The contribution of adenines in the catalytic core of 10-23 DNAzyme improved by the 6-amino group modifications

Article abstract of DOI:10.1016/j.bmcl.2016.07.076

In the catalytic core of 10-23 DNAzyme, its five adenine residues are moderate conservative, but with highly conserved functional groups like 6-amino group and 7-nitrogen atom. It is this critical conservation that these two groups could be modified for b

Full text of DOI:10.1016/j.bmcl.2016.07.076

Bioorganic & Medicinal Chemistry Letters 26 (2016) 4462–4465
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The contribution of adenines in the catalytic core of 10-23 DNAzyme
improved by the 6-amino group modifications
Junfei Zhu a, Zhiwen Li a, Qi Wang b,c, Yang Liu d, Junlin He b,
⇑
a College of Life Science, Guizhou University, Guiyang 550025, China
b Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China
c Hununbeier People’s Hospital, Hununbeier 021008, China
d Guangxi Medical University, Nanning 530021, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In the catalytic core of 10-23 DNAzyme, its five adenine residues are moderate conservative, but with
highly conserved functional groups like 6-amino group and 7-nitrogen atom. It is this critical conserva-
tion that these two groups could be modified for better contribution. With 20-deoxyadenosine analogues,
several functional groups were introduced at the 6-amino group of the five adenine residues. 3-
Aminopropyl substituent at 6-amino group of A15 resulted in a five-fold increase of kobs. More efficient
DNAzymes are expected by delicate design of the linkage and the external functional groups for this 6-
amino group of A15. With this modification approach, other functional groups or residues could be opti-
mized for 10-23 DNAzyme.
Received 15 May 2016
Revised 13 July 2016
Accepted 29 July 2016
Available online 30 July 2016
Keywords:
10-23 DNAzyme
Amino group
Imidazolyl group
Chemical modification
Adenine
Ó 2016 Elsevier Ltd. All rights reserved.
The appearance of noncoding nucleic acids and their critical
roles in modulating genetic process has initiated great interest in
exploring more natural and unnatural functions and their practical
applications,1–4 such as antisense,5 quadruplex,6 CpG motifs,7
ribozymes8 and deoxyribozymes,9 aptamers,10 siRNAs11 and
miRNAs,12 et al. In the efforts toward these purposes, understand-
ing the structural basis and optimization of their functions is thus
extremely required. Simple residue mutation on these functional
nucleic acids, 10-23 DNAzyme13,14 and 8-17 DNAzymes,15,16
ribozymes,17 TBA (thrombin-binding aptamer),18,19 and siRNAs,20
confirmed the residue conservation and the optimum selection
results by nature and in vitro selection. It is rational that both
the residues and the sequence composition contribute to the active
conformation and functions by base stacking and hydrogen bond-
ing. As revealed by NMR and X-ray analysis for TBA and its interac-
tion with thrombin, its eight guanine residues form a quadruplex
motif and the functional groups of the loop residues are responsi-
ble for the direct interactions with the active residues from throm-
bin.21,22 Fluorescence method determined that pKa shifts of
nucleobases are prevalent in DNA and RNA tertiary structures, this
kind of ionization led to activation of the functional groups of
nucleobases as general acid/base catalysts.23 Experimental evi-
dences suggested that cytidine,24 adenine,25 and guanine26 could
act as general acid–base in ribozyme catalysis. Therefore, both
base-stacking and the specific functional groups around the four
nucleobases are equally important for a specific functional nucleic
acid.27 In the optimization of functional nucleic acids, nucleobase
modification for an insight into the structural basis of nucleic acids
has been extensively studied.28 Especially, amino and imidazolyl
groups are most often used as external groups for an activation
of nucleic acids, and this kind of base modification has been trans-
ferred to nucleoside triphosphates used in in vitro selection, to find
the optimum disposition of the active functional groups and more
powerful nucleic acids.29
10-23 DNAzyme is an in vitro selected catalytic DNA molecule,
its catalytic function is performed by a 15-mer catalytic core,9a in
which every nucleobase is related to the reaction rate more or
less, but all are unique at their specific positions (Scheme 1). Our
studies revealed that the spacial location and functional groups
of five dA residues are both critical for their contribution.30 For
example, 7-nitrogen atom of nine purine residues were critical
for the catalytic activity, but its contribution at A9 could be opti-
mized with 8-aza-7-deaza-20-deoxyadenosine 1 and its 7-substi-
tuted derivative 2 (Scheme 2).30 Especially, some of the highly
conserved dG residues could be improved by its analogues. By this
way, the functional groups could be regarded as the origin of
mechanistic understanding and optimization of functional nucleic
acids. In adenine, its 6-amino group is an important functional
group, it was suggested to act as general acid or base in the
⇑
Corresponding author. Tel./fax: +86 10 6821 1656.
E-mail address: hejunlin@bmi.ac.cn (J. He).
http://dx.doi.org/10.1016/j.bmcl.2016.07.076
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

J. Zhu et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4462–4465
4463
of the whole conformation characteristic of B-DNA duplex (Fig. S1),
ensuring the DNAzyme-substrate complex formation.
5'- AGG TGC AGG rA UGG AGA GCA- 3'
DR19
3'- TCC ACG TCC
ACC TCT CGT- 5'
Firstly, the conservation of the 6-amino group of five adenine
residues (A5, A9, A11, A12 and A15) was studied by the deletion
of the 6-amino group with compound 3. Each replacement at these
positions led to a change of the catalytic activity. The negative
effect at A5, A9, A11 and A12 was observed for the modified DNA-
zymes (DZ-A5-3, DZ-A9-3, DZ-A11-3, and DZ-A12-3) compared to
the unmodified DZ01 (Table 1). But for A15, this deletion resulted
in a positive effect on the catalytic reaction (DZ-A15-3 vs DZ01),
the interaction of the 6-amino group of A15 with other residues
seems to be unfavorable for the catalytic reaction. These results
demonstrated that the 6-amino group of all adenines was closely
related to the activity, no matter whether they contribute to the
activity by constructing the catalytic conformation or take part in
the catalytic reaction directly (Fig. 1).
Based on these observations, compounds 4 and 5 were designed
for the possibility of improving the effect of the 6-amino group, in
which the 6-amino group was partially substituted with 2-ami-
noethyl group or imidazolylethyl group, respectively. By this
design, the partial hydrogen-bonding ability of 6-amino group
was kept, and the extra amino and imidazolyl groups were intro-
duced for more interactions and more influence on the catalytic
conformation.
15
A
G
G
C
T
G
C
12 A
11 A
10-23DZ
A 5
C
9
G
C
A
T
Scheme 1. 10-23 DNAzyme and its complementary DNA-RNA-DNA substrate
DR19, in which the bold letters are the ribonucleotide residues as the cleavage site,
as indicated by an arrow, and the adenine residues in the catalytic core are
numbered.
catalytic reactions of ribozymes and form active conformation with
other residues through hydrogen bonding.25 Here, compound 3
(Scheme 2)30b was designed as probe to study the importance of
the 6-amino group of five dA residues in the catalytic core of 10-
23 DNAzyme, and it was further activated by amino and
imidazolyl groups by compounds 4–6. Another two compounds 7
and 8 (Scheme 2) were designed for the evaluation of the 6-
amino group in the lead compound 1 and the active compound 2
for A9. All of them were the analogues of 20-deoxyadenosine, by
which the least interruption on the base occupation and stacking
was kept.
When 2-aminoethyl group was introduced to 6-amino group by
compound 4, it was not accommodated at A5 and A12, it meant
that 6-amino group of A5 and A12 is very conservative and
uniquely needed. Its effect was similar to that of 20-deoxyadeno-
sine at A9 (DZ-Z9-4), A11 (DZ-A11-4), and A15 (DZ-A15-4). When
compared to compound 3 with 6-hydrogen atom, the 6-substituent
led to a significant increase at A9 (DZ-A9-4). At A15, although 6-(2-
aminoethyl)amino group of compound 4 (DZ-A15-4) was not as
good as a hydrogen atom of compound 3 (DZ-A15-3), its effect
was still positive when compared with parent DZ01. These results
demonstrated that 6-amino group at A9 and A15 could be further
activated, either with a different functional group or a change of
All the nucleoside analogues were converted to their corre-
sponding phosphoramidites for solid-phase synthesis of the modi-
fied DNAzymes, in which each of the five dA residues were
replaced by the nucleosides 1–8, respectively. All the DNAzymes
were purified by denaturing electrophoresis and characterized by
MALDI-TOF or ESI MS (Supplementary materials, Table S1). Under
single-turnover conditions, the observed rate constants of the
DNAzymes (2 lM) were measured against the DNA-RNA-DNA sub-
strate DR19 (20 nM) (Scheme 1), in the presence of 2 mM Mg2+ (in
the buffer: 50 mM Tris-HCl, pH 7.5, 37 °C), by which the effect of
the modification on the catalytic core was evaluated, without con-
sideration of the binding process of the DNAzyme with the sub-
strate, and the leaving step of the cleaved products.30,31
the linkage for the amino group, for
substittuent.
a possible better 6-
Under the reaction conditions, the Tm results indicated that the
6-amino substituents had little effect on the base stacking of the
DNAzyme-substrate complexes (Table S2). CD spectra showed lit-
tle changes of the amplitude and location of the positive lobe (Sup-
plementary materials). Therefore, this kind of modifications
remained the stable complex formation without significant change
When an imidazolyl group was introduced by compound 5, a
different profile of the position-dependent effect was observed.
A5, A9 and A12 seem to be more appropriate for the imidazolyl
Table 1
The observed rate constants of modified 10-23 DNAzymes with nucleoside analogues
3–6 under single-turnover conditions
NH2
Name
Catalytic core residues
kobs (minꢀ1) (ꢁ10ꢀ3
)
NH2
HN
DZ01
50-d(GGC TAG CTA CAA CGA)-30
50-d(GGC T3G CTA CAA CGA)-30
50-d(GGC TAG CT3CAA CGA)-30
50-d(GGC TAG CTA C3A CGA)-30
50-d(GGC TAG CTA CA3CGA)-30
50-d(GGC TAG CTA CAA CG3)-30
50-d(GGC T4G CTA CAA CGA)-30
50-d(GGC TAG CT4CAA CGA)-30
50-d(GGC TAG CTA C4A CGA)-30
50-d(GGC TAG CTA CA4CGA)-30
50-d(GGC TAG CTA CAA CG4)-30
50-d(GGC T5G CTA CAA CGA)-30
50-d(GGC TAG CT5 CAA CGA)-30
50-d(GGC TAG CTA C5A CGA)-30
50-d(GGC TAG CTA CA5CGA)-30
50-d(GGC TAG CTA CAA CG5)-30
50-d(GGC T6G CTA CAA CGA)-30
50-d(GGC TAG CT6CAA CGA)-30
50-d(GGC TAG CTA C6A CGA)-30
50-d(GGC TAG CTA CA6CGA)-30
50-d(GGC TAG CTA CAA CG6)-30
5.1 0.7
0.78 0.06
0.30 0.01
0.37 0.06
0.90 0.14
12.7 0.5
0.4 0.1
6.0 0.5
4.6 0.3
0.36 0.06
8.50 0.06
8.9 0.5
8.9 0.5
1.40 0.01
1.30 0.05
1.40 0.01
nda
NH2
NH2
N
N
DZ-A5-3
DZ-A9-3
DZ-A11-3
DZ-A12-3
DZ-A15-3
DZ-A5-4
DZ-A9-4
DZ-A11-4
DZ-A12-4
DZ-A15-4
DZ-A5-5
DZ-A9-5
DZ-A11-5
DZ-A12-5
DZ-A15-5
DZ-A5-6
DZ-A9-6
DZ-A11-6
DZ-A12-6
DZ-A15-6
N
N
N
N
N
N
N
N
N
N
N
N
O
N
O
N
O
HO
HO
HO
HO
O
OH
1
OH
2
OH
OH
3
4
H
N
NH2
N
HN
NH2
HN
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
O
HO
HO
HO
HO
O
O
O
11.8 0.7
3.4 0.3
nda
OH
OH
OH
OH
5
6
7
8
26.0 0.6
Scheme 2. 20-Deoxyadenosine analogues for 6-amino group modification in the
a
nd: no significant reaction was observed under present conditions.
catalytic core of 10-23 DNAzyme.

4464
J. Zhu et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4462–4465
Table 2
The observed rate constants of modified 10-23 DNAzymes with nucleoside analogues
1, 2, 7, and 8 under single-turnover conditions
Name
Catalytic core residues
kobs (minꢀ1) (ꢁ10ꢀ3
)
DZ-A9-1
DZ-A9-2
DZ-A9-7
DZ-A9-8
DZ-A5-7
DZ-A11-7
DZ-A12-7
DZ-A15-7
DZ-A5-8
DZ-A11-8
DZ-A12-8
DZ-A15-8
50-d(GGC TAG CT1CAA CGA)-30
50-d(GGC TAG CT2CAA CGA)-30
50-d(GGC TAG CT7CAA CGA)-30
50-d(GGC TAG CT8CAA CGA)-30
50-d(GGC T7G CTA CAA CGA)-30
50-d(GGC TAG CTA C7A CGA)-30
50-d(GGC TAG CTA CA7CGA)-30
50-d(GGC TAG CTA CAA CG7)-30
50-d(GGC T8G CTA CAA CGA)-30
50-d(GGC TAG CTA C8A CGA)-30
50-d(GGC TAG CTA CA8CGA)-30
50-d(GGC TAG CTA CAA CG8)-30
8.9 0.8
45
nd
4
6.90 0.25
nda
nd
9.8 1.1
1.3 0.1
<0.0001
0.30 0.01
0.80 0.01
0.90 0.01
a
nd: no significant reaction was observed under present conditions.
only with the 7-substituent in this context, either its functional
group or the linkage could be optimized. At other four positions
(A5, A11, A12 and A15), these modifications always led to signifi-
cant negative effect, with only one exception for DZ-A12-7, in
which the effect of compound
deoxyadenosine.
7
is similar to that of 20-
From this research, we learnt that in the catalytic core of 10-23
DNAzyme, two residues A9 and A15 could be improved by its 6-
amino substituent and further optimized by changing the extra
functional group and the linkage.
With this kind of chemical modification on single functional
groups of canonical residues in functional nucleic acids, the specific
functional group could be easily identified for activation with pro-
tein-like functional groups, as an approach for optimization of
functional nucleic acids. By this way, a large space for chemical
modification of functional nucleic acids was created.
Acknowledgment
Financial support from the National Natural Science Foundation
of China (Grant No. 21572268) is gratefully acknowledged.
Figure 1. A comparison of compounds 3–6 at A9 and A15 for a positive effect on the
catalytic reaction of 10-23 DNAzyme measured under single-turnover conditions,
DNAzyme (2 lM) and the substrate (20 nM) were mixed in the buffer (50 mM Tris-
HCl, pH 7.5, 2 mM Mg2+, 37 °C), and monitored at certain time points. The samples
Supplementary data
were separated with denaturing PAGE (20%) and analyzed with PhosphoImager.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.07.
076.
group than hydrogen atom and 2-aminoethyl groups, but its effect
is just as 6-amino group of natural 20-deoxyadenosine. While at
A11 and A15, further negative effect produced by this substitution.
The different size and hydrogen bonding ability of the 6-positioned
extra amino and imidazolyl group induced different position-
dependent effect.
Compound 6 was designed for improving the effect of 6-substi-
tuted external amino group of compound 4 at A9 and A15. Com-
pared with compound 4, the effect of the extra amino group was
strengthened by the one-carbon longer linkage (Table 1). Com-
pared to parent DZ01, 2-fold and 5-fold increase of kobs was
observed for DZ-A9-6 and DZ-A15-6, respectively. While, further
negative effect was observed for the conservative A5 and A12,
except for A11 with the similar result (Fig. 1).
References and notes
1. Bumett, J. C.; Rossi, J. J. Chem. Biol. 2012, 19, 60.
2. Mondal, T.; Subhash, S.; Vaid, R.; Enroth, S.; Uday, S.; Reinius, B.; Mitra, S.;
Mohammed, A.; James, A. R.; Hoberg, E.; Moustakas, A.; Gyllensten, U.; Jones, S.
J. M.; Gustafsson, C. M.; Sims, A. H.; Westerlund, F.; Gorab, E.; Kanduri, C. Nat.
Commun. 2015. http://dx.doi.org/10.1038/ncomms8743.
3. Dong, H.; Lei, J.; Ding, L.; Wen, Y.; Ju, H.; Zhang, X. Chem. Rev. 2013, 113, 6207.
4. Kole, R.; Krainer, A. R.; Altman, S. Nat. Rev. Drug Disc. 2012, 11, 125.
5. Nishina, K.; Piao, W.; Yoshida-Tanaka, K.; Sujino, Y.; Nishina, T.; Yamamoto, T.;
Nitta, K.; Yoshioka, K.; Kuwahara, H.; Yasuhara, H.; Baba, T.; Ono, F.; Miyata, K.;
Miyake, K.; Seth, P. P.; Low, A.; Yoshida, M.; Bennett, C. F.; Kataoka, K.;
Mizusawa, H.; Obika, S.; Yokota, T. Nat. Commun. 2015. http://dx.doi.org/
10.1038/ncomms8969.
6. Collie, G. W.; Parkinson, G. N. Chem. Soc. Rev. 2011, 40, 5867.
7. Krieg, A. M. Nucleic Acid Ther. 2012, 22, 77.
8. Weng, D. E.; Masci, P. A.; Radka, S. F.; Jackson, T. E.; Weiss, P. A.; Ganapathi, R.;
Elson, P. J.; Capra, W. B.; Parker, V. P.; Lockridge, J. A.; Cowens, J. W.; Usman, N.;
Borden, E. C. Mol. Cancer Ther. 2005, 4, 948.
Compound 1 is the lead compound for A9 modification and
30
compound 2 gives a 9-fold increase of kobs
.
With compounds 7
and 8, the importance of their 6-amino group was investigated.
As listed in Table 2, the evaluation results indicated that the 6-
amino group was highly needed for their positive effect. It was sug-
gested that the 6-amino group and 8-N atom and 7-substituents in
compounds 1 and 2 are cooperative in inducing a favorable cat-
alytic conformation, and further improvement could be expected
9. (a) Santoro, S. W.; Joyce, G. F. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 4262; (b)
Hallett, M. A.; Dalal, P.; Sweatman, T. W.; Pourmotabbed, T. Nucleic Acid Ther.
2013, 23, 379.
10. Sun, H.; Zhu, X.; Lu, P. Y.; Rosato, R. R.; Tan, W.; Zu, Y. Mol. Ther. Nucleic Acids
2014, 3, e182.
11. Badrealam, K. F.; Zubair, S.; Owais, M. Curr. Gene Ther. 2015, 15, 201.

J. Zhu et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4462–4465
4465
12. Lam, J. K. W.; Chow, M. Y. T.; Zhang, Y.; Leung, S. W. S. Mol. Ther. Nucleic Acids
2015, 4, e252.
13. Zaborowska, Z.; Fürste, J. P.; Erdmann, V. A.; Kurreck, J. J. Biol. Chem. 2002, 277,
26. Liu, L.; Cottrell, J. W.; Scott, L. G.; Fedor, M. J. Nat. Chem. Biol. 2009, 5, 351.
27. (a) Bottaro, S.; Di Palma, F.; Bussi, G. Nucleic Acids Res. 2014, 42, 13306; (b)
Ponce-Salvatierra, A.; Wawrzyniak-Turek, K.; Steuerwald, U.; Hobartner, C.;
Pena, V. Nature 2016, 529, 231.
_
40617.
14. Smuga, D.; Majchrzak, K.; Sochacka, E.; Nawrot, B. New J. Chem. 2010, 34, 934.
15. Peracchi, A.; Bonaccio, M.; Clerici, M. J. Mol. Biol. 2005, 352, 783.
16. Rong, W.; Xu, L.; Liu, Y.; Yu, J.; Zhou, Y.; Liu, K.; He, J. Bioorg. Med. Chem. Lett.
2012, 22, 4238.
17. Grasby, J. A.; Mersmann, K.; Singh, M.; Gait, M. J. Biochemistry 1995, 34, 4068.
18. Smirnov, I.; Shafer, R. H. Biochemistry 2000, 39, 1462.
19. Aviñó, A.; Fàbrega, C.; Tintoré, M.; Eritja, R. Curr. Pharm. Design 2012, 18, 2036.
20. Peacock, H.; Kannan, A.; Beal, P. A.; Burrows, C. J. J. Org. Chem. 2011, 76, 7295.
21. Wang, K. Y.; McCurdy, S.; Shea, R. G.; Swaminathan, S.; Bolton, P. H.
Biochemistry 1993, 32, 1899.
28. (a) Wachowius, F.; Höbartner, C. J. Am. Chem. Soc. 2011, 133, 14888; (b) Lermer,
L.; Roupioz, Y.; Ting, R.; Perrin, D. M. J. Am. Chem. Soc. 2002, 124, 9960.
29. (a) Perrin, D. M.; Garestier, T.; Hélène, C. J. Am. Chem. Soc. 2001, 123, 1556; (b)
Sidorov, A. V.; Grasby, J. A.; Williams, D. M. Nucleic Acids Res. 2004, 32, 1591; (c)
Jäger, S.; Famulok, M. Angew. Chem., Int. Ed. 2004, 43, 3337; (d) Hipolito, C. J.;
Hollenstein, M.; Lam, C. H.; Perrin, D. M. Org. Biomol. Chem. 2011, 9, 2266; (e)
Hollenstein, M.; Hipolito, C. J.; Lam, C. H.; Perrin, D. M. ACS Comb. Sci. 2013, 15,
174; (f) Kielkowski, P.; Fanfrlík, J.; Hocek, M. Angew. Chem., Int. Ed. 2014, 53,
7552; (g) Rohloff, J. C.; Gelinas, A. D.; Jarvis, T. C.; Ochsner, U. A.; Schneider, D.
J.; Gold, L.; Janjic, N. Mol. Ther. Nucleic Acids 2014, 3, e201.
22. Padmanabhan, K.; Padmanabhan, K. P.; Ferrara, J. D.; Sadler, J. E.; Tulinsky, A. J.
Biol. Chem. 1993, 268, 17651.
30. (a) He, J.; Zhang, D.; Wang, Q.; Wei, X.; Cheng, M.; Liu, K. Org. Biomol. Chem.
2011, 9, 5728; (b) Zhu, J.; Li, Z.; Yang, Z.; He, J. Bioorg. Med. Chem. 2015, 23,
4256; (c) Liu, Y.; Li, Z.; Liu, G.; Wang, Q.; Chen, W.; Zhang, D.; Cheng, M.; Zheng,
Z.; Liu, K.; He, J. Chem. Commun. 2013, 5037; (d) Yang, X.; Xiao, Z.; Zhu, J.; Li, Z.;
He, J.; Zhang, L.; Yang, Z. Org. Biomol. Chem. 2016, 14, 4032.
31. (a) Santoro, S. W.; Joyce, G. F. Biochemistry 1998, 37, 13330; (b) Cairns, M. J.;
King, A.; Sun, L.-Q. Nucleic Acids Res. 2003, 31, 2883; (c) Räz, M. H.; Hollenstein,
M. Mol. BioSyst. 2015, 11, 1454; (d) Nawrot, B.; Widera, K.; Wojcik, M.;
Rebowska, B.; Nowak, G.; Stec, W. J. FEBS J. 2007, 274, 1062; (e) Ota, N.;
Warashina, M.; Hirano, K.; Hatanaka, K.; Taira, K. Nucleic Acids Res. 1998, 26,
3385.
23. Wilcox, J. L.; Bevilacqua, P. C. J. Am. Chem. Soc. 2013, 135, 7390.
24. Nakano, S.; Chadalavada, D. M.; Bevilacqua, P. C. Science 2000, 287, 1493.
25. (a) Muth, G. W.; Ortoleva-Donnelly, L.; Strobel, S. A. Science 2000, 289, 947; (b)
Košutic´, M.; Neuner, S.; Ren, A.; Flür, S.; Wunderlich, C.; Mairhofer, E.;
}
´
Vušurovic, N.; Seikowski, J.; Breuker, K.; Hobartner, C.; Patel, D. J.; Kreutz, C.;
Micura, R. Angew. Chem., Int. Ed. 2015, 54, 15128; (c) Liberman, J. A.; Guo, M.;
Jenkins, J. L.; Krucinska, J.; Chen, Y.; Carey, P. R.; Wedekind, J. E. J. Am. Chem. Soc.
2012, 134, 16933; (d) Kath-Schorr, S.; Wilson, T. J.; Li, N.-S.; Lu, J.; Piccirilli, J. A.;
Lilley, D. M. J. J. Am. Chem. Soc. 2012, 134, 16717; (e) Wilson, T. J.; Li, N.-S.; Lu, J.;
Frederiksen, J. K.; Piccirillib, J. A.; Lilley, D. M. J. Proc. Natl. Acad. Sci. U.S.A. 2010,
107, 11751.