Synthesis of ethynylbenzene-substituted glycol as a versatile probe for labeling oligonucleotides

Article abstract of DOI:10.1016/j.bmcl.2014.01.082

Positron emission tomography (PET) is a highly sensitive quantitative imaging technique for studying molecular pathways and interactions in vivo. This imaging technique plays a key role in drug discovery, pharmacokinetics, pharmacodynamics, and assessing in vivo distribution. In this study, we designed an ethynylbenzene-substituted glycol (M^E) as a versatile probe for PET labeling of oligonucleotides through a click reaction.

Full text of DOI:10.1016/j.bmcl.2014.01.082

Accepted Manuscript
Synthesis of ethynylbenzene-substituted glycol as a versatile probe for labeling
oligonucleotides
Qin Ren, Kana Tsunaba, Yoshiaki Kitamura, Remi Nakashima, Aya Shibada,
Masato Ikeda, Yukio Kitade
PII:
S0960-894X(14)00126-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.01.082
BMCL 21321
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
24 December 2013
26 January 2014
31 January 2014
Please cite this article as: Ren, Q., Tsunaba, K., Kitamura, Y., Nakashima, R., Shibada, A., Ikeda, M., Kitade, Y.,
Synthesis of ethynylbenzene-substituted glycol as a versatile probe for labeling oligonucleotides, Bioorganic &
Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.01.082
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Synthesis of ethynylbenzene-substituted glycol as a versatile probe for labeling
oligonucleotides
Qin Rena, Kana Tsunabab, Yoshiaki Kitamurab,c, Remi Nakashimaa, Aya Shibadab,c, Masato Ikedaa,b,c
and Yukio Kitadea,b,c,*
aUnited Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1
Yanagido, Gifu 501-1193, Japan
bDepartment of Biomolecular Science, Graduate School of Engineering, Gifu University, 1-1
Yanagido, Gifu 501-1193, Japan
cDepartment of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1
Yanagido, Gifu 501-1193, Japan
 To whom correspondence should be addressed:
United Graduate School of Drug Discovery and Medical Information Sciences
Gifu University
1-1 Yanagido, Gifu 501-1193, Japan
Tel.: +81-58-293-2640; Fax: +81-58-293-2640
E-mail: ykkitade@gifu-u.ac.jp

Keywords
Artificial nucleoside; Alkynes; Stereoselective synthesis; Huisgen cycloaddition; Click chemistry

Abstract
Positron emission tomography (PET) is a highly sensitive quantitative imaging technique for
studying molecular pathways and interactions in vivo. This imaging technique plays a key role in
drug discovery, pharmacokinetics, pharmacodynamics, and assessing in vivo distribution. In this
study, we designed an ethynylbenzene-substituted glycol (ME) as a versatile probe for PET labeling
of oligonucleotides through a click reaction.

Following the success of the Human Genome Project, nucleic acids have been investigated as new
drug targets1,2. Although nucleic acid drugs are highly specific and less toxic than conventional drugs,
there are problems with non-specific side effects, nuclease degradation, and low cell membrane
permeability. It has been proposed that developing artificial nucleic acids and better drug delivery
systems could improve the safety and efficacy of nucleic acid drugs. In addition, techniques for
evaluating the pharmacokinetics and efficacy of the drugs are important. Positron emission
tomography (PET) can serve as a non-invasive technique for visualizing pharmacokinetics in vivo.
Moreover, the low dose of radiation required for PET makes it particularly suitable for imaging the
pharmacokinetics of biomedicines, including nucleic acid drugs.
It has been proposed that artificial nucleic acids containing an ethynyl group on the sugar or base
moiety could be labeled with azide derivatives bearing a PET radiotracer through an azide-alkyne
cycloaddition, known as a click reaction. Click reactions have been used to label artificial nucleic
acids, because of their selectivity, reliability, and versatility. However, most studies have used an
expensive copper ligand tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) to prevent the
decomposition of the nucleic acid oligomers. Furthermore, these reaction conditions generally
require heating for efficient labeling3-8; therefore, these reaction conditions are too harsh for PET
labeling of chemically unstable nucleic acid oligomers. The labeling reaction must also reach
completion within minutes rather than hours because the radionuclides used for PET imaging are
short-lived positron emitters (t1/2 = 110 min for 18F).

Very recently, we have investigated a mild, rapid click reaction between a 4-fluorobenzylazide and
nucleic acid oligomers containing 5-ethynyl-1,3-benzenedimethanol (BE)9. Under our reaction
conditions, the click reaction was complete within several tens of minutes at room temperature, with
no copper ligands or heating. However, we found that double-stranded nucleic acids containing BE in
the middle of sequence showed low thermal stability (low melting temperature)10. Molecular force
field calculations showed that the distance between the two oxygen atoms of BE is two-fold greater
than that of 2-deoxy-D-ribose, which could disturb the double-stranded helical structure.
In this study, we have designed (S)-4-ethynylmandelol (ME) which has a glycol nucleic acid
(GNA) skeleton as a new sugar analogue moiety (Scheme 1). Because the distance between the two
oxygen atoms of ME is similar to that of 2-deoxy-D-ribose, we expected that ME would form more
stable double-stranded helical structures than BE. Furthermore, GNA has an asymmetric carbon in
the sugar moiety and the (S)-GNA enantiomer stabilizes double-stranded DNA11-15. In this work, we
introduced ME or BE at three different positions of an oligonucleotide (15-mer including ME or BE)
and carried out a click reaction between single-stranded or double-stranded oligonucleotides
containing ME or BE and 4-fluorobenzylazide under our conditions.
The synthesis of the ME began with the reaction of L-mandelic acid (2) with ethanol and sulfuric
acid to give ethyl ester 3. Ethyl ester 3 was selectively iodinated at the 4-position of the benzene ring
in the presence of silver trifluoromethanesulfonate, to give ethyl (S)-4-iodomandelate (4)16. The ester
was reduced with sodium borohydride to give the corresponding diol (5)17. Trimethylsilylacetylene

and diol 5 were reacted in piperidine to give acetylene derivative 6. The TMS group was removed
with potassium carbonate to give the ethynylbenzene-substituted glycol (ME, 1)18. The primary
hydroxyl group of ME was protected with a 4,4′-dimethoxytrityl (DMTr) group to give mono-DMTr
derivative 7, which was phosphorylated with 2-cyanoethyldiisopropylchlorophosphoramidite to
produce phosphoramidite 8. ME was incorporated at the 3′-end of the oligonucleotides by succinating
the mono-DMTr derivative (7) and the resulting succinate was linked to a controlled pore glass
(CPG) solid support (9).
Nucleic acid oligomers containing ME at the 5′-ends, the center, or the 3′-end (ON 12, 13, or 14)
were synthesized by an automated nucleic acid synthesizer using phosphoramidite derivative 8 or
solid supported 9. For comparison, nucleic acid oligomers containing BE (ON 15, 16, 17) were also
synthesized9. A complementary strand (ON 10) and a control strand (ON 11) without the artificial
nucleic acids (ME and BE) were also synthesized (Table 1). The structures of these nucleic acid
oligomers were confirmed by MALDI-TOF/MS analysis19.
First, the click reaction between the single-stranded DNA oligomers (ON 12–17) and
4-fluorobenzylazide was examined under optimized conditions9. The sequence of the corresponding
4-fluorobenzyltriazole (FBT)-appended DNA oligomers are depicted in Table 1. The reaction was
tracked by ion-pair reversed-phase (RP) HPLC using a standard ODS column. Typical reaction
scheme and the corresponding HPLC charts are shown in Scheme 2 and Figure 1, respectively. After
15 min, the peak (tR = 9.5 min) corresponding to ON 13 (dT7-ME-dT7) decreased and a new peak

appeared at 12.7 min. From MALDI-TOF/MS analysis, the new peak was assigned as a
4-fluorobenzyltriazole (FBT)-appended DNA oligomer ON 19 (dT7-MFBT-dT7), which was the
desired click reaction product (Figure 1). Because other intense peaks were not observed, the
consumption of the click reaction starting materials (ON 12–17) after 15 min was used to evaluate
the labeling efficiency. Figure 2 shows that the click reaction of ONs containing ME proceeded more
efficiently than that for ONs containing BE with the same sequences, indicating that ME itself is
suitable for click reaction compared to BE presumably due to its reduced steric hindrance.
Interestingly, we found that ON 13 (dT7-ME-dT7), which contained ME in the center of the sequence,
exhibited the most efficient labeling, suggesting that reducing of steric hindrance may help to
accelerate the click reaction9.
The thermal stability of double-stranded DNA oligomers was determined in order to evaluate the
click reaction (Figure 3). The stability of double-stranded DNA oligomers containing BE and ME at
the 3′ or 5′ ends was similar to that of a control double-stranded DNA oligomer ON 10 (dA15)/ON 11
(dT15). In contrast, the stability of double-stranded DNA oligomers containing BE and ME in the
center of the sequence ON 10 (dA15)/ON 13 (dT7-ME-dT7) or ON 10 (dA15)/ON 16 (dT7-BE-dT7) was
significantly reduced. However, the double-stranded DNA oligomer containing ME was more stable
compared with the BE oligomer for approximately 5 °C.
Finally, the click reaction between the double-stranded DNA oligomers and 4-fluorobenzylazide
was examined under the same conditions as the click reaction for the single-stranded DNA oligomers.

The ion-pair RP-HPLC analysis revealed that double-stranded DNA oligomers ON 10 (dA15)/ON 13
(dT7-ME-dT7) dissociated into two single-stranded DNA oligomers. A peak (tR = 11 min)
corresponding to the starting material ON 13 (dT7-ME-dT7) decreased and the product peak ON 19
(dT7-MFBT-dT7) appeared at 14.5 min (Scheme S1 and Figure S1). This suggests that the labeling
was efficient even for the double-stranded structure. Electrophoresis (native-PAGE) was used to
investigate whether the click reaction proceeded without dissociation of the double-stranded DNA
oligomer into the single-stranded DNA oligomers. Figure S2 shows that a band of the
double-stranded DNA oligomer was observed after the click reaction. Therefore, the click reaction
should proceed without the dissociation of the double-stranded structure.
In conclusion, we have stereoselectively synthesized an ethynylbenzene-substituted glycol, ME,
from L-mandelic acid and introduced it into DNA oligomers. Single-stranded DNA oligomers
containing ME can be labeled with 4-fluorobenzylazide more efficiently than those containing BE.
Although further investigation is required, ME should be a potentially suitable for PET labeling of
single-stranded nucleic acid oligomers. The thermal stability of the double-stranded DNA oligomer
containing ME was better than that containing BE, particularly when the artificial nucleic acid (BE
and ME) was introduced at the center of the sequence. Furthermore, the click reaction of the
double-stranded DNA oligomer containing ME was also successful. Although it is not necessary to
introduce PET labeling reagents at the center of the sequence, this finding could be useful for
chemical modification of double-stranded DNA oligomers and functionalization. We are currently

examining the use of ME for fluorescent labeling and for further chemical modification of nucleic
acid oligomers.
Acknowledgments
This work was supported in part by Grants-in-Aid for Scientific Research (B) from the Japan
Society for the Promotion of Science.

References and notes
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19. MALDI-TOF/MS analyses of ONs. The following spectra were obtained by MALDI-TOF/MS
(negative mode, 3-hydroxypicolinic acid as matrix). ON 10: calculated mass, 4632.9; observed
mass, 4633.3. ON 11: calculated mass, 4497.7; observed mass, 4496.8. ON 12: calculated mass,
4417.7; observed mass, 4418.7. ON 13: calculated mass, 4417.7; observed mass, 4418.7. ON
14: calculated mass, 4417.7; observed mass, 4418.2. ON 15: calculated mass, 4417.7; observed
mass, 4419.2. ON 16: calculated mass, 4417.7; observed mass, 4417.4. ON 17: calculated mass,
4417.7; observed mass, 4418.4.

LEGENDS
Scheme 1. Reagents and conditions: (a) H2SO4, EtOH, 70 °C, 97%; (b) I2, AgOTf, CH2Cl2, rt, 68%;
(c) NaBH4, EtOH, rt, 86%; (d) TMS-acetylene, PdCl2(PPh3)2, CuI, piperidine, rt, 85%; (e) K2CO3,
MeOH, H2O, rt, quant; (f) DMTrCl, pyridine, rt, 31%; (g) (i-Pr)2NP(Cl)O(CH2)2CN, i-Pr2NEt, THF,
rt, 79%; (h) i) succinic anhydride, DMAP, pyridine, rt; ii) CPG, EDCI, DMF, rt, 26.5 μmol/g.
Table 1. Sequences of single-stranded DNA oligomers used in this study.
Scheme 2. Click reaction between a single-stranded DNA oligomer bearing ME ON 13 (dT7-ME-dT7)
and 4-fluorobenzylazide to produce a FBT-appended DNA oligomer ON 19 (dT7-MFBT-dT7).
Figure 1. Chromatogram of the click reaction of single-stranded DNA oligomers. A: 5% MeCN/0.1
M TEAA buffer (pH 7.0), B: 50% MeCN/0.1 M TEAA buffer (pH 7.0). Gradient: A:B = 83:17 (0
min) → 60:40 (20 min); flow rate, 1 mL/min; measurement wavelength, 260 nm.
Figure 2. Efficiency of the click reaction (substrate consumption after 15 min) between
single-stranded DNA oligomers and 4-fluorobenzylazide.
Figure 3. Thermal stability (melting temperature) of double-stranded DNA oligomers.

Table 1
No.of ON
Sequence
ON 10 (dA15)
5'-d(AAAAAAAAAAAAAAA)-3'
5'-d(TTTTTTTTTTTTTTT)-3'
5'-d(METTTTTTTTTTTTTT)-3'
5'-d(TTTTTTTMETTTTTTT)-3'
5'-d(TTTTTTTTTTTTTTME)-3'
5'-d(BETTTTTTTTTTTTTT)-3'
5'-d(TTTTTTTBETTTTTTT)-3'
5'-d(TTTTTTTTTTTTTTBE)-3'
5'-d(MFBTTTTTTTTTTTTTTT)-3'
ON 11 (dT15)
ON 12 (ME-dT14)
ON 13 (dT7-ME-dT7)
ON 14 (dT14-ME)
ON 15 (BE-dT14)
ON 16 (dT7-BE-dT7)
ON 17 (dT14-BE)
ON 18 (MFBT-dT14)
ON 19 (dT7-MFBT-dT7) 5'-d(TTTTTTTMFBTTTTTTTT)-3'
ON 20 (dT14-MFBT
ON 21 (BFBT-dT14)
)
5'-d(TTTTTTTTTTTTTTMFBT)-3'
5'-d(BFBTTTTTTTTTTTTTTT)-3'
ON 22 (dT7-BFBT-dT7) 5'-d(TTTTTTTBFBTTTTTTTT)-3'
ON 23 (dT14-BFBT
5'-d(TTTTTTTTTTTTTTBFBT)-3'
)

Scheme 1

Scheme 2

Figure 1

Figure 2

Figure 3