Synthesis of aminoglycoside-3′-conjugates of 2′-O-methyl oligoribonucleotides and their invasion to a 19F labeled HIV-1 TAR model

Article abstract of DOI:10.1021/bc200101r

The potential of aminoglycosides to induce RNA-invasion has been demonstrated. For this purpose, aminoglycoside-3′-conjugates of 2′-O-methyl oligoribonucleotides have been synthesized entirely on a solid phase. The synthesis includes an automated oligonucleotide chain elongation to solid-supported neomycin, ribostamycin, and methyl neobiosamine, and a two-step deprotection/release of the solid-supported conjugate, which allows exploitation of a simple protecting group scheme. Conjugates have been targeted to a ¹⁹F labeled HIV-1 TAR RNA model (Trans Activation Response element of HIV), which allows monitoring of the invasion by ¹⁹F NMR spectroscopy. A remarkably enhanced invasion, compared to that resulting from the corresponding unmodified 2′-O-methyl oligoribonucleotide (5′-CAGGCUCA-3′), has been obtained by the neomycin conjugate. The increased affinity results from a cooperative binding of the neomycin moiety and hybridization, though the invasion may also follow a mechanism, in which the first molar equivalent of the conjugate induces hybridization of the second.

Full text of DOI:10.1021/bc200101r

ARTICLE
pubs.acs.org/bc
Synthesis of Aminoglycoside-30-Conjugates of 20-O-Methyl
Oligoribonucleotides and Their Invasion to a 19F labeled HIV-1 TAR
Model
Anu Kiviniemi and Pasi Virta*
Department of Chemistry, University of Turku, FIN-20014 Turku, Finland
S Supporting Information
b
ABSTRACT: The potential of aminoglycosides to induce RNA-invasion has been demon-
strated. For this purpose, aminoglycoside-30-conjugates of 20-O-methyl oligoribonucleo-
tides have been synthesized entirely on a solid phase. The synthesis includes an automated
oligonucleotide chain elongation to solid-supported neomycin, ribostamycin, and methyl
neobiosamine, and a two-step deprotection/release of the solid-supported conjugate, which
allows exploitation of a simple protecting group scheme. Conjugates have been targeted to a
19F labeled HIV-1 TAR RNA model (Trans Activation Response element of HIV), which allows monitoring of the invasion by 19
NMR spectroscopy. A remarkably enhanced invasion, compared to that resulting from the corresponding unmodified 20-O-methyl
oligoribonucleotide (50-CAGGCUCA-30), has been obtained by the neomycin conjugate. The increased affinity results from a
cooperative binding of the neomycin moiety and hybridization, though the invasion may also follow a mechanism, in which the first
molar equivalent of the conjugate induces hybridization of the second.
F
’ INTRODUCTION
design to shorter oligonucleotide sequences. Improvements
considering the cellular uptake may additionally be provided
since neomycin has been shown to assist the lipid-mediated
delivery of oligonucleotides.29 Pioneering work for the ami-
noglycoside-induced antisense oligonucleotides has already
been done. Neomycin has been covalently attached into an
oligodeoxyribonucleotide, complementary to a seven-bases-
long R-sarcin loop RNA sequence, and its ability to enhance
duplex formation has been studied.30 A 14-mer PNA-neamine
conjugate targeted to HIV-1 TAR has displayed anti-HIV-1
activity and sequence-specific metal-ion-independent cleavage
of the target RNA.31,32 A set of neamine- and ribostamycin-20-
O-methyl oligoribonucleotide conjugates has been prepared
in order to study their nuclease activity,33 and DNA-
duplex stabilization has been obtained by 40-C-conjugated
paromamine.34,35 It is worth noting that conjugation of nea-
mine with dinucleotides has been reported to remarkably
decrease affinity to HIV-1 TAR RNA, due to electrostatic
repulsion between the phosphates, whereas conjugates of the
corresponding PNA show about 2-fold binding affinities com-
pared to that of neamine.36
Aminoglycosides are antibiotics, which interact with a variety
of RNA targets. Binding to the ribosomal decoding site
(Aꢀsite) is the basis of their bactericidal effect,1ꢀ5 and com-
parable affinities to other RNA targets, e.g., to several
ribozymes6ꢀ16 and to important RNA motifs of HIV (TAR,
RRE and DIS),17ꢀ20 show their potential as more general
RNA-targeting drugs. Aminoglycosides are also able to stabi-
lize DNA- and RNA-triplexes, DNA-RNA hybrid triplexes, and
hybrid duplexes.21ꢀ23 The recognition of a certain RNA target
is usually based on a bulge or an internal loop, in which the
canonical base-pairing is disrupted by few mismatches.24 As an
example, the site-specific binding of neomycin to the HIV-1
TAR region occurs below the UCU bulge through the minor
groove.25 Although the nonionic interactions may play an
important role in the recognition, aminoglycosides’ affinity
to RNA mainly originates from electrostatic interaction of their
protonated amino groups with phosphodiester linkages. The
binding, hence, is promiscuous, which is the main bottleneck of
aminoglycoside-based RNA-targeting.24 In principle, the pro-
miscuity may be overcome by appropriate structural modifica-
tions, but conformational adaption of an RNA target upon
complex formation makes development of specific ligands
extremely complex.26 One option to increase the specificity
is the conjugation of aminoglycosides to other recognition
moieties. By conjugation to oligonucleotides or PNA, the
aminoglycoside’s binding may be cooperatively directed by
hybridization, and the specificity may be expectedly improved.
In view of the antisense approach,27,28 such conjugates are
attractive, since the binding of the covalently attached amino-
glycoside may enhance hybridization and direct the drug
HIV-1 TAR RNA is a potential target for antisense oligonu-
cleotides since it occurs at the 50-end of all HIV-1 RNA tran-
scripts and is regulated by the cognate protein Tat during
transcriptional elongation.37ꢀ40 The target region in HIV-1
TAR is a relatively short but chemically interesting RNA-
construct, which consists of a trinucleotide UCU bulge and a
Received: February 24, 2011
Revised:
June 14, 2011
Published: June 21, 2011
r
2011 American Chemical Society
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|

Bioconjugate Chemistry
ARTICLE
Scheme 1. Synthesis of Conjugates 10ꢀ16a
a Reagents and conditions: (i) TfaOMe, MeOH, Et3N; (ii) DMTrCl, Py, overall yields (from 1, 4 and 7), 3, 73%; 6, 59%; and 9, 51%; (iii)
succinanhydride, DMAP, pyridine; (iv) LCAA-CPG, HATU, DIEA, DMF; (v) benzylamine, HATU, DIEA, DMF; (vi) acetic anhydride, lutidine,
1-methyl imidazole, THF; (vii) standard automated RNA synthesis; (ix) 0.1 mol Lꢀ1 NaOMe in MeOH; (x) ammonolysis; (xi) RP HPLC conditions, a
gradient elution from 10% to 40% acetonitrile in 0.1 mol Lꢀ1 aq. Et3NH+AcOꢀ over 25 min, Thermo ODS Hypersil column (250 ꢁ 4.6 mm, 5μm) at a
flow rate of 1.0 mL minꢀ1
.
Scheme 2. Invasions of 8-Mer 20-O-Methyl Oligoribonucleotide (17) and Its Neomycin-30-conjugate (10) to the 19F Labeled HIV-
1 TAR RNA Model (A)
hairpin loop linked together by a tetranucleotide stem region.
The essential elements for HIV-1 TAR targeting may, however,
be applied more generally to oligonucleotide-based drug design
since related RNA-constructs exist in many biologically relevant
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ARTICLE
Figure 1. (a) 19F NMR spectra of A (20 μmol Lꢀ1 in 25 mmol Lꢀ1 NaH2PO4 buffered H2OꢀD2O (9:1, v/v, pH 6.5) in the presence of increasing
concentrations of 10. 5-[4,4,4,-Trifluoro-3,3,-bis(trifluoromethyl)but-ynyl]-20-deoxyuridine (10 μmol Lꢀ1) was used as an internal standard (std). (b)
Titration curves obtained by integrals of 19F resonances in different concentrations of 10 (b) and 17 (O), {[Cn] = [Atot] ꢁ a relative 19F peak area
Cn/(Cn + A). (c) 19F shift versus temperature profiles. A, B, C17, D17, and C/D10; cf. Schemes 1 and 2.
RNAs. Among them are microRNAs, which recently have been
reassessed as potential of antisense targets.41 We have recently
shown that neomycin induces invasion of a 20-O-methyl oligor-
ibonucleotide to a HIV-1 TAR model.42 In the present study,
neomycin, ribostamycin, and methyl neobiosamine have been
covalently attached to the 30-terminus of 20-O-methyl oligori-
bonucleotides (10ꢀ16), and influence of the conjugated
aminoglycosides on RNA invasion has been studied. As done
previously, a 19F labeled HIV-1 TAR RNA model A (Scheme 2)
has been used as a target, which allows monitoring of the
secondary structural arrangement upon invasion by 19F NMR
spectroscopy. For the preparation of the conjugates (10ꢀ16), a
straightforward procedure was applied (Scheme 1). Partially
protected aminoglycosides (3, 6, and 9), bearing the dimethox-
itrityl protected primary hydroxyl group and trifluoroacetyl
protected amino groups, were covalently attached to a LCAA
CPG-support via succinyl linker. After capping of the remaining
reactive functionalities, 20-O-methyl oligoribonucleotide se-
quences were automatically elongated to these solid-supported
aminoglycosides. A two-step procedure (1, NaOMe/MeOH; 2,
ammonolysis) was used for the deprotection/release, to avoid
OfN-acyl migration on the aminoglycoside moieties. A rela-
tively simple protecting group scheme may, hence, be utilized.
For Kd determinations, a mixture of the 19F labeled HIV-1 TAR
model was titrated by these conjugates. According to 19F NMR
spectroscopic monitoring (Figure 1a), the neomycin moiety
remarkably induces invasion of 20-O-methyl oligoribonucleotides
(Figure 1b). The right side (rings I, II, and III, i.e., ribostamycin)
of the neomycin moiety also resulted in a slight enhancement,
but influence of the left side (rings III and IV, i.e., biosamine)
remained marginal (Table 2). The enhanced invasion was
attributed to a formation of a stabilized macro-looped complex,
by concurrent hybridization and binding of the neomycin moiety
(Scheme 2 and Figure 1c). Interestingly, if the neomycin moiety
is not appropriately available, the invasion may also follow a
mechanism, in which the first molar equivalent of the conjugate
induces hybridization of the second.
’ EXPERIMENTAL PROCEDURES
General Remarks. The NMR spectra of 3, 6, and 9 were
recorded at 500 MHz. The chemical shifts are given in ppm from
internal TMS and coupling constants in hertz. Peak assignments
were supported by COSY and HSQC. The mass spectra of 3, 6,
and 9 and oligonucleotide conjugates 10ꢀ16 were recorded by
ESI ionization. Oligonucleotides 10ꢀ16 were assembled on an
Applied Biosystems 392 DNA synthesizer using commercially
available 20-O-methyl ribonucleoside phosphoramidites. Stan-
dard RNA coupling protocol was followed. The 19F labeled TAR
model (A) was prepared as previously described.42 A Thermo
Hypersil ODS column (250 ꢁ 4.6 mm, 5 μm) at a flow rate of
1 mL minꢀ1 was used for the RP HPLC purification of the
conjugates (10ꢀ16). A gradient elution from 10% to 40%
acetonitrile in 0.1 mol Lꢀ1 Et3NH+AcOꢀ-buffer over 25 min
was used.
19F NMR Spectroscopy. Oligonucleotides (triethylammonium
salts) were lyophilized to dryness and dissolved in 25 mmol Lꢀ1
NaH2PO4-buffer (pH 6.5) in D2OꢀH2O (1:9 v/v). For the titration
experiments, conjugates were added as 1 mmol Lꢀ1 solutions to a
20 μmol Lꢀ1 solution of 19F labeled HIV-1 TAR model (A). Prior to
each spectroscopic detection, the mixtures were heated to 90 °C for
1 min and then allowed to cool down to room temperature. Spectra
were recorded at a frequency of 376.1 MHz on a Bruker Avance
400 MHz spectrometer. Typical experimental parameters were
chosen as follows: 19F excitation pulse, 12.6 μs; acquisition time,
0.675 s; relaxation delay, 1.0 s; and usual number of scans, 2400 or
4800. 19F resonances were referenced relative to external CCl3F.
Sample temperature for the 19F shift versus temperature profiles
(cf. Figure 1c) was calibrated by using known shifts of ethylene glycol
at different temperatures. Kd values were calculated from relative peak
areas of 19F resonance signals of the free HIV-1 TAR model (A) and
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Bioconjugate Chemistry
ARTICLE
of the complexes (C) according to the following equations: Kd[C] =
[A][ON], [A] + [C] = [Atot], [ON] = [ONtot]-[C] f [C]2 ꢀ
([Atot] + [ONtot] + Kd)[C] + [ONtot][Atot] = 0, where [C] =
and 4.2 Hz), 1.75 (ddd, 1H, J = 12.8 Hz each). 13C NMR
(CD3OD, 125 MHz) δ: 158.7, 145.0, 136.0, 135.8, 130.02,
129.95, 128.0, 127.4, 126.3, 112.7, 109.2, 96.6, 86.7, 83.2, 76.4,
75.5, 72.9, 72.1, 70.9, 70.2, 70.1, 63.1, 54.3, 53.8, 49.7, 49.2, 40.5,
31.5 (Tfa related peaks not listed). HRMS(ESI): found
1163.2786, [M + Na]+ requires 1163.2771 (error 1.3 ppm).
Methyl-20,60-Bis-N-trifluoroacetyl Neobiosamine B (9).
Compound 9 was synthesized from 7 (as free amine, β:R =
0.77:0.23, 0.63 g, 1.9 mmol) as described for the neomycin
derivative 3 from 1 above, but 2 mol equiv of DMTrCl was used.
After silica gel chromatography (5% MeOH and 0.2% Et3N in
CH2Cl2), 0.63 g (51%) of 8 (pure β isomer) was obtained as
white foam. 1H NMR (CD3OD, 500 MHz) δ: 7.60 (m, 2H), 7.47
(m, 4H), 7.28 (m, 2H), 7.20 (m, 1H), 6.87 (m, 4H), 5.36 (b, 1H),
4.36 (d, 1H, J = 7.3 Hz), 4.34 (b, 1H), 4.27 (b, 1H), 4.20 (dd, 1H,
J = 6.8 and 6.7 Hz), 4.07 (b, 1H), 3.77 (s, 6H), 3.71 (dd, 1H, J =
12.8 and 8.8 Hz), 3.62 (b, 1H), 3.54 (b, 1H), 3.52 (dd, J = 12.8
and 4.9 Hz), 3.36 (s, 3H), 3.32 (b, 1H), 3.18 (m, 2H), 2.33 (b,
1H). 13C NMR (CD3OD, 125 MHz) δ: 157.4, 144.4, 135.3,
135.0, 128.7, 127.0, 125.9, 124.9, 111.3, 111.1, 100.2, 96.3, 85.4,
75.3, 69.9, 69.6, 67.8, 67.5, 65.6, 60.7, 54.1, 52.8, 50.1 (Tfa related
peaks not listed). HRMS(ESI): found 842.2415, [M + Na]+
requires 841.2383 (error 3.8 ppm).
Synthesis of the Conjugates 10ꢀ14. Succinanhydride (3.4
mg, 33 μmol) and a catalytic amount of DMAP were added to a
mixture of 3, 6, or 9 (33 μmol, each) in dry pyridine (0.50 mL).
The mixtures were stirred overnight at ambient temperature and
evaporated to dryness. (According to MS(ESI), the crude
product mixtures also contained multiply succinated side pro-
ducts in addition to monosuccinates.) The residues were dis-
solved in DMF (1.0 mL), and HATU (25 mg, 67 μmol), DIEA
(23 μL, 130 μmol), and LCAA-CPG support (100 mg) were added
to the mixtures. After overnight shaking, the suspensions were
filtered, and the supports were washed with DMF and CH2Cl2 and
dried under vacuum. The potential unreacted carboxylic acid
groups on the supports were capped by amide bond formation.
Accordingly, the supports were suspended in DMF (1.0 mL), and
benzylamine, HATU, and DIEA were added to the suspensions.
After overnight shaking, the supports were filtered, washed with
DMF and CH2Cl2, and dried under vacuum. The unreacted
hydroxyl groups of the solid supported aminoglycosides and the
unreacted amino groups of the initial support were then acetylated
by double syringe method using a mixture of acetic anhydride,
lutidine, and N-methyl imidazol in THF (5:5:8:82, v/v/v/v, for
15 min at 25 °C). After acetylation, the resins were washed with
THF and CH2Cl2 and dried under vacuum. A small aliquot of
each resin was analyzed by the DMTr-cation assay. Loadings of
17 μmol gꢀ1 (neomycin resin), 14 μmol gꢀ1 (ribostamycin
resin), and 19 μmol gꢀ1 (methyl neobiosamine resin) were
obtained. The appropriate oligonucleotide chains were then
assembled (0.5 μmol scale) by standard automated RNA proto-
col. After chain assembly, supports were treated with 0.1 M NaOMe
in MeOH (1.0 mL, for 2 h at 25 °C) and filtered. One molar
aqueous ammonium chloride (100 μL) was added to the filtrates,
and the mixtures were evaporated to dryness. Supports and the
filtrates were combined and treated with saturated ammonia for
15 h at 55 °C. Finally, the mixtures were filtered, evaporated to
dryness, dissolved in water, and purified by RP HPLC [a gradient
elution from 10% to 40% acetonitrile in 0.1 mol Lꢀ1 aq.
Et3NH+AcOꢀ over 25 min, Thermo ODS Hypersil column
(250 ꢁ 4.6 mm, 5 μm) at a flow rate of 1.0 mL minꢀ1; an
example of the RP HPLC chromatogram is shown in Scheme 1].
concentration of the invasion complex = X[Atot], X = a relative 19
F
signal peak area (cf. Figure 1a) of the invasion complex, [Atot] = the
total concentration of A, [A] = concentration of free A, [ONtot] =
total concentration of the conjugate, and [ON] = concentration
of the free conjugate. Titrations were additionally performed in
the presence of the 5-[4,4,4,-trifluoro-3,3-bis(trifluoromethyl)but-1-
ynyl]-20-deoxyuridine (10 μmol Lꢀ1) standard,43 to which the peak
areas were referenced.
Thermal UV Denaturation Studies. Absorbance versus tem-
perature profiles (Supporting Information) were recorded at
260 nm on a Perkin-Elmer Lambda 35 UVꢀvis spectrometer
equipped with a multiple cell holder and a Peltier temperature-
controller. The temperature was changed at a rate of 0.5 °C/min
(from 20 to 90 °C). The measurements were performed in
25 mM NaH2PO4 buffer (pH 6.5). Tm values were determined as
the maximum of the first derivate of the melting curve.
50-O-(4,40-Dimethoxytrityl)-1,3,20,60,2000,6000-hexa-N-trifluoro-
acetylneomycin (3). Triethylamine (3.6 mL, 26 mmol) and
methyl trifluoroacetate (2.6 mL, 26 mmol) were added to
neomycin trisulfate (7, 2.0 g, 2.2 mmol) in methanol (10 mL).
The mixture was stirred overnight at room temperature and
evaporated to dryness. Saturated NaHCO3 was added to the
residue, and the crude product (2) was extracted with ethyl
acetate. The organic layers were combined, dried over Na2SO4,
filtered, and evaporated to dryness. The residue was further dried
by coevaporation with dry pyridine and dissolved in dry pyridine,
and then 4,40-dimetoxytrityl chloride (0.80 g, 2.4 mmol) was
added to the mixture. After overnight reaction, saturated NaH-
CO3 was added, and the product was extracted with ethyl acetate.
The organic layers were combined, dried over anhydrous Na2SO4,
filtered, and evaporated to dryness. The residue was purified by
silica gel chromatography (10% MeOH, 1% Et3N in CH2Cl2) to
yield 2.35 g (73%) of the product (3) as white foam. 1H NMR
(CD3OD, 500 MHz) δ: 7.46 (m, 2H), 7.36ꢀ7.30 (m, 6H), 7.21
(m, 1H), 6.90 (m, 4H), 5.78 (d, 1H, J = 3.3 Hz), 5.16 (d, 1H, J =
4.6 Hz), 5.09 (d, 1H, J = 1.5 Hz), 4.29 (dd, 1H, J = 4.8 Hz, both),
4.21ꢀ4.20 (m, 2H), 4.14 (m, 1H), 4.11 (dd, 1H, J = 6.7 Hz,
both), 4.01 (m, 1H), 3.97 (m, 1H), 3.92 (m, 1H), 3.87 (dd, 1H,
J = 9.9 and 8.8 Hz), 3.80 (s, 6H), 3.75ꢀ3.63 (m, 5H), 3.60 (dd,
1H, J = 13.5 and 7.4 Hz), 3.56ꢀ3.51 (m, 2H), 3.35ꢀ3.31 (m,
3H), 3.19 (dd, 1H, J = 10.5 and 3.1 Hz), 2.00 (ddd, 1H, J = 12.9
Hz, 4.2 and 4.2 Hz), 1.77 (ddd, 1H, J = 12.7 Hz, each). 13C NMR
(CD3OD, 125 MHz) δ: 157.93, 157.85, 145.0, 136.0, 135.6,
130.2, 129.9, 128.0, 127.4, 126.3, 112.7, 109.0, 97.6, 96.3, 87.4,
86.3, 81.9, 76.9, 75.6, 74.8, 73.0, 72.1, 71.5, 70.3, 69.8, 69.2, 67.1,
62.5, 54.3, 53.5, 51.3, 49.6, 49.1, 40.4, 39.6, 31.5 (Tfa related
peaks not listed). HRMS(ESI): found 1515.3239, [M + Na]+
requires 1515.3265 (error 1.7 ppm).
50-O-(4,40-Dimethoxytrityl)-1,3,20,60-tetra-N-trifluoroace-
tylribostamycin (6). Compound 6 was synthesized from 4 (as a
disulfate salt, 0.25 g, 0.38 mmol) as described for the neomycin
derivative 3 from 1 above. After silica gel chromatography (5%
MeOH and 0.2% Et3N in CH2Cl2) 0.26 g (59%) of 6 was
obtained as white foam. 1H NMR (CD3OD, 500 MHz) δ: 7.46
(m, 2H), 7.34 (m, 4H), 7.29 (m, 2H), 7.20 (m, 1H), 6.86 (m,
4H), 5.67 (d, 1H, J = 2.7 Hz), 5.22 (d, 1H, J = 4.0 Hz), 4.18ꢀ4.10
(m, 3H), 4.02ꢀ3,97 (m, 3H), 3.92 (m, 1H), 3.81 (m, 1H), 3.78
(s, 6H), 3.76ꢀ3.64 (m, 4H), 3.60 (m, 2H), 3.30ꢀ3.25 (m, 2H),
3.23 (dd, 1H, J = 10.2 and 3.8 Hz), 2.01 (ddd, 1H, J = 12.9 Hz, 4.2
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Table 1
Table 2.a
conjugate
observed mass
calculated mass
oligonucleotide
Kd (μmol Lꢀ1
)
10
11
12
13
14
15
16
1646.9a
909.0b
1666.9c
1136.3a
816.7a
1566.9a
1501.8a
1646.4
909.0
10
0.59 ( 0.16
4.9 ( 1.5
11
1666.4
1136.3
816.7
12
0.32 ( 0.22
37.0 ( 2.0
3.8 ( 0.7
13
15
1566.8
1501.8
16
25.0 ( 0.4
18.5 ( 1.1
3.6 ( 0.8
17
a [M ꢀ 2H]2ꢀ. b [M ꢀ 4H]4ꢀ. c [M + K ꢀ 3H]2.
17 + 14 (1:1, n/n)
a Each Kd is simplified to stoichiometric (1:1) complexes.
The isolated products were desalted and lyophilized to dryness.
Authenticity of the isolated products 10ꢀ16 was verified by MS
(ESI) spectroscopy (Table 1).
for comparative evaluation of the affinities of different invader
oligonucleotides. A distinct 19F resonance signal from that
referring to the initial hairpin structure may be obtained, when
a structural change, such as invasion (C/D) or thermal denatura-
tion (B), takes place. Additionally, temperature-dependent
behavior of the complexes may be followed by 19F shift vs
temperature profiles since the potential macro-looped (C) and
the open-chain invasion complex (D) may, in a favorable case,
result in 19F signals with different resonance shifts (Figure 1c).
Note that there is a linear temperature-dependent 19F shift
downfield with the slope 0.014 ppm Kꢀ1. Deviations from this
passive shift refer to structural changes in RNA. In the present
study, model A was titrated with conjugates 10ꢀ13, (14 + 17),
15, and 16, and their invasion efficiency was evaluated by 19F
NMR spectroscopy. We recently showed that invasion of 20-O-
methyl oligoribonucleotide 17 (Scheme 2) to A may be induced
by the presence of neomycin (as a discrete molecule).42 There-
fore, neomycin, when covalently attached to the same 20-O-
methyl oligoribonucleotide (10), may be expected to result in a
cooperative effect, in which the complex is stabilized by con-
current hybridization and binding of the neomycin moiety. The
site-specific binding of neomycin occurs below the bulged
nucleotides of the native HIV-1 TAR region through the minor
groove.25 Stabilization of the complex should, hence, be based on
the formation of the macro-looped complex (C10) since the
open-chain invasion complex (D10) contains only the canonical
double strand with a modest affinity to aminoglycosides. 19F
NMR spectra during the titration of A with 10 is shown in
Figure 1a, and the titration curve recording the relative 19F peak
areas is shown in Figure 1b. As seen from Figure 1b, conjugate 10
provides a significantly enhanced invasion compared to that
obtained by unmodified 20-O-methyl oligoribonucleotide 17.
Fitting the titration curve to the stoichiometric (1:1) complex
formation, gave Kd = 0.59 ( 0.16 μmol Lꢀ1 with 10, whereas
Kd = 18.5 ( 1.1 μmol Lꢀ1 was obtained with 17. An interesting
observation was additionally done, when the mixture of the
complex C/D10 was heated. The 19F shift vs temperature profile
of the complex (C/D10) followed the prolonged downfield curve
of the complex (C/D17) with unmodified 20-O-methyl oligor-
ibonucleotide 17 (Figure 1c). We have previously concluded that
the downfield curve (r in Figure 1c) refers to the macrolooped
complex (C17) and the upfield curve (Δ in Figure 1c) to the
open-chain invasion complex (D17).42 The prolonged downfield
curve (b) refers to neomycin binding and is consistent with the
expectation that the enhanced invasion is related to the stabilized
macro-looped complex (C10).
’ RESULTS AND DISCUSSION
Synthesis of the Aminoglycoside 30-Conjugates of 20-O-
Methyl Oligoribonucleotides (10ꢀ16). Synthesis of the con-
jugates (10ꢀ16) is described in Scheme 1. The protecting group
scheme was simplified by using a one-pot anchoring/protection
procedure of partially protected aminoglycosides (3, 6, and 9).
Neomycin (1), ribostamycin (4), and methyl neobiosamine (7)
were N-trifluoroacetylated (2, 5, and 8), and the primary
hydroxyl group was selectively protected by the 4,40-dimethox-
ytrityl group. Compounds (3, 6, or 9) were then treated with one
molar equivalent of succinanhydride (iii), the crude products
were attached to the LCAA-CPG support (iv), and the potential
free carboxyl and hydroxyl groups on the supports were capped by
amide bond formation (v) and acetylation (vi), respectively. An
automated oligonucleotide chain elongation (vii) was success-
fully performed on the obtained aminoglycoside supports. In order
to avoid potential OfN-acyl migration on the aminoglycoside
moieties, a two-step deprotection-procedure was performed.
The solid-supported conjugates were subjected to a mixture of
NaOMe in methanol (ix, ester cleavage), and then the deprotec-
tion was continued by ammonolysis (x, amide cleavage). The
sodium methoxide pretreatment favors transesterification, in
which the trifluoroacetamides stay intact. According to RP
HPLC chromatograms (an example is shown in Scheme 1)
and MS (ESI) spectra (Table 1), products were successfully
obtained without OfN-acyl migration.
Invasion of the Conjugates to the 19F Labeled TAR Model
(A). 19F is biologically nonexistent and has an NMR-sensitive
nucleus (83% compared to 1H), which additionally offers wide
chemical shift dispersion. These properties make the 19F nucleus
a useful probe for the monitoring of conformational transitions in
RNA.42ꢀ51 Closely related to this work, 20-deoxy-20-fluoronu-
cleosides incorporated in RNA have been used for the monitor-
ing RNA-aminoglycoside interactions,47 and 2,4-difluorotoluene
nucleosides have been used for the quantification of hairpin/
hairpin and duplex/hairpin equilibria of self-complementary
RNAs.49 We have recently demonstrated the applicability of
19F NMR spectroscopy to the monitoring of RNA invasion, by
studying the binding of 20-O-methyl oligoribonucleotides to the
19F labeled HIV-1 TAR RNA model A.42 The 5-[4,4,4-trifluoro-
3,3-bis(trifluoromethyl)but-1-ynyl]-20-deoxyuridine sensor43
within the model decreases the stability of the four nucleotide
stem between the loop and the UCU-bulge (ΔTm = ꢀ2.7 °C),
slightly facilitating the invasion. The model (A) is, hence, useful
Invasion by the other conjugates was similarly studied (see
titration curves in the Supporting Information), and the Kd
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ARTICLE
values for each complex are listed in Table 2. Comparing Kd of
or a slightly decreased Tm = 59.2ꢀ60.6 °C was observed with
C
11 (4.9 ( 1.5 μmol Lꢀ1) to that of C10 (0.59 ( 0.16 μmol Lꢀ1
)
other conjugates.
shows that shifting of the 20-O-methyl oligoribonucleotide
sequence by one nucleotide (C at 50-end deleted and G at 30-
end added) significantly decreases the affinity. More interest-
ingly, the titration curve obtained by C11 hardly follows 1:1-
complex formation, but the saturation is deviated to a 2:1-
stoichiometry. An explanation to this phenomenon is that the
above-mentioned cooperative effect could not be gained with 11
but that the first molar equivalent of the conjugate may induce
invasion of the second. In order to evaluate the efficiency of this
ternary mechanism, titration was additionally performed with
a 1:1-mixture of unmodified octamer 17 and neomycin conju-
gate 14, which with the short CAG-strand cannot alone invade A
(cf. 13 below). Conjugate 14, however, does induce the invasion
of 17 of A, for which Kd = 3.6 ( 0.8 μmol Lꢀ1 (simplified to 1:1-
complex formation) may be determined. This verifies that
invasion with conjugate 11 may be induced in a ternary manner.
The ternary induction may partly take place also with 10 (or with
other conjugates as well), but the titration profile may be more
overlapped by that of the stoichiometric (1:1) complex forma-
tion. In order to avoid potential structural constraints for the
cooperative recognition, conjugate 12, bearing a flexible poly-
ethylene glycol spacer between the neomycin moiety and 20-O-
methyl oligoribonucleotide, was targeted to A. As expected, the
more flexible spacer facilitates cooperativity, and a higher affinity
(Kd = 0.32 ( 0.22 μmol Lꢀ1) compared to that of 10 was
obtained. Noteworthy, this strongly stoichiometric complex
formation approach's limitations can be reliably detected by
the adopted method (based on integrals of 19F resonance
signals). Importance of the left (biosamine) and right
(ribostamycin) sides of the neomycin moiety may be seen in
Kd values of C16 and C17. The affinities were expectedly weaker
than that of 10 (C10: Kd = 0.59 ( 0.16 μmol Lꢀ1), and the
ribostamycin moiety still promotes invasion (C13: Kd = 3.8 ( 0.7
μmol Lꢀ1), but influence of the biosamine moiety (C14: Kd =
’ CONCLUSIONS
Aminoglycoside (neomycin, ribostamycin, and methyl
neobiosamine) 30-conjugates of 20-O-methyl oligoribonucleo-
tides have been synthesized entirely on a solid phase, and their
invasion efficiency to a 19F labeled HIV-1 TAR model has been
evaluated by 19F NMR spectroscopy. In addition to the
determined Kd-values, 19F shift versus temperature profiles
of the complexes have been measured. A significantly en-
hanced invasion in the presence of the neomycin moiety
(conjugates 10 and 12) has been obtained. Enhancement
has been concluded to be involved in the stabilized macro-
looped complex, which allows concurrent hybridization and
binding of the neomycin moiety. Aminoglycoside-promoted
RNA-invasion has hence been verified, which may give new
ideas for the oligonucleotide-based RNA targeting.
’ ASSOCIATED CONTENT
Supporting Information. H NMR and 13C NMR spectra,
1
S
b
RP HPLC chromatograms, titration curves, 19F shift vs the
temperature profile, and UV-melting curves. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Fax: +35823336700. E-mail: pamavi@utu.fi.
’ ACKNOWLEDGMENT
The financial support from the Academy of Finland is grate-
fully acknowledged.
25 ( 0.4 μmol Lꢀ1) to that of 17 (C17: Kd = 18.5 ( 1.1 μmol Lꢀ1
)
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