Synthesis of cross-linked DNA containing oxidized abasic site analogues

Article abstract of DOI:10.1021/jo500944g

DNA interstrand cross-links are an important family of DNA damage that block replication and transcription. Recently, it was discovered that oxidized abasic sites react with the opposing strand of DNA to produce interstrand cross-links. Some of the cross-links between 2'-deoxyadenosine and the oxidized abasic sites, 5'-(2-phosphoryl-1,4-dioxobutane) (DOB) and the C4-hydroxylated abasic site (C4-AP), are formed reversibly. Chemical instability hinders biochemical, structural, and physicochemical characterization of these cross-linked duplexes. To overcome these limitations, we developed methods for preparing stabilized analogues of DOB and C4-AP cross-links via solid-phase oligonucleotide synthesis. Oligonucleotides of any sequence are attainable by synthesizing phosphoramidites in which the hydroxyl groups of the cross-linked product were orthogonally protected using photochemically labile and hydrazine labile groups. Selective unmasking of a single hydroxyl group precedes solid-phase synthesis of one arm of the cross-linked DNA. The method is compatible with commercially available phosphoramidites and other oligonucleotide synthesis reagents. Cross-linked duplexes containing as many as 54 nt were synthesized on solid-phase supports. Subsequent enzyme ligation of one cross-link product provided a 60 bp duplex, which is suitable for nucleotide excision repair studies.

Full text of DOI:10.1021/jo500944g

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pubs.acs.org/joc
Synthesis of Cross-Linked DNA Containing Oxidized Abasic Site
Analogues
Souradyuti Ghosh and Marc M. Greenberg*
Department of Chemistry, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, United States
S
* Supporting Information
ABSTRACT: DNA interstrand cross-links are an important family of
DNA damage that block replication and transcription. Recently, it was
discovered that oxidized abasic sites react with the opposing strand of
DNA to produce interstrand cross-links. Some of the cross-links
between 2′-deoxyadenosine and the oxidized abasic sites, 5′-(2-
phosphoryl-1,4-dioxobutane) (DOB) and the C4-hydroxylated abasic
site (C4-AP), are formed reversibly. Chemical instability hinders
biochemical, structural, and physicochemical characterization of these
cross-linked duplexes. To overcome these limitations, we developed methods for preparing stabilized analogues of DOB and C4-
AP cross-links via solid-phase oligonucleotide synthesis. Oligonucleotides of any sequence are attainable by synthesizing
phosphoramidites in which the hydroxyl groups of the cross-linked product were orthogonally protected using photochemically
labile and hydrazine labile groups. Selective unmasking of a single hydroxyl group precedes solid-phase synthesis of one arm of
the cross-linked DNA. The method is compatible with commercially available phosphoramidites and other oligonucleotide
synthesis reagents. Cross-linked duplexes containing as many as 54 nt were synthesized on solid-phase supports. Subsequent
enzyme ligation of one cross-link product provided a 60 bp duplex, which is suitable for nucleotide excision repair studies.
Scheme 2
INTRODUCTION
■
A number of cytotoxic pharmacological agents, such as nitrogen
mustards and mitomycin C, produce DNA interstrand cross-
links (ICLs).1,2 Other exogenous and endogenous bis-electro-
philes also produce ICLs by alkylating the nitrogenous DNA
bases in either the major or minor groove.3 ICLs are absolute
blocks to replication and transcription, making the agents that
produce them potent DNA damaging agents. Cells are
protected from the deleterious effects of ICLs by nucleotide
excision repair (NER).4 How ICLs are recognized and removed
by NER is an active area of research.5−7 The importance of
these subjects is exemplified by recent observations of ICLs that
are misrepaired by the bacterial NER system (UvrABC), which
converts them to double-strand breaks.8−10 A double-strand
break is the most deleterious form of DNA damage. Recently,
ICLs resulting from the reaction of three abasic lesions (AP,
C4-AP, DOB) with native nucleotides on the opposing strand
have been discovered.11−15 Cross-links 1 (Scheme 1) and 2
(Scheme 2) form from the reaction of the C4′-oxidized abasic
site (C4-AP) and 5′-(2-phosphoryl-1,4-dioxobutane) (DOB)
with the N6-amino group of the dA that is opposite a 3′-
adjacent thymidine.13−15 These ICLs revert to the oxidized
abasic lesions in phosphate buffer and decompose upon
reaction with NaBH3CN, which is often used as a stabilizing
agent. The chemical properties of 1 and 2 make character-
ization of their repair and other chemical and structural
properties impractical and motivated us to synthesize stable
analogues.
Scheme 1
C4-AP and DOB are oxidized abasic lesions that are
produced by several antitumor agents, as well as γ-radiolysis
following C4′- or C5′-hydrogen atom abstraction, respec-
tively.16 For instance, C4-AP is a major product formed by
bleomycin under O2 limiting conditions.17 The enediyne
Received: April 29, 2014
© XXXX American Chemical Society
A
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antitumor antibiotics produce C4-AP and DOB.18 These
oxidized abasic sites share a common 1,4-dicarbonyl structural
feature that is required for forming cross-links 1 and 2
(Schemes 1 and 2) and is responsible for other biochemically
interesting reactions. In duplex DNA, C4-AP (following
hydrolysis of its 5′-phosphate) and DOB efficiently, irreversibly
inactivate DNA polymerase β.19,20 In addition to inactivating
this vital enzyme involved in DNA repair, the oxidized abasic
sites also inactivate DNA polymerase λ, which is believed to be
a back-up of DNA polymerase β.21 C4-AP has also been shown
to react with histone proteins in nucleosome core particles.22
Histone proteins catalyze DNA cleavage at C4-AP lesions with
half-lives as short as ∼15 min. Furthermore, the lysine
residue(s) responsible for strand scission is modified in the
process (Scheme 3). Examination of these biochemical
acidic conditions used for detritylation and whose own cleavage
will not affect the β-cyanoethyl phosphate protecting group, the
amides used to protect nucleobase exocyclic amines, or the
ester linkages employed for capping aborted syntheses and
tethering the oligonucleotides to the solid phase support.38,39
Three orthogonal functional groups that have been successfully
used in oligonucleotide synthesis were initially considered.
These were the levulinyl (Lev) group mentioned above,37,40−42
the o-nitrobenzyl photoredox (ONV) reaction,43−45 and Pd(0)-
mediated cleavage of allyloxycarbonyl groups.46 After briefly
experimenting with allyloxycarbonyl chemistry (data not
shown), we chose to investigate the Lev and ONV groups
with respect to the synthesis of DOB ICLs (3).
Synthesis of Oligonucleotides Containing an Ana-
logue of a DOB ICL (3). We approached the synthesis of
oligonucleotides containing 3 (Scheme 4) by first preparing the
Scheme 3
Scheme 4
reactions was facilitated by the availability of synthetic
substrates containing DOB or C4-AP.14,23−25 As mentioned
above, ICLs 1 and 2 revert to the respective lesions. The half-
life for 1 at 25 °C is ∼10 h and that for 2 under the same
conditions is ∼3 h.13,15 Although ICLs 1 and 2 are stable
enough to be isolated and characterized by mass spectrometry,
they do not survive long enough for more thorough
characterization. Consequently, we pursued the synthesis of
stable analogues 3 and 4.
A number of strategies for DNA−DNA interstrand cross-link
synthesis have been reported. Some combine solid-phase
oligonucleotide synthesis with postsynthetic chemistry that
enables cross-linking by a latent electrophile upon hybridization
with a complementary oligonucleotide.26−33 Other methods
involve highly reactive intermediates that may produce mixtures
of products.34 Total synthesis of cross-linked DNA on solid-
phase support is an alternative, more linear approach. Some
methods take advantage of sequence symmetry to reduce the
number of steps.35,36 Complete control over the sequence of
the cross-linked product by solid-phase synthesis can be
achieved by using orthogonal protecting groups that enable
selective unmasking of appropriate nucleophiles. Recently,
Damha employed such an approach for preparing branched
RNA molecules.37 One advantage of this approach was that the
modified phosphoramidite was compatible with commercially
available solid-phase synthesis reagents. We have taken a similar
approach for synthesizing stabilized analogues of DOB and C4-
AP interstrand cross-links.
entire (“template”) strand containing the cross-linked dA (6)
using commercially available 5′-dimethoxytrityl-protected
phosphoramidites and 5a,b (Scheme 5). The secondary alcohol
of the DOB analogue containing the orthogonal protecting
group (7) was revealed following detritylation and capping of
the 5′-hydroxyl group of the template strand, and the solid-
phase synthesis was completed using 3′-dimethoxytrityl 5′-β-
cyanoethyl (“reverse”) phosphoramidites to obtain 8.
The requisite phosphoramidites were readily synthesized
from an activated form of 2′-deoxyinosine (9) using the
methodology established by Lakshman (Scheme 5).47 The
hydroxypyrrolidine (11) substitution product (10) was a
common precursor to the levulinylated (5a) and o-nitroveratrol
carbonate (5b) phosphoramidites. Following desilylation, the
nucleoside analogues (12a,b) were carried on to the
phosphoramidites via the dimethoxytritylated products
(13a,b) using standard procedures.
Oligonucleotides were prepared using 5a and standard
commercially available β-cyanoethyl phosphoramidites contain-
ing standard amine protecting groups, with the exception of dC.
N4-Benzoyl-dC was used instead of the N-acetyl analogue to
prevent deprotection during hydrazinolysis. A variety of
coupling conditions were experimented with, but ultimately
good yields (typically between 50−65%, but as high as 98%)
were obtained when the levulinylated phosphoramidite (5a,
0.15 M) was double-coupled for 15 min. The 5′-terminus of the
RESULTS AND DISCUSSION
■
To stabilize ICLs derived from the reaction of DOB or C4-AP
with the N6-amino group of dA, we targeted the lower
oxidation state analogues (3, 4, Schemes 1 and 2) that lack the
labile hemiaminal functional groups. These analogues could be
used to synthesize cross-linked oligonucleotides on solid-phase
supports by employing orthogonal protecting groups for the
respective hydroxyl groups. This approach requires one
orthogonal protecting group for preparing DOB ICLs (3,
Scheme 1) and two for C4-AP ICLs (4, Scheme 2). To
maximize use of the most common commercially available
reagents, protecting groups were considered that are stable to
B
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a
Scheme 5
a
Key: (a) 11, Cs2CO3, THF; (b) levulinic acid, EDCI, DMAP, DMF, or o-nitroveratryl chloroformate, DMAP, CH2Cl2; cc) Et3N·3HF, THF; (d)
DMTCl, pyridine; (e) 2-cyanoethyl chlorophosphoramidite, diisopropylethylamine, CH2Cl2.
template strand (8, Scheme 4) was manually capped with a
solution containing 5% (vol/vol) each of acetic anhydride, N-
methylimidazole, and pyridine in THF following detritylation.
The resin was typically incubated 10 times with the reagents for
4 min each cycle for oligonucleotides ≤25 nucleotides long.
Twenty-five cycles were used for longer oligonucleotides.
Complete capping and its stability to the delevulinylation
conditions were crucial to prevent unwanted extension during
opposing strand synthesis. Delevulinylation was carried out
using 0.25 M hydrazine in 3:2 pyridine/AcOH at room
temperature for 10 min for oligonucleotides ≤25 nucleotides
long and 25 min for longer substrates. Capping efficiency and
its resistance to cleavage during hydrazinolysis was examined by
determining extension in a model oligonucleotide (dT9) that
had been capped as described above. A portion of the resin was
subjected to additional automated synthesis with or without
prior treatment with the delevulinylation reagents. The resin
was subsequently deprotected and cleaved from the solid-phase
support using concentrated aqueous ammonia, and an aliquot
of the crude material was analyzed by denaturing PAGE
following 5′-32P-labeling. No extension was detected via
phosphorimaging analysis (data not shown). Following
delevulinylation, the second strand (“arm”) of the ICL was
synthesized using reverse phosphoramidites. The first phos-
phoramidite was double-coupled (15 min coupling time), and
subsequent phosphoramidites were single-coupled for the same
amount of time. The final products (14−20, Figure 1) were
obtained following deprotection and cleavage using concen-
trated aqueous ammonia, purified by denaturing PAGE, and
analyzed by either MALDI-TOF MS or ESI-MS. Using these
procedures cross-linked DNA containing 3 in a template strand
as long as 55 nt and an 8 nt long arm (20) was prepared. Cross-
linked molecules containing longer template strands (e.g., 19,
20) were synthesized without full-length arms in the
complementary strand. Due to the linear process of solid-
phase oligonucleotide synthesis, we anticipated using enzyme
ligation to assemble the products containing full-length
complementary strands (“arms”). This approach is demon-
Figure 1. Chemically synthesized oligonucleotides containing 3.
strated below for a cross-linked duplex containing C4-AP
analogue 4.
Although the levulinyl group was satisfactory for preparing
cross-linked oligonucleotides containing stabilized DOB
analogue 3, we also examined the utility of the photolabile
5b in ICL synthesis. This was done in part to establish the
viability of the venerable o-nitrobenzyl photoredox reaction for
preparing cross-links containing 4, which requires a phosphor-
amidite whose synthesis is longer than that of 5b. Thinking
ahead to the synthesis of 4, we recognized that the necessity of
the N4-benzoyl dC protecting group when synthesizing ICLs
using the levulinyl group created a conundrum because it was
previously found to be incompatible with the o-nitroveratrole
photolabile protecting group.48,49 Previous experiments were
C
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carried out using a UV light source with maximal output at 350
nm that emitted light close to 300 nm. Rather than search for
an alternative orthogonal protecting group, we explored
photolysis conditions that would be compatible with the
benzoyl protecting group on dC (and likely dA). To direct our
search, we measured the absorbance of solutions (52 μM) of an
o-nitroveratrole carbonate, as well as benzoyl-dC and benzoyl-
dA phosphoramidites between 350 and 400 nm (Figure 2).
Synthesis of Oligonucleotides Containing an Ana-
logue of a C4-AP ICL (4). Having established the
compatibility of the levulinyl and o-nitroveratrole groups with
solid-phase synthesis conditions, we sought to implement a
strategy for synthesizing ICLs containing 4 that utilizes these
orthogonal protecting groups (Scheme 6). We envisioned
synthesizing the template strands of the ICLs using standard
solid-phase synthesis and the capping conditions described for
the preparation of oligonucleotides containing the DOB
analogue (Scheme 4). The o-nitroveratrole group would be
removed from 22, and the primary alcohol extended using 5′-
dimethoxytrityl phosphoramidites. Following capping of 23, 24
would be delevulinylated and the 3′-arm of 25 extended using
reverse phosphoramidites. Finally, 26 would be cleaved from
the solid-phase support and 4 purified by denaturing PAGE.
The synthesis of 4 from 24 was anticipated to follow the
general procedure developed for preparing 3 (Scheme 4).
The strategy for the synthesis of the necessary phosphor-
amidite (32, Scheme 7) was based upon the chemistry
developed for the DOB analogue. One previously reported
procedure for pyrollidine 27 did not work in our hands.50
Instead, 27 was synthesized as described by Merino and reacted
with 9 to produce the fully functionalized cross-link analogue
(28).51 We were unable to selectively functionalize the primary
alcohol in 28 using o-nitroveratryl chloroformate. Hence, 29
was prepared in a straightforward, albeit circuitous, manner and
selectively detritylated to provide the primary alcohol necessary
for the formation of 30. Following desilylation, 31 was carried
on to phosphoramidite 32 via standard methods.
The utility of 32 for synthesizing cross-linked oligonucleo-
tides was demonstrated using one short (8 bp, 33, Figure 3)
duplex and two longer ones. In each instance, 32 was double-
coupled manually. The coupling yields ranged from 80 to
100%. All other phosphoramidites were double-coupled via
automated solid-phase synthesis when preparing the longer
template strands in 34 and 35, resulting in 98.2% average
stepwise yield. Following synthesis of the template strand, a
portion of the resin (typically 1/6 to 1/5 of the total amount of
resin present in a 1 μmol scale column) was photolyzed as
described above for 1.5−2.0 h. The “5′-arm” was then
synthesized manually. The 5′-termini were phosphorylated
Figure 2. Absorbance of N-benzoylphosphoramidites and o-nitro-
veratrole (ONV) protected 10 between 350 and 400 nm. All solutions
were 54 μM.
Absorption by the benzoyl group was negligible at 375 nm,
but the o-nitroveratrole carbonate modestly absorbed light at
this wavelength. The utility of the NVOC-protected phosphor-
amidite (5b) was demonstrated by synthesizing 21. Double-
coupling of 5b proceeded in 92% yield, and following
photolysis using a long pass filter (Newport no. CGA-375)
that had zero transmittance at 370 nm and 50% at 375 nm the
complementary strand was extended in 53% overall yield
(determined via trityl cation release). With the exception of the
first phosphoramidite, which was coupled for 15 min, standard
coupling cycles were used to prepare the cross-linked strand.
Approximately 4.5 nmol of 21 was isolated following
concentrated ammonium hydroxide deprotection and denatur-
ing PAGE purification from ∼1/5th of the resin from a 1 μmol
scale commercial column.
Scheme 6
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a
concentrated aqueous ammonium hydroxide at 55 °C over-
night and purified by denaturing PAGE.
Scheme 7
T4 DNA Ligase-Mediated Ligation of 34. To demon-
strate the feasibility of transforming cross-linked oligonucleo-
tides containing incomplete arms (e.g., 34, 35) into ones with
fully complementary strands, 40 was obtained from 34 and 36−
39 using T4 DNA ligase (Scheme 8). Cross-link product 40 is
sufficiently long (60 bp) to be used in nucleotide excision repair
studies. The one 5′-terminus of 34 that was not already
phosphorylated was phosphorylated using T4 polynucleotide
kinase and ATP. The 5′-terminus was of 38 also enzymatically
phosphorylated except γ-32P-ATP was used. These oligonucleo-
tides were hybridized using 34 as the limiting reagent and
ligated (16 °C, 12 h) using T4 DNA ligase and ATP. The
product was purified by denaturing PAGE and isolated by the
crush and soak method. Isolated yields were determined using
the specific activity of 38 and varied between 10% and 20%,
based upon the amount of 34 used in the reaction. Following
desalting and reannealing, the integrity of 40 was examined
using restriction enzymes.52
a
Key: (a) 9, Cs2CO3, THF; (b) DMTCl pyridine; (c) levulinic acid,
EDCI, DMAP, DMF; (d) AcOH, H2O, THF (3:1:1); (e) o-
nitroveratryl chloroformate, DMAP, CH2Cl2; (f) Et3N·3HF, THF;
(g) 2-cyanoethyl chlorophosphoramidite, diisopropylethylamine,
CH2Cl2.
SUMMARY
■
The methods described enable chemical synthesis of cross-
linked DNA. By utilizing orthogonal protecting groups, there
are no sequence limitations. By combining the chemical
synthesis with DNA ligase, cross-links that are sufficiently
long (e.g., 60 bp) for subsequent nucleotide excision repair
studies are accessible. In this particular study, stabilized
analogues of chemically labile DNA cross-links were synthe-
sized. The true cross-links (1, 2) contain epimerizable centers
at the cross-linked nucleotide. The analogues prepared (3 and
27) contain the stereochemistry of native DNA at the cross-
linked sites. These were chosen because it was anticipated that
these would be the least distorting and most stable stereo-
isomers. However, the method is amenable to synthesizing
other stereoisomers and is generally applicable to the synthesis
of cross-linked DNA (and RNA).
during solid-phase synthesis using a commercially available
phosphoramidite (Solid CPR II, Glen Research) to facilitate
subsequent T4 DNA ligase mediated ligation. All phosphor-
amidites were double-coupled except the first, which was
coupled four times. Coupling yields were determined by
measuring the amounts of dimethoxytrityl cation released and
calculated based upon the theoretical yield, which took into
account the amount of resin used, its loading, and the overall
yield of the template strand. This analysis indicated that the
first phosphoramidite coupled in between 63% and 90%.
However, subsequent phosphoramidites were coupled in as
high as 99.8% average stepwise yield for the synthesis of the 5′-
arm in 34 and as low as 97.9% in 35. To complete the
syntheses of the C4-AP cross-link analogue containing
oligonucleotides, the levulinyl group was removed as described
above for the DOB ICL analogue containing oligonucleotides.
The syntheses of the 3′-arms were then carried out manually
using the same strategy employed for preparing the 5′-arms.
Coupling yields for the 3′-arms could not be determined as
described for the 5′-arms because the delevulinylation
conditions cleaved some of the o-nitroveratrole group that
was not released upon photolysis. Consequently, partial
extension of this arm resulted in apparent coupling yields
>100%. Cross-linked products 33−35 were deprotected with
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).
Figure 3. Chemically synthesized oligonucleotides containing 4.
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Scheme 8
Oligonucleotides were synthesized via automated DNA synthesis on
an Applied Biosystems model 394 instrument. Commercially available
DNA synthesis reagents including phosphorylation reagent were
purchased from Glen Research. Phosphoramidites 5a, 5b, and 32 were
dried for 12 h in a lyophilizer prior to the experiment.
Oligonucleotides containing cross-link analogues were deprotected
using concentrated NH4OH at 55 °C for 12 h. Oligonucleotides
containing native nucleotides only were deprotected using 1:1 mixture
of aqueous methylamine (40%)−concentrated concentrated NH4OH
at 55 °C for 1 h.53 Oligonucleotides were purified by 20% denaturing
polyacrylamide gel electrophoresis (PAGE), isolated by the crush and
soak method, and desalted using C-18-Sep-Pak cartridges (Waters).
Oligonucleotides containing modified nucleotides were characterized
by MALDI-TOF MS or ESI-MS. Oligonucleotides were 5′-32P-labeled
by T4 polynucleotide kinase (New England Biolabs) and γ-32P-ATP
(Perkin Elmer) using standard protocols.54 Experiments involving
radiolabeled oligonucleotides were analyzed following PAGE using a
Storm 840 phosphorimager and Imagequant TL software. RSaI, TaqαI,
and CutSmart buffer were from NEB.
Synthesis of 10. The hydrochloride salt of 11 (667 mg, 5.5 mmol)
was added to a stirred solution of 9 (1.6 g, 2.7 mmol) in THF (50
mL), followed by addition of Cs2CO3 (4.2 g, 13.5 mmol). The
reaction was allowed to stir for 3.5 h at room temperature, at which
time the solvent was evaporated in vacuo. The crude material was
dissolved in DCM (100 mL) and washed with saturated sodium
bicarbonate (70 mL). The organic layer was collected, dried over
sodium sulfate, and concentrated. Flash chromatography (70% EtOAc
in DCM to 100% EtOAc) yielded 11.2 g of 10 (82%). Rf (5% MeOH
in DCM) = 0.3. 1H NMR (CDCl3): δ 8.3 (s, 1H), 8.0 (s, 1H), 6.41 (t,
1H, J = 6.4 Hz), 4.60 (m, 1H), 4.56 (m, 1H), 3.84−4.54 (m, 5H), 3.81
(dd, 1H, J = 3.9, 11 Hz), 3.72 (dd, 1H, J = 3.7, 11 Hz), 3.05 (bd s,
1H), 2.59 (dt, 1H, J = 5.6, 12.2 Hz), 2.38 (m, 1H), 2.13 (m, 2H), 1.89
(bd s, 1H), 0.88 (s, 18H), 0.074 (s, 6H), 0.070 (s, 6H). 13C NMR
(CDCl3): δ 153.2, 152.7, 149.8, 137.2, 120.8, 87.8, 84.2, 72.3, 63.1,
45.8, 41.1, 26.1, 25.9, 18.6, 18.2, 8.7, −4.5, −4.6, −5.2, −5.3. IR (neat):
3300 (bd), 2951, 2856, 1591, 1471, 1252, 1105, 1031 cm−1. HRMS
(ESI/APCI-TOF): m/z [M + H]+ calcd for C26H48N5O4Si2 550.3239,
found 550.3250.
Synthesis of Levulinyl-Protected 10. Levulinic acid (604 mg,
529 μL, 5.1 mmol), DMAP (731 mg, 5.9 mmol), and EDCI (989
mmol, 5.2 mmol) were added to a stirred solution of 10 (405 mg, 0.7
mmol) in DMF (9 mL). The reaction mixture was stirred for 4 h at
room temperature under inert atmosphere. The resulting light orange
mixture was diluted with EtOAc (80 mL) and then washed with water
(50 mL), saturated sodium bicarbonate (50 mL), and brine solution
(50 mL). The organic layer was dried over sodium sulfate,
concentrated in vacuo, and subjected to flash chromatography using
30% EtOAc in DCM to obtain the desired product (445 mg, 93%). Rf
(30% EtOAc in DCM) = 0.31. 1H NMR (CDCl3): δ 8.32 (s, 1H), 7.97
(s, 1H), 6.42 (t, 1H, J = 6 Hz), 5.45 (m, 1H), 4.56 (s, 1H), 3.97−4.54
(m, 5H), 3.76 (dd, 1H, J = 4.6, 10.8 Hz), 3.72 (dd, 1H, J = 2.5, 10.8
Hz), 2.69 (m, 2H), 2.59 (m, 1H), 2.52 (m, 2H), 2.37 (m, 1H), 2.19
(m, 2H), 2.12 (s, 3H), 0.88 (s, 18H), 0.07 (s, 6H), 0.04 (s, 6H). 13C
NMR (CDCl3): δ 206.4, 172.3, 153.1, 152.8, 150.1, 137.5, 120.8, 87.8,
84.1, 72.2, 62.9, 41.1, 37.9, 29.9, 28.2, 26.1, 25.8, 18.5, 18.1, −4.6, −4.7,
−5.3, −5.4. IR (neat): 2852, 2926, 2856, 1720, 1591, 1471, 1156,
1093, 834, 777 cm−1. HRMS (ESI/APCI-TOF): m/z calcd for [M +
H]+ C31H54N5O6Si2 648.3607, found 648.3616.
Synthesis of NVOC-Protected 10. A mixture of 10 (304 mg, 0.55
mmol), nitroveratryloxycarbonyl chloride (615 g, 2.22 mmol), and
(dimethylamino)pyridine (549 mg, 4.43 mmol) was stirred in DCM
(6 mL) at room temperature for 2 h, at which time the solution was
diluted with 50 mL of DCM. The diluted contents were washed with
saturated sodium bicarbonate (2 × 50 mL) followed by washing with
brine (50 mL). The combined aqueous layers were washed with DCM
(50 mL), and the combined organic layers were dried over sodium
sulfate. After the solvent was evaporated in vacuo, the product mixture
was dissolved in DCM. It was purified by flash chromatography (10%
EtOAc in DCM to 60% EtOAc in DCM) to yield the desired product
1
as a white solid (401 mg, 92%). Rf (50% EtOAc in DCM): 0.6. H
NMR (CDCl3): δ 8.35 (s, 1H), 7.99 (s, 1H), 7.72 (s, 1H), 7.02 (s,
1H), 6.45 (t, 1H, J = 8 Hz), 5.59 (s, 2H), 5.45 (m, 1H), 4.60 (m, 1H),
4.01- 4.38 (m, 5H), 3.94 (s, 3H), 3.93 (s, 3H), 3.83 (dd, 1H, J = 11.2,
4.8 Hz), 3.75 (dd, 1H, J = 3.2, 10.8 Hz), 2.63 (td, 1H, J = 6, 12.8 Hz),
2.41 (m, 1H), 2.33 (m, 2H), 0.914 (s, 9H), 0.911 (s, 9H), 0.11, (s,
6H), 0.08 (s, 6H). 13C NMR (CDCl3): δ 154.3, 153.8, 153.0, 152.8,
150.1, 148.4, 139.7, 137.6, 126.7, 120.8, 87.9, 84.1, 72.3, 66.6, 63.0,
56.7, 56.6, 41,2, 30.7, 26.1, 25.9, 18.6, 18.2, −4.5, −4.6, −5.2, −5.3. IR
(neat): 2953, 2928, 2856, 1749, 1591, 1523, 1470, 1253, 1219, 1064,
834, 776 cm−1. HRMS (ESI/APCI-TOF): m/z [M + H]+ calcd for
C36H57N6O10Si2 789.3669, found 789.3661.
Synthesis of 12a. Triethylamine trihydrofluoride (0.220 mg, 221
μL, 1.37 mmol) was added to a solution of levulinyl-protected 10 (310
mg, 0.47 mmol) in THF (3.0 mL). After being stirred at room
temperature for 17 h, TLC (5% MeOH in DCM) showed that a small
amount of starting material was still present. An additional equivalent
of desilylating reagent was added and the reaction stirred for 6
additional h at which time no starting material remained. The reaction
was evaporated to dryness in vacuo, and 140 mg of a white foamlike
product (71% yield) was collected after purification by flash
chromatography (2% MeOH in DCM to 5% MeOH in DCM). Rf
1
(5% MeOH in DCM) = 0.3. H NMR (MeOH-d4): δ 8.23 (s, 1H),
8.18 (s, 1H), 6.42 (t, 1H, J = 6.8 Hz), 5.44 (m, 1H), 4.88 (m, 4H),
4.56 (d, 1H, J = 3.6 Hz), 4.10−4.50 (1H), 4.05 (d, 1H, J = 2.4 Hz),
3.87−4.02 (1H), 3.84 (dd, 1H, J = 11.2, 2.6 Hz), 3.73 (dd, 1H, J =
12.4, 2.9 Hz), 2.70−2.86 (3H), 2.52 (t, 2H, J = 6.4 Hz), 2.37 (m, 1H),
2.24 (m, 2H), 2.06 (s, 3H). 13C NMR (MeOD-d4): δ 209.4, 174.0,
154.2, 153.2, 150.4, 140.6, 121.8, 89.8, 87.2, 73.1, 63.6, 42.6, 38.6, 32.9,
31.1, 29.5, 29.0. IR (neat): 3263, 2929, 1713, 1593, 1472, 1204, 1158.
1095 cm−1. HRMS (ESI/APCI-TOF): m/z [M + H]+ calcd for
C19H26N5O6 420.1878, found 420.1887.
Synthesis of 12b. Triethylamine trihydrofluoride (208 mg, 209
μL, 1.28 mmol) was added to a solution of NVOC protected 10 (202
mg, 0.26 mmol) in THF (2.7 mL) and the mixture was stirred
overnight at room temperature. After the solvent was evaporated in
vacuo, the residue was dissolved in DCM and purified by flash
chromatography (18 × 2.2 cm) (2% methanol in DCM to 4%
methanol in DCM) to yield the desired product as a white foam (131
1
mg, 89%). Rf (4% methanol in DCM) = 0.15. H NMR (CDCl3): δ
8.29 (s, 1H), 7.77 (s, 1H), 7.72 (s, 1H), 7.01 (s, 1H), 6.86 (d, 1H, J =
8 Hz), 6.31 (dd, 1H, J = 5.6, 8 Hz), 5.59 (s, 2H), 5.45 (d, 1H, J = 16
Hz), 4.8 (d, 1H, J = 4 Hz), 4.57 (m, 1H), 3.96−4.34 (m, 4H), 3.95 (s,
F
dx.doi.org/10.1021/jo500944g | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Featured Article
3H), 3.94 (s, 3H), 3.79 (t, 1H, J = 8 Hz), 3.14 (m, 1H), 2.63 (s, 1H),
2.28 (d, 1H, J = 5.2 Hz), 2.25 (d, 1H, J = 5.5 Hz). 13C NMR (CDCl3):
δ 154.3, 153.8, 153.4, 152.1, 149, 148.5, 139.8, 139.1, 126.5, 122.2,
110.2, 108.3, 89.9, 87.9, 77.4, 73.8, 66.6, 63.7, 56.6, 56.5, 54.7, 53.2,
46.5, 45.4, 40.6, 32.3, 30.2, 29.7, 8.9. IR (neat): 3332 (bd), 2934, 2270,
1750, 1596, 1522, 1471, 1387, 1332, 1277, 1220, 1064, 987, 792, 668
cm−1. HRMS (ESI/APCI-TOF): m/z [M + H]+ calcd for
C24H29N6O10 561.1940, found 561.1944.
(m, 2H), 3.29 (m, 2H), 2.79 (m, 1H), 2.73 (d, 1H, J = 6.4 Hz), 2.69
(d, 1H, J = 6.4 Hz), 2.63 (m, 1H), 2.55 (m, 2H), 2.12 (m, 2H), 2.14
(s, 3H), 1.07−1.19 (m, 12H). 31P NMR (CDCl3): 149.06, 148.95. IR
(neat): 2960, 1720, 1593, 1509, 1469, 1250, 1178, 1077 cm−1. HRMS
(ESI/APCI-TOF): m/z [M + H]+ calcd for C49H60N7O9P 922.4263,
found 922.4265.
Synthesis of 5b. DIPEA (94 mg, 0.73 mmol) was added to 13b
(118 mg, 0.13 mmol) that was azeotropically dried with pyridine,
followed by 1.5 mL of freshly distilled DCM. The solution was cooled
in an ice bath, and 2-cyanoethyl N,N-diisopropylchlorophosphor-
amidite (42.6 mg, 0.11 mmol) was added. After the reaction mixture
was stirred for 1 h, the reaction mixture was diluted with EtOAc (30
mL), washed with saturated sodium bicarbonate (20 mL), and then
dried over sodium sulfate. After the solvent was evaporated in vacuo,
the product mixture was dissolved in 60% ethyl in hexanes. A silica
column was packed with 1% Et3N in hexanes and then washed with 3
column volumes volume of 60% EtOAc in hexanes. The above
solution was loaded onto the column and separated (60% EtOAc in
hexanes to 90% EtOAc in hexanes) to yield the desired product (108
Synthesis of 13a. A pyridine (2 mL) solution of DMT-Cl (73 mg,
0.19 mmol) and azeotropically dried 12a (75 mg, 0.19 mmol) was
stirred overnight, at which time the reaction was quenched with
MeOH and evaporated to dryness in vacuo. The dry mass was
dissolved in EtOAc (20 mL) and washed with saturated sodium
bicarbonate (15 mL) and dried over sodium sulfate. A flash silica
column was prepared using 1% Et3N in hexanes that was washed with
5 column volumes of 1% MeOH in EtOAc prior to loading the
mixture. A solvent combination of 1% MeOH in EtOAc that was
increased to 3% MeOH in EtOAc was used to obtain 93 mg of product
1
(68%). Rf (5% MeOH in DCM) = 0.4. H NMR (CDCl3): δ 8.29 (s,
1
1H), 7.86 (s, 1H), 7.38 (d, 2H, J = 8.6 Hz), 7.14−7.34 (m, 7H), 6.78
(d, 4H, J = 8.6 Hz), 6.45 (t, 1H, J = 6.0 Hz), 5.48 (s, 1H), 4.64 (m,
1H), 4.15−4.55 (m, 2H), 4.12 (m, 1H), 3.72−4.09 (m, 2H), 3.78 (s,
3H), 3.75 (s, 3H), 3.36 (m, 2H), 3.05 (bd s, 1H), 2.68−2.85 (m, 3H),
2.44−2.59 (m, 3H), 2.21 (m, 2H), 2.15 (s, 3H). 13C NMR (CDCl3): δ
206.4, 172.3, 158.5, 153.0, 152.6, 149.8, 144.5, 137.2, 135.7, 130.0,
128.0, 127.9, 126.9, 120.7, 113.1, 86.5, 85.8, 83.9, 72.6, 63.8, 55.2, 54.0,
53.1, 47.0, 45.8, 40.3, 37.8, 30.7, 29.8, 28.0, 24.2. IR (neat): 3300 (bd),
2932, 2836, 1720, 1592, 1508, 1443 cm−1. HRMS (ESI/APCI-TOF):
m/z [M + H]+ calcd for C40H44N5O8 722.3184, found 722.3188.
Synthesis of 13b. A pyridine (3 mL) solution of DMT-Cl (108
mg, 0.26 mmol) and azeotropically dried (pyridine) 12b (131 mg, 0.23
mmol) was stirred overnight, at which time the reaction was diluted
with 50 mL of EtOAc. The diluted contents were washed with
saturated ammonium chloride (2 × 50 mL), followed by washing with
brine (50 mL). The aqueous layers were washed with EtOAc (50 mL),
and the combined organic layers were dried over sodium sulfate. After
the solvent was evaporated in vacuo, the product mixture was
dissolved in DCM containing a few drops of Et3N. A silica column
packed with 1% Et3N in DCM was used for separation. The crude
product was purified by flash chromatography (60% EtOAc in DCM
with a few drops of Et3N to 3% methanol with 90% EtOAc in DCM
with a few drops of Et3N) to yield the desired product as a faint yellow
foam (125 g, 64%). Rf (3% methanol and 90% EtOAc in DCM) = 0.3.
1H NMR (CDCl3): δ 8.30 (s, 1H), 7.88 (s, 1H), 7.72 (s, 1H), 7.41 (d,
2H, J = 5.2 Hz), 7.20−7.38 (m, 7H), 7.01 (s, 1H), 6.81 (d, 4H, J = 12
Hz), 6.45 (t, 1H, J = 6.4 Hz), 5.58 (s, 2H), 5.45 (m, 1H), 4.64 (m,
1H), 4.03−4.58 (m, 5H), 3.94 (s, 3H), 3.91 (s, 3H), 3.77 (s, 6H), 3.38
(m, 2H), 2.76 (m, 1H), 2.51 (m, 1H), 2.31 (bd s, 3H). 13C NMR
(CDCl3): δ 158.6, 154.3, 153.9, 153.1, 152.8, 150.0, 148.5, 144.7,
139.8, 137.5, 135.8, 131.1, 130.1, 128.9, 128.2, 128.0, 127.0, 126.6,
120.9, 113.3, 109.9, 108.3, 86.7, 85.9, 84.1, 72.8, 66.6, 63.9, 56.7, 56.5,
55.3, 46.2, 40.4, 38.6, 32.1, 30.5, 29.8, 26.1, 23.9, 23.1, 14.3, 11.1, 8.9.
IR (neat): 3314 (bd), 2926, 2853, 1749, 1593, 1508, 1250, 1219, 1064,
909, 729 cm−1. HRMS (ESI/APCI-TOF): m/z [M + H]+ calcd for
C45H47N6O12 863.3246, found 863.3236.
Synthesis of 5a. DIPEA (85 mg, 0.66 mmol) was added to 13a
(79.2 mg, 0.11 mmol) that was azeotropically dried with pyridine
followed by 0.2 mL of freshly distilled DCM. The solution was cooled
in an ice bath, and 2-cyanoethyl N,N-diisopropylchlorophosphor-
amidite (23 mg, 0.11 mmol) was added. After being stirred for 1 h, the
reaction mixture was diluted with EtOAc (20 mL), washed with
saturated sodium bicarbonate (15 mL), and then dried over sodium
sulfate. After the organic layer was concentrated in vacuo, the
compound was purified by flash chromatography (15 × 1.5 cm, packed
using 0.5% Et3N in hexanes and then washed with 5 column volumes
of EtOAc) using EtOAc as eluent to isolate 5a (79.6 mg, 78%). Rf
(100% EtOAc) = 0.5. 1H NMR (CDCl3): δ 8.31 (s, 1H), 7.90 (d, 1H,
J = 7.7 Hz), 7.35−7.46 (m, 2H), 7.13−7.32 (m, 7H), 6.72 (m, 4H),
6.44 (t, 1H, J = 5.1 Hz), 5.47 (s, 1H), 4.70 (m, 1H), 4.28 (m, 1H),
3.84−4.57 (m, 3H), 3.76 (s, 6H), 3.64 (m, 1H), 3.57 (m, 2H), 3.37
mg, 75%). Rf (90% EtOAc in hexanes) = 0.6. H NMR (CDCl3): δ
8.31 (s, 1H), 7.92 (d, 1H, J = 6.2 Hz), 7.74 (s, 1H), 7.39 (m, 2H, J =
11.2 Hz), 7.15−7.33 (m, 7H), 7.01 (s, 1H), 6.72−6.84 (m, 4H), 6.45
(t, 1H, J = 8 Hz), 5.59 (s, 2H), 5.45 (bd s, 1H), 4.72 (m, 1H), 4.00−
4.60 (m, 5H), 3.95 (s, 3H), 3.92 (s, 3H), 3.77 (s, 6H), 3.26−3.71 (m,
5H), 2.82 (m, 1H), 2.75 (t, 1H, J = 4 Hz), 2.52−2.67 (m, 2H), 2.44 (t,
1H, J = 4 Hz), 2.34 (bd s, 2H), 1.08−1.57 (m, 12H). 31P NMR
(CDCl3): δ 148.9, 148.7. IR (neat): 2925, 2852, 1749, 1592, 1509,
1467, 1249, 1065, 1033, 977, 793 cm−1. HRMS (ESI-TOF): m/z [M +
H]+ calcd for C54H64N8O13P 1063.4330, found 1063.4309.
Synthesis of 28. A DMF (130 mL) solution of (2R,3S)-2-
(hydroxymethyl)-3-hydroxypyrrolidine hydrochloride51 (27, 2.02 g,
13.1 mmol) and Cs2CO3 (17.1 g, 52.6 mmol) were stirred at room
temperatures for 30 min, at which time 9 (9.4 g, 15.7 mmol) was
added. The reaction was stirred overnight at room temperature
followed by dilution with 400 mL EtOAc. The diluted contents were
washed with saturated sodium bicarbonate (300 mL), followed by
washing with brine (2 × 200 mL). The combined aqueous layers were
washed with EtOAc (200 mL), and the combined organic layers were
dried over sodium sulfate. After the solvent was evaporated in vacuo,
the product mixture was dissolved in DCM. It was purified by flash
chromatography (30−80% EtOAc in DCM to 3% methanol and 7%
DCM in EtOAc) to yield the desired product as a white solid (3.54 g,
1
46%). Rf (90% EtOAc in DCM) = 0.2. H NMR (CDCl3): δ 8.27 (s,
1H), 8.04 (s, 1H), 6.42 (t, 1H, J = 8.4 Hz), 4.58 (bd s, 2H), 4.02−4.43
(m, 3H), 3.99 (dd, 1H, J = 4, 7.1 Hz), 3.85 (dd, 1H, J = 4.8, 11.9 Hz),
3.61−3.80 (m, 3H), 2.51−2.74 (m, 2H), 2.41 (m, 1H), 2.01−2.26 (m,
2H), 1.87 (bd s, 1H), 0.89 (s, 18H), 0.08 (s, 6H), 0.06 (s, 6H). 13C
NMR (CDCl3): δ 154.0, 152.5, 150.1, 137.8, 120.6, 88.0, 84.4, 84.4,
73.9, 72.1, 70.5, 65.5, 62.9, 41.3, 26.8, 26.2, 25.99, 25.97, 18.6, 18.2,
−4.4, −4.5, −5.1, −5.2. IR (neat): 3331 (bd), 2950, 2929, 2857, 1591,
1471, 1254, 1099, 1071, 836, 778 cm−1. HRMS (ESI/APCI-TOF): m/
z [M + H]+ C27H50N5O5Si2 calcd for 580.3345, found 580.3335.
Synthesis of DMT-Protected 28. After azeotropically drying with
pyridine, 28 (1.01 g, 1.7 mmol) was dissolved in a mixture of DMT-Cl
(859.6 mg, 2.09 mmol), and (dimethylamino)pyridine (43 mg, 0.348
mmol) in pyridine (20 mL) at 0 °C. The reaction mixture was allowed
to slowly warm to room temperature and then stirred overnight, at
which time it was diluted with 150 mL of EtOAc. The diluted contents
were washed with saturated ammonium chloride (2 × 200 mL) and
then with brine (100 mL). The combined aqueous layers were washed
once with EtOAc (100 mL), and the combined organic layers were
dried with sodium sulfate. After the solvent was evaporated in vacuo,
the product mixture was dissolved in DCM containing a few drops of
Et3N. A flash chromatography on a column packed with 1% Et3N in
DCM (30% EtOAc in DCM to 70% EtOAc in DCM, along with a few
drops of Et3N) yielded the desired product as a faint yellow foam (1.19
g, 72%). Rf (30% EtOAc in DCM) = 0.2. 1H NMR (CDCl3): δ 8.29 (s,
1H), 7.93 (s, 1H), 7.38 (d, 2H, J = 8.2 Hz), 7.10−7.29 (m, 7H), 6.70
(m, 4H), 6.43 (t, 1H, J = 6.8 Hz), 4.59 (m, 2H), 4.05−4.21 (m, 1H),
4.01 (dd, 1H, J = 4, 7.2 Hz), 3.81 (m, 1H), 3.75−3.80 (m, 4H), 3.74
G
dx.doi.org/10.1021/jo500944g | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Featured Article
(m, 5H), 3.58 (dd, 1H, J = 3.6, 9.2 Hz), 3.27 (dd, 1H, J = 9.2, 6.8 Hz),
2.59 (td, 1H, J = 7.2, 12.4 Hz), 2.39 (m, 1H), 2.33 (m, 1H), 2.00 (bd s,
1H), 1.75 (bd s, 1H), 0.92 (s, 9H), 0.90 (s, 9H), 0.11 (s, 6H), 0.08 (s,
3H), 0.07 (s, 3H). 13C NMR (CDCl3): δ 158.35, 158.33, 153.2, 152.4,
144.8, 137.1, 136.7, 136.2, 136.0, 130.0, 129.9, 128.2, 127.6, 126.6,
120.6, 112.96, 112.92, 87.7, 86.4, 84.1, 77.2, 74.5, 72.1, 67.0, 62.9, 60.3,
55.1, 46.9, 41.0, 32.3, 25.9, 25.7, 18.4, 18.0, −4.67, −4.79, −5.3, −5.4.
IR (neat): 3301 (bd), 3034, 2952, 2856, 1737, 1589, 1508, 1469, 1250,
1093, 1033, 834, 779 cm−1. HRMS (ESI/APCI-TOF): m/z [M + H]+
calcd for C48H68N5O7Si2 882.4652, found 882.4635.
mL). The aqueous layers were washed with DCM (50 mL). The
combined organic layers were dried over sodium sulfate. After the
solvent was evaporated in vacuo, the crude was dissolved in 30%
EtOAc in hexanes and purified by flash chromatography (30−70%
EtOAc in hexanes) to yield the desired product as a yellowish foam
(349 mg, 83%). Rf (50% EtOAc in hexanes) = 0.3. 1H NMR (CDCl3):
δ 8.35 (s, 1H), 8.01 (s, 1H), 7.71 (s, 1H), 7.02 (s, 1H), 6.43 (t, 1H, J =
8 Hz), 5.55 (s, 2H), 5.42 (bd s, 1H), 4.59 (m, 1H), 4.55 (m, 2H), 4.17
(m, 1H), 3.98 (m, 2H), 3.89−3.97 (m, 7H), 3.81 (dd, 1H, J = 4.4, 11.2
Hz), 3.74 (dd, 1H, J = 4.3, 11.2 Hz), 2.70 (m, 2H), 2.63 (m, 1H), 2.53
(m, 2H), 2.41 (m, 2H), 2.18 (m, 1H), 2.12 (s, 3H), 0.90 (s, 9H), 0.89
(s, 9H), 0.09 (s, 6H), 0.07 (s, 3H), 0.06 (s, 3H). 13C NMR (CDCl3): δ
206.2, 172.0, 154.4, 153.7, 153.0, 152.5, 150.2, 148.3, 139.6, 137.9,
126.6, 120.7, 109.8, 108.1, 87.8, 84.0,72.0, 66.4, 63.0, 62.8, 56.4, 56.3,
47.3, 40.9, 37.7, 29.9, 29.6, 29.6, 28.0, 25.9, 25.7, 18.3, 17.9, 18.3, 17.9,
15.3, −4.7, −4.8, −5.4, −5.5. IR (neat): 2953, 2928, 2856, 1738, 1587,
1524, 1466, 1278, 1253, 1221, 1065, 837, 780 cm−1. HRMS (ESI/
APCI-TOF): m/z [M + H]+ calcd for C42H65N6O13Si2 917.4143,
found 917.4128.
Synthesis of 31. A solution of triethylamine trihydrofluoride (291
mg, 293 μL, 1.8 mmol) and 30 (339 mg, 0.363 mmol) in THF (4 mL)
were stirred overnight at room temperature. After evaporating the
solvent in vacuo the residue was dissolved in dichloromethane and
purified by flash chromatography (2−4% methanol in DCM) to yield
the desired product as a white foam (271 mg, 93%). Rf (4% methanol
in DCM) = 0.15. 1H NMR (CDCl3): δ 8.31 (s, 1H), 7.78 (s, 1H), 7.70
(s, 1H), 7.01 (s, 1H), 6.62 (d, 1H, J = 12 Hz), 6.30 (dd, 1H, J = 6, 8.8
Hz), 5.54 (s, 2H), 5.42 (m, 1H), 4.65−4.86 (m, 2H), 4.55 (m, 2H),
4.19 (s, 1H), 3.86−4.01 (m, 9H), 3.76 (m, 1H), 3.10 (m, 1H), 2.72
(m, 2H), 2.37−2.59 (m, 3H), 2.17−2.33 (m, 3H), 2.13 (s, 3H). 13C
NMR (CDCl3): δ 206.3, 172.1, 154.4, 153.6, 153.4, 151.8, 149.3,
148.3, 139.6, 139.4, 129.4, 126.4, 110.0, 108.2, 89.6, 87.7, 77.6, 73.5,
66.5, 63.5, 63.2, 56.4, 56.3, 53.3, 40.4, 37.7, 29.7, 29.6, 27.9. IR (neat):
3341 (bd), 2927, 1717, 1588, 1523, 1468, 1330, 1277, 1220, 1158,
1096, 1065 cm−1. HRMS (ESI/APCI-TOF): m/z [M + H]+ calcd for
C30H37N6O13 689.2413, found 689.2445.
Synthesis of DMT-Protected 31. DMT-Cl (130 mg, 0.32 mmol),
(dimethylamino)pyridine (1 mg, 0.01 mmol), and pyridine (4 mL)
were added to azeotropically dried (pyridine) 31 (186 mg, 0.26 mmol)
at 0 °C. The reaction mixture was allowed to slowly warm to room
temperature and stir overnight, at which time it was diluted with 50
mL of ethyl acetate. The solution was washed with saturated
ammonium chloride (2 × 50 mL), followed by washing with brine
(50 mL). The combined aqueous layers were washed with ethyl
acetate (50 mL), and the combined organic layers were dried with
sodium sulfate. After the solvent was evaporated in vacuo, the crude
mixture was dissolved in dichloromethane containing a few drops of
Et3N. The product was purified by flash chromatography (20−60%
ethyl acetate in dichloromethane to 3% methanol in ethyl acetate, with
a few drops of Et3N) using a column packed with 1% Et3N in DCM.
The desired product was isolated as a faint yellow foam (182 mg,
64%). Rf (5% methanol in DCM) = 0.4. 1H NMR (CDCl3): δ 8.30 (s,
1H), 7.88 (s, 1H), 7.71 (s, 1H), 7.39 (d, 2H, J = 7.8 Hz), 7.19−7.33
(m, 7H), 7.03 (s, 1H), 6.79 (d, 4H, J = 9 Hz), 6.44 (t, 1H, J = 5.2 Hz),
5.55 (s, 2H), 5.43 (bd s, 1H), 4.63 (bd s, 1H), 4.25−4.82 (m, 5H),
4.07−4.19 (m, 2H), 3.95 (s, 3H), 3.94 (s, 3H), 3.77 (m, 7H), 3.40
(dd, 1H, J = 3.6, 10.8 Hz), 3.38 (dd, 1H, J = 5.4, 10.8 Hz), 2.67−2.80
(m, 3H), 2.36−2.53 (m, 3H), 2.23 (m, 1H), 2.12 (s, 3H). 13C NMR
(CDCl3): δ 206.2, 172.1, 158.5, 154.4, 153.7, 153.1, 152.5, 150.2,
148.3, 144.5, 139.6, 137.7, 135.7, 130.0, 128.0, 127.8, 126.9, 126.5,
120.8, 113.2, 109.9, 108.2, 86.6, 85.7, 83.9, 80.2, 77.7, 72.7, 66.4, 63.8,
63.1, 60.5, 56.5, 56.4, 55.2, 47.5, 40.1, 37.7, 30.6, 29.7, 28.0, 26.1. IR
(neat): 2982−2939 (bd), 1734, 1587, 1509, 1372, 1236, 1176, 1157,
1043, 1064 cm−1. HRMS (ESI/APCI-TOF): m/z [M + H]+ calcd for
C51H55N6O15 (M + H+) 991.3720, found 991.3726.
Synthesis of 29. 1-Ethyl-3-(3-(dimethylamino)propyl)-
carbodiimide hydrochloride (1.15 g, 6 mmol) and DMAP (655 mg,
5.26 mmol) in DMF (10 mL) were added to DMT-protected 28 (715
mg, 0.75 mmol). After the mixtur was stirred for 10 min at room
temperature, levulinic acid (608 mg, 533 μL, 5.25 mmol) was added.
The reaction mixture was stirred for 2 h followed by dilution with 70
mL of EtOAc. The solution was washed with saturated sodium
bicarbonate (1 × 70 mL) and then with brine (2 × 50 mL). The
combined aqueous layers were washed once with EtOAc (50 mL), and
the combined organic layers were dried with sodium sulfate. After the
solvent was evaporated in vacuo, the product mixture was dissolved in
DCM containing a few drops of Et3N. It was purified by flash
chromatography (10% EtOAc in DCM with few drops of Et3N to 35%
EtOAc in DCM with a few of drops of Et3N) to yield the desired
product as a white foam (714 mg, 89%). Rf (30% EtOAc in DCM) =
1
0.4. H NMR (CDCl3) at 39 °C: δ 8.27 (bd s, 1H), 7.91 (bd s, 1H),
7.15−7.26 (m, 9H), 6.66−6.71 (m, 4H), 6.43 (bd s, 1H), 5.51 (bd s,
1H), 4.62 (bd s, 1H), 4.03−5.21 (m, 1H), 4.13 (m, 1H), 4.02 (m,
2H), 3.82 (m, 1H), 3.79 (m, 1H), 3.75 (s, 3H), 3.74 (s, 3H), 3.57 (m,
2H), 2.60−2.78 (m, 3H), 2.47−2.59 (m, 3H), 2.43 (m, 1H), 2.13 (m,
4H), 0.94 (s, 9H), 0.91 (s, 9H), 0.11 (s, 6H), 0.08 (s, 6H). 13C NMR
(CDCl3) at 39 °C: δ 206.3, 172.3, 158.67, 158.64, 153.4, 152.7, 150.7,
145.1, 137.4, 136.4, 136.2, 130.29, 130.20, 128.4, 127.8, 126.8, 121.2,
113.2, 113.2, 88.0, 86.9, 84.3, 77.2, 72.5, 64.6, 63.2, 55.3, 41.2, 38.1,
29.9, 28.5, 26.3, 26.0, 18.6, 18.2, −4.4, −4.5, −5.1, −5.2. IR (neat):
2961, 2929, 2856, 1725, 1720, 1588, 1509, 1468, 1251, 1177, 1088,
1033, 835 cm−1. HRMS (ESI/APCI-TOF): m/z [M + H]+ calcd for
C53H74N5O9Si2 980.5020, found 980.5028.
Synthesis of DMT Deprotected 29. Acetic acid, water, and
tetrahydrofuran (3:1:1, 50 mL) were added to 29 (715 mg, 0.67
mmol). After the mixture was stirred for 22 min at room temperature,
methanol (2 mL) was added, and the solution was stirred for an
additional 8 min, at which time 300 mL of EtOAc was added. The
solution was washed with water (3 × 100 mL). The combined aqueous
layers were washed with fresh EtOAc (200 mL). The combined
organic layers were carefully washed with saturated sodium
bicarbonate (400 mL) and then dried over sodium sulfate. After the
solvent was evaporated in vacuo, the product mixture was dissolved in
30% EtOAc in hexanes and purified by flash chromatography (50%
EtOAc in hexanes to 80% EtOAc in hexanes) to yield the desired
product as a white foam (403 mg, 89%). Rf (70% EtOAc in hexanes) =
1
0.3. H NMR (CDCl3): δ 8.32 (s, 1H), 8.06 (s, 1H), 6.44 (t, 1H, J =
6.8 Hz), 5.22 (bd s, 1H), 4.75 (bd s, 1H), 4.60 (td, 1H, J = 5.2, 3.2
Hz), 4.23 (m, 2H), 3.99 (dt, 1H, J = 3.2, 7.1 Hz), 3.93 (m, 1H), 3.84
(dd, 1H, J = 4.3, 10.6 Hz), 3.75 (dd, 1H, J = 3.1, 10.6 Hz), 3.71 (dd,
1H, J = 7.1, 11.24 Hz), 2.69 (m, 2H), 2.61 (td, 1H, J = 6.4, 12.4 Hz),
2.52 (m, 2H), 2.42 (m, 1H), 2.14−2.39 (m, 3H), 2.12 (s, 3H), 0.92 (s,
18H), 0.10 (s, 6H), 0.08 (s, 6H). 13C NMR (CDCl3): δ 206.7, 206.2,
172.1, 153.7, 152.3, 150.1, 137.8, 87.8, 84.7, 77.2, 71.9, 67.0, 65.3, 62.7,
41.1, 37.7, 30.85, 30.81, 29.79, 29.73, 29.6, 28.0, 25.9, 25.7, 18.3, 17.9,
−4.6, −4.8, −5.4, −5.5. IR (neat): 3363 (bd), 2953, 2928, 2856, 1719,
1589, 1470, 1253, 1157, 1110, 1071, 1029, 836, 778 cm−1. HRMS
(ESI/APCI-TOF): m/z [M + H]+ calcd for C32H56N5O7Si2 678.3726,
found 678.3729.
Synthesis of 30. 4,5-Dimethoxy-2-nitrobenzyl chloroformate (326
mg, 1.18 mmol), DMAP (277 mg, 2.23 mmol), and DCM (6 mL)
were added to azeotropically dried (pyridine) DMT-deprotected 29
(310 mg, 0.447 mmol). After being stirred at room temperature for 2
h, the reaction mixture was diluted with 100 mL of DCM. The diluted
solution was washed with sodium bicarbonate (50 mL) and brine (50
Synthesis of 32. DMT-protected 31 (110 mg, 0.11 mmol) was
azeotropically dried with pyridine. 2-Cyanoethyl N,N-diisopropyl-
chlorophosphoramidite (42.2 mg, 44 μL, 0.19 mmol), diisopropyle-
thylamine (120 mg, 161 μL, 0.9 mmol), and dichloromethane (3 mL)
were added, and the reaction mixture was stirred for 45 min at room
H
dx.doi.org/10.1021/jo500944g | J. Org. Chem. XXXX, XXX, XXX−XXX

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Featured Article
temperature, at which time it was diluted with 60 mL of ethyl acetate.
The solution was washed with saturated sodium bicarbonate (25 mL),
followed by brine (25 mL), and then dried with sodium sulfate. After
the solvent was evaporated in vacuo, the mixture was dissolved in 60%
ethyl acetate in hexanes and purified (60−90% ethyl acetate in
hexanes) on a silica column packed with 1% Et3N in hexanes and
washed with 3 column volumes of 60% ethyl acetate in hexanes. The
yield of the desired product was 101.1 mg (78%). Rf (90% EtOAc in
hexanes) = 0.6. 1H NMR (CDCl3): δ 8.31 (s, 1H), 7.91 (d, 1H, J = 7.1
Hz), 7.72 (s, 1H), 7.40 (m, 2H), 7.15−7.34 (m, 7H), 7.04 (s, 1H),
6.79 (m, 4H), 6.44 (t, 1H, J = 6.4 Hz), 5.55 (s, 2H), 5.42 (bd s, 1H),
4.57−4.71 (m, 4H), 4.29 (m, 1H), 3.94 (m, 7H), 3.77 (s, 6H), 3.58
(m, 2H), 3.38 (m, 3H), 3.29 (m, 3H), 2.63−2.84 (m, 4H), 2.36−2.63
(m, 4H), 2.21 (bd s, 1H), 2.11 (s, 3H), 1.05−1.20 (m, 12H). 31P
NMR (CDCl3): δ 148.7, 148.5. IR (neat): 2965, 2932, 1736, 1586,
1623, 1508, 1465, 1364, 1329, 1278, 1246, 1221, 1178, 1156, 1065,
1032, 975, 823, 793, 727 cm−1. HRMS (ESI-TOF): m/z [M + H]+
calcd for C60H72N8O16P 1191.4804, found 1191.4780.
General Procedure for Synthesis of Template Strand
Containing 6 and 22. Native nucleotides were coupled using
standard synthesis cycles (25 s coupling, 5 s capping with acetic
anhydride, 15 s oxidation with 0.02 M I2 in THF/H2O/pyridine, 95 s
detritylation with 3% TCA in DCM) for all oligonucleotides except for
the 34 and 35, where double coupling was utilized. Oligonucleotides
15, 16, 19, 20, 34, and 35 were prepared on 1000 Å resin. All other
oligonucleotides were synthesized using 500 Å resin. Modified
phosphoramidites 5a and 5b were coupled using automated double
coupling cycles (15 min coupling time), while 32 was coupled
manually, as described below. The synthesizer was programmed to
pause when it reached the point where the manual coupling was
utilized.
T4 PNK buffer (70 mM TRIS-HCl pH 7.6, 10 mM MgCl2, 5 mM
DTT), 50 μCi γ-32P-ATP, and 30 U of T4 PNK at 37 °C for 1 h.
Excess g-32P-ATP was removed by passing the reaction through a 1
mL Sephadex G25 column. The specific activity was determined by
counting the radioactivity (using a liquid scintillation counter) and
measuring the concentration of 38 (A260). Separately, 34 (40 pmol)
was 5′-phosphorylated on the template strand in a 25 μL reaction
containing 1 × T4 PNK buffer, ATP (80 pmol), and 30 U of T4 PNK
at 37 °C for 1 h. Both phosphorylation reactions were stopped by
heating at 65 °C for 30 min. The phosphorylated products were
hybridized with 160 pmol each of 36, 37 ,and 39 in 10 mM sodium
phosphate (pH 7.2) and 100 mM sodium chloride solution by slowly
cooling from 90 to 16 °C. The reaction was incubated overnight at 16
°C in the presence of T4 DNA ligase (50 U) and 1 × ligase buffer (50
mM Tris-HCl pH 7.5, 10 mM MgCl2, 10 mM DTT, 1 mM ATP).
Formamide loading buffer (50 μL, 90% formamide, 10 mM EDTA, pH
8.0) was added, followed by heating at 90 °C for 5 min and cooling on
ice prior to purification by 20% denaturing PAGE (37 × 32 × 0.04
cm). The gel was run under limiting power (55 W) for 18 h. The
product band was excised from the gel and crushed, and the DNA was
eluted overnight in 5 mL of elution buffer (0.2 M NaCl and 1 mM
EDTA). The supernatant was desalted using a 100 mg C-18-Sep-Pak
cartridge to provide 40. The yield (10−20%) of 40 was determined
based on the specific activity of 5′-32P-38.
General Procedure for Restriction Enzyme Treatment. The
appropriate restriction enzyme (10 U) and 5′-32P-40 were incubated in
a 10 μL reaction containing of 1 X CutSmart buffer (50 mM potassium
acetate, 20 mM TRIS acetate, 10 mM magnesium acetate, 100 μg/mL
BSA, pH 7.9) at 37 °C (RsaI) or 65 °C (TaqαI) for 1 h, at which time
formamide loading buffer (2 μL, 90% formamide, 10 mM EDTA, pH
8.0) was added followed by heating at 90 °C and cooling on ice. The
reactions were analyzed by 20% denaturing PAGE.
General Procedure for Manual Coupling. Phosphoramidite 32
(13 mg, 11 μmol) was dissolved in 300 μL of activator solution (5-
ethylthio-1H-tetrazole in acetonitrile, 0.25 M). The solution was
repeatedly passed through a column for 10 min containing the partially
synthesized oligonucleotide on CPG resin that was fitted with two 1
mL plastic syringes. The resin was washed with dry acetonitrile (5 mL)
and dried under vacuum for 10 min. For manual double coupling, this
procedure was repeated using a fresh solution of phosphoramidite and
activator prior to carrying out the remaining solid-phase synthesis
steps on the automated synthesizer.
ASSOCIATED CONTENT
* Supporting Information
■
S
NMR spectra of new compounds and mass spectra of modified
oligonucleotides. Restriction enzyme cutting sites and auto-
radiograms of ligation to produce 40 and restriction cleavage.
This material is available free of charge via the Internet at
http://pubs.acs.org.
General Procedure for Trityl Quantification. The trityl output
collected from the DNA synthesis was diluted in a volumetric flask
using 0.1 M pTsOH in acetonitrile. The synthetic yield of the
corresponding step was calculated based on the absorbance of trityl
cation measured at 495 nm (ε495 = 70000).
General Procedure for Photolysis of Resin Containing ONV-
Protected 6 and 22. Photolysis of the resin was carried out in 6−8
mg portions in toluene for 2 h in a Pyrex tube using a Schoeffel 1000
W short-arc lamp (model LH151N/2) fitted with an optical filter
(Newport no. CGA-375). The resin was stirred using a pipe cleaner
(to prevent crushing the resin) during the photolysis. After the
photolysis, the resin was washed sequentially with hexanes (3 × 3 mL),
DCM (3 × 3 mL), and methanol (10 × 3 mL) within the Pyrex tube.
After drying on a rotovap, the resin was transferred to a DNA synthesis
column and dried under vacuum before any further synthetic
modification. Methanol wash and drying in the previous step was
repeated until complete recovery of resin was achieved.
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
■
We are grateful for generous support of this research from the
National Institute of General Medical Sciences (GM-063028).
We thank Dr. Jonathan Sczepanski (The Scripps Research
Institute) for helpful discussions and Dr. Michael Delaney
(Dharmacon Research) for advice on the manual coupling
procedure. We are grateful to Professor Steven Rokita (Johns
Hopkins University) for providing access to the Schoeffel 1000
W arc lamp.
General Procedure for Delevulinylation of Levulinyl-
Protected 6 and 24. A solution of 0.25 M hydrazine in pyridine/
acetic acid (3:2) was passed back and forth through the resin in a DNA
synthesis column as described above for the manual coupling
procedure for 10 min (for ≤25 nucleotide long) or 25 min (for
longer substrates). For longer treatment times, fresh hydrazine
solution was used every 6 min. At the end of the treatment, the
resin was washed sequentially with methanol (5 × 10 mL) and
acetonitrile (5 × 10 mL) and then dried under vacuum before
continuing the oligonucleotide synthesis.
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