Spectroscopic and molecular modeling methods to study the interaction between naphthalimide-polyamine conjugates and DNA

    Spectroscopic and molecular modeling methods to study the interaction between naphthalimide-polyamine conjugates and DNA Zhiyong Tian, Yingying huang, Yan Zhang, Lina Song, Yan Qiao, Xuejun Xu, Chaojie Wang PII: DOI: Reference:

S1011-1344(15)30139-1 doi: 10.1016/j.jphotobiol.2016.01.017 JPB 10235

To appear in: Received date: Accepted date:

9 November 2015 29 January 2016

Please cite this article as: Zhiyong Tian, Yingying huang, Yan Zhang, Lina Song, Yan Qiao, Xuejun Xu, Chaojie Wang, Spectroscopic and molecular modeling methods to study the interaction between naphthalimide-polyamine conjugates and DNA, (2016), doi: 10.1016/j.jphotobiol.2016.01.017

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ACCEPTED MANUSCRIPT Spectroscopic and molecular modeling methods to study the interaction between naphthalimide-polyamine conjugates and DNA Zhiyong Tian a, Yingying huang a, Yan Zhang a, Lina Song a,

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Yan Qiaob,c, Xuejun Xu *,b,d, Chaojie Wang *,d

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(a Institute of chemical biology, Henan University, Kaifeng 475004, china)

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(b Basic Medical College, Zhengzhou University, Zhengzhou, 475008,China) (c State Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China.)

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(d The Key Laboratory of Natural Medicine and Immuno-Engineering, Henan

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University, Kaifeng 475004, china)

* Corresponding author. Tel. +86 18621534352 (X. Xu), +86 13619810550

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Wang).

(C.

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E-mail addresses: [email protected] (X. Xu), [email protected] (C.

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Wang).

Abstract: The effect of polyamine side chains on the interaction between naphthalimide-polyamine conjugates (1~7) and herring sperm DNA was studied by

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UV/vis absorption and fluorescent spectra under physiological conditions (pH=7.4). The diverse spectral data and further molecular docking simulation in silico indicated that the aromatic moiety of these compounds could intercalate into the DNA base pairs while the polyamine motif might simultaneously locate in the minor groove. The triamine compound 7 can interact more potently with DNA than the corresponding diamine compounds (1~6). The presence of the bulky terminal group in the diamine side chain reduced the binding strength of compound 1 with DNA, compared to other diamine compounds (2~6). In addition, the increasing methylene number in the diamine backbone generally results in the elevated binding constant of compounds-DNA complex. The fluorescent tests at different temperature revealed that the quenching mechanism was a static type. The binding constant and thermodynamic parameter showed that the binding strength and the type of interaction force, 1

ACCEPTED MANUSCRIPT associated with the side chains, were mainly hydrogen bonding and hydrophobic force. And the calculated free binding energies of molecular docking are generally consistent with the stability of polyamine-DNA complexes. The circular dichroism

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assay about the impact of compounds 1-7 on DNA conformation testified the B to

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A-like conformational change.

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Keywords: naphthalimide, polyamine conjugates, DNA, spectroscopy, model docking.

1. Introduction

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The study on the interaction between small molecules and DNA has been caught people’s attention of recent research in the scope of life science, chemistry and clinical medicine [1–3]. As we now know, DNA is the carrier of genetic information

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and gene expression of the material basis, which plays an extremely important role in

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the process of human life for its abilities to interfere with transcription (gene expression and protein synthesis) and DNA replication, a major step in cell growth

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and division. Generally speaking, A variety of small molecules usually interacts reversibly with DNA in three primary ways: (1) intercalation of planar or

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approximately planar aromatic ring systems between base-pairs [4]; (2) groove binding in which the small molecules bound on nucleic acids are located in the major or minor groove [4]; (3) binding along the exterior of DNA helix that is through interactions which are generally nonspecific and are primarily electrostatic [5–8]. The 1, 8-naphthalimide derivatives are the DNA intercalating agents because of their consisting of a flat, generally p-π deficient aromatic system of which binds to DNA by insertion between the base pairs of the double helix [4]. They displayed good antitumor activity due to their intercalation causing the base pairs to separate vertically, so twisting the sugar phosphate backbone and changing the degree of rotation between successive base pairs [9–17]. Polyamines can bind to DNA by hydrogen bond or electrostatic interactions and cause DNA conformational changes [18–21]. Naphthalimide-polyamine conjugates have been also proved to display good 2

ACCEPTED MANUSCRIPT activity in vitro and intercalate into the DNA [22–26]. They could induce DNA conformational transition and the substituted groups linked to naphthalimide scaffold displayed some impacts on related interaction [26]. It is ever reported that

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naphthalimide-polyamine conjugates aren’t surely the DNA intercalators [27]. However, to date the side chain effects on the interactions between different

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naphthalimide-polyamine conjugates and DNA, including numbers of free nitrogen atoms, type of terminal amino group and numbers with position of methylene, have been reported rarely. Besides, types of DNA conformational transition and the

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interaction mode need to be clarified. The present work will address these issues by the interaction between naphthalimide-polyamine conjugates (1~7, Fig. 1) and herring

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sperm DNA. 2. Materials and methods

absorption

spectra

were

measured

on

a

Unicam

UV 500

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UV–vis

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2.1. Apparatus

spectrophotometer using a 1.0 cm cell. Fluorescence measurements were performed

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with a Cary Eclipse spectrofluorimeter. Circular dichroism spectrum measurements were performed on a Modle 420 SF (USA) automatic recording spectrophotometer in a 1 mm quartz cell.

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2.2. Materials

Naphthalimide-polyamine conjugates 1~7 were prepared previously [22–25]. Their solutions (2.00×10-4 mol ∙ L-1) were either prepared with the Tris–HCl (pH=7.4) buffer solution (UV and Fluorescence) or the phosphate buffer saline (PBS, pH=7.4) buffer solution (CD) and stored at 4 °C. Herring sperm DNA (Sino-American Biotechnology Company, Beijing, China) was used without further purification. And its stock solution was prepared either by dissolving an appropriate amount of DNA in Tris–HCl (pH=7.4) buffer solution (2.284×10-4 mol ∙ L-1, for UV and Fluorescence) or PBS (pH=7.4) buffer solution (4.568×10-4 mol ∙ L-1, for CD), stored at 4°C. Ethidium bromide (EB, Sigma Chem. Co., USA) stock solution (1.57×10-5 mol ∙ L-1) was prepared by dissolving its crystals with the Tris–HCl buffer solution and stored in a cool and dark place. 3

ACCEPTED MANUSCRIPT 2.3. Procedures 2.3.1 UV–Vis measurement 2 mL solution of compounds 1~7 (2.00×10-4 mol∙ L-1 in Tris-HCl (pH=7.4) were

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mixed with 0.0, 0.10, 0.20, 0.30, 0.60, 0.90 1.20, 1.50, 1.80, 2.10, 2.40, 2.70 and 3.0

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mL of herring sperm DNA (2.284×10-4mol∙ L-1) respectively. The mixture was diluted

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to 5mL with Tris-HCl (pH=7.4). Thus, two groups of samples were prepared in the concentration of DNA at 0.0, 4.56, 9.13, 13.69, 27.4, 41.08, 54.77, 68.46, 82.15, 95.84, 109.54, 123.23 and 136.92×10-6 mol∙ L-1. One contained only compounds 1~7

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(80×10-6 mol ∙ L-1) as control, the others contained different concentration of DNA but

30min. at room temperature. 2.3.2 Fluorescence measurement

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had the same concentration of compounds 1~7. All the above solution was shaken for

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2.3.2.1 Interaction of Compounds 1~7 with DNA

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Preparation of samples is the same as that of UV-Vis samples. Fluorescence wavelengths and intensity areas of samples 1~7 were measured at 298, 303 and 310 K

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in the wavelength range of 355–690 nm with exciting wavelength at 345 nm. 2.3.2.2 Interaction of Compounds 1~7 with DNA-EB complex 0.3 mL solution of herring sperm DNA (2.284×10-5 mol∙ L-1 in Tris-HCl (pH=7.4)

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and 0.4mL EB (1.57×10-5 mol∙ L-1) was mixed with 0.0, 0.10, 0.20, 0.30, 0.60, 0.90 1.20, 1.50, 1.80, 2.10, 2.40, 2.70 and 3.00 mL of compounds 1~7 (2.0×10-4mol∙ L-1) respectively. The mixture was also diluted to 5mL with Tris-HCl (pH=7.4). Thus, three groups of samples were prepared in the concentration of compounds 1~7 at 0.0, 4.0, 8.0, 12.0, 24.0, 36.0, 48.0, 60.0, 72.0, 84.0, 96.0 and 108.0 and 120.0×10-6 mol ∙L-1. One contained only DNA (13.7×10-6 mol∙ L-1) and EB (15.7×10-6 mol ∙ L-1) as control, the others contained different concentration of compounds 1~7 but had the same concentration of DNA and EB. All the above solution was shaken for 30min. at room temperature. Fluorescence wavelengths and intensity areas of samples 1~7 were measured at 298, 303 and 310 K in the wavelength range of 520–800 nm with exciting wavelength at 510 nm. 2.3.2.3 Iodide quenching 4

ACCEPTED MANUSCRIPT 0.5mL solution of compounds 1~7 (2.00×10-4 mol/L) and 0.5mL herring sperm DNA (22.84×10-4mol/L) in Tris-HCl (pH=7.4) were mixed with 0.0, 0.20, 0.40, 0.60, 0.80, 1.00 1.20, 1.40, 1.60, 1.80, and 2.00mL of KI (2.0×10-2 mol∙ L-1) respectively.

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Meanwhile, 0.5 mL solution of compounds 1~7 (2.00×10-4 mol/L) was only mixed with 0.0, 0.20, 0.40, 0.60, 0.80, 1.00 1.20, 1.40, 1.60, 1.80, and 2.00mL of KI

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(2.0×10-2 mol∙ L-1) respectively. The two kinds of mixtures were diluted to 5mL with Tris-HCl (pH=7.4) to possess the concentration of KI at 0.0, 400, 800, 1200, 2400, 3600, 4800, 6000, 7200, 8400, 9600, 10800, 12000×10-6mol ∙ L-1. The control groups

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contained only compounds 1~7 (20×10-6 mol ∙ L-1) and different concentration of KI, the other samples contained different concentration of KI and fixed concentrations of

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compounds 1~7 (20×10-6 mol ∙ L-1) and DNA (22.82×10-6 mol ∙ L-1). All the above solution was shaken for 30min. at room temperature. Fluorescence wavelengths and

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intensity areas of samples were the same as 2.3.2.1.

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2.3.2.4 Effect of ionic intensity on the interaction between compounds 1~7 and DNA 1.0mL solution of compounds 1~7 (2.00×10-4 mol∙ L-1) and herring sperm DNA

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1.0mL (2.284×10-4mol∙ L-1) in Tris-HCl (pH=7.4) were mixed with 0.0, 0.10, 0.20, 0.30, 0.60, 0.90 1.20, 1.50, 1.80, 2.10, 2.40, 2.70 and 3.00mL of NaCl (4.0×10-2 mol∙ L-1) respectively. The mixture was diluted to 5mL with Tris-HCl (pH=7.4), too. Thus,

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samples were prepared in the concentration of NaCl at 0.0, 800, 1600, 2400, 4800, 7200, 9600, 12000, 14400, 16800, 19200, 21600 and 24000×10-6 mol ∙ L-1. One contained only compounds 1~7 (40×10-6 mol ∙ L-1) and DNA (45.68×10-6 mol ∙ L-1) as control, the others contained different concentration of NaCl but had the same concentration of compounds 1~7 and DNA. All the above solution was shaken for 30min. at room temperature. Fluorescence wavelengths and intensity areas of samples were also the same as 2.3.2.1. 2.3.3 CD measurement 2mL solution of herring sperm DNA (9.128×10-4mol∙ L-1) in PBS (pH=7.4) was mixed with 0.0, 0.40, 0.80 and 1.20mL of compounds 1~7 (2.00×10-4 mol∙ L-1) respectively. The mixture was diluted to 5mL with PBS (pH=7.4). Thus, samples were prepared in the concentration of compounds 1~7 at 0.0, 16.0, 32.0 and 48.0×10-6 mol∙ 5

ACCEPTED MANUSCRIPT L-1. One contained only DNA (182.56×10-6 mol ∙ L-1) as control, the others contained different concentration of compounds 1~7 but had the same concentration of DNA. All the above solution was shaken for 30min. at room temperature.

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2.3.4 Model Docking

The crystal structure of DNA duplex (5′-GC|GTAC|GC-3′) was available in

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reference [28] (pdb code: 1AL9). Waters were removed from the DNA PDB file. Essential hydrogen atoms and charges of molecules including DNA and compounds 1~7 were added with aid of usinlide (Schrodinger LLC) software (Tripos, Inc., St.

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Louis, MO 63144) [29−34]. The experiments in silico of molecule modeling of compounds 1~7 in complex with the DNA were performed and the docking results

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were presented on the usinlide (Schrodinger LLC) platform. 3. Results and Discussion.

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3.1 UV spectroscopic characteristics

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As shown in Fig. 2, the UV spectrum of naphthalimide-polyamine conjugates (1~7) in the absence and presence herring sperm DNA was performed by Ultraviolet

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visible range spectrophotometer. It was observed that a continuous decrease in the absorbance of compounds 1~7 and a red shift were followed with the increasing concentration of DNA, suggesting compounds 1~7 could insert into the base pairs of

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DNA. As we know, the hypochromism and hyperchromism are spectral features of DNA concerning changes in its double helix structure. The hypochromic and hyperchromism effect of compounds 1~7 are thought due to the interaction between the electronic states of the intercalating chromophore and those of the DNA bases [35]. It is expected that the strength of this electronic interaction would decrease as the cube of the distance of separation between compounds 1~7 and the DNA bases [36]. So the hypochromism suggested the close proximity of compounds 1~7 to the DNA bases. The compounds 1~7 solution exhibited hypochromic effect and bathochromic shift in UV/Vis spectra upon binding to DNA, respectively, representing a characteristic of an intercalating mode [37]. 3.2 Fluorescence spectroscopy 3.2.1 Fluorescence quenching 6

ACCEPTED MANUSCRIPT In order to evaluate the DNA binding properties of naphthalimide-polyamine conjugates, the inherent fluorescence of compounds 1~7 allowed us to examine their interaction with herring sperm DNA by FL spectrometry. As shown in Fig. 3, the

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fluorescence of compounds 3~7 were quenched upon addition of DNA. The spectra of compounds 1~4, however, were rebounded irregularly upon addition of DNA from

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0.0 to 3 mL, and great rebounding was observed for compound 1 at 303 and 310K. These indicated that DNA is one potential target of compounds as expected. Fluorescence quenching could be explained by lack of H bonding of the amino group

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with solvent water molecules when they bind to DNA.

It is well-known that ethidium bromide (EB) is a DNA intercalator, which is

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usually used as a spectral probe to establish the mode of binding of small molecules to double-helical DNA [38]. The fluorescence of EB at about 605 nm increases after

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binding with DNA because of intercalation. Similarly to EB, if naphthalimides

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intercalates into the helix of DNA, it would compete with EB for its intercalation sites in DNA, and the displacement of EB from the DNA-EB complex cause a significant

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decrease in the fluorescence intensity of the DNA–EB complex [39–40]. As a consequence, herring sperm DNA-EB complex in the presence of increasing concentrations of naphthalimide-polyamine conjugates (1~7) were measured. As

the

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shown in Fig. 4, the fluorescence intensity of DNA-EB complex were decreased by gradually

growing

concentration

of

compounds

1~7,

deducing

that

naphthalimide-polyamine conjugates could intercalate into DNA and a new complex was possibly formed between compounds 1~7 and DNA, which provided further evidence that naphthalimide-polymine conjugates could change DNA conformation [20, 21, 41–43]. 3.2.2 Fluorescence quenching mechanism We know that fluorescence quenching can take place by different mechanisms, which are usually categorized as dynamic quenching and static quenching. Dynamic quenching depends on diffusion effects, thus, the diffusion coefficients and bimolecular quenching constants would be larger at higher temperatures. Static

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ACCEPTED MANUSCRIPT quenching nevertheless generates less fluorescent complex with decreased stabilities and static quenching constants accompanying by elevated temperature [44]. To clarify the quenching mechanism of the interaction between naphthalimide-polyamine

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conjugates and DNA (or DNA-EB), the fluorescence quenching tests were also carried out at 298, 303 and 310K, which could be described by Stern-Volmer equation

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[45–47].

The Stern–Volmer equation is F0 / F = 1+ K SV c (1), where F0 and F are the fluorescence intensities in the absence and presence of quencher (DNA for

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compounds 1~7 for DNA-EB, respectively), KSV is the Stern-Volmer quenching constant, [c] is the concentration of DNA (or compounds 1~7), Kq is the biomolecule

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quenching rate constant and Kq = KSV/τ0. τ0 is the average lifetime of the molecule without any quencher and the fluorescence lifetime of the biopolymer is around 10-8s

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[48]. The Stern–Volmer plots of F0/F versus [c] at the three temperatures were showed

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in Fig. 5~6, and the calculated KSV and Kq values were listed in Table 1~2. The values of quenching constant KSV were decreased with increasing temperature except

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compound 3-DNA and compounds 2~6-DNA-EB complex and the values of Kq were much greater than that of the maximum scattering collision quenching constant (2 .000 ×1010 L∙ mol-1), indicating that the fluorescence quenching mechanism of 1~7

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initiated by DNA and DNA-EB complex initiated by compounds 1 and 6~7 was a typical static quenching procedure [49–50]. As far as concerned compounds 2~5-DNA-EB complex were, because their values of Kq were much greater than 2 .000 ×1010 L∙ mol-1, their fluorescence quenching mechanism was a static quenching procedure. The values of quenching constant KSV of compound 3-DNA and compounds 2~5-DNA-EB complex, however, did not decrease with increasing temperature, revealing that their fluorescence quenching mechanism of DNA was not a typically static type [49–51]. It is reported that the static quenching constant does not necessarily decrease with the increasing of temperature, sometimes it increases or does not change [52−55]. So fluorescence quenching mechanism of compound 3 initiated by DNA or DNA-EB by compounds 2~5 was overall static. As shown in Table 1, the values of quenching constant KSV of compounds–DNA 8

ACCEPTED MANUSCRIPT complex were generally decreased in the following order: 7>6>5>4>3>2>1 at 310K, the temperature for general cell assay, which revealed that the impact of DNA on the triamine conjugate 7 is more than those of diamine conjugates. The value of Ksv of

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compound 1–DNA complex was the smallest among these compounds due to the bulky terminal dimethylamino group in the diamine backbone. In addition to the

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slight difference between compounds 4 and 5 with seven methylene units, the values of quenching constant KSV of compounds–DNA complex were also decreased in the following order: 4 (5-2) >3 (4-2) >2 (3-2) and 6 (4-4) > 5(4-3) >3 (4-2), which

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implied numbers and position of compounds–DNA complex also effected on the values of KSV. These results showed that there were the side chain effects among

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compounds 1~6 when they bound to DNA [26]. However, the ternary compounds-DNA-EB systems displayed the sequence of 7>4, 5, 6>3>2>1 at 310K,

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and the abnormal behavior of compounds 4~6 might result from both the side chain

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effects and the interference of EB (Table 2) [26]. In addition, the correlation coefficient of compound 1-DNA at 303 and 310K were very small in Table 2, which

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may be due to its weak stability.

3.2.3 Interaction mode between compounds 1~7 and DNA For a static quenching process, the binding constant (Kb) can be determined by

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the following equation when small molecules bind independently to a set of equivalent sites in a macromolecule such as DNA [56–57]: log[1/c] = log[F/(F0- F)] + log Kb

(2), where Kb means the binding constant for interaction of naphthalimide

–DNA, and F0, F, [c] have the same meanings as in Eq. 1. The values of Kb could be determined from the intercept and slope by plotting log [1/c] against log [F (F0 - F)] (intercept = log Kb) (Fig. 7-8), and the corresponding values of Kb were listed in Table 3-4. The changed trend of Kb with increasing temperature was in accordance with KSV’s dependence on temperature as mentioned above, revealing that the binding between naphthalimides and DNA was moderate, and there may be a reversible naphthalimides-DNA complex formed [58]. As illustrated in Table 3, the values of quenching constant Kb of binary compounds–DNA system were generally decreased in a similar order: 7>6>3, 5> 9

ACCEPTED MANUSCRIPT 4>2>1, 3 (4-2), 4 (5-2) >2 (3-2) and 6 (4-4) > 3(4-2), 5 (4-3) that is generally consistent with the values of KSV of those compounds-DNA complex at 310K, providing further evidences to the side chain effects [26]. As also illustrated in Table

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4, those of ternary compounds-DNA-EB complex were decreased in the following order: 7>4, 5, 6>3>2>1 at 310K, also adding more evidences to the side chain effects

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which is basically consistent with of those compounds-DNA complex though the distorted order of compounds 4~6 were observed [26]. Besides, the correlation coefficient of compound 1-DNA at 303 and 310K were very small in Table 3, which

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was also because its stability was too weak.

There are several acting forces between a small organic molecular and

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biomacromolecules, mainly include hydrophobic force, hydrogen bond, van der Waals force, electrostatic interactions. It is assumed that the interaction enthalpy change

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(∆H°) varies insignificantly over the limited temperature range studied, thus the

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thermodynamic parameters can be determined from the van’t Hoff equation: (3)

∆G° = -RT lnK = ∆H°-T∆S°

(4)

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Ln (K2/K1) = (1/T1-1/T2) ∆H°/R

In Eq. 3-4, K is analogous to the binding constant at the corresponding temperature and R is gas constant (8.314 J mol−1 •K−1). The enthalpy change (∆H°)

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and entropy change (∆S°) were calculated from the Eq. 3-4, and the corresponding results were presented in Table 3-4. Ross et al. depicted the sign and magnitude of thermodynamic parameter associated with various individual kinds of interaction, which may take place in DNA association processes, as characterized below [38]: (a) Positive ΔH and ΔS values are frequently regarded as evidence for typical hydrophobic interaction. (b) Negative ΔH and ΔS values arise from van der Waals force and hydrogen bonding formation. (c) A positive value of ΔS and a negative ΔH value are stated precisely as a specific electrostatic interaction between ionic species in aqueous solution. However, it is also reported that a positive value of ΔS and a negative ΔH value are taken as hydrogen bonds and hydrophobic forces [59]. From Table 3 and 4, it can be seen that the negative ∆H° and negative ∆S° values (compounds 1, 2, 4, and 6 -DNA or compounds 10

ACCEPTED MANUSCRIPT 1 and 5-DNA-EB) showed that the hydrogen bond and played a dominant role in the interactions between naphthalimides and DNA. At the same time, it can be also seen that the negative ∆H° and positive ∆S° values (compounds 5 and 7-DNA and

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compounds 2~3 or 6~7-DNA-EB) revealed that the hydrogen bonds and hydrophobic forces played a dominant part in the interactions between naphthalimides and DNA

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[60]. Again, it can be seen that the positive ∆H° and positive ∆S° values (compounds 3-DNA and 4-DNA-EB) revealed that the hydrophobic force played a dominant part in the interactions between naphthalimides and DNA. Therefore, it can be concluded

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that the hydrogen bonds and hydrophobic forces played a dominant part in the interactions between naphthalimides (compounds 1~7) and DNA, which are further

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supported by effects of ionic intensity. 3.2.4 Iodide quenching studies

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A highly negatively charged quencher is expected to be repulsed by the

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negatively charged phosphate backbone of DNA, therefore an intercalative bound drug molecules should be avoided to be quenched by anionic quencher, but the free

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aqueous complexes or groove binding drugs should be quenched readily by anionic quenchers. At the same time, whether the quencher accesses to fluorophore also plays a part in free and bound one [61–62]. Negatively charged iodide ion was selected for

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this purpose. The quenching constants (Ksv) were obtained from Stern–Volmer equation. The values of Ksv of compounds 1~7 by iodide ion in the absence and presence of DNA were shown in Fig. 9 and listed in Table 5. As shown in Fig. 9 and Table 5, It was apparent that iodide quenching effects was decreased when compounds 1, 3~7 were bound to DNA, which suggested that compounds 1, 3~7 intercalated into the base pairs of DNA while compound 2 might bind to DNA by groove because its decreasing rate of Ksv by iodide ion in the absence and presence of DNA was very small. It is deduced that compound 2 might bind to DNA by groove and intercalation into the base pairs of DNA because it could displace EB from DNA-EB complex. These results showed that compounds 1~7 also altered the B-DNA helical structure. As also shown in Table 5, the values of Ksv of compounds 1~7 by iodide ion in the absence and presence of DNA were decreased in the following order: 11

ACCEPTED MANUSCRIPT 7>5>4>3>2>1(except compound 6), adding more evidences to the side chain effects which is consistent with of those compounds-DNA complex on the whole [26]. 3.2.5 Effects of ionic intensity on the compounds 1~7 and DNA interaction

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DNA is an anionic polyelectrolyte with phosphate groups and monitoring the spectral change with different ionic strength is an efficient method to distinguish the

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binding modes between molecules and DNA. NaCl is used to control the ionic strength of the solutions. The addition of Na+ would attenuate the electrostatic interaction between molecules and DNA due to its competition for phosphate groups

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in DNA [63]. Therefore, the effect of NaCl on the fluorescence of DNA–compounds 1~7 system were studied. As shown in Fig. 10, the fluorescence intensity of

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compounds 1~7-DNA complex were not basically changed with the increasing concentration of NaCl. The results indicated that interaction between compounds 1~7

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and DNA could exclude the electrostatic interaction mode.

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3.4. Circular dichroism spectroscopy

It has been reported that polyamines (putrescine, spermidine, and spermine) can

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result in DNA conformational changes, B- to Z-DNA or B- to A-DNA transitions in specific sequences, and polyamine analogues have the capability of inducing DNA conformational conversions, B-DNA to A-DNA transitions and also causing

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aggregation and precipitation of highly polymerized DNA [20–21]. In addition, circular dichroism (CD) is a very powerful technique to monitor the conformational alteration in DNA upon interaction with exogenous substances

[64–67].

Naphthalimide-polyamine conjugates could induce DNA conformational transition but the transition type of DNA conformation was not clarified [26]. So CD spectral study was performed to investigate the interaction between herring sperm DNA and compounds 1~7. As shown in Fig. 11, The CD of the free DNA composed of four major peaks at 210 (positive), 221 (positive), 245 (negative), and 280 nm (positive), which is consistent with CD spectra of double helical DNA in B conformation [20, 68–71]. When compounds 1~7 were added, they could induce significant changes in a negative peak around 245 nm caused by the helical B conformation and in a positive 12

ACCEPTED MANUSCRIPT peak around 280 nm due to base stacking [72]. As is shown in Fig. 11, compounds 1~7 could induce significant changes in a positive peak around 210 and 220 nm due to base stack. The decreases in the intensity of the negative band on the whole of 1~7

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were observed when the concentration of compounds 1~7 increases, suggesting a similar manner as the B to A-like conformational change [13, 20, 63–71, 73–75].

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Besides, there were obvious blue shifts from 247 to 237nm with the increasing concentration of compounds 2, 3, 4 and 7. These results also proved the existence of side chain effects. At the same time, no induced new peak emerges, excluding the

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classic intercalation [76–77]. These results of CD studies show that the conformation of herring sperm DNA can be affected by compounds 1~7 and compounds 1~7

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intercalated into DNA. Moreover, compounds 1~7 are not the typical DNA intercalators [76–77]. With the evidences of polyamines (putrescine, spermidine, and

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spermine) binding to DNA by groove [21], it is rational to speculate that the

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interaction mode of naphthalimide-polyamine conjugates and DNA includes groove binding and intercalation.

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3.4. Model Docking

For full understanding of the interaction between naphthalimide-polyamine conjugates and DNA [26], we performed molecular docking calculations by usinlide

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(Schrodinger LLC) (Fig. 12) [29–32]. First, the crystal structure of DNA in complex with substrate was derived from Protein Data Bank (PDB ID: 1AL9) [33]. Hydrogen atoms and charges were added during a brief relaxation performed using the ―Protein Preparation Wizard‖ workflow in Maestro 9.0 (Schrodinger LLC) [34]. After the hydrogen bond network was optimized, the crystal structure was minimized until the root-mean-square deviation (rmsd) between the minimized structure and the starting structure reached 0.3 Å with OPLS 2005 force field. The grid-enclosing box was placed on the centroid of the binding ligand in the crystal structure, and a scaling factor of 1.0 was set to van der Waals (VDW) radii of those receptor atoms with partial atomic charges of less than 0.25. Standard precision (SP) approach of Glide was adopted to dock the molecules into the binding site with the default parameters, and the top-ranking poses of each molecule were retained. The 13

ACCEPTED MANUSCRIPT docking results suggested that a similar binding pattern is presented among compounds 1~7 with DNA, which is one intercalation between base pair of DNA and the planar portions of compounds 1~7 and another groove interaction between groove

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of DNA and the polyamine chains. The π–π stacking interactions were formed between naphthalimides and DNA base pair. The polyamine chains bind to the groove

SC R

through hydrophobic interaction, as well as had interaction to the backbone phosphate group of DNA duplex by through H-bonds. These findings are different from the similar work by Ahmed Ouameur et al in which polyamines bound to DNA only by

NU

groove because they are lack of planar aromatic ring systems [21, 78].

Besides, the

aminoalkyl chain establishes hydrophobic interaction and the amino groups interact

MA

with N3 of adenine, 2-deoxyribose group, cytidine-O2, thymine-O2 and the backbone phosphate group through hydrogen bond with nucleic acids. The difference of the

D

aminoalkyl chain, however, leads to the side chain effects (Table 6). The binding

TE

energies ∆G revealed the stability of the complexes formed: 7>6>5>3>2>1 (except compound 4), which was basically consistent with the stability of compounds

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1~7-DNA complexes (Kb).

4. Conclusion

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In this work, the effects of a naphthalimide pharmacophore coupled with diverse side chains on the interaction between naphthalimide-polyamine conjugates (1~7) with herring sperm DNA were studied by diverse spectroscopic methods and molecular docking. The triamine compound 7 can interact more potently with DNA than the corresponding diamine compounds (1~6). The presence of the bulky terminal group in the diamine side chain reduced the binding strength of compound 1 with DNA, compared to other diamine compounds (2~6). In addition, the increasing methylene number in the diamine backbone generally results in the elevated binding constant

of

compounds-DNA

complex.

The

main

interaction

of

naphthalimide-polyamine conjugates with DNA through intercalative mode and the quenching mechanism was a static type. Furthermore, the iodide quenching effect corroborated that the compounds 1, 3~7 were the DNA intercalator. In addition, the 14

ACCEPTED MANUSCRIPT fluorescence quenching data measured at different temperatures and additional experiment of NaCl suggested that the binding process was mainly driven by hydrogen bond and hydrophobic forces. And the calculated free binding energies of

IP

T

molecular docking are generally consistent with the stability of polyamine-DNA complexes. What’s more, the B to A-like conformational change can occur because of

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the binding of naphthalimide-polyamine conjugate to DNA, and compounds 1~7 was not the classic DNA intercalator which indicated compounds 1~7 have also the possibility of binding to DNA by groove, particular compound 2. In addition,

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molecular docking supported that compounds 1~7 could bind to DNA through one groove interaction and another intercalation.

MA

Acknowledgements

This work was supported by the China Postdoctoral Science Foundation Funded

D

Project (No. 20110490991), the Henan Natural Science Foundation (No.

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134200510009), the Henan Programs for Science and Technology Development (No. 132102310026) and the Henan Natural Science Foundation of Education (No.

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2011B3500001, 14A350004). We wish to thank Dr. Yuan Zhao (the Key Laboratory of Natural Medicine and Immuno-Engineering in Henan University) for virtual docking simulation work. We also wish to thank DDDC of State Key Laboratory of

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New Drug Design, School of Pharmacy, East China University of Science and Technology for providing us the Glide (Schrodinger LLC) software for the virtual docking simulation work.

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biogenic polyamines, RNA 16 (2010), 1968–1979.

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ACCEPTED MANUSCRIPT O N

N

NH

.2HCl

O

1

NH2

NH m

.2HCl

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N

T

O

n

2, m=1, n=1 3, m=2, n=1 4, m=3, n=1 5, m=2, n=2 6, m=2, n=3 O H N

N H

O

.3HCl

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7

NH2

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N

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O

Fig.1. Structures of naphthalimide-polyamine conjugates

1

2

D

2

0 300

400

Wavelength/nm

0 300

350

1 2 3 4 5 6 7 8 9 10 11 12 13

0 300

350

400

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2.0

400

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2.0

1

Compound 6

1.8

1.8 1.6

1

1.6 1.4

Absorbance

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Absorbance

2

13 13

1.4 1.2

Absorbance

350

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0 300

1

1

Absorbance

13

Compound 3

Compound 2

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Absorbance

Compound 1

Absorbance

2

1.0

13

0.8

1.2 1.0 0.8

0.6

13

0.6 0.4

0.4 0.2

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400

Wavelength/nm

Wavelength/nm

0.0 300

400

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Absorbance

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Fig. 2. UV absorption spectra of compounds 1~7 with herring sperm DNA. Numbers 1–13 indicated the DNA concentration: 0.0, 4.56×10-6, 9.13×10-6, 13.69×10-6, 27.4×10-6, 41.08×10-6, 54.77×10-6, 68.46×10-6, 82.15×10-6, 95.84×10-6, 109.54×10-6, 123.23×10-6 and 136.92×10-6 mol∙ L-1, respectively. Compounds 1~7 applied were 80×10-6 mol∙ L-1.

24

ACCEPTED MANUSCRIPT

250

200

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1 2 3 4 5 6 7 8 9 10 11 12 13

Compound 3 400

Relative Fluorscence

100

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Relative Fluorscence

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1 2 3 4 5 6 7 8 9 10 11 12 13

350

1 2 3 4 5 6 7 8 9 10 11 12 13

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1 Compound 7 500

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500

1

13

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Relative Fluorscence

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1 2 3 4 5 6 7 8 9 10 11 12 13

Compound 4 600

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550

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700

0 350

500

Relative Fluorscence

450

SC R

400

T

100 0 350

0

Fig. 3. Fluorescence spectroscopy of compounds 1~7 and herring sperm DNA. Numbers 1–13 indicated the DNA concentration: 0.0, 4.56×10-6, 9.13×10-6, 13.69×10-6, 27.4×10-6, 41.08×10-6,

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54.77×10-6, 68.46×10-6, 82.15×10-6, 95.84×10-6, 109.54×10-6, 123.23×10-6 and 136.92×10-6 mol∙ L-1, respectively. Compounds 1~7 applied was 80×10-6 mol∙ L-1. Scan condition of compounds 1~7: EX = 345 nm, EM scan is 355~600 nm; Slits of both EX and EM of compounds 1~7 were

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2.5 nm and 5 nm, respectively.

25

ACCEPTED MANUSCRIPT 300

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Relative Fluorscence

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700

750

Wavelength/nm

Wavelength/nm

300

Compound 7

200

150

100

800

650

700

750

800

Wavelength/nm

Compound 6 1

250

200

150

13 100

50

0 550

600

650

700

750

800

Wavelength/nm

MA

1

250

Relative Fluorscence

600

NU

550

1

Relative Fluorscence

200

Compound 5

SC R

Relative Fluorscence

300

1

Relative Fluorscence

Compound 4

250

1

250

IP

Relative Fluorscence

200

Compound 3

Compound 2

1000

T

1 2 3 4 5 6 7 8 9 10 11 12 13

Compound 1

Relative Fluorscence

250

13

50

550

600

650

700

750

800

TE

Wavelength/nm

D

0

Fig. 4. Fluorescence spectroscopy of compounds 1~7 with herring sperm DNA-EB Numbers 1–13 indicated the compounds concentration: 0.0, 4×10-6, 8×10-6, 12×10-6, 24×10-6, 36×10-6, 48×10-6,

CE P

60×10-6, 72×10-6, 84×10-6, 96×10-6, 108×10-6 and 120×10-6 mol∙ L-1, respectively. DNA and EB applied was 13.7×10-6 and 15.7 ×10-6 mol∙ L-1, respectively. Scan condition: EX = 510 nm, EM = 520~800 nm; Slits of both EX and EM of compounds 1~7 were 5 nm except compound 2 which

AC

slits of both EX and EM were 5 nm and 10 nm, respectively.

26

ACCEPTED MANUSCRIPT 1.40

298K 303K 310K

Compound 1 1.35

1.5

298K 303K 310K

Compound 2

1.6

298K 303K 310K

1.5 1.30

1.4

1.4

1.20

1.3

F0/F

F0/F

1.25

F0/F

Compound 3

1.2

1.15

1.3

1.2

1.10

1.1

1.1 1.0

1.0

1.00 20

40

60

80

100

120

140

0

20

40

-6

120

140

0

80

100

120

140

-6

298 303 310 Compound 6

3.0

2.5

F0/F

1.5

F0/F

60

IP 298K 303K 310K

Compound 5

1.6

1.6

40

3.5

1.7

Compound 4

20

CDNA(10 mol/L)

1.8

1.8

F0/F

100

CDNA(10 mol/L)

298K 303K 310K

2.0

80 -6

CDNA(10 mol/L)

2.2

60

SC R

0

T

1.05

1.4

2.0

1.3

1.4

1.2

1.5

1.2 1.1

1.0

1.0

1.0

20

40

60

80

100

120

140

0

-6

20

40

60

80

-6

CDNA(10 mol/L)

CDNA(10 mol/L)

3.5

298K 303K 310K

Compound 7

120

140

0

20

40

60

80

100

120

140

-6

CDNA(10 mol/L)

MA

3.0

100

NU

0

F0/F

2.5

2.0

1.5

0

20

40

60

80

100

120

140

-6

TE

CDNA(10 mol/L)

D

1.0

Fig. 5 Stern-Volmer plot of fluorescence quenching of compounds 1~7 by herring sperm DNA at

AC

CE P

different temperatures

27

ACCEPTED MANUSCRIPT 298K 303K 310K

1.5

Compound 1

1.7

298K 303K 310K

2.6

298K 303K 310K

1.6

2.4

Compound 3

Compound 2 2.2

1.4

1.5

2.0 1.4

F0/F

1.3

1.2

1.6 1.4

1.2

1.1

1.8

1.2

1.1 1.0

T

F0/F

F0/F

1.3

1.0 1.0 40

60

80

100

120

0

20

40

2.4

100

120

20

40

60

80

100

120

-6

298K 303K 310K

Compound 5

2.8

2.6

298K 303K 310K

Compound 6

2.4

2.6

2.2

2.2

2.0

F0/F

2.4

2.0

F0/F

2.2

1.8

2.0 1.8

1.6

1.8 1.6

1.6

1.4

1.4 1.4

1.2

1.2

1.2

1.0

1.0

1.0

20

40

60

80

100

120

0

20

-6

80

100

C5(10 mol/L)

298K 303K 310K

Compound 7

120

0

20

40

60

80

100

120

-6

C6(10 mol/L)

MA

3.5

60 -6

C4(10 mol/L)

4.0

40

NU

0

3.0

F0/F

0

C3(10 mol/L)

3.0

Compound 4

F0/F

80

C2(10 mol/L)

298K 303K 310K

2.6

60 -6

-6

C1(10 mol/L)

IP

20

SC R

0

2.5

2.0

1.5

0

20

40

60

80

100

120

-6

TE

C7(10 mol/L)

D

1.0

Fig. 6 Stern-Volmer plot of fluorescence quenching of herring sperm DNA-EB by compounds

AC

CE P

1~7 at different temperatures

28

ACCEPTED MANUSCRIPT 298 303 310

5.4

298 303 310

5.4

Compound 1 5.2

5.2

5.0

5.0

4.8

4.8

298K 303K 310K

5.4 5.2

4.6 4.4

4.4

4.2

4.2

4.2

4.0

4.0

3.8

3.8

1.2

1.4

1.6

1.8

3.8 0.8

5.2

5.2

Compound 4

Compound 5

5.0

log[1/cDNA]

4.8 4.6 4.4

4.8 4.6 4.4

4.2

4.2

4.0

4.0

3.8

3.8

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

0.0

log[F/(F0-F)]

5.4

4.6 4.4 4.2 4.0

0.6

0.4

0.6

0.8

1.0

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

1.2

1.4

1.6

1.8

2.0

298 303 310

5.4 5.2

Compound 6

5.0 4.8

298 303 310

4.6 4.4 4.2 4.0 3.8 -0.4

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

log[F/(F0-F)]

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

TE

log[F/(F0-F)]

D

3.8 0.2 0.4

0.2

MA

Compound 7

4.8

-0.6 -0.4 -0.2 0.0

0.2

log[F/(F0-F)]

298 303 310

5.2

1.6

NU

-0.2

1.4

log[F/(F0-F)]

298 303 310

5.4

5.0

5.0

1.2

log[F/(F0-F)]

298K 303K 310K

5.4

1.0

T

1.0

0.6

IP

0.8

0.4

SC R

0.6

4.0

0.2

log[F/(F0-F)]

log[1/cDNA]

4.6

4.4

0.4

log[1/cDNA]

4.8

log[1/cDNA]

4.6

5.0

log[1/cDNA]

log[1/cDNA]

log[1/cDNA]

Compound 3 Compound 2

Fig. 7 Plot of log [1/cDNA] versus log[F /(F0 – F)] of the interaction between compounds (1~7)

AC

CE P

and herring sperm DNA at different temperatures

29

ACCEPTED MANUSCRIPT

5.4

Compound 1

4.8

5.0

4.8

4.8 4.6

4.4

4.4

4.4

4.2

4.2

4.2

4.0

4.0

4.0 3.8

3.8

0.8

1.0

1.2

1.4

0.2

1.6

0.4

1.0

298 303 310

5.4

Compound 5

Compound 4

5.0

4.6

4.4

4.4

4.2

4.2

4.0

4.0

3.8 0.4

0.6

0.8

1.0

1.2

1.4

3.8 -0.4

1.6

log[F/(F0-F)]

5.4

0.6

0.8

1.0

1.2

1.4

298 303 310

5.4

Compound 6

5.0 4.8

4.4 4.2 4.0 3.8

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

log[F/(F0-F)]

MA

Compound 7

0.4

4.6

log[F/(F0-F)]

298 303 310

5.6

0.2

5.2

4.8

4.6

0.2

0.0

5.6

NU

4.8

0.0

-0.2

1.4

log[1/c6]

5.2

5.0

log[1/c5]

5.2

-0.2

1.2

log[F/(F0-F)]

5.6

5.4

5.2 5.0 4.8 4.6 4.4 4.2

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

TE

log[F/(F0-F)]

D

4.0 3.8 -0.6

0.8

log[F/(F0-F)]

298 303 310

5.6

0.6

IP

0.6

SC R

0.4

log[F/(F0-F)]

log[1/c4]

5.2

5.0

4.6

0.2

log[1/c7]

Compound 3

4.6

3.8

Plot of log [1/ccomp.] versus log[F /(F0 – F)] of the interaction between compounds 1~7

CE P

Fig. 8

5.4

log[1/c3]

5.0

298 303 310

5.6

Compound 2

5.2

log[1/c2]

5.2

log[1/c1]

298 303 310

5.6

5.4

T

298 303 310

5.6

AC

and herring sperm DNA-EB at different temperatures

30

ACCEPTED MANUSCRIPT 3.0

4.5

Compound 1 +KI Compound 1 +KI+DNA

2.8

5.0

Compound 2 +KI Compound 2 +KI+DNA

4.0

2.6

3.5 3.5

2.2 3.0

1.8

F0/F

2.0

F0/F

F0/F

Compound 3 +KI Compound 3 +KI+DNA

4.5 4.0

2.4

2.5

3.0 2.5

1.6 2.0

2.0

1.5

1.5

1.4 1.2 1.0 0.8 40

60

80

100

0.5

120

0

20

40

60

cKI

100

120

0

cKI

Compound 5 +KI Compound 5 +KI+DNA

6

Compound 4 +KI Compound 4 +KI+DNA

5

80

5

20

40

60

80

100

120

cKI

IP

20

6

SC R

0

T

1.0

1.0

Compound 6 +KI Compound 6 +KI+DNA

5

4

F0/F

4

F0/F

F0/F

4

3

3

3

2

2

1

2

1

20

40

60

80

100

120

1

0

20

Compound 7 +KI Compound 7 +KI+DNA

80

100

120

0

20

40

60

80

100

120

cKI

MA

6

5

F0/F

60

cKI

cKI

7

40

NU

0

4

3

2

0

20

40

60

80

100

120

TE

cKI

D

1

Fig. 9. Fluorescence quenching plots of compounds 1~7 by KI in the absence and presence of

CE P

DNA. c(compound) = 20×10-6 mol∙ L-1; c(DNA) = 22.84 ×10-6 mol∙ L-1; the KI concentration was

AC

4~120×10-3 mol∙L-1

31

ACCEPTED MANUSCRIPT 2.0

1.8

Compound 2

1.6

1.4

F/F0

1.2 1.0 0.8 0.6 0.4

1.4

1.2

1.2

1.0

0.0 0.000

0.005

0.010

0.015

0.020

0.025

0.8

0.6

0.6

0.4

0.4 0.2

0.0 0.000

0.005

0.010

0.015

0.020

0.0 0.000

0.025

1.4

1.2

1.2

1.0

F/F0

F/F0

F/F0

1.2

0.8 0.6

0.6

0.6 0.4

0.4

0.4 0.2

0.2

0.2

0.020

CNaCl(mol/L)

1.8

F/F0

1.2 1.0 0.8 0.6 0.4

0.010

0.015

0.020

0.025

0.0 0.000

0.005

0.010

0.015

CNaCl(mol/L)

0.020

0.025

TE

CNaCl(mol/L)

D

0.2

0.005

0.015

MA

Compound 7

1.4

0.0 0.000

0.010

CNaCl(mol/L)

2.0

1.6

0.005

0.025

NU

0.015

1.0 0.8

0.8

0.010

Compound 6

1.6

1.4

1.4

0.005

0.025

1.8

SC R

Compound 5 1.6

0.0 0.000

0.020

2.0

1.8

1.0

0.015

IP

2.0

1.8

Compound 4

0.010

CNaCl(mol/L)

2.0

0.0 0.000

0.005

CNaCl(mol/L)

CNaCl(mol/L)

1.6

1.0

0.8

0.2

0.2

Compound 3

1.6

1.4

F/F0

Compound 1

1.6

F/F0

2.0

1.8

1.8

T

2.0

AC

CE P

Fig. 10. Effects of NaCl on the fluorescence intensity of compounds 1~7-DNA system

32

0.020

0.025

ACCEPTED MANUSCRIPT

Compound 2 10

Ellipticity (mdeg)

8 6 4 2

6 4 2 0

-2

-2

-4

-4 240

260

280

300

320

340

360

380

200

0 16 32 48

20

Compound 3

240

260

280

300

320

340

Ellipticity (mdeg)

NU

5

MA

0

-5

-10 200

220

240

260

280

300

320

340

360

0 16 32 48

8 6 4 2 0 -2 -4 -6

380

200

220

240

260

280

300

320

340

360

380

Wavelength/nm

D

Wavelength/nm

380

Compound 4

10

10

360

Wavelength/nm

12

15

Ellipticity (mdeg)

220

SC R

220

Wavelength/nm

12

0 16 32 48

Compound 5

8

4 2 0 -2 -4 220

240

260

280

300

320

340

0 16 32 48

Compound 6

10 8 6 4 2 0 -2 -4

360

380 -6

AC

200

CE P

6

12

Ellipticity (mdeg)

TE

10

Ellipticity (mdeg)

8

0

200

0 16 32 48

12

0 16 32 48

Compound 1

T

Ellipticity (mdeg)

10

IP

12

200

Wavelength/nm

220

240

260

280

300

320

340

360

380

Wavelength/nm

0 16 32 48

14

Compound 7

12

Ellipticity (mdeg)

10 8 6 4 2 0 -2 -4 -6 200

220

240

260

280

300

320

340

360

Wavelength/nm

Fig. 11. Circular dichroism spectra of DNA (182.56×10-6 mol∙ L-1 and the compounds–DNA system at 0, 16, 32 and 48 ×10-6 mol∙ L-1.

33

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

34

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

Fig. 12. The models of molecule simulation in silico of compounds 1~7 binding to DNA. compounds 1~7 bind to DNA through groove interaction and intercalation. The DNA is represented by tube (colored by atom type: C, yellow; polar H, blue; Cl, green; N, dark blue; O, red; P, orange). 35

ACCEPTED MANUSCRIPT

Table 1 Quenching constants of the interaction between compounds 1~7 and herring sperm DNA -1

310 298 2

303 310 298 303 310 298 303 310 298

5

303

310 7

CE P

298

TE

298 303

303

2.560×10

3

3.660×10

3

3.350×10

3

3.480×10

3

6.690×10

3

5.690×10

3

4.140×10

3

5.000×10

3

4.880×10

3

4.230×10

3

1.551×10

4

1.280×10

4

8.640×10

3

1.526×10

4

1.418×10

4

1.240×10

4

AC

310

3.200×10

3

D

310 6

3.630×10

3

MA

4

7.585×10

2

36

0.9327

11

0.7819

10

0.7529

11

0.9747

11

0.9700

11

0.9682

11

0.9810

11

0.9893

11

0.9875

11

0.9752

11

0.9923

11

0.9650

11

0.9879

11

0.9879

11

0.9805

12

0.9648

12

0.9536

11

0.9800

12

0.9681

12

0.9658

12

0.9762

1.970×10 1.590×10

7.585×10 3.630×10

NU

3

1.590×10

3

3.200×10

2.560×10 3.660×10

3.350×10

3.480×10 6.690×10

5.690×10

r

11

T

303

1.970×10

3

Kq(L•mol )

IP

298 1

-1

Ksv(L•mol )

T(K)

SC R

Compound

4.140×10 5.000×10 4.880×10 4.230×10 1.551×10 1.280×10 8.640×10 1.526×10 1.418×10 1.240×10

ACCEPTED MANUSCRIPT Table.2 Quenching constants of the interaction between compounds 1~7 and herring sperm DNA-EB -1

1

303 310 298

2

303 310 298

3

303 298 303 310 298 303 310

TE

298 6

303 310

CE P

298

7

D

5

303

4.800×10

3

4.010×10

3

4.450×10

3

1.169×10

4

1.182×10

4

1.039×10

4

1.107×10

4

1.247×10

4

1.167×10

4

1.408×10

4

1.092×10

4

1.247×10

4

1.229×10

4

1.131×10

4

1.123×10

4

2.153×10

4

1.954×10

4

1.699×10

4

AC

310

2.260×10

3

MA

4

3.260×10

3

37

3.840×10

0.9805

3.260×10

11

0.9667

2.260×10

11

0.9433

11

0.9937

11

0.9873

11

0.9887

12

0.9894

12

0.9928

12

0.9939

12

0.9927

12

0.9909

12

0.9888

12

0.9875

12

0.9950

12

0.9940

12

0.9975

12

0.9924

12

0.9940

12

0.9977

12

0.9981

12

0.9990

4.800×10

NU

310

3.840×10

r

11

IP

298

3

-1

Kq(L•mol )

T

Ksv(L•mol )

T(K)

4.010×10

SC R

Compound

4.450×10 1.169×10 1.182×10 1.039×10 1.107×10 1.247×10 1.167×10 1.408×10 1.092×10 1.247×10 1.229×10 1.131×10 1.123×10 2.153×10 1.954×10 1.699×10

ACCEPTED MANUSCRIPT Table 3 Binding constants and thermodynamic parameters of the interaction between compounds (1~7) and herring sperm DNA

298 2

303 310 298

3

303 310 298

4

303 310 298

5

303 310 298

6

303 310

0.9115

−18.162

−51.876

−0.111

0.7200

2

−14.866

−51.876

−0.111

0.5952

3

−20.025

−44.653

−0.0826

0.9104

3

−19.612

−44.653

−0.0826

0.9262

3

−19.431

−44.653

−0.0826

0.9246

3

−20.298

11.338

0.106

0.9865

3

−19.770

11.338

0.106

0.9753

3

−21.570

11.338

0.106

0.9845

3

−21.881

−36.576

−0.0560

0.9555

3

−21.980

−36.576

−0.0560

0.9978

3

−21.208

−38.576

−0.0560

0.9844

−21.159

−20.536

0.00209

0.9917

−21.169

−20.536

0.00209

0.9942

−21.468

−20.536

0.00209

0.9821

1.142×10

−23.149

−30.011

−0.0230

0.9830

3

−23.033

−30.011

−0.0230

0.9847

3

−23.146

−30.011

−0.0230

0.9615

4

−23.157

−10.022

0.0441

0.9885

4

−23.376

−10.022

0.0441

0.9944

4

−23.783

−10.022

0.0441

0.9815

1.352×10 3.200×10 3.238×10 2.405×10 1.881×10 3.612×10 2.561×10 4.313×10 6.847×10 6.156×10 3.747×10

3

5.116×10

3

4.462×10

3

4.144×10

4

9.351×10 7.952×10 1.146×10

303 310

1.072×10 1.018×10

38

T

−0.111

AC

7

−51.876

CE P

298

−18.718

3

1.910×10

IP

310

r

SC R

303

∆S° (kJ∙mol-1)

NU

1

∆H° (kJ∙ mol-1)

3

MA

298

Kb (L•mol-1) ∆G°(kJ•mol-1)

D

T(K)

TE

Compound

ACCEPTED MANUSCRIPT

Table 4 Binding constants and thermodynamic parameters of the interaction between compounds and DNA-EB complex 3.637×103

−20.307

−30.460

303

2.960×10

3

−20.136

−30.460

7.176×10

2

−16.948

4.034×10

3

−20.684

3.185×10

3

−20.319

3.872×10

3

−21.293

1.419×10

4

−23.687

1.405×10

4

−24.509

1.136×10

4

1.214×10

4

1.462×10

4

1.364×10

4

1.924×10

4

1.223×10

4

1.286×10

4

298 303 310 298 4

303 310 298

5

303 310 298

6

303 298 303 310

−0. 0341

0.9524

−2.263

0.0614

0.9945

−2.263

0.0614

0.9912

−2.263

0.0614

0.9964

−14.269

0.0316

0.9945

−14.269

0.0316

0.9876

−24.066

−14.269

0.0316

0.9935

−23.300

−7.475

0.103

0.9931

−24.158

−7.475

0.103

0.9892

−24.539

−7.475

0.103

0.9925

−24.441

−25.785

−0.00451

0.9960

−23.708

−25.786

−0.00451

0.9880

IP

T

−30.460

−24.387

−25.786

−0.00451

0.9675

1.309×10

−23.487

−5.565

0.0601

0.9983

1.179×10

4

−23.616

−5.565

0.0601

0.9945

1.200×10

4

−24.209

−5.565

0.0601

0.9950

2.155×10

4

−24.721

−12.640

0.0405

0.9993

1.981×10

4

−24.924

−12.640

0.0405

0.9979

1.895×10

4

−25.386

−12.640

0.0405

0.9962

AC

7

0.9469

4

CE P

310

−0. 0341

SC R

310

0.9690

NU

303

r

−0.0341

MA

298

3

∆H° (kJ∙ mol-1) ∆S° (kJ∙mol-1)

298 310 2

Kb (L•mol-1) ∆G°(kJ•mol-1)

D

1

T(K)

TE

Compound

39

ACCEPTED MANUSCRIPT

Table 5 Dynamic quenching constants of compounds by KI in the absence and presence of DNA Ksv of absence of DNA (L•mol-1)

Ksv of presence of DNA (L•mol-1)

1

14.12

8.79

2

23.67

22.97

3

28.595

4

32.43

5

36.2

6

40.47

7

42.77

T

Compound

IP

23.620

NU

SC R

29.83 30.41 28.72 35.14

Table 6 Binding bites and the free binding energies of the interaction between the side chains of compounds 1~7 and DNA in model docking

4 5 6 7

MA

D

3

N3 of adenine N3 of adenine, thymine-O2 and the 2-deoxyribose group N3 of adenine and the 2-deoxyribose group N3 of adenine, and the the backbone phosphate 2-deoxyribose group group N3 of adenine and the Cytosine-O and the 2-deoxyribose group 2-deoxyribose group N3 of adenine and thymine-O2 N3 of adenine and the N3 of adenine and the 2-deoxyribose group 2-deoxyribose group

TE

2

N2 of side chain

CE P

1

N1 of side chain

AC

Compound

40

N3 of side chain ∆G°(kj•mol-1)

−9.95 −10.02 −10.04 −10.00 −10.08 −10.65 −11.26

ACCEPTED MANUSCRIPT Graphical Abstract Article (or other type)

Spectroscopic and molecular

interaction between

O

N H 7

naphthalimide-polyamine

NH2

IP

N

H N

SC R

modeling methods to study the

T

O

.3HCl

Compound 7

NU

Absorbance

1

13

0 300

350

400

1 Compound 7

500

400

300

13

200

100

0 350

400

450

500

550

600

Wavelength/nm

TE

D

MA

Wavelength/nm

Relative Fluorscence

600

conjugates and DNA

0 16 32 48

AC

Compound 7

12 10

Ellipticity (mdeg)

CE P

14

8 6 4 2 0 -2 -4 -6 200

220

240

260

280

300

320

340

360

Wavelength/nm

Zhiyong Tian Yingying huang,

The interaction between naphthalimide-polyamine conjugates with herring

Yan Zhang, Lina Song, Yan Qiao,

sperm DNA was studied by UV/vis absorption, fluorescent spectra CD and

Xuejun Xu*, Chaojie Wang *

model docking under physiological conditions.

41

ACCEPTED MANUSCRIPT Highlights

1. The binding strength was associated with side chain effects of polyamine.

IP

T

2. Polyamine conjugates could bind to herring sperm DNA by intercalation and groove.

SC R

3. Compounds 1~7 could cause the B to A-like DNA conformational change. 4. The type of interaction force was mainly hydrogen bonding and hydrophobic force.

AC

CE P

TE

D

MA

NU

5. The fluorescent quenching mechanism is a static type.

42