Activation of human RNase L by 2′- and 5′-O-methylphosphonate- modified oligoadenylates

Article abstract of DOI:10.1016/j.bmcl.2011.11.040

To determine the influence of internucleotide linkage and sugar ring conformation, and the role of 5′-terminal phosphate, on the activation of human RNase L, a series of 2′- and 5′-O-methylphosphonate-modified tetramers were synthesized from appropriate m

Full text of DOI:10.1016/j.bmcl.2011.11.040

Bioorganic & Medicinal Chemistry Letters 22 (2012) 181–185
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Activation of human RNase L by 20- and 50-O-methylphosphonate-modified
oligoadenylates
⇑
ˇ
Ondrej Páv, Natalya Panova, Jan Snášel, Eva Zborníková, Ivan Rosenberg
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo n. 2, 166 10 Prague 6, Czech Republic
a r t i c l e i n f o
a b s t r a c t
To determine the influence of internucleotide linkage and sugar ring conformation, and the role of 50-ter-
minal phosphate, on the activation of human RNase L, a series of 20- and 50-O-methylphosphonate-mod-
ified tetramers were synthesized from appropriate monomeric units and evaluated for their ability to
activate human RNase L. Tetramers pAAApcX modified by ribo, arabino or xylo 50-phosphonate unit pcX
activated RNase L with efficiency comparable to that of natural activator. Moreover, incorporation of
phosphonate linkages ensured the stability against cleavage by nucleases. The substitution of 50-terminal
phosphate for 50-terminal phosphonate in tetramer pcXAAA afforded tetramers with excellent activation
efficiency and with complete stability against cleavage by phosphomonoesterases.
Article history:
Received 22 September 2011
Revised 8 November 2011
Accepted 10 November 2011
Available online 23 November 2011
Keywords:
Human recombinant RNase L
Phosphonate
Oligoadenylate
FRET
Ó 2011 Elsevier Ltd. All rights reserved.
The short 20,50-oligoadenylates (2-5A) play a significant role
in the interferon-induced antiviral defence mechanism of cells.1,2
In the presence of double-stranded RNA, interferon induces expres-
sion of the 20,50-oligoadenylate synthetase utilizing ATP as a sub-
strate for the synthesis of 50-phosphorylated 2-5As, (pp)p50
Ap(Ap)nA20 (where n is mainly 1 or 2).3 These oligonucleotides bind
to a latent endoribonuclease RNase L and subsequently activate it.
The activated enzyme is capable of cleaving single-stranded RNA
and thus of preventing expression of viral proteins. Activity of RNase
L is regulated by a specific 20,50-exonuclease (20,50-phosphodiester-
ase) that cleaves 2-5A to AMP and ATP.4 It has been shown that 2-
5A also plays an important role in the regulation of cell growth
and differentiation, antiproliferative functions of interferons, and
in apoptosis.2,5
Successful use of RNase L pathway in targeting several viral
strains (HIV6; RSV7; EMCV8; VSV, VV, HPV9; Mengovirus10) has
been reported, making RNase L agonists candidates for antiviral
therapy. Moreover, a role of RNase L in suppression of prostate
cancer and leukemia has been reported.4,11 Abnormalities in the
RNase L pathway, its hyperactivity, and increased levels in blood
samples have been reported in patients with chronic fatigue syn-
drome (CFS). This also makes the RNase L antagonists of potential
interest.12
cific nuclease cleavage. Protection of the 50-phosphate group, a key
structural feature of 2–5A’s, against cleavage by phosphomonoes
terases is also a significant issue. In connection with these require-
ments, modified oligonucleotides bearing phosphonate intern-u-
cleotide linkages have been shown to offer remarkable protection
against nucleases.13 These compounds containing structurally
diverse types of phosphonate linkages have attracted our attention
for a long time.13
In our previous work,14 we presented a study on the influence of
the 20-O-methylphosphonate internucleotide linkage in 2-5A
analoges on the process of binding to, and activating of, murine
RNase L. We synthesized a library of 2-5A tetramers in which
one or more adenylate residues were replaced by either ribo Xa,
arabino Xb, or xylo Xc adenosine-20-phosphonate unit Xpc (Fig. 1).
We found that tetramers pAAA modified by ribo Xa and xylo Xc
20-phosphonate units activated RNase L with efficiency comparable
to that of natural pA4. Surprisingly, the tetramer pAAXpcA modified
by arabino Xb 20-phosphonate unit was unable to activate RNase L,
and seems to be a potent inhibitor of the enzyme. Moreover, the
phosphonate 2-5A’s exhibited increased stability against cleavage
by nucleases compared to the natural 2-5A.
Here we report an extended study on the synthesis and investi-
gation of biological properties of 20- and 50-O-methylphosphonate-
modified oligoadenylates as potential agonists and antagonists of
human RNase L (Figs. 1 and 2). We focused on the influence of
internucleotide linkage on the activation of human RNase L, and
the role of 50-phosphate and sugar ring conformation in these
processes.
Any successful biological application of oligoadenylates would
depend on the stability of these compounds in cells, particularly
on the resistance of the 2’,5’ internucleotide linkages against a spe-
First we prepared the appropriate monomers. Synthesis of
⇑
Corresponding author. Tel.: +420 220 183 381; fax: +420 220 183 531.
E-mail address: ivan@uochb.cas.cz (I. Rosenberg).
20-phosphonate monomers was already published in Pav et al.14
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.040

182
O. Páv et al. / Bioorg. Med. Chem. Lett. 22 (2012) 181–185
OH
P
O
oligoadenylates in the 50?20 direction. The BOMOM group was
O
OH 1st
O
recently introduced for the synthesis of RNA in reverse direction
by our group.15 Similarly, arabino nucleoside-50-phosphonate (2e)
was first dimethoxytritylated with dimethoxytritylchloride in the
presence of silver triflate and then protected with benzoyl group
to afford a mixture of regiomers 3e and 4e. Dimethoxytritylation
of xylo nucleoside-50-phosphonate 2f followed by reaction with
benzoyl cyanide afforded 20-dimethoxytrityl derivative 3f as the
only product. Due to the trans-20,30-diol configuration in arabino
and xylo monomers, there is no need to use any special protecting
group such as BOMOM. Thus, the benzoyl group which, in this par-
ticular case, is fully compatible with the solid phase synthesis and
deprotection cycle, was used.
unAit
A
A
O
2
nd unit
O
OH
O
O
OH
P
A
Xa
O
O
P
O
O
OH
O
O
OH
3rd unit
OH
O
O
P
O
O
O
A
O
O
O
Xb
Xc
P
OH
OH
OH
HO
A
O
4th unit
OH
O
A
O
P
O
O
O
OH
P
OH
O
O
Since the mixtures of 20,30-regioisomers 3d, 4d and 3e, 4e were
not separable by silica gel chromatography, the separation was
accomplished by preparative HPLC to afford the desired phospho-
nates 3d and 3e. The fully protected phosphonates 3 were treated
with bromotrimethylsilane and subsequently esterified with 4-
methoxy-1-oxido-2-pyridylmethanol (MOP-OH) in the presence of
2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane-2-oxide (CDDO)
and methoxypyridine-N-oxide (MPNO) to afford the appropriate
phosphonomethyl monomers 5.
O
OH OH
Figure 1. Oligoadenylate tetramers modified by ribo Xa, arabino Xb, and xylo Xc
adenosine-2’-phosphonate units.
O
P
OH
O
P
O
O
A
O
O
P
OH
OH
A
A
O
O
O
O
OH
Once all monomeric building blocks were prepared, various oli-
goadenylate tetramers were synthesised in both the 20?50 and the
50?20 directions (Table 1). Solid-phase synthesis was performed on
O
O
O
OH
Xe
OH
O
O
a 1
[2(3)-O-benzoyl-5-O-dimethoxytrityl-1-(6-N-benzoyladenin-9-
yl)-b-
-ribofuranos-3(2)-O-succinoyl]LCAA-CPG and in the 50?20
l
mol scale in the 20?50 direction (20-phosphonate series) using
Xd
Xf
Figure 2. Ribo Xd, arabino Xe, and xylo Xf adenosine-5’-phosphonate units.
D
direction (50-phosphonate series) using [2-[2-(4,4’-dimethoxytri-
tyloxy)ethylsulfonyl]ethyl-succinoyl]LCAA-CPG as solid supports.
In case of the synthesis in 20?50 direction, the 50-terminal phos-
phate was introduced by 2-[2-(4,40-dimethoxytrityloxy)-ethyl-
sulfonyl]ethyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite.
Synthesis, deprotection and purification of the modified tetramers
were performed using the protocols published earlier in Pav et al.15
As was mentioned above, one of the aims of the presented study
was to define the role of stereochemical factors influencing the
process of activation of human RNase L, especially those associated
with internucleotide linkage. Thus, we introduced the ribo, arabino,
50-O-Methylphosphonate monomers were prepared according to
Scheme 1. Suitably protected nucleosides 1 were phosphonylated
with diisopropyl tosyloxymethylphosphonate in the presence of
sodium hydride to afford deprotected 50-phosphonates 2 upon
acidic cleavage of ethoxymethylidene (1d) and tetrahydropyranyl
(1e, 1f) groups. A mixture of fully protected nucleotides 3d and
4d was prepared by the reaction of ribo 50-phosphonate 2d and
benzoyloxymethoxymethyl chloride (BOMOM–Cl) in the presence
of dibutyltin dichloride, followed by dimethoxytritylation with
dimethoxytrityl chloride in the presence of silver triflate. The
reason for the introduction of benzoyloxymethoxymethyl group
(BOMOM) emerges from the synthesis of 50-phosphonate-modified
and xylo-configured 20- and 50-phosphonate units Xa–c and Xd–f
,
respectively, (Figs. 1 and 2) into individual positions of oligoadeny-
O
HO
O
ABz
O
ABz
ABz
ABz
O
O
O
O
O
O
(iii)-(iv)
(vi)-(viii)
MOPO
(i)-(ii)
O
O
P
P
P
OiPr
OiPr
OH
BOMOMO
iPrO
iPrO
OR´ OR
O
O
HO
OH
ODMTr
2d
3d
4d
R=DMTr; R´=BOMOM
5d
1d
R=BOMOM; R´=DMTr
ABz
ABz
ABz
ABz
O
HO
O
O
THPO
O
HO
RO
DMTrO
(iv)-(v)
O
O
(i)-(ii)
O
O
(vi)-(viii)
MOPO
O
O
P
P
P
OiPr
iPrO
OiPr
OH
iPrO
HO
THPO
OR´
R=DMTr; R´=Bz
R=Bz; R´=DMTr 4e
BzO
1e
2e
5e
3e
ABz
(iv)-(v)
ABz
O
HO
ABz
(i)-(ii)
OBz
O
O
OTHP
O
OH
ABz
OBz
O
O
O
O
(vi)-(viii)
O
O
P
P
P
OiPr
iPrO
OiPr
OH
iPrO
MOPO
OTHP
OH
ODMTr
ODMTr
5f
2f
1f
3f
Scheme 1. Synthesis of 50-O-methylphosphonate derivatives of adenine nucleosides. Reagents and conditions: (i) TsOCH2PO(OiPr)2, NaH, DMF, rt; (ii) 80% AcOH, rt; (iii)
BOMOM–Cl, Bu2SnCl2, DIPEA, DCE, 80 °C; (iv) DMTr–Cl, CF3SO3Ag, py, rt; (v) BzCN, TEA, ACN, rt; (vi) Me3SiBr, 2,6-lutidine, ACN, rt; (vii) 4-methoxy-1-oxido-2-
pyridylmethanol, CDDO, MPNO, py, rt; (viii) PhSH, Et3N, DMF, rt.

O. Páv et al. / Bioorg. Med. Chem. Lett. 22 (2012) 181–185
183
Table 1
Evaluated 20- and 50-Phosphonate-modified oligoadenylates
lates (Table 1). The introduction of these units into oligoadenylates
variously changed the length of the internucleotide linkage, the
conformation of the sugar–phosphate backbone, the conformation
of the sugar part of the respective phosphonate unit, and distribu-
tion of a negative charge along the oligoadenylate strand in the
place of modified internucleotide linkage.
The ability of modified oligoadenylates to activate human
RNase L was monitored by the cleavage of FRET substrate [CY5]-
r(C11-UU-C7)-[BHQ2] using fluorescence reader. For more details
see Supplementary data. The natural activators pA4 (6) and pA3
exhibited identical EC50 values of 17 nM. The EC50 values of two
sets of phosphonate-modified tetramers summarized in Table 2
were calculated from regression curves (see Supplementary data).
The remaining tetramers containing two, or more phosphonate
ribo modification was readily tolerated whereas the arabino and
xylo modifications afforded tetramers with lower activation ability.
In contrast to murine RNase L where 20-phosphonate linkage was
very well tolerated,14 the 50-phosphonate linkage was better toler-
ated by human RNase L than the 20-phosphonate one. The intro-
duction of more than one phosphonate unit resulted in
significant decrease in activation ability.
a
Unlike murine RNase L which requires pA4 tetramer for the acti-
vation, pA3 trimer is able to activate human RNase L.2 Therefore,
the fourth unit is known for not being crucial for the activation
process. Nonetheless, modification of this unit may still afford a
tetramer with improved properties such as enhanced binding to
the active site or higher stability against cleavage by nucleases.
The incorporation of 50-phosphonate linkage between the third
and the fourth unit afforded tetramers (17d, 17e, 17f) with activa-
tion efficiency comparable or better to that of natural activator.
The 20-phosphonate tetramers 9a and 9b modified at the same site
retained a moderate activation ability. Importantly, both phospho-
units exhibited EC50 values above 10 lM.
In general, there was a strong relationship between the position
of modified linkage and the ability of modified 2-5A to activate the
enzyme. Comparing the influence of sugar part configuration, the
Table 2
EC50 values (nM) of the selected activators of human RNase L
a
Average value from three independent measurements (5–10)%.
Exonuclease cleaves the tetramers 8 and 16 to trimers pAXpcA and pAApcX, respectively.
Completely stable against exonucleases.
b
c

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O. Páv et al. / Bioorg. Med. Chem. Lett. 22 (2012) 181–185
2500
2000
1500
1000
500
phosphonate tetramer at a 1:1 ratio. None of the prepared oligoa-
6
denylates exhibited antagonist activity under these conditions.
In conclusion, we prepared two sets of oligoadenylates modified
by regioisomeric 20- and 50-phosphonate units. We found that tet-
ramers pAAApcX modified by ribo (17d), arabino (17e), and xylo
(17f) 50-phosphonate units activated RNase L with efficiency com-
parable to that of natural activator. Moreover, the modification at
this position provided tetramers possesing stability of the 2’-O-P-
CH2-O-5’ internucleotide linkage against cleavage by exonucleases
forming the 5’-nucleotides. Our earlier study on the modified oli-
gonucleotides containing the regioisomeric 3’-O-P-CH2-O-5’ link-
age showed their complete stability against nucleases of L1210
cell free extract and phosphodiesterase I.15,17,18 The substitution
of 50-terminal phosphate for 50-terminal phosphonate in tetramer
pcXAAA 14d afforded tetramer with excellent activation efficiency
and with complete stability against cleavage by phosphomonoes-
terases but not against exonucleases, however, there is no problem
to modify the 2’(3’)-end of pcXAAA 14d to prevent the exonuclease
cleavage. The improved stability observed with compounds 14d
and 17d–f is anticipated to facilitate in vivo evaluation of these
oligoadenylates.
14d
15d
16d
17d
0
0
100
200
Time (s)
300
400
Figure 3. Cleavage of FRET substrate in the presence of natural activator 6 and
tetramers modified by ribo 50-phosphonate unit Xd (14d, 15d, 16d, 17d).
Acknowledgements
nate linkages at this position ensure the stability against cleavage
by exonucleases.14,15 Interestingly, the position of the methylene
group in the phosphonate 2’,5’ internucleotide linkages of regioiso-
meric pairs of ribo tetramers (7a and 15d, 8a and 16d, and 9a and
17d) substantially influences their EC50 values as obvious from the
Table 2.
The support by the grant 202/09/0193 (Czech Science Founda-
tion) and Research centers KAN200520801 (Acad. Sci. CR) and
LC060061 (Ministry of Education, CR) under the Institute research
project Z40550506 is gratefully acknowledged.
Tetramers pAXpcAA 8 and pApcXAA 15 bearing ribo, arabino,
and xylo modifications at the second position exhibited a consider-
ably reduced activation ability. The second unit is essential for the
binding of a tetramer to RNase L2,16 and it seems that any modifi-
cation of the internucleotide linkage at this position has a detri-
mental effect. This finding is in agreement with the data we
obtained in our study dealing with murine RNase L.14
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.11.040.
References and notes
Interestingly, the tetramers pcXAAA 14 bearing 50-terminal
phosphonate instead of 50-terminal phosphate retained the activa-
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phosphate is a key structural feature of 2-5As and its cleavage by
phosphomonoesterases leads to an immediate loss of the activa-
tion ability of the tetramer. Thus, the presence of 50-terminal
phosphonate affords an activator with complete stability against
5’-dephosphorylation by phosphomonoesterases.17 However, this
tetramer 14 is not resistant against exonucleases because of the
presence of natural units at second, third, and fourth positions.
The incorporation of the ribo 5’-phosphonate unit (Xd) at the fourth
position of tetramer instead of the natural unit provided both
phosphomonoesterase and exonuclease stable ribo tetramer
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