Interstrand cross-link and bioconjugate formation in RNA from a modified nucleotide

Article abstract of DOI:10.1021/jo501982r

RNA oligonucleotides containing a phenyl selenide derivative of 5-methyluridine were chemically synthesized by solid-phase synthesis. The phenyl selenide is rapidly converted to an electrophilic, allylic phenyl seleneate under mild oxidative conditions. The phenyl seleneate yields interstrand cross-links when part of a duplex and is useful for synthesizing oligonucleotide conjugates. Formation of the latter is illustrated by reaction of an oligonucleotide containing the phenyl selenide with amino acids in the presence of mild oxidant. The products formed are analogous to those observed in tRNA that are believed to be formed posttranslationally via a biosynthetic intermediate that is chemically homologous to the phenyl seleneate. (Chemical Equation Presented).

Full text of DOI:10.1021/jo501982r

Article
pubs.acs.org/joc
Interstrand Cross-Link and Bioconjugate Formation in RNA from a
Modified Nucleotide
Jack L. Sloane and Marc M. Greenberg*
Department of Chemistry, Johns Hopkins University, 3400 N. Charles Street, Baltimore, Maryland 21218, United States
S
* Supporting Information
ABSTRACT: RNA oligonucleotides containing a phenyl
selenide derivative of 5-methyluridine were chemically
synthesized by solid-phase synthesis. The phenyl selenide is
rapidly converted to an electrophilic, allylic phenyl seleneate
under mild oxidative conditions. The phenyl seleneate yields
interstrand cross-links when part of a duplex and is useful for
synthesizing oligonucleotide conjugates. Formation of the
latter is illustrated by reaction of an oligonucleotide containing the phenyl selenide with amino acids in the presence of mild
oxidant. The products formed are analogous to those observed in tRNA that are believed to be formed posttranslationally via a
biosynthetic intermediate that is chemically homologous to the phenyl seleneate.
linking RNA via modified nucleotides that contain furan or
vinyl sulfides have been reported. Oxidation of the furan
appended through the 2′-hydroxyl of uridine or introduced as a
nonnucleotide spacer in an oligonucleotide produces an
electrophilic 1,4-dicarbonyl that reacts with a cytidine in the
opposing strand.18 Similarly, oxidation of the vinyl sulfide that
is an analogue of adenosine produces a more electrophilic vinyl
sulfoxide that reacts with the exocyclic amine of cytidine.19
We previously reported on the mild oxidation of 1 in duplex
DNA by a variety of oxidants, such as singlet oxygen, H2O2, and
NaIO4 (Scheme 1).20,21 The resulting phenyl selenoxide 2
undergoes rapid [2,3]-sigmatropic rearrangement to an electro-
philic species (3) that rearomatizes upon reaction with
nucleophiles. The 2′-deoxycytidine analogue 5 undergoes a
similar reaction to form 6.22 In duplex DNA electrophiles 3 and
6 react with nucleotides in the opposing strand to produce
cross-linked products (e.g., 4). Water slowly traps the
electrophiles in the absence of an appropriate nucleophilic
partner in DNA, and other nucleophiles such as azide can be
used as well. We anticipated that this reactivity pattern could be
extended to RNA where it might be useful for probing tertiary
interactions, as well as providing a convergent approach for
preparing RNA molecules containing C5-functionalized
uridines, a common modification in bacterial RNA.23
INTRODUCTION
■
The known roles of RNA in biology and biochemistry continue
to grow. Its newly discovered features include novel primary
and tertiary structures. One way in which organic chemistry can
contribute to this important area is by developing methods for
synthesizing and analyzing these novel structures. In this report
we describe the chemistry that can be used to synthesize RNA-
containing C5-substituted uridines and for producing inter-
strand cross-links in RNA. The latter function may prove useful
for detecting tertiary interactions in RNA.
A variety of enzymatic and chemical tools that are used for
studying DNA structure are also employed for examining the
more complex structure of RNA molecules.1 These tools are
being applied in the development of high-throughput methods
for RNA structure elucidation.2−4 The tools employed include
hydroxyl radical cleavage and chemical reagents that exploit the
differential reactivity of nucleotides based upon their solvent
exposure.4−7 Dimethyl sulfate and diethyl pyrocarbonate are
examples of the latter.1,8,9 In addition, Weeks has pioneered the
development of reagents that exploit the accessibility of the 2′-
hydroxyl group to extract RNA structural information from
differential reactivity.10−13 Each of these reagents provides
inferential structural information based on relative reactivity:
less-accessible nucleotides that are “buried” within the three-
dimensional RNA structure are less reactive.
In contrast, cross-linking agents detect proximal functional
groups. A number of cross-linking methods are available,
including photolabile nucleotides (e.g., 4-thiouridine, 6-
thioguanosine) and exogenous reagents (e.g., 1,4-phenyl
diglyoxal).14−16 Researchers have also utilized photochemistry
to produce reactive intermediates, such as carbenes and
nitrenes, although cross-link yields are often low in these
experiments.14 In another approach, cisplatin was elegantly
used in conjunction with phosphorothioated RNA to detect
short-range interactions.17 Recently, mild methods for cross-
RESULTS AND DISCUSSION
■
Synthesis of RNA Containing a Latent Electrophilic
C5-Modified Uridine. Phosphoramidite 12 was synthesized in
a manner similar to that previously described for 1 starting from
7 (Scheme 2).24−26 Although the intermediate bromide en
route to 8 could be isolated, this proved to be impractical, and
it was the bromination step that was responsible for the low
Received: August 27, 2014
Published: October 8, 2014
© 2014 American Chemical Society
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manually coupled for 10 min as previously described.28
Coupling yields for 12, as measured using dimethoxytrityl
cation detection, varied widely from as low as 40% to almost
quantitative. Oligonucleotides were deprotected using a
mixture of concentrated aqueous ammonia and methylamine
(25 °C, 4 h), followed by desilylation using Et3N·3HF.29,30
Carrying out the deprotection at higher temperatures (50−60
°C) led to decomposition of the phenyl selenide (9).
Anhydrous tert-butyl hydroperoxide was substituted for iodine
in a mixture of water and pyridine as the oxidant to minimize
premature oxidation of the phenyl selenide.31 Despite this
safeguard, it was necessary to purify oligonucleotides containing
the phenyl selenide 9 by a two-step procedure. Following
denaturing polyacrylamide gel electrophoresis, full-length
oligonucleotide containing 9 was separated from its more
polar product(s) (e.g., 17), believed to result from adventitious
oxidation, by reverse-phase HPLC. Purified oligonucleotides
were characterized by MALDI-TOF MS. We prepared three
oligonucleotides containing 9 for this study (Figure 1). In each
instance, a 2′-deoxynucleotide is introduced at the 3′-terminus.
This was performed for convenience, so that NaIO4 could be
used as an oxidant. However, H2O2 (vide infra) is also a
satisfactory oxidant and is compatible with 3′-terminal
ribonucleotides.
Scheme 1
a
Scheme 2
Rapid Formation of an Electrophile from 9 upon Mild
Oxidation. Mild oxidation of phenyl selenides 1 and 5 rapidly
produces the corresponding allylic phenyl seleneates (3, 6) via
the respective selenoxides (e.g., 2). Sodium periodate was the
oxidant of choice for the 2′-deoxyribonucleosides, but this
reagent is incompatible with the vicinal diol in ribonucleosides.
The oxidation of ribonucleosides with NaIO4 is so facile that
this reagent is used in conjunction with reductive amination to
produce 3′-oligonucleotide conjugates.32 Consequently, H2O2
1
was used. H NMR analysis (with water suppression) of the
oxidation carried out in deuterated phosphate buffer (50 mM,
pD 7.4) showed that 9 behaves in a very similar manner to 1
and 5 (Figure 2).20,22 The phenyl selenide 9 was consumed
within minutes, giving rise to a diastereomeric mixture of the
allylic phenyl seleneate (16, Figure 2B). The corresponding
selenoxide was not detected. In the absence of a nucleophile, 16
reacted slowly with H2O to form 17, which was only a minor
product after 18 h at 25 °C (Figure 2C). Phenyl seleneate 16
reacts more slowly with H2O in buffer than does 3 or 6, both of
which showed complete conversion to their respective
hydroxymethyl species (e.g., 17) after 18 h.20,22
a
Key: (a) NBS, AIBN, benzene; (b) (PhSe)2, NaBH4, DMF; (c) NH3,
MeOH; (d) DMTCI, pyridine; (e) TBDMSCI, AgN03, THF; (f) 2-
cyanoethyl N,N-diisopropylchlorophosphoramidite, DIPEA, CH2CI2.
yields. The reaction did not proceed to completion, and
attempts to push it further resulted in even lower yields.
Despite this, the reaction was scalable and 8 was obtained in
millimole quantities and carried forward to 10 via ammonolysis
product 9. Selective silylation of 10 using Ogilvie’s method
yielded only the desired 2′-silyl ether 11, which was then
phosphitylated to produce 12.27
Oligonucleotides containing 9 (molecules are referred to
using the same number whether they are present in a polymer
or as a monomer.) were prepared by automated solid-phase
synthesis (Figure 1). Standard coupling methods were
employed for all phosphoramidites, except for 12, which was
Interstrand Cross-Link Formation from 9 upon Mild
Oxidation. UV-melting experiments indicated that 9 did not
significantly destabilize duplex RNA. The Tm (10 mM
phosphate pH 7.4, 100 mM KCl, 2 mM MgCl2) for 19a
containing 9 (56.3 0.4 °C) was just slightly lower than that of
the analogous duplex containing uridine in place of the phenyl
selenide (19b, 58.7
0.3 °C).33 Stable hybridization is a
requirement for ICL formation. Initial cross-linking experi-
ments were carried out on 9 in a duplex composed of 2′-
deoxynucleotides (18, Figure 3). Treatment of 5′-32P-18 with
NaIO4 (5 mM) yielded 51% interstrand cross-linked (ICL)
product in 3 h at 25 °C and more than 45% in 30 min. This is
comparable to and even higher than the ICL yields obtained
from 1 and 5 under the same conditions.20,22 In contrast,
oxidation of 5′-32P-19a reliably produced less than 15% of the
cross-linked product at 25 °C and only 30 4% at 37 °C in 5
h. Similarly, oxidation of 5′-32P-19a by H2O2 (10 mM) at 37 °C
produced the cross-linked product in 26 1% yield. Hydroxyl
Figure 1. Sequences of oligonucleotides containing 9. Capital letters
indicate a 2′-deoxyribonucleotide and small letters a ribonucleotide.
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predominantly with the opposing adenine, producing the cross-
link product analogous to 4 (Scheme 1).
Kinetic analysis of the cross-linking of 19a correlated with
the observed lower yields (Figure 4). The first-order rate
1
Figure 2. H NMR analysis of H2O2 (50 mM) oxidation of 9 (50
Figure 4. Interstrand cross-link growth from 5′-32P-19a as a function
of time at 37 °C following oxidation by NaIO4 (5 mM).
mM) in deuterated phosphate buffer (50 mM, pD 7.4) at 25 °C. A.
Prior to H2O2 addition. B. Fifteen minutes after H2O2 addition. C.
Eighteen hours after H2O2 addition.
constant was 1.0 0.2 × 10−4 s−1 (t1/2 = 116 min). This is
more than 11 times slower than the rate constant for cross-
linking from 1 in a comparable DNA duplex.35 The slower
1
reactivity of 16 with water observed by H NMR is not the
source of decreased cross-linking rate constant. If it were, then
9 would also have reacted more slowly in the DNA duplex
(18). Subsequent cross-linking experiments were typically
carried out overnight at 37 °C to ensure complete reaction of
9 (or more accurately 16). In search of an explanation for why
the ICL yield from 19a was so much lower than in 18, we
focused on possible differences in the rotational barrier of the
glycosidic bond in 16 as a function of oligonucleotide sequence,
because ICL formation requires occupation of the syn-
conformation in which the allylic phenyl seleneate is oriented
toward the opposing strand in the duplex (Scheme 3). This
implicitly assumes that the rate-limiting step in cross-linking is
Scheme 3
Figure 3. Sequences of oligonucleotide duplexes containing 9. Capital
letters indicate a 2′-deoxyribonucleotide and small letters a
ribonucleotide.
radical cleavage indicated that the A opposite 9 (A24) was the
major cross-linking site, but a small amount of cross-linking also
occurred at A25.33,34 Mass spectrometry of the ICL confirmed
that the product corresponded to the mass of the duplex
following the loss of benzeneselenol. Upon the basis of these
observations and the precedent established by 1, we propose
that 23 corresponds to the major ICL product from oxidation
of 9 in 19a.21 Hence, 16 (produced from oxidation of 9) reacts
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adoption of the syn-conformation. This is unknown for 16 but
has been shown to be the case in related studies in which ICLs
are produced upon photolysis of 1.21,36
We initially examined the effects of mismatches at the
adjacent base pair (20a, 20b). Duplex destabilization in the
vicinity of the 9-adenosine base pair might facilitate adoption of
the syn-conformation by weakening base pairing. However, in
side-by-side reactions the ICL yields from 19a (22 2%), 20a
(21 2%), and 20b (22 5%) were within experimental error
of one another. Consequently, we must rely upon related
literature to rationalize the slower cross-linking by 16 than by 3
and the assumption. It is well established that the barrier for
rotation about the glycosidic bond in pyrimidines is lower when
the nucleoside is in the C2′-endo conformation than when it
populates the C3′-endo conformation (Scheme 3).37 2′-
Deoxynucleotides adopt the C2′-endo conformation in B-
form DNA whereas RNA duplexes typically adopt an A-form
structure in which the ribonucleotides are in the C3′-endo
conformation. Furthermore, recent calculations predict that 2′-
deoxy pyrimidine nucleosides populate the syn-conformational
isomer that is required for ICL formation more readily than do
the comparable ribonucleosides.38 Although ribonucleotides
typically adopt the C3′-endo conformation in duplex RNA
(e.g., 19a), structurally related 2′-fluorothymidine is believed to
populate the C2′-endo isomer when only one molecule of it is
present in a DNA duplex.39 We hypothesize that the
surrounding duplex environment influences the sugar pucker
of 9 (16) much the same way that the conformation of 2′-
fluorothymidine is affected. Lower ICL yields and slower cross-
linking are observed from 19a because 9 (16) exists
predominantly in the C3′-endo conformation, whereas the
C2′-endo conformation of the phenyl selenide should be
relatively preferred in the DNA duplex (18).
Figure 6. Interstrand cross-link yield from 9 in 19a as a function of salt
at 37 °C following oxidation by NaIO4 (5 mM). Note: [NaCl] = 100
mM as [MgCl2] is varied (left), and [MgCl2] = 0 as [NaCl] is varied
(right).
oxidations of 19a significantly lowered the cross-link yields, and
to a smaller extent so too did increasing the concentration of
NaCl from 100 mM to 250 mM. One possible explanation for
the reduced ICL yields with either increasing MgCl2 or NaCl
concentration is that the duplex is more stable in higher salt,
resulting in an increase in the barrier for rotation about the
glycosidic bond in 16 (Scheme 3). The larger decrease
observed by the addition of MgCl2 may also be explained by
the report that magnesium rigidifies the RNA.40 This too could
increase the barrier to forming the syn-conformation of 16 that
is required for cross-linking.
Synthesis of Amino Acid Conjugates from 9.
Oligonucleotide conjugates have useful applications in
biotechnology and as potential therapeutic agents.41−47 In
addition, RNA is often posttranslationally modified. Some
amino acid modifications at the C5-position of uridine are
proposed to proceed via electrophilic intermediates analogous
to 16 that result from cysteine addition to the C6-position.23
Consequently, we carried out conjugation reactions between 14
and amino acids to demonstrate the utility of phenyl selenide 9
(Scheme 4). The respective conjugates of 14 (10 μM) with
Cross-linking in duplexes containing 9 treated with NaIO4
was general and in many ways paralleled the reactivity of 1.21
For instance, the ICL yield declined to 14 2% when 9 was
flanked by adenosines (22) that provide greater π-stacking from
22
2% in 19a. We also observed that ICLs form more
Scheme 4
efficiently when 9 is opposite adenosine and cytidine than when
the phenyl selenide is opposed by guanosine or uridine (Figure
5). This could be the result of the positioning of the
glycine (24a, 59%) and phenylalanine (24b, 43%) were isolated
by reverse-phase HPLC and characterized by MALDI-TOF
MS, following reaction in the presence of the amino acid (10
mM) and NaIO4 (5 mM) at 37 °C for 3 h.
Figure 5. Interstrand cross-link yield from 9 as a function of opposing
nucleotide at 37 °C following oxidation by NaIO4 (5 mM).
electrophile in syn-16 with the relatively more nucleophilic
N1 and N3 of adenine (19a) and cytosine (21b), respectively,
compared to the N1 (21a) and N3 (21c) of guanine and uracil.
As was the case with 19a, hydroxyl radical cleavage of the
isolated ICLs indicated that the nucleotide opposite 9 (16) is
the major site of cross-linking.33 Salt content also had an effect
on ICL yields in 19a (Figure 6). Addition of MgCl2 to NaIO4
SUMMARY
■
Phenyl selenide 9 was incorporated into chemically synthesized
oligonucleotides. When part of a duplex, 9 yields ICLs
predominantly with the opposing nucleotide upon mild
oxidation by H2O2 or NaIO4 via the allylic phenyl seleneate
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above was dissolved in dry DMF (15 mL) and added to the solution.
The reaction was stirred overnight, yielding a light brown mixture. The
reaction was quenched by the addition of H2O (50 mL), whereupon
the mixture bubbled violently. The mixture was extracted with EtOAc
(4 × 50 mL). The organic layers were combined and split in half. Each
half was washed with NaHCO3 (60 mL) and brine (60 mL) and dried
over NaSO4. The solvent was evaporated to yield a yellow oil (3.25 g)
which was then purified by flash chromatography (EtOAc/DCM 2%−
(16). The allylic phenyl seleneate (16) is also useful for
forming oligonucleotide conjugates, as demonstrated by
reacting the single stranded oligonucleotide 14 with amino
acids. Conjugation with amino acids is particularly relevant to
the biosynthesis of tRNA.23 These reactions suggest that 9 may
be a useful tool for producing modified RNA molecules in a
convergent manner, providing novel platforms for the delivery
of nucleic acids to cells. This chemistry may also prove valuable
as a structural probe for detecting tertiary interactions in RNA
such as kissing loops and pseudoknots.48−52 If 9 can be
incorporated into RNA via its respective nucleotide triphos-
phate, as the respective 2′-deoxynucleotide phenyl selenide is, it
may also be useful for randomly probing RNA folding.2,3,53
1
10%) to give 8 as a white foam (805 mg, 32%): H NMR (CDCl3) δ
3.27 (d, 1H, J = 12.4 Hz), 3.54 (d, 1H, J = 12.4 Hz), 4.61 (m, 3H),
5.49 (t, 1H, J = 6 Hz), 5.75 (m, 1H), 6.30 (d, 1H, J = 6 Hz), 6.84 (s,
1H), 7.26 (m, 4H), 7.38 (m, 6H), 7.57 (m, 5H), 7.97 (m, 4H), 8.07
(dd, 2H, J = 6.8 Hz), 9.82 (bd s, 1H); 13C NMR (CDCl3), 14.4, 21.2,
23.3, 60.6, 64.1, 71.4, 73.8, 80.7, 87.3, 113.7, 128.3, 128.6, 128.75,
128.84, 129.0, 129.3, 129.48, 129.51, 129.8, 130.0, 130.1, 133.87,
133.94, 134.0, 135.27, 135.33, 150.0, 161.8, 165.4, 165.5, 166.1; IR
(KBr): 3060, 2923, 1726, 1601, 1452, 1378, 1315, 1266, 1178, 1070,
1025 cm−1; HRMS (ESI/APCI-TOF) m/z calculated for [M + H]+
C37H31N2O9Se 727.1195, found 727.1184.
EXPERIMENTAL SECTION
■
General Methods. Solvents used in reactions were purified and
dried (using CaH2 or Na/benzophenone) by distillation before use.
Reagents were purchased from commercial sources and were used
without further purification. Reactions were carried out under a
positive pressure of argon atmosphere and monitored by TLC on silica
gel G-25 UV254 (0.25 mm). Spots were detected using UV light and/
or by charring with a solution of either ammonium molybdate, ceric
ammonium sulfate in water and H2SO4, or p-anisaldehyde in ethanol
and H2SO4. Flash chromatography was performed on silica gel 60
(40−60 μm). The ratio between silica gel and crude product ranged
from 100:1 to 20:1 (w/w).
Preparation of 9. Gaseous ammonia was bubbled through a
mixture of 8 (800 mg, 1.1 mmol) in MeOH (8 mL) cooled to 0 °C for
40 min. As the solution became saturated with ammonia, the solid 8
slowly dissolved and the liquid became yellow. After the solution was
saturated with ammonia, the reaction flask was sealed and the mixture
was stirred overnight. After 30 min of reaction, all solid had dissolved
into solution and the mixture was a clear yellow color. To quench the
reaction, argon was bubbled through the solution for 30 min. The
solvent was evaporated to a yellow oil (700 mg). The resulting yellow
oil, which was insoluble in the chromatography solvent, was dissolved
in MeOH. Silica (3 g) was added to the mixture and the solvent was
evaporated, yielding yellow, free-floating silica powder. This powder
was added to the top of a flash column, and the compound was
purified by flash chromatography (MeOH/DCM 8%−12%), yielding 9
as a white foam (234 mg, 57%): 1H NMR (MeOH-d4) δ 3.72 (d, 1H, J
= 2.8 Hz), 3.75 (d, 1H, J = 3.2 Hz), 3.83 (m, 2H), 3.92 (t, 1H, J = 5.2
Hz), 4.04 (m, 2H), 5.91 (d, 1H, J = 4.4 Hz), 7.41 (m, 3H), 7.60 (m,
3H); 13C NMR (MeOH-d4), 14.6, 20.9, 23.8, 24.2, 24.5, 61.7, 63.4,
71.3, 75.9, 86.3, 90.7, 113.2, 109.0, 109.6, 130.5, 130.90, 135.89,
135.94, 136.0, 138.7, 152.3, 164.8, 210.2; IR (KBr): 3388, 2924, 1684,
1559, 1540, 1507, 1275, 1102 cm−1; HRMS (ESI/APCI-TOF) m/z
calculated for [M + H]+ C16H19N2O6Se 415.0403, found 415.0402.
Preparation of 10. The triol 9 (452 mg, 1.21 mmol) was
azeotropically dried twice from pyridine (2 mL). Dry 9 was dissolved
in pyridine (9 mL) and cooled to 0 °C. Vacuum-dried DMTCl (617
mg, 1.82 mmol) and DMAP (29 mg, 0.24 mmol) were added to the
cooled solution, which immediately became orange in color. The
mixture was allowed warm to room temperature and was stirred under
argon for 8 h. The reaction was quenched by the addition of NaHCO3
(15 mL), and the resulting solution was extracted with DCM (2 × 15
mL) and washed with brine (20 mL). The organic layer was
concentrated in vacuo to a yellow foam (1.11 g). Flash
chromatography (0.5% Et3N to 2% MeOH, 0.5% Et3N in DCM)
yielded 10 as a white foam (539 mg, 66%): 1H NMR (CDCl3) δ 3.10
(d, 1H, J = 12 Hz), 3.39 (m, 2H), 4.84 (d, 1H, J = 12 Hz), 3.76 (s,
6H), 4.13 (m, 1H), 4.19 (m, 2H), 5.84 (d, 1H, J = 2.8 Hz), 6.82 (m,
3H), 7.13 (m, 2H), 7.17 (m, 2H), 7.27 (m, 8H), 7.42 (d, 2H, J = 1.6
Hz), 7.46 (s, 1H); 13C NMR (CDCl3), 23.0, 55.5, 62.9, 70.3, 76.6,
77.4, 83.9, 87.0, 90.9, 112.4, 113.6, 127.3, 127.8, 128.3, 128.4, 129.2,
130.0, 130.3, 134.4, 135.5, 135.7, 136.6, 144.6, 150.9, 158.9, 163.1; IR:
3229, 3056, 2925, 1670, 1684, 1653, 1635, 1507, 1457, 1251, 1177,
1086, 1035 cm−1; HRMS (ESI/APCI-TOF) m/z calculated for [M +
Na]+ C37H36N2O8NaSe 739.1529, found 739.1528.
Oligonucleotides were synthesized on an Applied Biosystems
Incorporated 394 oligonucleotide synthesizer. Phenyl selenide
oligonucleotides were synthesized using 2′-O-TOM RNA phosphor-
amidites commercially available from Glen Research. Phosphoramidite
12 was coupled manually (10 min), as previously described.28
Commercially available THF/pyridine/acetic anhydride was used as
a capping reagent and 1 M tert-butyl hydroperoxide in toluene was
used as an oxidizing reagent. Oligonucleotides were deprotected using
concentrated AMA [50% NH4OH, 50% methylamine (40% in H2O)]
at room temperature for 4 h, desilylated in a 1.8 M TEA·3HF solution
(2:1:1.5 N-methylpyrrolidinone:TEA:TEA·3HF), purified by 20%
denaturing PAGE, isolated by the crush and soak method, desalted
using C-18-Sep-Pak cartridges (Waters), and characterized by MALDI-
TOF.29,30 Oligonucleotides containing the phenyl selenide modifica-
tion (9) were subjected to additional purification by HPLC on a RP-
C18 column, with monitoring carried out at 260 nm. The peak of
interest was collected at 20.1 min using the following gradient
conditions: 0−18 min 10−20% B in A, 18−23 min 20−80% B in A,
23−28 min, 80% B in A, 28−30 min 80−10% B in A, 30−50 min 10%
B in A, at a flow rate of 1.0 mL/min [A: 0.05 M TEAA (pH 7.0)/
MeCN 95:5; B: 0.05 M TEAA (pH 7.0)/MeCN 50:50].
Oligonucleotides were 5′-32P-labeled by T4 polynucleotide kinase
(New England Biolabs) and γ-32P-ATP (PerkinElmer) using standard
protocols.54 Experiments involving radiolabeled oligonucleotides were
analyzed following PAGE using a Storm 840 phosphorimager and
Imagequant TL software. Radiolabeled oligonucleotides were hybri-
dized with 5 equiv of complementary oligonucleotides in 10 mM
potassium phosphate (pH 7.2) and 100 mM NaCl at 65 °C for 15
min, slowly cooled to room temperature, and then stored at 4 °C
overnight.
Preparation of 8. N-Bromosuccinimide (NBS) was freshly
recrystallized from water and dried under vacuum overnight. A
solution of 7 (2 g, 3.5 mmol), NBS (934 mg, 5.25 mmol), and AIBN
(93 mg, 0.57 mmol) was suspended in dry benzene (30 mL). The
solution was a slight yellow color, and the solid did not completely
dissolve. The solution was heated to reflux for 6 h, whereupon the
solution turned from dark red to brown. The solvent was evaporated
in vacuo to yield a brown solid. Diphenyl diselenide (2.18 g, 7 mmol)
was dissolved in dry DMF (15 mL) to create an orange solution.
NaBH4 (530 mg, 14 mmol) was added slowly to the solution over 10
min. Upon each addition of NaBH4, the solution bubbled fiercely and
faded from an orange color to colorless. The brown residue from
Preparation of 11. Tritylated nucleoside 10 (450 mg, 0.66 mmol)
was azeotropically dried three times from pyridine (3 × 1.5 mL). Silver
nitrate (136 mg, 0.80 mmol) was suspended in a mixture of pyridine
(267 μL, 3.32 mmol) and THF (4.5 mL) and sonicated for 30 min.
TBDMSCl (121 mg, 0.80 mmol) was added to the contents in the
sonicated flask, creating a milky white solution with some undissolved
solid. Dried 10 was dissolved in dry THF (1.5 mL) and added to the
flask via syringe. No physical change was observed in reaction flask.
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The reaction was stirred under argon for 5 h. The contents of the
reaction flask consisted of a slightly yellow liquid layer and gray solid
collected at the bottom of the flask. The heterogeneous mixture was
filtered through Celite into a stirring 20% NaHCO3 solution (10 mL),
extracted with EtOAc (3 × 10 mL), and concentrated in vacuo to yield
a yellow foam (448 mg). Purification by flash chromatography (10%
EtOAc in DCM) yielded 11 as a white foam (337 mg, 64%): 1H NMR
(CDCl3) δ 0.13 (s, 6H), 0.92 (s, 10H), 2.59 (d, 1H, J = 5.2 Hz), 3.12−
3.10 (d, 1H, J = 11.6 Hz), 3.44−3.35 (ddd, 2H, J1 = 19.6, J2 = 10.4 Hz,
J3 = 3.2), 3.46−3.43 (d, 1H, 11.6 Hz), 3.77 (s, 6H), 4.10−4.07 (m,
2H), 4.27−4.25 (t, 1H, J = 4.4 Hz), 5.92−5.91 (d, 1H, J = 4.4 Hz),
6.84−6.81 (m, 4H), 7.21−7.15 (m, 4H), 7.34−7.29 (m, 9H), 7.42−
7.39 (dd, 2H, J1 = 3.2 Hz, J2 = 1.6 Hz), 7.50 (s, 1H), 8.37 (bd s, 1H);
13C NMR (CDCl3) −4.9, −4.5, 18.2, 22.8, 25.9, 29.9, 55.5, 63.9, 71.1,
76.3, 83.8, 87.3, 88.8, 112,6, 113.6, 127.4, 127.6, 128.3, 128.4, 129.1,
130.3, 130.35, 134.0, 135.7, 136.7, 144.6, 159.0; IR (KBr): 3193, 3056,
2927, 2856, 1700, 1684, 1653, 1507, 1458, 1252, 1176, 1121, 1035
cm−1; HRMS (ESI/APCI-TOF) m/z calculated for [M + Na]+
C43H50N2O8NaSiSe 853.2394, found 853.2395.
in 95% formamide loading buffer, and subjected to electrophoresis on
a 20% denaturing polyacrylamide gel. The alkali ladder was generated
by treating radiolabeled oligonucleotide with 0.2 M NaOH in 10 mM
EDTA at 90 °C for 15 s. The reaction was quenched by addition of 5
μL each of stop buffer (9.5 M urea, 85 mM NaOAc, 1% v/v AcOH)
and 95% formamide loading buffer. RNase A sequencing was
performed with 1 μU of enzyme in 5 μL of reaction buffer (300
mM NaCl, 5 mM EDTA, and 10 mM Tris-HCl (pH 7.5)) at 37 °C for
20 min. To this reaction was added 5 μL of 95% formamide loading
buffer.
Conjugation Reactions with Amino Acids. The phenyl selenide-
modified oligonucleotide (14, 10 μM) and the relevant nucleophile
(10 mM) were incubated at 37 °C in a mixture of 10 mM potassium
phosphate (pH 7.2), 100 mM NaCl, and 5 mM NaIO4 (total volume
10 μL). The resulting solution was diluted with the addition of extra
water (15 μL) and filtered through a 0.22 μm filter. The mixture was
subjected to UPLC analysis on a RP-C18 HPLC column with
monitoring carried out at 260 nm. Peaks were analyzed using the
following gradient conditions: 0−18 min 10−20% B in A, 18−23 min
20−80% B in A, 23−28 min, 80% B in A, 28−30 min 80−10% B in A,
30−50 min 10% B in A, at a flow rate of 1.0 mL/min [A: 0.05 M
TEAA (pH 7.0)/MeCN 95:5; B: 0.05 M TEAA (pH 7.0)/MeCN
50:50]. The relevant peak of interest was collected from the LC,
lyophilized to dryness, and analyzed by MALDI-TOF MS.
Preparation of 12. Silylated nucleoside 11 (101 mg, 0.13 mmol)
was azeotropically dried twice over pyridine (1 mL). Dried 11 was
dissolved in DCM (0.8 mL), and Hunig’s base was added (87 μL, 63.3
̈
mg, 0.49 mmol). 2-Cyanoethyl N,N-diisopropylchlorophosphorami-
dite (43 μL, 45 mg, 0.19 mmol) was added to the reaction flask via
syringe. The reaction was stirred under argon at room temperature for
2.5 h, yielding a clear yellow solution. The reaction mixture was diluted
with DCM (1 mL), quenched with NaHCO3 (2 mL), and extracted
with DCM (2 × 2 mL). The organic layer was concentrated to a
yellow oil. Flash chromatography (30% EtOAc in hexanes) yielded a
ASSOCIATED CONTENT
■
S
* Supporting Information
NMR spectra of monomers, mass spectra of oligonucleotides
containing nonnative nucleotides, hydroxyl radical cleavage
1
mixture of diastereomers of 12 as a white foam (102 mg, 81%): H
1
histograms of cross-linked RNA, and full H NMR spectra of
NMR (CDCl3) δ 0.01−0.08 (m, 12H), 0.89 (d, 20H, J = 7.2 Hz), 1.15
(d, 6H, J = 6.8 Hz), 1.18−1.13 (m, 17H), 1.27−1.24 (t, 8H, J = 7.2
Hz), 2.37−2.34 (t, 2H, J = 6.4 Hz), 2.67−2.63 (q, 2H, J = 6.4 Hz),
3.04 (d, 1H, J = 12.4 Hz), 3.09 (d, 1H, J = 12.4 Hz), 3.34−3.29 (dd,
1H, J1 = 16 Hz, J2 = 2.8 Hz), 3.31 (t, 1H, J = 2.8 Hz) 3.41−3.38 (dd,
1H, J1 = 8.4 Hz, J2 = 2.8 Hz), 3.57−3.51 (m, 7H), 3.76−3.74 (m,
12H), 3.89−3.86 (m, 1H), 3.99−3.90 (m, 1H), 4.28−4.21 (m, 3H),
4.35−4.33 (q, 1H, J = 4.4 Hz), 4.40 (t, 1H, J = 4.8 Hz), 4.49−4.46 (dd,
1H, J1 = 6.4 Hz, J2 = 4.8 Hz), 5.95−5.93 (d, 1H, J = 5.6 Hz), 6.05−
6.03 (d, 1H, J = 6 Hz), 6.84−6.79 (m, 8H), 7.16−7.12 (m, 6H), 7.33−
7.26 (m, 19H), 7.43−7.41 (m, 4H), 7.70 (s, 1H), 7.73 (s, 1H), 8.62
(br, 2H); 31P NMR (CDCl3) 149.1, 150.5; HRMS (ESI/APCI-TOF)
m/z calculated for [M + Na]+ C52H67N4O9NaSiPSe 1053.3472, found
1053.3489.
the oxidation of 9 (Figure 2). This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 410-516-8095. Fax: 410-516-7044. E-mail:
mgreenberg@jhu.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
1H NMR Study of Oxidation of 9 by H2O2. Nucleoside 9 was
dissolved in D2O (50 mM deuterated phosphate buffer, pD 7.4) to
make a 50 mM solution in an NMR tube. The mixture was analyzed
We are grateful for generous support of this research from the
National Institute of General Medical Sciences (GM-054996).
We thank Dr. Michael Delaney (Dharmacon Research), Dr.
Rakesh Paul (Johns Hopkins University), and Souradyuti
Ghosh (Johns Hopkins University) for advice on the manual
coupling procedure. We thank Dr. Cathy Moore (Johns
Hopkins University) for assistance with the water suppression
NMR experiment on 9.
1
via H NMR with water suppression, whereupon a 20× solution of
H2O2 was added to the tube via pipet. The final concentrations of 9
and H2O2 were 50 mM. The mixture was quickly agitated and placed
1
back in the NMR instrument for kinetic analysis. H NMR spectra (8
scans, with water suppression) were taken every minute for 60 min. An
additional spectrum was taken of the solution after incubation at room
temperature for 18 h.
Interstrand Cross-Link Formation with Duplex RNA. The 32P-
labeled oligonucleotide (0.3 μM) and its complementary sequence
(1.5 μM) were dissolved in 100 mM NaCl and 10 mM NaH2PO4 (pH
7.4). The solution was heated to 65 °C and allowed to cool to 4 °C
over the course of 1 h. NaIO4 (5 mM) and H2O2 (10 mM) reactions
of RNA duplexes (11 nM) were carried out in 10 mM sodium
phosphate (pH 7.2) and 100 mM NaCl. Aliquots were taken at
prescribed times, immediately quenched with an equal volume of 95%
formamide loading buffer, and stored at −20 °C until analysis by 20%
denaturing PAGE.
Fe(II)-EDTA Analysis of Cross-Linked DNA. Fe(II)-EDTA reactions
were carried out in 500 μM (NH4)2Fe(SO4)2, 500 μM EDTA, 2 mM
sodium ascorbate, 100 mM H2O2, 10 mM NaCl, 10 mM potassium
phosphate (pH 7.2) for 10 min at room temperature and were
quenched with 10 μL of excess thiourea (100 mM). Samples were
desalted via C-18 Sep-Pak cartridges (100 mg), lyophilized, suspended
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