Second Generation G-Quadruplex Stabilizing Trimethine Cyanines

Article abstract of DOI:10.1021/acs.bioconjchem.9b00571

G-Quadruplex DNA has been recognized as a highly appealing target for the development of new selective chemotherapeutics, which could result in markedly reduced toxicity toward normal cells. In particular, the cyanine dyes that bind selectively to G-quadruplex structures without targeting duplex DNA have attracted attention due to their high amenability to structural modifications that allows fine-tuning of their biomolecular interactions. We have previously reported pentamethine and symmetric trimethine cyanines designed to effectively bind G-quadruplexes through end stacking interactions. Herein, we are reporting a second generation of drug candidates, the asymmetric trimethine cyanines. These have been synthesized and evaluated for their quadruplex binding properties. Incorporating a benz[c,d]indolenine heterocyclic unit increased overall quadruplex binding, and elongating the alkyl length increases the quadruplex-to-duplex binding specificity.

Full text of DOI:10.1021/acs.bioconjchem.9b00571

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Article
Second Generation G-Quadruplex Stabilizing Trimethine Cyanines
Eric A. Owens, Hang T. Huynh, Ekaterina Stroeva, Arghya Barman, Kostiantyn
Ziabrev, Ananya Paul, Sarah V. Nguyen, Matthew David Laramie, Donald
Hamelberg, Markus W. Germann, W. David Wilson, and Maged Henary
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00571 • Publication Date (Web): 13 Sep 2019
Downloaded from pubs.acs.org on September 14, 2019
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Second Generation G-Quadruplex Stabilizing Trimethine Cyanines
Eric A. Owens1,2,#, Hang T. Huynh1,#, Ekaterina M. Stroeva1,, Arghya Barman, Kostiantyn
Ziabrev1, Ananya Paul, Sarah V. Nguyen, Matthew Laramie, Donald Hamelberg1,2,*, Markus
Germann1,3,*, W. David Wilson1,2,*, Maged Henary1,2,*
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1Department of Chemistry, Georgia State University, Petit Science Center,
100 Piedmont Ave SE. Atlanta GA 30303
2Center for Diagnostics and Therapeutics, Georgia State University, Petit Science Center,
100 Piedmont Ave SE, Atlanta, GA 30303, USA
3Department of Biology, Georgia State University, Petit Science Center,
100 Piedmont Ave., Atlanta, GA 30303, USA
*Corresponding Authors’ E-Mail Addresses: dhamelberg@gsu.edu (D.H.); dmwg@gsu.edu
(M.G.); wdw@gsu.edu (W.D.W.); mhenary1@gsu.edu (M.H.).
#Authors contributed equally to this work.
Abstract. G-quadruplex DNA has been recognized as a highly appealing target for the
development of new selective chemotherapeutics, which could result in markedly reduced toxicity
towards normal cells. In particular, the cyanine dyes that bind selectively to G-quadruplex
structures without targeting duplex DNA have attracted attention due to their high amenability to
structural modifications that allows fine tuning of their biomolecular interactions. We have
previously reported pentamethine and symmetric trimethine cyanines designed to effectively bind
G-quadruplexes through end stacking interactions. Herein, we are reporting a second generation
of drug candidates, the asymmetric trimethine cyanines. These have been synthesized and
evaluated for their quadruplex binding properties. Incorporating a benz[c,d]indolenine
heterocyclic unit increased overall quadruplex binding and elongating the alkyl length increases
the quadruplex-to-duplex binding specificity.
Keywords. G-quadruplex, cyanine, thermal melting, circular dichroism, mass spectrometry,
nuclear magnetic resonance
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Introduction. Effective drug receptors that can be selectively and strongly targeted are a
requirement for the rational design of new therapeutics for improving human health. DNA has
remained an appealing target due to its involvement in fundamental biological processes in both
native and diseased cells. Selectively inhibiting the replication and/or transcription of nucleic acids
would target processes that eventually lead to diseased states.1,2 Alternative folding patterns of
DNA, such as Holliday junctions and quadruplexes, offer a potential, favorable route toward
effectively targeting the nucleic acid machinery of diseased cells.1-3 In line with this current trend,
we have been exploring ligands that bind and stabilize DNA G-quadruplex structures. A
quadruplex is formed from a stacked tetrad of self-associated guanine nucleotides that assemble
around a stabilizing cation. Quadruplex-forming sequences are present in oncogene promoter
regions and within telomeres. Both classes of quadruplex structures have been investigated in
several laboratories for the design of new chemotherapeutics.4,5 Either the transcription of
oncogene associated proteins or the enzymatic inhibition of telomerase, which is overexpressed in
cancer cells and is responsible for continued replication processes that require telomere elongation,
can potentially be targeted.
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Developing compounds that strongly and selectively bind and stabilize relevant quadruplex
sequences could lead to potent chemotherapeutics. With the goal of design of such compounds,
numerous small molecule scaffolds have been investigated. Among them, cyanine dyes have
shown a great deal of promise. Cyanines were already known to bind to several forms of DNA6-9,
and the first report using cyanines to identify G-quadruplex DNA took advantage of the unique
spectroscopic changes which occurred upon binding of the dye DODC to dimeric hairpin
quadruplexes.10 While the spectral response was specific for quadruplexes, DODC was found to
bind to all forms of DNA tested. Further exploration of the scaffold has led to the generation of
cyanines which display negligible binding to duplex and triplex DNA structures. The high
amenability of the cyanine pharmacophore to structural modification has driven the interest in
exploiting this class of compounds. Indeed, numerous reports have been published using cyanine-
quadruplex interactions to for basic detection of quadruplex structures11-14, detecting specific
analytes15-17, and, as we have previously reported, potential therapeutic use18,19. However, in many
cases, the interactions of cyanines with G-quadruplex DNA are not specific over interactions with
dual or single stranded DNA, but only result in specific and detectable changes to the optical
properties of the dye.14 We have previously reported the synthesis and biophysical evaluation of
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several symmetric tricationic cyanines with steric restraints to inhibit duplex interactions but which
allow binding to quadruplex structures.18 Considering the diversity and various groove
arrangements present in quadruplex structures an asymmetric cyanine could be well suited for
improving quadruplex interactions and selectivity. Asymmetric cyanines are widely employed as
nucleic acid labels and noncovalent probes after systematically manipulating their core structure
to suit individualized needs.7-9,20,21 Their remarkable amenability to these diverse modifications
furthers their applicability in nucleic acid targeting. Through a reverse engineering of these
compounds, we have drastically reduced duplex binding affinity through well-placed substituents
to provide steric restraints. A planar heterocyclic moiety was incorporated to facilitate potential
end-stacking with the quadruplex. The binding affinity and binding mode to quadruplex structures
occurring in telomeres and promoters are assessed by an array of biophysical techniques against
both human telomeric (Tel22) and cMyc (Myc19) sequences. In the presence of intracellular
concentrations of K+, both of these sequences form a G-quadruplex structure with the G bases
arranged in a tetrad orientation. While in Myc19 all strands in the quadruplex have a parallel
orientation in Tel22 only 3 of the 4 strands are parallel (3+1).22,23 Comparing the affinities of the
cyanine compounds will reveal differences and common targeting features that may be exploited.
A duplex forming DNA sequence will be used as a negative control.
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Results and Discussion
Synthesis. Our effective route toward second generation trimethine cyanine quadruplex binding
agents begins with the commercially available benz[c,d]indol-2(1H)-one and is outlined in Scheme
1. Treatment of this reagent with phosphorous pentasulfide effectively yields the corresponding
thione, which is activated through simple alkylation using iodomethane. Meldrum’s acid in a
highly basic environment will replace the methanethiol affording the enamine intermediate. At this
point, the heterocyclic nitrogen is activated to perform SN2 chemistry in basic media. Alkylation
was performed using either iodomethane or iodobutane to afford the tertiary amine. The reactive
and acidic methyl group was obtained through thermal and acidic decomposition of the protecting
Meldrum’s acid group leaving the terminal methyl moiety for further modification. Subjecting the
reactive methyl group to the Vilsmeier formylation reaction followed by alkaline cleavage affords
the intermediate for half of the final binding ligand. The alternate half is formed through the well-
established Fischer-indole reaction with halogenated phenyl hydrazine salts and 3-methyl-2-
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butanone which, in the presence of refluxing acetic acid, effectively affords the bicyclic products
through a hydrazone intermediate followed by a pericyclic rearrangement as shown in Scheme 2.
Alkylation of this heterocyclic precursor in dry acetonitrile with the corresponding bromopropyl
trimethylammonium bromide affords the highly hygroscopic dicationic salts for the second half of
the target binding agents. Combining each of the heterocyclic precursors in acetic anhydride yields
the final bright blue colored asymmetric cyanine binding ligands shown in Scheme 3.
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O
O
O
O
O
O
P2S5, Py
reflux
CH3I
O
EtOH
Et3N
S
S
O
NH
N
N
N
O
H
H
2
3
1
R-I
K2CO3
DMF
O
O
O
AcOH, KI
HCl
1. POCl3/DMF
O
2. Toluene
40% KOH
N I
R
N
N
R
O
R
8
9
: R = Me
: R = Bu
4
5
: R = Me
: R = Bu
6
7
: R = Me
: R = Bu
Scheme 1. Formation of the aldehyde intermediate of the benz[c,d]indolenine heterocyclic unit for
reaction to form the final quadruplex binding ligands.
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O
5
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Br
N
X
Br
X
X
HCl
NH2
AcOH
reflux
MeCN
reflux
N
N
N
H
Br
14 X
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N
Br
:
= H
= F
= Cl
= Br
10 X
:
= H
= F
= Cl
= Br
15 X
:
11 X
:
16 X
:
12 X
:
17 X
:
13 X
:
Br
Br2
AcOH
O
O
Br
N
Br
O
O
O
O
O
O
1. DMF, K2CO3
45 oC, 12h
N
MeCN
reflux
NH2
N
Br
2.p-TSA, toluene
125 oC, 18h
N
Br
18
19
Br
N
Br
S
N
S
MeCN
reflux
N
Br
N
Br
20
Scheme 2. Preparation of dicationic salts 14-17, 19, 20.
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X
X
Ac2O
O
R
R
= Bu
= Me
21
22
23
24
: X = H
: X = F
: X = Cl
: X = Br
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28
: X = H
: X = F
: X = Cl
: X = Br
N
N
R
Br
N
N
R
Br
N
Br
N
21-28
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8 R
9 R
:
:
= Me
= Bu
14 X
:
= H
= F
Br
15 X
:
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17 X
:
:
= Cl
= Br
O
N
R
R
= Bu
= Me
O
O
O
Ac2O
29
30
N
N
R
N
Br
Br
N
29, 30
19
Br
Br
S
N
Ac2O
R
R
= Bu
= Me
S
31
32
N
N
R
Br
Br
Br
N
Br
N
20
31, 32
Scheme 3. Final synthetic step for compounds 21-32.
H
1. ACN, Et3N
1. P2S5, Py
BuI
2. NaClO4
EtOH
O
O
S
2. CH3I
N
N
N
N
N
I
H
ClO4
N
ClO4
34
35
33
Scheme 4. Preparation of the monomethine cyanine compound for quadruplex binding.
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1.
O
O
5
6
7
8
O
O
EtOH
Et3N
Br
Br
1. P2S5, Py
2. CH3I
Br
Br2, CHCl3
O
S
O
2.
AcOH, KI
HCl
9
N
N
N
N
I
I
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Br
Ph
N
Ph
X
Ph
N
N
X
39
Pyridine
H
X
1:1
N
O
N
Ac2O
Br
N
Br
N
N
Br
Br
Br
N
N
14 X
17 X
:
:
= H
= Br
40 X
42 X
: = H
:
:
= H
41 X
43 X
: = Br
= Br
Scheme 5. Synthetic route for the brominated benz[c,d]indole ring and formation of the trimethine
cyanine structure.
Routes to two synthetic precursors were employed to generate potential quadruplex binding
agents based on our results in previous publications. We have seen that the length between
heterocycles can influence the binding interactions; additionally, heterocyclic halogenations were
observed to increase the binding strength to the quadruplex. We were interested in whether
shortening the polymethine bridge by one vinylene (-CH=CH-) unit would make a substantial
change in the quadruplex binding as determined by thermal melting. The benzo[c,d]indole-
intermediate was alkylated and in basic conditions, the dicationic salt 14 is nucleophilic enough to
displace the thiomethane group to afford the monomethine cyanine shown in Scheme 4.
Surprisingly, the binding was abolished when shortening the compound by 2.96 angstroms (as
determined by measurements of the lowest energy structures minimized using DFT calculations
with the B3LYP basis set), likely due to the loss of co-planarity of the heterocyclic moieties as
illustrated in Figure 1. We therefore we discontinued the pursuit of monomethine cyanines as
strong quadruplex interacting agents. While the trimethine cyanine compounds are planar steric
repulsion causes a twist in the monomethine compounds.
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In order to strengthen the quadruplex interactions, incorporating heterocyclic
halogenations on the trimethine benz[c,d]indolenine heterocyclic portion using the chemistry
shown in Scheme 5 should yield an increase in binding interaction, as we previously found.
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Figure 1. Molecular depictions of the most energetically favorable state for monomethine
compound compared to trimethine analog showing drastically different twisting associated with
the reduction of the polymethine-bridge and corresponding ΔTm measurements at 4:1
compound:Tel22 ratio.
The synthesis of brominated compounds (monobromo 42 and dibromo 43) involves a varied
synthetic route starting with the alkylation of the thioester compound affording the butyl
derivative—as it showed improved G4 binding compared to the methyl counterpart. The
heterocyclic portion, para to the tertiary amide nitrogen, was found to be sufficiently activated for
effective bromination. This intermediate was prepared and reacted in similar fashion as previously
described to afford the methylene intermediate. Correspondingly, the reaction of dicationic salts
(14 and 17) with diphenylformamidine in a 1:1 molar ratio in boiling acetic anhydride with careful
reaction monitoring affords the half-reacted compound which undergoes loss of acetanilide by
nucleophilic displacement by the brominated benzo[c,d]-containing analog.
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Biophysical Evaluation. As an initial screen to test selective binding to quadruplex over duplex
DNA, we utilized UV-thermal melting with a telomere model DNA of 22 bases (Tel22).22,24
Following the initial screen more quantitative surface plasmon resonance binding studies were
done with compounds that selectively stabilize quadruplex DNA over duplex DNA. In the SPR
studies a 19 mer model sequence for the Myc oncogene (Myc19) was also used to test compound
binding to different G-quadruplexes.23,25 A duplex DNA (AATT) was used as a control. In addition
to binding affinity and stoichiometry, SPR also provides binding kinetics. Finally, circular
dichroism (CD) spectroscopy was performed with representative compounds 26 and 28 to gain
insight into the binding mode of the compounds to the quadruplex structure.
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Table 1: General molecular structures, and UV-vis thermal melting Tm changes of new asymmetric
trimethine cyanine binding agents with telomeric quadruplex (Tel22) and a control hairpin duplex
DNA sequence (AATT)
X
X
X
X
O
O
S
N
N
N
N
N
N
R
R
R
N
N
N
31, 32
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Tel22: AGGGTTAGGGTTAGGGTTAGGG-3'
Myc19: AGGGTGGGGAGGGTGGGGA-3'
C
5-CCAATTCG
3-GGTTAAGC
T
C
AATT :
T
Heterocyclic
Modifications
Tel22a
(Compound:DNA)
AATTb
(Compound:DNA)
ID
Benz[c,d]
Other
-H
-F
-Cl
-Br
-H
(1:1)
4.4
4.3
0.0
2.7
2.1
1.1
(2:1)
9.9
9.9
7.4
9.0
7.8
2.9
(4:1)
17.4
18.0
16.5
16.4
20.6
19.8
(6:1)
22.5
22.0
22.1
21.8
26.3
25.3
(4:1)
3.6
3.4
3.5
3.3
2.3
1.1
21
22
23
24
25
26
Methyl, -H
Methyl, -H
Methyl, -H
Methyl, -H
Butyl, -H
Butyl, -H
-F
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29
Butyl, -H
Butyl, -H
Methyl, -H
-Cl
-Br
-O-CH2-O-
4.2
2.5
2.2
9.2
11.2
5.7
22.8
20.9
6.3
23.3
23.3
---
2.2
2.2
---
7
8
9
30
Butyl, -H
-O-CH2-O-
1.1
4.1
---
24.8
2.0
31
32
35
42
43
Methyl, -H
Butyl, -H
Butyl, -H
Butyl, -Br
Butyl, -Br
-S-
-S-
-H
-H
-Br
2.5
2.5
1.0
1.0
1.1
6.1
9.5
0.5
2.0
4.3
---
---
0.9
8.7
---
---
---
1.4
---
---
5.6
---
0.3
2.3
0.6
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aTel22: 3µM G-quadruplex DNA, 50 mM TRIS buffer with a KCl concentration of 50 mM, pH
7.5. At this salt concentration Tel22 shows a melting temperature of 60 0C.
bAATT: 6 µM hairpin duplex DNA, 50 mM TRIS buffer with a potassium concentration of 50
mM. At this salt concentration the sequence shows a melting temperature of 74 0C.
Following thermal melting evaluation (Table 1) SPR was employed to obtain more direct
binding information, Myc19 has high Tm and cannot be used in a screen but the Tel22 and Myc19
are used in SPR. When the methylated compounds, 21-24, were examined, the SPR response
indicated strong binding affinity to both Myc19 and Tel22; however, the butylated counterparts
revealed higher affinity for Myc19 compared to the methyl analogs. Two representative
sensorgrams for methylated compound 24 and butylated compound 28 are shown in Figure 2.
Additionally, the butyl-substituted ligand displayed selectivity with the binding affinity to Myc19
being higher (Table 2). These results support the Tm findings and offer more concrete and
quantitative insight into the binding strength of these asymmetric trimethine compounds to the two
different quadruplex forming sequences.
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Figure 2. Representative Surface Plasmon Resonance-Biosensor Sensorgrams. The
brominated G4 binding agents 24 (methyl) and 28 (butyl) were examined with immobilized Tel22
and Myc19 DNA. Compounds were in the flow solution and concentrations are color coded as in
the Figure. The SPR RU response clearly shows that there are two binding sites with different KD
values. We fit the curves to a two binding site equation.
Table 2. Equilibrium binding constants of the asymmetric cyanine analogs with Tel22 and
Myc19 quadruplex sequences obtained from SPR studies.
ID
21
22
23
24
25
26
Myc19 (K1 ; K2) M-1
5.3 x 106 ; 6.1 x 105
4.9 x 106 ; <105
Tel22 (K1 ; K2) M-1
3.6 x 106 ; 9.1 x 105
6.9 x 106 ;<105
2.0 x 107 ; 6.4 x 105
5.5 x 106 ; <105
1.2 x 107 ; 3.2 x 106
7.9 x 106 ; <105
2.9 x 107 ; 8.1 x 106
3.5 x 107 ; 2.8 x 106
6.0 x 106 ; 2.0 x 106
8.4 x 106 ; 2.9 x 105
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1.4 x 107 ; 4.3 x 105
4.4 x 106 ; 8.1 x 105
2.6 x 106 ; <105
NB
1.1 x 108 ; 3.8 x 106
NB
9
5.1 x 106 ; 3.5 x 105
7.1 x 106 ; 6.6 x 105
5.5 x 106 ; < 105
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4.4 x 106 ; 1.8 x 106
Experiments have the reproducibility within 10%, and the two
K values were obtained by fitting the results with the two site
equation in the Experimental section
NB: no binding based on Tm
The two observed KA values in Table 2 differ over a range of around 3 to quite large. They may
represent compound binding at the two ends of the quadruplex which provide different
environment.
A. 28_Myc19
A. 28_Tel22
Figure 3. UV-vis absorbance spectra of 2 μM 28 titrated with Myc19 (A) and Tel22 (B) in 50
mM K+ buffer. Experiments were done with DNA in Tris–HCl/ 50 mM K+ buffer at pH 7.4.
Titration was done until no further changes occur. Break down of 28_Myc19 titration A1) 0.0 –
6.0 μM and A2) 7.5 - 30.0 μM Myc19.
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To test the model with two KA values an absorption titration of compound 28 was done with Myc19
(Figure 3). Tel22 shows a monophasic titration profile with an isosbestic point at 670 nm. The
spectra are red shifted and show 35% hypochromicity upon drug binding. Similar shifts and
hypochromicity are also observed for Myc19. However, there was a loss of isobestic behavior as
the concentration of the quadruplex was increased as shown in inset A1 and A2, which supports
two different binding events. This suggests that there are two different binding constants of 28 on
the DNA in agreement with SPR results.
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Figure 4. CD spectra of the cyanine compound 26 (panel A) and compound 28 (panel B) with
Tel22 and cMYC19 G-quadruplex sequences. The ligands were titrated into the G-quadruplex
solutions (5 µM) until no further change in ICD signals were obtained. The insets show the mole
ratio of drug:DNA.
To further elucidate how these compounds are binding to G-quadruplex DNA, CD studies were
conducted to qualitatively evaluate the possible binding modes of the cyanine analogs with the G-
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quadruplex structures. Upon complex formation with biomolecules, achiral ligands exhibit an
induced CD (ICD) signal which is generally indicative of the binding mode of the ligand 26. Weak
ICD signals are characteristic of G-quadruplex end-stackers, whereas, strong ICD signals are
characteristic of ligands with groove interactions 26,27. Figure 4 shows CD spectra for 26 and 28
with both the human telomeric and cMYC G-quadruplex sequences titrated until no further change
in the CD signal is observed. Compound 26 and 28 exhibit strong ICD signals at the compound
absorbance region (~520-760 nm) along with G-quadruplex DNA CD signal change (240–300 nm)
upon complex formation with Tel22 G-quadruplex sequence suggesting that with the end staking
at the terminal tetrads, these two compounds also form stacked complexes in multiple grooves of
the telomeric G-quadruplex sequence (Figure 4A and Figure 4B). In the cMYC sequence, both
compounds show strong G-quadruplex DNA CD signal change (240–280 nm), indicates that the
strong end staking interaction. The lack or weak ICD signal for both compounds, 26 and 28, with
cMYC sequence point out no groove interaction.
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Nuclear Magnetic Resonance Spectroscopy. NMR studies for Tel24 (Figure 5) were carried out
to locate the binding site(s) and to guide subsequent molecular modeling studies. The imino
protons of Tel24 were previously assigned23,28 and serve as convenient monitors of the binding
event. Tel24 was selected for NMR studies because it can form quadruplex structures in solution
that are more amenable for detailed NMR studies. We note that Tel22 and Tel24 quadruplexes
exhibit similar behavior in the UV melting screen (Supporting Information) indicating similar
binding affinity and binding modes.
The imino proton NMR spectra in Figure 5 shows the titration of Tel22 with compound 22 and 26.
In both cases, there are several imino protons that are broadened and shifted upon binding of the
ligands which localizes the primary binding location. For both compounds, imino protons
corresponding to G3, G9 and G17 indicated by the arrows are affected which reveals that the
bottom tetrad, the 3’ side, is the major binding site. Interestingly, the presence of the butyl group
in 26 results in a different perturbation map on the bottom tetrad suggesting different binding
orientation. In addition, and in contrast to compound 22 there is no perturbation of the top tetrad
up to a 1:1 titration for compound 26. Based on the SPR data the secondary binding interaction for
26 is >10 fold weaker and would therefore not be significantly populated at a 1:1 ligand quadruplex
ratio. For 22 the binding is weaker and therefore binding to the top tetrad becomes more likely.
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For both compounds, if the ligands are in excess of a 1:1 ratio the remaining resonances get less
intense and much broader suggesting exchange broadening on binding of the second ligand.
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Figure 5. Imino proton spectra of the Tel24 quadruplex titrated with compound 22 (left) and
26 (right). Ligands were added at the ratio indicated on the plot, where 1:0 ratio corresponds to
the imino proton spectrum of Tel24 in absence of ligand. The assignment of the imino protons is
indicated by the numbers on the quadruplex schematics and color coded according to the effect of
the ligand on the imino protons. Red residues are strongly affected, yellow are somewhat affected,
and green are not affected.
Molecular Modeling. The SPR results (Table 2) show almost all the compounds bind to the Tel
and Myc sequences with similar affinities, except for Compound 28, which binds to Myc19
approximately two orders of magnitude stronger than to Tel. It is not clear from an atomistic
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perspective why compound 28 is more selective for Myc than Tel. The NMR results suggest that
the compounds primarily stack at the end of the quadruplex (Figure 5). Using the NMR results as
a guide, we carried out multiple molecular dynamics simulations to examine the binding mode and
exact points of contact with the quadruplex using compounds 24 and 28 (Figures 6 and 7).
Compound 24 binds Myc and Tel with similar affinities, while compound 28 binds Myc better
than Tel. We see that the planar benz[c,d] heterocyclic portion inserts under the quadruplex loop
which is logical based on the steric restraints between the tetraplex surface and the dynamic look
for both compounds. The alternate heterocyle bearing the ammonium arm aligns itself well with
the edge of the quadruplex and the quaternary ammonium group inserts itself in the available
groove of the quadruplex. This binding paradigm is conserved in both the methylated and butylated
compounds.
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Interestingly, the quaternary ammonium group of compound 24 interacts with the groove
of Myc and Tel similarly (Figures 6A and 7A). On the other hand, the quaternary ammonium group
of compound 28 makes more contacts with the groove of Myc than Tel, suggesting that more
favorable contact is made between compound 28 and Myc than between compound 28 and Tel.
These results complement the binding results from SPR, suggesting that the extra interaction of
compound 28 with the group of Myc is responsible for the higher binding affinity. The results
suggest that the binding affinity of the compounds could be enhanced with that quadruplex by
taking advantage of the additional interaction with the groove.
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Figure 6. Molecular dynamics snapshots for the binding of compounds 24 and 28 to Myc
DNA. (A) compound 24 with Myc, (B) compound 28 with Myc.
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Figure 7. Molecular dynamics snapshots for the binding of compounds 24 and 28 to Tel
DNA. (A) Compound 24 with Tel, (B) Compound 28 with Tel.
Conclusion. Through an optimized synthetic route, we have prepared second generation
trimethine cyanines that display high binding affinity to quadruplex DNA with high selectivity
over duplex sequences. The benz[c,d]indole displays a highly planar surface that could interact by
stacking with the quadruplex ends, especially when the heterocyclic nitrogen atom bears a single
methyl group. SPR data of these compounds binding to quadruplexes indicate more than one site
of interaction where the primary site is stronger followed by another 10-fold weaker binding site.
These SPR data also showed selectivity in that this second-generation trimethine cyanines
demonstrates stronger binding and stability with parallel Myc19 over the 3+1 quadruplex structure
Tel22. When the preferred binding site becomes saturated, the compounds interact with the second
site, which has a lower binding constant, as seen in Figures 2-5. We have observed that by
elongating the alkyl chain duplex DNA binding is suppressed, probably due to the steric repulsion
arising from the butyl chain. This prevents binding to duplex DNA but allows favorable
interactions with quadruplex grooves. SPR data reveal that compounds that have butyl chains also
preferentially bind with parallel quadruplex structures. NMR guided molecular modeling suggest
an interesting end stacking binding mode and additional groove interactions facilitated by the
quaternary ammonium arm. These NMR results explain the two binding modes with different K
values observed in SPR experiments. These structures suggest that developing asymmetric binding
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ligands may help take advantage of the asymmetry involved in many quadruplex forming
sequences.
7
8
9
Materials and Methods. All starting reagents were obtained commercially from Sigma Aldrich
or Matrix Scientific and were used without purification. Nuclear magnetic resonance experiments
were performed on a Bruker Avance 400 MHz spectrometer and processed using Topspin 3.1.
Chemical shift values are reported versus tetramethylsilane as an internal standard purchased in
the commercially obtained deuterated solvents (Cambridge Isotope Laboratory). The reactions
were followed using silica gel 60 F254 thin layer chromatography plates (Merck EMD Millipore,
Darmstadt, Germany). Open column chromatography was utilized for the purification of all final
compounds using 60-200u, 60A, classic column silica gel (Dynamic Adsorbents, Norcross, GA).
UV-Vis/NIR absorption spectra were recorded on a Varian Cary 50 spectrophotometer. High-
resolution accurate mass spectra (HRMS) were obtained either at the Georgia State University
Mass Spectrometry Facility using a Waters Q-TOF micro (ESI-Q-TOF) mass spectrometer or
utilizing a Waters Micromass LCT TOF ES+ Premier Mass Spectrometer. Liquid chromatography
utilized a Waters 2487 single wavelength absorption detector with wavelengths set between 640
and 700 nm depending on the particular photophysical properties. The column used in LC was a
Waters Delta-Pak 5 uM 100A 3.9 x 150 mm reversed phase C18 column. Evaporative light
scattering detection analyzes trace impurities that cannot be observed by alternate methods; a
SEDEX 75 ELSD was utilized in tandem with liquid chromatography to confirm purity (>95% as
determined by LC-ELSD-MS).
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Synthesis.
S
N
H
1
Benzo[cd]indole-2(1H)-thione (1): Freshly distilled pyridine (200 ml) was added to
commercially obtained benzo[c,d]indol-2(1H)-one (53.9 g, 0.24 mol) and phosphorus pentasulfide
(80 g, 0.36 mol). This mixture was heated at 110 °C for 2h and then cooled to room temperature.
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DI water (650 ml) was added to the reaction mixture and the resulting suspension was allowed to
stand for 1h. An oily residue was obtained which was extracted with diethyl ether (3 x 350 ml) and
the organic layer was washed with 1M HCl solution followed by brine. The organic layer was
collected and dried over MgSO4. The solvent was evaporated to afford the product as orange oil
(53.1 g) which was used in the next step without additional purification.
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2-(methylthio)benzo[c,d]indole (2) and 5-(benzo[cd]indol-2(1H)-ylidene)-2,2-dimethyl-
1,3-dioxane-4,6-dione (3) were synthesized as previously reported.29,30
O
O
O
N
O
R
4
5
: R = Me
: R = Bu
A mixture of compound 3 (10.2 mmol), alkyl iodide (30.6 mmol) and K2CO3 (30.6 mmol) were
heated in DMF (40 mL) at 90 °C for 18 h under a nitrogen atmosphere. The mixture was cooled
to room temperature and then filtered. The filtrate was concentrated under reduced pressure. The
residue was purified on silica gel (flash column chromatography, using EtOAc–hexanes, 1:2 as an
eluent).
Quaternized benzo[c,d]indolinium salts 6 and 7 were prepared as previously described.29,30
N I
R
6
7
: R = Me
: R = Bu
Ester 4 or 5 (2.9 mmol) was dissolved in acetic acid (4 mL) and the mixture was refluxed
for 20 min. Concentrated HCl (4 mL) was added dropwise to the refluxing mixture until the color
changed from red to green. The mixture was cooled to room temperature, and saturated KI solution
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was added until the product started to precipitate. The product was filtered off, washed with ether,
5
6
and dried in vacuo affording quaternary ammonium salts 6 and 7 in good yield.
7
8
The benzo[c,d]indolenine aldehydes 8 and 9 were prepared using previously published
9
procedures.31
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O
N
8
(E)-2-(1-methylbenzo[cd]indol-2(1H)-ylidene)acetaldehyde (8): Yield 20%; Mp 149-151
°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.48 (s, 3H), 5.79 (d, J = 7.6 Hz, 1H), 7.15 (m, 1H), 7.51-
7.70 (m, 2H), 7.71 (t, J = 8.9 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 8.58 (d, J = 7.2 Hz, 1H), 10.30 (d,
J = 7.6 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 30.08, 102.92, 105.17, 119.29, 125.74, 126.52,
129.54, 129.57, 130.11, 130.26, 143.16, 156.32, 186.25.
O
N
9
(E)-2-(1-butylbenzo[cd]indol-2(1H)-ylidene)acetaldehyde (9): Yield 41%; Mp 178-180
°C; 1H NMR (400 MHz, DMSO-d6) δ: 0.19 (t, J = 7.6 Hz, 3H), 1.36-1.41 (m, 2H), 1.64 (p, J = 7.2
Hz, 2H), 3.98 (t, J = 7.6 Hz, 2H), 5.83 (d, J = 7.6 Hz, 1H), 7.13 (t, J = 7.2 Hz, 1H), 7.50 (s, 2H),
7.72 (t, J = 7.6 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 8.59 (d, J = 7.2 Hz, 1H), 10.32 (d, J = 7.6 Hz,
1H); 13C NMR (100 MHz, DMSO-d6) δ: 14.22, 20.07, 29.82, 42.60, 102.96, 105.34, 119.27,
126.70, 129.59, 130.17, 130.38, 142.82, 155.37, 186.36.
Heterocyclic salts 14-20 were prepared according to previously published procedures and
the spectra were in agreement with the previous results.32
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N
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N
Br
14
2,3,3-Trimethyl-1-(3-(trimethylammonio)propyl)-3 H-indol-1-ium bromide (14): Yield 61
%; Mp 197-199 °C.
F
N
Br
N
Br
15
5-Fluoro-2,3,3-trimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium bromide (15):
Yield 90%; Mp 237-240 °C.
Cl
N
Br
N
Br
16
5-Chloro-2,3,3-trimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium bromide (16):
Yield 82%; Mp 215–217 °C.
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N
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N
Br
17
5-Bromo-2,3,3-trimethy`l-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium bromide (17):
Yield 81%; Mp 225–227 °C.
O
O
N
Br
Br
N
19
6,7,7-trimethyl-5-(3-(trimethylammonio)propyl)-7H-[1,3]dioxolo[4,5-f]indol-5-ium
bromide (19): Yield 70%; Mp 255-257 °C; 1H NMR (400 MHz, D2O) δ: 1.48 (s, 6H), 2.40-2.45
(m, 2H), 3.14 (s, 9H), 3.59 (t, J = 8.8 Hz, 2H), 4.44 (t, J = 8.0 Hz, 2H), 6.07 (s, 2H), 7.16 (s, 1H),
7.31 (s, 1H); 13C NMR (100 MHz, D2O) δ: 21.50, 21.85, 44.45, 53.26, 54.48, 62.56, 62.59, 96.56,
103.02, 103.84, 134.31, 137.03, 148.49, 149.67, 195.93.
S
N
Br
N
Br
20
2-methyl-3-(3-(trimethylammonio)propyl)benzo[d]thiazol-3-ium bromide (20): Yield
93%; Mp >260 °C; 1H NMR (400 MHz, DMSO-d6) δ: 2.35 (bs, 2H), 3.07 (s, 9H), 3.19 (s, 3H),
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3.62 (t, J = 8.4 Hz, 2H), 4.73 (t, J = 8.0 Hz, 2H), 7.76 (t, J = 7.6 Hz, 1H), 7.86 (t, J = 7.6 Hz, 1H),
13
8.31 (t, J = 7.6 Hz, 1H); C NMR (100 MHz, DMSO-d6) δ: 17.54, 22.04, 46.12, 53.23, 62.26,
116.95, 124.69, 128.80, 129.18, 130.23, 140.94, 177.87.
7
8
9
Synthesis of Final Trimethine Cyanines 21-32. Individual dicationic salt 14-17, 19 or 20
was added followed by acetic anhydride (3 mL). The slurry was allowed to warm to 40 °C while
stirring. After 30 minutes, benz[c,d]indolenine aldehyde 8 or 9 was added to the mixture. The
reaction progress was monitored by UV-Vis spectrophotometry and using neutral alumina TLC
eluting with 1% methanol in DCM. After the reaction was finished, the mixture was poured into a
mixture of ethyl acetate (40 mL) and diethyl ether (10 mL) under heavy stirring. The resulting
solid was filtered and dried in vacuo. The final products were purified by flash column
chromatography using neutral alumina eluting with a mixture of MeOH and DCM.
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H
N
N
Me
Br
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21
Br
3,3-Dimethyl-2-((1E,3E)-3-(1-methylbenzo[cd]indol-2(1H)-ylidene)prop-1-en-1-yl)-1-(3-
(trimethylammonio)propyl)-3H-indol-1-ium bromide (21): Yield 33%; Mp >245-247 °C; 1H NMR
(400 MHz, DMSO-d6) δ: 1.80 (s, 6H), 2.23 (brs, 2H), 3.14 (s, 9H), 3.66 (brs, 2H), 3.87 (s, 3H),
4.28 (brs, 2H), 7.04 (t, J = 13 Hz, 2H), 7.40 (t, J = 7 Hz, 1H), 7.53 (t, J = 7 Hz, 1H), 7.65-7.72 (m,
4H), 7.84 (d, J = 8 Hz, 1H), 8.02 (t, J = 7 Hz, 1H), 8.33 (d, J = 8 Hz, 1H), 8.39 (d, J = 7 Hz, 1H),
13
8.74 (t, J = 13 Hz, 1H); C NMR (100 MHz, MeOD-d4) δ: 21.22, 23.22, 27.77, 40.91, 49.67,
52.52, 52.55, 52.57, 52.60, 62.96, 105.74, 108.48, 109.82, 111.37, 122.34, 122.40, 122.93, 123.10,
125.88, 126.76, 127.75, 128.83, 129.29, 129.54, 129.96, 131.47, 141.38, 141.55, 149.66, 149.68,
149.70, 174.83; HRMS (ESI) calculated for [C31H37N3]2+ m/z 225.6488, found m/z 225.6478.
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Bioconjugate Chemistry
1
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F
5
6
7
8
9
N
N
Me
Br
10
11
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13
14
15
16
17
18
19
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22
23
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26
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N
22
Br
5-Fluoro-3,3-dimethyl-2-((1E,3E)-3-(1-methylbenzo[cd]indol-2(1H)-ylidene)prop-1-en-
1-yl)-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium bromide (22): Yield 49%; Mp >260 °C; 1H
NMR (400 MHz, MeOD-d4) δ: 1.88 (s, 6H), 2.41 (bs, 2H), 3.17 (bs, 2H), 3.29 (s, 9H), 3.83 (s,
3H), 4.36 (t, J = 7 Hz, 2H), 7.10 (t, J = 15 Hz, 2H), 7.28 (t, J = 9 Hz, 1H), 7.43 (d, J = 7 Hz, 1H),
7.49 (d, J = 8 Hz, 1H), 7.60-7.66 (m, 2H), 7.74 (d, J = 8 Hz, 1H), 7.86 (t, J = 8 Hz, 1H), 8.16 (d,
J = 8 Hz, 1H), 8.21 (d, J = 7 Hz, 1H), 8.79 (t, J = 13 Hz, 1H); 19F NMR (375 MHz, DMSO-d6) δ:
114.1; HRMS (ESI) calculated for [C31H36FN3]2+ m/z 234.6441, found m/z 234.6430.
Cl
N
N
Me
Br
N
23
Br
5-Chloro-3,3-dimethyl-2-((1E,3E)-3-(1-methylbenzo[cd]indol-2(1H)-ylidene)prop-1-en-
1-yl)-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium bromide (23): Yield 76%; Mp >260 °C; 1H
NMR (400 MHz, DMSO-d6) δ: 1.80 (s, 6H), 2.22 (bs, 2H), 3.16 (s, 9H), 3.75 (t, J = 8 Hz, 2H),
3.89 (s, 3H), 4.25 (t, J = 7 Hz, 2H), 7.16 (d, J = 13 Hz, 1H), 7.28 (d, J = 13 Hz, 1H), 7.57 (d, J =
8 Hz, 1H), 7.66-7.74 (m, 3H), 7.86 (d, J = 8 Hz, 1H), 7.90 (s, 1H), 8.02 (t, J = 8 Hz, 1H), 8.33 (d,
J = 8 Hz, 1H), 8.40 (d, J = 7 Hz, 1H), 8.71 (t, J = 13 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ:
21.46, 28.44, 49.91, 53.09, 62.08, 62.64, 106.83, 11123, 113.82, 123.62, 123.77, 124.75, 129.05,
129.88, 130.12, 130.47, 130.70132.28, 141.04, 141.78, 143.73, 149.56, 156.44, 174.23; HRMS
(ESI) calculated for [C31H36ClN3]2+ m/z 242.6293, found m/z 242.6283.
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Bioconjugate Chemistry
Page 26 of 42
1
2
3
4
Br
5
6
7
8
9
N
N
Me
Br
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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31
32
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34
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49
50
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60
N
24
Br
5-Bromo-3,3-dimethyl-2-((1E,3E)-3-(1-methylbenzo[cd]indol-2(1H)-ylidene)prop-1-en-
1-yl)-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium bromide (24): Yield 24%; Mp >260 °C; 1H
NMR (400 MHz, DMSO-d6) δ: 1.79 (s, 6H), 2.19 (bs, 2H), 3.12 (s, 9H), 3.65 (t, J = 8 Hz, 2H),
3.89 (s, 3H), 4.23 (bs, 2H), 7.03 (d, J = 13 Hz, 1H), 7.11 (d, J = 13 Hz, 1H), 7.60 (d, J = 8 Hz,
1H), 7.72-7.76 (m, 3H), 7.88 (d, J = 7 Hz, 1H), 8.02-8.06 (m, 2H), 8.36 (d, J = 8 Hz, 1H), 8.42 (d,
J = 7 Hz, 1H), 8.72 (t, J = 13 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 22.57, 26.19, 29.21,
30.44, 32.12, 42.42, 50.86, 51.01, 54.11, 64.41, 106.95, 110.22, 112.13, 114.29, 119.98, 125.23,
126.25, 127.30, 128.85, 130.73, 130.89, 131.19, 131.49, 133.22, 133.47, 142.45, 142.84, 144.86,
151.32, 158.77, 175.22; HRMS (ESI) calculated for [C31H36BrN3]2+ m/z 264.6041, found m/z
264.6041.
H
N
N
Bu
Br
N
25
Br
2-((1E,3E)-3-(1-butylbenzo[cd]indol-2(1H)-ylidene)prop-1-en-1-yl)-3,3-dimethyl-1-(3-
(trimethylammonio)propyl)-3H-indol-1-ium bromide (25): Yield 54%; Mp >164-165 °C; 1H NMR
(400 MHz, CDCl3) δ: 0.99 (t, J = 7 Hz, 3H), 1.61 (sextet, J = 7 Hz, 2H), 1.79 (s, 6H), 1.94 (quint,
J = 7 Hz, 2H), 2.44 (bs, 2H), 3.47 (s, 9H),4.37 (t, J = 7 Hz, 2H), 4.47 (t, J = 8 Hz, 2H), 4.64 (t, J
= 8 Hz, 2H), 7.18 (d, J = 7 Hz, 1H), 7.28 (t, J = 7 Hz, 1H), 7.38 (d, J = 7 Hz, 1H), 7.52 (t, J = 8
Hz, 1H), 7.57 (t, J = 8 Hz, 1H), 7.65 (d, J = 8 Hz, 1H), 7.80-7.85 (m, 2H), 7.98 (d, J = 13 Hz, 1H),
8.09 (d, J = 8 Hz, 1H), 8.16 (d, J = 7 Hz, 1H), 8.26 (d, J = 8 Hz, 1H), 8.80 (t, J = 13 Hz, 1H); 13C
NMR (100 MHz, CDCl3) δ: 13.61, 19.94, 21.46, 28.69, 30.90, 40.99, 44.28, 48.92, 53.85, 61.90,
107.24, 108.77, 110.09, 113.14, 121.34, 122.28, 125.10, 125.60, 125.81, 129.02, 129.07, 129.39,
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Page 27 of 42
Bioconjugate Chemistry
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5
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129.75, 130.71, 140.05, 141.02, 141.11, 149.16, 155.06, 173.29; HRMS (ESI) calculated for
[C34H43N3]2+ m/z 246.6723, found m/z 246.6005.
7
8
F
9
10
11
12
13
14
15
16
17
18
19
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21
22
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24
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N
N
Bu
Br
N
26
Br
2-((1E,3E)-3-(1-butylbenzo[cd]indol-2(1H)-ylidene)prop-1-en-1-yl)-5-fluoro-3,3-
dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium bromide (26): Yield 18%; Mp >181-
183 °C; 1H NMR (400 MHz, DMSO-d6) δ: 0.93 (t, J = 7 Hz, 3H), 1.48 (q, J = 7 Hz, 2H), 1.80 (s,
6H), 2.23 (bs, 2H), 3.18 (s, 9H), 3.83 (bs, 2H), 4.28 (bs, 2H),4.36 (bs, 2H), 7.34-7.41 (m, 3H),
7.62-7.82 (m, 6H), 8.02 (bs, 1H), 8.34 (bs, 2H), 8.73 (bs, 1H); 19F NMR (375 MHz, DMSO-d6) δ:
116.04; HRMS (ESI) calculated for [C34H42FN3]2+ m/z 255.6676, found m/z 255.6664.
Cl
N
N
Bu
Br
N
27
Br
2-((1E,3E)-3-(1-butylbenzo[cd]indol-2(1H)-ylidene)prop-1-en-1-yl)-5-chloro-3,3-
dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium bromide (27): Yield 67%; Mp >260
°C; 1H NMR (400 MHz, DMSO-d6) δ: 0.93 (t, J = 7 Hz, 3H), 1.49 (q, J = 7 Hz, 2H), 1.80 (s, 6H),
2.22 (bs, 2H), 3.08 (m, 2H) 3.15 (s, 9H), 3.78 (bs, 2H), 4.25 (t, J = 7 Hz, 2H), 4.38 (t, J = 7 Hz,
2H), 7.29 (d, J = 13 Hz, 1H), 7.42 (d, J = 13 Hz, 1H), 7.58 (d, J = 9 Hz, 1H), 7.67-7.74 (m, 3H),
7.86 (d, J = 8 Hz, 1 H), 7.90 (s, 1H), 8.03 (t, J = 8 Hz, 1H), 8.35 (d, J = 8 Hz, 1H), 8.41 (d, J = 7
Hz, 1H), 8.75 (d, J = 13.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 14.32, 20.07, 21.49, 28.50,
31.32, 41.86, 44.29, 49.89, 53.08, 62.61, 107.16, 109.42, 111.25, 113.85, 123.61, 123.68, 124.80,
128.29, 129.04, 129.59, 129.99, 130.17, 130.46, 130.74, 132.25, 141.05, 141.28, 143.78, 149.71,
155.59, 174.20; HRMS (ESI) calculated for [C34H42ClN3]2+ m/z 263.6528, found m/z 263.6518.
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Bioconjugate Chemistry
Page 28 of 42
1
2
3
4
Br
5
6
7
8
9
N
N
Bu
Br
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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N
28
Br
5-Bromo-2-((1E,3E)-3-(1-butylbenzo[cd]indol-2(1H)-ylidene)prop-1-en-1-yl)-3,3-
dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium bromide (28): Yield 50%; Mp >260
°C; 1H NMR (400 MHz, DMSO-d6) δ: 0.93 (t, J = 7 Hz, 3H), 1.50 (q, J = 7 Hz, 2H), 1.80 (s, 8H),
2.22 (s, 2H), 3.16 (s, 9H), 3.80 (bs, 2H), 4.25 (bs, 2H), 4.39 (bs, 2H), 7.31 (d, J = 14.0 Hz, 1H),
7.46 (d, J = 13.0 Hz, 1H), 7.69-7.72 (m, 3H), 7.86 (d, J = 8 Hz, 1H), 8.01 (m, 1H), 8.34 (d, J = 8.0
Hz, 1H), 8.41 (d, J = 7.0 Hz, 1H), 8.75 (t, J = 13.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ:
14.30, 20.08, 21.46, 28.50, 31.30, 41.80, 44.33, 49.88, 53.09, 62.71, 107.01, 109.35, 111.36,
114.21, 118.52, 123.77, 124.78, 126.35, 128.36, 129.60, 129.99, 130.18, 130.76, 131.88, 132.3,
141.28, 141.47, 144.05, 149.73, 155.70, 174.03; HRMS (ESI) calculated for [C34H42BrN3]2+ m/z
286.6265, found m/z 286.6253.
O
O
N
N
Me
Br
N
29
Br
7,7-dimethyl-6-((1E,3E)-3-(1-methylbenzo[cd]indol-2(1H)-ylidene)prop-1-en-1-yl)-5-(3-
(trimethylammonio)propyl)-7H-[1,3]dioxolo[4,5-f]indol-5-ium bromide (29): Yield 36%; Mp
251-253 °C; 1H NMR (400 1MHz, DMSO-d6) δ: 1.75 (s, 6H), 2.20 (s, 3H), 3.13 (s, 9H), 3.63 (bs,
2H), 3.82 (bs, 2H), 4.24 (bs, 2H), 6.16 (s, 2H), 7.10-6.92 (m, 2H), 7.47 (s, 1H), 7.50 (s, 1H), 7.69
(bs, 1H), 7.77 (t, J = 8.0 Hz, 1H), 7.99 (bs, 1H), 8.30 (q, J = 8.0 Hz, 2H), 8.63 (t, J = 12.0 Hz, 1H);
13C NMR (100 MHz, DMSO-d6) δ: 21.68, 24.68, 28.53, 42.20, 50.18, 52.76, 53.12, 62.77, 95.49,
102.71, 104.30, 107.68, 109.66, 122.59, 130.10, 130.55, 131.43, 135.91, 136.23, 147.00, 147.94,
148.47, 175.34; HRMS (ESI) calculated for [C32H37N3O2]2+ m/z 247.6471, found m/z 247.6426.
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Page 29 of 42
Bioconjugate Chemistry
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7
8
9
O
N
O
N
N
Bu
Br
10
11
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13
14
15
16
17
18
19
20
21
22
23
24
25
26
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Br
6-((1E,3E)-3-(1-butylbenzo[cd]indol-2(1H)-ylidene)prop-1-en-1-yl)-7,7-dimethyl-5-(3-
(trimethylammonio)propyl)-7H-[1,3]dioxolo[4,5-f]indol-5-ium bromide (30): Yield 63%; Mp
>260 °C; 1H NMR (400 MHz, DMSO-d6) δ: 0.93 (t, J = 7 Hz, 3H), 1.46-1.50 (m, 2H), 1.75-1.79
(m, 8H), 2.21 (bs, 2H), 3.15 (s, 9H), 3.74 (d, J = 7 Hz, 2H), 4.28 (t, J = 9 Hz, 2H), 6.15 (s, 2H),
7.18-7.28 (m, 2H), 7.47 (s, 1H), 7.53 (d, 11 Hz, 2Hz), 7.67 (t, J = 8 Hz, 1H), 7.75 (d, J = 8 Hz,
13
1H), 7.98 (t, J = 7 Hz, 1H), 8.25-8.31 (m, 2H), 8.64 (t, J = 12 Hz, 1H); C NMR (100 MHz,
MeOD-d4) δ: 12.94, 19.90, 21.31, 27.81, 30.83, 41.35, 43.71, 49.77, 52.62, 62.86, 94.11, 102.36,
103.14, 106.48, 107.82, 109.15, 122.35, 125.01, 126.33, 129.28, 129.46, 129.61, 130.20, 130.93,
135.52, 135.56, 141.22, 147.16, 148.30, 148.78, 154.79, 174.94; HRMS (ESI) calculated for
[C35H43N3O2]2+ m/z 268.6672, found m/z 268.6661.
S
N
N
Me
Br
N
31
Br
2-((1E,3E)-3-(1-methylbenzo[cd]indol-2(1H)-ylidene)prop-1-en-1-yl)-3-(3-
(trimethylammonio)propyl)benzo[d]thiazol-3-ium bromide (31): Yield 37%; Mp 230-232 °C; 1H
NMR (400 MHz, DMSO-d6) δ: 2.28 (bs, 2H), 3.05 (s, 2H), 3.16 (s, 9H), 3.72 (s, 2H), 4.62 (t, J =
7 Hz, 2H), 6.81 (d. J = 13 Hz, 1H), 7.41 (d, J = 6 Hz, 1H), 7.59 (d, J = 13 Hz, 1H), 7.59 (d, J = 13
Hz, 1H); 7.71-7.75(m, 4H), 7.87 (t, J = 8 Hz, 1H), 8.12 (d, J = 8 Hz, 1H), 8.20 (t, J = 8 Hz, 2H),
8.45 (d, J = 6 Hz, 1H), 8.59 (t, J = 13 Hz, 1H); 13C NMR (100 MHz, MeOD-d4) δ: 21.71, 29.52,
43.59, 52.22, 52.59, 62.56, 105.28, 107.66, 113.78, 121.34, 122.80, 124.89, 126.05, 126.15,
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