Activation of murine RNase L by isopolar 2′-phosphonate analogues of 2′,5′ oligoadenylates

Article abstract of DOI:10.1021/jm050401v

To determine the influence of methylene group insertion in the internucleotide linkage on the binding process of 2′,5′- oligoadenylates to RNase L, a series of 2′-phosphonate-modified trimers and tetramers were synthesized from appropriate monomeric units

Full text of DOI:10.1021/jm050401v

J. Med. Chem. 2006, 49, 3955-3962
3955
Activation of Murine RNase L by Isopolar 2′-Phosphonate Analogues of 2′,5′ Oligoadenylates
Ondrej Pav, Eva Protivinska, Martina Pressova, Michaela Collinsova, Jiri Jiracek,† Jan Snasel,‡ Milena Masojidkova,
Milos Budesinsky, and Ivan Rosenberg*
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 166 10 Prague 6, Czech Republic
ReceiVed April 27, 2005
To determine the influence of methylene group insertion in the internucleotide linkage on the binding process
of 2′,5′-oligoadenylates to RNase L, a series of 2′-phosphonate-modified trimers and tetramers were
synthesized from appropriate monomeric units and evaluated for their ability to bind to murine RNase L.
Tetramers pAAXA modified by ribo-, arabino-, or xylo-2′-phosphonate unit X in the third position were
capable of binding to RNase L in nanomolar concentrations. The replacement of the first residue (pXAAA),
or both the first and the third residues (pXAXA), was also tolerated by the enzyme. In contrast, in all cases,
the replacement of the second residue (pAXAA) resulted in the significant decrease of binding ability.
Additionally, no more than two phosphonate modifications in the tetramer were allowed to retain the binding
affinity to the enzyme. Although all three tetramers pAAXA were found to be potent enzyme binders, only
tetramers modified by ribo- and xylo-2′-phosphonate unit X activated the RNase L-catalyzed cleavage of
the RNA substrate. Surprisingly, tetramer pAAXA, modified by arabino-2′-phosphonate unit X, did not
activate the enzyme and can be considered a potent antagonist. In comparison with their natural counterpart,
the phosphonate analogues of the pA4 exhibit superior resistance toward nucleases present in the murine
spleen homogenate.
Introduction
internucleotide linkage. Among these, chiral phosphorothioate
2-5A trimers7-10 are capable of binding to RNase L and
activating the enzyme in the order of pA(RP)A(RP)A > pA-
(SP)A(RP)A > pA(RP)A(SP)A, with an efficiency comparable
to that of natural pAAA. However, the pA(SP)A(SP)A trimer as
well as a nonchiral phosphorodithioate trimer11 bind to RNase
L but do not activate it (RP and SP denote the configuration of
the phosphorus atom of phosphorothioate internucleotide link-
age).
The biological application of an antisense oligonucleotide
covalently linked with 2′,5′-oligoadenylate remarkably increased
the antisense effect of the oligonucleotide. Under these condi-
tions, target viral mRNA was cleaved with RNase L, the
cleaving activity of which was stimulated by the 2-5A part of
the oligonucleotide chimeras. In contrast to the RNase H
mechanism of antisense oligonucleotide action, there is no
limitation concerning the structure of the antisense oligonucle-
otide.12,13 These anticancer and antiviral oligonucleotide chi-
meras demonstrated the possibility of the successful application
of the antisense strategy combined with the recruitment and
activation of RNase L to the destruction of an antisense
oligonucleotide-selected RNA. In particular, the potent inhibition
of RSV and tumor proliferation was reported.5,14-18
Any successful biological application of oligoadenylates
would depend on the stability of these compounds in cells, in
fact, on the resistance of internucleotide linkage against nuclease
cleavage. In connection with this requirement, the modified
oligonucleotides bearing the O-P-C and C-P-O internucle-
otide linkages were shown to offer absolute protection against
nucleases releasing the 3′(or 2′-)- and 5′-nucleotide units,
respectively. These compounds containing structurally diverse
types of isosteric, nonisosteric, or chiral phosphonate linkages
have attracted our attention for many years.19-25
The short 2′,5′-oligoadenylates (2-5A) play an important role
in the interferon-induced antiviral defense mechanism of cells.1,2
In the presence of double-stranded RNA, interferon induces the
expression of the 2′,5′-oligoadenylate synthetase utilizing ATP
as a substrate for the synthesis of the 5′-phosphorylated 2-5As,
(pp)p5′Ap(Ap)nA2′ (where n ) 1-8, but mainly 3 or 4).3 These
oligonucleotides bind to RNase L, a latent endoribonuclease
and, subsequently, activate it. This enzyme, in its active form,
is capable of cleaving single stranded RNA and thus preventing
the expression of viral or bacterial proteins. The activity of
RNase L is terminated/regulated by a specific 2′,5′-exonuclease
that cleaves 2-5A to AMP and ATP. It has been speculated that
2-5A may also play an additional role in the regulation of cell
growth and differentiation and apoptosis.2,4
The effort to develop 2-5A analogues capable of activating
RNase L, at least as potent as the natural 2-5A itself, has led to
the definition of enzyme structural requirements related to the
particular parts of the 2-5A. It was found that every functionality
of each individual nucleotide moiety of 2-5A contributed highly
specifically to the binding to RNase L and its activation. Thus,
the conformation of ribose moieties, the nature of the adenine-
based nucleobase, the 2′,5′-phosphate backbone, and the pres-
ence of 5′-(poly)phosphate group were determined the most
important factors influencing RNase L activity.2,5 Recently, the
resolution of the crystal structure of the ankyrin domain of
RNase L in a complex with 2-5A trimers provided more detailed
information about the mode of the binding process and
confirmed some experimentally obtained structure-activity
relationships.6
Surprisingly, among a number of prepared oligoadenylate
analogues, there are only two types of 2-5As with modified
This work deals with the role of the one type of C-
phosphonate internucleotide linkage in 2-5A analogues in the
process of binding to and the activation of murine RNase L.
We synthesized three sets of trimeric and tetrameric 2-5A
* Corresponding author. Tel: +420 220 183 381. Fax: +420 220 183
531. E-mail: ivan@uochb.cas.cz.
† E-mail: jiracek@uochb.cas.cz.
‡ E-mail: snasel@uochb.cas.cz.
10.1021/jm050401v CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/07/2006

3956 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
PaV et al.
linkages into the 2-5A chain was seen as a factor for the
remarkable increase in stability of the modified 2-5As against
nucleases.
Results and Discussion
Preparation of Monomers. Phosphonate monomers 4a-c
(Scheme 1) were prepared by multistep synthesis from 6-N-
benzoyl derivatives of adenosine (ribo-A), arabinofuranosylad-
enine (ara-A), and xylofuranosyladenine (xylo-A). The dimethoxy-
tritylation of ribo-A and ara-A derivatives resulted in a mixture
of 2′(3′),5′-bis-O-dimethoxytrityl derivatives, which afforded,
after resolution on silica gel,26 approximately 40% of the desired
3′,5′-bis-O-dimethoxytrityl derivatives 1a (ribo) and 1b (ara-
bino), respectively, as the starting compounds for the phospho-
nylation reaction. In the xylo series, the more readily accessible
3′,5′-O-ethoxymethylene derivative 5 was prepared from xylo-A
and triethyl orthoformate. The protected nucleosides with free
2′-hydroxy groups 1a, 1b, and 5 were phosphonylated with
diisopropyl-tosyloxymethylphosphonate. Upon acidic deprotec-
tion, they provided the N-protected 2′-phosphonates 2a-c. 5′-
O-Dimethoxytritylation of these compounds followed by ben-
zoylation of the 3′-hydroxy group yielded the fully protected
phosphonates 3a-c. The deprotection of isopropyl ester groups
with bromotrimethylsilane and subsequent esterification of free
phosphonic acids with 4-methoxy-1-oxido-2-pyridylmethanol in
the presence of DCC afforded appropriate ribo-4a, arabino-
4b, and xylo-4c monomers.
Figure 1. Binding affinities of modified oligoadenylates (% at 1 µM
concentration) to murine RNase L related to natural pA4 (6). (For values
see Table 1.)
Table 1. Binding Affinities (% at 1 µM concentration) of Phosphonate
Oligoadenylates to Murine RNase L Related to Natural pA4 (6)
binding affinity (%)
2′-5′ oligoadenylate
unit X sugar configuration
no.
abbreviation
ribo-(a)
arabino-(b)
xylo-(c)
6
7
8
pAAAA
pXAAA
pAXAA
pAAXA
pXXAA
pAXXA
pXAXA
pXXXA
pXAA
100
81
53
99
25
44
92
28
39
0
40
37
99
24
33
51
0
49
33
0
86
67
97
51
88
93
43
64
41
45
9
10
11
12
13
14
15
16
Synthesis of Oligoadenylates. The synthesis of the library
of 2-5A analogues was performed by sequential replacement
of adenosine 2′-phosphate units in oligoadenylates pA4 and pA3
by ribo (Xa)-, arabino (Xb)-, or xylo (Xc)-2′-phosphonate units
(Figure 1, Table 1).
pAXA
pXXA
25
analogues in which one or more adenylate residues were
replaced by either ribo-, arabino-, or xylo-adenosine-2′-phos-
phonate unit X (a-c) (Figure 1, Table 1). The newly formed
2′-5′ linkage, isopolar to the phosphodiester one but nonisosteric
to it, contains an extra methylene group inserted between the
phosphorus atom and the 2′-oxygen atom of the sugar residue.
The presence of the bridging methylene group makes the linkage
one atom longer in comparison with phosphodiester one, and
these compounds thus exhibit more conformational flexibility
than natural pA4. Moreover, the introduction of phosphonate
The oligonucleotides were synthesized from the 2′- to the
5′-end on CPG-riboA5′-OH (loading 25 µmol/g) on a 0.5 µmol
scale using the GenSyn V02 DNA/RNA synthesizer. The
protected adenosine 2′-phosphoramidite and the appropriate 2′-
phosphonates 4a-c were introduced into the oligonucleotide
chain by phosphoramidite and phosphotriester methods, respec-
tively. The 5′-terminal phosphate group was introduced by 2-[2-
(4,4′-dimethoxytrityloxy)ethylsulfonyl]ethyl-(2-cyanoethyl)-
(N,N-diisopropyl)-phosphoramidite (DESEP). Protocols for the
synthesis and deprotection procedure are provided in the
Scheme 1a
a (i) (a) TsOCH2PO(OiPr)2, NaH, DMF, r.t.; (b) 80% AcOH, r.t. (ii) (c) DMTr-Cl, py, r.t; (d) BzCN, Et3N, CH3CN, r.t. (iii) (e) Me3SiBr, 2,6-lutidine,
CH3CN, r.t.; (f) 4-methoxy-1-oxido-2-pyridylmethanol, DCC, py, r.t.

ActiVation of Murine RNase L
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3957
Table 2. EC50 Values of the Most Potent Phosphonate-Modified
Oligoadenylates
What is the explanation for this low binding affinity to the
enzyme? The second adenylate unit seems to be crucial for the
binding process. The only data in the literature related to the
modification of the sugar component of the second unit concerns
the 3′-hydroxy group, which is the essential structural motif for
binding of the oligoadenylates to RNase L.2,6 The removal of
the 3′-hydroxyl or its replacement with fluoro or methoxy
moieties were found to result in the inability of the respective
oligoadenylates to bind to and activate RNase L.28-31 The
changes in the sugar conformation of the second unit exerted a
similar effect. This finding could explain the low affinity of
the xylo tetramer pAXAA (8c) in which the configuration
(spatial orientation) of the 3′-hydroxyl differs from that of the
ribo configuration. Surprisingly, the ribo tetramer pAXAA (8a)
was also found inactive, despite the correct configuration on
the C3′ atom bearing the hydroxy group. In addition, the NMR
study aimed at the determination of the C3′-endo and C2′-endo
conformations and performed with the modified ribo dimer
(2′,5′)-XA and the unmodified ribo dimer (2′,5′)-AA revealed
the conformational identity of both dimers.32 These findings
suggest that the mutual orientation of the second and third units
as well as the spatial shift of the charged phosphorus moiety in
the pAXAA tetramer, given by the conformation of the
methylene-extended internucleotide linkage C2′-O-CH2-P-O-
C5′′, also plays an important role in the binding process. In
addition, the hydroxyls of the phosphodiester group connecting
the second and third units of 2-5A interact highly specifically
with amino acid residues in the binding pocket.6 The spatial
shift of the charged phosphorus moiety may disrupt these
interactions, and this effect may thus result in the decrease of
the binding affinity to the enzyme. We also cannot exclude a
steric conflict of the 2′-O-methylene group with amino acid
residues in the RNase L binding pocket. In this respect,
interesting results could be obtained with oligoadenylates with
a ribo-configured second unit but with regioisomeric phospho-
nate internucleotide linkage C2′-O-P-CH2-O-C5′′ connecting the
second and third units. Finally, the reasons for the low binding
activity in the case of arabino tetramer pAXAA (8b) could be
seen both in the changed conformation of the sugar component
of the second unit that may distort the 3′-hydroxy group and in
the geometry of the modified internucleotide linkage in which
the phosphorus moiety is oriented in the direction opposite to
that in the ribo compound. No more than two phosphonate
modifications in the oligoadenylate can be made to retain its
binding affinity to the enzyme. Fully modified oligoadenylates
pXXXA (13) and pXXA (16) showed considerably reduced
binding ability. In addition, trimeric oligoadenylates 14-16 were
unable to compete effectively with labeled pA4pCp.
2′-5′ oligoadenylate
no.
abbreviation
pAAAA
EC50a (nM)
0.9
6
7a
7c
pXAAA
396
421
9a
9b
9c
pAAXA
8.3
3.6
1.3
11c
pAXXA
pXAXA
393
12a
12c
267
323
a EC50 represents the concentration of the oligoadenylate analogue that
gives a 50% displacement of radioactive pA4[32P]pCp; for details, see the
Experimental Section.
Experimental Section and the Supporting Information. All
prepared oligoadenylates 6-16 were purified by HPLC, and
the purity was checked by RP HPLC in 0.1 M TEAA and 0.1%
TFA. The molecular weights of the products were confirmed
by MALDI-TOF MS.
Binding of Modified Oligoadenylates to Murine RNase
L. As was mentioned above, one of the aims of the presented
study was to define the role of stereochemical factors influencing
the process of binding of oligoadenylates to RNase L, especially
those associated with internucleotide linkage. Thus, we intro-
duced the ribo, arabino, and xylo-configured 2′-phosphonate
units X (a-c) (Figure 1) into individual positions of oligoad-
enylates. The introduction of these units into oligoadenylates
variously changed the length of the internucleotide linkage, the
conformation of the sugar-phosphate backbone, and the
conformation of the sugar part of the respective phosphonate
unit.
The affinities of natural 2-5A and its phosphonate-modified
analogues to RNase L were examined in a radiobinding assay
according to Silverman et al.27 This assay is based on the ability
of modified oligoadenylates to compete with a [32P]-labeled pA4-
pCp probe for the specific binding to RNase L. The assay was
performed with a 126 000g supernatant prepared from the
murine spleen homogenate. The results are illustrated in Figure
1 and summarized in Table 1. For the eight most potent
compounds, we determined the EC50 values (Table 2).
The data shows that the binding affinity of modified 2-5A to
murine RNase L is dependent more on the position and the
number of modified units X (a-c) than on the conformation
and/or configuration of the appropriate sugar part. Phosphonate
tetramers pAAXA (9a-c) modified in the third position are able
to bind to RNase L with an efficiency comparable to that of
natural pAAAA (6) (Table 2). Also, tetramers pXAAA (7a, 7c),
pAXXA (11c), and pXAXA (12a, 12c) containing modifications
(i) at the first position (7a, 7c), (ii) both at the second and the
third positions (11c), and (iii) both at the first and the third
positions (12a, 12c) are still efficient competitors of the natural
pAAAA (6).
All modified oligoadenylates without the 5′-terminal phos-
phate exhibited considerably reduced binding to RNase L
(structures and data not shown). This finding is consistent with
an earlier conclusion that at least one 5′-terminal phosphate
group is necessary for the effective activation of RNase L.33,34
RNase L-Catalyzed Cleavage of 5′-[32P]-r(C11U2C7). The
activation of murine RNase L by selected binders (6, 7a, 7c,
9a-c, 11c, 12a, 12c) was determined by monitoring the ability
of the enzyme to cleave the specific radiolabeled substrate 5′-
[32P]-r(C11U2C7). For the cleavage assay, we modified the
procedure described by Silverman.35 In our methodology,
streptavidin-coated magnetic beads modified by biotinylated pA4
17 were used as an affinity matrix for the selective immobiliza-
tion and purification of RNase L from the murine spleen
homogenate.
However, tetramers pAXAA (8a-c), pXXAA (10a-c),
pAXXA (11a, 11b), and pXXXA (13a-c), bearing modification
at the second position, exhibited a binding affinity below 80%,
except for pAXXA (11c) (Table 1). Under experimental condi-
tions, the 80% binding affinity at 1 µM concentration ap-
proximately corresponds to the EC50 value, which is three orders
of magnitude higher than the EC50 value of potent RNase L
activators (data not shown).

3958 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
PaV et al.
Figure 2. The time-dependent cleavage of 5′-[32P]-r(C11U2C7) to 5′-
[32P]-r(C11Up) by murine RNase L in the presence of 1 µM pA4 (6)
and 1 µM modified tetramers pAAXA (9a-c) and in the absence of
the added activator (blank).
Figure 3. Degradation of 2-5A and its analogues in the murine spleen
supernatant.
resistance in the murine spleen supernatant. No cleavage of the
internucleotide linkages in compounds 9a-c and 12a, 12c was
observed. In this case, only slow 5′-dephosphorylation caused
by a phosphomonoesterase took place. These findings suggest
that under experimental conditions an exonuclease releasing 5′-
mononucleotides from the 3′-end of oligonucleotide is probably
responsible for the cleavage of compounds 7a, 7c and 6. This
exonuclease did not cleave oligoadenylates 9a-c and 12a, 12c
because of the presence of the noncleavable C2′-O-CH2-P-O-
C5′′ internucleotide linkage in the cleavage site (the enzyme
cannot cleave off of the first 5′-mononucleotide).
We found that the incubation of magnetic bead-bound RNase
L with the 5′-[32P]-r(C11U2C7) substrate in the absence of free
oligoadenylate resulted in only 15% cleavage (see blank, Figure
2) of the substrate after 90 min. However, a considerable
enhancement of specific RNase L cleaving activity was achieved
in the presence of 1 µM natural activator pA4 6 (Figure 2). Also,
tetramers pAAXA 9a and 9c modified by ribo- and xylo-
phosphonate units, respectively, were found to be potent
activators of RNase L-catalyzed cleavage. Moreover, tetramer
9c seems to be a more potent activator than natural pA4 (Figure
2). Surprisingly, the closely related arabino tetramer 9b was
found to be completely inactive in the assay (the extent of
substrate cleavage did not surpass a blank experiment; Figure
2), and therefore, tetramer 9b can be considered an inhibitor of
RNase L. In all of these cases, the only product of cleavage
was 5′-[32P]-r(C11Up). This finding is in agreement with that
obtained with the human enzyme as reported by Carroll et al.36
Conclusion
In summary, we prepared a series of phosphonate-modified
oligoadenylate trimers and tetramers and examined their binding
affinity to murine RNase L. We found that the replacement of
the first (pXAAA 7a, 7c), the third (pAAXA 9a-c), and both
the first and the third natural residues (pXAXA 12a, 12c) by
the modified ones preserved the binding affinity to RNase L.
In this study, we have selected several potent binders of murine
RNase L. We also examined the ability of these selected
compounds to activate RNase L. Tetramers pAAXA 9a and 9c
modified by ribo- and xylo-2′-phosphonate units, respectively,
activated RNase L with an efficiency comparable to that of
natural pA4. Surprisingly, tetramer pAAXA 9b modified by the
arabino-2′-phosphonate unit was unable to activate RNase L
and seems to be an inhibitor of the enzyme. The binders with
EC50 values of about 300 nM were unable to activate RNase L
under experimental conditions. The introduction of the phos-
phonate internucleotide linkage of the C2′-O-CH2-P-O-C5′′ type
remarkably increased the stability of the modified oligoadenyl-
ates against the nucleases of the murine spleen homogenate in
comparison with natural 2-5A. The findings achieved in this
work seem to be a good starting point for further research in
this area. We have already started the work on the synthesis of
modified oligoadenylates containing the regioisomeric C2′-O-
P-CH2-O-C5′′ type of phosphonate internucleotide linkage,
which differs from the one described here in the position of the
extra methylene group within the linkage. Our attention will
be focused on the modification of the 5′-terminal phosphate
group of oligoadenylates to eliminate its instability against
phosphomonoesterases.
Tetramers pAAXA 9a-c differ from natural oligoadenylates
in the conformation of the sugar component of the third unit
and in stereochemical arrangement of the internucleotide linkage.
Thus, it seems that these stereochemical changes in the third
position could be responsible for the properties that classify
modified oligoadenylates as either agonists or antagonists in
the activation process of RNase L. Concerning compound 9b,
it is very probably a potent antagonist. Other selected binders
(7a, 7c, 11c, 12a, 12c) with EC50 values of about 300 nM were
unable to activate RNase L under experimental conditions (data
not shown).
Nuclease Stability of Modified Oligoadenylates. A rapid
degradation of phosphodiester oligonucleotides by nucleases in
body fluids is one of the most important limitations to their in
vivo use. The increase of the half-life values of these compounds
could thus bring the remarkable benefit of a prolonged antiviral
effect. In connection with this, the stability of 2-5A phosphonate
analogues against the nucleases of the murine spleen supernatant
was examined under the conditions of the binding assay. The
course of cleavage was monitored by HPLC, and the results
are summarized in Figure 3. Phosphodiester-linked pAAAA (6)
and oligoadenylates pXAAA (7a, 7c) are rapidly degraded in
the murine spleen supernatant (half-life for pA4 is approximately
10 h at 4 °C). In contrast, the most potent oligoadenylates
pAAXA (9a-c) and pXAXA (12a, 12c) (see Table 2), with
EC50 values in the nanomolar range, exhibited superior nuclease
Experimental Section
The course of the reactions was checked by TLC on silica gel
HPTLC, Alufolien Kieselgel 60 F254 (Merck) foils, and the

ActiVation of Murine RNase L
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3959
products were detected by UV light or by spraying with 5%
ethanolic solution of sulfuric acid and subsequent heating (detection
of O-DMTr derivatives, deep orange spots). Preparative column
chromatography (PLC) was carried out on 40-60 µm spherical
silica gel (Fluka). The sorbent was neutralized with Et3N (0.1%,
v/v). Elution was performed at the rate of 40 mL/min. PLC and
TLC were carried out with the following solvent systems (v/v):
chloroform-ethanol 9:1 (C-1), ethyl acetate-toluene 1:1 (T-1), ethyl
acetate-acetone-ethanol-water 4:1:1:1 (H-1), and ethyl acetate-
acetone-ethanol-water 12:2:2:1 (H-2). HPLC analysis was per-
formed on a column of reverse-phase (4.6 × 150 mm) Nucleosil
100-5 C18 (Macharey-Nagel), either isocratically at various con-
centrations of methanol in 0.1 M TEAA or by the gradient of
methanol in the same buffer. Mass spectra were recorded on a ZAB-
EQ (VG Analytical) instrument, using the FAB (ionization by Xe,
accelerating voltage 8 kV) technique with glycerol and thioglycerol
as matrixes. MALDI TOF MS was performed on a Reflex IV
(Bruker-Daltonics) instrument. 1H and 13C NMR spectra were
measured on a Varian UNITY-500 instrument (1H at 500 MHz,
13C at 125.7 MHz) in d6-DMSO or CDCl3 and were referenced
either to TMS as internal standard (1H in CDCl3) or to the solvent
signal using the following relationships: δH(DMSO) ) 2.50, δC-
(DMSO) ) 39.70, and δC(CDCl3) ) 77.00.
General Procedures. Method A. Preparation of 5′-O-
Dimethoxytrityl 2′-phosphonates. Dimethoxytrityl chloride (1.1
mmol) was added under stirring to a solution of 2′-phosphonate
(2a-c) (1.0 mmol) in pyridine (10 mL), and the mixture was stirred
overnight at r.t. (TLC in system C-1). The reaction was quenched
by the addition of Et3N (1 mL) and methanol (5 mL), and the
mixture was concentrated under reduced pressure. The residue was
dissolved in chloroform (50 mL) and extracted with water (3 × 20
mL). The organic layer was dried over anhydrous sodium sulfate
and evaporated. The product was purified by chromatography on
silica gel (elution with a gradient of 0-8% ethanol in chloroform).
Method B. Preparation of 3′,5′-O-Bis-dimethoxytrityl Nucleo-
sides 1a, b. Dimethoxytrityl chloride (1.1 mmol) was added under
stirring to a solution of 6-N-benzoyl adenosine (1.0 mmol) in
pyridine (10 mL), and the mixture was stirred overnight at r.t. (TLC
in system C-1). After that, additional dimethoxytrityl chloride (1.3
mmol) and Et3N (2.0 mmol) were added, and the mixture was set
aside for 1 day at r.t. (TLC in system T-1) to produce a mixture of
2′,5′- and 3′,5′-bis-dimethoxytrityl derivatives. The reaction was
quenched by methanol (5 mL), and the mixture was concentrated
under reduced pressure. The residue was dissolved in chloroform
(50 mL) and extracted with water (3 × 20 mL), and the organic
layer was dried over anhydrous sodium sulfate and evaporated. The
product was chromatographed on silica gel (elution with a gradient
of 0-50% ethyl acetate in toluene) to afford the 2′,5′- and 3′,5′-
bis-dimethoxytrityl derivatives as faster and slower TLC com-
pounds, respectively. Only the 3′,5′-regioisomer was used for further
reactions.
Method E. Removal of Isopropyl Ester Groups. Bromotri-
methylsilane (4 mmol) was added to a solution of phosphonate
diester (3a-c) (1.0 mmol) and 2,6-lutidine (8 mmol) in acetonitrile
(10 mL). The reaction mixture was left aside overnight at r.t. and
then concentrated under reduced presure (TLC in system H-1). The
residue was treated with 2 M TEAB (5 mL) and methanol (10 mL),
and the solution was evaporated and the residue co-distilled with
ethanol and toluene. The crude nucleoside phosphonic acids were
used for subsequent steps without further purification.
Method F. Preparation of MOP Monoesters 4a-c. DCC (5.0
mmol) was added to a solution of nucleoside phosphonic acid (1.0
mmol) and 4-methoxy-1-oxido-2-pyridylmethanol (3.0 mmol) in
pyridine (5 mL), and the reaction mixture was stirred for 3 days at
r.t. (TLC in system H-1). The reaction mixture was diluted with
water (3.3 mL) to achieve a 60% concentration of pyridine, heated
overnight at 50 °C to cleave off one ester group, and concentrated
under reduced pressure. The product was purified by chromatog-
raphy on silica gel (elution gradient from ethyl acetate to 100%
H-2 and then from H-2 to 100% H-1) and freeze-dried from
dioxane.
Method G. Removal of 3′,5′-O-Bis-dimethoxytrityl and 3′,5′-
Ethoxymethylene Protecting Groups. Bis-dimethoxytrityl and
ethoxymethylene derivatives (1.0 mmol) were treated with 80%
acetic acid (10 mL) for 12 and 48 h, respectively, at r.t. (TLC in
system C-2) and then concentrated under reduced presure. The
product was purified by chromatography on silica gel (elution with
a linear gradient of 0-10% ethanol in chloroform).
6-N-Benzoyl-9-(3,5-O-Bis-dimethoxytrityl-â-D-ribofurano-
syl)adenine (1a). Compound 1a was prepared using method B from
6-N-benzoyl-9-(â-D-ribofuranosyl)adenine (3.5 g; 9.5 mmol). Chro-
matography on silica gel afforded 4.8 g (52%) of the faster TLC
compound (2′,5′-derivative) and 3.4 g (36%) of the slower TLC
compound (3′,5′-derivative).
Diisopropyl-[1-(6-N-benzoyladenin-9-yl)-â-D-ribofuranos-2-
O-yl]methylphosphonate (2a). Compound 2a was prepared using
method C from 6-N-benzoyl-9-(3,5-O-bis-dimethoxytrityl-â-D-
ribofuranosyl)adenine (1a) (1.6 g; 1.6 mmol) followed by method
G. Yield: 0.67 g (75%).
Diisopropyl-[3-O-benzoyl-1-(6-N-benzoyladenin-9-yl)-5-O-
dimethoxytrityl-â-D-ribofuranos-2-O-yl]methylphosphonate (3a).
Compound 3a was prepared using method A from diisopropyl-[1-
(6-N-benzoyladenin-9-yl)-â-D-ribofuranos-2-O-yl]methylphospho-
nate (2a) (0.67 g; 1.2 mmol) followed by method D. Yield: 0.98
g (85%).
4-Methoxy-1-oxido-2-picolyl-[3-O-benzoyl-1-(6-N-benzoylad-
enin-9-yl)-5-O-dimethoxytrityl-â-D-ribofuranos-2-O-yl]meth-
ylphosphonate (4a). Compound 4a was prepared using method E
from diisopropyl-[3-O-benzoyl-1-(6-N-benzoyladenin-9-yl)-5-O-
dimethoxytrityl-â-D-ribofuranos-2-O-yl]methylphosphonate (3a) (0.88
g; 0.9 mmol) followed by method F. Yield: 0.54 g (59%).
6-N-Benzoyl-9-(3,5-O-bis-dimethoxytrityl-â-D-arabinofurano-
syl)adenine (1b). Compound 1b was prepared using method B from
6-N-benzoyl-9-(â-D-arabinofuranosyl)adenine (6.7 g; 18.0 mmol).
Chromatography on silica gel afforded 4.9 g (28%) of the faster
TLC compound (3′,5′-derivative) and 8.3 g (47%) of the slower
TLC compound (2′,5′-derivative).
Diisopropyl-[1-(6-N-benzoyladenin-9-yl)-â-D-arabinofuranos-
2-O-yl]methylphosphonate (2b). Compound 2b was prepared
using method C from 6-N-benzoyl-9-(3,5-O-bis-dimethoxytrityl-
â-D-arabinofuranosyl)adenine (1b) (3.8 g; 3.9 mmol) followed by
method G. Yield: 1.6 g (76%).
Method C. Preparation of 2′-O-Diisopropyl-phosphonomethyl
Nucleosides 2a, b. Sodium hydride (3.0 mmol) was added at 0 °C
to a stirred solution of 3′,5′-bisdimethoxytrityl derivative (1a, b)
(1.0 mmol) and diisopropyl-tosyloxymethylphosphonate (2.0 mmol)
in DMF (10 mL), and the reaction mixture was left to warm
gradually to r.t. and then stirred overnight (HPLC in the system
with 75%-100% MeOH). The reaction was quenched by the
addition of glacial acetic acid (0.11 mL; 2.0 mmol) at 0 °C, and
the mixture was concentrated under reduced pressure. The product
was purified by chromatography on silica gel (elution with a
gradient of 0-50% ethyl acetate in toluene).
Method D. Preparation of 3′-O-Benzoyl Nucleosides 3a-c.
Benzoyl cyanide (1.5 mmol) was added under stirring to a solution
of 5′-dimethoxytrityl phosphonate derivative (1.0 mmol) and
triethylamine (0.2 mmol) in acetonitrile (10 mL), and the mixture
was stirred overnight at r.t. (TLC in system T-1). The reaction was
quenched by methanol (5 mL), and the mixture was concentrated
under reduced pressure. The product was purified by chromatog-
raphy on silica gel (elution with a gradient of 0-50% ethyl acetate
in toluene).
Diisopropyl-[3-O-benzoyl-1-(6-N-benzoyladenin-9-yl)-5-O-
dimethoxytrityl-â-D-arabinofuranos-2-O-yl]methylphosphon-
ate (3b). Compound 3b was prepared using method A from
diisopropyl-[1-(6-N-benzoyladenin-9-yl)-â-D-arabinofuranos-2-O-
yl]methylphosphonate (2b) (1.6 g; 2.9 mmol) followed by method
D. Yield: 2.3 g (82%).
4-Methoxy-1-oxido-2-picolyl-[3-O-benzoyl-1-(6-N-benzoylad-
enin-9-yl)-5-O-dimethoxytrityl-â-D-arabinofuranos-2-O-yl]meth-
ylphosphonate (4b). Compound 4b was prepared using method E
from diisopropyl-[3-O-benzoyl-1-(6-N-benzoyladenin-9-yl)-5-O-

3960 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
PaV et al.
dimethoxytrityl-â-D-arabinofuranos-2-O-yl]methylphosphonate (3b)
(2.2 g; 2.3 mmol) followed by method F. Yield: 1.5 g (65%).
Diisopropyl-[1-(6-N-benzoyladenin-9-yl)-â-D-xylofuranos-2-
O-yl]methylphosphonate (2c). Toluenesulfonic acid (2.5 mL of
10% solution in dioxane) was added under stirring to a solution of
6-N-benzoyl-9-(â-D-xylofuranosyl)adenine (0.9 g; 2.5 mmol) and
triethyl orthoformate (0.5 mL; 3.0 mmol) in DMF (20 mL), and
the mixture was stirred for 5 h at r.t. (TLC in system C-1). The
reaction was quenched by the addition of 2 M TEAB (5 mL), and
the mixture was concentrated under reduced pressure. The residue
was dissolved in chloroform (100 mL) and extracted with saturated
sodium bicarbonate (3 × 20 mL). The organic layer was dried over
anhydrous sodium sulfate and evaporated. Crude 3′,5′-ethoxy-
methylene derivative 5 was then converted, according to method
C followed by method G, to phosphonate 2c. Yield: 0.8 g (60%).
Diisopropyl-[3-O-benzoyl-1-(6-N-benzoyladenin-9-yl)-5-O-
dimethoxytrityl-â-D-xylofuranos-2-O-yl]methylphosphonate (3c).
Compound 3c was prepared using method A from diisopropyl-[1-
(6-N-benzoyladenin-9-yl)-â-D-xylofuranos-2-O-yl]methylphospho-
nate (2c) (0.8 g; 1.5 mmol) followed by method D. Yield: 1.1 g
(76%).
4-Methoxy-1-oxido-2-picolyl-[3-O-benzoyl-1-(6-N-benzoylad-
enin-9-yl)-5-O-dimethoxytrityl-â-D-xylofuranos-2-O-yl]meth-
ylphosphonate (4c). Compound 4c was prepared using method E
from diisopropyl-[3-O-benzoyl-1-(6-N-benzoyladenin-9-yl)-5-O-
dimethoxytrityl-â-D-xylofuranos-2-O-yl]methylphosphonate (3c)
(1.1 g; 1.1 mmol) followed by method F. Yield: 0.7 g (64%).
Synthesis and Deprotection of Oligoadenylates 6-16. Oli-
goadenylates 6-16 were synthesized using the oligoadenylate solid-
phase synthesis protocol (Supporting Information). The 5′-terminal
phosphate was introduced by 2-[2-(4,4′-dimethoxytrityloxy)-
ethylsulfonyl]ethyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphor-
amidite (DESEP).
The deprotection of 6-16 was achieved in three steps. The
4-methoxy-1-oxido-2-picolyl (MOP) ester group of phosphonate
moieties was removed by treatment with thiophenol in DMF
(PhSH-Et3N-DMF 1:1.4:2) at r.t. for 6 h. The removal of the
2-cyanoethyl protecting groups of the phosphodiester residues, 2-(2-
hydroxyethylsulfonyl)ethyl ester group of the 5′-phosphate moiety,
N-benzoyl groups of adenine residues as well as the release of
oligomers from CPG was achieved in 8 M ethanolic methylamine
at r.t. for 16 h. The 2′-O-silyl protecting groups were cleaved by
treatment with 1 M TBAF in THF at r.t. for 16 h. Desalting and
prepurification on Oligopure cartridges (Hamilton) gave deprotected
oligoadenylates, which were finally purified by the RP HPLC using
a linear gradient of acetonitrile (0-10%, 50 min) in 0.1 M TEAB
or TEAA and freeze-dried.
pCp, and 2.5 nmol of ATP were mixed with 30 U (0.75 µL) of T4
RNA ligase (Amersham Biosciences) in a total volume of 50 µL
of 100 mM HEPES/NaOH (pH 7.6), 15 mM MgCl2, 6.6 mM DTT,
and 20% (v/v) DMSO (ligation buffer). The reaction proceeded at
4 °C, and after 15 h, additional 10 nmol of pCp, 2.5 nmol of ATP,
and 20 U of T4RNA ligase in 8 µL of ligation buffer were added.
The reaction was checked on a Luna C18 column (5 µm, 150 ×
4.6 mm, Phenomenex) at 260 nm using a linear gradient of
acetonitrile in aqueous 0.1 M TEAB (0-12% in 70 min) at a flow
rate of 1 mL/min. The same conditions were used for the isolation
of the product (pA4pCp). The molecular weight of the products
was confirmed by MALDI-TOF MS.
32
Synthesis of p-5′-(2-5)A
4-[ P]pC-3′-p. The synthesis of 32P-
32
labeled, radioactive pA
4-[ P]pCp was also performed according
to Silverman et al. with several modifications.27 Thus, 0.275 nmol
of pA4, 0.275 nmol of ATP, and 22 U of T4 RNA ligase in a total
volume of 17 µL of ligation buffer were added to 83 pmol (250
µCi, 9.25 MBq) of freeze-dried [32P]pCp (Amersham Biosciences).
The reaction proceeded at 4 °C for 3 days, and the radiolabeled
product was purified by HPLC using the conditions described above.
The product was freeze-dried, dissolved in water, and stored at -70
°C. The purity of the product was checked using the HPLC system
with radiodetection.
Binding Affinity Assay. The soluble mouse spleen homogenate
(20 µL), prepared as described above, was mixed in a final volume
of 25 µL with pA4 or its analogue and pA4-[32P]pCp to give about
20 000 dpm per test tube (about 0.4 nM pA4-[32P]pCp). The final
concentration of pA4 or its analogue was set to 1 µM for the
determination of percent inhibition. The competition curves for the
determination of the EC50 values of the oligoadenylate analogues
were measured at several different analogue concentrations at
otherwise identical conditions. The samples were allowed to stand
at 4 °C for 60 min and then filtered through nitrocellulose filters.
The filters were thoroughly washed with 20 mM Tris/HCl (pH 7.6),
100 mM KCl, 5 mM MgCl2, and 5% glycerol (5 × 1 mL). The
adsorbed radioactivity was measured, after drying, using a toluene
scintillation cocktail. All samples were assayed in triplicate, and
the values were reproducible within (15%.
Modification of Magnetic Beads. The modification of magnetic
beads (Sigma) was performed according to MagSelect SA instruc-
tions. The resin slurry (250 µL) was transferred to a sample tube
and washed with PBS at pH 7.4 (5 × 1 mL) using a magnetic
separator. After that, the magnetic beads were incubated with
biotinylated pA4 17 (60 nmol) in PBS (0.5 mL) under shaking at
r.t. for 2 h and finally washed with PBS (3 × 1 mL). The
measurement of absorbance of the supernatant at 260 nm revealed
an almost quantitative binding yield (>95%). The modified beads
were stored in PBS with 0.01% NaN3 as a preservative (final
volume 750 µL) at 2 °C.
RNase L Purification and Cleavage Activity Assay. Prior to
the assays, a slurry of pA4-modified beads (6 µL, about 0.5 nmol
of bound pA4) was diluted with the unmodified resin slurry (10
µL) and washed 3 times with the buffer A (0.2 mL, 25 mM HEPES/
NaOH at pH 7.6, 100 mM KCl, 5 mM Mg(OAc)2, 5 mM DTT,
1.2 mM ATP, 1 mM PMSF, 25 µg/mL of pepstatin, and 100 µg/
mL of leupeptin).
Then, the murine spleen homogenate (0.3 mL, 7.4 mg of proteins
per ml) was added, and the suspension of beads was shaken at 5
°C for 2 h. Afterward, the magnetic beads with bound RNase L
were washed 10 times with ice-cold buffer A (0.5 mL) and finally
resuspended in 90 µL of buffer A containing BSA (0.1 mg/mL).
The cleavage assay (in a final volume of 100 µL) was started by
the addition of pA4 or its modified analogue (final concentration 1
µM) and the 5-[32P]-r(C11U2C7) substrate (specific activity 3 Ci/
mmol, suplemented with r(C11U2C7) to the final concentration of
10 µM). The reactions were shaken at 37 °C for 90 min. Aliquots
(10 µL) of the reaction mixture were taken after 0, 10, 20, 30, 40,
60, 75, and 90 min, and the reaction was stopped by adding 10 µL
of the Maxam-Gilbert loading buffer (98% deionized formamide,
10 mM EDTA, 0.025% xylene cyanol, and 0.025% bromophenol
blue) and boiling for 5 min. In a blank assay, the pA4 was omitted.
The purity of prepared oligonucleotides 6-16 was checked by
RP HPLC in 0.1 M TEAA and 0.1% TFA and found to be higher
than 95% (based on an HPLC chromatogram).
Preparation of Soluble Mouse Spleen Homogenate. Male
inbred BALB/c mice weighing approximately 22-27 g were
decapitated, and their spleens were perfused with cold saline,
excised, and immediately frozen at -72 °C. All of the following
procedures were carried out at 4 °C. When needed, mouse spleens
were thawed, minced with scissors, and homogenized in 25 mM
HEPES/NaOH (pH 7.6), 5 mM Mg(OAc)2, 100 mM KCl, 5 mM
DTT, 1 mM PMSF, 25 mg/mL of pepstatin, 100 mg/mL of
leupeptin A, and 1.2 mM ATP (1.5 mL per one spleen) using a
Teflon-glass homogenizer. The homogenate was sonicated twice
on ice for 15 s. The homogenate was centrifuged at 600g for 20
min. The pellet was discarded, and the supernatant was centrifuged
at 17 000g for 20 min. The pellet was discarded again, and the
supernatant was ultracentrifuged at 126 000 g for 90 min. The
protein concentration in the supernatant was measured by the
procedure of Bradford37 using bovine serum albumin as a standard.
The resulting supernatant was supplemented with glycerol (10%
v/v), stored at -72 °C, and later used as a source of RNase L.
Synthesis of p-5′-(2-5)A4-pC-3′-p. The synthesis of nonlabeled
p-5′-(2-5)A4-pC-3′-p was performed according to Silverman et al.
with several modifications.27 Thus, 2.5 nmol of pA4, 10 nmol of

ActiVation of Murine RNase L
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3961
(10) Charachon, G.; Sobol, R. W.; Bisbal, C.; Salehzada, T.; Silhol, M.;
Charubala, R.; Pfleiderer, W.; Lebleu, B.; Suhadolnik, R. J.
Phos-phorothioate analogues of (2′,5′) (A)4: agonist and
antagonist activities in intact cells. Biochemistry 1990, 29, 2550-
2556.
(11) Beigelman, L.; Matulic-Adamic, J.; Haeberli, P.; Usman, N.; Dong,
B.; Silverman, R. H.; Khamnei, S.; Torrence, P. F. Synthesis and
biological activities of a phosphorodithioate analog of 2′,5′-oligoad-
enylate (2-5A). Nucleic Acids Res. 1995, 23, 3989-3994.
(12) Cramer, H.; Player, M. R.; Torrence, P. F. Discrimination between
ribonuclease H- and ribonuclease L-mediated RNA degradation by
2′-O-methylated 2-5A-antisense oligonucleotides. Bioorg. Med.
Chem. Lett. 1999, 9, 1049-1054.
(13) Zhou, L. H.; Civitello, E. R.; Gupta, N.; Silverman, R. H.; Molinaro,
R. J.; Anderson, D. E.; Torrence, P. F. Endowing RNase H-inactive
antisense with catalytic activity: 2-5A-morphants. Bioconjugate
Chem. 2005, 16, 383-390.
(14) Leaman, D. W.; Longano, F. J.; Okicki, J. R.; Soike, K. F.; Torrence,
P. F.; Silverman, R. H.; Cramer, H. Targeted therapy of respiratory
syncytial virus in African Green Monkeys by intranasally adminis-
tered 2-5A antisense. Virology 2002, 292, 70-77.
The samples were electrophoresed in a denaturing 20% polyacryl-
amide gel (7 M urea, 0.09 M Tris-borate at pH 8.3, 2 mM EDTA,
and 20% acrylamide). The gels were dried and analyzed using a
Phosphor Imager (Molecular Dynamics). The scanned gels were
quantitated with ImageQuant software (Molecular Dynamics).
Stability of pA4 and Its Analogues in Soluble Mouse Spleen
Homogenate. The resistance of oligonucleotides toward degradation
by nucleases from soluble mouse spleen homogenate was checked
on a Luna C18 column (5 µm, 150 × 4.6 mm, Phenomenex) at
260 nm, using a linear gradient of acetonitrile in aqueous 0.1%
(v/v) TFA (0-12% in 70 min) at a flow rate of 1 mL/min. The
soluble mouse spleen homogenate (9 µL) was mixed with 2.5 nmol
(1 µL) of pA4 or its analogue. The reaction mixtures were kept at
4 °C and periodically checked by HPLC (after mixing the sample
with 10 µL of aqueous 0.1% (v/v) TFA and centrifugation at
14 000g for 10 min). The peaks of oligonucleotides and their
degradation products were integrated using CSW 1.6 software (Data
Apex, Prague).
(15) Xu, Z.; Kuang, M.; Okicki, J. R.; Cramer, H.; Chaudhary, N. Potent
inhibition of respiratory syncytial virus by combination treatment
with 2-5A antisense and ribavirin. AntiViral Res. 2004, 61, 195-
206.
(16) Leaman, D. W.; Cramer, H. Controlling gene expression with 2-5A
antisense. Methods: A Companion to Methods in Enzymology 1999,
18, 252-265.
(17) Kondo, Y.; Koga, S.; Komata, T.; Kondo, S. Treatment of prostate
cancer in Vitro and in ViVo with 2-5A-anti-telomerase RNA compo-
nent. Oncogene 2000, 19, 2205-2211.
(18) Xiao, W.; Li, G.; Player, M. R.; Maitra, R. K.; Waller, C. F.;
Silverman, R. H.; Torrence P. F. Nuclease-resistant composite 2′,5′-
oligoadenylate-3′,5′-oligonucleotides for the targeted destruction of
RNA: 2-5A-iso-antisense. J. Med. Chem. 1998, 41, 1531-
1539.
Acknowledgment. This work is dedicated to our colleague
and excellent team member Martina Pressova, Ph.D., who
passed away last year. We gratefully acknowledge the financial
support by grants No. A4055101 and No. 203/05/P557 (both
GA Acad. Sci., Czech Rep.) and Network of Excellence EMIL
(sixth Framework) under research project Z40550506 (Acad.
Sci., Czech Rep.). We are indebted to Dr. Zdenek Tocik (IOCB)
for valuable discussions, Dr. Marcela Novotna (IOCB) for the
purification of modified oligoadenylates, and the staff of the
Department of Organic Analysis of IOCB for the measurements
of mass spectra.
(19) Endova, M.; Masojidkova, M.; Budesinsky, M.; Rosenberg, I. 2′,3′-
O-Phosphonoalkylidene derivatives of ribonucleotides: synthesis and
reactivity. Tetrahedron 1998, 54, 11151-11186.
(20) Endova, M.; Masojidkova, M.; Budesinsky, M.; Rosenberg, I. 3′,5′-
O-Phosphonoalkylidene derivatives of 1-(2-deoxy-â-D-threo-pento-
furanosyl)thymine: synthesis and reactivity. Tetrahedron 1998, 54,
11187-11208.
(21) Kralikova, S; Budesinsky, M.; Masojidkova, M.; Rosenberg, I.
Efficient synthesis of 2′-deoxynucleoside 3′-C-phosphonates: reactiv-
ity of geminal hydroxyphosphonate moiety. Nucleosides, Nucleotides
& Nucleic Acids 2000, 19, 1159-1183.
(22) Kralikova, S; Budesinsky, M.; Masojidkova, M.; Rosenberg I.
Geminal hydroxy phosphonate derivatives of nucleosides: a novel
class of nucleoside 5-monophosphate analogues. Tetrahedron Lett.
2000, 41, 955-958.
Supporting Information Available: 1H, 13C NMR, and HR
FAB data for compounds 1-4, MALDI-TOF MS, purity data, and
retention time data (tR) for oligoadenylates 6-16, protocol for the
synthesis of oligoadenylates, synthesis of biotinylated pA4 17, and
the time-dependent profiles of 5′-[32P]-r(C11U2C7) cleavage by
murine RNase L. This material is available free of charge via the
Internet at http://pubs.acs.org.
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(32) Results of NMR conformational analysis: (2′,5′)-AA: PN ) 0, Φm(N)
) 36, XN ) 0.58, PS ) 162, Φm(S) ) 36, XS ) 0.42. (2′,5′)-XA:
PN ) 18, Φm(N) ) 36, XN ) 0.55, PS ) 189, Φm(S) ) 38, XS
0.45.
)
(33) den Hartog, J. A.; Wijnands, R. A.; van Boom, J. H. Synthesis of
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