Conjugates between minor groove binders and Zn(II)-tach complexes: Synthesis, characterization, and interaction with plasmid DNA

Article abstract of DOI:10.1016/j.tet.2017.04.013

A new family of conjugates between a Zn(II)-tach complex and (indole)₂ or benzofuran-indole amide minor groove binders connected through alkyl or oxyethyl linkers of different lengths has been prepared. The conjugates bind strongly to DNA. However, the complexation to DNA to promote the Zn(II) catalyzed hydrolytic cleavage of the DNA results instead in its inhibition. This inhibition effect has been confirmed also using Cu(II). Modeling studies suggest that in the most stable complex conformation, the minor groove binder and the linker lie in the minor groove hampering the interaction between the metal complex and the phosphate backbone of DNA. Therefore, the linear arrangement of minor groove binder-linker-metal complex appears to be effective to ensure tight binding but unproductive from a hydrolytic point of view.

Full text of DOI:10.1016/j.tet.2017.04.013

Tetrahedron xxx (2017) 1e11
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Conjugates between minor groove binders and Zn(II)-tach complexes:
Synthesis, characterization, and interaction with plasmid DNA
,
Claudia Sissi a **, Luca Dovigo a, Maria Laura Greco a, Antonella Ciancetta b,
,
***
c
c
d
Stefano Moro b , Jakub W. Trzcinski , Fabrizio Mancin , Paola Rossi ,
ꢀ
Giampiero Spalluto d ****, Paolo Tecilla d
,
, *
a Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo 5, Padova, Italy
b Molecular Modeling Section (MMS), Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo 5, Padova, Italy
c Department of Chemical Sciences, University of Padova, Via Marzolo 1, Padova, Italy
d Department of Chemical and Pharmaceutical Sciences, University of Trieste, Via Giorgieri 1, Trieste, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A new family of conjugates between a Zn(II)-tach complex and (indole)2 or benzofuran-indole amide
minor groove binders connected through alkyl or oxyethyl linkers of different lengths has been prepared.
The conjugates bind strongly to DNA. However, the complexation to DNA to promote the Zn(II) catalyzed
hydrolytic cleavage of the DNA results instead in its inhibition. This inhibition effect has been confirmed
also using Cu(II). Modeling studies suggest that in the most stable complex conformation, the minor
groove binder and the linker lie in the minor groove hampering the interaction between the metal
complex and the phosphate backbone of DNA. Therefore, the linear arrangement of minor groove binder-
linker-metal complex appears to be effective to ensure tight binding but unproductive from a hydrolytic
point of view.
Received 27 December 2016
Received in revised form
28 March 2017
Accepted 10 April 2017
Available online xxx
Keywords:
Synthetic nuclease
DNA
Minor groove binder
Tach
© 2017 Elsevier Ltd. All rights reserved.
Zn(II) complexes
1. Introduction
Zn(II)5 has been less investigated. In recent years, several ap-
proaches have been exploited in order to develop more potent and
The first report of a metal ion complex able to hydrolytically
cleave DNA was published more than 25 years ago.1 Nevertheless,
the interest in synthetic DNA hydrolytic agents based on metal
complexes is still increasing due to the promising applications of
such systems in DNA manipulation, a key tool in the field of
biotechnology.2 On the other hand, some issues about activity and
sequence selectivity are still partly unresolved. Indeed, with the
notable exception of Ce(IV) based catalysts,3 many of the reported
cleavage agents show moderate activity at low concentration and/
or scarce sequence selectivity. Moreover, many complexes are
based on metal ions such as Cu(II),4 in which oxidative cleavage
often competes with the desired hydrolysis of DNA. The use of
intrinsically less reactive but hydrolytically more stable ions such as
selective artificial nucleases. As an example, di-metallic or poly-
metallic complexes have been used in order to increase the cata-
lytic activity.6,7 Alternatively, conjugation of the metal complexes
with DNA binders has been considered to promote interaction of
the cleaving agents with the substrate. This last approach is
particularly interesting because
a tailored DNA binder may
contemporaneously ensure high affinity and sequence selectivity.
Following this rationale, several systems have been reported in
which the hydrolytically active metal ion complex is conjugated to
base pairs intercalators,8e12 major groove binders,13 nucleo-
bases,14,15 DNA or PNA fragments,16,17 simple positively charged
group,18,19 etc. In general, but not always, the increased affinity of
the conjugate for the substrate ensures higher activity with respect
to the isolated metal complex. These studies pointed out the
importance of a correct design because, for example, factors such as
the incorrect length of the linker between the metal complex and
the DNA binder may lead to unaltered or even decreased
reactivity.8,9
* Corresponding author.
** Corresponding author.
*** Corresponding author.
**** Corresponding author.
E-mail address: ptecilla@units.it (P. Tecilla).
Among the different types of DNA ligands, minor groove binders
http://dx.doi.org/10.1016/j.tet.2017.04.013
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Please cite this article in press as: Sissi C, et al., Conjugates between minor groove binders and Zn(II)-tach complexes: Synthesis,
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2
C. Sissi et al. / Tetrahedron xxx (2017) 1e11
appear quite interesting because they bind tightly to DNA with
some sequence selectivity. Pioneering work of Dervan on N-
methylpyrrole/N-methylimidazole polyamides (Py/Im) has shown
that these ligands bind strongly to the minor groove of DNA
selectively recognizing C-G rich sequences.20 This sequence selec-
tivity can be modulated by changing the structure of the ligand and
Im/Py and Py/Py polyamides target G-C and A-T (or T-A) rich se-
quences, respectively.21 Moreover, work from the group of Boger
has demonstrated that minor groove binders based on oligoindole
polyamides ensure high DNA affinity and T-A sequence selec-
tivity.22 Conjugation of oxidative metal complexes23 or of alkylating
reactive centers24 to such minor groove ligands led to synthetic
agents able to damage DNA with high potency and sequence
selectivity. Somewhat surprisingly, this approach has been almost
overlooked in the design of hydrolytic synthetic nucleases and the
examples reported in the literature are very rare. The group of Qiao
and Zhao, has reported a mononuclear Zn(II) complex appended to
Py-based oligopolyamides of different length which, although at
high concentration, showed enhanced reactivity respect to the
Zn(II) complex alone and some selectivity toward A-T rich se-
quences.25 More recently the same group has described a conjugate
between the same minor groove ligand and a bi-metallic Zn(II)
complex which shows a marked preference for double strand with
respect to random single strand cleavage. This is a consequence of
the tight binding to DNA which fixes the hydrolytic catalyst in a
certain site increasing the probability of double strand cleavage.26
On this premise and following our interest in the design of
synthetic nucleases,27 we decided to explore further the potential
of minor groove ligands as elements to improve activity and
selectivity of the hydrolytic catalyst. As a DNA ligand we chose the
(indole)2 or benzofuran-indole amides which were appended to a
Zn(II)-triaminocyclohexane complex using flexible linkers of
different length and nature. Here we report the synthesis, charac-
terization and DNA interaction studies of this new class of com-
pound. Somehow disappointingly our compounds bind tightly to
DNA but they are inactive in its cleavage. An interpretation of this
behavior based on molecular modeling studies will be presented.
Fig. 1. Structures of the minor groove binder tach conjugates prepared.
The general synthetic route for the synthesis of the conjugates
1e7 is shown in Scheme 1. Appropriate amine counterparts 9e14,
2. Result and discussion
2.1. Design and synthesis of the minor groove binder tach
conjugates
The structures of the minor groove binder/tach conjugates
investigated in this work are reported in Fig. 1. The DNA ligand is in
each case an (indole)2 amide except in compound 5 which features
a benzofuran-indole amide. These ligands have been chosen
because they combine relatively simple synthetic accessibility with
a tight binding in the DNA minor groove with sequence selectivity
for A-T rich regions. Moreover, studies on duocarmycin models in
which the DNA ligand portion have been systematically varied have
shown that an indole dimeric structure is long enough to maintain
the binding and DNA damaging ability of the native drug thus
avoiding the need for the synthesis of longer polyamides.22,24 The
cis,cis-triaminocyclohexane (tach) metal ion chelating subunit was
chosen on the basis of our previous studies which have shown that
its Zn(II) complex is a rather efficient catalyst for the cleavage of
model phosphate esters and plasmid DNA.8 The DNA binder and
the catalytic subunits were connected through a flexible linker of
different length and polarity with the aim to get insight into the
role played by the linker length and nature in determining the
reactivity of the systems. Finally compound 8 was designed with a
hairpin structure that should ensure a correct and more fixed
positioning of the tach-Zn(II) complex in the proximity of the
phosphate backbone of DNA.
Scheme 1. Reagents: i: CH3CN, DIPEA, reflux; ii: CH3OH, H2, PdeC 10%, iii: DMF, EDC,
Et3N, rt 72 h; iv: CH2Cl2-TFA 1:1, rt 4 h.
Please cite this article in press as: Sissi C, et al., Conjugates between minor groove binders and Zn(II)-tach complexes: Synthesis,
characterization, and interaction with plasmid DNA, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.04.013

C. Sissi et al. / Tetrahedron xxx (2017) 1e11
3
were obtained by alkylation with bromoalkyl(oxy)amines 15e20,
protected at the amino functions as benzyloxycarbonyl derivatives,
of tach di-Boc (21), in the presence of DIPEA at reflux to give
compounds 22e27, as previously reported.8 Then, treatment of
compounds 22e27, with hydrogen in the presence of PdeC affor-
ded the desired amine derivatives 9e14. Compounds 1e7 were
easily obtained as trifluoroacetate salts by coupling amino de-
rivatives 9e14 with the bis-indole (31) or benzofuran-indole (32)
carboxylic acids28 in the presence of EDC at room temperature to
afford amido compounds 33e39, followed by deprotection at the
amino functions by treatment with TFA in CH2Cl2 at room
temperature.
Scheme 3. Formation of the metal complexes of compounds 1e8.
peak at m/z ¼ 652.3 observed in the case of Zn(II) is attributed to a
dimeric structure with two methoxides bridging the two metal
ions.
The synthesis of the hairpin derivative 8 follows similar pro-
cedures and is shown in Scheme 2. The mono-BOC-protected de-
rivative of the triaminocyclohexane (28) was reacted with two
equivalents of 19 affording the corresponding bis-alkylated deriv-
ative 29, that after hydrogenolysis gave the desired amino com-
pound 30. This latter was coupled with 31 in the presence of EDC
affording derivative 40 which after N-Boc deprotection with TFA
gave final compound 8 as the trifluoroacetate salt.
The Zn(II) and Cu(II) complexes of the tach conjugates were
finally prepared following a simple procedure previously optimized
by us.8 In this method the complexes are directly prepared by
addition of one equivalent of Zn(NO3)2 or Cu(NO3)2 water solution
to millimolar solutions of the ligands in water/MeOH mixtures and
by adjusting the final pH to 7.0 with NaOH (Scheme 3). The solution
obtained is then directly used as the mother solution in the studies
with DNA.
2.2. Interaction of the tach conjugates with DNA
The ability of the Zn(II) complexes of the tach-DNA minor
groove binder conjugates to bind DNA was preliminarily and
qualitatively evaluated by electrophoretic measurements.
Increasing amounts of the tested Zn(II) complexes were added to a
solution of pBR332 plasmid DNA in HEPES buffer at pH 7.1. After
mixing, the solutions were immediately loaded on an agarose gel
without any incubation time. Fig. 3 shows the results obtained with
The formation of the metal complexes was confirmed by ESI-MS
and Fig. 2 reports as a representative example the results obtained
with compound 4. The mass spectra show the formation of the (4-
Hþ)$Zn(II) and (4-Hþ)$Cu(II) complex at m/z ¼ 620.2 and 619.2,
respectively. The small peak at m/z ¼ 558.3, visible in the spectra, is
the residual free ligand. The insets of Fig. 2 show the calculated
isotopic cluster distributions which match very well the experi-
mentally observed distributions for the two metal complexes. The
Fig. 2. ESI-MS spectra of the complexes between compound 4 and Zn(NO3)2 (top) and
Cu(NO3)2 (bottom). The inset shows the calculated isotopic cluster distributions for the
complexes with formula (4-Hþ)$Zn(II) (top) and (4-Hþ)$Cu(II) (bottom).
Scheme 2. Reagents: i: CH3CN, DPEA, reflux; ii: CH3OH, H2, CePd 10%; iii: DMF, EDC,
Et3N, rt 72 h; iv: CH2Cl2-TFA 1:1, rt 4 h.
Please cite this article in press as: Sissi C, et al., Conjugates between minor groove binders and Zn(II)-tach complexes: Synthesis,
characterization, and interaction with plasmid DNA, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.04.013

4
C. Sissi et al. / Tetrahedron xxx (2017) 1e11
Fig. 4. Agarose gel electrophoresis of pBR322 DNA (12
mM) in the presence of the
indicated concentration of the Zn(II) complexes of ligands 1e5 and tach in 20 mM
HEPES containing EDTA and SDS, pH 7.1, 37 ꢀC. Lane* are the ligands in the absence of
metal ion. Lane C is the DNA control in the absence of additives. The gel has been
loaded immediately after preparation of the different solution without incubation
time.
Fig. 3. Agarose gel electrophoresis of pBR322 DNA (12
mM) in the presence of the
indicated concentration of the Zn(II) complexes of ligands 1e5 and tach in 20 mM
HEPES, pH 7.1, 37 ꢀC. Lane* are the ligands in the absence of metal ion. Lane C is the
DNA control in the absence of additives. The gel has been loaded immediately after
preparation of the different solution without incubation time.
particular, we monitored the absorption properties of our conju-
gates in the presence of increasing concentrations of calf thymus
DNA (ctDNA). An example is reported in Fig. 5.
the control compound tach and the ligands 1e5 in the presence/
absence of 1 eq. of Zn(II).
By increasing the concentration of the Zn(II) complexes, the
signal corresponding to DNA gradually disappears from the gel.
This behavior is likely a consequence of the binding of the conju-
gates to the DNA which induces aggregation/precipitation of the
plasmid, thus preventing its entrance in the gel. Interestingly, the
same effect is observed also in the absence of metal ion at least at
the highest tested conjugate concentration Additionally, Zn(II)-tach
complex, which binds to DNA only through electrostatic in-
teractions, requires a higher concentration to exert the same effect.
Taken together, this experimental evidence suggests the involve-
ment of the minor groove binder in the process. Among the tested
Zn(II) complexes the ranking order 2 ~ 3 > 4 ~ 5 > 1 describes the
efficiency of the compounds in inducing the DNA fading effect,
which should, at least qualitatively, correlate to their ability to bind
to DNA leading to its precipitation. This corresponds to a higher
efficiency for conjugates with a 4 and 6 methylene linker and, at
parity of linker length, for the (indole)2 vs benzofurane-indole
moiety.
Addition of the nucleic acid to the tested binders reduced the
intensity of the absorption band of the ligands either in the pres-
ence or in the absence of coordinated metal ions (Zn(II) or Cu(II)).
The affinity of the binder for the nucleic acid was remarkable
although the binding event is complex as suggested by the different
shape of the binding isotherms (Fig. 5B). This may be the result of
several factors such as, for example, different binding stoichiometry
or different aggregation state of the ligands which were not further
investigated. Therefore, for a meaningful comparison we report in
Table 1 the concentration required to induce 50% of the maximal
observed absorbance variation at a constant wavelength, which we
can be referred as an effective concentration at 50% of the binding
event (EC50).
Data in Table 1 show that metal ion coordination actually im-
proves the recognition of the double helix lowering the EC50 by a
factor of about 2. On the other hand, compounds 4 and 5 exhibited
comparable EC50. This suggests that the substitution of the
(indole)2 amide with a benzofuran-indole amide doesn't impair the
formation of the complex. Conversely, the presence of the oxy-
ethylene linker reduces the affinity of the ligand (compare com-
pounds 4 and 7). Interestingly, moving from one (indole)2 binding
moiety (compound 7) to two (compound 8) a two fold reduction of
the EC50 was determined, thus ruling out cooperative binding of the
two minor groove moieties to DNA.
All these data correlate with the different behavior of our con-
jugates on DNA migration, thus supporting the observed fading of
plasmid DNA in the gel being related to the interaction of our li-
gands with the nucleic acid.
To assess the DNA binding mode of the conjugates, we acquired
the CD spectra of ctDNA in the presence of 4, Zn(II)-4 or Cu(II)-4
(Fig. 6). In agreement with the above described efficient recognition
of the macromolecule, addition of 4 leads to a significant change in
the dichroic features of the nucleic acid. In particular, a relevant
positive induced CD contribution appears in the ligand absorption
region. This is indicative of its binding in the minor groove. It is
The loss of bands in the gel was induced also by conjugates 6e8
(data not shown). However, in this case higher concentrations
(20e50 mM) were required to fully prevent DNA visualization. This
supports a contribution of the hydrophobic linker to the process
which becomes less relevant when the more polar oxyethylene
linker is included. Noteworthy, the presence of two (indole)2 sub-
units in compound 8 reduced the concentration at which DNA is
not any more detectable.
Fortunately, the observed fading effect of DNA was due to a
reversible binding of our conjugates on the nucleic acid. Indeed,
results shown in Fig. 4 clearly support the observation that the
addition to the samples of an excess of EDTA and SDS before loading
hampers the complexation of the conjugates, and restores the
normal mobility of the plasmid DNA.
To confirm a correlation between the observed effect on the
electrophoretic mobility of the DNA with the binding of our ligands
to the macromolecule, we performed UV-VIS titrations. In
Please cite this article in press as: Sissi C, et al., Conjugates between minor groove binders and Zn(II)-tach complexes: Synthesis,
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C. Sissi et al. / Tetrahedron xxx (2017) 1e11
5
Fig. 6. CD spectra of ctDNA alone and in the presence of 4, Zn(II)-4 or Cu(II)-4 recorded
in 20 mM HEPES, pH 7.1.
should result in a decrease of the intensity of the band corre-
sponding to the supercoiled DNA and in the appearance of the spots
of nicked and eventually linear DNA. Disappointingly, the Zn(II)
complexes of the tested conjugates did not show any ability to
cleave DNA up to a concentration of 200 mM. This is shown in Fig. 7
(top) for complexes 6e8 but comparable results were obtained
with complexes 1e5. The activity of the Zn(II) complexes of the
conjugates is even lower than that of the Zn(II)-tach complex which
is known to cleave plasmid DNA at concentrations above 100 mM
under the same experimental conditions.8 Therefore, the conjuga-
tion of the Zn(II)-tach to the minor groove binders led not to an
increase, but to an inhibition of the hydrolytic activity of the metal
complex. To confirm this effect, the DNA cleavage produced by the
complex of ligands 6e8 with the more active Cu(II) metal ion was
investigated. The results shown in Fig. 7 (bottom) showed cleavage
activity only at high concentration while, in the same experimental
conditions, the Cu(II)-tach complex is reported to fully nick plasmid
at concentration below 20 m
M.29 Therefore, also in this case, the
Fig. 5. Variation of the absorption spectrum of 4 (37
mM) induced by addition of
conjugation of the metal complex to the minor groove binder is
detrimental for its DNA cleaving activity.
ctDNA in 20 mM HEPES, pH 7.1 (PANEL A). Arrow indicates the effect of increasing
ctDNA concentrations. In PANEL B the relative variation of the absorbance of 4 alone,
Zn(II)-4 or Cu(II)-4 recorded at 305 nm is reported as a function of nucleic acid
concentration.
worth noting that complexation of 4 with Zn(II) or Cu(II) did not
significantly rearrange the ligand within the groove. Indeed, the
positive signal is preserved in each case.
Once we demonstrated that the Zn(II) complexes of the conju-
gates bind DNA, their ability to cleave DNA was investigated. We
checked this activity by following the formation of a cleaved site on
pBR322 upon a 24 h incubation at 37 ꢀC in HEPES buffer pH 7.1 and
by resolving the reaction products by agarose gel electrophoresis in
the presence of SDS to dissociate the metal complexes from the
nucleic acid. In these experimental conditions, DNA cleavage
Table 1
EC50 (mM) values determined for the tested de-
rivatives by UV-VIS titrations with ctDNA in 20 mM
HEPES, pH 7.1.
EC50 (mM)
Fig. 7. Top: Agarose gel electrophoresis of pBR 322 DNA (12
with the indicated concentration of the Zn(II) complexes of ligands 6e8 in 20 mM
HEPES, pH 7.1, 37 ꢀC. Bottom: Agarose gel electrophoresis of pBR 322 DNA (12
M) after
3 h incubation with the indicated concentration of the Cu(II) complexes of ligands 6e8
in 20 mM HEPES, pH 7.0, 37 ꢀC. Lane C is DNA incubated in the absence of complexes.
Before loading, samples were added of EDTA and SDS. S and N refer to the supercoiled
and nicked plasmid DNA, respectively.
mM) after 24 h incubation
4
2.60 0.27
1.45 0.10
1.49 0.13
2.70 0.09
3.13 0.05
1.44 0.07
Zn(II)-4
Cu(II)-4
5
7
8
m
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C. Sissi et al. / Tetrahedron xxx (2017) 1e11
2.3. Molecular modeling of DNA conjugates complexes
The data illustrated above indicate that the Zn(II) complexes of
the tach conjugates are able to bind but not cleave plasmid DNA. To
get insight into such behavior, molecular mechanics calculations
were performed on the complexes between B-DNA and Zn(II)-3 and
Zn(II)-8. In the case of Zn(II)-3 (Fig. 8) the optimized structure of the
complex shows the (indole)2 subunit deeply inserted in the DNA
minor groove. The C6-linker which is attached to one extremity of
the (indole)2 follows the minor groove curvature, and it is also
deeply inserted in the groove establishing stabilizing hydrophobic
interactions between the alkyl chain and the DNA. In this confor-
mation the appended Zn(II)-tach complex is not able to get close to
the phosphate backbone, and this unfavorable geometry hampers
the interaction between the metal ion and the phosphate group
leading to inhibition of the cleavage process. Therefore the design
of the conjugates in which the binder, the linker and the metal
complex are arranged in a linear fashion seems to lead to unpro-
ductive binding modes, because the conjugate is buried in the
minor groove and the metal complex is not able to reach the
phosphate backbone.
The less predictable result obtained in the cleavage of DNA was
the inactivity of the hairpin derivative Zn(II)-8. This dimeric com-
pound was indeed prepared with the hope, based on modeling
studies, that the tweezer like binding mode of the two (indole)2
subunits should force the Zn(II)-tach complex close to the phos-
phate groups of DNA. This is illustrated in the modeled structure of
Fig. 9 (left), in which the favorable orientation of the metal complex
is evident. However, the same calculation suggested the possibility
of a less favorable binding mode in which the metal complex is
turned away from the DNA pointing toward the exterior of the
minor groove well distant from the phosphate backbone as shown
in Fig. 9 (right). Clearly, the experimental results indicate that this
second unproductive binding mode prevails.
Fig. 9. Modeled structures of two different complexes between Zn(II)-8 and DNA. DNA
structure is represented using its Connolly's surface.
inhibition with the Zn(II)-tach conjugates being less effective than
the isolated Zn(II)-tach complex, regardless of the length and na-
ture of the linker. This inhibition effect has been confirmed also
using Cu(II) as metal ion. Modeling studies suggest that in the most
stable complex conformation the minor groove binder and the
linker lie in the minor groove hampering the interaction between
the metal complex and the phosphate backbone of DNA. Interest-
ingly, the hydrophobic interaction of the linker with the minor
groove is stronger than the electrostatic/coordination interaction of
Zn(II) with the phosphate groups. Therefore, the linear arrange-
ment of minor groove binder-linker-metal complex appears to be
unproductive from a hydrolytic point of view. This may also explain
the relatively low reactivity of the system reported by Qiao and
Zhao which, although with different minor groove binders and
metal complexes, presents the same linear arrangement of the
catalyst components25,26 and also suggests a strategy for increasing
the DNA affinity of minor groove binders. On this basis, we believe
that active hydrolytic catalysts may be obtained by changing the
topology of the conjugates and in particular by attaching the linker
to a position on the minor groove binder subunit such as to force
the linker itself to exit the minor groove allowing the positioning of
the metal complex close to the phosphate group. Work is progress
to prepare new conjugates in order to verify this hypothesis.
3. Conclusions
In this paper we have presented the synthesis of a new class of
4. Experimental section
conjugates between
a Zn(II)-tach complex and (indole)2 or
benzofuran-indole amide minor groove binders connected through
alkyl or ethoxyethyl linkers. The conjugates strongly bind DNA and
the binding strength is influenced by the nature and length of the
linkers. This suggests that not only the tach unit, which both in the
free and in the Zn(II) complex form can provide additional elec-
trostatic interaction with the phosphate backbone, but also the
linker can have an effect on the DNA affinity of the conjugates.
Surprisingly, the complexation to DNA instead of promoting the
Zn(II) catalyzed hydrolytic cleavage of the DNA results in its
All commercially available reagents were purchased from
Aldrich, Fluka and Strem Chemicals and used without purification
unless otherwise mentioned. Solvents were purchased from
Aldrich, VWR, Fluka and Riedel, and deuterated solvents from
Cambridge Isotope Laboratories and Aldrich. Reactions were
routinely monitored by thin-layer chromatography (TLC) on silica
gel (precoated F254 Merck plates). Chromatography was performed
on Merck silica gel 60F-254 (230÷400 Mesh) and the solvents
employed were of analytical grade. NMR spectra were recorded on
a Jeol 400 spectrometer (operating at 400 MHz for proton and at
100 MHz for carbon) or on Jeol X-270 (operating at 270 MHz for
proton and at 67.8 MHz for carbon). Chemical shifts (d) are reported
in ppm using the solvent residual signal as an internal reference.
Coupling constants (J) are quoted in Hz. The letter in brackets in the
13C NMR spectra description is the multiplicity of the carbon atom
as determined from DEPT experiments (s ¼ singlet, d ¼ doublet,
t ¼ triplet, q ¼ quartet). Electrospray mass spectra were recorded
on a Perkin Elmer APII spectrometer. HRMS spectra were recorded
on a Perceptive Biosystems ABI Mariner ESI-TOF instrument.
Infrared spectra (IR) were recorded with a Nicolet Nexus 870 in-
strument operating in the ATR mode on a diamond crystal. All-cis-
3,5-N,N-di-tert-butyloxycarbonyl-1-N-(4-aminobutyl)-1,3,5-
triaminocyclohexane8
(10),
All-cis-3,5-N,N-di-tert-butylox-
Fig. 8. Modeled structure of the complexes between Zn(II)-3 and DNA. DNA structure
is represented using its Connolly's surface while the interacting molecule as stick (left)
and Connolly's surface (right).
ycarbonyl-1-N-(6-aminohexyl)-1,3,5-triaminocyclohexane8 (11),
All-cis-3,5-N,N-di-tert-butyloxycarbonyl-1-N-(8-aminooctyl)-1,3,5-
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C. Sissi et al. / Tetrahedron xxx (2017) 1e11
7
triaminocyclohexane (12),8 N-Benzyloxycarbonyl-2-bromo-ethyl-
70.1 (t); 70.3 (t); 71.1 (t); 128.0 (dd); 128.4 (d); 136.5 (s); 156.3 (s).
ESI-MS (m/z): 348.2 [M þ Hþ]; 369.9 [M þ Naþ]; 386.1 [M þ Kþ];
364.9 [M þ NHþ4 ].
amine
(15),30
All-cis-3,5-N,N-di-tert-Butyloxycarbonyl-1,3,5-
triaminocyclohexane (21, tach-DIBOC8), All-cis-5-N-tert-Butylox-
ycarbonyl-1,3,5-triaminocyclohexane (28, tach-MONOBOC8), 5-
[((1H-Indol-2'-yl)carbonyl)amino]-lH-indole-2-carboxylic acid28
4.2. General synthesis of All-cis-3,5-N,N-di-tert-butyloxycarbonyl-
(31),
5-[((Benzofuro-2'-yl)carbonyl)amino]-1H-indole-2-
1-N-[u- N-(benzyloxycarboyl)aminoalkyl/alkyloxy]-1,3,5-
triaminocyclohexane derivatives (22e27)
carboxylic acid28 (32), were synthesized as reported.
4.1. N-Benzyloxycarbonyl-
u-bromo-1-alkyloxyamine 19e20
To a solution of tach-DIBOC (21, 250 mg, 0.76 mmol) in dry
CH3CN (18 mL) were added 1.3 eq. of the appropriate amino bromo
The desired aminoalcohol (17 mmol, 2-(2-aminoethoxy)ethanol
or 2-[2-(2-aminoethoxy)ethoxy]ethanol) was dissolved in CH3OH
(10 mL) and TEA (5.45 mL, 39.2 mmol) was added. To this solution,
stirred at room temperature, was added dropwise a solution of
benzyloxycarbonyl chloride (ZCl, 3.2 g, 18.77 mmol) in CH3OH
(5 mL). After the addition was complete the reaction mixture was
stirredat room temperature for 20 h, protected from moisturewith a
CaCl2 drying tube. The solvent was then evaporated and the crude
was dissolved in CH2Cl2 (150 mL) and washed first with a 5% aqueous
solution of KHSO4 (2 ꢁ 50 mL) and then with water (2 ꢁ 50 mL). The
evaporation of the dried solvent (Na2SO4) afforded the product as a
clear oil pure enough to be used in the following steps.
derivative (15e20) and N-diisopropylethylamine (DIPEA, 132 ml,
0.76 mmoli). The obtained mixture was refluxed for 8 days and the
reaction monitored by TLC (CHCl3/MeOH/NH3 9.5:0.5:0.1). Then the
solvent was removed under reduced pressure, the residue dis-
solved in CHCl3 (35 mL) and washed three times with saturated
NaHCO3 (10 mL). The organic phase was dried over Na2SO4,
concentrated and the crude residue was purified by flash chroma-
tography (SiO2, CHCl3/MeOH/NH3 98:1:1) to afford the final com-
pounds in a good yield.
4.2.1. All-cis-3,5-N,N-di-tert-butyloxycarbonyl-1-N-[2- N-
(benzyloxycarboyl)aminoethyl]-1,3,5-triamino-cyclohexane (22)
Yield 42%; white solid, mp 188 ꢀC; Rf ¼ 0.6 (CHCl3/MeOH/NH3
4.1.1. 2-(N-Benzyloxycarbonyl-2-aminoethoxy)ethanol31
9.5:0.5:0.1); 1H NMR (CDCl3)
d
: 0.86 (dd, 3H, J1 ¼ J2 ¼ 11.8 Hz); 1.35
Yield 85%; Rf ¼ 0.35 (CH2Cl2/CH3OH 9.5:0.5); 1H NMR (CDCl3)
d
:
(s, 18H); 2.13e2.24 (m, 3H); 2.52e2.60 (m, 1H); 2.74 (q, 2H,
J ¼ 5.5 Hz); 3.25 (q, 2H, J ¼ 5.5 Hz); 3.52 (br, 2H); 4.42 (br, 2H); 5.09
2.58 (br, 1H); 3.39 (q, 2H, J ¼ 5.6 Hz); 3.52e3.59 (m, 4H); 3.69e3.76
(m, 2H); 5.09 (s, 2H); 5.42 (br, 1H); 7.31e7.35 (m, 5H); 13C NMR
(s, 2H); 5.18 (brs, 1H); 7.22e7.39 (m, 5H); 13C NMR (CDCl3)
d: 28.4
(CDCl3)
d
: 40.8 (t); 61.6 (t); 66.7 (t); 70.1 (d); 72.2 (d); 128.1 (d);
(q); 39.9 (t); 41.3 (t, 3C); 46.3 (t); 46.7 (d, 2C); 53.4 (d); 66.7 (t); 79.4
(s); 128.1 (d, 2C); 128.2 (d); 128.5 (d, 2C); 136.5 (s); 154.9 (s); 156.5
(s); ESI-MS (m/z): 507.2 [M þ Hþ].
128.5 (d); 136.4 (s); 156.6 (s); ESI-MS (m/z): 240.0 [M þ Hþ]; 262.0
[M þ Naþ]; 278.0 [M þ Kþ]; 257.0 [M þ NHþ4 ].
4.1.2. 2-[2-(N-Benzyloxycarbonyl-2-aminoethoxy)ethoxy]ethanol32
4.2.2. All-cis-3,5-N,N-di-tert-butyloxycarbonyl-1-N-[2-(N-
Benzyloxycarbonyl-2-aminoethoxy)ethyl]-1,3,5-
triaminocyclohexane (26)
Yield 90%. Rf ¼ 0.30 (CH2Cl2/CH3OH 9.5:0.5); 1H NMR, (CDCl3)
d:
2.38 (br, 1H); 3.41 (q, 2H, J ¼ 6.2 Hz); 3.54e3.61 (m, 8H); 3.75e3.83
(m, 2H); 5.10 (s, 2H); 5.39 (br, 1H); 7.32e7.38 (m, 5H); 13C NMR,
Yield 42%; white solid; Rf ¼ 0.22 (CHCl3/CH3OH/NH3 9:1:0.1); 1H
(CDCl3)
d
: 40.8 (t); 61.5 (t), 66.6 (t); 69.8 (t); 71.4 (t); 128.1 (d); 128.4
NMR (CDCl3)
d
: 0.84 (dd, 3H, J1 ¼ J2 ¼ 10.9 Hz); 1.45 (s, 18H); 2.15
(d); 136.5 (s); 156.4 (s); ESI-MS (m/z): 284.3 [M þ Hþ]; 306.0
(bs, 3H); 2.56 (t, 1H, J ¼ 11.2 Hz); 2.75 (t, 2H, J ¼ 4.8 Hz); 3.35 (q, 2H,
[M þ Naþ]; 322.3 [M þ Kþ]; 301.2 [M þ NHþ4 ].
J ¼ 4.8 Hz); 3.52 (br, 6H); 4.40 (bs, 2H); 5.09 (s, 2H); 5.54 (bs, 1H);
The above protected aminoalcohol (2 mmol) was dissolved in
CH2Cl2 (15 mL) and CBr4 (0.990 g, 2.98 mmol) and Ph3P (0.783 g,
2.98 mmol) were added to the stirred solution at 0 ꢀC. The solution
was warmed at room temperature and the reaction was followed by
TLC (Petroleum ether/EtOAc 7/3). After 90 min at r.t. water was
added in the reaction flask. The solution was diluted with CH2Cl2
(110 mL) and the organic phase was washed twice with water and
brine, dried over Na2SO4 and the solvent evaporated to dryness
under vacuum. The crude material was purified by flash chroma-
tography (SiO2, petroleum ether/AcOEt 90/10, gradient 2%) to give
the bromide as a transparent oil.
7.24e7.40 (m, 5H); 13C NMR, (CDCl3)
d: 28.3 (q); 39.5 (2t, 2C); 41.0
(2t, 2C); 47.2 (2d, 2C); 47.3 (t); 53.5 (d); 66.5 (t); 69.6 (t); 70.2 (t);
79.3 (s); 128.0 (d, 2C); 128.1 (d, 1C); 128.4 (d, 2C); 136.5 (s, 1C);
154.9 (s, 2C); 156.4 (s); ESI-MS (m/z): 552.2 [M þ Hþ]; 573.8
[M þ Naþ]; 589.4 [M þ NHþ4 ].
4.2.3. All-cis-3,5-N,N-di-tert-butyloxycarbonyl-1-N-{2-[2-(N-
Benzyloxycarbonyl-2-aminoethoxy)ethoxy]ethyl}-1,3,5-
triaminocyclohexane (27)
Yield 49%; white solid, Rf ¼ 0.35 (CHCl3/CH3OH/NH3 9:1:0.1); 1H
NMR, (CDCl3)
d
: 0.84 (dd, 3H, J1 ¼ J2 ¼ 11.5 Hz); 1.50 (s, 18H);
2.11e2.19 (m, 3H); 2.57 (t, 1H, J ¼ 11.2 Hz); 2.76 (t, 2H, J ¼ 4.6 Hz);
3.38 (q, 2H, J ¼ 5.0 Hz); 3.51e3.62 (m, 10H); 4.40 (bs, 2H); 5.08 (s,
4.1.3. 2-(N-Benzyloxycarbonyl-2-aminoethoxy)-1-bromo-ethane
(19)
2H); 5.63 (bs, 1H); 7.28e7.37 (m, 5H); 13C NMR, (CDCl3)
d: 27.8 (q);
Yield 77%; Rf ¼ 0.4 (petroleum ether/AcOEt 6:4); 1H NMR,
39.1 (2t, 2C); 40.3 (2t, 2C); 46.3 (2d, 2C); 46.4 (t); 52.8 (d); 66.1 (t);
69.5 (t, 2C); 70.0 (t, 2C); 78.6 (s); 128.1 (dd, 5C); 136.2 (s, 1C); 154.9
(s, 2C); 156.0 (s); ESI-MS (m/z): 595.4 [M þ Hþ]; 617.3 [M þ Naþ].
(CDCl3)
d
: 3.41 (q, 2H, J ¼ 5.2 Hz); 3.45 (t, 2H, J ¼ 6.0 Hz); 3.57 (t, 2H,
J ¼ 5.2 Hz); 3.76 (t, 2H, J ¼ 6.0 Hz); 5.11 (s, 2H); 7.32e7.36 (m, 5H);
13C NMR, (CDCl3)
d: 30.3 (t); 40.7 (t); 66.5 (t); 69.7 (t); 70.6 (t); 127.9
(d); 128.2 (d); 136.4 (s); 156.3 (s). ESI-MS (m/z): 304.0 [M þ Hþ];
4.3. Synthesis of All-cis-5-N-tert-butyloxycarbonyl-1,3-N,N-di-[2-
(N-Benzyloxycarbonyl-2-aminoethoxy)ethyl]-1,3,5-triamino-
cyclohexane (29)
324.1 [M þ Naþ]; 340.0 [M þ Kþ]; 318.9 [M þ NHþ4 ].
4.1.4. 2-[2-(N-Benzyloxycarbonyl-2-aminoethoxy)ethoxy]-1-
bromo-ethane (20)
To a solution of tach-MONOBOC (28, 121 mg, 0.53 mmol) in dry
CH3CN (10 mL) were added 2 eq. of Bre(CH2)2eOe(CH2)2eNH-Z
(19) and DIPEA (185 ml, 1.06 mmol). The obtained mixture was
refluxed for 4 days and the reaction monitored by TLC (CHCl3/
MeOH/NH3 9:1:0.2). Then the solvent was removed under reduced
Yield 64%; Rf ¼ 0.35 (petroleum ether/AcOEt 6:4); 1H NMR,
(CDCl3)
d
: 3.39 (q, 2H, J ¼ 5.0 Hz); 3.45 (t, 2H, J ¼ 6.1 Hz); 3.57 (t, 2H,
J ¼ 5.0 Hz); 3.60e3,67 (m, 4H); 3.79 (t, 2H, J ¼ 6.1 Hz); 5.14 (s, 2H);
7.28e7.39 (m, 5H). 13C NMR, (CDCl3),
d
: 30.2 (t); 40.8 (t); 66.6 (t);
Please cite this article in press as: Sissi C, et al., Conjugates between minor groove binders and Zn(II)-tach complexes: Synthesis,
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8
C. Sissi et al. / Tetrahedron xxx (2017) 1e11
pressure, and the residue dissolved in CHCl3 (30 mL) and washed
three times with 5% NaHCO3 (10 mL) and one time with brine
(10 mL). The organic phase was dried, concentrated and the crude
residue was purified by flash chromatography (SiO2, CHCl3/MeOH/
NH3 100:0.5:1) to afford 107 mg of the final compound (29) as a
yellow oil with a 30% yield. Rf: 0.8 (CHCl3/MeOH/NH3 9.5:0.5:0.1);
4.5.1. (Indole)2-C2-tach-DIBOC (33)
Yield 30.6%; pale yellow solid; mp 276 ꢀC; Rf ¼ 0.6 (CH2Cl2/
MeOH/NH3 8.5:1.5:0.1); 1H NMR (DMSO)
d: 0.84 (dd, 2H,
J1 ¼ J2 ¼ 11.6 Hz); 0.99 (dd, 1H, J1 ¼ J2 ¼ 11.6 Hz); 1.37 (s, 18H); 1.81
(d, 1H, J ¼ 11.4 Hz); 1.92 (d, 2H, J ¼ 11.4 Hz); 2.50e2.61 (m, 1H);
2.65e2.76 (m, 2H); 3.19e3.39 (m, 4H); 6.86 (d, 1H, J ¼ 8.0 Hz); 7.06
(t, 1H, J ¼ 8.0 Hz); 7.09 (s, 1H); 7.21 (t, 1H, J ¼ 8.0 Hz); 7.37e7.53 (m,
3H); 7.67 (d, 1H, J ¼ 8.0 Hz); 8.10 (s, 1H); 8.43 (bs, 1H); 10.13 (s, 1H);
1H NMR, (CDCl3),
d
: 0.84 (dd, 3H, J1 ¼ J2 ¼ 11.2 Hz); 1.41 (s, 9H), 2.08
(d, 1H, J ¼ 11.4 Hz); 2.16 (d, 2H, J ¼ 11.4 Hz); 2.54 (t, 2H, J ¼ 11.2 Hz);
2.72e2.80 (m, 4H); 3.33e3.56 (m, 13H); 4.35 (bs, 1H); 5.10 (s, 4H);
11.58 (bs, 1H); 11.70 (bs, 1H); 13C NMR (DMSO)
d: 28.4 (q); 46.0 (t);
5.35 (bs, 2H); 7.25e7.38 (m, 10H); 13C NMR, (CDCl3),
d
: 28.4 (q);
46.4 (d, 2C); 53.3 (d); 77.5 (s, 2C); 102.5 (d), 103.4 (d); 112.3 (d);
112.4 (d); 112.8 (d); 118.5 (d); 119.9 (d); 121.7(d); 123.6 (d); 126.9
(s); 127.2 (s); 131.5 (s); 132.0 (s); 133.3 (s); 133.6 (s); 136.7 (s); 154.8
(s, 2C); 159.5 (s); 161.2 (s); ESI-MS (m/z): 674.4 [M þ Hþ].
39.65 (t, 2C); 39.85 (t); 40.86 (t); 46.43 (t); 46.74 (d); 53.73 (d, 2C);
66.61 (t, 2C); 69.72 (t, 2C); 70.26 (t, 2C); 79.3 (s); 128.27 (dd);
136.58 (s, 1C); 155.07 (s); 156.4 (s); ESI-MS (m/z): 672.6 [M þ Hþ];
694.5 [M þ Naþ].
4.5.2. (Indole)2-C4-tach-DIBOC (34)
Yield 20.6%; pink solid; mp 225 ꢀC; Rf ¼ 0.6 (CH2Cl2/MeOH/NH3
4.4. General procedure for Z-deprotection (9e14, 30)
8.5:0.5:0.1); 1H NMR (CD3OD)
d
: 0.99 (dd, 2H, J1 ¼ J2 ¼ 11.6 Hz); 1.06
To a methanolic (15 mL) solution containing 0.2 mmol of the
appropriate Z-protected ligands (22e27, 29) was added a catalytic
amount (20 mg) of 5% PdC. The resulting mixture was stirred under
hydrogen atmosphere for 2 h. The reaction was monitored by TLC
(CHCl3/MeOH/NH3 9:1:0.1). Then the catalyst was filtered off and
the solvent removed under reduced pressure affording the desired
final compounds (9e14, 30) in a good yield.
(dd, 1H, J1 ¼ J2 ¼ 11.6 Hz); 1.39 (s, 18H); 1.56e1.72 (m, 4H); 2.01 (d,
1H, J ¼ 11.4 Hz); 2.11 (d, 2H, J ¼ 11.4 Hz); 2.64e2.72 (m, 3H);
3.32e3.44 (m, 4H); 7.01e7.12 (m, 2H); 7.20 (t, 1H, J ¼ 8.0 Hz); 7.26
(s, 1H); 7.36e7.48 (m, 3H); 7.61 (d, 1H, J ¼ 8.1 Hz); 7.98 (s, 1H); 13
C
NMR (CD3OD) d: 27.5 (t); 28.5 (t); 28.9 (q); 38.9 (t); 40.3 (t, 3C); 47.3
(t); 48.0 (d, 2C); 55.0 (d); 80.2 (s, 2C); 104.3 (d); 105.0 (d); 113.2 (d,
2C); 115.5 (d); 120.8 (d); 121.3 (d); 123.0 (d); 125.3 (d); 129.1 (s, 2C);
132.4 (s, 2C); 133.4 (s); 135.9 (s); 138.6 (s); 157.7 (s, 2C); 162.6 (s);
164.1 (s); MS-ESI (m/z): 702.4 [M þ Hþ].
4.4.1. All-cis-3,5-N,N-di-tert-butyloxycarbonyl-1-N-(2-
aminoethyl)-1,3,5-triamino-cyclohexane (9)
Yield 76%; white solid; Rf ¼ 0.1 (CHCl3/MeOH/NH3 9:1:0.1); 1H
4.5.3. (Indole)2-C6-tach-DIBOC (35)
NMR (DMSO)
d
: 1.17 (dd, 3H, J1 ¼ J2 ¼ 11.9 Hz); 1.36 (s, 18H); 1.92
Yield 18%; white solid; mp 221 ꢀC; Rf ¼ 0.6 (CH2Cl2/MeOH/NH3
(dd, 3H, J1 ¼ J2 ¼ 11.9 Hz); 3.07e3.16 (m, 3H); 3.27e3.38 (m, 4H);
8.5:1.5:0.1); 1H NMR (CD3OD)
d
: 0.92 (dd, 2H, J1 ¼ J2 ¼ 11.7 Hz); 1.03
7.04 (bs, 2H); ESI-MS (m/z): 373.3 [M þ Hþ].
(dd, 1H, J1 ¼ J2 ¼ 11.7 Hz); 1.20e1.51 (m, 24H); 1.56e1.65 (m, 2H);
1.92e2.08 (m, 3H); 2.49e2.61 (m, 3H); 3.30e3.42 (m, 4H);
6.95e7.14 (m, 2H); 7.20 (t, 1H, J ¼ 7.9 Hz); 7.27 (s, 1H); 7.34e7.51 (m,
4.4.2. All-cis-3,5-N,N-di-tert-butyloxycarbonyl-1-N-[2-(2-
aminoethoxy)ethyl]-1,3,5-triaminocyclohexane (13)
3H); 7.60 (d, 1H, J ¼ 7.9 Hz); 7.98 (s, 1H); 13C NMR (CD3OD)
d: 22.0
Yield 84%; white solid; 1H NMR (CDCl3)
d: 0.91 (dd, 3H,
(t); 27.4 (q); 28.9 (t); 29.1 (t, 2C); 37.7 (t); 37.8 (t); 39.0 (t, 2C); 46.2
(t); 46.6 (d, 2C); 53.5 (d); 78.6 (s, 2C); 102.8 (d); 103.5 (d); 111.8 (d,
2C); 114.0 (d); 119.2 (d); 119.8 (d); 121.4 (d); 123.7 (d); 127.7 (s, 2C);
131.0 (s, 2C); 132.0 (s); 134.4 (s); 137.1 (s); 156.2 (s, 2C); 162.5 (s,
2C); ESI-MS (m/z): 629.4 [Mþ-2BOC]; 730.5 [M þ Hþ].
J1 ¼ J2 ¼ 11.5 Hz); 1.37 (s, 18H); 2.14e2.26 (m, 3H); 2.58e2.66 (m,
1H); 2.74e2.83 (m, 2H); 2.86 (t, 2H, J ¼ 5.3 Hz); 3.47 (t, 2H,
J ¼ 5.3 Hz); 3.51e3.62 (m, 4H); 4.42 (bs, 2H).
4.4.3. All-cis-3,5-N,N-di-tert-butyloxycarbonyl-1-N-{2-[2-(2-
aminoethoxy)ethoxy]ethyl}-1,3,5-triaminocyclohexane (14)
Yield 76%; white solid; 1H NMR (CDCl3)
d: 0.88 (dd, 3H, J1 ¼ J2
4.5.4. (Indole)2-C8-tach-DIBOC (36)
Yield 22.2%; white solid; mp 208 ꢀC, Rf ¼ 0.6 (CH2Cl2/MeOH/NH3
11.7 Hz); 1.36 (s, 18H); 2.13e2.25 (m, 3H); 2.59 (t, 1H, J ¼ 11.2 Hz);
2.78 (t, 2H, J ¼ 5.15 Hz); 3.48 (t, 2H, J ¼ 5.13 Hz); 3.51e3.65 (m,10H);
4.42 (bs, 2H).
8.5:1.5:0.1); 1H NMR (CD3OD)
d: 0.89e1.04 (m, 3H); 1.34e1.59 (m,
28H); 1.64e1.71 (m, 2H); 2.02e2.23 (m, 3H); 2.67e2.79 (m, 3H);
3.39e3.51 (m, 4H); 7.08e7.16 (m, 2H); 7.25e7.37 (m, 2H); 7.51e7.55
(m, 2H); 7.67e7.73 (m, 2H); 8.10 (s, 1H); 13C NMR (CD3OD)
d: 27.5 (t,
4.4.4. All-cis-5-N-tert-butyloxycarbonyl-1,3-N,N-di-[2-(2-
2C); 28.8 (q); 29.3 (t, 2C); 29.4 (t); 30.6 (t); 39.0 (t); 40.0 (t, 3C); 47.2
(t); 47.7 (d, 2C); 54.9 (d); 79.9 (s, 2C); 104.1 (d); 104.8 (d); 113.3 (d,
2C); 115.1 (d); 120.5 (d); 121.3 (d); 122.9 (d); 125.2 (d); 129.1 (s, 2C);
132.6 (s, 2C); 133.5 (s); 135.6 (s); 138.4 (s); 157.4 (s, 2C); 163.7 (s,
2C); ESI-MS (m/z): 758.4 [M þ Hþ].
aminoethoxy)ethyl]-1,3,5-triamino-cyclohexane (30)
Yield 71%, white solid; 1H NMR (CDCl3)
d: 0.87 (dd, 3H,
J1 ¼ J2 ¼ 11.5 Hz); 1.40 (s, 9H); 2.10 (dd, 3H, J1 ¼ J2 ¼ 11.4); 2.53 (t,1H,
J ¼ 11.5); 2.72e2.83 (m, 6H); 3.40e3.54 (m, 12H); 4.71 (bs, 1H).
4.5. General synthesis of conjugates between minor groove binders
and tach derivatives (33e39)
4.5.5. Benzofuran-indole-C4-tach-DIBOC (37)
Yield 20%; yellow solid; mp 183 ꢀC; Rf ¼ 0.7 (CH2Cl2/MeOH/NH3
A DMF (3 mL) solution of acid derivatives (31, 32, 0.188 mmol,1.2
eq), EDCI (60 mg, 2 eq) TACH derivative (9e14, 0.156 mmol) and
8.5:1.5:0.1); 1H NMR (CD3OD)
d: 1.01e1.14 (m, 3H); 1.38 (s, 18H);
1.61e1.70 (m, 4H); 1.98 (d, 1H, J ¼ 11.6 Hz); 2.19 (d, 2H, J ¼ 11.6 Hz);
2.81e2.92 (m, 3H); 3.33e3.43 (m, 4H); 7.03 (s, 1H); 7.29 (t, 1H,
J ¼ 7.9 Hz); 7.38e7.46 (m, 3H); 7.57 (s, 1H); 7.61 (d, 1H, J ¼ 8.0 Hz);
Et3N (21
mL) was stirred at room temperature for 2 days and
monitored by TLC (CH2Cl2/MeOH/NH3 8.5:1.5:0.1). Then the solvent
was removed in vacuo, and the residue treated with water (40 mL).
The aqueous solution was extracted with EtOAc (3 ꢁ 25 mL), and
the combined organic phases dried and concentrated under
reduced pressure. The crude residue was purified by flash chro-
matography (CH2Cl2/MeOH/NH3 100:0.5:1) to afford the final
compounds 33e39 in moderate yield.
7.71 (d, 1H, J ¼ 7.9 Hz); 8.02 (s, 1H); 13C NMR (CD3OD)
d: 22.6 (t);
28.3 (t); 28.9 (q); 39.9 (t); 42.2 (t, 3C); 46.7 (t); 47.8 (d, 2C); 55.1 (d);
70.6 (s, 2C); 104.5 (d); 112.0 (d); 113.3 (d, 2C); 115.8 (d); 120.8 (d);
124.0 (d); 125.2 (d); 128.5 (d); 129.1 (s, 3C); 133.5 (s); 136.2 (s);
156.8 (s); 157.7 (s); 159.6 (s, 2C); 164.2 (s, 2C); ESI-MS (m/z): 703.2
[M þ Hþ].
Please cite this article in press as: Sissi C, et al., Conjugates between minor groove binders and Zn(II)-tach complexes: Synthesis,
characterization, and interaction with plasmid DNA, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.04.013

C. Sissi et al. / Tetrahedron xxx (2017) 1e11
9
4.5.6. (Indole)2-NHeCH2eCH2eOeCH2eCH2eNH-tach-DIBOC (38)
4.7.2. (Indole)2-C4-tach (2)
Yield 17%; white solid; Rf: 0.39 (CH2Cl2/MeOH/NH3 8.5:1.5:0.1);
Yield 96%; yellow solid; mp 227 ꢀC; Rf ¼ 0.0 (CH2Cl2/MeOH/NH3
1H NMR (CD3OD)
d
: 0.96 (dd, 3H, J1 ¼ J2 ¼ 11.5 Hz); 1.40 (s, 18H);
8.5:1.5:0.1); nmax 3360e3110 (br), 1669, 1630, 1546, 1421, 1313, 1181,
1.96 (d, 1H, J ¼ 11.6 Hz); 2.15 (d, 2H, J ¼ 11.6 Hz); 2.49 (t, 1H,
J ¼ 11.5 Hz); 2.77 (t, 2H, J ¼ 5.15 Hz); 3.24e3.40 (m, 2H); 3.51e3.65
(m, 6H); 7.02e7.11 (m, 2H); 7.22 (t, 1H, J ¼ 7.9 Hz); 7.30 (s, 1H);
7.42e7.51 (m, 3H); 7.64 (d, 1H, J ¼ 8.0 Hz); 8.01 (s, 1H); 13C NMR
1115; 1H NMR (CD3OD)
d
: 1.60 (dd, 3H, J1 ¼ J2 ¼ 11.7 Hz); 1.65e1.80
(m, 4H); 2.40 (d, 1H, J ¼ 11.7 Hz); 2.49 (d, 2H, J ¼ 11.6 Hz); 3.07e3.13
(m, 2H); 3.29e3.45 (m, 5H); 6.99e7.05 (m, 2H); 7.19 (t, 1H,
J ¼ 7.8 Hz); 7.25 (s, 1H); 7.37e7.45 (m, 3H); 7.59 (d, 1H, J ¼ 7.8 Hz);
7.97 (s, 1H); ESI-MS (m/z): 502.3 [M þ Hþ]. HRMS (ESI): [M þ Hþ],
found 502.2947. C28H36N7O2 requires 502.2930.
(CD3OD) d: 32.7 (t) 40.2 (t); 43.6 (d, 2C); 45.9 (d, 2C); 46.9 (t, 2C);
50.0 (d); 66.9 (t); 71.6 (t); 79.2 (s) 104.8 (d); 105.1 (d); 113.1 (d);
113.4 (d); 115.7 (d); 121.1 (d); 121.3 (d); 122.9 (d); 125.3 (d); 129.1
(s, 2C); 132.4 (s, 2C); 133.4 (s); 136.0 (s); 138.6 (s); 155.4 (s); 160.5
(s); 162.1 (s); ESI-MS (m/z): 718.5 [M þ Hþ].
4.7.3. (Indole)2-C6-tach (3)
Yield 89%; white solid; mp 204 ꢀC; Rf ¼ 0.0 (CH2Cl2/MeOH/NH3
8.5:1.5:0.1); nmax 3316, 3305e3120 (br),1669, 1635,1548,1418,1311,
4.5.7. (Indole)2-NHe(CH2eCH2eO)2eCH2eCH2eNH-tach-DIBOC
(39)
1180, 1125; 1H NMR (CD3OD)
d: 1.37e1.47 (m, 4H); 1.52e1.71 (m,
7H); 2.33e2.51 (m, 3H); 2.95e3.03 (m, 2H); 3.28e3.40 (m, 5H);
6.98e7.06 (m, 2H); 7.19 (t,1H, J ¼ 7.6 Hz); 7.25 (s, 1H); 7.36e7.44 (m,
3H); 7.60 (d, 1H, J ¼ 7.6 Hz); 7.97 (s, 1H); ESI-MS (m/z): 530.2
[M þ Hþ]. HRMS (ESI): [M þ Hþ], found 530.3294. C30H40N7O2 re-
quires 530.3243.
Yield 18%; pale yellow solid; Rf: 0.4 (CH2Cl2/MeOH/NH3
8.5:1.5:0.1); 1H NMR (CD3OD/CDCl3)
d:
0.93 (dd, 3H,
J1 ¼ J2 ¼ 11.5 Hz); 1.31 (s, 18H); 1.93e1.99 (m, 3H); 2.40e2.50 (m,
1H); 2.69 (t, 2H, J ¼ 5.4 Hz); 3.26e3.34 (m, 2H); 3.55e3.68 (m,10H);
7.07 (t, 1H, J ¼ 8.0 Hz); 7.10 (s, 1H); 7.23 (t, 1H, J ¼ 8.1 Hz); 7.31 (s,
1H); 7.42e7.50 (m, 3H); 7.64 (d, 1H, J ¼ 8.0 Hz); 7.84 (s, 1H); 13C
4.7.4. (Indole)2-C8-tach (4)
NMR (CD3OD/CDCl3)
d: 28.4 (t); 38.5 (t); 39.0 (t, 2C); 40.5 (d, 2C);
Yield 96%; white solid; mp 224 ꢀC; Rf ¼ 0.0 (CH2Cl2/MeOH/NH3
8.5:1.5:0.1); nmax 3350e3180 (br),1668,1633,1544,1420,1310,1179,
45.3 (t); 45.6 (d); 48.0 (t); 69.1 (t, 2C); 69.7 (t, 2C); 79.2 (s); 103.6
(d); 103.9 (d); 111.9 (d); 112.1 (d); 114.0 (d); 120.2 (d); 120.3 (d);
121.5 (d); 123.9 (d); 127.1 (s); 127.2 (s); 130.3 (s); 131.1 (s); 133.9 (s);
136.3 (s); 136.7 (s); 155.4 (s); 160.5 (s); 162.1 (s); ESI-MS (m/z):
762.4 [M þ Hþ].
1125; 1H NMR (CD3OD)
d: 1.20e1.42 (m, 8H); 1.54e1.69 (m, 7H);
2.34e2.50 (m, 3H); 2.96e3.04 (m, 2H); 3.29e3.42 (m, 5H); 6.87 (s,
1H); 6.99e7.07 (m, 2H); 7.19 (t, 1H, J ¼ 7.8 Hz); 7.26 (s, 1H);
7.37e7.44 (m, 2H); 7.60 (d, 1H, J ¼ 7.8 Hz); 7.98 (s, 1H); 13C NMR
(CD3OD) d: 27.5 (t, 2C); 30.7 (t); 31.0 (t); 32.9 (t); 34.6 (t); 40.7 (t,
4.6. Synthesis of di-[(Indole)2-NHeCH2eCH2eOeCH2eCH2eNHe]-
tach-MONOBOC (40)
4C); 46.7 (t); 46.9 (d, 2C); 53.6 (d); 104.5 (d); 105.1 (d); 113.3 (d, 2C);
115.6 (d); 120.9 (d); 121.4 (d); 123.1 (d); 125.4 (d); 129.3 (s); 132.6
(s, 2C); 133.5 (s); 136.0 (s); 138.7 (s); 162.7 (s); 164.1 (s); ESI-MS (m/
z): 558.3 [M þ Hþ]. HRMS (ESI): [M þ Hþ], found 558.3708.
A DMF (10 mL) solution of acid derivative 31 (0.489 mmol, 4 eq),
EDCI (125 mg, 0.81 mmol, 6 eq), TACH derivative 30 (0.109 mmol, 1
C32H44N7O2 requires 558.3556.
eq) and Et3N (61 mL) was stirred at room temperature for 2 days and
monitored by TLC (CH2Cl2/MeOH/NH3 8.5:1.5:0.1). Then the solvent
was removed in vacuo and the residue treated with water (10 mL).
The suspension was extracted with EtOAc (3 ꢁ 25 mL), and the
combined organic phases dried and concentrate under reduced
pressure. The crude residue was purified by flash chromatography
(CH2Cl2/MeOH/NH3 100:0.5:1) to afford the final compound 40 as a
pale yellow solid (yield 17%). Rf: 0.39 (CH2Cl2/MeOH/NH3
4.7.5. Benzofuran-indole-C4-tach (5)
Yield 97%, dark yellow solid; mp 166 ꢀC; Rf ¼ 0.0 (CH2Cl2/MeOH/
NH3 8.5:1.5:0.1); nmax 3220e3000 (br), 1668, 1628, 1557, 1446, 1259,
1178, 1126; 1H NMR (CD3OD)
d
: 1.61 (dd, 3H, J1 ¼ J2 ¼ 11.6 Hz);
1.67e1.81 (m, 4H); 2.40 (d, 1H, J ¼ 11.6 Hz); 2.51 (d, 2H, J ¼ 11.6 Hz);
3.08e3.15 (m, 2H); 3.34e3.46 (m, 5H); 7.05 (s, 1H); 7.31 (t, 1H,
J ¼ 7.7 Hz); 7.41e7.57 (m, 3H); 7.58 (s, 1H); 7.62 (d, 1H, J ¼ 7.6 Hz);
8.5:1.5:0.1); 1H NMR (CD3OD/CDCl3)
d:
1.05 (dd, 3H,
7.72 (d, 1H, J ¼ 7.7 Hz); 8.04 (s, 1H); 13C NMR (CD3OD)
d: 24.8 (t);
J1 ¼ J2 ¼ 11.7 Hz); 1.34 (s, 9H); 1.78e184 (m, 3H); 2.22e2.33 (m,
6H); 2.60 (bs, 1H); 3.27e3.33 (m, 4H); 3.41e3.53 (m, 8H); 7.00e7.05
(m, 4H); 7.21e7.27 (m, 4H); 7.38e7.50 (m, 6H); 7.57e7.63 (m, 2H);
7.99 (s, 2H). ESI-MS (m/z): 1006.6 [M þ Hþ].
28.0 (t); 34.6 (t); 39.6 (t, 3C); 46.3 (t); 47.0 (d, 2C); 53.6 (d); 104.7 (d,
C3); 112.0 (d); 113.3 (d, 2C); 115.8 (d); 121.0 (d); 124.0 (d); 125.2 (d);
128.1 (d); 129.1 (s); 131.8 (s, 2C); 133.4 (s); 136.2 (s); 156.8 (s); 159.6
(s); 163.5 (s); 164.4 (s); ESI-MS (m/z): 503.2 [M þ Hþ]. HRMS (ESI):
[M þ Hþ], found 503.2819. C28H35N6O3 requires 503.2771.
4.7. General procedure for N-Boc deprotection (1e8)
4.7.6. (Indole)2-NHeCH2eCH2eOeCH2eCH2eNH-tach (6)
Yield 92%; brown solid; Rf ¼ 0.0 (CH2Cl2/MeOH/NH3 8.5:1.5:0.1);
nmax 3305e3105 (br), 1670, 1629, 1546, 1434, 1312, 1180, 1124; 1H
N-Boc protected derivative (33e40, 0.03 mmol) was dissolved in
CH2Cl2 (1 mL) and trifluoroacetic acid (1 mL) was added. The
mixture was stirred at room temperature for 2 h and the reaction
monitored by TLC (CH2Cl2/MeOH/NH3 8.5:1.5:0.1). Then the solvent
was removed under reduced pressure and the obtained salts (1e8)
were characterized and utilized without any further purification.
NMR (CD3OD)
d
: 1.44 (dd, 3H, J1 ¼ J2 ¼ 11.7 Hz); 2.23 (d, 1H,
J ¼ 11.7 Hz); 2.41 (d, 2H, J ¼ 11.7 Hz); 2.61e2.74 (m, 3H); 2.92e3.02
(m, 2H); 2.98e3.47 (m, 4H); 3.55e3.61 (m, 2H); 6.89 (t, 1H,
J ¼ 7.9 Hz); 6.93 (s, 1H), 7.04 (t, 1H, J ¼ 8.0 Hz); 7.12 (s, 1H);
7.22e7.34 (m, 3H); 7.44 (d, 1H, J ¼ 7.9 Hz); 7.83 (s, 1H); 13C NMR
4.7.1. (Indole)2-C2-tach (1)
(CD3OD) d: 40.2 (t); 43.6 (d, 2C); 46.0 (d, 2C); 46.9 (t, 2C); 50.0 (d);
Yield 80%; yellow solid; mp 229 ꢀC; Rf ¼ 0.0 (CH2Cl2/MeOH/NH3
8.5:1.5:0.1); nmax 3314e3095 (br), 1668, 1648, 1543, 1422, 1313,
66.9 (t); 71.6 (t); 104.8 (d); 105.1 (d); 113.1 (d); 113.4 (d); 115.7 (d);
121.1 (d); 121.3 (d); 122.9 (d); 125.3 (d); 129.2 (s); 132.4 (s, 2C);
133.4 (s); 136.0 (s); 138.6 (s); 160.8 (s); 162.7 (s); ESI-MS (m/z):
518.2 [M þ Hþ]. HRMS (ESI): [M þ Hþ], found 518.2893. C28H36N7O3
requires 518.2880.
1180, 1125; 1H NMR (CD3OD)
d: 1.53e1.70 (m, 3H); 2.41 (d, 1H,
J ¼ 11.6 Hz); 2.51 (d, 2H, J ¼ 11.6 Hz); 3.26e3.52 (m, 5H); 3.65e3.75
(m, 2H); 7.04 (t, 1H, J ¼ 7.7 Hz), 7.06 (s, 1H); 7.20 (t, 1H, J ¼ 7.7 Hz);
7.26 (s, 1H); 7.37e7.44 (m, 3H); 7.60 (d, 1H, J ¼ 7.7 Hz); 8.00 (s, 1H);
ESI-MS (m/z): 474.1 [M þ Hþ]; HRMS (ESI): [M þ Hþ], found
474.2663. C26H32N7O2 requires 474.2617.
4.7.7. (Indole)2-NHe(CH2eCH2eO)2eCH2eCH2eNH-tach (7)
Yield 94%; yellow solid; Rf ¼ 0.0 (CH2Cl2/MeOH/NH3 8.5:1.5:0.1);
Please cite this article in press as: Sissi C, et al., Conjugates between minor groove binders and Zn(II)-tach complexes: Synthesis,
characterization, and interaction with plasmid DNA, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.04.013

10
C. Sissi et al. / Tetrahedron xxx (2017) 1e11
nmax 3325e3065 (br), 1671, 1615, 1547, 1436, 1312, 1180, 1124; 1H
NMR (CD3OD)
J1 ¼ J2 ¼ 11.9 Hz); 2.41e2.49 (m, 1H); 2.83e2.92 (m, 2H); 3.27e3.32
(m, 2H); 3.59e3.70 (m, 10H); 7.08 (t, 1H ¼ J ¼ 7.2 Hz); 7.1 (s, 1H);
7.24 (t, 1H, J ¼ 7.3 Hz); 7.48 (s, 1H); 7.42e7.48 (m, 3H); 7.63 (d, 1H,
J ¼ 7.2 Hz); 8.02 (s, 1H); ESI-MS (m/z): 562.3 [M þ Hþ]. HRMS (ESI):
[M þ Hþ], found 562.3128. C30H40N7O4 requires 562.3141.
4.11. Molecular modeling studies
d: 0.88 (dd, 3H, J1 ¼ J2 ¼ 11.9 Hz); 1.59 (dd, 3H,
Zn(II)-tach complexes were optimized using Hartree-Fock cal-
culations with the 6e311þþG(d,p) basis set. A water molecule
coordinated to the apical position of Zn metal ion was used. This
water molecule was used to saturate, at least partially, the coordi-
nation sphere of the zinc metal ion. Quantum chemistry calcula-
tions were carried out with Gaussian 03.33 Harmonic vibrational
frequencies were obtained from RHF/6e311þþG(d,p) calculations
and used to characterize local energy minima (all frequency real).
Atomic charges were calculated by fitting to electrostatic potential
maps (CHELPG method). Structures of metal complex- conjugates
were built using the “Builder” module of Molecular Operation
Environment (MOE, version 2013.08).34 Conjugates complexes
were minimized using MMFF94 force field. All bond distances
involving Zn(II) metal ion are freeze during the optimization step.
Charges for the Zn(II)-triaminocyclohexane complexes were im-
ported from the Gaussian output files.
4.7.8. di-[(Indole)2-NHeCH2eCH2eOeCH2eCH2eNHe]-tach (8)
Yield 97%; orange solid; Rf
8.5:1.5:0.1); nmax 3350e3085 (br), 1671, 1636, 1545, 1416, 1310, 1177,
1124; 1H NMR (CD3OD)
¼
0.0 (CH2Cl2/MeOH/NH3
d
: 0.89 (dd, 3H, J1 ¼ J2 ¼ 10.6 Hz); 1.61e1.67
(m, 3H); 2.48e2.56 (m, 2H); 2.81e2.89 (m, 8H); 3.50e3.62 (m, 9H);
7.02e7.09 (m, 4H); 7.21-7.29 (m, 4H); 7.39e7.48 (m, 6H); 7.58e7.64
(m, 2H); 8.01 (s, 2H); 13C NMR (CD3OD)
d: 40.1 (t); 43.6 (d, 2C); 45.9
(d, 2C); 46.9 (t, 2C); 50.0 (d); 66.9 (t); 71.6 (t); 104.9 (d, 2); 105.1 (d,
2C); 113.1 (d, 2C); 113.3 (d, 2C); 115.7 (d, 2C); 121.1 (d, 2C); 121.3 (d,
2C); 122.9 (d, 2C); 125.3 (d, 2C); 129.2 (s, 4C); 132.4 (s, 4C); 133.1 (s,
2C); 136.0 (s, 2C); 138.6 (s, 2C); 162.6 (s); 162.9 (s); ESI-MS (m/z):
The present study involved the use of consensus dinucleotide
intercalation geometries d(ApT) and d(GpC) initially obtained
using NAMOT2 (Nucleic Acid MOdeling Tool, Los Alamos National
Laboratory, Los Alamos New Mexico), software.35 d(ApT) and
d(GpC) intercalation sites were contained in the centre of a dec-
anucleotide duplex of sequences d(50-ATATA-30)2 and d(50-
GCGCG-30)2 respectively. Decamers in B-form were built using the
“DNA Builder” module of MOE. Decanucleotides were minimized
using Amber94 all-atom force field,36 implemented by MOE
modeling package, until the rms value of Truncated Newton
944.9 [M
þ
Kþ]. HRMS (ESI): [M
þ
Hþ], found 906.4589.
C
50H56N11O6 requires 906.4415.
4.8. Electrophoretic procedure for DNA binding/cleavage
experiments
Stock solutions of the tach minor groove binders conjugates 1e8
and tach (0.5e1 mmol) were prepared in H2O/CH3OH 2:1 (ligands
1e3, 5e8) and H2O/CH3OH 2:3 (ligand 4). The ligand was diluted in
the proper solvent mixture, one equivalent of Zn(NO3)2 or Cu(NO3)2
in water was added and the pH was corrected to 7 by addition of
NaOH. The methanol content in the final reaction mixtures was
always lower than 4%. DNA cleavage experiments were performed
using pBR 322 (Gibco BRL) in 20 mM HEPES, pH 7.1. Reactions were
method (TN) was <0.001 kcal molꢂ1
Å
ꢂ1. The dielectric constant
was assumed to be distance independent with a magnitude of 4.
The metal complex-minor groove binder conjugates (3 and 8)
were docked into both intercalation sites using flexible MOE-Dock
methodology. The purpose of MOE-Dock is to search for favorable
binding configurations between a small, flexible ligand and a rigid
macromolecular target. Searching is conducted within a user-
specified 3D docking box, using “tabù search” protocol37 and
performed by incubating DNA (12 m
M base pairs) at 37 ꢀC in the
presence/absence of increasing amounts of metal complex for the
indicated time. Reaction products were resolved on a 1% agarose gel
in TAE buffer (40 mM TRIS base, 20 mM, acetic acid, 1 mM EDTA)
containing 1% SDS to dissociate the ligands from DNA. The resolved
bands were visualized by ethidium bromide staining and photo-
graphed. The relative amounts of different plasmid structures were
quantified using a BioRad Gel Doc 1000 apparatus interfaced to a PC
workstation. DNA binding experiments were performed as re-
ported in the previous paragraph but the reaction mixture was
loaded immediately on the agarose gel without incubation time
and the TAE buffer did not contain EDTA and SDS.
MMFF94 force field.38 MOE-Dock performs
a user-specified
number of independent docking runs (55 in our specific case)
and writes the resulting conformations and their energies to a
molecular database file. The resulting DNA-minor groove binder
complexes were subjected to MMFF94 all-atom energy minimi-
zation until the rms of conjugate gradient was <0.1 kcal molꢂ1 Åꢂ1
.
Also in these step, all bond distances involving Zn(II) metal ion are
freeze during the energy optimization. To model the effects of
solvent more directly, a set of electrostatic interaction corrections
are used. MOE suite implemented a modified version of GB/SA
contact function described by Still and co-authors.39 These terms
model the electrostatic contribution to the free energy of solva-
tion in a continuum solvent model. The interaction energy values
were calculated as the energy of the complex minus the energy of
4.9. UV-VIS titrations
Spectrophotometric titrations were performed with a Perkin-
Elmer
l20 in 20 mM HEPES, pH 7.1. Binding was followed by
addition of increasing amounts of ctDNA to a freshly prepared
ligand solution in 20 mM HEPES, pH 7.1. Experimental data were
analyzed in terms of relative variation of the absorbance as a
function of DNA concentration.
the ligand, minus the energy of DNA: DEinter ¼ E(complex)
-
(E(L) þ E(rDNA)).
Acknowledgments
4.10. CD measurements
The authors thank dr. Tiziana De Luca and dr. Silvia Masetto
for their involvement in synthetic work. This work was sup-
ported by Fondazione Beneficentia Stiftung, MIUR-PRIN
2010JMAZML and UNITS-FRA_2015 projects. The molecular
modeling work coordinated by S.M. has been carried out with
financial support from the University of Padova, Italy, and from
the MIUR, PRIN 2010e2011, 20103W4779_004 project. S.M. is
also very grateful to Chemical Computing Group for the scientific
and technical partnership.
Circular dichroism spectra were recorded at 25 ꢀC using 1 cm
path length quartz cells on a Jasco J 810 spectropolarimeter
equipped with a NESLAB temperature controller. CD spectra were
recorded on samples containing 80
mM ctDNA and 40 ligands
concentrations in 20 mM HEPES, pH 7.1. The reported spectrum of
each sample represents the average of 3 scans. Observed elliptic-
ities were converted to mean residue ellipticity [
q
] ¼ deg x cm2
x
dmolꢂ1 (Mol. Ellip.).
Please cite this article in press as: Sissi C, et al., Conjugates between minor groove binders and Zn(II)-tach complexes: Synthesis,
characterization, and interaction with plasmid DNA, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.04.013

C. Sissi et al. / Tetrahedron xxx (2017) 1e11
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Please cite this article in press as: Sissi C, et al., Conjugates between minor groove binders and Zn(II)-tach complexes: Synthesis,
characterization, and interaction with plasmid DNA, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.04.013