Small Sequence-Sensitive Compounds for Specific Recognition of the G?C Base Pair in DNA Minor Groove

A Journal of

Accepted Article

Title: Small Size Sequence-sensitive Compounds for Specific

Recognition of G·C Base Pair in DNA Minor Groove.

Authors: Abdelbasset Ahmed Farahat, Pu Guo, Hadir Shoeib, Ananya

Paul, David Boykin, and W.David Wilson

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To be cited as: Chem. Eur. J. 10.1002/chem.201904396

Link to VoR: http://dx.doi.org/10.1002/chem.201904396

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Small Size Sequence-sensitive Compounds for Specific Recognition

of G·C Base Pair in DNA Minor Groove.

Abdelbasset A. Farahat^1,2,*, Pu Guo¹, Hadir Shoeib¹, Ananya Paul¹, David W.

Boykin¹, W. David Wilson¹*

1. Department of Chemistry and Center for Diagnostics and Therapeutics Georgia

State University, 50 Decatur St SE, Atlanta, GA 30303, USA.

2. Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy,

Mansoura University, Mansoura 35516, Egypt

* Corresponding authors emails. A. A. Farahat (afarahat@gsu.edu ),

W.D. Wilson (wdw@gsu.edu)

Abstract.

A novel series of small size diamidines with thiophene and modified N-

alkylbenzimidazole σ-hole module represent specific binding to single G·C base pair

(bp) DNA sequence. The variation of N-alkyl or aromatic rings were sensitive to

microstructure of DNA minor groove. Thirteen new compounds were synthesized

to test their binding affinity and selectivity. The dicyanobenzimidazoles needed to

synthesize the target diamidines were made via condensation/cyclization reactions

of different aldehydes with different 3-amino-4-(alkyl- or phenyl-amino)

benzonitriles. The final diamidines were synthesized using lithium bis-

trimethylsilylamide (LiN[Si(CH₃)₃]₂) or Pinner methods. The newly synthesized

compounds showed strong binding and selectivity to AAAGTTT compared to

similar sequences AAATTT and AAAGCTTT investigated by several biophysical

methods including biosensor-SPR, fluorescence spectroscopy, DNA thermal

melting, ESI-MS spectrometry, Circular dichroism, and Molecular Dynamics. The

binding affinity results determined by fluorescence spectroscopy are in accordance

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with those obtained by biosensor-SPR. These small size single G·C bp highly

specific binders extend the compound database for future biological applications.

Keywords.

Biosensor-SPR, Diamidines, Minor Groove Binders, Molecular Dynamic

Simulation, N-alkylbenzimidazole G·C Base Pair.

1. Introduction.

Minor groove binders that can recognize mixed base pairs (bps) DNA with sequence

selectivity are of interest for a variety of research areas ^[1]. The finding that minor

groove binding compounds can be very effective inhibitors of major groove binding

proteins is particularly important for the use of these compounds as therapeutic

agents as well as biological probes ^[2-5]. The heterocyclic diamidine minor groove

binders that have been designed and prepared in our group have been demonstrated

to get into a variety of cell types and to have clinically useful biological activity with

low toxicity ^[6-8]. In addition, diamidines have been shown to be excellent inhibitors

[9,10]

of transcription factors (TFs) both in vitro

and in vivo ^[11,12]. The diamidine

minor groove binders synthesized previously have been essentially pure AT

interacting compounds. To recognize and inhibit a broad range of TFs, however, it

is essential to develop compounds that bind to the DNA sequence that contain G·C

as well as A·T bps. To do this, we have initiated a project to incorporate to G·C bp

recognizing modules into heterocyclic diamidines.

Ligands that can recognize mixed base pair sequences of DNA containing

A·T and G·C bps are an important development area for minor groove binders. At

first, the development took a major step forward with modification of the minor

groove binders, netropsin (Nt.) and distamycin (Dst.) ^[13-15]

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The proposal was to replace one or more pyrrole groups in Nt. and Dst. with N-

methylimidazole groups ^[13-15]. The extra N in N-methylimidazole relative to pyrrole

should allow that group to H-bond with the G-NH₂that protrudes into the minor

groove. Although several groups have worked with synthetic polyamides, solution

difficulties have limited their applications ^[16-18]

.

Our goal was to prepare entirely new types of compounds without the amide

group that could bind to mixed A·T and G·C bps containing sequences but would

also have varied chemical and structural properties that would allow them to

specifically bind to target sequences and effectively target a variety of different cell

types. The initial research in the area has produced quite different compounds that

are undergoing additional development to increase affinity and selectivity for the

target sequence type with a single G·C flanked by A·T bps ^[19-21]. Among them, the

N-MeBI-Thiophene containing compounds were found to be effectively modified to

improve their binding affinity and specificity ^[22]

.

Incorporating the N-MeBI thiophene unit into a heterocyclic cation base

structure, as in Figure 1, resulted in DB2429 which can recognize a target G·C bp

in an AT sequence ^[21]. Exploration of the importance of the N-MeBI-thiophene

module to binding and specificity shows that both the N-MeBI and thiophene are

essential for the strong, specific interactions with the target sequence. Replacing the

thiophene or N-MeBI with other similar structures decreased the binding affinity and

specificity. Compounds with the N-MeBI replaced by benzimidazole (BI), for

example, bind much more strongly to pure AT DNA sequences than to any sequence

with a G·C bp ^{[21, 22]}. For the design of strong binders in DNA minor groove,

compounds should have, for example, the appropriate curvature to fit the convex

shape of the minor groove. ^[23-27]

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One or two cationic groups are necessary for new compounds to aid solubility and

increase the electrostatic interaction with the negative DNA backbone ^{[23, 28, 29]}. For

example, amidine cationic groups, which have been quite successful for targeting

DNA, provide a hydrogen-bond-donating planar surface for optimizing interactions

with A·T bps at flanking sites of G·C bps. Also, the twist dihedral between amidine

and phenyl ring would help the compound structure to fit the helical curvature of the

minor groove ^[30,31]. Moreover, the groove binding ligand should include a H-bond

acceptor (N-MeBI) to recognize G-NH₂. In order to improve binding specificity, it

is important to attempt to take advantage differences in groove-width of targeted

sequences. Based on Rohs ^[25]and our work ^[22], the wider DNA minor groove at GC

versus AT sequences can be targeted. So, strategic structural modification of the

groove-binders could lead to optimization of binding specificity. The significant

improvement of binding specificity with such small changes in the overall

compound structure ^[21]encouraged us to design new agents to specifically recognize

mixed sequence DNA.

Figure 1. A) Chemical structure of a reported single G·C bp binder, DB2429, and the modified

compounds from DB2429 used in this study. B) The DNA sequences used in this study; DNA

sequences used for SPR studies were labeled with 5’-biotin.

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We have recently shown that larger compounds with the N-MeBI-thiophene

module can be modified to have significant improvements in binding specificity for

a target sequence with a single G·C bp ^[22]. This is a very significant step forward in

preparation of biologically useful compounds. For drug design, however, there is a

significant improvement in synthetic ease and cost by using smaller compounds such

as DB2429 and its analogs. In making these small molecules we used readily

available starting materials, avoided using the Palladium catalyzed expensive

organometallic reactions and the final products were obtained after few synthetic

steps. There is also a general improvement in cell uptake with smaller compounds

^{[30, 16-18]}. A problem with DB2429, however, is that its ratio of binding to a target

single G·C bp sequence over an equivalent pure AT sequence is only about a factor

of 10. A concept for improving this ratio is that by designing DB2429 analogs with

a variety of substituents, local variations in DNA microstructure could be recognized

better with higher binding selectivity^[32].

The first set of compounds that were prepared had modifications at the N-BI

position of DB2429 (Scheme 1, Table 1). We wanted to increase the size of the alkyl

group to reduce binding to narrow DNA sequences like AAATTT, and bigger size

substituents may fit the wider DNA minor groove such as AAAGTTT, for example,

methyl < ethyl < isopropyl < isobutyl < neopentyl substituents. We also employed

the N-trifluoroethyl DB2716 to test the different influence between the trifluoroethyl

and ethyl substitutions. The linear group with a more polar substituent as in the N-

propylmethoxy DB2722 was prepared by the same methods (Scheme 1). To

dramatically change the shape of the alkyl group, compounds with N-cyclobutyl

(DB2720), N-cyclopentyl (DB2713) and N-cyclohexyl (DB2721) substituents were

prepared. Finally, the substituent was significantly changed with a phenyl (Ph)

substituent on the BI group, N-Ph (DB2739). Additional compounds were prepared

with substitutions designed to test different σ-hole systems. The σ-hole system

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preorganizes the thiophene-N-XBI module for G·C bp interaction in the minor

groove. The C−S single bonds of thiophene ring presents a relatively positive

electrostatic potential that can form an interaction with electron-donating atoms such

as the unsubstituted N of N-XBI. The interaction is based on the presence of low

lying thiophene C−S σ* orbitals on S that give rise to the positive electrostatic

potential or a σ-hole ^[33]. One compound, with an N-MeBI was prepared with the

thiophene S replaced by Se to evaluate the effect of size and changes in polarizability

[34, 35]

at that position,

as well as, DB2591 with a thiazole structure replacing the

original thiophene ring. A control compound for the N-Me DB2591, the NH DB2652,

without an N-BI substituent, was also prepared. Brief synthetic details are given here

for the synthesis of the new compounds along with Scheme 1.

2. Results and Discussion

2. 1. Chemistry

Scheme 1 outlines the synthesis of the final thiophene-benzimidazole diamidines 6a-

n. Different alkyl and arylamines underwent nucleophilic substitution reaction with

4-fluoro-3-nitrobenzonitrile 1 to afford the alkyl and aryl substituted nitro

benzonitrile 2a-k. The previous nitro compound was reduced using stannous

chloride in ethanol to produce the diamine 3a-k. The diamine 3a-k underwent

oxidative condensation and cyclization with the thiophene, selenophene or thiazole

aldehydes 4a-d using sodium metabisulphite in DMSO to afford the prefinal

bisnitriles 5a-n. The final diamidines 6a-n were produced by allowing the bisnitriles

to react with either lithium bistrimethylsilylamide or ethanolic/HCl (Pinner

methodology).

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2.2. DNA Binding.

2.2.1. DNA Thermal Melting: Screening for mixed sequence binding.

We have used changes in DNA thermal melting temperature (T_m) to provide an

effective way for initial testing of compounds for binding affinity with different

DNA sequences. We have used the three sequences shown in Figure 1 for

comparative experiments. Most of the dicationic amidines reported so far have

shown pure AT sequence binding activity and a DNA with the binding site -

AAATTT- was used to test for AT binding affinity. Our primary interest was

studying DNA that has a single G·C bp with the binding sequence –AAAGTTT-.

The third sequence with two G·C bps was also used to test for binding affinity and

selectivity versus AAAGTTT.

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Table 1. Thermal Melting (ΔT_m, ℃)^a, Fluorescence Titration (K_D, nM )^b,

and SPR (K_D, nM)^cof Studied Compounds with Pure AT and Mixed DNA

Sequences.

ΔT_m(^oC) ^a

SPR AAAGTTT

Experiments

Sequences

AAA AAA

(K_D, nM) / AAATTT

(K_D, nM) /

FL (K_D,

AAA

TTT

64.5

(^oC)

G

GC

TTT

70.5

(^oC)

nM) ^b

TTT

66.5

(^oC)

Selectivity Ratio

vs AAATTT

Compounds

5

3

2

11

10

5

2

45 ± 2

44 ± 2

21 ± 2

51 / 522 / 10

49 / NB / NB

20 / NB / NB

6a

6b

6c

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2

9

2

33 ± 3

36 / NB / NB

6d

6e

30 ± 2

--

2

10

2

19 ± 2

6f

3

2

10

9

4

2

29 ± 2

21 ± 1

--

6g

6h

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2

3

9

2

3

15 ± 2

17 ± 1

10 ± 4

21 / NB / NB

6i

10

11

--

6j

8 / 1330 / 166

6k

5

13

3

8

7

9

3

4

3

59 ± 2

82 ± 4

83 ± 2

--

6l

6m

6n

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a. ΔT_m= T_m(the complex) - T_m(the free DNA). 3 µM DNA sequences were tested in TNE100

buffer with the ratio of 2:1 [ligand] / [DNA]. An average of two independent experiments with a

reproducibility of 0.5 ℃.

b. Binding affinity (K_D, nM) calculated from fluorescence experiments for 20 nM compound

titrated with sequence AAAGTTT in TNE100 buffer at 25 °C. The excitation wavelength is 337

nm. Two independent experiments were studied. The errors represent the standard errors of the

mean.

c. SPR binding affinity (K_D, nM) were investigated in Tris-HCl buffer (50 mM Tris-HCl, 100

mM NaCl, 1 mM EDTA, 0.05% P20, pH 7.4) at a 100 μL·min-1 flow rate. “--” means not done,

“NB” means binding between ligands and sequence AAATTT is no detectible binding.

The lead compound, DB2429, with an N-Me substituent was previously

reported ^[21]and has strong binding to the single G·C bp sequence, the ΔT_mis 11 ℃,

with lower ΔT_mand weaker binding to the AT and two G·C bps sequences, 5 and

5 ℃ respectively. Except for the N-MeBi-selenophene DB2665 and the NH DB2652,

the rest of the tested compounds with different bulky alkyl and aromatic groups

showed similar binding to DB2429 with AAAGTTT and much weaker binding to

both the AAATTT and AAAGCTTT sequences. Most of the N-substitutions

modified compounds showed improved selectivity to AAAGTTT as planned (Table

1). DB2665 with selenophene as a spacer will be helpful to future cell uptake studies,

however, showed less selectivity towards AAAGTTT. Since the bigger size of

selenium over sulfur, the entire structure of DB2665 is more linear than DB2429 and

does not to fit the minor groove curvature well. DB2652 with thiazole as a spacer

and unsubstituted NH of benzimidazole showed strong binding to AAATTT

sequence than other tested sequences and this agrees with other previously tested

NH-benzimidazole compounds prepared in our lab ^[36]. DB2591 with thiazole as a

spacer and N-Me substituted benzimidazole still shows strong binding and

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selectivity towards AAAGTTT sequence compared to other tested sequences and

this indicates that changing the spacer from thiophene to thiazole preserves but

weakens the target DNA binding activity and selectivity. We conclude that changing

the alkyl substituents on the benzimidazole moiety has no large effect on the

AAAGTTT binding activity but has a great effect on increasing the selectivity

towards that sequence. The presence of these alkyl substituents is crucial for binding

and selectivity towards AAAGTTT and its absence leads to compounds with strong

binding to pure AT sequences.

To test the sequence sensitivity of the compounds, the wider flanking

sequence with single G·C bp ATAGTAT was used in DNA melting experiments

(Table S4). Comparing to ATAGTAT and AAAGCTTT, the sequence AAAGTTT

is still the strongest binding target for this type of compound. Since the G·C bp is

present in the ATAGTAT sequence, the binding affinity is stronger than two G·C

bps sequence. Depending on the different microstructure on flanking part between

AAAGTTT and ATAGTAT, the bigger group modification of N-MeBI only

improves the G-binding part but not the AT-flanking part.

2.2.2. Fluorescence Emission Spectroscopy: Binding Affinity

The aim of binding assays is to measure quantitatively the interactions between two

molecules, such as a small molecule binding with DNA. If a change in the

fluorescence intensity accompanies the binding of two species, this can be used to

quantitatively monitor the binding interaction and determine the stoichiometry of

binding using equilibrium titration methods. Such methods have been used

extensively to examine numerous drug–DNA interactions in solution including

dicationic amidines-DNA interactions. Generally, if the molecule changes its

fluorescence, then this species is titrated with the non-fluorescent molecule (e.g.,

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DNA) and the change in fluorescence intensity is monitored as a function of total

DNA concentration ^[12,30]

.

Figure 2. A, C, E) Fluorescence Emission spectra for 20 nM DB2429, DB2713 and DB2717

titrated with sequence AAAGTTT in TNE100 buffer at 25 °C. The excitation wavelength is 337

nm. The slit width is [10, 5 nm]; B, D, F) Fluorescence binding curve between 20 nM DB2429,

DB2713 and DB2717 and sequence AAAGTTT in TNE100 buffer to determine equilibrium

constant; G) Representative SPR sensorgrams for DB2713 in the presence of AAAGTTT

sequence, concentrations of DB2713 from bottom to top are 2-500 nM.; H) SPR steady-state

binding plots for DB2713 with AAAGTTT sequence. The data are fitted to a steady-state binding

function using a 1:1 model to determine equilibrium binding constants.

The fluorescence intensity of our tested compounds decreased on titration

with increasing concentrations of the sequence AAAGTTT. The binding affinities

(K_Dvalues) were determined by fluorescence titration experiments are shown in

Table 1. Generally, the N-alkyl-substituted benzimidazole diamidines bind strongly

to the DNA sequence AAAGTTT as it appears from the K_D. The N-phenyl

substituted benzimidazole diamidine DB2739 is the strongest binder to DNA with

K_Dof 10 nM. Compounds with alkyl substitutions on the benzimidazole bind

strongly to DNA such as the N-isopropyl DB2692, N-isobutyl DB2710, and N-

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methyl with selenophene DB2665 except for the methyl and ethyl substituted

DB2429, DB2689 that showed slightly weaker binding. In the meantime,

compounds with cycloaliphatic substitutions such as N-cyclopentyl (DB2713) and

N-cyclohexyl (DB2721) showed very good binding. To test the effect of increasing

the length and bulkiness of the N-alkyl substituent, we prepared the N-isobutyl

(DB2710), N-trifluoroethyl (DB2716), N-tert-butyl (DB2717), and N-

propylmethoxy (DB2722) derivatives, these new derivatives still show high DNA

affinity as seen from the K_D(19-35 nM). For selected new compounds, the K_D

obtained from the SPR binding affinity studies compared closely with the values

obtained from the fluorescence titrations. Compounds containing either thiophene

or selenophene as spacer binds strongly to DNA while DB2652 and DB2591 that

contains thiazole as a spacer has weaker binding to DNA (K_D= 82 nM and 141 nM

respectively).

We further tested the fluorescence activity of six selected compounds against

two other DNA sequences namely, ATAGTAT and AAAGCTTT (Table S1). The

tested compounds showed moderate binding with the sequence ATAGTAT,

compound N-propylmethoxy DB2722 is the strongest binder with K_Dof 58 nM while

the same compound showed K_Dof 19 nM on binding to AAAGTTT, this

demonstrates that DB2722 is more specific towards the sequence AAAGTTT than

ATAGTAT. After studying the K_Dvalues of the tested compounds against

ATAGTAT, we realized that in general these six compounds are more specific

binders with AAAGTTT than ATAGTAT. These six tested compounds showed no

binding to AAAGCTTT. Although sequence AAAGTTT and ATAGTAT look

similar, there is a large difference (almost 2 Å) of minor groove width between

AAAGTTT and ATAGTAT according to high-throughput DNA shape

prediction^[32].Our N-substituents compounds are so sensitive to the variations of the

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microstructure of DNA that they bind to AAAGTTT and ATAGTAT with different

binding affinities.

2.2.3. Biosensor-SPR: Methods for Quantitative Binding

Biosensor-SPR methods provide an excellent way to quantitatively evaluate the

interaction of small organic molecules with immobilized biomolecules ^[38]. SPR

provides sensitive, real-time progress of the binding reaction as well as the

equilibrium binding affinity, kinetics, and stoichiometry of complex formation ^{[23, 38]}

.

Based on the DNA thermal melting and fluorescence spectroscopy results, the

interactions of selected compounds, DB2429, DB2689, DB2692, DB2710, and

DB2713, with AAAGTTT were evaluated by SPR (Table S2). In according to the

fluorescence spectroscopy experiments, SPR results indicate the similar binding

affinity to sequence AAAGTTT (Table 1, Table S2). Representative SPR

sensorgrams for DB2713 with N-cyclopentyl is shown in Figure 2G. Moreover, the

data are fitted to a steady-state binding function using a 1:1 model to achieve the

binding affinity (K_D= 19 nM) with the sequence AAAGTTT. Consistent with the

fluorescence quenching experiment of sequence AAAGTTT titrated into DB2713

(Figure 2C and D), the binding affinity is nearly the same (K_D= 21 nM). As shown

in Table S2, we studied these selected compounds on sequence specificity with

sequences AAATTT, AAAGTTT, and AAAGCTTT. The parent compound,

DB2429, binds to AAAGTTT as 51 nM K_D. The selectivity ratios for AAATTT and

AAAGCTTT are 10 and 20 respectively. When we change the N-substitution to

ethyl (DB2689), isopropyl (DB2692), isobutyl (DB2710) and cyclopentyl (DB2713),

the sequence specificity was improved. DB2689 and DB2710 show similar binding

to the sequence AAAGTTT but decreases binding to no detectible binding to the

pure AT sequence and two G·C bps sequences. However, DB2692 and DB2713 even

improve the binding two times to our target single G sequence with extremely high

sequence selectivity. All the N-alkyl except N-Me compounds in SPR studies show

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no detectible binding to the narrowest minor groove of pure AT sequence because

of their bulky substitution. Moreover, the minor groove of two G·C bps sequence is

wider than the single G sequence. The only one G-NH₂recognition unit (N of N-

substitution benzimidazole) and the appropriate size of entire structure lead to much

weaker or no detectible binding to AAAGCTTT. The binding affinity with target

sequence AAAGTTT and the selectivity ratio compared with a similar sequence

AAATTT are shown in Table 1 to indicate the selectivity of each tested compound.

The original compound DB2429 with small Me group can fit into narrow minor

groove like AAATTT and results in poorer selectivity (10 fold) than the compounds

with bigger bulk substituents such as ethyl (DB2689), isopropyl (DB2692), isobutyl

(DB2710), and cyclopentyl (DB2713), The bigger bulk would allow ligands to fit

into the wider minor groove microenvironments as in AAAGTTT but blocks them

from binding to narrow minor groove sequences as in AAATTT ^[32]. DB2739 with

N-phenyl substituent is the strongest binder in this series but with worse selectivity

since the phenyl ring could rotate freely around the C-N bond to adjust the structure

to fit different widths of the minor groove.

2.2.4. Circular dichroism (CD): Binding Mode

CD titration experiments are a reliable method for monitoring the saturation limit

for compounds binding with DNA sequences and the binding mode. Spectra of CD

monitor the asymmetric environment of the compounds that bind to DNA and

therefore can be used to give information regarding the binding mode ^{[39, 40]}. Free

diamidines have no CD signals but on the titration of the diamidines into DNA,

substantial positive CD signals (ICD) shown in the absorption region between 240

and 480 nm. These positive ICD signals confirm a minor-groove binding mode by

these molecules, as previously reported with similar structures. The induced positive

CD, consistent with binding in the minor groove, can be explained by nondegenerate

exciton coupling with transitions in the nucleobases ^[41]. ICD signals at the range of

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320 to 440 nm were observed due to the gradual increase of N-isopropyl DB2692,

N-cyclopentyl DB2713, and N-cyclobutyl DB2710 to the AAAGTTT sequence. As

shown from Figure 3A, 3B and3C of DB2692, DB2710 and DB2713 bind in the

minor groove of the AAAGTTT sequences, and this agrees with fluorescence and

SPR results. Finally, the CD titration experiments prove a minor-groove binding

mode for the compounds in Figure 3.

Figure 3. Circular dichroism spectra of the titration of representative compounds, A) DB2692,

B) DB2713, and C) DB2710 with a 5 µM AAAGTTT sequence in the TNE100 buffer at 25 °C .

Arrows indicate the changes.

2.2.5. Competition electrospray ionization mass spectrometry (ESI−MS):

Binding Stoichiometry and Relative Binding Affinity

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Figure 4. ESI-MS negative mode spectra of the competition binding of sequences (A and B)

AAATTT (5’-CCAAATTTGCCTCTCGAAATTTGG-3’), AAAGTTT (5’-

GCCAAAGTTTGCCTCTGCAAACTTTGGC-3’) and AAAGCTTT (5’-

CCAAAGCTTTGCTCTCAAAGCTTTGG-3’) (10 µM each); (C and D) ATATGTAT (5’-

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CCATAGTATGCTCTCATACTATGG-3’), AAAGTTT (5’-

GCCAAAGTTTGCCTCTGCAAACTTTGGC-3’), and AAAGCTTT (5’-

CCAAAGCTTTGCTCTCAAAGCTTTGG-3’) (10 µM each); with 20 µM DB2713 in buffer (50

mM ammonium acetate with 10% methanol (v/v), pH 6.8). (A and C) The ESI-MS spectra of

free DNA mixture. (B and D) The ESI-MS spectra of DNA mixture with DB2713. The ESI-MS

results shown here are deconvoluted spectra, and molecular weights are shown with each peak.

Full DNA sequences are in Table S3.

Competition ESI-mass spectrometry (MS) provides a direct analysis of relative

binding affinity, stoichiometry, and specificity for small molecule DNA complexes

^[42]. The use of some DNA sequences simultaneously mixed with a ligand creates a

competitive binding environment for direct comparison of DNA interactions. The

competition ESI-MS analysis results of the N-cyclopentyl DB2713 with DNA

sequences AAATTT, AAAGTTT, ATAGTAT, and AAAGCTTT, are shown in

Figure 4. Since the same molecular weight between sequence AAATTT and

ATAGTAT which we used for MS experiments, we divide the experiments into two

parts. One is shown in Figure 4 A and 4 B with sequences AAATTT, AAAGTTT,

and AAAGCTTT. The other one is shown as Figure 4 C and 4 D with sequences

ATAGTAT, AAAGTTT, and AAAGCTTT. The A and C plots show three peaks for

the three DNA sequences as shown in the figure. On addition of the N-cyclopentyl

DB2713, the peak for AAAGTTT (8539) decreases with the simultaneous

appearance of a new peak at m/z = 8967 that is characteristic of a 1:1 AAAGTTT–

DB2713 complex (Figure 4 B). There is no appearance of any complex peak with

the other DNA sequences. In Figure 4D, with the addition of DB2713, the peak for

AAAGTTT (8539) decreases a lot compared to the other two sequences with a new

peak of AAAGTTT – DB2713 complex (8967) appearing. However, another peak

at m/z = 7731 also appears as 1:1 complex of ATAGTAT and DB2713. Referring to

the binding affinity results in Table S1, DB2713 binds to ATAGTAT (K_D= 95 nM)

weaker than AAAGTTT (K_D= 15 nM) but much stronger than AAAGCTTT. In

agreement to binding results from other experiments, results for competition ESI-

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MS analysis of DB2713 with sequences AAATTT, ATAGTAT, AAAGTTT, and

AAAGCTTT are done to test the sequence specificity.

2.2.6. Molecular Dynamic Simulation (MD).

To help better understand the structural basis of molecular recognition of DNA

sequences with a single G·C bp in an AT context, molecular dynamics (MD)

simulations for the representative N-isopropyl compound, DB2692, were conducted.

The DB2692-A3GT3 bound complex showed that DB2692 is selectively bound to

the minor groove of the AAAGTTT binding site and makes excellent van der Waals

interactions with the walls of the minor groove (Figure 5 A). There are three

optimum H-bonds in the complex. Both amidine groups form −N−H to T=O H-

bonds (Figure 5 B) that are an average of 2.03 Å in length. The amidines also form

numerous highly dynamic H-bonds to water molecules. These water molecules

frequently also form H-bonds with A·T bps at the floor of the minor groove and help

link the compound to the specific binding site in the groove and stabilize the complex.

The third strong H-bond with 1.97 Å bond length is from the central G-NH that

projects into the minor groove to the unsubstituted imidazole N in the BI group of

DB2692, Figure 5 C, to account for binding selectivity of DB2692. Additional

selectivity in binding is provided by the −CH group of the six-member ring of BI

that points into the minor groove. This −CH forms dynamic close interactions with

the N3 of dA at the floor of the minor groove (Figure 5 C). Additional direct

stabilizing interactions are formed by –CH of the phenyl group that point to the floor

of the minor groove with dA-N3 groups on the bases at the floor of the groove.

Extensive interactions are formed by the conjugated aromatic-system of DB2692

with the sugar−phosphate back bone of the minor groove. In addition to H-bonds

with the A·T bps, the amidines of DB2692 also form electrostatic interactions with

the backbone phosphates.

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2.05 Å

B

A

1.77 Å

2.03 Å

3.62 Å

C

1.97 Å

3.57 Å

1.97 Å

2.04 Å

1.69 Å

Figure 5. A, B, C. Molecular Dynamics (MD) model of DB2692 bound to an AAAGTTT site: (A)

A space-filling model viewed into the minor groove of the AAAGTTT binding site with bound

DB2692. The DNA bases are represented in tan-white-red-blue-orange (C−H−O−N−P) color

scheme and DB2692 is magenta-white-blue-yellow (C−H−N−S) color scheme. The important

interactions between different sections of the DB2692-DNA complex are illustrated in (B) and (C).

The terminal amidine group forms a strong hydrogen bond with the carbonyl group of a dT (yellow

dashed lines), and another direct stabilizing interaction is observed from a Ph−CH group of

DB2692 which points to N3 of a dA at the floor of the minor groove (B). In (C), the imidazole-N

makes a strong hydrogen bond interaction with the exocyclic NH of dG. The BI−C−H that points

to the floor of the minor groove forms strong interactions with N3 of a dA.

To understand more about the sequence specificity of DB2692, we did

detailed MD simulation studies with both pure AT and two GC bps containing

sequences from the experimental studies. The DB2692-A3T3 bound complex

showed that due to the absence of H-bond donor group at the minor groove of A3T3

sequence and also partial hydrophobic nature of the minor groove in AT sequence

the unsubstituted imidazole N in the BI group of DB2692 rotates to the outside of

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the minor groove. The hydrophobic bulky isopropyl group of DB2692 partly fits

near the minor groove floor. This unusual orientation of the BI group places the

adjacent amidine group out of the minor groove which leads to loss of the favorable

amidine binding interactions.

DB2692-A3GCT3 MD simulated result showed that DB2692 does not fit tightly at

the wider minor groove of A3GCT3 sequence. Due to the poor stacking interaction,

the –CHs from N-Me BI and Ph make weak bonding interactions with DNA bases

and loses several favorable biding interactions. The single G·C bp mismatch at the

minor groove also caused unfavorable weak amidine NH-O=dC interaction and

again the complex loss some favorable binding enthalpy. The MD simulation results

with three sequences with different minor groove widths not only validate the

experimental results on the sequence-specific binding of DB2692 at the groove of

single GC bp containing sequence but also provides very important information

about rationally designed organic small molecules for sequence-specific DNA

recognition.

3. Conclusion.

In the current investigation, we have prepared thirteen new small size compounds of

DNA sequence-specific recognition diamidines based on the structure of DB2429 to

function as specific mixed DNA sequence binders. The new ligands contain N-alkyl-

BI-thiophenes which are preorganized by both a σ-hole interaction to fit the shape

of the DNA minor groove and H-bond to the –NH of G·C bps that project into the

minor groove. We prepared a series of diamidines with different size alkyl groups

on the benzimidazole nitrogen to determine if we could take advantage of the

difference in groove width created by incorporating G·C bps to the AAATTT

sequences with the goal of increasing the sequence specificity of minor groove

binders. All the alkyl substituents on the N-alkyl BI produced a recognizable

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increase of selectivity for the single G·C bp sequence (Table 1) with the N-phenyl

substituted benzimidazole diamidine DB2739 being the strongest binder to DNA

with K_Dof 10 nM with high selectivity to single G·C bp sequence. These results

were confirmed by using different DNA binding techniques namely, Fluorescence

emission spectroscopy, Δ Tm, SPR, CD, mass spectrometry, and molecular

dynamics.

4. Experimental.

4.1. DNA Melting experiments

DNA thermal melting experiments were performed on a Cary 300 Bio UV-vis

spectrophotometer (Varian). The concentration of each hairpin DNA sequence was

3 μM in Tris-HCl buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, pH 7.4)

using 1 cm quartz cuvettes. The mixture solutions of DNA and ligands were tested

with the ratio of 2:1 [ligand/DNA]. All samples were annealed prior to each

experiment. The spectrophotometer was set at 260 nm with a 0.5 °C/min increase

beginning at 25 °C, which is below the DNA melting temperature and ending above

it at 95 °C. The absorbance of the buffer was subtracted, and a graph of normalized

absorbance vs. temperature was created using KaleidaGraph 4.0 software. The ΔTm

values were calculated using a combination of the derivative function and estimation

from the normalized graphs.

4.2. Fluorescence Titrations

Fluorescence spectra were recorded on a Cary Eclipse Spectrophotometer, with

excitation and emission slit width fixed at [10, 5 nm] depending on the

concentrations of ligands. The free compound solutions at different concentrations

were prepared in an appropriate buffer in Tris-HCl buffer (50 mM Tris−HCl, 100

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mM NaCl, 1 mM EDTA, pH 7.4), and DNA sequence (AAAGTTT) aliquots were

added from a concentrated stock. All titration spectra were collected after allowing

an incubation time of 10 min. All compounds were excited at 350 nm based on

molecular absorbance from UV-vis spectroscopy. Emission spectra of these

compounds were monitored from 400 to 620 nm wavelength. All the fluorescence

titrations were performed at 25 °C. Then the Fluorescence Titration equation was

made in Kaleidagraph 4.0 to determine the K_Dvalue_.

4.3. Biosensor-Surface Plasmon Resonance (SPR).

SPR measurements were performed with a four-channel Biacore T200 optical

biosensor system (GE Healthcare, Inc., Piscataway, NJ). A streptavidin-derivatized

(SA) CM5 sensor chip was prepared for use by conditioning with a series of 180 s

injections of 1 M NaCl in 50 mM NaOH (activation buffer) followed by extensive

washing with HBS buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05%

P20, pH 7.4).

Biotinylated-DNA samples (AAAATTTT, AAAAGTTTT,

AAAGAAGTTT, AAAGAAACTTT, AAAGAAAACTTT, and AAGAAAAACTT

hairpins, Table S1) of 25-30 nM were prepared in HBS buffer and immobilized on

the flow cell surface by noncovalent capture as previously described. ^[43]Flow cell 1

was left blank as a reference, while flow cells 2−4 were immobilized separately by

manual injection of biotinylated-DNA stock solutions (flow rate of 1 μL/min) until

the desired amount of DNA response units (RU) was obtained (250−300 RU).

Ligand solutions were prepared with degassed and filtered TNE 100 with 0.05%

(v/v) surfactant P20 by serial dilutions from a concentrated stock solution. Typically,

a series of different ligand concentrations (2 nM to 500 nM) were injected over the

DNA sensor chip at a flow rate of 100 μL/min for 180 s, followed by buffer flow for

ligand dissociation (600−1800 s). After each cycle, the sensor chip surface was

regenerated with a 10 mM glycine solution (pH 2.5) for 30 s followed by multiple

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buffer injections to yield a stable baseline for the following cycles. The reference

response from the blank cell was subtracted from the response in each flow cell

containing DNA to give a signal (RU, response units) that is directly proportional to

the amount of bound compound. The predicted maximum response per bound

compound in the steady-state region (RU_max) was determined from the DNA

molecular weight, the amount of DNA on the flow cell, the compound molecular

weight, and the refractive index gradient ratio of the compound and DNA. RU was

plotted as a function of free ligand concentration (C_free), and the equilibrium binding

constants were determined with a one-site binding model (K₂= 0).

r = (K₁·C_free+ 2K₁·K₂·C_free²)/(1 + K₁·C_free+ K₁·K₂·C_free²)………………………………..(1)

where r represents the moles of bound compound per mole of DNA hairpin duplex,

K₁and K₂are macroscopic binding constants (for a single-site or 1:1 model K₂= 0),

and C_freeis the free compound concentration in equilibrium with the complex.

RUmax in the equation was used as a fitting parameter, and the obtained value was

compared to the predicted maximal response per bound ligand to evaluate the

stoichiometry. Kinetic analysis was performed by globally fitting the binding results

for the entire concentration series using a standard 1:1 kinetic model with integrated

mass transport-limited binding parameters as described previously ^[44]

.

4.4. Circular dichroism.

CD titration experiments were performed on a Jasco J-1500 CD spectrometer in 1

cm quartz cuvette at 25 °C. Tris-HCl buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM

EDTA, pH 7.4) was used. First, this buffer was added in 1 cm cuvette then buffer

scan was collected before the titration experiments and then the hairpin DNA

sequence AAAGTTT (5 μM) in the same cuvette and then the ligand was added to

the DNA solution and incubated for 10 min to reach equilibrium binding for the

DNA-ligand complex formation. With each addition of the ligand, scan was

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collected. For each titration point, three spectra were averaged from 480 to 240 nm

wavelength with scan speed 50 nm/min, with a response time of 1s. Buffer scan was

used as baseline, subtracted from the scan of following samples. Baseline subtracted

graphs were done using the KaleidaGraph 4.0 software

4.5. Competition Electrospray Ionization Mass Spectrometry (ESI-MS).

Electrospray Ionization Mass Spectrometry (ESI-MS) analyses were performed on

a Waters Q-TOF micro Mass Spectrometer (Waters Corporate, Milford, MA)

equipped with an electrospray ionization source (ESI) in a negative ion mode. DNA

sequences AAATTT, ATAGTAT, AAAGTTT and AAAGCTTT, Table S1, for ESI-

MS experiments were purified by dialyzing it in 50 mM ammonium acetate buffer

(pH 6.7) at 4 ºC with 3x buffer exchange. Test samples were prepared in 50 mM

ammonium acetate with 10% v/v methanol at pH 6.7 and introduced into the ion

source through direct infusion at 5 µl/min flow rate. The competitive experiments

were done by mixing a ligand and DNAs with different sequences at different ratios.

The instrument parameters were typically as follows: capillary voltage of 2800 V,

sample cone voltage of 30 V, extraction cone voltage of 1.0 V, desolvation

temperature of 70 °C, and source temperature of 100 °C. Nitrogen was used as

nebulizing and drying gas. A multiply charged spectra were acquired through a full

scan analysis at mass range from 300-2500 Da and then deconvoluted to the spectra

presented. MassLynx 4.1 software was used for data acquisition and deconvolution.

4.6. Molecular Dynamic (MD) Simulation.

Structure optimization of DB2692 was performed by using DFT/B3LYP theory with

the 6-31+G* basis set in Gaussian 09 (Gaussian, Inc., 2009, Wallingford, CT) with

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Gauss-view 5.09 ^[45]. Partial charges were derived using the RESP fitting method

(Restrained Electrostatic potential) ^{[46, 47]}. AMBER 14 (Assisted Model Building

with Energy Refinement) software suite was used to perform molecular dynamic

(MD) simulations ^[48]. Canonical B-form ds[(5’-CCAAAGTTTGG-3’)(5’-

CCAAACTTTGG-3’)] DNA was built in Nucleic Acid Builder (NAB) tool in

AMBER. AMBER preparation and force field parameter files required to run

molecular dynamic simulations for DB2692 molecule were produced using

ANTECHAMBER ^[49]. Specific atom types assigned for DB2692 molecule were

adapted from the ff99 force field. Most of the force field parameters for DB2692

molecule were derived from the existing set of bonds, angles and dihedrals for the

similar atom types in parm99 and GAFF force fields ^[50]. Some dihedral angle

[51,52]

parameters were obtained from previously reported parametrized data

.

Molecular structure with specific atom types used for the DB2692 molecule is shown

in Figure S1. Parameters of DB2692 in frcmod file are listed at the Table S5.

AutoDock Vina program was used to dock the DB2692 in the minor groove

of DNA to obtain the initial structure for DB2692-DNA complex ^[53]. MD

simulations were performed in explicit solvation conditions where the DNA-

DB2692 complex was placed in a truncated octahedron box filled with TIP3P water

using xleap program in AMBER. Sodium ions were used to neutralize the system.

A 10 Å cutoff was applied on all van der Waals interactions. The MD simulation

was carried out using the Sander module with SHAKE algorithm applied to constrain

all bonds. Initially, the system was relaxed with 500 steps of steepest-descent energy

minimization. The temperature of the system was then increased from 0 K to 310 K

for over 10 ps under constant-volume conditions. In the final step, the production

run on the system was subsequently performed for 500 ns under NPT (constant-

pressure) conditions.

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4.7. Chemistry.

All commercial reagents were used without further purification. All melting points

were determined on a Mel-Temp 3.0 melting point instrument, and are uncorrected.

TLC analysis was carried out on silica gel 60 F254 precoated aluminum sheets using

UV light for detection. ¹HNMR and ¹³CNMR spectra were recorded on a Bruker 400

MHz and 100 MHz spectrometer respectively using the indicated solvents. Mass

spectra were obtained from the Georgia State University Mass Spectrometry

Laboratory, Atlanta, GA. Elemental analysis were performed by Atlantic Microlab

Inc., Norcross, GA. Compounds 5a and 6a (DB 2429) was prepared according to the

procedure published in reference 21.

Synthesis of 4-(alkylamino)-3-nitrobenzonitrile (2a-k)

Amines (40 mmol) were added to 4-fluoro-3-nitrobenzonitrile (3.32 gm, 20 mmol)

suspension in ethanol (20 ml) and stirred at room temperature for 24 h. The

precipitated yellow solid was filtered and washed with cooled ethanol and dried.

In case of aniline, the reaction mixture was heated at 80 ^oC for 24h.

4-(Methylamino)-3-nitrobenzonitrile (2a)

Orange solid (3.39 gm 89 %), mp 168-169 ^oC (as reported, ^[54]. ¹HNMR (DMSO-d₆):

δ 8.64 (br s, 1 H), 8.49 (s, 1 H), 7.83 (d, J = 9 Hz, 1 H), 7.09 (d, J = 9 Hz, 1 H), 2.99

(s, 3 H); ESI-HRMS: m/z calculated for C₈H₈N₃O₂: 177.0533, found: 177.0524 (M⁺

+ 1).

4-(Ethylamino)-3-nitrobenzonitrile (2b).

Orange solid (3.39 gm 89 %), mp 134-135 ^oC (as reported, ^[54]. ¹HNMR (DMSO-d₆):

δ 8.56 (br s, 1 H), 8.49 (d, J = 1.2 Hz, 1 H), 7.80 (d, J = 9.2 Hz, 1 H), 7.16 (d, J =

9.2 Hz, 1 H), 3.45 (q, J = 6.4 Hz, 2 H), 3.45 (t, J = 7.2 Hz, 3 H); ESI-HRMS: m/z

calculated for C₉H₁₀N₃O₂: 192.0768, found: 192.0761 (M⁺+ 1).

4-(Isopropylamino)-3-nitrobenzonitrile (2c).

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o

[55]

1

Orange solid (3.36 gm 82 %), mp 111-112 C (Lit. mp 110-113 C,

. HNMR

(DMSO-d₆): δ 8.49 (s, 1 H), 8.20 (d, J = 7.2 Hz, 1 H), 7.81 (d, J = 9.2 Hz, 1 H), 7.21

(d, J = 9.2 Hz, 1 H), 4.01 (m, 1 H), 1.27 (d, J = 6 Hz, 6 H); ¹³CNMR (DMSO-d₆):

145.5, 137.1, 131.3, 130.3, 117.5, 115.4, 96, 43.7, 21.4; ESI-HRMS: m/z calculated

for C₁₀H₁₁N₃O₂: 228.0749, found: 228.0759 (M⁺+ Na).

4-(Isobutylamino)-3-nitrobenzonitrile (2d).

Orange solid (3.47 gm 79 %), mp 77-78 ^oC. ¹HNMR (DMSO-d₆): δ 8.61 (br s, 1 H),

8.49 (s, 1 H), 7.79 (d, J = 8.8 Hz, 1 H), 7.19 (d, J = 8.8 Hz, 1 H), 3.25 (t, J = 6.4 Hz,

2 H), 1.94 (m, 1 H), 0.94 (d, J = 6.4 Hz, 6 H); ESI-HRMS: m/z calculated for

C₁₁H₁₄N₃O₂: 220.1081, found: 220.1070 (M⁺+1).

4-(Neopentylamino)-3-nitrobenzonitrile (2e).

Orange solid (3.3 gm 71 %), mp 126-127 ^oC. ¹HNMR (DMSO-d₆): δ 8.52 (m, 2 H),

7.81 (dd, J = 1.2, 9 Hz, 1 H), 7.93 (d, J = 9 Hz, 1 H), 3.27 (d, J = 6 Hz, 2 H), 0.98

13

(s, 9H); CNMR (DMSO-d₆): 147.1, 137.1, 131.3, 130.2, 117.5, 115.4, 96.1, 53,

31.4, 26.5; ESI-HRMS: m/z calculated for C₁₂H₁₆N₃O₂: 234.1237, found: 234.1226

(M⁺+ 1).

3-nitro-4-((2,2,2-trifluoroethyl)amino)benzonitrile (2f).

Orange solid (3.82 gm 79 %), mp 87-88 ^oC. ¹HNMR (DMSO-d₆): δ 8.72(m, 1 H),

8.56 (s, 1 H), 7.93 (d, J = 9.2 Hz, 1 H), 7.45 (d, J = 9.2 Hz, 1 H), 4.43 (m, 2H); ESI-

HRMS: m/z calculated for C₉H₇FN₃O₂: 246.0458, found: 246.0476 (M⁺+ 1).

4-((3-Methoxypropyl)amino)-3-nitrobenzonitrile (2g).

Orange solid (3.52 gm 75 %), mp 68-69 ^oC. ¹HNMR (DMSO-d₆): δ 8.76 (br s, 1 H),

8.49 (s, 1 H), 7.82 (d, J = 8.8 Hz, 1 H), 7.15 (d, J = 8.8 Hz, 1 H), 3.45 (m, 4 H), 3.26

(s, 3 H), 1.86 (t, J = 5.6 Hz, 2 H); ¹³CNMR (DMSO-d₆): 146.3, 137.1, 131.3, 130.2,

117.7, 115, 95.7, 69.5, 57.5, 40.2, 27.5; ESI-HRMS: m/z calculated for C₁₁H₁₄N₃O₃:

236.1030, found: 236.1019 (M⁺+ 1).

4-(Cyclobutylamino)-3-nitrobenzonitrile (2h).

29

Article Doi

Small Sequence-Sensitive Compounds for Specific Recognition of the G?C Base Pair in DNA Minor Groove

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