NDI and DAN DNA: Nucleic acid-directed assembly of NDI and DAN

Article abstract of DOI:10.1021/jo402704z

Two novel DNA base surrogate phosphoramidites 1 and 2, based upon relatively electron-rich 1,5-dialkoxynaphthalene (DAN) and relatively electron-deficient 1,4,5,8-naphthalenetetracarboxylic diimide (NDI), respectively, were designed, synthesized, and incorporated into DNA oligonucleotide strands. The DAN and NDI artificial DNA bases were inserted within a three-base-pair region within the interior of a 12-mer oligonucleotide duplex in various sequential arrangements and investigated with CD spectroscopy and UV melting curve analysis. The CD spectra of the modified duplexes indicated B-form DNA topology. Melting curve analyses revealed trends in DNA duplex stability that correlate with the known association of DAN and NDI moieties in aqueous solution as well as the known favorable interactions between NDI and natural DNA base pairs. This demonstrates that DNA duplex stability and specificity can be driven by the electrostatic complementarity between DAN and NDI. In the most favorable case, an NDI-DAN-NDI arrangement in the middle of the DNA duplex was found to be approximately as stabilizing as three A-T base pairs.

Full text of DOI:10.1021/jo402704z

Article
pubs.acs.org/joc
NDI and DAN DNA: Nucleic Acid-Directed Assembly of NDI and DAN
Brian A. Ikkanda, Stevan A. Samuel, and Brent L. Iverson*
Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States
S
* Supporting Information
ABSTRACT: Two novel DNA base surrogate phosphoramidites 1 and 2,
based upon relatively electron-rich 1,5-dialkoxynaphthalene (DAN) and
relatively electron-deficient 1,4,5,8-naphthalenetetracarboxylic diimide (NDI),
respectively, were designed, synthesized, and incorporated into DNA
oligonucleotide strands. The DAN and NDI artificial DNA bases were
inserted within a three-base-pair region within the interior of a 12-mer
oligonucleotide duplex in various sequential arrangements and investigated
with CD spectroscopy and UV melting curve analysis. The CD spectra of the
modified duplexes indicated B-form DNA topology. Melting curve analyses
revealed trends in DNA duplex stability that correlate with the known
association of DAN and NDI moieties in aqueous solution as well as the
known favorable interactions between NDI and natural DNA base pairs. This
demonstrates that DNA duplex stability and specificity can be driven by the
electrostatic complementarity between DAN and NDI. In the most favorable
case, an NDI−DAN−NDI arrangement in the middle of the DNA duplex was found to be approximately as stabilizing as three
A−T base pairs.
units that promote zipperlike, stacked assembly22 have also
INTRODUCTION
■
been used in the design of artificial DNA structures.
The DNA double helix is a molecular architecture endowed
We23−25 and others26,27 have been exploring the use of
with numerous properties that enable it to serve as an ideal
relatively electron-rich 1,5-dialkoxynaphthalene (DAN) and
scaffold for precise arrangement of aromatic moieties.1−3 Non-
covalent interactions between aromatic nucleobases, combined
relatively electron-deficient 1,4,5,8-naphthalenetetracarboxylic
diimide (NDI) (Figure 1) derivatives in aqueous solution to
create novel folded and assembled structures based on
with desolvation effects, contribute to the DNA duplex
structure, specificity, and stability.4,5 In particular, the
alternating face-centered stacking of the DAN and NDI
remarkable specificity of complementary oligonucleotide
strands derived from Watson−Crick hydrogen bonding can
be exploited to arrange various non-natural DNA base
units.23 Importantly, in strongly interacting solvents such as
water, NDI and DAN have an association constant that is 1 or 2
orders of magnitude larger than the self-association constant of
surrogates in a highly predictable fashion. Because base pairs
either NDI or DAN, respectively.28 This specificity is thought
are stacked in a ladderlike fashion, properties such as
fluorescence6 and electron transfer7,8 as well as various complex
to be due to complementary electrostatic interactions that can
best be rationalized by focusing on the local and direct
interactions of the highly polarized substituents (i.e., the
supramolecular architectures9 can be investigated. The
advanced nature of automated DNA synthesis greatly simplifies
diimide carbonyl groups of NDI and the ether oxygen atoms of
the placement of novel DNA base surrogates in a strand at any
DAN) on the periphery of the aromatic rings.29 Such
chosen location(s) within a sequence.
considerations explain why DAN and NDI adopt a fully face-
Several non-natural DNA base surrogates based on moieties
centered stacking geometry in the solid state (Figure 2a) while
with particular structure and function have been designed and
synthesized that alter DNA stability and structure,10−13
NDI self-stacks in an offset mode (Figure 2b) and DAN does
not prefer to self-stack but rather adopts a herringbone
geometry in the solid state (Figure 2c).30 In the context of
desolvation, these preferred electrostatic-driven geometries
would favor DAN−NDI association over NDI or DAN self-
association in water. Interestingly, in previous work involving
duplex assembly from relatively flexible amide-linked chains of
DAN and NDI units,25 the free energy of duplex formation
including efforts to expand the genetic code with designed
nucleobases.14−17 Through these studies, a great deal has been
learned about requirements for successful DNA duplex
formation in the presence of non-natural aromatic moieties.
For example, designed nucleobases with alternative patterns of
hydrogen bonding have been successfully incorporated into
DNA duplexes, demonstrating that the hydrogen-bonding
pattern found in natural DNA bases can be altered and still
produce stable duplexes.18,19 Non-hydrogen-bonding yet
isosteric DNA bases20,21 as well as other non-natural aromatic
Received: December 5, 2013
Published: February 6, 2014
© 2014 American Chemical Society
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Figure 1. Structures and electrostatic potential surfaces calculated for (left) NDI and (right) DAN using DFT (B3LYP/6-31G*) as implemented in
Spartan (Wavefunction, Inc.).
Figure 2. X-ray crystal structures of (a) DAN−NDI face-centered stacked monomers,32 (b) NDI−NDI offset stacked monomers,33 and (c) DAN
herringbone geometry in the solid state.33
decreased only slightly as the temperature increased, demon-
strating an apparent enthalpy−entropy compensation effect.31
Herein is described the synthesis of novel DAN and NDI
DNA base surrogate phosphoramidites and their incorporation
into DNA oligonucleotides. The stabilities and structures of
various DAN- and NDI-modified oligomers were investigated.
Complementary oligonucleotides that assembled to allow for
alternating NDI−DAN−NDI stacking proved to be more
stable than any of the other combinations investigated,
demonstrating, to the best of our knowledge, for the first
time that a stacking preference based on electrostatic
complementarity can drive duplex stability and specificity.
RESULTS
■
Design of Modified Phosphoramidites. The NDI and
DAN building blocks 1 and 2 were specifically designed to
provide the appropriate flexibility and spacing needed to align
Figure 3. Model depicting the predicted NDI−DAN interaction: (left)
NDI and DAN units in a face-centered stack at the center of the
side view; (right) top-down view. NDI and DAN are shown in blue
DNA duplex structure (Figure 3). Both 1 and 2 utilize the
simplified (S)-GNA backbone (Figure 4), which is known to
and red, respectively.
promote interstrand stacking, leading to relatively stable
duplexes.34 The (S)-GNA backbone was used instead of its
block 3 based on (S)-1,2-propanediol (Figure 4) had to be
enantiomeric analogue particularly because of its known
double-helical structure based on X-ray crystallographic studies,
which may help explain the modified duplex melting temper-
ature.35,36 On the basis of qualitative computer models (Figure
3), linkers of two methylene units for NDI and three methylene
units for DAN appeared to be optimum. These linker lengths
were judged to be long enough to allow exactly the same face-
centered stacking geometry seen in previous foldamers in
aqueous solution23,24 as well as in alternating stacks observed in
the solid state.32,33 It should be noted that rather than base
pairing per se, the DAN and NDI units are intended to stack in
more of a zipper-type arrangement, reminiscent of the systems
reported by Leumann and co-workers.22 The unique aspect of
the present approach is the well-documented preference for
alternating stacking between DAN and NDI units, adding a
new specificity element to the duplex design. For the successful
formation of our designed duplex, however, a “spacer” building
placed across from each modified base to maintain proper
backbone spacing.
Synthesis of the Phosphoramidites. The DAN
phosphoramidite 1 was synthesized starting with the mono-
methylation of 1,5-dihydroxynaphthalene utilizing a stoichio-
metric amount of methyl iodide (Scheme 1). The product was
further alkylated with 3-bromopropan-1-ol to yield alcohol 6.
Intermediate 6 was then reacted with (R)-(+)-glycidol through
a regio- and enantiospecific epoxide opening mediated by
DIBALH to give diol 7 in 29% yield with an enantiomeric
excess of 99% (chiral HPLC).37 Diol 7 was selectively protected
on the primary hydroxyl group using 4,4′-dimethoxytrityl
chloride and subsequently transformed into phosphoramidite 1
using standard conditions.38
The synthesis of the NDI phosphoramidite 2 could not
proceed by a similar epoxide-opening step because the imide
carbonyls of NDI are sensitive to DIBALH, so an alternative
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Figure 4. Structures of the phosphoramidite monomers.
a
Scheme 1
a
Reagents and conditions: (a) MeI, K2CO3, MeCN, 82 °C, 37%; (b) 3-bromopropan-1-ol, K2CO3, MeCN, 82 °C, 75%; (c) (R)-(+)-glycidol,
DIBALH, DCM, 0 °C → rt, 29%; (d) DMTrCl, DMAP, pyridine, rt, 35%; (e) (i-Pr)2NP(Cl)OCH2CH2CN, DIPEA, DCM, rt, 73%.
a
Scheme 2
a
Reagents and conditions: (a) (R)-(+)-glycidol, CsF, 130 °C, 24%; (b) DMTrCl, pyridine, rt, 89%; (c) MeNH2, EtOH, 78 °C, 96%; (d) MeNH2,
DMF, μwave, 75 °C → 140 °C, 83%; (e) 12, Et3N, DMF, μwave, 140 °C, 22%; (f) (i-Pr)2NP(Cl)OCH2CH2CN, DIPEA, DCM, rt, 83%.
route was necessary (Scheme 2). This route involved
phthalimide-protected ethanolamine 9, which was used to
open (R)-(+)-glycidol under mediation by CsF, giving diol 10
in 24% yield with an enantiomeric excess of 89% (chiral
HPLC).39 Because this was an initial study of the use of our
new surrogate bases within this oligonucleotide system, the
synthesis was carried on without separation of the enantiomers.
Diol 10 was selectively protected on the primary hydroxyl
group using 4,4′-dimethoxytrityl chloride under standard
conditions.38 The phthalimide protecting group was then
removed using methylamine to yield free primary amine 12. A
microwave procedure was used to append the two primary
amines to 1,4,5,8-tetracarboxylic acid dianhydride (13), first
using methylamine to obtain the methyl monoimide and then
using the free amino group of 12 to achieve the asymmetric
NDI intermediate 14.40 Intermediate 14 was converted to the
protected phosphoramidite 2 using standard conditions.38
As shown in Scheme 3, the spacer phosphoramidite 3 was
synthesized starting from compound 15 using conditions
similar to those in previous syntheses.38,41
Sequence Design. In order to investigate the DAN and
NDI artificial DNA bases, a three-base-pair region was inserted
into the interior of duplex 1, a control DNA sequence of 12
base pairs (Figure 5). Four single-stranded 15-mer oligonucleo-
tides with NDI and DAN modifications were designed to
generate duplexes 5−8 as well as the oligonucleotides for the
a
Scheme 3
a
Reagents and conditions: (a) (i-Pr)2NP(Cl)OCH2CH2CN, DIPEA,
DCM, rt, 79%.
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Figure 5. Modified DNA base surrogates and a cartoon representing the control DNA duplex 1 as well as the insertions of natural DNA bases
(duplex 2 and duplex 3), spacer units (duplex 4), and the four NDI- and DAN-modified units (duplexes 5−8).
a
three control duplexes with three A−T, G−C, or spacer base
pairs (duplexes 2−4, respectively). The control duplexes
duplex 2 and duplex 3 both contain a deoxyribose backbone
as a reference in order to compare our modified duplexes to
natural DNA duplexes, noting that substituting a GNA
backbone within a sequence of natural bases has been shown
to destabilize the DNA duplex melting temperature.42 Duplex 5
was designed to examine the stability provided by stacking of
three DAN units, while duplex 6 was designed to examine the
stability provided by alternating DAN−NDI−DAN stacking.
Duplex 7 was designed to examine the stability provided by
stacking of three NDI units, while duplex 8 was designed to
examine the stability provided by alternating NDI−DAN−NDI
stacking. NDI is a known strong DNA intercalator43,44 and has
been shown to have relatively high affinity for G-quadruplex
DNA,45,46 and therefore, it is predicted to have greater
association with natural DNA bases than DAN. This led to
the prediction that duplex 8 (with two NDI−nucleobase
contacts) would provide greater stability than duplex 6 (with
two DAN−nucleobase contacts). For the same reason, and
additionally because NDI is known to produce relatively stable
self-stacks compared to DAN self-stacks, duplex 7 was
predicted to provide significantly greater stability than duplex
5. Taken together, analysis of the relative stabilities of duplexes
1−8 allowed an assessment of the role that the electrostatic
complementarity of DAN and NDI plays in stabilizing DNA
duplexes and how such stability compares with those provided
by natural A−T and G−C sequences.
Synthesis of Oligonucleotides. Oligonucleotides were
synthesized on an automated nucleic acid synthesizer according
to standard automated oligonucleotide synthesis protocols,
except for those utilizing the building blocks 1, 2, and/or 3.
These modified phosphoramidites were dissolved in a 3:1
CH2Cl2/CH3CN solution to solubilize the monomers
adequately. To avoid aminolysis of the imide functional
group in NDI, oligonucleotides containing NDI were
synthesized utilizing UltraMild synthesis and deprotection
methods from Glen Research, which avoid the concentrated
aqueous ammonia cleavage step. All of the oligonucleotides
were characterized by HRMS-ESI (negative mode, CH3CN/
aqueous ammonium carbonate).
Table 1. Tm Data for the DNA Duplexes
a
DNA melting experiments were carried out at a duplex concentration
of 1.5 μM (pH 7, 100 mM NaCl, 10 mM NaH2PO4, 0.1 mM EDTA).
for duplex 3 containing three G−C base pairs, which displayed
a melting temperature 11 °C higher than that of the control
duplex 1. The next most stable duplex was duplex 2 containing
A−T base pairs, which exhibited a 4 °C increase in melting
temperature compared with duplex 1. Duplex 4 containing
three spacer units instead of any bases or aromatic units was by
far the least stable duplex examined (its melting temperature
was 30 °C lower than that of the control duplex 1), indicating
that duplex 4 should probably be viewed as containing two
hexamer duplexes that melt more or less independently.
As expected, the modified duplex with the highest melting
temperature was duplex 8, which showed an increase in
thermal stability of 3 °C compared with the control duplex 1. It
should be noted that this melting temperature increase is
comparable to that afforded by three A−T base pairs (duplex
2) but not as large as that seen with three G−C base pairs
(duplex 3). Comparing duplex 7 with duplex 8 reveals that a 4
°C increase in melting temperature was afforded by changing
the central NDI unit to a more electrostatically complementary
DAN moiety in an NDI−DAN−NDI arrangement. Similarly,
Thermal Denaturing Studies. Thermal denaturing studies
were performed to quantify the influence of DAN and NDI
stacking patterns on the duplex stability (Table 1). The highest
melting temperature for any of the duplexes studied was seen
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Figure 6. CD spectra of duplexes 1−8. All spectra were were recorded at a duplex concentration of 1.5 μM (pH 7, 100 mM NaCl, 10 mM
NaH2PO4, 0.1 mM EDTA).
comparing duplex 5 to duplex 6 shows that a homo-DAN
arrangement is also significantly less stabilizing than a DAN−
NDI−DAN alternating duplex, in this case by 10 °C. Finally, as
expected, NDI−nucleobase interactions appear to be preferred
over DAN−nucleobase interactions, as duplex 8 exhibited a 5
°C higher melting point than duplex 6.
NDI and especially DAN−DAN−DAN self-stacking within a
DNA duplex. Upon comparison of the melting temperatures of
duplex 5 and duplex 6, an impressive 10 °C increase in melting
temperature was observed upon replacement of the central
DAN with NDI to create an alternating DAN−NDI−DAN
arrangement. Consistent with this trend, comparing the melting
temperatures of duplex 7 and duplex 8 shows that there was a
4 °C increase upon exchange of the central NDI to a DAN to
create an alternating NDI−DAN−NDI arrangement. It is
reasonable that the latter difference is less significant than the
former because NDI is known to self-stack with relatively high
stability, albeit in an offset stacking mode, while DAN self-
stacking is not known to be favorable.28 It thus appears that the
known electrostatic complementarity between these units is
stabilizing in the context of DNA duplexes, thereby exerting
control over duplex specificity without the use of specific
hydrogen-bonding patterns.
The NDI−DAN−NDI arrangement in duplex 8 provided
the most significant stability of any of the modified base
sequences examined, as its melting temperature was 5 °C
higher than that of the corresponding DAN−NDI−DAN
duplex 6. This is as expected because NDI is a known DNA
intercalator, so the two NDI−nucleobase interactions predicted
to occur at either end of the NDI−DAN−NDI segment within
duplex 8 should be energetically favorable compared with the
two DAN−nucleobase interactions predicted to occur at either
end of the DAN−NDI−DAN segment within duplex 6.
Circular Dichroism Spectra of the Duplexes. In order to
investigate overall duplex structure, the duplexes were analyzed
by CD spectroscopy (Figure 6). Each of the modified duplexes
showed spectral features consistent with an overall B-form
DNA topology, exhibiting a bisignate spectrum with a null
occurring near λ = 260 nm. In addition, CD spectra at various
temperatures ranging from 15 to 70 °C demonstrated behavior
similar to that the corresponding duplex 1 CD spectra, which
further supports its B-form structure. Although this is not a
high-resolution technique, these results do rule out any
substantial deviations from B-form structure in any of the
duplexes. Unfortunately, little to no insight can be gained
regarding the exact stacking topologies of the modified bases in
duplexes 4−8 from the CD spectra alone. Even at duplex
concentrations of 10 μM, the relative concentration of NDI is
too small to detect any significant CD signal, let alone changes
within CD spectra, resulting from the NDI absorbance in the
300−400 nm range.
DISCUSSION
■
Taken together, our results indicate a strong preference for
alternating NDI−DAN−NDI stacking relative to NDI−NDI−
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(R)-3-(3-((5-Methoxynaphthalen-1-yl)oxy)propoxy)propane-
1,2-diol (7). To a clean dry 50 mL round-bottom flask were added 6
(0.5211 g, 2.245 mmol) and CH2Cl2 (15 mL). The reaction mixture
was cooled to 0 °C with an ice bath, and DIBALH (1.25 mL of a 1.5 M
solution in toluene, 1.875 mmol) was added. The reaction mixture was
taken off the ice, allowed to warm to room temperature, and stirred for
30 min, and then (R)-(+)-glycidol (0.1 mL, 1.506 mmol) was added
dropwise. The reaction mixture was stirred for 72 h at room
temperature. Potassium sodium tartrate (0.5525 g, 1.959 mmol)
dissolved in a minimal amount of water was added, and the reaction
mixture was stirred for 30 min and then extracted three times with
ethyl acetate. The resulting organic layers were combined and washed
with water and brine. The product mixture was then dried over
Na2SO4 and concentrated in vacuo. The product was purified by silica
gel column chromatography (4% methanol in CH2Cl2) to give a
Although no direct spectroscopic evidence for NDI and
DAN stacking in any of the duplexes was obtained, the
predictable differences in stabilities, consistent with the known
preference for alternating NDI−DAN stacking, provides
supporting evidence that especially in duplex 8 the NDI and
DAN units are stacked in, or close to, the preferred face-
centered geometry. On the basis of CD analysis, we do know
that the modified bases do not cause any significant deviations
from the B-form structure of the entire duplex. Overall, these
considerations indicate the design of non-natural base
surrogates 1 and 2 was able to facilitate the desired NDI−
DAN stacking within the context of a B-form DNA helix.
CONCLUSION
1
brown solid (0.133 g, 0.434 mmol, 29% yield). Mp 48−50 °C. H
■
NMR (400 MHz, CDCl3, ppm) δ 7.84 (d, J = 8.3 Hz, 2H), 7.36 (dd, J
= 14.5, 7.8 Hz, 2H), 6.82 (d, J = 7.6 Hz, 2H), 4.16 (t, J = 6.1 Hz, 2H),
3.97 (s, 3H), 3.88−3.81 (m, 1H), 3.71 (t, J = 6.3 Hz, 2H), 3.65 (dd, J
= 11.5, 3.6 Hz, 1H), 3.56 (dd, J = 11.5, 6.0 Hz, 1H), 3.49−3.46 (m,
2H), 3.25 (s, 2H), 2.15 (p, J = 6.2 Hz, 2H). 13C NMR (400 MHz,
CDCl3, ppm) δ 155.1, 154.2, 126.5, 125.1, 114.1, 114.0, 105.4, 104.4,
72.2, 70.6, 68.2, 64.7, 63.9, 55.4, 29.4. HRMS-ESI (m/z) calcd for
Two new DNA base surrogate building blocks based on DAN
and NDI have been synthesized and incorporated into DNA
oligonucleotide strands. In the most favorable arrangement, an
NDI−DAN−NDI sequence in the middle of a DNA duplex
was found to be approximately as stabilizing as three A−T base
pairs. The results demonstrate that electrostatic complemen-
tarity between DAN and NDI can be used to drive the
specificity and stability of DNA duplexes. Future work will
explore how NDI and DAN units behave in alternate patterns
within a DNA duplex, including their effect at the terminal
positions of oligonucleotide duplexes as well as the
thermodynamics of longer stretches of NDI and DAN within
DNA.
+
C17H22NaO5 [M + Na]+ 329.1359, found 329.1355. [α]2D2 −7.3 (c
0.50, CHCl3). 99% ee as determined by HPLC (Chiralcel ODH
column, 0.46 cm I.D. × 25 cm long; eluent, hexane/i-PrOH 95:5 v/v;
flow rate, 1.0 mL/min; UV at 254 nm; room temperature; see the
Supporting Information).
(S)-1-(Bis(4-methoxyphenyl)(phenyl)methoxy)-3-(3-((5-me-
thoxynaphthalen-1-yl)oxy)propoxy)propan-2-ol (8). In a clean,
dry 50 mL round-bottom flask, 7 (0.4012 g, 1.3096 mmol) and DMAP
(0.0160 g, 0.131 mmol) were dissolved in pyridine (13.1 mL). The
atmosphere was purged with argon, and the mixture was stirred. Next,
4,4′-dimethoxytrityl chloride (0.4508 g, 1.330 mmol) was slowly
added to the reaction mixture. The mixture was stirred overnight, and
the pyridine was then removed in vacuo. The product was purified by
silica gel column chromatography (1:1 hexanes/ethyl acetate with
0.1% triethylamine) to yield a light-tan oil (0.2793 g, 0.459 mmol, 35%
yield). 1H NMR (400 MHz, CDCl3, ppm) δ 7.83 (d, J = 8.0 Hz, 2H),
7.42 (d, J = 7.1 Hz, 2H), 7.38−7.33 (m, 2H), 7.33−7.27 (m, 6H),
7.23−7.17 (m, 1H), 6.87−6.77 (m, 6H), 4.18 (t, J = 6.1 Hz, 2H), 3.99
(s, 3H), 3.98−3.93 (m, 1H), 3.76 (s, 6H), 3.75−3.71 (m, 2H), 3.56
(ddd, J = 16.0, 9.7, 5.3 Hz, 2H), 3.18 (dd, J = 5.5, 2.9 Hz, 2H), 2.42 (s,
1H), 2.17 (p, J = 6.2 Hz, 2H). 13C NMR (400 MHz, CDCl3, ppm) δ
155.2, 154.3, 126.6, 125.1, 114.2, 114.0, 105.4, 104.4, 72.4, 70.5, 68.3,
EXPERIMENTAL SECTION
■
5-Methoxynaphthalen-1-ol (5). In a 100 mL round-bottom flask
with a stir bar, 1,5-dihydroxynaphthalene (1.00 g, 6.2 mmol) was
dissolved in 32 mL of acetonitrile. To the reaction mixture were added
K2CO3 (0.95 g, 6.8 mmol) and CH3I (0.39 mL, 6.2 mmol). A reflux
condenser was fitted to the reaction flask, and the reaction vessel was
purged with argon. The reaction mixture was stirred at reflux overnight
and then cooled to room temperature, and the acetonitrile was
removed in vacuo. The thick black reaction mixture was then dissolved
in CHCl3 and filtered through Celite. The filtrate was washed three
times with saturated NaHCO3 and three times with brine and then
dried over Na2SO4. The CHCl3 was removed in vacuo, and the crude
product was purified with silica gel column chromatography (2%
acetone in CHCl3) to give a tan solid (0.41 g, 2.4 mmol, 37% yield).
Mp 128−132 °C. 1H NMR (400 MHz, CDCl3, ppm) δ 7.85 (d, J = 8.5
Hz, 1H), 7.74 (d, J = 8.5 Hz, 1H), 7.40 (t, J = 7.7 Hz, 1H), 7.30 (t, J =
7.5 Hz, 1H), 6.85 (d, J = 7.4 Hz, 2H), 5.23 (s, 1H), 4.00 (s, 3H). 13C
NMR (400 MHz, CDCl3, ppm) δ 155.3, 151.1, 126.9, 125.3, 125.1,
+
64.8, 64.0, 55.5, 53.4, 29.6. HRMS-ESI (m/z) calcd for C38H40NaO7
[M + Na]+ 631.2666, found 631.2670.
(S)-1-(Bis(4-methoxyphenyl)(phenyl)methoxy)-3-(3-((5-me-
thoxynaphthalen-1-yl)oxy)propoxy)propan-2-yl (2-Cyanoeth-
yl) N,N-Diisopropylphosphoramidite (1). In a dry 15 mL round-
bottom flask, 8 (0.2793 g, 0.459 mmol) was dissolved in CH2Cl2 (8.2
mL), and N,N-diisopropylethylamine (0.51 mL, 2.963 mmol) was
added. The reaction mixture was stirred and purged with argon, and 2-
cyanoethyl N,N-diisopropylchlorophosphoramidite (0.2 mL, 0.918
mmol) was added dropwise. The reaction mixture was stirred for 3 h at
room temperature, poured into saturated aq. NaHCO3, washed three
times with CH2Cl2, and dried over Na2SO4. The CH2Cl2 was removed
in vacuo, and the product was purified by silica gel column
chromatography (4:1 hexanes/ethyl acetate with 0.1% triethylamine)
to yield a mixture of diastereomers as a faint-yellow oil (0.270 g, 0.334
+
114.7, 113.6, 109.4, 104.4, 55.5. HRMS-CI (m/z) calcd for C11H11O2
[M + H]+ 175.0754, found 175.0760.
3-((5-Methoxynaphthalen-1-yl)oxy)propan-1-ol (6). In a 250
mL three-neck round-bottom flask with a stir bar, 5 (1.47 g, 8.42
mmol) was dissolved in 84 mL of acetonitrile. To the reaction mixture
were added K2CO3 (1.28 g, 9.27 mmol) and 3-bromopropan-1-ol
(1.32 mL, 15.10 mmol). The flask was fitted with a condenser and
septa and purged with argon. The reaction mixture was heated at reflux
for 24 h and then cooled to room temperature, and the acetonitrile
was removed in vacuo. The thick black reaction mixture was dissolved
in CH2Cl2 and filtered through Celite. The CH2Cl2 was removed in
vacuo, and the reaction mixture was purified by silica gel column
chromatography (5% acetone in CH2Cl2) to give a light-tan solid (1.47
1
mmol, 73% yield). H NMR (400 MHz, CDCl3, ppm) δ 7.86 (t, J =
7.5 Hz, 2H), 7.52−7.44 (m, 2H), 7.41−7.17 (m, 11H), 6.88−6.75 (m,
6H), 4.18 (dt, J = 17.1, 6.0 Hz, 3H), 4.00 (s, 3H), 3.80 (s, 2H), 3.76 (s,
6H), 3.74−3.69 (m, 2H), 3.69−3.60 (m, 3H), 3.60−3.48 (m, 1H),
3.30 (dd, J = 9.4, 4.4 Hz, 1H), 3.26−3.19 (m, 1H), 3.19−3.12 (m,
1H), 2.57−2.35 (m, 2H), 2.16 (tt, J = 12.4, 6.1 Hz, 2H), 1.20 (d, J =
6.8 Hz, 6H), 1.16 (d, J = 6.7 Hz, 3H), 1.05 (d, J = 6.7 Hz, 3H). 13C
NMR (400 MHz, CDCl3, ppm) δ 158.5, 158.3, 155.1, 154.3, 144.9,
139.4, 136.1, 136.0, 130.0, 129.0, 128.2, 128.1, 127.8, 127.7, 127.6,
127.0, 126.6, 126.5, 125.2, 125.0, 117.7, 117.6, 114.1, 114.0, 113.9,
1
g, 6.33 mmol, 75% yield). Mp 100−102 °C. H NMR (400 MHz,
CDCl3, ppm) δ 7.83 (dd, J = 14.0, 8.9 Hz, 2H), 7.42−7.33 (m, 2H),
6.86 (t, J = 7.7 Hz, 2H), 4.27 (t, J = 5.8 Hz, 2H), 3.99 (s, 3H), 3.96 (t,
J = 6.0 Hz, 2H), 2.18 (p, J = 6.0 Hz, 2H), 1.83 (s, 1H). 13C NMR (400
MHz, CDCl3, ppm) δ 155.2, 154.3, 126.6, 126.5, 125.2, 125.1, 114.3,
114.0, 105.5, 104.5, 65.6, 60.5, 55.5, 32.1. HRMS-CI (m/z) calcd for
+
C14H16O3 [M]+ 232.1099, found 232.1101.
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The Journal of Organic Chemistry
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113.1, 112.9, 105.3, 104.4, 104.3, 85.9, 85.8, 72.7, 72.5, 72.3, 72.1, 71.8,
67.9, 67.8, 64.8, 64.1, 63.9, 58.4, 58.2, 55.4, 55.1, 43.1, 43.0, 42.9, 29.7,
29.6, 24.6, 24.5, 24.4, 20.1, 20.0. 31P NMR (400 MHz, CDCl3, ppm) δ
149.4, 149.3. HRMS-ESI (m/z) calcd for C47H58N2O8P+ [M + H]+
809.3925, found 809.3930.
phenanthroline-1,3,6,8(2H,7H)-tetraone (14). In a clean, dry two-
neck round-bottom flask, 1,4,5,8-naphthalenetetracarboxylic dianhy-
dride (2.04 g, 7.61 mmol) was suspended in DMF (40 mL), and
CH3NH2 (2 M in THF, 3.75 mL, 7.5 mmol) was added. The reaction
mixture was sonicated for 5 min and then stirred and heated under
microwave irradiation in the open reaction vessel fitted with a reflux
condenser at 75 °C for 5 min and then 140 °C for 5 min. The reaction
temperature was monitored by an internal probe. The reaction vessel
was cooled to room temperature, and the solvent was removed in
vacuo. The dark-brown solid was suspended in acetone, and the
suspension was added to vigorously stirring 1 N HCl. The product was
filtered, washed with water, and dried overnight in vacuo to yield a tan
solid that was not purified any further (1.74 g, 6.17 mmol, 83% crude
2-(2-Hydroxyethyl)isoindoline-1,3-dione (9). Compound 9
was synthesized according to a protocol previously reported in the
literature.47 Mp 118−120 °C. H NMR (400 MHz, CDCl3, ppm) δ
1
7.85−7.77 (m, 2H), 7.73−7.66 (m, 2H), 3.91−3.79 (m, 4H), 2.67 (s,
1H). 13C NMR (400 MHz, CDCl3, ppm) δ 168.8, 134.0, 131.9, 123.3,
+
60.8, 40.7. HRMS-CI (m/z) calcd for C10H10NO3 [M + H]+
192.0655, found 192.0659.
(R)-2-(2-(2,3-Dihydroxypropoxy)ethyl)isoindoline-1,3-dione
(10). To a 50 mL round-bottom flask were added CsF (0.081 g, 0.053
mmol) and 9 (4.7 g, 24.6 mmol). The reaction mixture was purged
with argon, stirred, heated to 135 °C, and stirred for 45 min, and then
(R)-(+)-glycidol (1.48 mL, 22.3 mmol) was added dropwise. The
mixture was stirred overnight, cooled to room temperature, and
dissolved in 4% methanol in CH2Cl2. The product was purified by
silica gel column chromatography (4% methanol in CH2Cl2) to yield a
1
yield). H NMR (400 MHz, DMSO-d6, ppm) δ 8.73−8.65 (m, 4H),
+
3.43 (s, 3H). HRMS-CI (m/z) calcd for C15H8NO5 [M + H]+
282.0397, found 282.0395.
In a clean oven-dried microwave reaction vessel, the resulting crude
product (0.2031 g, 0.7222 mmol) and 12 (0.2420 g, 0.5531 mmol)
were dissolved in DMF (5 mL), and triethylamine (0.08 mL) was
added. The reaction vessel was sealed and sonicated for 5 min. The
reaction mixture was stirred and heated under microwave irradiation at
140 °C for 5 min and then allowed to cool to room temperature. The
solvent was removed in vacuo, and the product was purified by silica
gel column chromatography (1% methanol in CH2Cl2 with 0.2%
triethylamine) to yield a tan frothy solid (0.0834 g, 0.1191 mmol, 22%
yield). 1H NMR (400 MHz, CDCl3, ppm) δ 8.65 (d, J = 1.0 Hz, 4H),
7.39 (d, J = 7.2 Hz, 2H), 7.25 (dd, J = 13.4, 8.2 Hz, 6H), 7.16 (t, J =
7.2 Hz, 1H), 6.76 (d, J = 8.7 Hz, 4H), 4.42 (t, J = 5.7 Hz, 2H), 3.94−
3.88 (m, 1H), 3.84 (td, J = 5.7, 1.8 Hz, 2H), 3.74 (s, 6H), 3.67 (dd, J =
9.6, 3.8 Hz, 1H), 3.60 (dd, J = 9.6, 6.6 Hz, 1H), 3.56 (s, 3H), 3.18−
3.08 (m, 2H), 2.73 (d, J = 4.2 Hz, 1H). 13C NMR (400 MHz, CDCl3,
ppm) δ 162.8, 158.4, 144.8, 136.0, 131.0, 130.9, 130.0, 128.1, 127.8,
126.7, 126.5, 126.4, 126.3, 113.0, 86.0, 72.7, 69.9, 68.2, 64.4, 55.2, 39.8,
27.4. HRMS-ESI (m/z) calcd for C41H36N2O9Na+ [M + Na]+
723.2313, found 723.2317.
(S)-1-(Bis(4-methoxyphenyl)(phenyl)methoxy)-3-(2-(7-meth-
yl-1,3,6,8-tetraoxo-7,8-dihydrobenzo[lmn][3,8]phenanthrolin-
2(1H,3H,6H)-yl)ethoxy)propan-2-yl (2-Cyanoethyl) N,N-Diiso-
propylphosphoramidite (2). In a dry 5 mL round-bottom flask, 11
(0.0565 g, 0.0806 mmol) was dissolved in CH2Cl2 (1.5 mL), and N,N-
diisopropylethylamine (0.09 mL, 0.517 mmol) was added. The
reaction mixture was stirred and purged with argon, and 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite (0.05 mL, 0.2241 mmol) was
added dropwise. The reaction mixture was stirred for 2 h at room
temperature, poured into saturated aq. NaHCO3, washed three times
with CH2Cl2, and dried over Na2SO4. The CH2Cl2 was removed in
vacuo, and the product was purified by silica gel column
chromatography (1:1 hexanes/ethyl acetate with 0.1% triethylamine)
to yield a mixture of diastereomers as a faint-yellow oil (0.0605 g,
0.0671 mmol, 83% yield). 1H NMR (400 MHz, CDCl3, ppm) δ 8.72−
8.56 (m, 4H), 7.40 (t, J = 7.9 Hz, 2H), 7.31−7.19 (m, 6H), 7.15 (dd, J
= 14.0, 7.1 Hz, 1H), 6.82−6.68 (m, 4H), 4.48−4.33 (m, 2H), 4.14−
4.02 (m, 1H), 3.90−3.76 (m, 4H), 3.74 (d, J = 3.6 Hz, 6H), 3.71−3.62
(m, 3H), 3.56 (s, 3H), 3.51−3.42 (m, 1H), 3.23 (dd, J = 9.6, 4.6 Hz,
1H), 3.20−3.11 (m, 1H), 3.04 (dd, J = 9.3, 6.4 Hz, 1H), 2.64 (tq, J =
7.6, 3.7 Hz, 1H), 2.49−2.32 (m, 1H), 1.12 (dd, J = 6.7, 3.8 Hz, 6H),
1.08 (d, J = 6.8 Hz, 3H), 0.96 (d, J = 6.8 Hz, 3H). 13C NMR (400
MHz, CDCl3, ppm) δ 162.8, 162.5, 158.2, 144.8, 136.0, 135.9, 130.8,
130.7, 129.9, 129.8, 128.1, 128.0, 127.5, 126.5, 126.4, 126.3, 126.2,
117.9, 117.6, 112.8, 85.8, 72.6, 72.5, 72.2, 72.0, 71.9, 67.8, 64.0, 63.8,
58.4, 58.3, 58.2, 58.1, 55.0, 43.0, 42.9, 42.8, 39.7, 39.5, 27.2, 24.5, 24.4,
24.3, 24.2, 20.2, 20.1, 20.0. 31P NMR (400 MHz, CDCl3, ppm) δ
149.5, 149.1. HRMS-ESI (m/z) calcd for C50H53N4O10PNa+ [M +
Na]+ 923.3392, found 923.3375.
1
white solid (1.25 g, 4.70 mmol, 24% yield). Mp 59−61 °C. H NMR
(400 MHz, CDCl3, ppm) δ 7.84−7.78 (m, 2H), 7.72−7.66 (m, 2H),
3.87 (t, J = 5.5 Hz, 2H), 3.77 (dt, J = 9.7, 4.8 Hz, 1H), 3.73−3.64 (m,
2H), 3.61 (dd, J = 11.5, 4.1 Hz, 1H), 3.58−3.49 (m, 3H), 3.30 (s, 1H),
2.83 (s, 1H). 13C NMR (400 MHz, CDCl3, ppm) δ 168.5, 134.0,
131.8, 123.3, 72.4, 70.4, 68.7, 63.6, 37.4. HRMS-CI (m/z) calcd for
C13H16NO5+ [M + H]+ 266.1023, found 266.1024. [α]2D4 −8.7 (c 0.73,
CHCl3). 89% ee as determined by HPLC (Chiralcel ODH column,
0.46 cm I.D. × 25 cm long; eluent, hexane/i-PrOH 95:5 v/v; flow rate,
1.0 mL/min; UV at 254 nm; room temperature; see the Supporting
Information)
(S)-2-(2-(3-(Bis(4-methoxyphenyl)(phenyl)methoxy)-2-
hydroxypropoxy)ethyl)isoindoline-1,3-dione (11). In a dry 15
mL round-bottom flask, 10 (0.3526 g, 1.33 mmol) was dissolved in
pyridine (6 mL). The mixture was purged with argon and stirred, and
then 4,4′-dimethoxytrityl chloride (0.5435 g, 1.604 mmol) was slowly
added. The mixture was stirred for 4 h, and the pyridine was removed
in vacuo. The product was purified by silica gel column
chromatography (1:1 hexanes/ethyl acetate with 0.1% triethylamine)
1
to yield an off-white oil (0.6715 g, 1.183 mmol, 89% yield). H NMR
(400 MHz, CDCl3, ppm) δ 7.82 (dd, J = 5.4, 3.1 Hz, 2H), 7.70 (dd, J
= 5.5, 3.0 Hz, 2H), 7.41 (d, J = 7.2 Hz, 2H), 7.28 (dd, J = 14.0, 8.4 Hz,
6H), 7.19 (t, J = 7.2 Hz, 1H), 6.81 (d, J = 8.9 Hz, 4H), 3.95−3.90 (m,
1H), 3.88 (t, J = 5.7 Hz, 2H), 3.78 (s, 6H), 3.71 (td, J = 5.8, 2.8 Hz,
2H), 3.62 (dd, J = 9.6, 3.8 Hz, 1H), 3.53 (dd, J = 9.6, 6.7 Hz, 1H), 3.14
(qd, J = 9.4, 5.6 Hz, 2H), 2.65 (d, J = 4.6 Hz, 1H). 13C NMR (400
MHz, CDCl3, ppm) δ 168.3, 158.3, 144.8, 135.9, 133.9, 131.9, 129.9,
128.0, 127.7, 126.7, 123.2, 113.0, 85.9, 72.5, 69.7, 68.3, 64.3, 55.1, 37.4.
HRMS-ESI (m/z) calcd for C34H33NO7Na+ [M + Na]+ 590.2149,
found 590.2150.
(S)-1-(2-Aminoethoxy)-3-(bis(4-methoxyphenyl)(phenyl)-
methoxy)propan-2-ol (12). In a dry 250 mL round-bottom flask, 11
(1.0084 g, 1.7765 mmol) was dissolved in ethanol (15 mL), and
CH3NH2 (33% in ethanol, 30 mL, 141 mmol) was added. The
reaction flask was fitted with a condenser and purged with argon, and
the reaction mixture was heated to reflux for 2.5 h. The mixture was
cooled to room temperature, and the solvent was removed in vacuo.
The product was purified by silica gel column chromatography (10%
methanol in CH2Cl2 with 0.1% triethylamine) to yield an off-white oil
1
(0.4859 g, 1.111 mmol, 96% yield). H NMR (400 MHz, CDCl3,
ppm) δ 7.43 (d, J = 7.3 Hz, 2H), 7.32 (d, J = 8.8 Hz, 4H), 7.25 (t, J =
7.5 Hz, 2H), 7.16 (t, J = 7.3 Hz, 1H), 6.80 (d, J = 8.9 Hz, 4H), 3.94
(td, J = 9.2, 5.8 Hz, 1H), 3.72 (s, 6H), 3.59 (dd, J = 10.0, 3.3 Hz, 1H),
3.53−3.41 (m, 3H), 3.30 (s, 2H), 3.15 (ddd, J = 21.1, 9.3, 5.7 Hz, 2H),
2.78 (t, J = 5.0 Hz, 2H). 13C NMR (400 MHz, CDCl3, ppm) δ 158.2,
144.6, 135.8, 129.8, 127.9, 127.5, 126.5, 112.8, 85.7, 72.7, 72.0, 69.4,
64.3, 63.2, 54.9, 41.0. HRMS-ESI (m/z) calcd for C26H31NO5Na+ [M
+ Na]+ 460.2094, found 460.2095.
(S)-1-(Bis(4-methoxyphenyl)(phenyl)methoxy)propan-2-ol
(15). Compound 15 was synthesized according to a protocol
previously reported in the literature.41 1H NMR (400 MHz, CDCl3,
ppm) δ 7.44 (d, J = 7.1 Hz, 2H), 7.31 (dd, J = 15.4, 8.4 Hz, 6H),
7.25−7.16 (m, 1H), 6.84 (d, J = 9.0 Hz, 4H), 4.03−3.93 (m, 1H), 3.80
(s, 6H), 3.13 (dd, J = 9.2, 3.4 Hz, 1H), 3.00 (dd, J = 9.2, 7.9 Hz, 1H),
(S)-2-(2-(3-(Bis(4-methoxyphenyl)(phenyl)methoxy)-2-
hydroxypropoxy)ethyl)-7-methylbenzo[lmn][3,8]-
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2.42 (s, 1H), 1.11 (d, J = 6.4 Hz, 3H). 13C NMR (400 MHz, CDCl3,
ppm) δ 158.4, 144.8, 136.0, 130.0, 129.1, 128.1, 127.8, 126.8, 113.1,
86.0, 68.8, 67.1, 55.2, 18.9. HRMS-ESI (m/z) calcd for C24H26O4Na+
[M + Na]+ 401.1723, found 401.1727.
small compared with the amounts of naturally occurring DNA bases, it
is unlikely that this approximation caused any major discrepancies in
DNA concentration. The melting studies were performed in a Teflon-
stoppered 1 cm path length quartz cell on a UV−vis spectropho-
tometer equipped with a thermoprogrammer. Each melting temper-
ature run involved combining 0.5 mL of complementary strand
samples to obtain 1.5 μM duplex in solution. The samples were
initially heated to 85 °C for 5 min and then cooled from 85 to 70 °C at
a rate of 1 °C/min and then from 70 to 5 °C at a rate of 0.5 °C/min.
The absorbance at 260 nm was monitored. Two runs of these
experiments were carried out per sample and averaged.
CD Spectroscopy. CD spectra were recorded on a circular
spectropolarimeter, and all of the spectra were measured in a 1 cm
path length quartz cell. Samples were prepared from UV melting
temperature studies (1.5 μM in duplex concentration) in a phosphate
buffer (pH 7, 100 mM NaCl, 10 mM NaH2PO4, 0.1 mM EDTA).
Samples were initially heated to 85 °C for 5 min and allowed to cool to
room temperature for 45 min before the CD data were collected. The
experiments were carried out at 25 °C.
(S)-1-(Bis(4-methoxyphenyl)(phenyl)methoxy)propan-2-yl
(2-Cyanoethyl) N,N-Diisopropylphosphoramidite (3). In a dry
50 mL round-bottom flask, 15 (0.2608 g, 0.6891 mmol) was dissolved
in CH2Cl2 (12.3 mL), and N,N-diisopropylethylamine (0.77 mL,
4.4102 mmol) was added. The reaction mixture was stirred and purged
with argon, and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite
(0.31 mL, 1.3897 mmol) was added dropwise. The reaction mixture
was stirred for 3.5 h at room temperature, poured into saturated aq.
NaHCO3, washed three times with CH2Cl2, and dried over Na2SO4.
The CH2Cl2 was removed in vacuo, and the product was purified by
silica gel column chromatography (1:4 hexanes/ethyl acetate with
0.1% triethylamine) to yield a mixture of diastereomers as a faint-
1
yellow oil (0.3146 g, 0.5437 mmol, 79% yield). H NMR (400 MHz,
CDCl3, ppm) δ 7.48 (d, J = 8.3 Hz, 2H), 7.36 (dd, J = 8.9, 3.7 Hz,
4H), 7.28 (dd, J = 14.6, 7.4 Hz, 2H), 7.20 (dd, J = 14.7, 8.6 Hz, 1H),
6.83 (t, J = 8.5 Hz, 4H), 4.21−4.04 (m, 1H), 3.91−3.82 (m, 1H), 3.79
(d, J = 3.5 Hz, 6H), 3.77−3.72 (m, 1H), 3.70−3.51 (m, 2H), 3.22−
3.14 (m, 1H), 3.02 (dd, J = 9.4, 4.6 Hz, 1H), 2.91 (dd, J = 9.0, 5.7 Hz,
1H), 2.62 (t, J = 6.5 Hz, 1H), 2.56−2.41 (m, 1H), 1.28 (t, J = 5.3 Hz,
3H), 1.24−1.16 (m, 9H), 1.11 (d, J = 6.8 Hz, 3H). 13C NMR (400
MHz, CDCl3, ppm) δ 158.3, 145.0, 136.3, 136.2, 130.1, 130.0, 129.1,
128.2, 127.7, 127.6, 126.6, 126.5, 117.7, 113.1, 112.9, 85.8, 85.7, 70.0,
69.8, 68.0, 67.8, 67.8, 58.5, 58.3, 58.1, 57.9, 55.1, 43.0, 42.9, 24.7, 24.6,
24.5, 24.4, 20.4, 20.3, 20.2, 19.7, 19.6. 31P NMR (400 MHz, CDCl3,
ppm) δ 147.7, 147.4. HRMS-ESI (m/z) calcd for C33H44N2O5P+ [M +
H]+ 579.2982, found 579.2977.
Oligonucleotide Synthesis. Unmodified oligonucleotides were
synthesized on an automated nucleic acid synthesizer using a standard
protocol for 2-cyanoethyl phosphoramidites (0.067 M) on a Glen
UnySupport expedite format column on a 1 μmol scale. Because of
their poor solubility in CH3CN, all of the modified-base phosphor-
amidites were diluted with 3:1 CH2Cl2/CH3CN. The oligonucleotides
were synthesized using a trityl-on synthesis and cleaved from the resin
with 1 mL of concentrated aqueous ammonia at room temperature for
12−24 h. The cleaved oligonucleotides were diluted with 1 mL of
NaCl solution (100 mg/mL) and then semipurified by application to a
Glen Pak purification column.
To avoid aminolysis, oligonucleotides containing NDI were
synthesized utilizing UltraMild synthesis and deprotection methods
from Glen Research. UltraMild-compatible phosphoramidites (Pac-
dA-CE, Ac-dC-CE, and iPr-Pac-dG-CD phosphoramidites) and the
Universal Support III expedite format column on a 1 μmol scale were
used. The oligonucleotides were cleaved from the resin using the
UltraMild deprotection solution (0.05 M K2CO3 in methanol) for 12 h
and then diluted with 1 mL of 0.1 M TEAA, neutralized with 6 μL of
glacial acetic acid, and lyophilized. The resulting dried crude trityl-on
oligonucleotides were dissolved in 2 mL of 0.1 M TEAA and then
applied directly on a Glen Pak purification column. The standard
protocol for trityl-on oligonucleotides was used for the Glen Pak
purification columns to afford the semipurified detritylated oligonu-
cleotides. All of the semipurified oligonucleotides were last purified by
reversed-phase HPLC using a C18 peptide semipreparatory reversed-
phase column with 0.1 M aqueous TEAA (pH 7) and CH3CN as the
eluent. All of the oligonucleotides were characterized by HRMS-ESI
(negative mode, CH3CN/aqueous ammonium carbonate) (see the
Supporting Information).
ASSOCIATED CONTENT
■
S
* Supporting Information
General procedures for synthetic methods; chiral HPLC
1
chromatograms of 7 and 10; H, 13C, and 31P NMR spectra
of synthesized compounds; molecular masses of individual
oligonucleotides measured by ESI high-resolution mass
spectrometry; HPLC chromatograms of modified oligonucleo-
tides; methods for determining extinction coefficients of 1 and
2; and UV melting temperature curves. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: iversonb@austin.utexas.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Sean Kerwin for access to a UV−vis
spectrophotometer and Dr. Andrew Ellington for access to
the automated DNA synthesizer. This work was supported by
the Robert A. Welch Foundation (F1188) and the National
Institutes of Health (GM-069647).
REFERENCES
■
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