Probing the activation site of ribonuclease L with new N6-substituted 2′,5′-adenylate trimers

Article abstract of DOI:10.1016/S0968-0896(03)00060-9

2-5A trimer [5′-monophosphoryladenylyl(2′-5′)adenylyl (2′-5′)adenosine] activates RNase L. While the 5′-terminal and 2′-terminal adenosine N⁶-amino groups play a key role in binding to and activation of RNase L, the exocyclic amino function of the second adenylate (from the 5′-terminus) plays a relatively minor role in 2-5A's biological activity. To probe the available space proximal to the amino function of the central adenylate of 2-5A trimer during binding to RNase L, a variety of substituents were placed at that position. To accomplish this, the convertible building block 5′-O-dimethoxytrityl-3′-O-(tert-butyldimethylsilyl)-6-(2,4- dinitrophenyl)thioinosine 2′-(2-cyanoethylN,N-diisopropylphosphoramidite) was prepared as a synthon to introduce 6-(2,4-dinitrophenyl)thioinosine into the middle position of the 2-5A trimer during automated synthesis. Post-synthetic treatment with aqueous amines transformed the (2,4-dinitrophenyl)thioinosine into N⁶-substituted adenosines. Assays of these modified trimers for their ability to bind and activate RNase L showed that activation activity could be retained, albeit with some sacrifice compared to unmodified p5′A2′p5′A2′p5′A. Thus, the spatial domain about thisN⁶-amino function could be available for modifications to enhance the biological potency of 2-5A analogues and to ligate 2-5A to targeting vehicles such as antisense molecules.

Full text of DOI:10.1016/S0968-0896(03)00060-9

Bioorganic & Medicinal Chemistry 11 (2003) 2041–2049
Probing the Activation Site0of Ribonuclease L with New
N6-Substituted 20,5 -Adenylate Trimers
Ursula Munch,a,b Ling Chen,a,b Suzanne F. Baylya,b and Paul F. Torrencea,b,
*
aSection on Biomedical Chemistry, Laboratory of Medicinal Chemistry,
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0805, USA
bDepartment of Chemistry, Northern Arizona University, Flagstaff, AZ 86011-5698, USA
Received 13 November 2002; accepted 27 December 2002
Abstract—2-5A trimer [50-monophosphoryladenylyl(20-50)adenylyl(20-50)adenosine] activates RNase L. While the 50-terminal and 20-
terminal adenosine N6-amino groups play a key role in binding to and activation of RNase L, the exocyclic amino function of the
second adenylate (from the 50-terminus) plays a relatively minor role in 2-5A’s biological activity. To probe the available space
proximal to the amino function of the central adenylate of 2-5A trimer during binding to RNase L, a variety of substituents were
placed at that position. To accomplish this, the convertible building block 50-O-dimethoxytrityl-30-O-(tert-butyldimethylsilyl)-6-
(2,4-dinitrophenyl)thioinosine 20-(2-cyanoethylN,N-diisopropylphosphoramidite) was prepared as a synthon to introduce 6-(2,4-
dinitrophenyl)thioinosine into the middle position of the 2-5A trimer during automated synthesis. Post-synthetic treatment with
aqueous amines transformed the (2,4-dinitrophenyl)thioinosine into N6-substituted adenosines. Assays of these modified trimers for
their ability to bind and activate RNase L showed that activation activity could be retained, albeit with some sacrifice compared to
unmodified p50A20p50A20p50A. Thus, the spatial domain about thisN6-amino function could be available for modifications to
enhance the biological potency of 2-5A analogues and to ligate 2-5A to targeting vehicles such as antisense molecules.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
phorothioate linkages7,12 or the introduction of a single
terminal hydroxyprolinol moiety.14 Ligation of 2-5A to
peptide nucleic acid (PNA) not only provided enhance
resistance to degradation, but also increased affinity to
target RNA.13,16 Modification of the 2-5A 50-terminal
The
2-5A-antisense
[2-5A,
general
formula
pn50A20(p50A20)mp50A] strategy offers a great potential
approach to therapeutic agents capable of control of gene
expression.1ꢀ7 The chimeric antisense compound, which
consists of a 50-phosphorylated 2-5A moiety and a cova-
lently attached 30,50-nucleic acid, peptide nucleic acid
(PNA) or PNA-nucleic acid domain, effects the selective
and specific cleavage of RNA both in cell-free systems
and in intact cells.1,8ꢀ16 The antisense domain binds to a
complementary RNA sequence, while the 2-5A portion
activates RNase L for cleavage of the targeted RNA
substrate to which the 2-5A chimera is bound.
monophosphate group to
a
monothiophosphate
reduced to likelihood of phosphatase inactivation of the
2-5A-antisense chimera construct.8 While the foregoing
modifications addressed affinity and enzymatic inacti-
vation issues, the inherent reduction of 2-5A biological
potency resulting from 2-5A-antisense construction has
not been the subject of structural modification studies.
Thus, previous reports have shown that there is an
approximate 10- to 50-fold reduction in ability to acti-
vate human 2-5A-dependent RNase L when 2-5A is
ligated to a oligonucleotide (or analogue) of usual anti-
sense length (12–18-mers).9,13,16 This is most probably a
result of the 2-5A-dependent RNase L structural
requirements for maximal activation by 20,50-oligoade-
nylates. The usually employed activators of RNase L
are trimeric and tetrameric 20,50-oligoadenylates, but we
have found recently that longer 20,50-oligoadenylates are
significantly less active than their counterparts of
shorter lengths. (S. Bayly and P. F. Torrence, unpub-
To enhance the biological activity of 2-5A-antisense
oligonucleotides, several structural modifications have
been examined. Thus, a 30-30-terminal phosphodiester
bond in the antisense domain6 reduced exonuclease
degradation, as did modification with 30-terminal phos-
*Corresponding author. Tel.: +1-520-523-3008; fax: +1-520-523-
8111; e-mail: paul.torrence@nau.edu; website: http://www.nau.
edu/nchem/faculty.html
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00060-9

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U. Munch et al. / Bioorg. Med. Chem. 11 (2003) 2041–2049
¨
lished observations). If the 20-terminus of 20,50-oligoa-
denylates is indeed limiting for full expression of the
activation of RNase L, might another mode of con-
jugation of the antisense oligonucleotide domain lead to
more biologically potent 2-5A-antisense chimeras? In
this study, we have explored the adenine base of 2-5A as
a potential point for linkage to antisense molecules.
whereby a phosphoramidite of a versatile base is intro-
duced into the oligomer sequence. After synthesis, the
DNA is treated with appropriate nucleophiles, which
displaces the leaving group of the modified base and
converts the oligomer into a series of different sub-
stituted products (Scheme 1).23ꢀ31 In 1992, Xu et al.28
presented
the
incorporation
of
6-(2,4-dini-
trophenyl)thiodeoxyguanosine phosphoramidite as a
convertible monomer into oligomers and their conver-
sion into DNA containing deoxyguanosine modified at
the 6-position with S-, N- and O-derivatives. This leav-
ing group is reported to be stable towards the reagents of
automated DNA synthesis and can be displaced under
mild condition of room temperature. Subsequently, the
same working group has extended this strategy for the
preparation of correspondingN6-substituted deox-
yadenine analogues, using the phosphoramidite mono-
mer of 6-(2,4-dinitrophenyl)thiodeoxyinosine.29
Various analogues of 2-5A have been synthesized to
examine the crucial structural parameters of 2-5A for
interaction with RNase L.17,18 The adenine rings of 2-5A
play a critical and differential role in the interaction of
RNase L with 2-5A. Exchange of the first (from the 50-
terminus) adenine base with inosine to yield
ppp50I20p50A20p50A led to a large decrease both in acti-
vation ability (200-fold loss) and in RNase L binding
affinity. The replacement of the 20-terminal adenine base
by hypoxanthine to form ppp50A20p50A20p50I resulted in
a 1000-fold drop in activation ability with little change in
RNase L binding ability. Finally, the exchange of the
middle nucleoside by inosine to give ppp50A20p50I20p50A
effected only a minor drop both in RNase L activation
(20-fold loss) and binding ability (2–3-fold decrease).19,20
A model for 2-5A interaction with RNase L was devel-
oped17,18,20,21 that posited separate binding and activa-
tion steps associated with nuclease activation, with the
various functional groups of 2-5A playing different roles
in these separable steps. Thus, the N6-amino function of
the first nucleotide of 2-5A was essential for RNase L
binding, without which no activation would be possible.
The exocyclic amino function of the last adenosine (20-
terminal) was crucial for the activation of RNase L, but
not for the separable binding step. In other words,
without the 20-terminal adenineN6-amino group, a 2-5A
congener might bind quite well to RNase L, but still not
activate its nuclease function. In contrast, theN6-amino
function of the middle adenosine was much less critical
for the biological activity of the 2-5A system, both for
activation and for binding, so that this amino function
might be a candidate for chemical modification.
Herein, we have employed the (2,4-dinitrophenyl)thio
leaving group for the generation of central adenylate-
modified 2-5A trimers, which are subsequently r0eferred
to with the general formula p50A20p50ANRR 20p50A
0
(ANRR , N6-R,R0-substituted adenosine) (Fig. 1). This
required the preparation of the versatile phosphor-
amidite 7, its incorporation into RNA during auto-
mated synthesis giving p50A20p50AS-dnPh20p50A (AS-dnPh
,
6-(2,4-dinitrophenyl)thioinosine)0 (8) and the conversion
of 8 into trimers p50A20p50ANRR 20p50A upon treatment
with aqueous amine.
Monomer synthesis
The synthesis of phosphoramidite 7 (Scheme 1) began
with the introduction of the 2,4-dinitrophenyl group
into 6-mercaptopurine riboside (1) according to Xu et
al.28 The reaction of commercially available compound
1 with 2,4-dinitrofluorobenzene in presence of triethyla-
mine resulted in 6-(2,4-dinitrophenyl)thioinosine (2) in
91% yield. Derivative 2 was then tritylated in the 50-
position with dimethoxytrityl chloride in the presence of
DMAP/triethylamine to give 50-O-dimethoxytrityl-6-
(2,4-dinitrophenyl)thioinosine (3) in 84% yield. Sub-
sequent silylation of compound 3 with tert-butyldi-
methylsilyl chloride and imidazole in DMF led to a
mixture of all three possible products 4–6, from which
the 20,30-bis-O-substituted derivative 6 was isolated after
silica gel column purification in 8% yield. For the
separation of the mono-protected isomers 4 and 5, a
second column purification had to be performed. The
Additional studies with uridylate-substituted 2-5A con-
geners confirmed these observations with inosinate-sub-
stituted oligonucleotides.22 Moreover, Player et al.21
showed that the 50-terminal adenosine purine N-1moi-
ety was key for RNase L binding while the 20-terminal
adenosine exocyclic N6-amino group was critical for
RNase L activation.
To probe the spatial relationships around this 2-5A
central adenylate N6-amino group during the interac-
tion of 20,50-oligoadenylates with RNase L, we have
synthesized a variety of analogues bearing N6-amino-
substituents that vary in steric bulk and hydrophobic
properties. Evaluation of the RNase L binding and
activation abilities of these congeners has provided a
clearer picture of ligand–receptor contacts.
desired 30-O-tert-butyldimethylsilyl product
4 was
obtained in a yield of 51% while the yield of the 20-O-
tert-butyldimethylsilyl side product was 27%. The
assignments of structures of the respective 30-O-
TBDMS- and 20-O-TBDMS-substituted isomers 4 and 5
1
were effected by H NMR spectra irradiation experi-
ments. By irradiation of the resonance frequency of the
H–C(10) proton, the signal of the neighbouring H–C(20)
proton was simplified. Subsequent irradiation of the
resonance frequency of the H–C(20) proton led to a
simplification of the signals of the H–C(10) and H–C(30)
protons. In this way, the assignments of the chemical
shifts to the corresponding H–C(20) or H–C(30) protons
Results and Discussion
A simplified chemical approach to these various trimers
recommends
a post-synthetic conversion strategy,

U. Munch et al. / Bioorg. Med. Chem. 11 (2003) 2041–2049
¨
2043
Scheme 1. Synthesis of 50-O-dimethoxytrityl-6-(2,4-dinitrophenyl)-30-O-(tert-butyldimethyl-silyl)thioinosine 20-(2-cyanoethyl N,N-diisopropylphos-
phoramidite) (7): (a) 2,4-dinitrofluorobenzene, Et3N, CH2Cl2, 91%; (b) DMTrCl, DMAP, Et3N, pyridine, 84%; (c) TBDMSCl, imidazole, DMF,
51% (4), 27% (5), 8% (6); (d) 2-cyanoethyl tetraisopropylphosphorodiamidite, tetrazole, CH2Cl2, 77%.
were unambiguous. By comparison of the chemical
shifts for the H–C(20) and H–C(30) protons of the sily-
lated isomers 4 and 5, and of the unsilylated compound
phosphoramidite chemistry as described earlier.32ꢀ35
The convertible phosphoramidite 7, which was prepared
as a 0.1M solution in dry CH 2Cl2, was employed with-
out modification to the coupling protocol. The stepwise
condensation yield, as monitored by the released DMTr
cation, generally ranged from 96% to 100%.
1
3, it was possible to determine the H NMR spectra of
the respective 30-O-TBDMS- and 20-O-TBDMS-sub-
stituted isomers 4 and 5. Thereby silylation at a carbo-
hydrate hydroxyl group led to a downfield movement of
the chemical shift for the proton, which stands geminal
to this silylated hydroxyl group. Irradiation of the
resonance frequency of the H–C(20) or H–C(30) proton,
respectively, resulted in a singlet for an eventually gem-
inal standing OH-function instead of a doublet. Finally,
phosphoramidite 7 was obtained in 77% yield by treat-
The structure of the resulting convertible p50A20p50AS-
dnPh20p50A trimer 8 is illustrated in Scheme 2. In this
form, oligomer 8 was protected at the exocyclic amino
functions, the 30-hydroxyl groups and at the inter-
nucleotide phosphate linkages, and was still attached to
the solid support. The resin-bound intermediate 8 was
treated with different aqueous base solutions at room
temperature for 1–2 days. This base treatment effected
simultaneously the displacement of the 6-(2,4-dini-
trophenyl)thio leaving group, the cleavage of the oligo-
mer from the solid support and, except for the TBDMS
group, complete deblocking of the protection groups to
give the functionalized trimers 9–17. For displacement
reactions involving volatile amines, the excess of base
ment of compound
4
with 2-cyanoethyl tetra-
isopropylphosphorodiamidite in presence of tetrazole in
CH2Cl2.
Trimer synthesis and derivatization
The convertible trimer p50A20p50AS-dnPh20p50A (8) was
synthesized on a DNA/RNA synthesizer using standard

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U. Munch et al. / Bioorg. Med. Chem. 11 (2003) 2041–2049
¨
Scheme 2. Derivatization and deprotection of convertible trimer 8.
RNase L activation by 2-5A analogues
was removed simply by lyophilization. In all other
cases, the reaction mixtures had to be neutralized with
aqueous AcOH to avoid degradation of the RNA
internucleotide linkages during evaporation. The result-
ing salts were removed with a C-18 SepPac cartridge,
and the product-containing fractions combined and
reduced to dryness. Subsequent cleavage of the 30-O-
TBDMS protecting group by treatment with TBAF/
THF and semi-preparative purification of the products
on a PRP-1HPLC column using standard methods,34ꢀ37
led to the converted trimers 18–26. In order to obtain
completely pure 2-5A analogues for biological testing
(oligomers 21 and 26 were especially problematical), an
additional Dionex HPLC at pH 8 with a Tris–HCl/
ammonium chloride gradient were performed. A final
desalting step led to the desired trimers. The purity of
oligonucleotides was analysed using both ion-pair
exchange and reversed phase chromatography, while
their identity was confirmed by mass spectrometry.
The ability of the N6-substituted 2-5A analogues to
activate RNase L for the degradation of poly(U) was
determined in an in vitro assay, employing poly(U) as
the substrate to be degraded. The concentrations EC50,
which cause cleavage of 50% of the poly(U) are listed in
Table 1. To be able to compare the respective EC50
values, each was divided by the EC50 of the unsub-
stituted 2-5A trimer to give the term Crel (Table 1). This
term expresses the relative concentration of the other
trimers needed to effect 50% cleavage. The higher the
Crel, the less effective the activation compared to the
effectiveness of the standard 2-5A trimer, the Crel of
which is defined as 1.
Overall, the modified 2-5A trimers display a trend of
decreased activity as the substituents at the amino
function increased in steric bulk. Thus, the N6-mono-

U. Munch et al. / Bioorg. Med. Chem. 11 (2003) 2041–2049
¨
2045
Table 1. Relative activities Crel of the N6-functionalized trimers to
unsubstituted 2-5A compound in cleavage activity and binding assay
ever, both analogues are significantly less effective acti-
vators than other monosubstituted derivatives This
results suggest that a hydrophilic group could be less
compatible with the 2-5A activator site than a more
hydrophobic substituent.
b
d
Compd
R
R0
EC50 (nM)a Crel IC50 (nM)c Crel
Series 1
18
19
Me
Me
Bz
H
0.44ꢁ0.05 (4)
1.5
26 (2)
2.6
Me 2.2ꢁ0.4 (4)
7.6 53–58 (2)
4.3 16–22 (2)
2.2 27–33 (2)
1.0 9.2–11 (2)
5.5
1.9
3.0
1.0
20
21
H
H
1.2ꢁ0.4 (3)
0.65ꢁ0.20 (4)
0.29ꢁ0.01 (4)
Binding assays
Et
p50A20p50A20p50A
The ability of certain of the modified 2-5A trimers to
bind to RNase L was examined in a radiobinding
assay for the set of compounds 18–21. The assay
ascertained the ability of the0 trimer to displace the
radiolabelled probe p50A20(p5 A20)2p50A30[32P]p50C30p
from RNase L. The IC50 values, listed in Table 1, give
the concentration required to inhibit radioactive probe
binding by 50%. Here again, the data were normal-
ized to the 2-5A standard, whose relative IC50 is
defined as 1. According to the Crel values, allN6-sub-
stituted trimers 18–21 showed a decrease in binding
ability compared to the unmodified 2-5A. There was a
fair correlation between loss of RNase L binding and
loss of ability to activate RNase L for the oligonu-
cleotides 18, 19, and 21. However, analogue 20, the
benzyl congener, displayed a contrary behavior with
an activation ability that was less than would have
been expected, based on the behavior of 18, 19, and
21. Binding assay were not conducted on compounds
22–26.
Series 2
22
23
24
25
Et
Et
H
H
H
3.1ꢁ0.4 (4)
17
8.5
13
(CH2)2NH2
(CH2)6NH2
Cyclopentyl
1
ꢁ0.5.4 (4)
2.4ꢁ0.3 (4)
0.34ꢁ0.03 (4)
1.9
26
1.8ꢁ0.6 (4)
0.18ꢁ0.04 (5)
10.2
1.0
p50A20p50A20p50A
These values are given as the mean ꢁ standard deviation, with the
number of experiments (n) given in parentheses.
aEC50 is defined as the concentration of trimer required to cleave 50%
of poly(U).
bCrel is the EC50 value divided by the EC50 of the 2-5A standard, with
2-5A arbitrarily assigned a Crel of 1.
cIC50 is defined as the concentration of trimer required to displace
50% of bound radioactive probe to RNase L.
dCrel is the IC50 value divided by the IC50 of the 2-5A standard, with
2-5A arbitrarily assigned a Crel of 1.
methylated trimer 18 possessed a Crel of 1.5 while the
dimethylated congener 19 displayed a decrease in acti-
vation ability with a Crel of 7.6, a 5-fold activity drop.
Similarly, the monoethylated trimer 21 showed a Crel
value of 2.2, only slightly less effective than unmodified
2-5A. In contrast, the diethylated analogue 22 was
about 8-fold less active than 21 with a Crel of 17. The
trend continued with the monobenzyl-substituted com-
pound 20 that showed a 4.3 reduction in activation
ability compared to parent 2-5A trimer. However, a
single N6-cyclopentyl group (25) had less effect than the
single benzyl substitution with a diminution in activity
(Crel=1.9) that was similar to that of the methyl or
ethyl substitutions. This result may imply that the lone
cyclopentyl moiety (in compound 25) can be accom-
modated in the RNase L 2-5A activation site, but that
the increased bulk of the benzyl group could not. That
some critical steric threshold is involved is suggested by
the significant decreases in activation upon dimethyl or
diethyl substitution. In addition, the analogue 26
(Crel=10.2) also demonstrated a major activity loss;
however, its activity was significantly greater than the
diethyl derivative 22. It might be speculated that the
restriction or ‘tying back’ of the methylene groups in the
piperidine ring structure reduces the entropy cost asso-
ciated with the accommodation in the RNase L activa-
tion site; however, its chair-like conformation would
require greater space accommodation than the cyclo-
pentyl substituent (Crel=1.9).
Conclusions
The foregoing experiments show
a tendency to
decreased RNase L activation ability as the N6-sub-
stituents of the central adenylate of 2-5A increase in
steric bulk. In addition, the data suggest a preference
for hydrophobic substituents at that site. Thus, 2-5A
trimers, which are appropriately substituted in the N6-
position of the central adenylate, are still capable of
productive binding and activation 2-5A-dependent
RNase L. Consequently, further modifications at this
position could be possible, such as attachment of anti-
sense deoxyoligonucleotides. 2-5A-Antisense chimeras
with such a mode of linkage between the 2-5A moiety
and the antisense domain have not yet been examined
for their biological properties. To date, 2-5A-antisense
chimeras have been constructed only through ligation
of 2-5A either at the 50-terminus or the 30-terminus of
the antisense chain. Since attachment of antisense to the
central adenylate likely would alter the spatial orienta-
tion of RNase L relative the antisense-hybridized RNA
target, this structural alteration may bring about differ-
ent sites of RNA cleavage and could provide access to
RNA sequences not available to 2-5A-antisense based
on 50- or 30-terminus ligation. In any case, the results of
this study open a path to a novel form of 2-5A-anti-
sense; however, the present study suggests that while
RNase L-active 2-5A-antisense may be generated by
linking an antisense chain to the N6-amino of the cen-
tral adenylate of 2-5A, such chimeric molecules, like
their 2-5A 20-terminus-conjugatde counterparts,9,13,16
would be significantly muted in their RNase L activa-
tion potency.
The N6-aminoethyl (23, Crel=8.5) and N6-aminohexyl
(24, Crel=13) congeners also suggest a role for steric
bulk in the determination of activator potency. How-

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U. Munch et al. / Bioorg. Med. Chem. 11 (2003) 2041–2049
¨
Experimental
H)+: 451.06; found: 451. Anal. calcd for C16H14N6O8S
(450.39): C 42.67, H 3.13, N 18.66; found: C 42.62, H
3.09, N 18.45.
Monomer synthesis
Chemical reagents were obtained from Aldrich (Mil-
waukee, WI, USA). All reactions were performed under
an inert atmosphere of argon in anhydrous solvents
(SureSeal packing, Aldrich) and used without further
purification or treatment. Analytical thin layer chroma-
tography was run on precoated plates of Whatman
silica gel, 60 A on polyester with fluorescence indicator
(Whatman, Clifton, NJ, USA). The compounds were
localized at 254 nm using a UV lamp and in case of the
tritylated products by staining with TFA gas. Flash
column chromatography was run on Merck silica gel 60
(0.040–0.063 mm). The melting point was measured on a
Laboratory Devices, Mel-Temp II apparatus and is
uncorrected. UV spectra were determined on a Hewlett
Packard 8452 spectrophotometer. 1H NMR spectra
were recorded on a Varian Gemini 300 MHz, the 31P
NMR spectra was obtained on a Varian Mercury
300 MHz spectrometer. The chemical shifts are reported
in parts per million (ppm) relative to internal tetra-
methylsilane in CDCl3 or DMSO-d6 (1H NMR) or
relative to H3PO4 (31P NMR). The coupling constants
(J) are reported in Hertz (Hz). Chemical ionisation (CI)
mass spectrum using NH3 was recorded on a Finnigan
MAT 4600 apparatus. Fast atom bombardment (FAB)
mass spectra were performed with nitrobenzylalcohol
(NBA) as a matrix on an Extrel EL 400 spectrometer.
Elemental analysis were carried out at Atlantic Micro-
lab, Inc. (Norcross, GA, USA).
50 - O - Dimethoxytrityl - 6 - (2,4 - dinitrophenyl)thioinosine
(3). Compound 2 (1.65 g, 3.66 mmol) was co-evapo-
rated with pyridine (3 ꢂ 10 mL) and taken up in pyri-
dine (30 mL). After addition of dimethoxytrityl chloride
(1.51 g, 4.46 mmol), DMAP (23 mg, 0.19 mmol) and
Et3N (715 mL, 5.15 mmol), the solution was stirred for
6 h. An additional amount of dimethoxytrityl chloride
(370 mg, 1.09 mmol) was added and after another 16.5 h
of stirring, the reaction mixture was diluted with
CH2Cl2 (80 mL) and washed with saturated NaHCO3
(100 mL). The aqueous layer was separated, extracted
with CH2Cl2 (3 ꢂ 100 mL) and the combined organic
layer dried (Na2SO4), evaporated and co-evaporated
with toluene (4 ꢂ 10 mL). The residue was purified by
flash chromatography (silica gel, 50 g, 3.5 ꢂ 16 cm,
CH2Cl2 (200ꢂmL), CH2Cl2/MeOH 100:1 (202 mL),
100:2 (204 mL), 100:3 (206 mL), 100:4 (208 mL)) to give
2.31g (84%) of product 3. Orange foam. Rf 0.55 (tolu-
ene/AcOEt/MeOH 5:4:1). UV (MeOH) lmax 342, 274,
1
230 (sh) nm. H NMR (CDCl3, 300 MHz) d (ppm) 9.01
(d, J=1.8 Hz, 1H, H–C(3) of 2,4-dinitrophenyl); 8.71 (s,
1H, H–C(2)); 8.33 (s, 1H, H–C(8)); 8.26 (dd, J=8.7,
J=3 Hz, 1H, H–C(5) of 2,4-dinitrophenyl); 7.88 (d,
J=8.7 Hz, 1H, H–C(6) of 2,4-dinitrophenyl); 7.27–7.14
(m, 9H, DMTr); 6.75–6.72 (m, 4H, o to MeO); 6.04 (d,
J=5.7 Hz, 1H, H–C(10)); 5.06 (d, J=3 Hz, 1H, OH–
C(20)); 4.97–4.92 (m, 1H, H-C(20)); 4.50–4.48 (m, 1H,
H–C(30)); 4.45 (m, 1H, H–C(40)); 3.77 (s, 6H, 2 MeO);
3.45 (dd, J=10.8, J=3.9 Hz, 1H, H-C(50)); 3.33 (dd,
J=10.8, J=3 Hz, 1H, H–C(50)); 2.92 (broad s, 1H, OH-
C(30)). FAB-MS calcd for (C37H32N6O10S–H)+: 753.20;
6-(2,4-Dinitrophenyl)thioinosine (2). To a suspension of
6-mercaptopurine riboside (1; 2.00 g, 7.04 mmol) in
CH3CN (135 mL) was added Et3N (6 mL, 43 mmol) and
2,4-dinitrofluorobenzene (1.06 mL, 8.38 mmol). After
stirring 1h at room temperature, the then clear solution
was concentrated under reduced pressure to a yellow
oil, which was co-evaporated with CH2Cl2 (3 ꢂ 30 mL).
The hereby precipitated solid was filtered by suction,
washed with CH2Cl2 and diethylether, and dried in
vacuo to give 2.40 g of 2. The filtrate was concentrated
to dryness and the resulting yellow oil was purified by
flash chromatography [silica gel, 30 g, 2 ꢂ 16 cm,
CH2Cl2 (100 mL), CH2Cl2/MeOH 100:1 (101 mL), 100:2
(102 mL), 100:3 (103 mL), 100:4 (208 mL), 100:5
(210 mL), 100:6 (212 mL)]: additional 480 mg of 2. Total
yield 2.88 g (91%). Compound 2 was employed in next
reaction step without further purification, for elemental
analysis 52 mg of 2 were recrystallized in CH2Cl2/
MeOH 19:5 (6 mL). Slight yellow solid. Mp 182–185 ꢃC
(dec.). Rf 0.35 (toluene/AcOEt/MeOH 5:4:1). UV
(MeOH) lmax 336, 276 nm. 1H NMR (DMSO-d6,
300 MHz) d (ppm) 8.93 (d, J=1.8 Hz, 1H, H–C(3) of
2,4-dinitrophenyl); 8.89 (s, 1H, H–C(2)); 8.77 (s, 1H, H–
C(8)); 8.47 (dd, J=8.7, J=2.9 Hz, 1H, H–C(5) of 2,4-
dinitrophenyl); 8.10 (d, J=9 Hz, 1H, H–C(6)0 of 2,4-
dinitrophenyl); 6.05 (d, J=5.1Hz, 1H, H–C(1 )); 5.57
(d, J=6 Hz, 1H, OH–C(20)); 5.27 (d, J=6 Hz, 1H, OH–
C(30)); 5.10 (t, J=5.4 Hz, 1H, OH–C(50)); 4.65–4.59 (m,
1H, H–C(20)); 4.22–4.18 (m, 1H, H–C(30)); 4.01–3.98
(m, 1H, H–C(40)); 3.74–3.67 (m, 1H, H–C(50)); 3.62–
3.34 (m, 1H, H–C(50)). CI-MS calcd for (C16H14N6O8S–
.
found: 753. Anal. calcd for C37H32N6O10S 1=2 H2O
(761.77): C 58.34, H 4.37, N 11.03; found: C 58.14, H
4.38, N 10.77.
50 -O-Dimethoxytrityl-30 -O-(tert-butyldimethylsilyl)-6-
(2,4-dinitrophenyl)thioinosine (4), 50-O-dimethoxytrityl-20
-O-(tert-butyldimethylsilyl)-6-(2,4-dinitrophenyl)thioino-
sine (5) and 50-O-dimethoxytrityl-20,30-bis-O-(tert-butyl-
dimethylsilyl)-6-(2,4-dinitrophenyl)thioinosine (6).
A
solution of compound 3 (2.29 g, 3.04 mmol), imidazole
(505 mg, 7.42 mmol) and tert-butyldimethylsilyl chloride
(590 mg, 3.91mmol) in DMF (5 mL) was stirred at
room temperature overnight (17 h). After dilution with
CH2Cl2 (80 mL), the solution was washed with satu-
rated NaCl (100 mL). The aqueous layer was separated,
extracted with CH2Cl2 (3 ꢂ 100 mL) and the combined
organic layer dried (Na2SO4), evaporated and co-eva-
porated with toluene (4 ꢂ 10 mL). Separation of pro-
duct mixture was achieved by flash chromatography
[silica gel, 60 g, 3.5 ꢂ 20 cm, toluene (200 mL), toluene/
AcOEt 18:1 (190 mL), 17:1 (180 mL), 16:1 (170 mL),
14:1 (150 mL), 12:1 (130 mL), 10:1 (110 mL), 9:1
(100 mL), 8:1(90 mL), 6:1(70 mL)]. First 244 mg (8%)
of the 20,30-bis-O-substituted derivative 6 was eluted,
then compound 5 and finally the 30-O-substituted pro-
duct 4. A mixture of isomers 4 and 5 was separated by a
second flash chromatography [silica gel, 15 g, 1.4 ꢂ
19 cm, toluene (100 mL), toluene/AcOEt 18:1 (95 mL),

U. Munch et al. / Bioorg. Med. Chem. 11 (2003) 2041–2049
¨
2047
17:1 (90 mL), 16:1 (85 mL), 14:1 (75 mL), 12:1 (65 mL)]
to give a total yield of 1.34 g (51%) of 4 and 716 mg
(27%) of 5.
propylphosphoramidite) (7). A mixture of compound 4
(1.30 g, 1.50 mmol), tetrazole (58 mg, 0.83 mmol) and
2-cyanoethyl tetraisopropylphosphorodiamidite (0.7 mL,
2.20 mmol) was stirred in CH2Cl2 (10 mL) under argon
at room temperature overnight (17 h). After dilution
with CH2Cl2 (80 mL), the solution was washed with
saturated NaHCO3 (100 mL). The aqueous phase was
separated, extracted with CH2Cl2 (3 ꢂ 100 mL) and the
combined organic layer dried (Na2SO4), evaporated and
the crude product purified by flash chromatography
(silica gel, 50 g, 3 ꢂ 13 cm, petroleum ether/acetone 9:1
(200 mL), 8:1(180 mL), 7:1(160 mL), 6:1(140 mL), 5:1
(120 mL), 9:2 (220 mL), 4:1 (200 mL), 7:2 (180 mL)):
1.23 g (77%) of 7. Yellow foam. Rf 0.24 (toluene/AcOEt
9:1), 0.43 (petroleum ether/acetone 3:1). UV (MeOH)
lmax 344, 276, 230 (sh) nm. 1H NMR (CDCl3,
300 MHz) d (ppm) 8.98–8.97 [m, 1H, H–C(3) of 2,4-
dinitrophenyl); 8.78, 8.74 (2s, 1H, H-C(2)]; 8.39, 8.37
(2s, 1H, H–C(8)); 8.04, 7.99 (2dd, J=9, J=8.7, J=3,
J=2.9 Hz, 1H, H–C(5) of 2,4-dinitrophenyl); 7.63, 7.56
(2d, J=9, J=9 Hz, 1H, H–C(6) of 2,4-dinitrophenyl);
7.38–7.18 (m, 9H, DMTr); 6.80–6.76 (m, 4H, o to
MeO); 6.32, 6.23 (2d, J=3.9, J=3.9 Hz, 1H, H–C(10));
5.13–5.09, 5.01–4.95 (2m, 1H, H–C(20)); 4.73, 4.67 (2t,
J=4.8, J=4.5 Hz, 1H, H–C(30)); 4.25–4.15 (m, 1H, H–
C(40)); 3.78, 3.78 (2s, 6H, 2 MeO); 3.68–3.44 (m,
CNCH2CH2, 2 NCHR2, H–C(50)); 3.23 (dd, J=11.1,
4. Yellow foam. Rf 0.19 (toluene/AcOEt 9:1). UV
1
(MeOH) lmax 340, 276, 228 (sh) nm. H NMR (CDCl3,
300 MHz) d (ppm) 8.99 (d, J=3 Hz, 1H, H–C(3) of 2,4-
dinitrophenyl); 8.75 (s, 1H, H–C(2)); 8.33 (s, 1H, H–
C(8)); 8.15 (dd, J=8.7, J=3 Hz, 1H, H–C(5) of 2,4-
dinitrophenyl); 7.72 (d, J=8.7 Hz, 1H, H–C(6) of 2,4-
dinitrophenyl); 7.37–7.18 (m, 9H, DMTr); 6.79–6.77 (m,
4H, o to MeO); 6.08 (d, J=4.8 Hz, 1H, H–C(10)); 4.86
(t, J=5 Hz, 1H, H–C(20)); 4.69 (t, J=4.4 Hz, 1H, H–
C(30)); 4.19–4.17 (m, 1H, H–C(40)); 3.78 (s, 6H, 2 MeO);
3.53 (dd, J=10.8, J=3 Hz, 1H, H–C(50)); 3.25 (dd,
J=11.1, J=3.5 Hz, 1H, H–C(50)); 3.10 (d, J=6 Hz, 1H,
OH–C(20)); 0.91(s, 9H, CMe ); 0.12 (s, 3H, SiMe); 0.04
3
(s, 3H, SiMe). FAB-MS calcd for (C43H46N6O10SSi–
H)+: 867.28; found: 867. Anal. calcd for
C43H46N6O10SSi (867.02): C 59.57, H 5.35, N 9.69;
found: C 59.66, H 5.47, N 9.58.
5. Yellow foam. Rf 0.28 (toluene/AcOEt 9:1). UV
1
(MeOH) lmax 342, 276, 226 (sh) nm. H NMR (CDCl3,
300 MHz) d (ppm) 9.01(d, J=1.8 Hz, 1H, H–C(3) of
2,4-dinitrophenyl); 8.66 (s, 1H, H–C(2)); 8.33 (s, 1H, H–
C(8)); 8.35–8.32 (m, 1H, H–C(5) of 2,4-dinitrophenyl);
7.91(d, J=8.7 Hz, 1H, H–C(6) of 2,4-dinitrophenyl);
7.44–7.18 (m, 9H, DMTr); 6.83–6.80 (m, 4H, o to
J=3.5 Hz, 1H, H–C(50)); 2.52, 2.41(2t,
J=6.5,
J=6.5 Hz, 2H, CNCH2CH2); 1.33–1.10 (m, 12H, 2
CMe2); 0.86, 0.85 (2s, 9H, CMe3); 0.16, 0.06 (2s, 3H,
SiMe); 0.13, 0.06 (2s, 3H, SiMe). 31P NMR (CDCl3,
121.4 MHz) d (ppm) 151.14, 150.39. FAB-MS calcd for
(C52H63N8O11PSSi–H)+: 1067.39; found: 1067. Anal.
calcd for C52H63N8O11PSSi (1067.24): C 58.52, H 5.95,
N 10.50; found: C 58.56, H 6.28, N 10.42.
0
MeO); 6.12 (d, J=5.1Hz, 1H, H–C(1 )); 4.99 (t,
J=4.8 Hz, 1H, H–C(20)); 4.42–4.38 (m, 1H, H–C(30));
4.30–4.28 (m, 1H, H–C(40)); 3.79 (s, 6H, 2 MeO); 3.55
(dd, J=10.8, J=3 Hz, 1H, H–C(50)); 3.41(dd, J=10.8,
J=3.9 Hz, 1H, H–C(50)); 2.67 (d, J=3.9 Hz, 1H, OH–
C(30)); 0.84 (s, 9H, CMe3); 0.01(s, 3H, SiMe); ꢀ0.13 (s,
3H, SiMe). FAB-MS calcd for (C43H46N6O10SSi–H)+:
867.28; found: 867. Anal. calcd for C43H46N6O10SSi
(867.02): C 59.57, H 5.35, N 9.69; found: C 59.40, H
5.43, N 9.64.
Synthesis and derivatization of p50A20p50AS-dnPh20p50A
The synthesis of the 2-5A trimer followed the strategy
described earlier.32ꢀ35 All DNA synthesis reagents, the
long chain alkylamino-controlled pore glass solid sup-
port (20 -O-acetyl-50 -O-dimethoxytrityl-N6 -phenoxy-
acetyladenosine-30-lcaa-CPG,
500 A),
and
the
6. Yellow foam. Rf 0.66 (toluene/AcOEt 9:1). UV
1
phosphorylation reagent for the 50-terminus of the tri-
mer (2-[2-O-(4,40-dimethoxytrityl)ethylsulfonyl]ethyl-20-
(cyanoethyl N,N-diisopropylphosphoramidite)) were
purchased from Glen Research (Sterling, VA, USA).
The building block for the 20,50-adenylate domain (50-O-
dimethoxytrityl-30 -O-(tert-butyldimethylsilyl)-N6-phe-
noxyacetyladenosine 20-(2-cyanoethyl N,N-diisopropyl-
phosphoramidite)) was obtained from Pharmacia
Biotech (Piscataway, NJ, USA). All phosphoramidites
were used as 0.1M solutions in CH 3CN, except for the
functionalized phosphoramidite 7, which was for solu-
bility reasons employed as a 0.1M solution in CH 2Cl2
and connected at the optional port.
(MeOH) lmax 344, 276, 226 (sh) nm. H NMR (CDCl3,
300 MHz) d (ppm) 9.01(d, J=3 Hz, 1H, H–C(3) of 2,4-
dinitrophenyl); 8.68 (s, 1H, H–C(2)); 8.35 (s, 1H, H–
C(8)); 8.24 (dd, J=8.7, J=3 Hz, 1H, H–C(5) of 2,4-
dinitrophenyl); 7.80 (d, J=7.8 Hz, 1H, H–C(6) of 2,4-
dinitrophenyl); 7.45–7.19 (m, 9H, DMTr); 6.83–6.80 (m,
4H, o to MeO); 6.07 (d, J=4.8 Hz, 1H, H–C(10)); 4.87
(t, J=5 Hz, 1H, H–C(20)); 4.33–4.31(m, 1H, H-C(3 0));
4.26–4.25 (m, 1H, H–C(40)); 3.79 (s, 6H, 2 MeO); 3.57
(dd, J=10.8, J=3.9 Hz, 1H, H-C(50)); 3.32 (dd,
J=10.8, J=3.9 Hz, 1H, H–C(50)); 0.87 (s, 9H, CMe3);
0.78 (s, 9H, CMe3); 0.07 (s, 3H, SiMe); 0.00 (s, 3H,
SiMe);ꢀ0.03 (s, 3H, SiMe);ꢀ0.28 (s, 3H, SiMe). FAB-
MS calcd for (C49H60N6O10SSi2–H)+: 981.37; found:
981. Anal. calcd for C49H60N6O10SSi2 (981.29): C 59.98,
H 6.16, N 8.56; found: C 60.19, H 6.20, N 8.40.
The trimer p50A20p50AS-dnPh20p50A (8) was synthesized
on a 1.0 mM scale on a DNA/RNA synthesizer (ABI
392) under 4,5-dicyanoimidazole activation using stan-
dard reaction cycles: 1.0 mm RNA for chain elongation
(600 s coupling time) and 1 mM Phosphorylation for 50-
terminal phosphorylation (60 s condensation time).
50 -O-Dimethoxytrityl-30 -O-(tert-butyldimethylsilyl)-6-
(2,4-dinitrophenyl)thioinosine 20-(2-cyanoethyl N,N-diiso-

2048
U. Munch et al. / Bioorg. Med. Chem. 11 (2003) 2041–2049
¨
After the synthesis in the trityl-off modus, the oligonu-
cleotide was washed with CH3CN and dried under a
stream of argon. The solid support with the attached
trimer was treated with 2 mL of aqueous base solution
in a screwed capped glass vial for 1–2 days at room
temperature (base concentrations and reaction times are
given in detail in Scheme 2). In the cases of methyla-
mine, dimethylamine and ethylamine, the displacement
mixture was separated from the resin by filtration over
cotton wool and the excess of base was removed by
lyophilization. All other amines were neutralized (pH 7)
with 50% aqueous acetic acid under ice-bath cooling
(about 1equiv AcOH to base) before separation from
resin and evaporation on a Speed-vac concentrator. The
solutions were desalted using C-18 SepPak cartridges
(12 g per 0.5 mmol scale). The loaded cartridge was
washed with H2O (40 mL), 5% MeOH/H2O (20 mL),
10% MeOH/H2O (20 mL) and 50% MeOH/H2O (70–
80 mL). 10 mL fractions were pooled and the oligomer
was usually found in the fractions of 50% MeOH/H2O,
seldom of 10% MeOH/H2O. For TBDMS cleavage,
1M TBAF/THF (1mL per 0.5 mmol scale) was added to
the concentrated residues 9 to 17 for about 20 h at room
temperature, then the reaction was stopped by addition
of 1mL H 2O under ice-bath cooling and the completely
unprotected trimers 18–26 were lyophilized. The crude
product was purified on a semi-prep Hamilton PRP-1
column (7 ꢂ 300 mm) (Hamilton, Reno, NV, USA):
Solvent A: 10 mM tetrabutylammonium phosphate
(TBAP), pH 7.5 in H2O; solvent B: 10 mM TBAP, pH
7.5 in CH3CN/H2O 8:2; gradient: convex from 5% B to
95% B in 50 min; flow rate: 5 mL/min. Purified products
were desalted with C-18 SepPak cartridges. The loaded
cartridge was washed with H2O (20 mL), 5% MeOH/
H2O (10 mL), 10% MeOH/H2O (10 mL) and 50%
MeOH/H2O (20 mL). Solvents were collected in 5-mL
fractions and the oligomer, usually found in fractions 9
and 10 (50% MeOH/H2O) was concentrated on a
Speed-vac concentrator. For additional purification, a
Dionex NucleoPac PA-100 (9 ꢂ 250) column was used
(Dionex, Sunnyvale, CA, USA): Solvent A: 25 mM
Tris–HCl, pH 8 in H2O; solvent B: 25 mM Tris and 1M
ammonium chloride, pH 8 in H2O; gradient: isocratic
0% B for 2 min, linear from 0% B to 50% B in 38 min,
linear from 50% B to 100% B in 10 min, isocratic 100%
B for 5 min, linear from 100% B to 0% B in 3 min and
isocratic 0% B for 5 min; flow rate: 1mL/min. Trimers
were finally desalted on a semi-prep Hamilton PRP-1
column (7 ꢂ 300 mm): Solvent A: 0.1M triethylammo-
nium acetate (TEAAc), pH 7 in H2O; solvent B: MeOH;
gradient: isocratic 0% B for 10 min, convex from 0% B
to 100% B in 30 min; flow rate: 5 mL/min. The solutions
of the resulting 2-5A trimers were quantitated as OD
A260 by UV spectrophotometry using approximately the
extinction coefficient for unmodified p50A20p50A20p50A
as reported earlier.19
Analysis of trimers
The ion-exchange method employed a Dionex Nucleo-
Pac PA-100 (4 ꢂ 250 mm) column: Solvent A: 25 mM
Tris–HCl, pH 8 in H2O; solvent B: 25 mM Tris and 1M
ammonium chloride, pH 8 in H2O; gradient: isocratic
0% B for 2 min, linear from 0% B to 50% B in 18 min,
linear from 50% B to 100% B in 10 min, isocratic B for
5 min, linear from 100% B to 0% B in 3 min and iso-
cratic 0% B for 5 min; flow rate: 1mL/min. For RP-18
analysis a Beckman Ultrasphere ODS (4.6 ꢂ 250 mm)
column was used (Beckman, Fullerton, CA, USA): Sol-
vent A: 50 mM ammonium phosphate, pH 7; solvent B:
MeOH/H2O 1:1; gradient: linear from 0% B to 50% B
in 50 min, linear from 50% B to 0% B in 10 min, iso-
cratic 0% B for 5 min; flow rate: 1mL/min. The mole-
cular weights were detected by electrospray mass
spectrometry on a HP 1100 MSD and correspond to the
calculated molecular masses for trimers 18 to 26. The
analytical data are listed in Table 2.
RNase L activation and cleavage of polyuridylic acid
[Poly(U)] substrate
Pure recombinant human RNase L was prepared by a
modification of a previously described procedure.38
Poly(U) was obtained commercially as a mixture of high
molecular weight uridine polymers. Using T4 RNA
ligase, the poly(U) was 30-labeled with 50-[32P]pCp and
then HPLC purified. This procedure and the assay have
been described previously.38,39 2 mL of a 1 0ꢂ cleavage
buffer (100 mM HEPES, pH 7.5, 1.0 M KCl, 50 mM
Mg(OAc)2, 10 mM ATP, and 143 mM 2-mercaptoetha-
nol) and 12–16 mL of RNase-free water were used in
each cleavage reaction. To this, 2 mL of a 1 0ꢂ solution
of 2-5A analogue (final concentrations 10ꢀ5–10ꢀ10 M)
and recombinant RNase L enzyme (final concentration
100 nM) were added, and lastly 2 mL of poly(U)-
[32P]pCp substrate (final concentration 10 mM in UMP
equivalents) to make a final volume of 20 mL. After a
15-min incubation at 30 ꢃC, 4 volumes of 5 mg/mL car-
Table 2. Physical data of trimers
Compd
Dionex pH 8
RP-18 pH 7
Empirical formula
ESPRAY-MS
Rt (min)
Rt (min)
Calcd
Found
18
19
20
21
22
23
24
25
26
9.3
9.2
9.9
9.3
9.3
7.5
7.4
9.4
9.3
16.7
19.9
42.5
21.4
34.0
15.6
26.2
37.1C
36.8
C31H40N15O19P3
C32H42N15O19P3
C37H44N15O19P3
C32H42N15O19P3
C34H46N15O19P3
C33H45N15O19P3
C37H53N15O19P3
35H46N15O19P3
1019.18
1033.20
1095.21
1033.20
1061.23
1048.22
1104.28
1073.23
1073.23
1019.50
1033.36
1095.4
1033.6
1061.49
1048.43
1104.49
1073.47
1073.47
C35H46N15O19P3

U. Munch et al. / Bioorg. Med. Chem. 11 (2003) 2041–2049
¨
2049
rier (yeast) RNA was added, and then 10 M ammonium
acetate to a final concentration of 2–2.5 M. After mixing
with 2 volumes of cold ethanol, the reaction mixtures
were left on ice for 30 min, and the precipitated RNA
pelleted with a brief spin at 4 ꢃC (12,000 g for 2 min).
The presence of cleaved fragments of poly(U)-[32P]pCp
was assessed by counting aliquots of the supernatant in
scintillation fluid.
10. Cirino, N. M.; Li, G.; Xiao, W.; Torrence, P. F.; Sil-
verman, R. H. Proc. Natl. Acad. Sci. U.S.A. 1997, 94,
1937.
11. Xiao, W.; Li, G.; Maitra, R. K.; Maran, A.; Silverman,
R. H.; Torrence, P. T. J. Med. Chem. 1997, 40, 1195.
12. Player, M. R.; Barnard, D. L.; Torrence, P. F. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 8874.
13. Wang, Z.; Chen, L.; Bayly, S. F.; Torrence, P. F. Bioorg.
Med. Chem. Lett. 2000, 10, 1357.
14. Verheijen, J. C.; van Roon, A.-M. M.; Meeuwenoord,
N. J.; Stuivenberg, H. R.; Bayly, S. F.; Chen, L.; van der
Marel, G. A.; Torrence, P. F.; van Boom, J. H. Bioorg. Med.
Chem. Lett. 2000, 10, 801.
15. Cramer, H.; Player, M. R.; Torrence, P. F. Bioorg. Med.
Chem. Lett. 1999, 9, 1049.
16. Verheijen, J. C.; van der Marel, G. A.; van Boom, J. H.;
Bayly, S. F.; Player, M. R.; Torrence, P. F. Bioorg. Med.
Chem. 1999, 7, 449.
17. Torrence, P. T.; Xiao, W.; Li, G.; Khamnei, S. Curr. Med.
Chem. 1994, 1, 176.
18. Player, M. R.; Torrence, P. F. Pharmacol. Ther. 1998, 78,
55.
19. Imai, J.; Torrence, P. F. J. Org. Chem. 1985, 50, 1418.
20. Imai, J.; Lesiak, K.; Torrence, P. F. J. Biol. Chem. 1985,
260, 1390.
21. Player, M. R.; Kalinichenko, E. N.; Podkopaeva, T. L.;
Mikhailopulo, I. A.; Seela, F.; Torrence, P. F. Biochem. Bio-
phys. Res. Commun. 1998, 245, 430.
22. Kitade, Y.; Alster, D. K.; Pabuccuoglu, A.; Torrence, P. T.
Bioorg. Chem. 1991, 19, 283.
Radiobinding assays
Crude lysate from insect cells (SF21), expressing a low
concentration of recombinant human RNase L, was
prepared according to previously described methodol-
ogy.40
The
radiobinding
assay
probe
p50A20(p50A20)2p50A30[32P]p50C30p, was synthesized by
the T4 RNA ligase-catalyzed addition of [32P]50pCp to
the 30 end of 2-5A, using a published procedure with
subsequent HPLC purification.38,39 To assay a given 2-
5A analogue, serial dilutions were prepared in water.
Each binding assay consisted of 5 mL of 2-5A analogue,
10 mL of SF21cell lysate, and 10 mL of a freshly pre-
pared master mix that consisted of concentrated Tris
buffer, ATP stock (pH 7.0), and sufficient 2-5A-
[32P]pCp probe to give final reaction concentrations of
10 mM Tris–HCl (pH 7.6), 85 mM KCl, 2 mM
Mg(OAc)2, 0.2 mM ATP, 5% (v/v) glycerol and
ꢄ0.1nM probe. The order of addition for each assay
was 2-5A or analogue, then master mix, then lysate.
Assay mixtures were incubated at 4 ꢃC for 2 h, after
which they were applied to nitrocellulose filters pre-
rinsed in 1 ꢂ Tris buffer (described above). Samples
were left to stand for ꢄ3 min, then washed (3 ꢂ) with
water. The filters were placed in scintillant and counted
in a liquid scintillation counter.38,39
23. MacMillan, A. M.; Verdine, G. L. J. Org. Chem. 1990, 55,
5931.
24. MacMillan, A. M.; Verdine, G. L. Tetrahedron 1991, 47,
2603.
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