mail: jrobles@ub.edu
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201000182.
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Aminoglycoside–Dinucleotide and –diPNA Conjugates
tions with RNA secondary structures and to offer new in-
sights into the design of novel RNA ligands. Currently, we
are evaluating these molecules as A-site ribosomal RNA
ligands and results will be reported elsewhere.
Scheme 2. Synthesis of aminoglycoside–dinucleotide and aminogly-
coside–diPNA conjugates via CuAAC.
As aminoglycoside precursors, azides 10, 12 and 14 were
prepared from neomycin and paromomycin, respectively
(Scheme 3), in order to explore derivatization of the amino-
glycoside backbone in three different rings and with a dif-
ferent number of amine groups. The synthesis of 5ЈЈ-azido-
5ЈЈ-deoxyneomycin (10) was carried out essentially as de-
scribed from neomycin (four steps, 37%).[27] Paromomycin
derivative 12 (Scheme 3, b) was prepared by sodium azide
substitution on hexa-O-acetyl-4Ј-O-benzoyl-6Ј-bromo-
pentakis(N-tert-butoxycarbonyl)-6Ј-deoxyparomomycin[33]
and subsequent deprotection with aq. conc. ammonia and
trifluoroacetic acid, resulting in a global 60% yield. Deriva-
tive 14 that contained an azide group at the idose ring was
prepared (Scheme 3, c), by selective monoacylation of 6ЈЈЈ
primary amine of paromomycin with succinimidyl azido-
acetate. Product was temporally protected with Boc groups
to allow purification by chromatography, and deprotected
by treatment with trifluoroacetic acid. It was obtained in
28% yield from paromomycin.
The corresponding O-alkynyl dinucleotides (15a–15d,
Scheme 4, a) were solid-phase synthesized by the standard
phosphoramidite method, and the hexyn-5-yl appendage
was incorporated via 5Ј-phosphorylation.[22,31] Alkynyl di-
nucleotides were finally deprotected and cleaved from the
Scheme 1. Molecules synthesized in the present work via CuAAC:
a) aminoglycoside–dinucleotide conjugates, b) and c) aminoglycos-
ide–diPNA conjugates.
readily obtained by solid-phase synthesis,[31] and several resin by treatment with aq. conc. ammonia, and as they
methods are reported to yield azido-aminoglycosides.[27] were sufficiently pure (Ͼ80% after HPLC analysis), they
Furthermore, due to the orthogonality of azides and alkyne were used without further purification in the following
functions, no additional protection was needed for the syn- CuAAC step. Our results below confirm that intermediate
thetic precursors and ligation could be performed in water, purification steps can be minimized, without significantly
where the reagents and products are highly soluble. In com- compromising the scope of the ligation reaction. The corre-
parison with alternative metal-free ligations,[32] the triazole sponding diPNAs 16–19 (Scheme 4 parts b–c) were as-
ring produced by the copper-catalyzed reaction has shown sembled by solid-phase synthesis on a Sieber resin, by use
a convenient covalent knot for conjugates acting as RNA of standard Fmoc/Bhoc methodology. The hexynyl append-
ligands,[29] by low steric hindrance and potentially forming age of diPNAs 16a–16b was incorporated after the removal
polar and stacking interactions. Here, this approach was of the N-terminal Fmoc group, by reaction with 5-hexyn-1-
tested by preparing 5ЈЈ-neomycin, 6Ј- or 6ЈЈЈ-paromomycin yl p-nitrophenyl carbonate at room temperature for 2 h. To
conjugates of dinucleotides dTT, dCC, dAA, dGG, and synthesize diPNAs 17–19 and 17ac–19ac, the alkynyl ap-
also, of PNA analogues H-tt-NH2 and H-aa-NH2 pendage was introduced via Nα-(2-Fmoc-aminoethyl)-Nα-
(Scheme 1). These conjugates that combine diversed amino- (3-pentynoyl)glycine[34] under the same conditions as the
glycosides, nucleobase sequences, oligonucleotide back- nucleobase monomers. At the end of the synthesis, the N-
bones and linkers were devised to explore specific interac- terminal Fmoc group was first removed, in order to pro-
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J. Robles et al.
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Scheme 4. Dinucleotide and diPNA precursors: a) 5Ј-O-hexynyl
dinucleotides (15a–15d). b) N-hexynyloxycarbonyl diPNAs (16a,
16b). c) diPNAs (17–18 and 17ac–18ac) containing Nα-(2-Fmoc-
aminoethyl)-Nα-(3-pentynoyl)glycine.
0.1 µmol of hexynyl oligonucleotide 15a reacted with an ex-
cess of 5ЈЈ-azido-5ЈЈ-deoxyneomycin 10 (1.67 equiv.). To
avoid the formation of potentially degradative CuII-amino-
glycoside species,[9a] CuI was first obtained from the reac-
tion of CuSO4 and sodium ascorbate (Asc), followed by the
addition of the dinucleotide and the aminoglycoside. After
some preliminary experiments, only moderate cycloaddition
was produced (Table 1, assay 1.3) by heating a solution con-
taining 0.24 m dinucleotide, 0.40 m azido-aminoglycos-
ide, CuI catalyst generated from 0.05 m CuSO4 and
1.2 m ascorbate, and 0.05 m of tris(benzyltriazolyl-
methyl)amine[36] (TBTA) to 70 °C for 24 h. Previously,
when the reaction was tested in the absence of TBTA, no
significant conjugation was observed after 24 h at 70 °C
(assay 1.1). Thus, TBTA, which acted here as CuI chelator,
Scheme 3. Azido-aminoglycosides 10–14. a) 5ЈЈ-Azido-5ЈЈ-deoxy-
neomycin (10). b) Synthesis of 6Ј-azido-6Ј-deoxyparomomycin (12).
c) Synthesis of 6ЈЈЈ-N-azidoacetylparomomycin (14). Reagents and
conditions: i) NaN3, DMF, 80 °C, 4 h, 75%; ii) NH3 in CH3OH,
60 °C, 4 h + 40% TFA in CH2Cl2, 2 h, 80%; iii) N-succinimidyl
azidoacetate, NaHCO3 10%, water/DMF + Boc2O; iv) 40% TFA
in CH2Cl2, 2 h, 28% (from paromomycin).
duce the free amine derivatives 17–19 or subsequently acet- was a determinant factor to enhance conjugation and limit
ylated to obtain 17ac–19ac. Modified PNA 17–19 were de- the degradation of the oligonucleotide, in accordance with
vised to evaluate the effect of i) the ligation position of the other previous reports.[24a,37] Copper concentration should
aminoglycoside to the polyamide backbone and ii) the pres- also be conveniently optimized. Without TBTA, the in-
ence of a free amine or an acetamide group at the N-ter- crease of copper concentration from 0.05 m to 0.1 m was
minus in the affinity properties of conjugates. Alkynyl found to be counterproductive, as the conjugate was pro-
diPNAs were finally cleaved from the resin and deprotected duced in low amounts and accompanied of several de-
by treatment with TFA/m-cresol (19:1) for 10 min at room graded products (assay 1.2). MW was also assayed, as it
temperature. Just as with the dinucleotide derivatives, no was recently reported to be compatible with the synthesis
further purification of diPNAs was neeeded before per- of oligonucleotide derivatives.[23a,24,25] Notably, MW pro-
forming the ligation step.
duced an extraordinary acceleration of the reaction and im-
Our ligation approach was first assayed in the prepara- proved the efficiency of conjugation, but only if TBTA was
tion of neomycin-TT conjugate (1a). Among the plethora present (assay 1.3). In the absence of chelator, by MW irra-
of methods known to generate the CuI catalyst,[35] we opted diating at 80 °C, very low conversion and degradation were
for the in situ reduction of CuSO4 with sodium ascorbate. observed after 1 h, even if CuI concentration was increased
The results of our assays are summarized in Table 1 (see (assay 1.4). When 0.07 m TBTA was present, the irradia-
HPLC profiles in Supporting Information). In every assay, tion produced a significant increase of conversion; up to
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Aminoglycoside–Dinucleotide and –diPNA Conjugates
62% after 30 min and up to 72% after 1 h (assays 1.5–1.6). significant differences in yields were observed between dinu-
Finally, optimal conversion into 1a was observed if irradia- cleotide and diPNA conjugates of the same sequence. With
tion was conducted at 100 °C for 1 h (assay 1.7). Most respect to conjugates synthesized from diPNAs containing
probably, in the presence of a chelating agent as TBTA that Nα-aminoethyl-Nα-(3-pentynoyl)glycine and 5ЈЈ-azido-5ЈЈ-
stabilizes CuI and prevents complexation with amino- deoxyneomycin (10), we observed that free amine PNAs
glycosides, MW experiments benefited from the high heat- 17–19 produced conjugates with lower conversion and
ing rate of water, the selective activation of the polar inter- lower purity than their acetylated counterparts 17ac–19ac.
mediates involved in CuAAC, and that reactions can be Although we could not ascertain the exact reasons for that,
carried out at higher temperatures than by conventional it clearly related to the presence of a N-terminal amine, with
heating.
independence of the ligation position of aminoglycoside.
Their lower conversions could be related to unfavourable
electrostatic between both protonated PNA and aminogly-
coside in the course of the ligation reaction. No significant
improvements could be obtained by extending reaction
times nor increasing temperatures. Conjugates 8 and 9 were
synthesized by ligation of 6ЈЈЈ-N-azidoacetylparomomycin
14 and diPNAs 16a and 18ac. Here, ligations required
longer times of microwave irradiation (2 h) than those car-
ried out with the other two aminoglycosides.
Table 1. Assays[a] of synthesis of 5ЈЈ-neomycin-5ЈdTT (1a).
15a
10
CuII Asc
TBTA T
Time Conv.
[h]
[mm] [m] [m] [m] [m] [°C][b]
[%][c]
1.1 0.24
1.2 0.24
1.3 0.24
1.4 0.24
1.5 0.34
1.6 0.34
1.7 0.34
0.40 0.05 1.2
0.40 0.1 2.4
0.40 0.05 1.2
0.40 0.1 2.4
0.60 0.07 1.2
0.60 0.07 1.2
0.60 0.07 1.2
–
–
70
70
24
52
24
1
n.d.
n.d.
moderate
n.d.
51
0.05 70
80 MW
–
0.07 80 MW 0.5
0.07 80 MW
0.07 100 MW
1
1
61
73
Table 2. Synthesis[a] of conjugates 1–4.
[a] Assays were performed with 0.1 µmol dinucleotide 15a (the
HPLC profiles are shown in ESI). [b] MW means microwaves irra-
diation. [c] Conversion of dinucleotide 15a into conjugate accord-
ing to the HPLC analyses.
Aminoglycoside +
dinucleotide/diPNA
Conjugate
T
Time Crude Isolated
[h]
yield[c] yield[d]
[°C][b]
[%]
[%]
10 + 15a
10 + 15b
10 + 15c
10 + 15d
10 + 16a
10 + 16b
12 + 15a
12 + 15b
12 + 15c
12 + 15d
12 + 16a
12 + 16b
10 + 17
10 + 17ac
10 + 18
10 + 18ac
10 + 19
10 + 19ac
14 + 16a
14 + 18ac
1a
1b
1c
1d
3a
3b
2a
2b
2c
2d
4a
4b
5
5ac
6
6ac
7
7ac
8
9
100 (MW) 1.0
80 (MW) 1.0
80 (MW) 1.0
80 (MW) 1.0
120 (MW) 1.5
120 (MW) 1.5
100 (MW) 1.0
80 (MW) 1.0
80 (MW) 1.0
80 (MW) 1.0
120 (MW) 1.5
120 (MW) 1.5
120 (MW) 1.5
120 (MW) 1.5
120 (MW) 1.5
120 (MW) 1.5
120 (MW) 1.5
120 (MW) 1.5
120 (MW) 2.0
120 (MW) 2.0
80
55
43
63
68
65
56
70
27
28
14
11
21
19
33
9
Based on the conditions of assay 1.5, which efficiently
rendered 1a, we continued by synthesizing the other conju-
gates in Scheme 1. The results are summarized in Table 2.
The temperature should be optimized to maximize conver-
sion: the optimal temperature was found to be 100 °C for
TT conjugates (1a, 2a), and 80 °C for other dinucleotide
conjugates (1b–1d, 2b–2d). Notably, we managed to increase
the scale from 0.1 µmol to 0.5–1.0 µmol without significant
differences in conversion or conjugate purity. The same pro-
cedure was also successfully applied for the preparation of
PNA conjugates 3a–3b and 4a–4b although reactions were
heated to 120 °C and extended to 1.5 h to maximize conver-
sion. Probably, conjugations were slower than in dinucleo-
tide derivatives due to the absence of favourable electro-
static interactions between aminoglycosides and diPNAs.
Conversion into conjugates was mostly higher than 70%
according to the HPLC analyses, except for conjugates 5–7
and 5ac–7ac, which were obtained in lower yields. However,
low amounts of fragmented products were inevitably ob-
served, probably caused by heating at the high temperature
needed to maximize conjugations. At room temperature,
conjugates shown to be stable for extended times (1–2 d)
in the presence of copper and ascorbate reagents. All the
conjugates were conveniently purified by reversed-phase li-
60
77
30
5
76
25
20
15
22
13
38
18
40
20
17
65
n.d.[e]
36
n.d.[e]
55
81
64
62
55
[a] Experiments were performed at 0.5–1 µmol scale. [b] MW means
microwaves irradiation. [c] Percentages of conjugates in synthesis
crudes according to HPLC analysis. [d] Isolated yield after purifica-
tion. [e] Not determined because of overlapping of chromato-
graphic bands.
Conclusions
To summarize, we have reported a straightforward pro-
quid chromatography and identified by mass spectrometry. cedure for the synthesis of aminoglycoside–dinucleotide
Regardless of high conversions, the presence of impurities conjugates derived from neomycin and paromomycin, for
chromatographically close to our desired products made the first time by a combination of the Huisgen alkyne–azide
difficult their purification, and it was probably the reason cycloaddition and microwaves irradiation. Notably, li-
for some of the low-to-moderate yields shown in Table 2. gations can be performed in aqueous solution, in very short
Notably, TT and AA conjugates were generally obtained times and from readily prepared precursors. In spite of what
with better yields than their CC and GG counterparts. No could be expected from the known degradative properties
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J. Robles et al.
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(m, 1 H, H3Ј), 4.09 (t, J = 3.1 Hz, 1 H, H3ЈЈЈ), 4.07 (m, 1 H, H4ЈЈ),
3.89 (dd, J = 9.9, 9.2 Hz, 1 H, H4 or H6), 3.75–3.81 (m, 3 H, H5,
H5Ј, H5aЈЈ), 3.65–3.71 (m, 3 H, H4ЈЈЈ, H6aЈ, H6bЈ), 3.62 (dd, J = 12.4,
5 Hz, 1 H, H5bЈЈ), 3.55 (dd, J = 10.5, 9.2 Hz, 1 H, H4 or H6), 3.38–
3.50 (m, 4 H, H6Ј, H2ЈЈЈ, H6bЈЈЈ, H5ЈЈЈ); 3.31 (dd, J = 10.7, 4.0 Hz, 1
H, H2Ј), 3.18–3.29 (m, 3 H, H4Ј, H1, H3), 2.36 (dt, J = 4, 12.4 Hz,
1 H, H2a), 1.74 (q, J = 12.4 Hz, 1 H, H2b) ppm. 13C NMR (D2O,
100 MHz): δ = 118.9, 115.0, 110.2, 95.6, 95.4, 84.5, 81.5, 81.3, 76.7,
76.5, 75.2, 73.5, 72.6, 72.5, 70.3, 70.2, 69.8, 68.8, 67.7, 67.4, 60.2,
53.8, 50.9, 50.8, 49.8, 49.5, 48.8, 40.5, 30.6, 28.1, 25.1, 21.3 ppm.
of aminoglycosides and their copper complexes, ligations
were straightforwardly achieved by CuI catalysis in the pres-
ence of a CuI chelator such as TBTA under an inert atmo-
sphere. The same method could also be applied to the prep-
aration of diPNA conjugates, just by extending reaction
times. The aminoglycoside conjugates that were prepared in
this work are currently being tested as selective effectors of
ribosomal RNA and the results will be reported elsewhere.
IR (KBr): ν = 2926, 2116, 1676 cm–1. MALDI-TOF MS (positive
˜
mode) m/z: 641.6 [M + H]+ (calcd. for C23H45N8O13+, 641.3), 663.7
[M + Na]+ (calcd. for C23H44N8O13Na+, 663.3), 679.7 [M + K]+
(calcd. for C23H44N8O13K+, 679.3). ESI HRMS (positive mode)
m/z: 641.3115 [M + H]+ (calcd. for C23H45N8O13+, 641.3100;
Experimental Section
General: Unless otherwise indicated, all chemicals were purchased
from commercial suppliers (reagent grade) and used without purifi-
cation. Dry CH3CN was obtained by distillation over CaH2 and
storage over CaH2 lumps. CH2Cl2 was neutralized and dried by
passing through basic Al2O3 and storage over CaH2. Nitrogen was
bubbled through DMF in order to remove volatile contaminants,
then the DMF was dried by storage over CaH2. NMR spectra were
recorded with a Varian Mercury 400 MHz. MALDI-TOF spectra
of oligonucleotides were recorded in a Perseptive Biosystems
Voyager DE-RP instrument, by using 2,4,6-trihydroxyaceto-
phenone (THAP) as a matrix.
differential 2.2 ppm), 663.2929 [M
+
Na]+ (calcd. for
C23H44N8O13Na+, 663.2920; differential 1.3 ppm), 321.1598 [M +
H]2+ (calcd. for C23H46N8O132+, 321.1586; differential 3.5 ppm).
6ЈЈЈ-N-Azidoacetylparomomycin (14): N-Succinimidyl azidoacetate
was prepared by reaction of azidoacetic acid[41] (540 mg,
5.4 mmol), N-hydroxysuccinimide (402 mg, 5.4 mmol) and dicyclo-
hexylcarbodiimide (720 mg, 5.4 mmol) in anhydrous DMF (10 mL)
for 1 h at 5 °C. The mixture was filtered through glass wool and
added drop by drop, in three portions, to a solution of paromomy-
cin. H2SO4 (1.25 g, 1.8 mmol) in 0.1  NaHCO3/DMF (4:3,
50 mL), and left to react for 18 h. Dioxane was added (200 mL),
and the pH was adjusted to 10 by addition of NaHCO3. Boc2O
was added (2.4 g, 10.8 mmol), and the mixture heated at 60 °C.
Two additional portions of Boc2O (1.3 g, 5.4 mmol) were added
after 12 h and 25 h, respectively. After 3 d of reaction, the solvent
was evaporated to near dryness, and redissolved in ethyl acetate
and water. The organic layer was separated, washed twice with
water, dried on Na2SO4 and evaported to dryness. Product was
purified by medium pressure chromatography (MPLC), by em-
ploying a 20–90% linear gradient of 0.045% TFA/water and
0.045% TFA/CH3CN. The fractions corresponding to 6ЈЈЈ-N-azi-
doacetyl-tetrakis(N-tert-butoxycarbonyl)paromomycin (II) were
pooled, evaporated and freeze-dried, to yield a white solid. TLC
(CH2Cl2/MeOH, 9:1): 0.58. 1H NMR ([D6]DMSO, 400 MHz); only
6Ј-Azido-6Ј-deoxyparomomycin (12): Aminoglycoside 11[33]
(300 mg, 0.195 mmol) and sodium azide (26 mg, 0.39 mmol) were
dissolved in 12 mL of anhydrous DMF, and the mixture was heated
at 80 °C for 4 h. The solvent was removed by evaporation and co-
evaporation with CH3CN, to obtain a solid residue. The residue
was redissolved in AcOEt and it was washed twice with water, dried
on MgSO4 and the solvent was evaporated. Hexa-O-acetyl-6Ј-
azido-4Ј-O-benzoyl-pentakis(N-tert-butoxycarbonyl)-6Ј-deoxy-
paromomycin (I) was obtained as a yellowish solid. No further
purification was needed according to TLC and NMR analyses;
yield 215 mg, 75%. TLC (CH2Cl2/CH3OH, 95:5) Rf = 0.42; m.p.
1
168–173 °C. H NMR ([D6]DMSO, 400 MHz); only most relevant
signals: δ = 7.84 (d, J = 7.7 Hz, 2 H, NHBoc), 7.65 (t, J = 7.3 Hz,
1 H, NHBoc), 7.50 (t, J = 7.7 Hz, 2 H, NHBoc), 6.87 (d, J =
8.4 Hz, 1 H, NHBoc), 6.82 (d, J = 9.5 Hz, 1 H, NHBoc), 6.20 (d,
J = 9.5 Hz, 1 H, NHBoc), 5.54 (d, J = 3.4 Hz, 1 H), 5.40 (d, J =
9.8 Hz, 1 H), 5.54 (br. s, 1 H), 5.39 (d, J = 9.6 Hz, 1 H), 5.19 (br.
s, 1 H), 5.13 (t, J = 9.2 Hz, 1 H), 5.05 (t, J = 9.2 Hz, 1 H), 4.82
most relevant signals:[39]
δ = 8.06 (t, J = 3.6 Hz, 1 H,
NHCOCH2N3), 6.80 (d, J = 8 Hz, 1 H, NHBoc), 6.64 (d, J = 6 Hz,
1 H, NHBoc), 6.19 (d, J = 9.6 Hz, 1 H, NHBoc), 5.86 (d, J =
10 Hz, 1 H, NHBoc), 1.37 (br. s, 36 H, CH3 tBu) ppm. MALDI-
TOF MS (positive mode) m/z: 1121.7 [M + Na]+ (calcd. for
(br. s, 1 H), 4.57 (m, 2 H), (m, 45 H, CH tBu) ppm. IR (KBr): ν
˜
3
= 3379, 2978, 2933, 2107, 1747 cm–1. MALDI-TOF MS (positive
mode) m/z: 1519.0 [M + Na]+ (calcd. for C67H100N8O30Na+,
1519.6), 1534.0 [M + K]+ (calc for C67H100N8O30K+, 1535.6). ESI
HRMS (positive mode) m/z: 1497.6572 [M + H]+ (calcd. for
C67H101N8O30+, 1497.6618; differential –3.1 ppm), 1519.6396 [M +
Na]+ (calc for C67H100N8O30Na+, 1519.6437; differential
–2.7 ppm).
C45H78N8O23Na+, 1122.1), 1137.7 [M
C45H78N8O23K+, 1138.2).
+
K]+ (calc for
Aminoglycoside II was treated with 20 mL of 40% TFA in CH2Cl2
for 2 h at room temperature. The solution was evaporated to near
dryness, and a solid was precipitated by addition of Et2O. Centrifu-
gation and Et2O washings rendered aminoglycoside 14 as a white
solid, pure enough not to be further purified. Yield (from paromo-
mycin): 350 mg, 28%. TLC (CH2Cl2/MeOH, 8:2): baseline;[38] m.p.
Aminoglycoside I (200 mg, 0.134 mmol) was dissolved in 15 mL
of NH3 in CH3OH (aprox. 7 ), the solution was transferred into
a screw-thread vial and heated at 60 °C for 9 h. The solvent was
evaporated to obtain a solid residue, which was subsequently
1
141–142 °C. H NMR (D2O, 400 MHz):[40] δ = 5.81 (d, J = 4 Hz,
1 H, H1Ј), 5.40 (d, J = 2.4 Hz, 1 H, H1ЈЈ), 5.22 (d, J = 1.6 Hz, 1 H,
treated with 20 mL of 40% TFA in CH2Cl2 for 2 h at room tem- H1ЈЈЈ), 4.44 (t, J = 6.40 Hz, 1 H, H3ЈЈ), 4.37 (dd, J = 3.8, 2 Hz, 1
perature. Then, the solution was evaporated to near dryness, and a
solid was precipitated by addition of Et2O. Centrifugation and
Et2O washings rendered aminoglycoside 12 as a brownish solid,
pure enough not to be further purified; yield 68 mg, 80%. TLC
(CH2Cl2/MeOH, 8:2): baseline;[38] m.p. 172–177 °C. 1H NMR
(D2O, 400 MHz):[40] δ = 5.78 (d, J = 4.4 Hz, 1 H, H1Ј), 5.26 (d, J
= 2.4 Hz, 1 H, H1ЈЈ), 5.15 (d, J = 1.6 Hz, 1 H, H1ЈЈЈ), 4.38 (dd, J =
H, H2ЈЈ), 4.27 (t, J = 3.1 Hz, 1 H, H3ЈЈЈ), 4.22 (m, 1 H, H4ЈЈ), 4.15
(m, 1 H, H3Ј), 4.08 (s, 2 H, CH2N3), 3.89–4.03 (m, 7 H, H5, H5aЈЈ
,
H5ЈЈЈ, H6a, H6bЈ, H4 or H6), 3.68–3.76 (m, 5 H, H4Ј, H4ЈЈЈ, H4 or H6,
H5bЈЈ, H6aЈЈЈ), 3.57 (s, 1 H, H2ЈЈЈ), 3.44–3.52 (m, 4 H, H2Ј, H1 or H3,
H5Ј, H6bЈЈЈ), 3.31–3.40 (m, 2 H, H1 or H3), 2.49 (dt, J = 4, 12.4 Hz,
1 H, H2a), 1.84 (q, J = 12.8 Hz, 1 H, H2b) ppm. 13C NMR (D2O,
100 MHz): δ = 170.8, 110.1, 96.2, 95.7, 84.7, 81.6, 75.7, 73.9, 73.7,
6.5, 4.8 Hz, 1 H, H3ЈЈ), 4.25 (dd, J = 4.8, 2.3 Hz, 1 H, H2ЈЈ), 4.17 72.7, 72.7, 69.4, 69.0, 67.9, 66.5, 63.7, 60.5, 54.0, 52.1, 51.1, 50.0,
3106
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© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 3102–3109

Aminoglycoside–Dinucleotide and –diPNA Conjugates
49.0, 39.8 ppm. MALDI-TOF MS (positive mode) m/z: 699.6 [M
cresol and freeze-dried. Crude diPNAs could be readily used in the
following ligation step. Products were quantified by UV absorption
+ H]+ (calcd. for C25H47N8O15+, 699.7), 721.6 [M + Na]+ (calcd.
for C25H46N8O15Na+, 721.7), 737.5 [M
+
K]+ (calc for
at 260 nm, considering the calculated values of ε260 (ε260 =
C25H46N8O15K+, 737.8). ESI HRMS (positive mode) m/z: 699.3156
17200 –1 cm–1 for dithymine PNAs and ε260 = 30800 –1 cm–1 for
16b). Aliquots were purified by HPLC for characterization pur-
poses. Table 4 summarizes the characterization data for diPNAs.
+
[M
+
H]+ (calcd. for C25H47N8O15
,
699.3155; differential
–0.1 ppm).
5Ј-O-Hexynyl Dinucleotides (15a–15d): Dinucleotides were as-
sembled on a CPG resin using standard phosphite triester method-
ology (ABI 381 synthesizer). The phosphoramidite derivatives of
2Ј-deoxynucleosides (ABz, CBz, GiBu and T), the corresponding nu-
cleoside-controlled pore glass supports and reagents and solvents
for solid-phase oligonucleotide synthesis were from Glen Research.
O-Hexynyl phosphoramidite was prepared by reaction of 5-hexyn-
1-ol and 2-cyanoethoxy-bis(diisopropylamino)phosphane, and in-
corporated to the 5Ј-end of oligonucleotides by extending the cou-
pling time to 15 min. Oligonucleotides were deprotected and
cleaved from the resin by treatment with aq. conc. ammonia at
60 °C for 6 h, except for 15a, which was treated for 2 h at room
temperature. As HPLC analyses showed purities Ͼ75%, products
were readily used in the following ligation step. Products were
quantified by UV absorption at 260 nm by considering the experi-
mental ε260 values of the corresponding dinucleotides. The follow-
ing Table 3 summarizes the characterization data of the dinucleo-
tides that were synthesized.
Table 4. Characterization data of alkynyl diPNAs.
diPNA
MALDI-TOF[a]
HPLC[b] tR [min]
[M + H]+ m/z
16a
16b
17
17ac
18
18ac
19
19ac
674.1 (674.3)
693.5 (691.3)
730.4 (730.7)
772.7 (772.8)
730.9 (730.7)
772.9 (772.8)
730.8 (730.7)
772.8 (772.8)
13.7[b]
10.5[c]
10.2[b]
11.8[b]
10.3[b]
11.8[b]
10.3[b]
11.5[b]
[a] PNAs were characterized by MALDI-TOF mass spectrometry
(positive and reflector mode) in a Perseptive Biosystems Voyager
DE-RP instrument, by using THAP matrix. The corresponding
monoisotopic masses are shown in parenthesis. [b] Analysis condi-
tions: Linear gradient from 0 to 100% B in 30 min, where solvent
A was 0.045% TFA/water and solvent B was 0.036% TFA/CH3CN.
[c] Analysis conditions: linear gradient from 10 to 30% B in 30 min,
where solvent A was 0.045% TFA/water and solvent B was 0.036%
TFA/CH3CN.
Table 3. Characterization data of 5Ј-O-hexynyl dinucleotides.
Dinucleotides
MALDI-TOF[a]
HPLC[b] tR
[min]
ε260
Aminoglycoside–Dinucleotide and Aminoglycoside–diPNA Conju-
gates: In a Biotage microwave vial (a 0.5 mL vial for the 0.1 µmol
scale or a 5 mL vial for the 0.5–1 µmol scale), 1:1 volumes of
0.48 m CuSO4·5H2O (0.2 equiv.) and 12 m sodium ascorbate
[M – H]– m/z
15a
15b
15c
15d
704.2 (705.2)
723.1 (723.2)
674.3 (675.2)
754.2 (755.2)
16.5
13.3
12.2
11.7
16800
27400
14600
21600
Table 5. Characterization data of aminoglycoside conjugates.
[a] MALDI-TOF spectra were obtained in a Perseptive Biosystems
Voyager DE-RP instrument (negative and reflector mode) by using
THAP/ammonium citrate matrix. The corresponding calculated
monoisotopic masses are shown in parenthesis. [b] Oligonucleotides
were analyzed by reversed-phase HPLC, by using a linear gradient
of 0 to 30% B in 20 min, where solvent A was 0.05  triethylammo-
nium acetate and solvent B was CH3CN.
Conjugates
MALDI-TOF[a]
HPLC tR
[min]
[M + H]+ m/z
1a
1b
1c
1d
3a
3b
2a
2b
2c
2d
4a
4b
5
5ac
6
6ac
7
7ac
8
9
1347.1 (1346.5)
1364.8 (1364.5)
1316.7 (1316.5)
1397.3 (1396.5)
1314.7 (1313.6)
1333.3 (1331.6)
1348.1 (1347.5)
1365.4 (1365.5)
1318.1 (1317.5)
1397.7 (1397.5)
1315.5 (1314.6)
1331.5 (1330.6)
1370.3 (1370.4)
1411.8 (1413.4)
1370.3 (1370.4)
1411.9 (1413.4)
1370.4 (1370.4)
1412.6 (1413.4)
1374.7 (1372.6)
1471.1 (1471.7)
8.6[b]
8.2[b]
6.8[b]
7.3[b]
12.5[c]
12.9[c]
10.0[b]
9.3[b]
Alkynyl diPNAs (16a–16b, 17–19 and 17ac–19ac): diPNAs
(40 µmol) were manually assembled on a commercially available
Fmoc-XAL-polyethyleneglycol-polystyrene resin (Novabiochem)
following standard procedures. Fmoc/Bhoc monomers were pur-
chased from Applied Biosystems, with the exception of Nα-(2-
Fmoc-aminoethyl)-Nα-(3-pentynoyl)glycine which was prepared es-
sentially as described.[34] To prepare N-hexynyloxycarbonyl
diPNAs 16a–16b, after the removal of the Fmoc group (20% piper-
idine in DMF), the resin was treated with 10 equiv. of 5-hexyn-1-yl
p-nitrophenyl carbonate [freshly prepared by reaction of 5-hexyn-1-
ol with bis(p-nitrophenyl) carbonate in anhydrous CH2Cl2 and
DIEA, quantitative yield] and 10 equiv. of DIEA, in NMP at room
temperature for 2 h. With respect to diPNAs containing Nα-(2-ami-
noethyl)-Nα-(3-pentynoyl)glycine, the Fmoc group was removed to
produce free N-terminal amino diPNAs 17–19 or after the Fmoc
removal, the resin was subsequently acetylated by treatment with
Ac2O/DIEA in DMF in the case of diPNAs 17ac–19ac. To produce
cleavage and deprotection, the corresponding resin was washed and
treated with TFA/m-cresol, 19:1 for 10 min at room temperature,
solvents were removed by evaporation and coevaporation with an-
hydrous ether. The resulting residue was resuspended with aq. 0.1%
TFA and filtered to separate the resin beads. The aqueous solution
that contained the diPNA was washed with AcOEt to remove m-
6.4[b]
8.5[b]
14.3[c]
13.5[c]
14.3[c]
17.3[c]
14.7[c]
16.4[c]
14.0[c]
15.9[c]
20.9[c]
17.9[c]
[a] MALDI-TOF spectra were obtained in a Perseptive Biosystems
Voyager DE-RP instrument (reflector mode) by using THAP/am-
monium citrate for the analysis of oligonucleotide conjugates, or
THAP matrices for the analysis of PNA conjugates. The corre-
sponding calculated monoisotopic masses are shown in parenthesis.
[b] Analysis conditions: linear gradient of 0 to 30% B in 20 min,
where solvent A was 0.05  triethylammonium acetate and solvent
B was CH3CN. [c] Analysis conditions: linear gradient from 0 to
20% B in 30 min, where solvent A was 0.045% TFA/water and
solvent B was 0.036% TFA/CH3CN.
Eur. J. Org. Chem. 2010, 3102–3109
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3107

J. Robles et al.
FULL PAPER
(5 equiv.) were reacted under argon for 5 min to produce the CuI
catalyst. Then, 0.48 m TBTA in water/MeOH (0.2 equiv.), 1 m
hexynyl dinucleotides or diPNAs (1 equiv.) and 2 m azido-amino-
glycosides (2 equiv.) in water were added. The vial was purged with
argon, sealed and irradiated (Biotage InitiatorTM) in the conditions
mentioned in the main text (see Table 1 and Table 2) and by select-
ing the following additional parameters: constant temperature, non
pre-stirring, high absorption level and fixed hold time. During the
reaction, the irradiation power was 25–40 W and the pressure in-
creased up to 2–3 bar. The resulting crude was freeze-dried and
purified by either HPLC or medium pressure chromatography, by
employing linear gradients of 0.05  triethylammonium acetate
(pH 7.0) and CH3CN, or 0.045% TFA/water and 0.036% TFA/
CH3CN. Conjugates were quantified by UV absorption at 260 nm,
by considering the corresponding ε260 values of the unmodified di-
nucleotides and diPNAs, and characterized by mass spectrometry.
Table 5 summarizes the characterization data of the conjugates de-
scribed in this study.
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Supporting Information (see also the footnote on the first page of
this article): HPLC profiles of the experiments of Table 1, NMR
spectra of aminoglycosides 12 and 14, and HPLC and mass spectra
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Acknowledgments
[21]
[22]
This work was supported by funds from the Spanish Ministerio de
Educación y Ciencia (MEC) (CTQ2007-68014-C02-01/BQU) and
the Generalitat de Catalunya (2005SGR00693). J. A. and A. C.
acknowledge predoctoral fellowships from the University of Barce-
lona (BRD and IBUB programs). The authors also wish to
acknowledge the Mass Spectrometry (Dr. Irene Fernández and
Laura Ortiz) and NMR facilities (Drs. Ma. Antònia Molins and
Francisco Cárdenas) of the Scientific-Technical Services of the Uni-
versity of Barcelona for technical support, and Ana Soriano for
her help with the preparation of bis(p-nitrophenyl) carbonate and
its use in the synthesis of alkynyl diPNAs.
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© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 3102–3109

Aminoglycoside–Dinucleotide and –diPNA Conjugates
Köster, N. Metzler-Nolte, Bioconjugate Chem. 2009, 20, 1578– [39] We could not identify the signal of CH2N3 protons in the spec-
1586.
trum of II, because of extensive signal overlapping in the region
in which it was expected (δ ≈ 3.5–4.0 ppm). However, CH2N3
signal was readily identified as a singlet at δ = 4.08 ppm in the
spectrum of aminoglycoside 14, obtained by deprotection of II.
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experiment and comparison with the spectra of other amino-
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127, 8600–8601.
[38] We assayed other TLC solvents to obtain a more convenient
Rf value for aminoglycosides 12 and 14, but we did not succeed.
We observed that for these and other aminoglycoside deriva-
tives synthesized in our group, MALDI-TOF was useful for
crude analyses and purity estimation.
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Received: February 10, 2010
Published Online: April 22, 2010
Eur. J. Org. Chem. 2010, 3102–3109
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3109