Conjugatable and Bioreduction Cleavable Linker for the 5′-Functionalization of Oligonucleotides

Article abstract of DOI:10.1021/acs.joc.6b02527

An efficient conjugatable and bioreduction cleavable linker was designed and synthesized for the 5′-terminal ends of oligonucleotides. A phosphoramidite reagent bearing this linker was successfully applied to solid phase synthesis and incorporated at the 5′-terminal ends of oligonucleotides. The controlled pore glass (CPG)-supported oligonucleotides were subsequently conjugated to a diverse range of functional molecules using a CuAAC reaction. The synthesized oligonucleotide conjugates were then cleaved using a nitroreductase/NADH bioreduction system to release the naked oligonucleotides.

Full text of DOI:10.1021/acs.joc.6b02527

Note
pubs.acs.org/joc
Conjugatable and Bioreduction Cleavable Linker for the 5′-
Functionalization of Oligonucleotides
Hisao Saneyoshi,* Yuta Yamamoto, Kazuhiko Kondo, Yuki Hiyoshi, and Akira Ono*
Department of Material and Life Chemistry, Faculty of Engineering, Kanagawa University, 3-27-1 Rokkakubashi, Kanagawa-ku,
Yokohama 221-8686, Japan
S
* Supporting Information
ABSTRACT: An efficient conjugatable and bioreduction cleavable linker was designed and synthesized for the 5′-terminal ends
of oligonucleotides. A phosphoramidite reagent bearing this linker was successfully applied to solid phase synthesis and
incorporated at the 5′-terminal ends of oligonucleotides. The controlled pore glass (CPG)-supported oligonucleotides were
subsequently conjugated to a diverse range of functional molecules using a CuAAC reaction. The synthesized oligonucleotide
conjugates were then cleaved using a nitroreductase/NADH bioreduction system to release the naked oligonucleotides.
he conjugation of oligonucleotides with functional
cleaved by a reductive-elimination reaction in cancer cells or
Tmolecules is an important strategy for the construction bacterial cells.28−30
Herein, we report the design and synthesis of a
of modified oligonucleotides with various applications in
medicinal chemistry, diagnostics, and biological science.1−6
Functional molecules can be attached to other compounds
using a postsynthetic modification strategy, which requires the
presence of a reactive group in the parent compound as a
handle for the attachment of the functional molecule. This
strategy has been widely used to attach a broad range of
functional molecules (i.e., from small molecules to macro-
molecules) to various compounds and represents a simple and
low-cost method for introducing functionality.5−7 Several
postsynthetic modification reactions have been reported in
the literature,5,6 including, most notably, the Cu(I)-catalyzed
alkyne azide cycloaddition (CuAAC) reaction,8−10 which has
been used extensively in the field of nucleic acid chemistry11−19
because of its high reaction efficacy and orthogonal reactivity
profile.
Stimulus-responsive linkers are highly desired for the
development of drug delivery systems for nucleic acid-based
drugs.20,21 To date, various cleavable linkers have been reported
with numerous applications in the life sciences.22,23 One of the
most commonly used types of cleavable linkers are disulfide
linkers, which can be cleaved by intracellular glutathione,24 and
linkers belonging to this class have been applied to several
nucleic acid-based drugs.20,25,26 As part of our research toward
the development of new reduction-responsive cleavable linkers
for oligonucleotides, we became interested in 4-nitrobenzyl-
type structures27 because we envisaged that they would be
phosphoramidite reagent bearing a 4-nitrobenzyl-type linker
with an alkyne moiety. This reagent was used in a final capping
reaction at the 5′-terminal end of an oligonucleotide prepared
using a DNA/RNA synthesizer. The subsequent CuAAC
reaction of this moiety with various functional molecules6
could then be used to achieve the efficient preparation of
oligonucleotide conjugates (Figure 1). These procedures could
therefore be used to synthesize a diverse range of 5′-modified
oligonucleotides from one parent oligonucleotide precursor.
Furthermore, this linker can be readily cleaved under
bioreductive conditions to release the naked oligonucleotide.
The synthesis of our phosphoramidite bearing a clickable/
biocleavable linker started from the commercially available
aldehyde 1, which was reduced with NaBH4 to give the known
alcohol 2.31 The phenolic group of 2 was selectively alkylated
with propargyl bromide in the presence of K2CO3 to give
compound 3, which was converted to phosphoramidite 4 for
solid phase DNA synthesis using standard procedures (Scheme
1).
Phosphoramidite 4 was incorporated at the 5′-end of a series
of model oligonucleotides (oligothymidylate and mixed
sequence) using standard phosphoramidite chemistry (Scheme
2). Next, prior to the release and deprotection, we conducted
Received: October 18, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.joc.6b02527
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Note
Figure 1. Conjugation/bioreductive cleavage strategy for nucleic acid.
Scheme 1. Synthesis of Phosphoramidite and Its Incorporation in Oligonucleotides
Scheme 2. Conjugation with Various Functional Molecules Using a CuAAC Reaction on the CPG Support
B
DOI: 10.1021/acs.joc.6b02527
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Note
Figure 2. RP HPLC profiles of the crude conjugated ODNs before HPLC purification, which were prepared using an on-solid support CuAAC
reaction. Panel A-1: ODN 1; Panel A-2: ODN 2; Panel B-1: ODN 7; Panel B-2: ODN 9. HPLC conditions: A buffer (0.1 M TEAA containing 5%
CH3CN), B buffer (CH3CN), gradient (B) 0 to 100% (60 min). Flow rate: 1 mL/min.
an on-solid support CuAAC reaction11,15,18 to allow for the
Scheme 3. Release of the Naked Oligonucleotide via a Nitro-
Reduction Reaction
attachment of several functional molecules, including biotin,32
α-tocopherol,33 N-acetylgalactosamine (GalNAc),34 and poly-
ethylene glycol (PEG)35 to the oligonucleotide. These
molecules were selected because they have been reported to
enhance cellular uptake and prolong blood circulation. With the
exception of the commercially available materials, the
procedure used to prepare the azide described in this study is
shown in Scheme S1. Upon completion of the reaction (48 h,
r.t.), the remaining reagents were washed out of the reaction
mixture, and the conjugated oligonucleotides were released
from the CPG support and deprotected with aqueous NH4OH.
The resulting oligonucleotides (ODNs) were analyzed by
reversed-phase HPLC (RP-HPLC), and the typical results are
shown in Figure 2.
Panels A-1 and B-1 show the HPLC chromatograms of the
nonconjugated oligonucleotides ODN 1 and ODN 7 before
HPLC purification, which were used as controls. These
chromatograms revealed that the linker was stable to the
DNA synthesis and deprotection conditions. Panels A-2 and B-
2 show the HPLC profiles of the conjugated oligonucleotides
ODN 2 and ODN 9 before HPLC purification, respectively.
The coupling yields for these reactions were generally good,
and several other conjugates were also prepared in good yield,
as described in Figure S1. The crude compound mixtures were
purified by preparative HPLC, and the structures of the pure
compounds were confirmed by MALDI-TOF mass spectros-
copy (Figure S2).
We then proceeded to investigate the cleavability of the
linker to determine whether the functional molecules could be
readily separated from the conjugated oligonucleotides by
bioreduction (Scheme 3). The conjugates were treated with
nitroreductase (from Escherichia coli) and NADH, which were
selected as model reductive conditions, and the reaction was
monitored by HPLC. Time-course profiles for the deprotection
reaction are shown in Figure 3. Panels A (bottom) and B
(bottom) show the peaks for the DNA conjugates ODN 2 and
ODN 9 as well as the internal standard (asterisk) and NADH
(5−12 min). The addition of the enzyme led to the cleavage of
the linker to give the corresponding 5′-phosphorylated ODNs
(Panel A, upper; Panel B, upper) in a time-dependent manner.
The conversion yields were estimated from the HPLC data, and
the results were determined to be as follows: 15 min, 35%; 30
min, 53%; 60 min, 69%; 120 min, 85% for panel A and 15 min,
22%; 30 min, 32%; 60 min, 38%; 120 min, 46% for panel B.
The newly formed peak (naked ODN, as indicated by the black
allow) was isolated by preparative HPLC, and its structure was
confirmed by MALDI-TOF mass spectroscopy. The linker was
stable in a buffer containing NADH or enzyme (Figure S3).
These results indicated that the reduction-elimination reaction
performed effectively for the elimination of the functionalized
molecules attached to the oligonucleotides. Several other
conjugate molecules (ODN 3−6 and 8) were also cleaved
under the same conditions, as shown in Figure S4. To assess
the applicability of these functionalized oligonucleotides to in
vivo systems, we need to investigate the cleavage reaction under
the hypoxic conditions typically observed in tumor cells. These
studies go beyond the scope of the current study but are
currently in progress in our laboratory and will be reported
elsewhere in due course.
To summarize, a phosphoramidite monomer bearing a
conjugatable and bioreduction cleavable linker was prepared
to functionalize the 5′-terminal ends of oligonucleotides. This
system was successfully applied to solid phase synthesis and
incorporated at the 5′-terminal ends of oligonucleotides. The
C
DOI: 10.1021/acs.joc.6b02527
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Note
Figure 3. Time-dependent RP-HPLC profiles for the nitroreductase-triggered cleavage of the linkers in the ODNs: Panel A: ODN 2; Panel B: ODN
9; *internal standard (N6-Bz-2′-dA). HPLC conditions: Panel A: A buffer (0.1 M TEAA containing 5% CH3CN), B buffer (CH3CN), gradient (B) 0
to 60% (36 min). Flow rate: 1 mL/min. Panel B: A buffer (0.1 M TEAA containing 5% CH3CN), B buffer (CH3CN), gradient (B) 0 to 20% (20
min)and then 20 to 100% (40 min). Flow rate: 1 mL/min.
to give (967 mg, 94%) as a yellow solid: mp 96.5−97.0 °C; 1H NMR
subsequent conjugation of these CPG-supported oligonucleo-
tides with a diverse range of functional molecules was achieved
using an on-CPG CuAAC reaction. The resulting oligonucleo-
tide-functionalized conjugates were successfully cleaved using a
nitroreductase/NADH system to release the naked oligonu-
cleotides. This new method could be used for the
(600 MHz, CDCl3) δ 10.66 (1H, s), 8.10 (1H, d, J = 8.3 Hz), 7.18
(1H, s), 6.98 (1H, dd, J = 8.2, 1.4 Hz), 4.77 (2H, d, J = 5.5 Hz), 1.89
(1H, t, J = 5.5 Hz).
(4-Nitro-3-(prop-2-yn-1-yloxy)phenyl)methanol 3. Com-
pound 2 (580 mg, 3.43 mmol) and K2CO3 (1.42 g, 10.3 mmol)
functionalization of oligonucleotides and bioactive nucleotides
at their 5′-termini, and the biocleavable properties could be
useful for medicinal chemistry and biotechnology applications.
For example, the linker reported in this study could be used like
a prodrug for bioreductive cleavage leading to the release of a
5′-phosphorylated guide strand RNA, which is a component of
were dissolved in DMF (10 mL), and a solution was stirred at 60 °C
for 1 h. To the solution was added propargyl bromide (368 μL, 4.12
mmol), and the solution was stirred at 60 °C for 1 h. The solution was
diluted with EtOAc and washed with H2O (twice). The organic
solution was dried (MgSO4), filtered, and evaporated in vacuo. The
residue was purified by column chromatography on a silica gel eluted
with hexane:EtOAc (80:20 → 70:30, v/v) to give 3 (670 mg, 94%) as
a pale yellow solid: mp 83.5−84.5 °C; 1H NMR (600 MHz, CDCl3) δ
7.86 (1H, d, J = 8.6 Hz), 7.28 (1H, s), 7.05 (1H, d, J = 8.3 Hz), 4.86
(2H, d, J = 2.4 Hz), 4.79 (2H, d, J = 5.5 Hz), 2.60 (1H, t, J = 2.4 Hz),
2.23 (1H, t, J = 5.8 Hz); 13C NMR (151 MHz, CDCl3) δ 151.2, 148.2,
139.0, 126.1, 118.9, 113.0, 77.2, 77.1, 64.0, 57.2.; HRMS (ESI-TOF)
the siRNA used for selective RNA interference in cancer cells.
EXPERIMENTAL SECTION
■
General Experimental Procedure. Chemicals were purchased
from a commercial supplier and used without further purification.
NMR spectra were recorded at 500 or 600 MHz for 1H NMR, 126 or
151 MHz for 13C NMR, and 243 MHz for 31P NMR. Chemical shifts
were measured from tetramethylsilane for H NMR spectra and 13C
1
NMR spectra and 85% phosphoric acid (0.0 ppm) for 31P NMR
spectra. The coupling constant (J) was reported in hertz.
Abbreviations for multiplicity were: s, singlet; d, doublet; t, triplet;
sext, sextet; m, multiplet; br, broad. Column chromatography was
carried out with a silica gel C-60 or NH silica gel. Thin-layer
chromatography (TLC) analyses were carried out on preparative TLC.
5-(Hydroxymethyl)-2-nitrophenol31 2. 3-Hydroxy-4-nitroben-
zaldehyde (1.02 g, 6.11 mmol) was dissolved in MeOH (20 mL). To
+
m/z: [M + Na+] calcd for C10H9N NaO4 : 230.0424, found: 230.0426.
2-Cyanoethyl (4-Nitro-3-(prop-2-yn-1-yloxy)benzyl) Diiso-
propylphosphoramidite 4. Compound 3 (52 mg, 250 μmol) was
coevaporated with pyridine (5 times) and toluene (twice) and
dissolved in CH2Cl2 (5 mL). To the solution were added iPr2NEt (87
μL, 500 μmol) and 2-cyanoethyl N,N-diisopropylchlorophosphorami-
dite (83 μL, 375 μmol) under argon. The mixture was stirred at room
temperature for 20 min and diluted with CH2Cl2, dried (Na2SO4),
filtered, and evaporated in vacuo. The residue was purified by column
chromatography on a NH-silica gel eluted with hexane:EtOAc (50:50,
the solution was carefully added NaBH4 (462 mg, 12.2 mmol), and the
solution was stirred at room temperature for 30 min. The solution was
quenched with 1 M HCl aq, evaporated in vacuo, dissolved in EtOAc,
and washed with H2O. The organic solution was dried (Na2SO4),
filtered, and evaporated in vacuo. The residue was purified by column
chromatography on a silica gel eluted with CHCl3/MeOH (95:5, v/v)
1
v/v) to give 4 (52 mg, 53%) as a clear oil; H NMR (500 MHz,
D
DOI: 10.1021/acs.joc.6b02527
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Note
CDCl3) δ 7.87 (1H, d, J = 8.6 Hz), 7.29 (1H, brs), 7.05−7.03 (1H,
m), 4.86 (2H, d, J = 2.6 Hz), 4.83−4.73 (2H, m), 3.95−3.89 (1H, m),
3.86−3.80 (1H, m), 3.74−3.63 (2H, m), 2.67 (2H, t, J = 6.6 Hz), 2.59
(1H, t, J = 2.3 Hz), 1.23−1.21 (12H, m); 13C NMR (126 MHz,
CDCl3) δ 151.1, 146.52, 146.47, 139.1, 125.9, 119.1, 117.6, 113.3,
77.26, 77.02, 64.5, 64.3, 58.5, 58.3, 57.2, 43.4, 43.3, 24.74, 24.68, 24.6,
20.52, 20.46; 31P NMR (243 MHz, CDCl3) δ 149.7 HRMS (ESI-
TOF) m/z: [M + Na+] calcd for C19H26N3NaO5P+: 430.1502, found:
430.1506.
2-(2-(2-(((R)-2,5,7,8-Tetramethyl-2-((4S,8S)-4,8,12-
trimethyltridecyl)chroman-6-yl)oxy)ethoxy)ethoxy)ethyl 4-
N-(2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethyl)-11-oxo-
2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f ]pyrido[3,2,1-ij]-
quinoline-9-carboxamide 9. Coumarin 343 (50 mg, 0.175 mmol)
and 1-amino-11-azido-3,6,9-trioxaundecane (50 mg, 0.229 mmol)
were dissolved in CH2Cl2 (2 mL). To the solution was added EDC·
HCl (38 mg, 0.198 mmol). The resulted mixture was stirred at room
temperature for 18 h. The mixture was evaporated in vacuo. The
residue was purified by column chromatography on a silica gel eluted
with CHCl3/MeOH (50:1, v/v) to give 9 (83 mg, 98%) as a yellow
1
syrup. H NMR (600 MHz, CDCl3) δ 9.08−9.07 (1H, m), 8.58 (1H,
s), 7.00 (1H, s), 3.70−3.63 (14H, m), 3.40 (2H, t, J = 4.8 Hz), 3.34
(4H, t, J = 6.9 Hz), 2.89 (2H, t, J = 6.2 Hz), 2.78 (2H, t, J = 6.2 Hz),
2.00 (4H, sext, J = 6.9 Hz); 13C NMR (151 MHz, CDCl3) δ 163.4,
162.9, 152.7, 148.1, 148.0, 127.0, 119.6, 109.0, 108.2, 105.6, 70.74,
70.68, 70.6, 70.0, 69.9, 50.7, 50.2, 49.8, 39.4, 27.5, 21.1, 20.2, 20.1;
Methylbenzenesulfonate 6. α-Tocopherol (312 mg, 0.724 mmol)
was dissolved in THF (10 mL) under argon. To the solution was
added 55% NaH (65 mg, 1.46 mmol), and the solution was stirred at
room temperature for 5 min. To the mixture was added triethylene
glycol bis(p-toluenesulfonate) (995 mg, 2.17 mmol), and it was stirred
at room temperature for 5 h. Crushed ice was added to the reaction
mixture, which was extracted with EtOAc. The organic solution was
washed with brine, dried (Na2SO4), filtered, and evaporated in vacuo.
The residue was purified by column chromatography on a silica gel
eluted with hexane:EtOAc (90:10 → 80:20, v/v) to give 6 (495 mg,
95%) as a yellow syrup. 1H NMR (600 MHz, CDCl3) δ 7.81 (2H, d, J
= 8.3 Hz), 7.33 (2H, d, J = 8.3 Hz), 4.18 (2H, t, J = 4.8 Hz), 3.80−3.77
(4H, m), 3.74 (1H, t, J = 4.8 Hz), 3.69−3.67 (2H, m), 3.65−3.63 (2H,
m), 2.57 (2H, t, J = 6.2 Hz), 2.43 (3H, s), 2.16 (3H, s), 2.12 (3H, s),
2.07 (3H, s), 1.83−1.72 (2H, m), 1.56−1.04 (24H, m), 0.87−0.84
(12H, m); 13C NMR (151 MHz, CDCl3) δ 148.0, 147.8, 144.8, 133.0,
129.8, 128.0, 127.8, 125.8, 122.8, 117.5, 74.8, 72.1, 70.9, 70.6, 69.3,
68.8, 40.1, 39.4, 37.49, 37.47, 37.44, 37.3, 32.8, 32.7, 31.3, 28.0, 24.8,
24.5, 23.9, 22.7, 22.6, 21.6, 21.1, 20.6, 19.8, 19.7, 12.7, 11.84, 11.79.;
HRMS (ESI-TOF) m/z: [M + Na+] calcd for C42H68NaO7S+:
739.4578, found: 739.4588.
+
HRMS (ESI-TOF) m/z: [M + Na+] calcd for C24H31N5NaO6 :
508.2167, found: 508.2174.
Compound 11.34 D-Galactosamine pentaacetate 10 (2.00 g, 5.14
mmol) was dissolved in 1,2-dichloroethane (20 mL) under argon.
TMSOTf (1 mL, 5.53 mmol) was added to the mixture, which was
stirred at 50 °C for 9 h. Saturated NaHCO3 aq was added to the
mixture, which was washed with saturated NaHCO3 aq (twice) and
brine (twice). The organic solution was dried (Na2SO4), filtered, and
evaporated in vacuo. The residue was purified by column
chromatography on a silica gel eluted with CHCl3/MeOH (50:1, v/
1
v) to give oxazoline derivative 11 (925 mg, 54%) as a clear syrup. H
(R)-6-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)-2,5,7,8-tetra-
methyl-2-((4S,8S)-4,8,12-trimethyltridecyl)chromane 7. Com-
NMR (600 MHz, CDCl3) δ 6.00 (1H, d, J = 6.9 Hz), 5.47 (1H, t, J =
3.1 Hz), 4.92 (1H, dd, J = 7.6, 3.4 Hz), 4.26 (1H, td, J = 6.9, 2.8 Hz),
4.21 (1H, dd, J = 11.7, 7.6 Hz), 4.13 (1H, dd, J = 11.7, 6.2 Hz), 4.01
(1H, td, J = 7.6, 1.4 Hz), 2.13 (3H, s), 2.07 (6H, s), 2.06 (3H, d, J =
1.4 Hz).
2-(2-(2-Azideethoxy)ethoxy)ethyl-2-acetamido-3,4,6-tri-O-
acetyl-2-deoxy-α-D-galactopyranoside 12.34 Compound 11 (847
pound 6 (120 mg, 0.167 mmol) was dissolved in DMF (2 mL). To
a solution was added NaN3 (109 mg, 1.68 mmol), and the solution
was stirred at 60 °C for 2 h. The reaction mixture was diluted with
EtOAc and washed with H2O and brine. The organic solution was
dried (Na2SO4), filtered, and evaporated in vacuo. The residue was
purified by column chromatography on a silica gel eluted with
hexane:EtOAc (9:1, v/v) to give azide derivative 7 (79 mg, 81%) as a
yellow syrup. 1H NMR (600 MHz, CDCl3) δ 3.83 (4H, s), 3.77−3.76
(2H, m), 3.73−3.71 (4H, m), 3.41 (2H, t, J = 4.8 Hz), 2.58 (2H, t, J =
6.9 Hz), 2.18 (3H, s), 2.14 (3H, s), 2.07 (3H, s), 1.83−1.72 (2H, m),
1.56−1.04 (24H, m), 0.87−0.83 (12H, m); 13C NMR (151 MHz,
CDCl3) δ 148.1, 147.8, 127.8, 125.8, 122.8, 117.5, 74.8, 72.1, 71.0,
70.8, 70.7, 70.2, 50.8, 40.1, 39.4, 37.5, 37.4, 37.3, 32.8, 32.7, 31.3, 28.0,
24.8, 24.5, 23.9, 22.7, 22.6, 21.0, 20.7, 19.8, 19.7, 12.7, 11.84, 11.79.;
mg, 2.57 mmol) and 0.5 M 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol in t
-butyl methyl ether (7.7 mL, 3.35 mmol) was coevaporated with
pyiridine (thrice) and toluene (thrice) and dissolved in 1,2-
dichloroethane (10 mL) under argon. To the solution was added 4
Å molecular sieves (1 g), and the mixture was stirred at room
temperature for 30 min. TMSOTf (232 μL, 1.29 mmol) was added,
and the mixture was stirred at room temperature for 19 h, and
TMSOTf (232 μL, 1.29 mmol) was further added. The mixture was
stirred at same temperature for 8 h, and Et3N was added. The mixture
was evaporated in vacuo. The residue was purified by column
chromatography on a silica gel eluted with hexane:EtOAc (1:9, v/v) to
give 12 (796 mg, 61%) as a clear syrup. 1H NMR (600 MHz, CDCl3)
+
HRMS (ESI-TOF) m/z: [M + Na+] calcd for C35H61N3NaO4 :
610.4554, found: 610.4569.
E
DOI: 10.1021/acs.joc.6b02527
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Note
δ 6.35 (1H, d, J = 8.9 Hz), 5.34 (1H, d, J = 3.4 Hz), 5.10 (1H, dd, J =
11.0, 3.4 Hz), 4.78 (1H, d, J = 8.3 Hz), 4.24−4.11 (3H, m), 3.93−3.91
(2H, m), 3.88−3.84 (1H, m), 3.75−3.64 (9H, m), 3.50−3.48 (2H, m),
2.17 (3H, s), 2.05 (3H, s), 2.00 (3H, s), 1.99 (3H, s).
Oligonucleotide Synthesis and On-CPG Conjugation by
CuAAC Reaction. Oligonucleotide synthesis was carried out on the
Applied Biosystems 394 DNA/RNA Synthesizer according to the
manufacturer’s recommendations. 2-Cyanoethyl phosphoramidite
(dT, dC, dA, and dG) and phosphoramidite 4 were used as 0.1 M
solutions in dry acetonitrile. Synthesized solid-supported ODNs were
used for conjugation as described below.
Condition A (5′-XTTTTTT-3′). CPG support (135 nmol on the 5 mg
CPG), azide (1 μmol), copper(II) sulfate pentahydrate (10 nmol),
Tris[1-benzyl-1H-1,2,3-triazol-4-yl]methyl]amine (10 nmol), and
sodium ascorbate (200 nmol) in 100 μL of a mixed solution
(MeOH:THF:H2O:tBuOH (50:25:24.5:0.5)) were allowed to stand at
room temperature for 48 h.
ACKNOWLEDGMENTS
■
This work was supported by the Strategic Research Base
Development Program for Private Universities (Kanagawa
University, 2012−2016) from the Ministry of Education,
Culture, Sports, Science and Technology, Japan (A.O.) and
the Takahashi Industrial and Economic Research Foundation
(H.S.).
REFERENCES
■
(1) Juliano, R. L.; Ming, X.; Nakagawa, O. Acc. Chem. Res. 2012, 45,
1067.
(2) Jeong, J. H.; Mok, H.; Oh, Y.-K.; Park, T. G. Bioconjugate Chem.
2009, 20, 5.
(3) Olejnik, J.; Krzymanska-Olejnik, E.; Rothschild, K. J. Nucleic Acids
Res. 1996, 24, 361.
(4) Kolpashchikov, D. M. Chem. Rev. 2010, 110, 4709.
(5) Singh, Y.; Murat, P.; Defrancq, E. Chem. Soc. Rev. 2010, 39, 2054.
(6) Winkler, J. Ther. Delivery 2013, 4, 791.
(7) Patterson, D. M.; Nazarova, L. A.; Prescher, J. A. ACS Chem. Biol.
2014, 9, 592.
Condition B (5′-YCACTCGATTGGTCAC-3′). CPG support (135
nmol on the 5 mg CPG), azide (1 μmol), copper(II) sulfate
pentahydrate (144 nmol), Tris[1-benzyl-1H-1,2,3-triazol-4-yl]methyl]-
amine (430 nmol), and sodium ascorbate (430 nmol) in 100 μL of a
mixed solution (MeOH:THF:H2O:tBuOH (50:25:21:4)) were
allowed to stand at room temperature for 48 h.
(8) Huisgen, R. Angew. Chem. 1963, 75, 604.
(9) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002,
67, 3057.
(10) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596.
(11) Bouillon, C.; Meyer, A.; Vidal, S.; Jochum, A.; Chevolot, Y.;
Cloarec, J. P.; Praly, J. P.; Vasseur, J. J.; Morvan, F. J. Org. Chem. 2006,
71, 4700.
(12) Gogoi, K.; Mane, M. V.; Kunte, S. S.; Kumar, V. A. Nucleic Acids
Res. 2007, 35, e139.
(13) Nakane, M.; Ichikawa, S.; Matsuda, A. J. Org. Chem. 2008, 73,
1842.
(14) Lonnberg, H. Bioconjugate Chem. 2009, 20, 1065.
(15) Jayaprakash, K. N.; Peng, C. G.; Butler, D.; Varghese, J. P.;
Maier, M. A.; Rajeev, K. G.; Manoharan, M. Org. Lett. 2010, 12, 5410.
(16) Pujari, S. S.; Xiong, H.; Seela, F. J. Org. Chem. 2010, 75, 8693.
(17) Pourceau, G.; Meyer, A.; Chevolot, Y.; Souteyrand, E.; Vasseur,
J. J.; Morvan, F. Bioconjugate Chem. 2010, 21, 1520.
(18) Yamada, T.; Peng, C. G.; Matsuda, S.; Addepalli, H.;
Jayaprakash, K. N.; Alam, M. R.; Mills, K.; Maier, M. A.; Charisse,
K.; Sekine, M.; Manoharan, M.; Rajeev, K. G. J. Org. Chem. 2011, 76,
1198.
(19) Willibald, J.; Harder, J.; Sparrer, K.; Conzelmann, K. K.; Carell,
T. J. Am. Chem. Soc. 2012, 134, 12330.
(20) Jeong, J. H.; Mok, H.; Oh, Y. K.; Park, T. G. Bioconjugate Chem.
2009, 20, 5.
(21) Ji, Y.; Yang, J.; Wu, L.; Yu, L.; Tang, X. Angew. Chem., Int. Ed.
2016, 55, 2152.
After the conjugation reaction was completed, excess reagents were
washed out using CH3CN (1 mL × 5) and CH2Cl2 (1 mL × 5). Then,
CPG-supported ODN was dried in vacuo and released from the CPG
support and deprotected by NH4OH at room temperature for 1 h
(ODN 1−6) or at 55 °C for 5 h (ODN 7−9). ODNs were purified by
HPLC with a reversed-phase silica gel column.
The structure of each ODN was confirmed by MALDI-TOF mass
spectrometry using refection negative mode. (Matrix for ionizing
samples was used as a mixture (10:1:1; saturated 3-hydroxy-2-picolic
acid, 2-picolic acid/H2O (50 mg/mL), and ammonium citrate/H2O
(50 mg/mL)). MALDI-TOF mass data: ODN 1: calcd [M − H]:
2030.32; found: 2030.16; ODN 2: calcd [M − H]: 2515.55; found
2514.81; ODN 3: calcd [M − H]: 2474.54; found 2474.88; ODN 4:
calcd [M − H]: 3830−4230; found 3761−4250; ODN 5: calcd [M −
H]: 2617.79; found 2618.37; ODN 6: calcd [M − H]: 2408.50; found
2408.20; ODN 7: calcd [M − H]: 4793.80; found 4795.80; ODN 8:
calcd [M − H]: 5238.01; found 5240.54; ODN 9: calcd [M − H]:
5381.25; found 5384.89
Bioreductive Cleavage of Linker for Releasing Naked
Oligonucleotides. ODN (10 μM), NADH (10 mM), and nitro-
reductase (20 or 80 μg for ODN 4) in 200 μL of 50 mM sodium
phosphate (pH 7.0) were incubated at 37 °C. Aliquots of sample
solution were analyzed by reverse-phase HPLC at appropriate times.
The appeared peak was fractionized and identified by MALDI-TOF
mass spectroscopy. 5′-PO4-TTTTTT-3′ (calcd [M − H]: 1841.28;
found: 1841.73). 5′-PO4-CACTCGATTGGTCAC-3′ (calcd [M −
H]: 4604.75; found: 4597.49).
(22) Leriche, G.; Chisholm, L.; Wagner, A. Bioorg. Med. Chem. 2012,
20, 571.
ASSOCIATED CONTENT
■
(23) Wong, P. T.; Choi, S. K. Chem. Rev. 2015, 115, 3388.
(24) Saito, G.; Swanson, J. A.; Lee, K. D. Adv. Drug Delivery Rev.
2003, 55, 199.
(25) Mintzer, M. A.; Simanek, E. E. Chem. Rev. 2009, 109, 259.
(26) Lee, Y. S.; Kim, S. W. J. Controlled Release 2014, 190, 424.
(27) Yamazoe, S.; McQuade, L. E.; Chen, J. K. ACS Chem. Biol. 2014,
9, 1985.
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.joc.6b02527.
HPLC profiles, stability under various conditions,
synthetic routes, and copies of H, 13C, and 31P NMR
1
spectra (PDF)
(28) Lin, T. S.; Wang, L.; Antonini, I.; Cosby, L. A.; Shiba, D. A.;
Kirkpatrick, D. L.; Sartorelli, A. C. J. Med. Chem. 1986, 29, 84.
(29) Wilson, W. R.; Hay, M. P. Nat. Rev. Cancer 2011, 11, 393.
(30) Wong, R. H.; Kwong, T.; Yau, K. H.; Au-Yeung, H. Y. Chem.
Commun. (Cambridge, U. K.) 2015, 51, 4440.
(31) Katayama, S.; Ae, N.; Kodo, T.; Masumoto, S.; Hourai, S.;
Tamamura, C.; Tanaka, H.; Nagata, R. J. Med. Chem. 2003, 46, 691.
(32) Ren, W. X.; Han, J.; Uhm, S.; Jang, Y. J.; Kang, C.; Kim, J. H.;
Kim, J. S. Chem. Commun. (Cambridge, U. K.) 2015, 51, 10403.
(33) Nishina, K.; Unno, T.; Uno, Y.; Kubodera, T.; Kanouchi, T.;
Mizusawa, H.; Yokota, T. Mol. Ther. 2008, 16, 734.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: saneyoshih@kanagawa-u.ac.jp.
*E-mail: akiraono@kanagawa-u.ac.jp.
ORCID
Hisao Saneyoshi: 0000-0003-4061-101X
Notes
The authors declare no competing financial interest.
■
F
DOI: 10.1021/acs.joc.6b02527
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Note
(34) Prakash, T. P.; Graham, M. J.; Yu, J.; Carty, R.; Low, A.;
Chappell, A.; Schmidt, K.; Zhao, C.; Aghajan, M.; Murray, H. F.;
Riney, S.; Booten, S. L.; Murray, S. F.; Gaus, H.; Crosby, J.; Lima, W.
F.; Guo, S.; Monia, B. P.; Swayze, E. E.; Seth, P. P. Nucleic Acids Res.
2014, 42, 8796.
(35) Iversen, F.; Yang, C.; Dagnaes-Hansen, F.; Schaffert, D. H.;
Kjems, J.; Gao, S. Theranostics 2013, 3, 201.
G
DOI: 10.1021/acs.joc.6b02527
J. Org. Chem. XXXX, XXX, XXX−XXX