Development of thiol-responsive amide bond cleavage device and its application for peptide nucleic acid-based DNA releasing system

Article abstract of DOI:10.1016/j.tetlet.2010.03.006

To develop a thiol-responsive DNA-releasing system, a thiol-responsive amino acid capable of inducing an amide bond cleavage in the presence of a thiol was developed. It was successfully combined with peptide nucleic acid (PNA), and thiol-induced release of DNA from the thiol-responsive PNA/DNA complex was observed.

Full text of DOI:10.1016/j.tetlet.2010.03.006

Tetrahedron Letters 51 (2010) 2525–2528
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Development of thiol-responsive amide bond cleavage device
and its application for peptide nucleic acid-based DNA releasing system
*
*
Akira Shigenaga , Jun Yamamoto, Hiroko Hirakawa, Keiji Ogura, Nami Maeda, Ko Morishita, Akira Otaka
Institute of Health Biosciences and Graduate School of Pharmaceutical Sciences, The University of Tokushima, Shomachi, Tokushima 770-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
To develop a thiol-responsive DNA-releasing system, a thiol-responsive amino acid capable of inducing
an amide bond cleavage in the presence of a thiol was developed. It was successfully combined with pep-
tide nucleic acid (PNA), and thiol-induced release of DNA from the thiol-responsive PNA/DNA complex
was observed.
Received 10 February 2010
Revised 24 February 2010
Accepted 1 March 2010
Available online 3 March 2010
Ó 2010 Elsevier Ltd. All rights reserved.
In the field of gene therapy and genetic recombination, develop-
ment of a non-viral methodology for controlled delivery of DNA or
RNA to prevent potential infection resulting from the use of viral
vectors has been attracting increasing attention.1 In this context,
release of an entrapped or encapsulated nucleic acid by a non-viral
vector only after entering target cells is indispensable. An intracel-
lular reductive environment, mainly due to the presence of thiols
like glutathione, is one of the most attractive triggering substances
for intracellular DNA/RNA releasing systems2 because the concen-
tration of thiols is significantly different between the intracellular
and extracellular environments (concentration of glutathione: 0.2–
First, we attempted to synthesize model peptide 2, possessing a
thiol-responsive amino acid, to examine its reactivity and selectiv-
ity against thiol. Scheme 2 shows the synthesis of thiol-responsive
model peptide 2. The phenolic hydroxyl group on substrate 34b was
sulfonylated with a p-nitrobenzenesulfonyl chloride in the pres-
ence of K2CO3 to afford pNs derivative 4. The TBS group on 4 was
removed under acidic conditions to afford alcohol 5, and it was oxi-
dized with PCC to give aldehyde 6. After subsequent oxidation of
aldehyde 6 with NaClO2, followed by Boc removal by the action
of TFA, the generated amine was re-protected with the Fmoc group
to afford Fmoc-protected thiol-responsive amino acid 7. The total
yield of Fmoc derivative 7 amounted to 64% over six steps begin-
ning from phenol 3. Finally, incorporation of amino acid derivative
7 into a peptide by Fmoc solid phase peptide synthesis (Fmoc SPPS)
afforded thiol-responsive model peptide 2.9
To examine the reactivity and selectivity of model peptide 2
against thiol, we tested the processing reaction outlined in Scheme
3. Diastereomerically purified peptide 2 (0.02% w/v) in 30% v/v ace-
tonitrile/phosphate buffer (20 mM, pH 7.6 or 9.0) was incubated
with or without 0.1% w/v nucleophiles including thiol and amine
at 37 °C for 24 h. The reaction progress was monitored by HPLC,
and the resulting peptides were characterized by electrospray ion-
ization mass spectrometry (ESI-MS). Yields of processing products
were calculated based on peak areas in the HPLC chart, and the re-
sults are summarized in Table 1. In the presence of sodium 2-mer-
captoethanesulfonate 11,10 processing products 8, 80 (an epimer of
8 at a carbon asterisked in Scheme 3),11 and 9 were obtained at
both pH 7.6 and 9.0 (entries 1 and 2, respectively). In these reac-
tions, pNs-deprotected intermediate 10 was not observed because
lactonization was possibly faster than removal of the pNs group.4a
The yield of processing products at pH 9.0 was higher than that at
pH 7.6, presumably due to an efficient generation of a nucleophilic
thiolate anion under basic conditions. To accelerate the processing
reaction under physiological conditions, introduction of the
electron-withdrawing group on pNs group or treatment of peptide
10 mM in cytoplasm; 2 l
M in plasma).3 Previously, we reported a
stimulus-responsive peptide which can induce a processing (pep-
tide bond cleavage) reaction after stimulus-induced removal of a
phenolic protective group followed by lactonization of the tri-
methyl lock moiety.4,5 In this Letter, we report the development
of a thiol-responsive amino acid and its application for a thiol-
responsive DNA releasing system.
It is known that a nitrobenzenesulfonyl amide and ester can be
easily cleaved in the presence of thiol,6 and thiol-induced cleavage
of an aryl nitrobenzenesulfonate has been successfully applied as a
quantitative thiol probe.7 With this in mind, we designed thiol-
responsive peptide nucleic acid (PNA8) 1 possessing a p-nitro-
benzenesulfonyl (pNs) group on the phenolic hydroxyl group of
the stimulus-responsive amino acid (Scheme 1). Addition of PNA
1 to a solution of complementary DNA should induce hybridization
to form a stable PNA/DNA double strand. In the presence of thiol,
removal of the pNs group followed by a lactonization of the tri-
methyl lock moiety should cause fragmentation of PNA 1 at the
C-terminal position of the thiol-responsive amino acid releasing
the complementary DNA.
* Corresponding authors.
E-mail addresses: ashige@ph.tokushima-u.ac.jp (A. Shigenaga), aotaka@ph.toku
shima-u.ac.jp (A. Otaka).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.03.006

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A. Shigenaga et al. / Tetrahedron Letters 51 (2010) 2525–2528
Scheme 1. Design of thiol-responsive peptide nucleic acid (PNA).
Scheme 2. Reagents and conditions: (i) p-nitrobenzenesulfonyl chloride (pNsCl), K2CO3, acetone, reflux, quant; (ii) AcOH, H2O, THF, quant; (iii) PCC, CH2Cl2, 91%; (iv) NaClO2,
NaH2PO4, 2-methyl-2-butene, tert-BuOH, H2O, acetone; (v) HCl, AcOEt; (vi) FmocOSu, Na2CO3, H2O, MeCN, 70% (three steps); (vii) Fmoc SPPS; (viii) TFA/triethylsilane/
H2O = 95:2.5:2.5 v/v/v.
Table 1
Effects of pH and nucleophile on the processing reaction of peptide 2 as shown in
Scheme 3
Entry
Nucleophile
pH
Yield (%)
1
2
3
4
HS(CH2)2SO3Na (11)
11
H2N(CH2)2SO3H
None
7.6
9.0
9.0
9.0
37
68
0
0
Next, we monitored the time course of the thiol-responsive pro-
cessing reaction of peptide 2 in the presence of 0.1% w/v thiol 11 at
pH 9.0 (Fig. S1). After 24 h of incubation at 37 °C, the yield of pro-
cessing products almost reached a plateau. It might be caused by
oxidative decomposition of thiol 11 under basic conditions to gen-
erate a disulfide by-product. In order to complete the processing
reaction, we tried stepwise addition of thiol in the two-portion
manner described below. Peptide 2 (0.02% w/v) in 30% v/v acetoni-
trile/phosphate buffer (pH 9.0, 20 mM) was incubated with 0.1% w/
v thiol 11 at 37 °C for 24 h, and additional 0.1% w/v thiol 11 was
added to the reaction mixture. It was incubated at the same tem-
perature for an additional 24 h, and the reaction progress was
monitored by HPLC (Fig. 1). Whereas the substrate remained after
incubation with 0.1% w/v thiol 11 at 37 °C for 24 h (Fig. 1b),
Scheme 3. Reagents and conditions: (i) 0.1% w/v nucleophile, phosphate buffer
(20 mM), 30% v/v MeCN, 37 °C, 24 h under Ar. Peptide 80 is an epimer of 8 at the
asterisked carbon.
2 with glutathione S-transferase7b is in progress. When peptide 2
was incubated without nucleophile or with a taurine as an amine
nucleophile, the processing products were not detected (entries 3
and 4). These results suggest that the processing reaction of model
peptide 2 occurs thiol-selectively.

A. Shigenaga et al. / Tetrahedron Letters 51 (2010) 2525–2528
2527
substrate 2 completely disappeared after stepwise addition of thiol
11 to afford processing products 8, 80 and 9 (Fig. 1c). When 0.2% w/
v thiol 11 was added in one portion, the reaction did not finish
within 48 h of incubation, and the yield of the processing products
was 76%. Therefore we decided to use the two-portion protocol for
a thiol treatment in subsequent experiments.
These results encouraged us to develop a thiol-responsive PNA.
PNA 12 depicted in Scheme 4 was designed to possess a Gly-thiol-
responsive amino acyl residue with length similar to that of thymine
derived t residue (Fig. 2). Additionally, a lysine residue was incorpo-
rated, which was expected to provide high solubility in water and
to interact with negatively charged DNA. Fmoc SPPS using PyBOP
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophos-
phate), HCTU (O-(6-chlorobenzotriazol-1-yl)-N,N,N0,N0-tetramethyl-
uronium hexafluorophosphate), or HATU (O-(7-azabenzotriazol-1-
yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate) allowed
PNA 1212 to be synthesized.13 Then we examined a thiol-responsive
processing reaction of PNA 12 in phosphate buffer (pH 9.0) (Scheme
4). After treatment with thiol 11 in the two-portion manner, PNA
12 completely disappeared and processing products 13 and 14 were
obtained in good purity (Fig. S2). Next we examined the ability of
PNA 12 to hybridize with complementary DNA and to release the
DNA from the complex by treatment with a thiol. Melting tempera-
ture (Tm) of PNA or DNA/DNA complex was measured in phosphate
buffer (10 mM phosphate, 100 mM NaCl, pH 9.0);14 the results are
summarized in Table 2. As a thiol-treated sample (depicted as ‘thiol
+’ in Table 2), hybridized nucleic acids were incubated with 0.1% w/
v of sodium 2-mercaptoethanesulfonate 11at37 °Cfor24 h, andwere
subsequently incubated with additional thiol 11 (0.1% w/v) at the
same temperature for 24 h. Melting curves were recorded using a
fluorescence spectrometer (kex = 525 nm, kem = 600 nm) in the
presence of ethidium bromide.15 In the absence of thiol, the melting
curve of PNA 12/DNA(A9) complex was observed under these
conditions, and Tm was estimated as 25.0 °C. (DNA(N9) refers to 50-
d(NNNNNNNNN)-30). As expected, Tm of PNA 12/DNA(A9) complex
was lowered in the presence of thiol (entries 1 and 2).16 In contrast,
DNA(T9)/DNA(A9) hybridization was not affected by thiol treatment
(entries 3 and 4). Furthermore, Tm of PNA 12/DNA(A9) complex was
Figure 1. HPLC profiles (a) before treatment with thiol 11, (b) after 24 h incubation
with 0.1% w/v thiol 11 at 37 °C under Ar, and (c) after 24 h incubation with 0.1% w/v
thiol 11 at 37 °C under Ar, followed by subsequent incubation with additional 0.1%
w/v thiol 11 at 37 °C for 24 h under Ar. Peptides were detected by UV absorbance at
220 nm. Asterisked peak is not a peptidic compound.
Figure 2. Structural comparison of Gly-thiol-responsive amino acyl residue and t
residue.
Scheme 4. Reagents and conditions: (i) 0.1% w/v thiol 11, phosphate buffer (pH 9.0, 20 mM), 30% v/v MeCN, 37 °C, 24 h under Ar. Subsequent addition of 0.1% w/v thiol 11,
37 °C, 24 h under Ar, as per the two-portion manner.

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A. Shigenaga et al. / Tetrahedron Letters 51 (2010) 2525–2528
Table 2
References and notes
Results of melting temperature experimentsa
1. Mintzer, M. A.; Simanek, E. E. Chem. Rev. 2009, 109, 259–302.
Entry
Nucleic acid
Thiol
Tm (°C)
2. Recent reviews covering a thiol-responsive gene delivery system: (a) Lin, C.;
Engbersen, J. F. J. J. Controlled Release 2008, 132, 267–272; (b) Ganta, S.;
Devalapally, H.; Shahiwala, A.; Amiji, M. J. Controlled Release 2008, 126, 187–
204; (c) Harada, A.; Kataoka, K. Prog. Polym. Sci. 2006, 31, 949–982.
3. (a) Anderson, E. Chem. Biol. Interact. 1998, 112, 1–14 and references therein; (b)
Jones, D. P.; Carlson, J. L.; Samiec, P. S.; Sternberg, P.; Mody, V. C.; Reed, R. L.;
Brown, L. A. S. Clin. Chem. Acta 1998, 275, 175–184.
1
2
3
4
PNA 12/DNA(A9)
PNA 12/DNA(A9)
DNA(T9)/DNA(A9)
DNA(T9)/DNA(A9)
ꢁ
+
25.0
<10.0
18.5
ꢁ
+
18.5
a
Conditions: 4.0 lM concentration for each strand, 10 mM phosphate buffer (pH
4. (a) Shigenaga, A.; Yamamoto, J.; Hirakawa, H.; Yamaguchi, K.; Otaka, A.
Tetrahedron 2009, 65, 2212–2216; (b) Shigenaga, A.; Tsuji, D.; Nishioka, N.;
Tsuda, S.; Itoh, K.; Otaka, A. ChemBioChem 2007, 8, 1929–1931.
5. Trimethyl lock system: (a) Jung, M. E.; Piizzi, G. Chem. Rev. 2005, 105,
1735–1766. and references therein; (b) Amsberry, K. L.; Borchardt, R. T.
J. Org. Chem. 1990, 55, 5867–5877; (c) Caswell, M.; Schmir, G. L. J. Am.
Chem. Soc. 1980, 102, 4815–4821; (d) Milstien, S.; Cohen, L. A. J. Am.
Chem. Soc. 1972, 94, 9158–9165; (e) Borchardt, R. T.; Cohen, L. A. J. Am.
Chem. Soc. 1972, 94, 9166–9174; (f) Milstien, S.; Cohen, L. A. Proc. Natl.
Acad. Sci. U.S.A. 1970, 67, 1143–1147.
9.0), 100 mM NaCl. Thiol ꢁ: The sample was not treated with thiol 11. Thiol +: The
sample was treated with thiol 11 (0.1% w/v at 37 °C for 24 h under Ar, followed by
subsequent treatment with thiol 11 (0.1% w/v) again at 37 °C for an additional 24 h
under Ar. Melting curves were recorded in the presence of ethidium bromide
(kex = 525 nm, kem = 600 nm). Tms are the average of two runs. DNA(N9) denotes 50-
d(NNNNNNNNN)-30.
higher than that of corresponding DNA(T9)/DNA(A9) complex (entries
1 and 3). These results suggest that PNA 12 is potentially applicable to
a thiol-responsive DNA releasing system.
6. (a) Kan, T.; Fukuyama, T. Chem. Commun. 2004, 353–359; (b) Fukuyama, T.;
Jow, C. K.; Cheung, M. Tetrahedron Lett. 1995, 36, 6373–6374.
7. (a) Yang, X.-F.; Wang, L.; Xu, H.; Zhao, M. Anal. Chim. Acta 2009, 631,
91–95; (b) Zhou, W.; Shultz, J. W.; Murphy, N.; Hawkins, E. M.; Bernad,
L.; Good, T.; Moothart, L.; Frackman, S.; Klaubert, D. H.; Bulleit, R. F.;
Wood, K. V. Chem. Commun. 2006, 4620–4622; (c) Maeda, H.; Matsuno,
H.; Ushida, M.; Katayama, K.; Saeki, K.; Itoh, N. Angew. Chem., Int. Ed.
2005, 44, 2922–2925.
8. (a) Nielsen, P. E. Chem. Biodivers. 2007, 4, 1996–2002; (b) Nielsen, P. E.; Michael,
E. In Peptide Nucleic Acid; Nielsen, P. E., Ed., second ed.; Horizon Bioscience:
Wymondham, 2004; pp 1–36.
In conclusion, we developed a thiol-responsive self-processing
amino acid which induces an amide bond cleavage reaction after
treatment with thiol. With its successful combination with a pep-
tide nucleic acid, thiol-responsive PNA was generated. Melting
temperature experiments clarified that thiol-responsive PNA 12
can bind to complementary DNA(A9), and the Tm of the thiol-
responsive PNA 12/DNA(A9) complex was drastically decreased
by treatment with thiol. These results suggest that thiol-responsive
PNA is potentially applicable as a DNA-binding moiety of a thiol-
responsive gene delivery system. Development of the thiol-respon-
sive PNA with sequence specificity and high sensitivity to thiols,
and its application for gene delivery system are in progress.
9. Thiol-responsive model peptide 2: Analytical HPLC condition (Cosmosil
5C18-AR-II analytical column (Nacalai Tesque, 4.6 ꢀ 250 mm, flow rate:
1 mL/min)): linear gradient of solvent
B in solvent A, 10–80% over
30 min. Retention time = 22.6 and 23.2 min, respectively for each
diastereomer of peptide 2. MS (ESI-IT) calcd for C68H88N13O17
S
([M+H]+): 1390.6, found; 1390.7 and 1390.7.
10. Because glutathione and its disulfide derivative make HPLC chart complicated,
thiol 11 was used as a model thiol.
11. Time dependent epimerization of processing product 8 to its epimer 80 was
observed under reaction conditions.
Acknowledgments
12. Thiol-responsive PNA 12: Analytical HPLC condition (Cosmosil 5C18-AR-II
analytical column (Nacalai Tesque, 4.6 ꢀ 250 mm, flow rate: 1 mL/min)):
We thank Dr. M. Nishida (Osaka University) for valuable discus-
sion. This research was supported in part by a Grant-in-Aid for Sci-
entific Research (KAKENHI), Takeda Science Foundation, The
Science and Technology Foundation of Japan, and Nagase Science
and Technology Foundation.
linear gradient of solvent
B in solvent A, 1–50% over 30 min. Retention
time = 22.0 min. MS (ESI-IT) calcd for C115H152N38O40S ([M+2H]2+): 1369.1,
found; 1369.1.
13. Synthesis of Fmoc-t-OH, see: Thomson, S. A.; Josey, J. A.; Cadilla, R.; Gaul, M. D.;
Hassman, C. F.; Luzzio, M. J.; Pipe, A. J.; Reed, K. L.; Ricca, D. J.; Wiethe, R. W.;
Noble, S. A. Tetrahedron 1995, 51, 6179–6194.
14. Rahman, S. M.; Seki, S.; Obika, S.; Yoshikawa, H.; Miyashita, K.; Imanishi, T. J.
Am. Chem. Soc. 2008, 130, 4886–4896.
Supplementary data
15. Because UV-absorption of thiol or disulfide was observed at 260 nm, standard
UV-absorption-based method was not applicable to this experiment.
16. For prediction of Tm value of PNA, see: Giesen, U.; Kleider, W.; Berding,
C.; Geiger, A.; Orum, H.; Nielsen, P. E. Nucleic Acids Res. 1998, 26, 5004–
5006.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2010.03.006.