Azide-alkyne cycloaddition for universal post-synthetic modifications of nucleic acids and effective synthesis of bioactive nucleic acid conjugates

Article abstract of DOI:10.1039/c4ob01132e

The regioselective post-synthetic modifications of nucleic acids are essential to studies of these molecules for science and applications. Here we report a facile universal approach by harnessing versatile phosphoramidation reactions to regioselectively incorporate alkynyl/azido groups into post-synthetic nucleic acids primed with phosphate at the 5′ termini. With and without the presence of copper, the modified nucleic acids were subjected to azide-alkyne cycloaddition to afford various nucleic acid conjugates including a peptide-oligonucleotide conjugate (POC) with high yield. The POC was inoculated with human A549 cells and demonstrated excellent cell-penetrating ability despite cell deformation caused by a small amount of residual copper chelated to the POC. The combination of phosphoramidation and azide-alkyne cycloaddition reactions thus provides a universal regioselective strategy to post-synthetically modify nucleic acids. This study also explicated the toxicity of residual copper in synthesized bioconjugates destined for biological systems. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob01132e

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Organic & Biomolecular Chemistry
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DOI: 10.1039/C4OB01132E
Azide-alkyne cycloaddition for universal post-synthetic modifications of nucleic acids
and effective synthesis of bioactive nucleic acid conjugates
Yu-Chih Su,a Yu-Lun Lo,a Chi-Ching Hwang,b Li-Fang Wang,a Min Hui Wu,a Eng-Chi
Wang,a Yun-Ming Wangc and Tzu-Pin Wang*,a
aDepartment of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung,
80708, Taiwan
bDepartment of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical
University, Kaohsiung, 80708, Taiwan
cDepartment of Biological Science and Technology, National Chiao Tung University,
Hsin-Chu, 300, Taiwan
*Corresponding authors: tzupinw@cc.kmu.edu.tw (T.-P. Wang)
*Contact address: Department of Medicinal and Applied Chemistry, Kaohsiung Medical
University, Kaohsiung, 80708, Taiwan.
Tel: +886-7-312-1101, ext. 2756, Fax: +886-7-312-5339.
1

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ABSTRACT
The regioselective post-synthetic modifications of nucleic acids are essential to studies of
these molecules for science and applications. Here we report a facile universal approach by
harnessing versatile phosphoramidation reactions to regioselectively incorporate
alkynyl/azido groups into post-synthetic nucleic acids primed with phosphate at the 5’ termini.
With and without the presence of copper, the modified nucleic acids were subjected to
azide-alkyne cycloaddition to afford various nucleic acid conjugates including a
peptide-oligonucleotide conjugate (POC) with high yield. The POC was inoculated with
human A549 cells and demonstrated excellent cell-penetrating capability despite cell
deformation caused by a small amount of residual copper chelated to the POC. The
combination of phosphoramidation and azide-alkyne cycloaddition reactions thus provides a
universal regioselective strategy to post-synthetically modify nucleic acids. This study also
explicated the toxicity of residual copper in synthesized bioconjugates destined for biological
systems.
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INTRODUCTION
Regioselective modifications of biomolecules with tag, probe or other biological molecules
have been a critical tool which significantly advanced biomolecule studies for fundamental
research and clinical application. In nucleic acids, site-specific modifications of smaller
DNA/RNA such as oligonucleotides can be achieved by phosphoramidite chemistry to link
predefined chemical moieties to positions in specific nucleotides during solid-phase
oligonucleotide synthesis.1 However, the solid-phase chemistry approach for regioselective
modifications of oligonucleotides suffers from inherited drawbacks including limits on the
length of synthesized oligonucleotides and on the variety of their incorporated chemical
functionality.
To complement the shortcomings of solid-phase oligonucleotide synthesis, many
site-specific post-synthetic modification methods for nucleic acids have been studied and
adapted to any size of nucleic acid and a broad diversity of chemical groups integrated into
the nucleic acids.2-5 Nevertheless, recently developed post-synthetic modification methods for
nucleic acids rely on enzyme catalysis to carry out chemical transformations but are unable to
provide a universal strategy for both DNA and RNA modifications. Moreover, the required
expensive enzymes and specific substrates in enzymatic reactions further stymie the efforts to
modify nucleic acids with various chemical entities within reasonable costs.
Here we report a universal, economical post-synthetic modification method for nucleic
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acids made possible by orthogonal azide-alkyne cycloaddition reactions. The powerful
copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction,6, 7 a prototype of click
chemistry,8 has found broad applications in science ranging from material studies to
biomolecule research.9 In addition, the copper-free variant of the CuAAC reaction,
strain-promoted azide-alkyne cycloaddition (SPAAC), harnesses excellent reactivity of
cyclooctyne derivatives and drastically expands the biocompatibility of the 1,3-dipolar
cycloaddition reaction for studying biomolecules in vivo.10, 11 In the current study, we
exploited the optimized site-specific two-step phosphoramidation reactions12-14 to facilitate
the introduction of azido and alkynyl groups to DNA/RNA. The acquired azide- and
alkyne-modified nucleic acids set the stage for azide-alkyne cycloaddition and allowed
effective and efficient conjugations with derivatives of biotin, fluorescein, and a cell
penetrating peptide (CPP) the Tat peptide. As proof of the concept, we further demonstrated
that the CuAAC-synthesized peptide-oligonucleotide conjugate (POC) was bioavailable and
successfully trafficked into human cells.
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RESULTS AND DISCUSSION
Potential of maleimide-assisted Michael addition for effective POC synthesis
Synthesis of POCs is an important strategy for the developments of nucleic acid-based
pharmaceuticals.15 Short nucleic acids including antisense oligonucleotides, siRNA, miRNA
and splice switching oligonucleotides have shown promise as therapeutic agents with the
ability to modulate gene expression with excellent specificity.15-17 However, the inability of
oligonucleotides to permeate membranes and the propensity of these molecules to be
entrapped in endosomes has raised concerns about the efficacy of DNA/RNA for clinical
applications.18, 19 The conjugation of nucleic acids with CPPs in order to synthesize POCs
could mitigate the delivery problems of oligonucleotides by promoting their translocation
across membranes and their escape from endosomal entrapment.15, 20, 21 CPPs enhance the cell
permeability of POCs and significantly improve the uptake efficiency of oligonucleotides in
POCs, qualities essential for POC applications in science and medicine.
We initially studied the phosphoramidation reaction-based Michael addition to achieve a
more effective synthesis of nucleic acid conjugates such as POCs. Previous studies indicated
that higher POC yield could be acquired by phosphoramidation, amidation and Michael
addition reactions in sequence.14 The successful POC preparation by the
phosphoramidation-based Michael addition reactions inspired us to pursue the strategy for
more effective synthesis of POCs. We first synthesized several N-maleoyl amino acid
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succinimidyl esters (3a-d; Scheme 1) which were reported to be more reactive,
hetero-bifunctional, cross-linkers in Michael addition than 3e used in a previous study.2, 14 We
thus anticipated that the 3a-d-assisted Michael addition reactions would deliver a better yield
of nucleic acid conjugates. Unexpectedly, none of 3a-d provided a POC yield better than 3e
(Fig. S1A). Moreover, up to four of the cysteine-containing Tat peptide molecules were
conjugated to DNA without prior modifications with cystamine and maleimides (the far right
lane in Fig. S1A) as indicated by MALDI-TOF MS analysis (Fig. S1B). The observation of
ineffective 3a-d-based POC synthesis and the occurrence of multiple peptides conjugated to
the DNA is perplexing as maleoyl-assisted Michael addition reactions are considered to be
one of the major routes to modify biomolecules with thiols, a popular method of nucleic acid
derivative synthesis for years.2, 22, 23
Optimal azide-alkyne cycloaddition for universal modifications of nucleic acids
Since the N-maleoyl amino acid succinimidyl ester 3a-d failed to provide a more effective
approach for nucleic acid modifications, we turned our attention to the CuAAC reaction to
acquire nucleic acid conjugates with higher yield (Scheme 2). We began with synthesis of
additional hetero-bifunctional cross-linkers, Cu(I) ligands and other
azido-/alkynyl-containing molecules for performing nucleic acid modifications by the
CuAAC reactions. For example, in order to have azide-modified nucleic acids in one of the
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CuAAC reactions (Scheme 2A), we synthesized several azido carboxylic acid succinimidyl
esters (6a-d; Scheme 3) to determine the appropriate azides in 6a-d and provide the best yield
for the CuAAC reaction. In addition, we synthesized
tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) and
tris(benzimidazol-2-ylmethyl)amine (NTB) as Cu(I)-stabilizing ligands to accelerate the
CuAAC reactions and prevent biomolecule degradation.24, 25 However, studies of THPTA and
NTB indicated that only THPTA was capable of stabilizing DNA/RNA structures in the
CuAAC reactions (Fig. S2) and THPTA was thus the sole ligand used in subsequent CuAAC
reactions for nucleic acid modifications. NTB was not a good ligand in CuAAC for nucleic
acid modifications because nucleic acids were completely degraded by the end of the reaction
(Fig. S2). To evaluate the effectiveness of the CuAAC reactions, we also synthesized alkynyl
and azido derivatives of the tag molecule biotin (10 and 12; Scheme 4) and the fluorophore
fluorescein (15-16; Scheme 5). Finally, 19 was synthesized (Scheme 6) to enable preparation
of alkyne-modified nucleic acids required in the other CuAAC reaction (Scheme 2B).
The optimal CuAAC reactions for azide- and alkyne-modified DNA/RNA derived from
the two-step phosphoramidation reactions were effectively developed with all the required
reagents at hand. We first determined that 6c and 6d were better electrophiles to react with
amino-nucleic acids and provided higher yields of azide-modified nucleic acids. For the
synthesis of alkyne-modified DNA/RNA, we originally used the commercially available
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propargylamine (9) as the substrate for nucleic acid modifications. The 9-modified
DNA/RNA, however, resulted in sluggish CuAAC reactions with low yield. We envisioned
that the reaction yield could be improved by moving the alkynyl group away from the nucleic
acids and introducing an electron-withdrawing group adjacent to the alkynyl group. Indeed,
when substituting 19 for 9 in nucleic acid conjugates, the CuAAC reactions provided far
better yield (results not shown). We then systematically surveyed the effects of nucleic acid
concentration, the concentrations of 6c/6d and 19, copper concentration, THPTA
concentration, reaction pH, the azide : alkyne ratio (Fig. S2), and the Cu : THPTA ratio to
obtain the optimal reaction conditions for the conjugation of azide-/alkyne-modified nucleic
acids with corresponding alkyne/azide substrates.
The optimal CuAAC reactions were successfully exploited to synthesize various nucleic
acid conjugates. For instance, the azide-modified 3’ primer DNA were effectively conjugated
with several alkynyl-containing substrates (Fig. 1A and Scheme 2A). Similar results were
acquired by using azide-modified RNA in the optimal CuAAC reactions (Fig. 1B). Moreover,
the alkyne-modified 3‘ primer DNA was also effectively conjugated with azido-containing
substrates by the other optimized CuAAC reaction (Fig. 2A and Scheme 2B). Again, a similar
high yield was observed in the optimized CuAAC reaction when azides were reacted with the
alkyne-modified 17-mer RNA (Fig. 2B). The presence of biotin and fluorescein moieties in
the CuAAC triazole products was confirmed by streptavidin (SAv) gel-shift analysis and
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fluorescence imaging after electrophoresis (Figs. 2B and S3). In addition, the modDifOiIe: 1d0.1039/C4OB01132E
nucleic acids were gel-purified and analyzed by MALDI-TOF MS to demonstrate the
production of the expected DNA and RNA conjugates (Electronic Supplementary Information,
ESI). However, as revealed in Figs. 1 and 2, nucleic acids suffered noticeable degradation in
the optimized CuAAC reactions likely the result of the presence of Cu(I)-derived reactive
species. We have ruled out the role of reactive oxygen species to trigger the undesired nucleic
acid degradation because similar degradation of nucleic acid was detected when performing
the CuAAC in the absence of oxygen (Fig. S4). Nevertheless, the triazole nucleic acids were
clearly the major products in the optimized CuAAC reactions.
As copper-induced nucleic acid degradation can compromise efforts to accurately
quantify nucleic acids in vivo by the optimized CuAAC reactions, we thus explored the
potential of the copper-free SPAAC reaction for nucleic acid modifications. We again
employed the two-step phosphoramidation reactions to easily synthesize azido-containing
nucleic acids which could react with cyclooctynes in the SPAAC reaction (Scheme 2A).
Congruent with the expectation, the cyclooctyne Alkyne MegaStokes dye 608 (DNA
conjugation substrate 3 in Fig. 1A) was smoothly labelled to the azide-modified 3’ primer
DNA without incurring detectable DNA degradation.5 The presence of the fluorophore and
structural integrity of the DNA in the SPAAC triazole product was also visualized and
confirmed by fluorescence imaging analysis after gel electrophoresis (Fig. S5). Moreover, the
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identity of the gel-purified MegaStokes dye 608-modified DNA was affirmed by
MALDI-TOF MS analysis (ESI). The results clearly explicate the reactivities of
phosphoramidation reaction-derived azido-containing nucleic acids in the CuAAC and
SPAAC reactions to provide the desired modified nucleic acids with good yield.
CuAAC for POC synthesis and causing undesirable cytotoxicity
Finally, we employed the optimized CuAAC reaction to more effectively synthesize POCs
(Scheme 2C) and demonstrate the cell-penetrating ability of the acquired POCs. Click
chemistry has been applied for POC synthesis.26, 27 However, past POC synthesis studies
depended on solid-phase phosphoramidite chemistry to afford alkynyl- or amino-containing
oligonucleotides and the CuAAC reaction to acquire POCs. Intriguingly, these synthesized
POCs were never administered to biological systems nor did they demonstrate
cell-penetrating activity. Here we successfully exploited the optimized CuAAC reaction and
conjugated the azide-modified FITC-labelled 3‘ primer DNA with an alkynyl-containing Tat
peptide (DNA conjugation substrate 5 in Fig. 1A) with excellent yield (Fig. 1A). However,
when inoculating human A549 cells with the gel-purified CuAAC-synthesized POC,
significant cell death was observed (Fig. 3). As phosphoramidation bonds and the Tat
peptides at the studied concentrations are non-toxic to human cells,14 the detected
cytotoxicity was attributed to a residual amount of copper catalyst in the POC or the newly
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formed triazole ring in the POC.
We further investigated the role of copper in cell death as copper ions have been
documented to trigger nucleic acid modifications and cleavage.28 We first confirmed the
presence of copper in the POC by ICP-MS analysis, and determined that only 3.1% of total
copper catalyst [1.91 nmol out of 60 nmol (acquired from 6 times of the scale-up 10X
CuAAC reaction)] was retained in the purified POC (1.60 nmol). In addition, similar levels of
cell death were observed when inoculating A549 cells with the Tat peptide and Cu(II) in
which the concentrations of the peptide and copper were equal to those in the POC (Fig. 3).
The results strongly supported that the residual copper from the CuAAC reaction trafficked
into the human A549 cells through chelation as the cause of the significant cytotoxicity and
cell death. Indeed, when extensively washing the POC with EDTA, we removed an additional
26% of copper in the POC and enhanced the viability of the POC-inoculated human A549
cells (Fig. 3). Moreover, we demonstrated translocation of the EDTA-treated POC into
human A549 cells after inoculation by flow cytometry (Fig. S6) and confocal laser scanning
microscopy (Fig. 4) even though the deformation of A549 cells was visible (PC in Fig. 4A).
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EXPERIMENTAL
Materials and Methods
All reagent-grade chemicals were purchased from commercial sources (Sigma-Aldrich, Acros,
Alfa Aesar, and Mallinckrodt Baker) except where noted, and were further purified as
necessary. The standard Tat peptide (being the 48th to 57th amino acid residues in the Tat
protein and having the sequence in H2N-GRKKRRQRRR-COOH; each bold and capitalized
letter standing for a specific amino acid residue)14 and its alkynyl version (amidation with
5-hexynoic acid at the N terminus of the Tat peptide) were purchased from Peptide 2.0
(Chantilly, VA, USA) and used without further purification. THPTA was synthesized
according to the method of Hong et al.24 1H and 13C NMR spectra were recorded using either
a Varian 200 or 400 MHz spectrometer (Varian, Inc., Palo Alto, CA, USA) at Kaohsiung
Medical University, Taiwan (KMU). NMR samples were prepared in CD3OD, D2O or CDCl3,
and the chemical shifts of 1H signals were given in parts per million downfield from TMS.
13C signals were given in parts per million based on the internal standard of each deuteriated
solvent. ESI high resolution mass spectra were acquired from Department of Chemistry,
National Sun Yat-Sen University, Taiwan (NSYSU-Chemistry) on a Bruker APEX II
Fourier-transfer mass spectrometry (FT-MS; Bruker Daltonics Inc., Taiwan). ICP-MS
analysis for quantification of copper in POCs was also performed by NSYSU-Chemistry on a
PE-SCIEX ELAN 6100 DRC mass spectrometer (PerkinElmer Taiwan, Kaohsiung, Taiwan).
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Radio-labeled or biotin-/fluorophore-modified nucleic acid conjugates were analyzed by urea
polyacrylamide gel electrophoresis (urea-PAGE) or SAv gel shift assay in urea-PAGE,
visualized and quantified by an Amersham Typhoon PhosphorImager (GE Healthcare
Bio-Sciences AB, Uppsala, Sweden) at KMU. Molecular mass of purified nucleic acid
conjugates was determined by an Autoflex III TOF/TOF analyzer (Bruker Daltonics) located
at KMU.14 POC uptake by human A549 cells was analyzed by a BD FACSCalibur cytometer
(BD, Franklin Lakes, NJ, USA) and a FluoView 1000 confocal laser scanning microscope
(Olympus, Tokyo, Japan) also resided at KMU.
Synthesis of N-maleoyl amino acid succinimidyl esters (3)
Compound 3a was synthesized according to previously reported procedures.29 Synthesis of
3b-d was also attained by following a published method.30
Synthesis of azidocarboxylic acid succinimidyl esters (6)
Synthesis of 6a and its precursor 3-azidopropionic acid (5a) from 3-bromopropionic acid (4a),
and synthesis of 6b from 4-bromobutyric acid (4b) were achieved by following the
procedures of Grandjean et al.31 The synthesis of 6c generally adhered to the method of Seo
et al.32
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6-Azido-hexanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (6d)
Synthesis of 6d was achieved by modifying a published method30 and briefly described below.
First, the required 6-azidohexanoic acid (5d) was synthesized by dissolving and reacting
6-bromohexanoic acid (4d; 3.0 g, 15.4 mmol) with sodium azide (2.0 g, 30.8 mmol) in DMF
(10 mL) at 85 °C for 3 h. The resulting reaction mixture was diluted with DCM, extracted
with 0.1 N HCl, dried over Na2SO4, and concentrated under reduced pressure to give
colorless oil of 5d (80%).
Without further purification, the acquired 5d (1.352 g, 8.8 mmol) was dissolved and
stirred in a DMF solution (20 mL) submerging in an ice-water bath, followed by slow
addition of sym-collidine (2.5 mL, 18.5 mmol) in 10 min to give the Flask A solution.
Immediately, the Flask B solution was prepared by dissolving NHS (4.048 g, 35.2 mmol) in
the other DMF solution (20 mL) also immersed in an ice-water bath, following by slowly
adding TFAA (4.93 mL, 35.2 mmol) while stirring for 10 min, and finally drop-wisely adding
sym-collidine (4.66 mL, 34.5 mmol) in 10 min to give the solution. The Flask B solution was
then slowly dripped into the Flask A solution in 1.5 h while maintaining both solutions in 0 °
C. The resulting mixture was returned to rt and stirred overnight. The final reaction mixture
was diluted with DCM (60 mL), extracted with 1 N HCl (50 mL) three times, dried over
Na2SO4, concentrated under reduced pressure, and further washed with Et2O (40 mL) three
times to afford the white-colored solid 6d (2.14 g, 76%). 1H NMR (400 MHz) (CDCl3) :
14

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3.30 (t, 2H), 2.84 (br s, 4H), 2.64 (t, 2H), 1.79 (q, 2H), 1.68-1.60 (m, 2H), 1.55-1.48 (m, 2H).
13C NMR (100.67 MHz) (CDCl3) : 169.1, 168.4, 51.0, 30.7, 28.3, 25.8, 25.5, 24.1. HRMS
(ESI) calculated for C10H14N4O4, [M+Na]+ 277.09073 (calcd.), 277.09081 (found).
Synthesis of 2’-aminoethyl 5-azido-pentanamide, TFA salt (7)
A reaction mixture for 7 synthesis was prepared by dissolving 6c (0.29 g, 1.2 mmol) in DCM
(2 mL) first and following by addition of Et3N (0.16 mL, 1.16 mmol) and
mono-t-Boc-ethylenediamine (0.24 g, 1.5 mmol)14 to the DCM solution. After reacting at rt
for 7 h, the final reaction mixture was diluted with DCM, extracted with 1 N HCl, 5%
NaHCO3 and sat. NaCl, sequentially. The resulting organic phase was dried over Na2SO4 and
concentrated under reduced pressure to give the Boc-protected 7 which was deprotected by
dissolving in 1 mL of TFA while stirring at 0 °C for 1 h, removed TFA under reduced
pressure, washed with Et2O to afford the colorless oil-like 7 (0.17 g, 75%). 1H NMR (400
MHz) (CDCl3) : 3.32 (t, 2H), 2.41 (t, 2H), 1.78-1.60 (m, 4H). 13C NMR (100.67 MHz)
(CDCl3) : 178.1, 51.0, 33.2, 28.3, 28.2, 21.8. HRMS (ESI) calculated for C7H16N5O,
[M+H]+ 186.13494 (calcd.), 186.13490 (found).
Synthesis of biotin derivatives, 10 and 12
5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoic acid prop-2-ynylamide (10)
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The method of Poole et al.33 was adopted to synthesize 10.
5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-pentanoic acid
[2-(5-azido-pentanoylamino)-ethyl]-amide (12)
The reaction mixture for 12 synthesis was prepared by dissolving 7 (0.098 g, 0.58 mmol),
Et3N (82 L, 0.58 mmol) and (+)-biotin N-hydroxysuccinimide ester13 [11; 0.19 g, 0.55 mmol;
synthesized from (+)-biotin (8)] in 5 mL of DMF while stirring at rt for 5 h. The final reaction
mixture was concentrated under reduced pressure, recrystallized in IPA, and washed with EA
to give the white-colored solid 12 (0.371 g, 80%). 1H NMR (400 MHz) (CD3OD) : 4.49 (1H,
dd), 4.31 (1H, dd), 2.93 (1H, dd), 2.70 (1H, d), 1.60-1.31 (4H, m), 1.25 (2H, q). 13C NMR
(100.67 MHz) (CD3OD) : 176.4, 176.1, 63.4, 61.6, 57.0, 52.1, 47.9, 41.0, 40.1, 36.8, 36.5,
29.8, 29.5, 29.4, 26.8, 24.1. HRMS (ESI) calculated for C17H29N7O3, [M+Na]+ 434.1950
(calcd.), 434.1947 (found).
Synthesis of fluorescein derivatives, 15 and 16
5(6)-(N-Propargyl)amidofluorescein (15)
5(6)-Carboxyfluorescein (13; 1.13 g, 3 mmol) was first dissolved in 10 mL of THF and
followed by slow addition of a 10-mL THF solution containing N-hydroxysuccinimide (0.414
g, 3.6 mmol) and DCC (0.70 g, 3.6 mmol). The resulting reaction mixture was stirred at rt for
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2 h, concentrated under reduced pressure, and resuspended in pentane (5 mL) to precipitated
the orange-red-colored 5(6)-carboxyfluorescein N-hydroxysuccinimide ester (14; 0.145 g, 0.3
mmol). Without further purification, the acquired 14 was dissolved in 10 mL THF, and
followed by addition of propargylamine (9; 39 L, 0.6 mmol) and Et3N (45 L, 0.32 mmol)
to initiate the reaction at rt for 3 h. The final reaction mixture was concentrated under reduced
pressure, redissolved in EA (10 mL) and extracted with 1 N HCl, water and sat. NaCl,
sequentially. The afforded organic phase was dried over Na2SO4 and concentrated under
reduced pressure to give 15 (0.08 g, 59%). 1H NMR (400 MHz) (CD3OD) : 8.01 (2H, s),
7.68 (1H, s), 7.33 (2H, dd), 6.58-6.57 (4H, m), 4.13 (2H, d), 2.59 (1H, t). 13C NMR (100.67
MHz) (CD3OD) : 181.4, 160.5, 135.7, 132.3, 131.2, 130.6, 129.7, 129.2, 129.0, 123.7, 113.6,
72.2, 34.8. HRMS (ESI) calculated for C24H15NO6, [M+Na]+ 436.0797 (calcd.), 436.0795
(found).
5(6)-[N-(5-azido-N’-ethylpentamido)]amidofluorescein (16)
Synthesis of 16 started from crude 14 which was synthesized as described above and also
used without further workup. A reaction mixture for 16 synthesis was prepared by dissolving
crude 14 (260 mg, 0.55 mmol), 7 (0.98 mg, 0.58 mmol) and Et3N (82 L, 0.58 mmol) in a
6-mL DCM/DMF (5:1) solution while stirring at rt for 4 h. The final reaction mixture was
concentrated under reduced pressure, redissolved in DCM (20 mL) and extracted with 1 N
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HCl, water and sat. NaCl, sequentially. The resulting organic phase was dried over Na2SO4
and concentrated under reduced pressure to give 16 (0.08 g, 61%). 1H NMR (400 MHz)
(CD3OD) : 8.47 (s, 1H), 8.11 (s, 1H), 7.63 (s, 1H), 7.32 (d, 1H), 6.69 (t, 4H), 6.60 (d, 1H),
6.58 (t, 2H), 6.56 (d, 1H), 4.17-4.04 (m, 2H), 2.35 (t, 2H), 2.27- 2.22 (m, 2H), 1.70-1.56 (m,
2H). 13C NMR (100.67 MHz) (CD3OD) : 103.8, 71.1, 66.5, 64.0, 52.1, 36.5, 34.9, 33.1, 30.8,
29.4, 26.7, 26.0. HRMS (ESI) calculated for C28H25N5O7, [M+Na]+ 566.16462 (calcd.),
566.16482 (found).
Synthesis of 6-propynoylamino-hexanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (19)
Propiolic acid (17; 98.4 L, 1.6 mmol) were activated in an EDC (0.33 g, 1.6
mmol)-dissolved DMF solution (2 mL) at 0 °C while stirring for 15 min, and followed by
adding a 6-aminohexanoic acid (0.212 g, 1.61 mmol)-containing 1 M Na2CO3/DMF mixture
(1 M Na2CO3/DMF = 1:2; 3 mL) and reacting at rt for 3 h. The final reaction mixture was
diluted with DCM, extracted with water twice and sat. NaCl once. The resulting organic
phase was dried over Na2SO4 and concentrated under reduced pressure to give light
orange-colored oily crude 18 (0.183 g, 1 mmol). Without further purification, the obtained 18
was dissolved in a DCM solution (5 mL) containing EDC (0.24 g, 1.2 mmol) and followed by
addition of NHS (0.18 g, 1.2 mmol) to have reaction at rt while stirring for 12 h. The final
reaction mixture was diluted with DCM, extracted with water twice and sat. NaCl once. The
18

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DOI: 10.1039/C4OB01132E
afforded organic phase was again dried over Na2SO4 and concentrated under reduced
pressure to provide golden-colored 19 (0.31 g, 82%). 1H NMR (400 MHz) (D2O) : 3.11 (t,
2H), 3.08-2.98 (m, 4H), 2.79 (s, 4H), 2.69 (s, 1H), 1.84-1.77 (m, 2H), 0.98 (t, 2H). 13C NMR
(100.67 MHz) (D2O) : 176.7, 174.5, 160.7, 55.4, 42.8, 36.5, 35.1, 25.5, 25.3, 25.1, 14.5.
HRMS (ESI) calculated for C13H16N2O5, [M+Na]+ 303.09514 (calcd.), 303.09503 (found).
Nucleic acid preparation and radio-labeling
The single-stranded 3’ primer DNA (5'-TACCCCTTGGGGATACCACC-3') was purchased
from Purigo Biotech, Inc., Taiwan and purified by 20% urea-PAGE. The TW171-17 RNA
(5’-GGGAUCGUCAGUGCAUU-3’), which is the first 17 nucleotides in the TW17 RNA,34
was purchased from Bioneer (Daejeon, South Korea) and used without purification. Both the
the 3’ primer DNA and the TW171-17 RNA were 32P-labeled at the 5’-end according to the
previously reported methods.14
Modifications of nucleic acids with azides and alkynes
DNA
As prepared in N-hydroxysuccimide esters, either azides 6 (Scheme 3) or alkynes (9 and 19;
Scheme 6) were covalently linked to the ethylenediamine-modified 3’ primer DNA by the
amidation reaction reported previously.14 The ethylenediamine-conjugated 3’ primer DNA
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was synthesized according to the optimized two-step phosphoramidation reaction DoOfI:D10N.10A39i/Cn4OB01132E
which ethylenediamine was the nucleophile in the reaction.12, 14 Acquired DNA conjugates
were purified by ethanol precipitation and analyzed by 20% urea-PAGE.
RNA
Similarly, N-hydroxysuccimide esters of either azides (6) or alkynes (9 and 19) were coupled
to the cystamine-modified TW171-17 RNA by the same amidation reaction for DNA
modifications indicated above.14 The cystamine-conjugated TW171-17 RNA was also prepared
by the optimized two-step phosphoramidation reaction of RNA in which cystamine served as
the nucleophile in the reaction.14 Acquired RNA conjugates were again purified by ethanol
precipitation and analyzed by 20% urea-PAGE.
Copper-catalyzed 1,3-dipolar azide-alkyne cycloaddition for nucleic acid modifications
The previously reported CuAAC reaction24 was further studied to modify nucleic acids with
appropriate substrates and to attain the best yield. The acquired optimal CuAAC reactions
were briefly stated below. Either azido- or alkynyl-modified DNA/RNA (80 pmol) and
corresponding alkynyl- or azido-containing molecules (1.6 nmol) were dissolved in 8.85 L
phosphate buffer (100 mM potassium phosphate, pH 7.0) and followed by adding in 0.15 L
of a CuSO4-THPTA premix solution (prepared by mixing one part of 20 mM CuSO4 in water
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and two parts of 50 mM THPTA in water), 0.5 L of 100 mM aminoguanidine, anDdO0I:.150.103L9/oC4fOB01132E
100 mM fresh-prepared sodium ascrobate in sequence to give the final reaction mixture of 10
L. After the reaction at rt for 1 h, the reaction products were purified by ethanol
precipitation and analyzed by 20% urea-PAGE.
In addition, the optimal CuAAC reaction was scaled up ten times to more efficiently
synthesize enough quantities of the Tat peptide-3’ primer DNA conjugate (a POC) for
bioactivity studies of the POC in human A549 cells. The scale-up CuAAC reaction (10X)
generally gave the triazole product yield similar to that of the optimal CuAAC reaction (1X).
Copper-free strain-promoted 1,3-dipolar azide-alkyne cycloaddition for the
modification of azido nucleic acids with the cyclooctyne substrate
The method of Winz et al.5 was studied to modify the azido nucleic acids with the
cyclooctyne Alkyne MegaStokes dye 608 and was briefly described below. The
azide-conjugated DNA/RNA (80 pmol) was dissolved in 76 L of phosphate buffer (50 mM
potassium phosphate, pH 7.0) to give an 1 M nucleic acid solution. The SPAAC reaction
was initiated by adding in 0.4 L of Alkyne MegaStokes dye 608 (10 mM in DMSO) and
proceeded at 35 °C for 2 h. The final reaction products were purified by ethanol precipitation
and analyzed by 20% urea-PAGE.
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DOI: 10.1039/C4OB01132E
Cytotoxicity by MTT assay
The cytotoxicity of inoculates was determined by MTT assays against A549 cells. In briefly,
A549 cells were seeded in 96-well tissue culture plates at a density of 5  103/well in medium
containing 10% FBS before treating inoculates. The cytotoxicity of inoculates was evaluated
by determining cell viability after 24 h of incubation with various concentrations of
inoculates (1-10 M). The number of viable cells was acquired by estimating their
mitochondrial reductase activity using the tetrazolium-based colorimetric method (MTT
conversion test).35
Flow cytometry analysis of cellular uptake
In order to observe the cellular uptake efficiency of inoculates, A549 cells were seeded in
6-well culture plates at a density of 2  105/well in medium containing 10% FBS for 24 h.
The medium containing of 5 M concentration of inoculates were added to cells. After 24 h
of incubation, cells were washed, trypsinized, centrifuged, and resuspended in 1 mL of cold
PBS, and then analyzed using the flow cytometer. The fluorescein-labeled inoculates (peptide,
DNA and POC) used in flow cytometry and confocal laser scanning microscopy were
prepared according to the published methods14 and the method developed in the current study.
Confocal laser scanning microscopy (CLSM)
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DOI: 10.1039/C4OB01132E
The intracellular delivery of inoculates was observed using CLSM. A549 cells were seeded at
a density of 1.0  105/well in 12-well plates containing one glass coverslip/well in RPMI
supplemented with 10% FBS, and then incubated for 24 h. Each inoculate of 5 M was added
to cells for 24 h at 37 °C. After incubation, the inoculate-containing medium was removed
and washed gently with 1 mL of 0.1 M PBS at pH 7.4. The cell nuclei was then stained with 5
g/mL Hoechst 33342 (Invitrogen, Carlsbad, CA) for 30 min. The cells on the coverslips
were washed 3 times with 0.1M PBS and mounted with a fluorescent mounting medium on
glass slides. Cell imaging was obtained by CLSM (Fv 1000; Olympus, Tokyo, Japan) and
analyzed using Olympus CLSM software.
23

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DOI: 10.1039/C4OB01132E
DISCUSSION
In summary, a novel strategy consisting of the versatile two-step phosphoramidation reactions
and the powerful CuAAC and SPAAC reactions enabled effective and selective modifications
of post-synthetic DNA and RNA, and has produced various nucleic acid conjugates including
a POC. The POC was effectively trafficked into human cells with the potential to alleviating
the delivery challenge of therapeutic nucleic acids in clinical applications. The combination
of the phosphoramidation and azide-alkyne cycloaddition reactions thus establishes a
universal regioselective approach to modify any 5’ phosphate-primed nucleic acids with
diverse molecules ranging from proteins and peptides to lower molecular mass tags and
probes. The effective modification strategy will facilitate administration, detection and
quantification of nucleic acids in vivo and in vitro. However, the results of this study have
raised concerns about the use of the CuAAC reaction for synthesis of bioconjugates such as
POCs destined to be delivered into biological systems. Residual copper chelated to the
bioconjugates may cause damage and reduce cell viability. Ongoing research is in progress to
extend azide-alkyne cycloaddition for more effective synthesis of modified nucleic acids with
bioactivity not adversely affected by a residual copper catalyst.
24

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DOI: 10.1039/C4OB01132E
ACKNOWLEDGEMENTS
We thank Dr. Susan Fetzer for critically reading the manuscript and Dr. Maria Linder for
advice on copper cytotoxicity issues. We also thank technical and instrumental assistance of
Center for Research Resources and Development of KMU. This work was supported with
grants from Ministry of Science and Technology of Taiwan awarded to T.-P. W.
(101-2113-M-037-005- and 102-2113-M-037-017-). W. M. H. W. is supported by the
undergraduate research program funded by Ministry of Science and Technology of Taiwan
(101CFD2100044).
25

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NOTES AND REFERENCES
a Department of Medicinal and Applied Chemistry, Kaohsiung Medical University,
Kaohsiung, 80708, Taiwan; tzupinw@cc.kmu.edu.tw (T.-P. W.).
b Department of Biochemistry, Kaohsiung Medical University, Kaohsiung, 80708, Taiwan.
c Department of Biological Science and Technology, National Chiao Tung University,
Hsin-Chu, 300, Taiwan.
Electronic Supplementary Information (ESI) available: Full details of figures referenced in
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MS spectra for gel-purified nucleic acid conjugates. See DOI: 10.1039/c000000x/
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DOI: 10.1039/C4OB01132E
SCHEMES
O
N
O
N
O
O
O
O
O
O
a
HO
NH2
b
n
O
N
HO
NH2
O
N
n
n
1b
= 2,
= 4, 1c
1a
O
O
3a
O
n
3b
= 4, 3c
= 2,
O
O
O
N
O
NH2
O
N
O
b
HO
O
N
N
O
O
O
O
1d
O
O
3d
O
3e
Scheme 1. Synthesis of the N-maleoyl amino acid succinimidyl esters (3). Synthesis of 3e
was reported in our previous study.14 a: 1) maleic anhydride (2), DMF, rt, 2 h, 2) NHS, DCC,
1-25 oC, 16 h. b: 1) Flask A: 2, DMF, rt, 3 h; sym-collidine, 0 oC, 2) Flask B: NHS, TFAA,
sym-collidine, 0 oC; 3) mixing solutions in Flasks A and B within 1-2 h, 0 oC; 4) rt, 16 h.
O
(A) The CuAAC and SPAAC reactions with azide-modified nucleic acids
N
O
O
R3 N3
O
P
O
P
O
5'
5'
5'
NH2 Phosphoramidation
NH2
+
H2N
3'
N
3'
3'
O
Amidation
n = 1 or 3
n = 1 or 3
H
O
O
H
O
P
R4
H
N
O
R3 N3
R3
N
5'
N
3'
P
N
N
N
R4
n = 1
H
n = 1
CuAAC
H
O
O
O
O
or 3
N
or 3
MegaStokes 608
3'
H
N
O
SPAAC
R3
5'
P
N
N
n = 13O
N
H
O
N
or
MegaStokes 608
O
(B)
The CuAAC reaction with alkyne-modified nucleic acids
N
O
R5
O
O
O
5'
5'
5'
O
NH2 Phosphoramidation
NH2
3'
+ H2N
O
P
N
3'
P
Amidation
n = 1 or 3
n = 1 or 3
H
O
O
H
O
P
H
N
O
R6
R5
5'
R5
N3
N
N
3'
N
3'
P
N
n = 13O
H
n = 1
N
CuAAC
H
O
O
O
or
N
R6
or 3
O
N
O
(C)
The combination of phosphoramidation and CuAAC reactions for synthesis of POCs
O
N3
O
P
O
P
O
5'
5'
NH2
Phosphoramidation
NH2
3'
O
+
H2N
3'
N
Amidation
H
O
O
N
N
Tat peptide
H
N
O
P
Tat peptide
H
N
O
P
5'
N
5'
N3
3'
N
H
3'
N
n = 4
n = 4
CuAAC
H
O
O
POC
O
O
29