Synthesis of [12]aneN3-dipeptide conjugates as metal-free DNA nucleases

Article abstract of DOI:10.1016/j.bmcl.2012.01.075

In this Letter, a series of macrocyclic polyamine [12]aneN ₃-dipeptide conjugates as a new type of metal-free nucleases were synthesized and fully characterized with ¹H NMR, ¹³C NMR, IR, and HR-MS. Results indicate that th

Full text of DOI:10.1016/j.bmcl.2012.01.075

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2303–2307
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of [12]aneN3–dipeptide conjugates as metal-free DNA nucleases
⇑
Zhi-Fen Li, Hua-Long Chen, Li-Jun Zhang, Zhong-Lin Lu
College of Chemistry, Beijing Normal University, Xinjiekouwai Street 19, 100875 Beijing, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this Letter, a series of macrocyclic polyamine [12]aneN3–dipeptide conjugates as a new type of metal-
free nucleases were synthesized and fully characterized with 1H NMR, 13C NMR, IR, and HR-MS. Results
indicate that these conjugates can bind to calf thymus DNA mainly through electrostatic interaction and
Received 21 November 2011
Revised 16 January 2012
Accepted 20 January 2012
Available online 3 February 2012
can cleave the plasmid DNA at 200
pathway.
l
M (pH 7.2, 37 °C), with an acceleration of 106-fold via hydrolytic
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Macrocyclic polyamine
[12]aneN3
Dipeptides
DNA cleavage
Gel-electrophoresis
Intensive work has been focused on the development of artifi-
cial nucleases for the cleavage of phosphodiester bonds in RNA,
DNA, and their model compounds.1–9 These studies are beneficial
for shedding light on understanding the chemistry of the corre-
sponding hydrolytic enzymes, providing artificial restriction en-
zymes for molecular biology which would be highly valuable in
the manipulation of DNA, and developing the antibiotic and che-
motherapeutic drugs. With the consideration that a lot of nucleas-
es have active sites comprising two or more metal ions, many
studies have been devoted to the design and synthesis of dinuclear
or polynuclear metal complexes.10–13 Encouraged by the structure
characterization of the active site found in staphylococcal nuclease
(SNase), which is metal-free and imparts 1016-fold rate accelera-
tion for DNA hydrolysis,14 metal-free artificial nucleases have been
received much attention more recently. Göbel15 and Breslow16
have pioneered in this field. Organic molecules such as guanidi-
nium derivatives,17,18 polyamines19 and macrocyclic poly-
amines,20–24 cyclodextrin derivatives,25 hydroxylamine,26 and
various peptides27–30 have shown the ability to cleave RNA, DNA,
and their model compounds. Among them, peptides and macrocy-
clic polyamines have received special attention. For example, Ser-
His dipeptide was proved to cleave the double-stranded DNA or
circular plasmid DNA via a hydrolytic mechanism.27 Linear peptide
and cyclopeptide were also used as metal-free DNA nucleases.28–30
Macrocyclic polyamines derivatives such as 1,4,7-triazacyclo-
nonane ([9]aneN3) containing guanidinoethyl and hydroxyethyl
side arms,21 1,4,7,10-tetraazacyclododecane ([12]aneN4) com-
pounds containing urea-bridge22 and [12]aneN4 with peptide nu-
cleic acid (PNA) units23,24 have shown efficient activity on the
cleavage of plasmid and genetic DNA. Compared to metal com-
plexes, metal-free cleavage reagents can be considered safer and
have potential clinical applications because of avoiding metal labil-
ity and toxicity, including uncontrolled complicated reaction trig-
ged by redox active metal ions, such as Cu(II) ion. However, the
cleavage of DNA with most metal-free artificial nucleases devel-
oped is either at higher concentration (10 mM)27 or non-physiolog-
ical condition,28 especially in the hydrolytic cleavage process.
Design and synthesis of new efficient artificial metal-free nucleases
are highly desired.
In this work, we combined macrocyclic polyamine [12]aneN3
with different dipeptides and investigated their feasibility in the
hydrolytic cleavage of DNA. A series of [12]aneN3–dipeptide conju-
gates were synthesized (Scheme 1) and fully characterized, their
binding ability with DNA and their catalytic activity in the cleavage
of plasmid DNA were measured. It demonstrated that these
[12]aneN3–dipeptide conjugates can hydrolytically cleave DNA at
200 lM concentration (pH 7.2, 37 °C).
Macrocyclic polyamine [12]aneN3 and dipeptides are connected
together through the –CH2CO– linkage in the target conjugates
6a–h. Two different synthetic routes by using 1,5,9-triazatricy-
clo[7.3.1.05.13]tridecane31 ([12]aneN3 precursor) 2 as common
starting material were designed: (i) N-alkylation of 2 with N-
bromoacetyl dipeptides, followed by acidic hydrolysis; (ii) amida-
tion of Boc-protected [12]aneN3 derived acid with NH2-dipeptide,
followed by deprotection of Boc. For the first route, it was found
that the purification of dipeptide halides and the target compounds
were very difficult. The second route proved to be mild in reaction
⇑
Corresponding author. Tel./fax: +86 10 58801804.
E-mail address: luzl@bnu.edu.cn (Z.-L. Lu).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.075

2304
Z.-F. Li et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2303–2307
O
R2
H
N
Boc
OH
Boc
O
OH
N
N
N
N
N
H
ii)
iii)
NH
N
R1
O
O
O
H
O
N
N
N
i)
Boc
Boc
5a-h
3
4
iv)
N
N
R2
N
O
R2
O
v)
H
N
H
O
N
O
O
2
NH
N
N
H
N
N
N
H
O
R1
O
H
O
R1
O
+
N
N
nHCl
6a-h
Br-
R2
R2
O
R2
O
H
H
vii)
vi)
H2N
O
N
O
N
O
Br
N
N
N
Boc
H
H
H
R1
O
R1
O
R1
-Boc-dipeptides
O
O
N
1a h
-
dipeptide
BrCH2CO-dipeptides
a R1 =H,
R2 =H
e R1 = CH 2CH(CH3)2
R2 = CH2C6H5
b R1 = CH2CH(CH3)2 R2 = H
c R1 = CH2CH(CH3)2 R2 = CH3
d R1 = CH2CH(CH3)2 R2 = CH2OH
f
R2 = CH2C6H4OH
1 = CH 2CH(CH3)2
R
g R1 = CH 2CH(CH3)2
h R1 = CH 2CH(CH3)2
R2 = CH2C8H5NH
R2 = (CH2)4NHFmoc (1h/5h)
R2 = (CH2)4NH2 (6h)
Scheme 1. Synthesis of the conjugates 6a–h. (i) ethyl chloroacetate, CH3CN, reflux; then 1.5 M HCl, relux; (ii) (Boc)2O, NaOH, THF, H2O; (iii) 1a–h, HATU, DIEA, CH2Cl2; (iv)
MeOH/HCl; 50% piperidine, DMF, then MeOH/HCl for 6h; (v) BrCH2CO-dipeptides, CH3CN, reflux; (vi) MeOH/HCl; (vii) bromoacetyl bromide, Na2CO3, NaHCO3, CH2Cl2, H2O,
ꢁ5 to 0 °C.
C(EB) = 10 lM (the concentration of EB in buffer solution), C50 is the
condition and easy handling during the purification process, thus it
was chosen to be the procedure of our synthesis. Thus 1,5,9-triaz-
atricyclo[7.3.1.05.13]tridecane 2 was first reacted with ethyl 2-chlo-
roacetate in acetonitrile under reflux overnight, the resulted
mixture was then concentrated and hydrolyzed with 1.5 M HCl
to afford a new artificial amino acid 3. Further protection of 3 with
two equivalent di-tert-butyldicarbonate resulted in 4, which is an
important building block in the construction of [12]aneN3 deriva-
tives. The total yield is 56% for the two steps.
All eight dipeptides were obtained by solution-phase synthesis,
the yields were approximately 50–80% (see Supplementary data).
Leucine is mainly used as the nitrogen end of the dipeptides, the
carbon end of the dipeptides varied from glycine, alanine, serine,
phenylalanine, tyrosine, tryptophan to lysine.
Acid 4 was coupled with different (NH2-)dipeptides in the pres-
ence of HATU to afford compound 5a–h with yields from 40–60%.
The target compounds 6a–h were quantitatively obtained by the
deprotection of Boc moieties in 5a–g with HCl in methanol at
0 °C. For compound 6h, it was prepared by the removal of Fmoc
in compound 5h with 50% piperidine in DMF (v/v) and then the
deprotection of Boc in methanolic hydrogen chloride solution. All
new compounds were characterized by 1H NMR, 13C NMR, IR,
ESI-MS or HR-MS. The detailed data were listed in the Supplemen-
tary data.
To evaluate the DNA binding ability of the conjugates 6a–h,
ethidium bromide (EB) displacement experiments were carried
out (Fig. S1). It can be seen that the fluorescence intensity of the
EB-DNA system gradually decreased upon the addition of various
amount of 6a–h in the buffer (5 mM Tris–HCl/50 mM NaCl, pH
7.2) at room temperature. The apparent binding constants of the
final concentration at which a 50% reduction of the fluorescence had
occurred. The Kapp data of 6a–h are listed in the Table 1, which are
of the same order. The apparent binding constants imply that the
conjugates 6a–h are mainly attributed to electrostatic interactions
of [12]aneN3 moiety and amino acid residues, since these data are
lower than those obtained for typical intercalators (Kapp ꢂ 106 M
ꢁ1) and groove binders (Kapp ꢂ 105 Mꢁ1).
Firstly, we screened the cleavage abilities of the conjugates 6a–
h by using agarose gel electrophoresis, the results are shown in
Figure 1. All pUC 18 DNA were supplied by Solarbio company,
which contains 2686 base pairs with the MCS region reversed.
The cleavage reactions were studied in 20 mM HEPES buffer at
pH 7.2, 37 °C. It is obvious that the eight conjugates cleaved the
plasmid DNA and showed almost same activities, approximately
50–60% of DNA were cleaved. It was found that the positions of
the new bands did not vary at different concentrations of the con-
jugates and different reaction time (see the following results),
which indicates that the new bands are not relaxed DNA. The
supercoiled DNA (Form I) can be cleaved to the open circular
DNA (Form II) and linear DNA (Form III). To clarify the new bands
that appeared in Figure 1 and other electrophoresis diagrams in
this work, we have measured the cleavage of plasmid DNA in the
presence of Hind III, which is an endonuclease for the production
of Form III. The result (see Fig. S2) clearly indicated that the new
band produced by the conjugate 6c is different from Form III. With
the above considerations, the new bands can be assigned to be
Table 1
conjugates 6a–h, Kapp
,
were calculated from the following
The apparent binding constants of compounds 6a–ha
equation.30,32
Compound
6a
6b
6c
6d
6e
6f
6g
6h
104 Kapp
4.2
4.0
5.9
7.2
6.6
6.4
4.7
7.1
Kapp ¼ KðEBÞ ꢀ C =C50
ðEBÞ
where K(EB) = 1.0 ꢀ 107 Mꢁ1 (the binding constant of EB to calf-thy-
a
The condition for the measurement: kex = 537 nm, [EB] = 10
[6a–h] = 0–2.6 mM, 25 °C.
lM, [DNA] = 100 lM,
mus DNA (CT-DNA), which was purchased from Solarbio Company,

Z.-F. Li et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2303–2307
2305
80
60
Form II%
1
2
3
4
5
6
7
8
9
40
20
0
Form II
Form I
1
2
3
4
5
6
7
8
9
Lane
a
b
Figure 1. Effect of different [12]aneN3–dipeptide 6a–h (200 lM) on the cleavage reactions of pUC 18 DNA (9 lg/mL) in a HEPES buffer (20 mM, pH 7.2, 20 mM NaCl) at 37 °C
for 48 h. (a) Agarose gel electrophoresis diagram: lane 1, DNA control; lanes 2–9 6c, 6b, 6d, 6e, 6h, 6a, 6f, 6g. (b) Quantitation of % plasmid relaxation relative to pUC 18 DNA
per lane corresponding to (a).
Form II, which are also consistent with the results of the DNA
cleavage in the presence of macrocyclic polyamines.21,22 Note,
the DNA cleavage by the conjugates 6a–h is highly selective, only
Form II was produced, no further cleavage was found.
Then the concentration and time dependences of the DNA
cleavage were investigated by using 6c and 6h as catalysts, the re-
sults were shown in Figures 2 and 3, respectively.
the combination of [12]aneN3 and dipeptides clearly results in new
reactivity. To avoid any effect of trace metal ions involved in DNA
cleavage, the control experiment using chelating agent EDTA
(1 mM) was also checked (Fig. 5), it was found that the conjugates
could cleave DNA without the participation of any metal ions. To
be noticed, EDTA has a stronger coordination ability with transition
metal ions when compared to macrocyclic polyamine [12]aneN3.
The binding constants of EDTA with Zn(II) and Cu(II) are around
With the increase of concentration of the catalysts from 50 to
500
l
M, the yields of Form II increased. The kinetic plots indicate
1017 Mꢁ1 36
, while those of [12]aneN3 with Zn(II) and Cu(II)
that the extent of supercoiled DNA cleavage varies exponentially
with the reaction time giving pseudo first-order kinetics with an
apparent initial first-order rate constant 0.016 0.001 and
0.014 0.001 hꢁ1 for 6c and 6h, respectively. The catalytic rates
are about 106 times higher than that of DNA cleavage in the ab-
sence of catalyst (10ꢁ8 hꢁ1).33–35
To confirm the catalytic activity of 6a–h, several controlled
experiments were carried out. DNA cleavage in the presence of
respective dipeptides and [12]aneN3 were measured (Fig. 4). It
was found that no obvious DNA cleavage was observed at the con-
appeared to be about 108 and 1012 Mꢁ1, respectively.37–39
There are two possible pathways for the cleavage of DNA with
artificial nucleases: hydrolytic and oxidative mechanisms. To ex-
clude an oxidative mechanism, reactive oxygen species (ROS) scav-
engers were added to the incubated samples (6c/6h + DNA). As
shown in Figure 6, the addition of hydroxyl radical scavengers
(DMSO, t-BuOH), singlet oxygen scavenger (NaN3), and superoxide
scavenger (KI) had no effect on the cleavage of DNA with com-
pounds 6c and 6h. In all cases, the plasmid DNA was cleaved as ob-
served for the controlled samples (lines 2 and 7) without evident
inhibition. The results clearly proved that a hydrolytic mechanism
centration of 200
l
M of [12]aneN3 and of various dipeptides. Thus
% Form II
80
(A)
1
2
3
4
5
6
7
60
40
20
0
Form II
Form I
1
2
3
4
5
6
7
lane
a
b
80
60
40
20
0
Form II%
1
2
3
4
5
6
7
(B)
Form II
Form I
1
2
3
4
5
6
7
lane
b
a
Figure 2. Concentration effect of compounds 6c (A) and 6h (B) on the cleavage reactions of pUC 18 DNA (9
Agarose gel electrophoresis diagram: lane 1, DNA control; lanes 2–7, [6c or 6h] = 50, 100, 200, 300, 400 and 500
l
g/mL) in a HEPES buffer (20 mM, pH 7.2) at 37 °C for 48 h. (a)
M, respectively. (b) Quantitation of % plasmid relaxation
l
relative to plasmid DNA per lane corresponding to (a).

2306
Z.-F. Li et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2303–2307
4.4
1
2
3
4
5
6
7
8
(A)
4.0
3.6
3.2
Form II
Form I
0
20
40
60
80
Time, h
a
b
4.4
1
2
3
4
5
6
7
8
(B)
4.0
3.6
3.2
Form II
Form I
0
20
40
60
80
Time, h
a
b
Figure 3. Time course of pUC 18 DNA (9
lg/mL) cleavage promoted by 6c (A) and 6h (B) (200 lM). (a) Agarose gel of the time-variable reaction products: lane 1, DNA control,
72 h; lanes 2–8, reaction times of 6, 12, 24, 40, 48, 64 and 72 h, respectively. (b) Plot of ln(% pUC 18 DNA) versus reaction time.
through electrostatic interaction. Agarose gel electrophoresis at
various conditions clearly indicate that these conjugates can cleave
1
2
3
4
5
6
7
8
9
10
the plasmid DNA at 200 l
M with an acceleration of 106-fold (pH
7.2, 37 °C) via hydrolytic pathway. The work showed that the com-
bination of macrocyclic polyamine and peptides can afford the
effective artificial metal-free nucleases. Further studies to improve
the catalytic efficiency and explore the relationship between the
structure and activity are undergoing in our lab.
FormII
FmorI
Figure 4. Agarose gel electrophoresis assays for the effect of dipeptide and
[12]aneN3 on the cleavage reaction of pUC 18 DNA (9 g/mL) in a HEPES buffer
(20 mM, pH 7.2) at 37 °C for 48 h. Lane 1, DNA control; lanes 2–9, 200 M 1a–h;
l
l
Acknowledgements
lane 10, 200
l
M [12]aneN3.
The authors gratefully acknowledge the financial assistances
from Program for New Century Excellent Talents at Universities
(NCET-08-0054), the Ministry of Education of China; and the
Nature Science Foundation of China (20972019); the Fundamental
Research Funds for the Central Universities (2009SC-1); Beijing
Municipal Commission of Education.
1
2
3
4
5
6
FormII
FmorI
Supplementary data
Figure 5. Agarose gel of pUC 18 DNA (9
lg/mL) incubated in HEPES buffer (20 mM,
pH 7.2) at 37 °C for 48 h. Lane 1, DNA control; lane 2, 1 mM of EDTA; lane 3, 200
6c; lane 4, 200 lM 6c + 1 mM EDTA; lane 5, 200 lM 6h; lane 6, 200 lM 6h + 1 mM
EDTA.
l
M
Supplementary data (details of synthesis and characterization
of compounds 6a–h, and DNA binding and gel electrophoresis
experiments) associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmcl.2012.01.075.
1
2
3
4
5
6
7
8
9
10
11
References and notes
Form II
Form I
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