The effects of varying the substituent and DNA sequence on the stability of 4-substituted DNA-naphthalimide complexes

Article abstract of DOI:10.1016/j.bpc.2018.04.008

DNA duplexes are stabilized by many interactions, one of which is stacking interactions between the nucleic acid bases. These interactions are useful for designing small molecules that bind to DNA. Naphthalimide intercalators have been shown to be valuable anti-cancer agents that stack between the DNA bases and exhibit stabilizing effects. There is a continued need to design intercalators that will exhibit these stabilizing effects while being more selective toward DNA binding. This work investigates 4-substituted naphthalimides with varying functional groups and their interactions with nucleic acid duplexes. Mode of binding was determined via wavelength scans, circular dichroism, and viscosity measurements. Optical melting experiments were used to measure the absorbance of the sample as a function of temperature. The T_m values derived from the DNA duplexes were subtracted from the T_m values derived from the DNA-intercalator complexes, resulting in ΔT_m values. The ΔT_m values demonstrated that the substituents on the intercalator affect the stability of the DNA-intercalator complex. From the results of this study and comparison to results from previous work, we conclude that the substituent type and position on the core intercalator molecule affect the stability of the complex it forms with DNA.

Full text of DOI:10.1016/j.bpc.2018.04.008

Accepted Manuscript
The effects of varying the substituent and DNA sequence on the
stability of 4-substituted DNA-naphthalimide complexes
Elizabeth A. Jolley, Laura K.E. Hardebeck, Yi Ren, Miranda S.
Adams, Michael Lewis, Brent M. Znosko
PII:
DOI:
Reference:
S0301-4622(18)30036-X
doi:10.1016/j.bpc.2018.04.008
BIOCHE 6088
To appear in:
Biophysical Chemistry
Received date:
Revised date:
Accepted date:
7 February 2018
22 April 2018
26 April 2018
Please cite this article as: Elizabeth A. Jolley, Laura K.E. Hardebeck, Yi Ren, Miranda S.
Adams, Michael Lewis, Brent M. Znosko , The effects of varying the substituent and DNA
sequence on the stability of 4-substituted DNA-naphthalimide complexes. The address
for the corresponding author was captured as affiliation for all authors. Please check if
appropriate. Bioche(2018), doi:10.1016/j.bpc.2018.04.008
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ACCEPTED MANUSCRIPT
The effects of varying the substituent and DNA sequence on the stability of 4-substituted DNA-
naphthalimide complexes
Elizabeth A. Jolley, Laura K. E. Hardebeck, Yi Ren, Miranda S. Adams, Michael Lewis, and
Brent M. Znosko*
*Department of Chemistry, Saint Louis University, 3501 Laclede Ave., St. Louis, MO 63103,
United States

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ABSTRACT
DNA duplexes are stabilized by many interactions, one of which is stacking interactions between
the nucleic acid bases. These interactions are useful for designing small molecules that bind to
DNA. Naphthalimide intercalators have been shown to be valuable anti-cancer agents that stack
between the DNA bases and exhibit stabilizing effects. There is a continued need to design
intercalators that will exhibit these stabilizing effects while being more selective toward DNA
binding. This work investigates 4-substituted naphthalimides, with varying functional groups,
and their interactions with nucleic acid duplexes. Mode of binding was determined via
wavelength scans, circular dichroism, and viscosity measurements. Optical melting experiments
were used to measure the absorbance of the sample as a function of temperature. The Tm values
derived from the DNA duplexes were subtracted from the Tm values derived from the DNA-
intercalator complexes, resulting in ΔTm values. The ΔTm values demonstrated that the
substituents on the intercalator affect the stability of the DNA-intercalator complex. From the
results of this study and comparison to results from previous work, we conclude that the
substituent type and position on the core intercalator molecule affect the stability of the complex
it forms with DNA.

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1. INTRODUCTION
Nucleic acids are attractive target molecules for therapeutics as they direct replication,
transcription, and translation.1 The ability to rationally design drugs that target nucleic acids,
therefore, is important in developing pharmaceuticals to combat cancer, tumor growth, etc.2,3 As
small molecule therapeutics that target nucleic acids continue to be developed, two classes of
agents have proven most promising, groove binders and intercalators.4 Groove binders bind
noncovalently to DNA and interact with either the sugar-phosphate backbone, the major groove,
or the minor groove.1 Intercalators also bind noncovalently to DNA and interact with the helix
by inserting between the bases.1 Intercalator structures generally consist of a polycyclic aromatic
core and a branching moiety and have been shown upon insertion to distort, by unwinding or
lengthening, the DNA helix without disrupting the hydrogen bonding of the base pairs.1,4-6
Naphthalimides are DNA intercalating agents that are useful as therapeutic agents due to
their conjugated pi system and ability for moiety attachment.3,7 Derivatives of 3-amino-1,8-
naphthalic anhydride were synthesized by Braña et al.8 and tested for activity against HeLa
cells.8,9 Derivatives with an amino group (amonafide) or nitro group (mitonafide) at the 3-
position (Figure 1), both with an ethylene linking chain between the core naphthalimide and a
terminal amine, were found to be most active.9 The therapeutic success of naphthalimide
intercalators stems from their ability to inhibit topoisomerase II activity.5,7,10 Due to the success
of the originally studied 1,8-naphthalimides, including the amonafide and mitonafide derivatives,
different analogues continue to be investigated for therapeutic activity.5,10-15
Figure 1. 3-substituted 1,8-naphthalimides amonafide (left) and mitonafide (right).
A significant amount of evidence shows that naphthalimides bind to nucleic acids via
intercalation. Waring et al. (1979) collected significant data to verify that mitonafide and
amonifide bound via intercalation.2 Tandon et al. (2017) review the discovery and development
of the 1,8-naphthalimide moiety and discuss that imide and ring-substituted naphthalimides,
bisnaphthalimides, and naphthalimide-metal complexes bind DNA via intercalation.15 More
specifically, they present previously published data showing that naphthalimide-polyamine
derivatives, naphthalimide derivatives containing hydroxyl-alkylamines, naphthalimide-
polyamine conjugates with amine substitutions, piperazine and piperidine conjugates of
naphthalimide, 5- or 6-substituted naphthalimide derivatives, 5-aromatic substituted
naphthalimides, naphthalimide derivatives with a flexible leucine chain, 2-allyl-6-substituted

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naphthalimide derivatives, as well as a variety of bis-naphthalimide derivatives and
naphthalimide-heterocycle hybrids bind DNA via intercalation. Johnson et al. (2015) previously
used multiple techniques to confirm that 3-substituted naphthalimides bind via intercalation.16
Most related to the 4-substitutents used in this study, Quintana-Espinoza et al. (2013) provided
data to show that 4-arylnaphthalimide derivatives bind via intercalation.17
Intercalation can be sequence specific. The general 5’-pyrimidine-purine-3’ preference
exhibited by simple monointercalators is supported by ample experimental evidence.18-20 In
general, simple intercalators show a preference for a CpG step. It has been suggested that this
preference arises from the lower energy required for unstacking this sequence relative to
others.21-23 Ample experimental evidence has also shown that the bis-intercalators echinomycin
and triostin A bind DNA by bisintercalation, with a preference for CpG steps.7,24-25 Evidence
also show that actinomycin has a marked preference for a GpC step.26 Previous experiments
show that GpT or TpG steps are the preferred binding step for naphthalimides. Bailly et al.
(1996) have shown that amonafide and mitonafide have a sequence preference for TpG and GpT
steps.27
Structural information offers insight into the characteristics essential for effective
intercalators. Currently, there is no solved structure of a mono-naphthalimide complexed with
DNA. However, along with ongoing research with mono-naphthalimides, there has been
developing work with bis-naphthalimides.28 There is one NMR structure in the Protein Data
Bank (PDB) containing a DNA duplex and bis-naphthalimide intercalator.26 This bis-
naphthalimide contains an aminoalkyl linker bridging the two-naphthalimide core molecules.
The structure shows the bisnaphthalimide binding to DNA at two TpG steps. The aminoalkyl
linker of the bis-naphthalimide is thought to play a major role in the intercalator approaching via
the major groove.27 Although the study reported that there appeared to be no effect on DNA
intercalation based on the substituent of the bis-naphthalimide ring,27 this could be masked by
the effect of the linker arm constraining the two moieties.
With the ongoing study of naphthalimide intercalators and their derivatives, there is still a
need to understand these molecules and their ability to interact with DNA. While most studies
focus on developing new intercalating compounds, few studies focus on the effects of small
changes to the existing intercalator structures.10,29,30 Also, most studies of naphthalimide
derivatives use either calf thymus30-33 or salmon testes DNA5,34 as the target nucleic acid. By
using such large DNA, it is difficult to understand DNA-intercalator interactions that are a result
of the specific DNA sequence. Johnson et al.16 recognized the need to understand intercalator
interactions with short oligonucleotides. A systematic study was conducted with 11 different
substituents at the 3-position of a naphthalimide intercalator with seven DNA duplexes and two
RNA duplexes. The results of the Johnson et al.16 study illustrated that nucleic acid sequence
and intercalator substituent do affect the stability of the DNA-intercalator complexes. By using
the same naphthalimides with both DNA and RNA, they were able to show that the
naphthalimide intercalators do not bind to the RNA duplexes. By studying the same intercalator
with different DNA duplexes of variable sequence composition, they were able to show stability
differences stemming from the differing sequence compositions. Similarly, using different
substituents attached to the 3-position of the naphthalimide and studying them complexed to the

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same DNA duplexes attributed the stability differences directly to effects from the substituents.16
What is still unclear is if the substituent is moved from the 3-position to another position on the
naphthalimide core, would the same trends be observed. Also, are the properties of
naphthalimides dictated by the naphthalimide core, the identity of the substituents, and/or the
location of the substituents?
To further evaluate the interactions important in DNA-intercalator complexes, herein we
report effects on stability of 4-substituted naphthalimide intercalators complexed with DNA.
Specifically, this study shows effects based on substituent identity, position of substituent, and
sequence composition. The DNA sequences and naphthalimide substituents used in this study
were the same as those used by Johnson et al.,16 with a difference in position of the substituent
(4- instead of 3-substituted). This work will provide a framework for more detailed studies of
other known intercalators, as well as aid in the rational design of novel intercalators that may
serve as therapeutic agents.
2. MATERIALS AND METHODS
2.1 Preparation of Oligonucleotides
The duplexes studied were also used in a previous study with 3-substituted
naphthalimides.16 Oligonucleotides were designed to have a mixture of sequence compositions,
with some A-T rich sequences and some G-C rich sequences giving a variety of possible binding
steps. These oligonucleotides were purchased from Integrated DNA Technologies (Coralville,
IA) and purified following previous procedures.35 The following duplexes were obtained: d(5’-
ATATATATATAT-3’)2 (A), d(5’-ATATATGATATA-3’)/(3’-TATATCATATAT-5’) (B), d(5’-
ATATAGTATATA-3’)/(3’-TATATACTATAT-5’) (C), d(5’-GTAATATTAC-3’)2 (D), d(5’-
GCGCGCGC-3’)2 (E), d(5’-GTCCGCGGAC-3’)2 (F), d(5’-GTCCGTCGGAC-3’)/(3’-
CAGGCAGCCTG-5’) (G), r(5’-GUCCGCGGAC-3’)2 (H), and r(5’-GCGCGCGC-3’)2 (I).
2.2 Preparation of Naphthalimide Derivatives
Naphthalimide intercalators with a total of eight different substituents at the 4-position
(Figure 2) were synthesized (as shown in Schemes 1-4). The functional groups appended
(similar to those used in a previous study with 3-substituted naphthalmides16) include: -H (HN), -
NO2 (NN), -NH2 (AN), -Cl (CN), -Br (BN), -I (IN), -NHCH(CH3)2 (IPN), and -NH(COCH3)
(NAN). The compounds 1,8-naphthalic anhydride; 4-nitro-1,8-naphthalic anhydride; 4-chloro-
1,8-naphthalic anhydride; and 4-bromo-1,8-naphthalic anhydride were purchased from Sigma-
Aldrich (St. Louis, MO). The 4-amino-1,8-naphthalic anhydride was synthesized from a
palladium reduction reaction with 4-nitro-1,8-naphthalic anhydride (Scheme 1). These five
naphthalic anhydride compounds gave the naphthalimide intercalators HN, NN, AN, CN, and
BN through condensation reactions with N,N-dimethylethylenediamine (Scheme 2).36 The IN
naphthalimide intercalator was synthesized via the Sandmeyer reaction with 4-amino-N-(2-
dimethylaminoethyl)-1,8-naphthalimide (Scheme 3).37 The IPN and NAN naphthalimide
intercalators were synthesized from 4-amino-N-(2-dimethylaminoethyl)-1,8-naphthalimide with
Na2CO3 and 2-iodopropane for IPN and with triethylamine and acetyl chloride for NAN
(Scheme 4).38,39 These intercalators were prepared as 0.01 M stock solutions in DMSO. The
successful synthesis of each intercalator was confirmed by NMR spectroscopy and mass
spectrometry.

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Figure 2. Naphthalimide intercalator core with substituents at 4-position where X = -H (HN), -NO2 (NN), -NH2
(AN), -Cl (CN), -Br (BN), -I (IN), -NHCH(CH3)2 (IPN), or -NHCOCH3 (NAN).
2.2.1 Synthesis of 4-amino-naphthalic anhydride (Scheme 1)
Scheme 1. Reduction of 4-nitro-1,8-naphthalic anhydride to 4-amino-1,8-naphthalic anhydride.
4-nitro-1,8-naphthalic anhydride (1.26 g, 5.20 mmol) was transferred to a three-necked
round bottom flask and dissolved in 165 mL of acetonitrile until the solid completely dissolved
to a brown-red solution. Palladium on activated carbon (0.18 g) was added to a three-neck round
bottom flask, which was sealed and evacuated. The solution stirred under H2 for 72 h at room
temperature and under atmospheric pressure. The reaction solution was then filtered, resulting in
the crude product, which was a dark yellow solid, as well as the catalyst. The crude product was
refluxed in 250 mL of acetone and filtered hot. This process was repeated and the aliquots of
acetone combined until all of the crude product was dissolved in acetone and the palladium
catalyst had been removed via filtration. The acetone solution was then filtered through Celite©
and removed via rotary evaporation to yield the purified product. (yellow solid, 1.11 g, 98%)
1H NMR (400 MHz, d6-DMSO) 6.88 (d, J=8 Hz, 1H), 7.69 (t, J=7 Hz, 1H), 7.79 (s, 2H),
8.19 (d, J=8 Hz, 1H), 8.44 (dd, J=1, 7 Hz, 1H), 8.70 (dd, J=1,8 Hz, 1H).
2.2.2 Synthesis of HN (Scheme 2)
The synthesis of HN was reported previously.40

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Scheme 2. Condensation of 1,8-naphthalic anhydrides where R = H, NO2, NH2, Cl, or Br with N,N-
dimethylethylenediamine to yield HN, NN, AN, CN, or BN.
2.2.3 Synthesis of NN (Scheme 2)
4-nitro-1,8-naphthalic anhydride (2.00 g, 8.22 mmol) was transferred to a round bottom
flask and dissolved in 150 mL of toluene. A slight excess of N,N-dimethylethylenediamine
(1.40 mL, 12.82 mmol) was added to the 4-nitro-1,8-naphthalic anhydride solution drop-wise.
The solution refluxed at T = 115~120 °C for 48 h. An additional 150 mL of MeOH was added to
the solution before rotavap. The resulting solid was dissolved in CH2Cl2 and washed with
saturated NaHCO3 and brine. Anhydrous MgSO4 was added to the organic layer to dry the
solution before rotavap. Filtration was performed to obtain the final product, which was
confirmed by LCMS and NMR spectra. (dark brown solid, 1.52 g, 59%)
1H NMR (400 MHz, d6-DMSO) 2.22 (s, 6H), 4.17 (t, J=7 Hz, 2H), 8.11 (t, J=8 Hz, 1H),
8.58 (s, 1H), 8.62 (s, 1H), 8.65 (dd, J=1,8 Hz, 1H), 8.73 (dd, J=1,9 Hz, 1H). EI+ ; M+ calculated
313.1063, found 313.1057 (C16H15N3O4).
2.2.4 Synthesis of AN (Scheme 2)
4-amino-1,8-naphthalic anhydride (0.44 g, 2.07 mmol) was added to a round bottom flask
and dissolved in 290 mL of 200 proof ethanol, resulting in a clear, yellow-orange solution. The
round bottom flask was transferred to a sand bath and heated until boiling. N,N-
dimethylethylenediamine (0.45 mL, 4.14 mmol) was added drop-wise to the naphthalimide
solution while then stirred at reflux for 2 h. The solution then stirred at room temperature for an
additional 48 h, and the solvent was removed via rotary evaporation to yield the final product.
(brown solid, 0.58 g, 91.2%)
1H NMR (400 MHz, d6-DMSO) 2.19 (s, 6H), 4.12 (t, J=7 Hz, 2H), 6.84 (d, J=8 Hz, 1H),
7.44 (s, 2H), 7.65 (t, J=8 Hz, 1H), 8.19 (d, J=8 Hz, 1H), 8.43 (dd, J=1,7 Hz, 1H), 8.61 (dd, J=1,8
Hz, 1H). 13C NMR (400 MHz, d6-DMSO) 30.6, 39.9, 56.6, 107.5, 108.2, 119.3, 121.7, 124.0,

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129.3, 129.7, 130.8, 131.0, 134.0, 162.9, 163.8. FAB+ ; (M+H)+ calculated 284.1400, found
284.1393 (C16H18N3O2).
2.2.5 Synthesis of CN (Scheme 2)
4-chloro-1,8-naphthalic anhydride (0.50 g, 2.15 mmol) was dissolved in 70 mL of 200
proof EtOH. N,N-dimethylethylenediamine (0.5 mL, 4.58 mmol) was added to the 4-chloro-1,8-
naphthalic anhydride solution drop-wise. The solution stirred at room temperature for 5 h before
evaporating EtOH and producing the LCMS and NMR confirmed product. (yellow solid, 0.36 g,
55%)
1H NMR (400 MHz, d6-DMSO) 2.21 (s, 6H), 4.16 (t, J=7 Hz, 2H), 8.02 (dd, J=2,9 Hz,
1H), 8.06 (d, J=8 Hz, 1H), 8.45 (d, J=8 Hz, 1H), 8.61 (m, J=1,10 Hz, 2H). 13C NMR (400 MHz,
d6-DMSO) 30.7, 45.4, 56.3, 121.4, 122.7, 127.7, 128.4, 128.5, 128.6, 130.1, 130.9, 131.6, 137.5,
162.7, 163.0. EI+ ; M+ calculated 302.0822, found 302.0821 (C16H15Cl35N2O2).
2.2.6 Synthesis of BN (Scheme 2)
4-bromo-1,8-naphthalic anhydride (0.60 g, 2.17 mmol) was dissolved in 70 mL of 200
proof EtOH. N,N- dimethylethylenediamine (0.5 mL, 4.58 mmol) was added to the 4-chloro-
1,8-naphthalic anhydride solution drop-wise. The solution stirred at room temperature for 5 h
before evaporating EtOH and producing the LCMS and NMR confirmed product. (brown solid,
0.22 g, 29%)
1H NMR (400 MHz, d3-ACN) 2.94 (d, J=5 Hz, 6H), 3.44 (q, J=5 Hz, 2H), 4.42 (t, J=5
Hz, 2H), 6.94 (d, J=8 Hz, 1H), 7.71 (t, J=8 Hz, 1H), 8.33 (d, J=8 Hz, 1H), 8.40 (dd, J=1,8 Hz,
2H), 8.56 (dd, J=1,7 Hz, 2H). 13C NMR (400 MHz, d6-DMSO) 34.8, 42.8, 55.3, 107.5, 108.3,
119.3, 121.7, 124.0, 129.5, 129.9, 131.3, 134.2, 153.1, 163.3, 164.4. EI+ ; M+ calculated
346.0317, found 346.0309 (C16H15Br79N2O2).
2.2.7 Synthesis of IN (Scheme 3)
4-amino-naphthalimide (0.20 g, 0.71 mmol) was added to a round bottom flask and
dissolved in 55 mL of a 3.25 M HI solution, resulting in a orange-brown solution. The
naphthalimide solution was cooled to 0ºC in a salt-ice bath. A solution of NaNO2 (0.5 g, 0.71
mmol in 1 mL of DI H2O) was cooled to 0ºC and added drop-wise to the stirring naphthalimide
solution to yield the diazonium intermediate. A solution of CuI (0.13 g in 5 mL of conc. HI) was
cooled to 0ºC as well and added drop-wise to the stirring diazonium solution. The reaction
stirred at 0ºC for 1 h and was then removed from the salt-ice bath to warm to room temperature.
The reaction stirred at room temperature for 18 h before being filtered, resulting in the product.
(dark brown solid, 0.14 g, 49%)

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Scheme 3. Synthesis of IN from AN via a Sandmeyer reaction.
1H NMR (400 MHz, d6-DMSO) 2.21 (s, 6H), 4.16 (t, J=7 Hz, 2H), 8.02 (t, J=8 Hz, 1H),
8.25 (d, J=8 Hz, 1H), 8.37 (d, J=8 Hz, 1H), 8.60 (m, J=1,8 Hz, 2H). 13C NMR (400 MHz, d6-
DMSO) 30.7, 45.4, 56.3, 122.0, 122.7, 128.3, 128.9, 129.1, 129.8, 131.1, 131.4, 131.7, 132.7,
162.8, 162.9.
2.2.8 Synthesis of IPN (Scheme 4)
Scheme 4. Synthesis of IPN and NAN from AN, via alkylation and acylation protocols, respectively.
4-amino-naphthalimide (0.36 g, 1.28 mmol) was added to a round bottom flask and was
dissolved in 75 mL of 200 proof ethanol, which partially dissolved the starting material. Upon
heating the solution to reflux, the starting material fully dissolved to a brown-orange solution. A
Na2CO3 solution (0.32 g, 2.99 mmol in 2.5 mL DI H2O) was added drop-wise to the stirring
naphthalimide solution. 2-iodopropane (1.5 mL, 15 mmol) was added drop-wise to the stirring
solution which was then heated at 80ºC for 30 h. Upon reaching room temperature, the crude
product precipitated out of solution and was filtered to isolate. The crude product was washed
with a 1:1 methanol-dichloromethane solution and filtered, resulting in the purified product.
(yellow solid, 0.10 g, 35%)
1H NMR (400 MHz, d6-DMSO) 1.37 (d, J=6 Hz, 4H), 3.09 (s, 4H), 3.50 (t, J=8 Hz, 2H),
3.89 (m, J=6 Hz, 1H), 4.43 (t, J=7 Hz, 2H), 6.87 (d, J=8 Hz, 1H), 7.58 (s, 1H), 7.69 (t, J=8 Hz,
1H), 8.23 (d, J=8 Hz, 1H), 8.47 (dd, J=1,7 Hz, 1H), 8.66 (dd, J=1,8 Hz, 1H). 13C NMR (400
MHz, d6-DMSO) 16.5, 33.2, 47.6, 59.1, 64.8, 107.4, 108.9, 119.9, 121.9, 122.3, 124.5, 130.3,
130.6, 132.0, 132.7, 134.9, 163.4, 164.4. FAB+ ; (M+H)+ calculated 326.1870, found 326.1869
(C19H24N3O2).

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2.2.9 Synthesis of NAN (Scheme 4)
4-amino-naphthalimide (0.22 g, 0.79 mmol) was added to a three-necked round bottom
flask and was dissolved in 75 mL of dry acetonitrile. The flask was evacuated and refilled with
nitrogen gas before being placed in an ice bath and cooled to 0ºC. Triethylamine (0.22 mL, 1.58
mmol) was added drop-wise via a syringe followed by acetyl chloride (0.0789 mL, 1.11 mmol),
which was also added in the same manner. The solution stirred at 0ºC for 15 min and at room
temperature for 4 h. The solution was then filtered, resulting in the final product. (yellow solid,
0.09 g, 35.8%)
1H NMR (400 MHz, d6-DMSO) 2.29 (s, 3H), 2.89 (s, 6H), 4.39 (t, J=6 Hz, 2H), 7.92 (t,
J=8 Hz, 1H), 8.35 (d, J=8 Hz, 1H), 8.51 (d, J=8 Hz, 1H), 8.56 (dd, J=1,7 Hz, 1H), 8.78 (dd,
J=1,9 Hz, 1H), 10.48 (s, 1H). 13C NMR (400 MHz, d6-DMSO) 24.4, 35.1, 42.8, 54.9, 117.5,
119.3, 122.4, 124.0, 126.4, 128.5, 129.6, 131.0, 131.8, 140.6, 163.5, 164.1, 169.7. FAB+ ;
(M+H)+ calculated 326.1506, found 326.1506 (C18H20N3O3).
2.3 UV-Vis Absorption Spectra Studies
UV-Vis absorption scans for each of the nine oligonucleotides with each of the eight
naphthalimide intercalators (72 complexes) were performed. Naphthalimide intercalator
concentration was kept constant at 0.25 mM and aliquots of increasing DNA or RNA
concentration (12.5, 20.0, 25.7, 35.0, and 42.5 μM in 10 mM NaH2PO4 and 1 mM Na2EDTA at
pH 7.0) were titrated into the samples. New samples were prepared for each concentration
increment. Absorbance was measured from 800 to 200 nm in a quartz cuvette using a Beckman-
Coulter DU800 spectrophotometer.
2.4 Circular Dichroism
A subset of duplexes and naphthalimide intercalators were studied by circular dichroism
(CD). The complexes studied were: Duplex D with AN, Duplex E with NAN, Duplex F with
NN, and Duplex G with CN. Oligonucleotides were prepared as 50 μM duplex concentration
samples in 10 mM NaH2PO4 and 1 mM Na2EDTA at pH 7.0. Duplexes were combined with a
naphthalimide intercalator to give duplex:intercalator molar ratios of 1:2. CD experiments were
performed on a Jasco J1500 spectropolarimeter at 10°C. The spectra of four scans from 350 to
230 nm were averaged to give one spectrum for each complex.
2.5 Viscosity Measurements
Viscosity experiments followed standard protocols.41-43 Briefly, a Cannon-Manning
semi-micro viscometer was used at room temperature. The flow time of each sample was
monitored using a digital stop watch, and each sample was tested three times to calculate the
average flow time. Each sample had a constant DNA concentration of 50 M with a
naphthalimide concentration range of 0-30 M. The observed flow times of each sample were
corrected by the flow time of the buffer alone to give each viscosity value.
2.6 Optical Melting Experiments
The samples were prepared in 10 mM NaH2PO4 and 1 mM Na2EDTA at pH 7.0 with the
duplex concentration constant at 50 μM and a 1:1 duplex:intercalator molar ratio. A total of

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three samples were made for each combination of nucleic acid and intercalator, with each sample
volume giving three runs, resulting in nine melt curves. Following previous procedures,16 optical
melting experiments were performed on all nine sequences with each of the eight intercalators.
Briefly, the experiments were run on a Beckman-Coulter DU800 spectrophotometer with a
Beckman-Coulter high performance temperature controller so as to measure absorbance at a rate
of 1°C/min at 260 nm for DNA and 280 nm for RNA.
The melt curves were analyzed using Meltwin44 and fit to a two-state model. The two-
state model assumes linearly sloping baselines and temperature-independent ΔH° and ΔS°
values.44,45 The mid-point of the resultant melt curve, or the melting temperature (Tm),
represents the point where half of the DNA is single stranded and half is double stranded. The
thermodynamic parameters, ΔG°37, ΔH°, and ΔS°, were derived from the melt curve analysis.
From these parameters, the average Tm value was calculated at a total strand concentration of 1 x
10-4 M. The average Tm value was calculated for each duplex alone and subtracted from the
average Tm value calculated for each complex, resulting in ΔTm values. Any ΔTM value ≤ 1°C
was assumed to have no effect on the stability of the DNA or RNA.
3. RESULTS AND DISCUSSION
3.1 Binding Mode Determination
Although there is an abundance of previous evidence showing that naphthalimide
derivatives bind DNA via intercalation (see above), additional evidence was collected with these
4-substituted naphthalimides to confirm binding via intercalation. Three different techniques
were used independently to determine the binding mode of the naphthalimide compounds.2
Changes in the UV absorption of a molecule in the presence of increasing concentrations of
DNA are indicative of intercalation. The absorption spectrum of all intercalators with each of
the duplexes was collected. Figure 3 shows the absorption spectra of AN in the presence of
increasing concentrations of Duplex E. There is a slight red shift and decrease in the maximum
absorption peak for AN as the concentration of DNA increases, indicating intercalation.4,31-33
Similar results were seen for all intercalators with each duplex (data not shown).
3.5
0.18
0.12
0.06
0
3
AN
AN+12.5 μM DNA
AN+20.0 μM DNA
AN+25.7 μM DNA
AN+35.0 μM DNA
AN+42.5 μM DNA
2.5
2
1.5
1
350
370
390
410
430
450
470
490
0.5
0
200
240
280
320
360
400
440
480
520
560
600
Wavelength (nm)

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Figure 3. Absorption spectra of AN (0.25 mM) with Duplex E (0-42.5 μM in 10 mM NaH2PO4 and 1 mM
Na2EDTA at pH 7.0).
CD was also used to ensure that the novel derivatives synthesized for this study were
binding to DNA through intercalation. The CD spectra of a subset of intercalators complexed
with DNA (Duplex D with 4AN, Duplex E with 4NAN, Duplex F with 4NN, and Duplex G with
4CN) were collected. Figure 4 shows the spectra of Duplex D with and without AN. The
increase in the positive band and decrease in the negative band for the CD of the DNA were
observed, indicating intercalation of the compound with DNA.4,5,31,32 Similar results were seen
for the other three complexes tested, and those complexes not tested were assumed to display
similar behavior.
Figure 4. CD spectra of Duplex D (50 μM in 10 mM NaH2PO4 and 1 mM Na2EDTA at pH 7.0) with and without
AN (1:2 molar ratio).
Ligand binding via intercalation results in the lengthening and unwinding of the DNA
helix, which causes the solution viscosity to increase. Solution viscosity measurements are often
used to differentiate intercalators and groove binders, as solution viscosity should not be affected
by a groove binder.42,43 Viscosity experiments were completed with duplex B with 4NN and
duplex C with 4AN. As the concentration of 4NN and 4AN increased from 0-30 M, the
viscosity increased from 0.08603 mm2/s to 0.1697 mm2/s for the duplex B solution and from
0.1006 mm2/s to 0.2565 mm2/s for the duplex C solution. The increase in solution viscosity as
4NN and 4AN is added to each DNA sample is consistent with studies of other intercalative
compounds27,41,46 and provides additional evidence for intercalative binding. Together, the UV
absorption, CD, and viscosity results verify that the naphthalimide compounds intercalate into
the short oligonucleotides used in this study.
3.2 Optical Melting Experiments
In order to further the understanding of substituent effects on nucleic acid binding, the
naphthalimide intercalators were complexed with DNA or RNA and studied using optical

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melting experiments. As samples of DNA with and without intercalator are thermally denatured,
the absorbance at 260 nm increases. In the presence of intercalator, an increase in the melting
temperature represents a stabilizing effect on the duplex. The melting data was collected in
triplicate, and Tm values from all runs tend to be within 1 ºC, consistent with the Tm error (1 ºC)
for the melting of the same DNA sequence by separate laboratories.47 Table 1 lists the ΔTM
values for each duplex-intercalator complex, calculated by subtracting the Tm of the DNA, or
RNA, alone from the Tm of the complex. The ΔTm values range from 0 to 17.6°C. The increase
in energy required to thermally denature a nucleic acid is evidenced by an increased Tm value.
Thus, ΔTm values are representative of the binding of the intercalator to the nucleic acid.
Comparing values given in Table 1 with respect to each intercalator, or with respect to each
duplex, gives meaningful insight into the effects the substituents and sequences have on binding.
3.2.1 Sequence Composition Effects
By using short oligonucleotides of varying sequence composition, preferences for
binding was elucidated. Duplexes A-D are AT-rich sequences, with Duplex A having an all AT
sequence, Duplexes B and C having one G-C pair inserted in the middle of the duplex giving a
TpG step (Duplex B) or GpT step (Duplex C), and Duplex D having one G-C pair at each
terminus. Duplexes E-G are GC-rich sequences, with Duplex F having two A-T pairs and
Duplex G having three A-T pairs. Duplexes H and I are the RNA equivalents of Duplexes F and
E, respectively. Table 1 shows the results from the optical melting experiments for each of these
duplexes. The all (AT) sequence Duplex A showed no effect from any of the naphthalimide
intercalators. This could be due to the sequence being the least stable in its uncomplexed form.48
With fewer hydrogen bonds, and therefore weaker duplex, the naphthalimides may not find a
stable conformation within the bases. Duplexes B and C with the single G-C pair in the middle
of a poly (AT) sequence had the largest average ΔTm values, 9.0°C and 8.0°C, respectively. The
introduction of the G-C base pair may provide enough stability to the duplex to allow the
insertion of the naphthalimide without too much disruption to the helical structure. Incorporating
a G-C pair into the poly (AT) sequence also introduces a TpG step (Duplex B) or GpT step
(Duplex C), depending on the location of the pair in the duplex. Previous work has shown that
naphthalimide intercalators have an affinity for TpG and GpT steps in AT rich sequences.27,34
The location of the GpT step within the duplex does make a difference in the duplex’s ability to
accommodate an intercalator. There is a difference when moving from one G-C pair in the
middle of a poly (AT) sequence (Duplex C) to one G-C pair at each terminus of the same
sequence (Duplex D). The average ΔTm value of 4.0°C for Duplex D, which has the G-C pairs at
the ends of the duplex, is a decrease from the average values for those duplexes with the G-C
pairs in the middle (Duplexes B and C).
Table 1. ΔTm values (°C) for naphthalimide intercalator compounds complexed with DNA or RNA.a
Duplexes
Compoun
-R
Average
d
A
B
C
D
E
F
G
H
I
b
14.
7
5.
5
4.
0
6.
2
5.
8
6.
3
5.
7
N.E N.E N.E
HN
NN
-H
6.0
5.4
7.7
5.4
N.E.c
N.E.
.
N.E
.
.
.
N.E
.
-NO2
6.1
1.3

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10.
3
2.
2
2.
9
3.
2
2.
3
4.
9
6.
7
4.
0
5.
2
6.
1
6.
2
4.
0
4.
7
6.
4
5.
6
3.
4
3.
2
3.
0
4.
0
3.
7
5.
1
4.
3
N.E N.E N.E
AN
CN
-NH2
-Cl
-Br
-I
N.E.
N.E.
N.E.
N.E.
N.E.
N.E.
0
5.6
6.3
6.8
8.0
5.3
5.5
4.7
4.7
8.4
7.6
.
.
.
N.E N.E N.E
8.9
4.4
5.2
.
.
N.E
.
.
BN
1.3
1.2
N.E N.E N.E
IN
.
.
.
-
11.
0
13.
6
17.
6
N.E N.E N.E
IPN
NAN
NHCH(CH3)2
.
.
.
N.E N.E N.E
-NHCOCH3
6.2
.
.
.
Averaged
9.0
8.0
0
0
0
aIndividual melting experiments were done in triplicate and resulting Tm values were typically
within 1°C. bAverage values for each compound complexed with duplexes B-F. cN.E. = no
effect. dAverage values for duplexes B-F with each compound.
The results from Duplexes A-D show that the naphthalimide derivatives studied here do
not stabilize poly (AT) sequences unless at least one G-C pair is added to the sequence.
Furthermore, the addition of two G-C pairs to the ends of a poly (AT) sequence is only half as
stabilizing as the addition of a G-C pair to the center of the helix. Similarly, 3-substituted
naphthalimide intercalators do not stabilize poly (AT) sequences except with the addition of at
least one G-C pair.16 Duplex B, with one G-C pair at the center of the helix, had the largest
average ΔTm value for both the 3- and 4-substituted naphthalimides.16
The poly (GC) sequence, or Duplex E, has an average ΔTm value of 5.6°C. This average
for an all (GC) sequence is in stark contrast with the average for the all (AT) sequence, which
showed no stabilization with any of the compounds. Upon addition of two A-T pairs near the
terminus of the poly (GC) duplex (Duplex F), the average ΔTm value is 4.3°C. This addition
created a GpT step at the end of the duplex, as is seen in Duplex D; however, the stem of Duplex
F is poly (GC), and the stem of Duplex D is poly (AT). Although both duplexes have the same
GpT step, the average ΔTm value for Duplex F is slightly larger than the average ΔTm value for
Duplex D. This result shows that although naphthalimide intercalators may have preferences to
bind at specific steps, the entire sequence composition is important for the binding affinity.
Interestingly, with the addition of three A-T pairs, there is a loss of effect, or stabilization, from
all intercalators except one (BN) for Duplex G, although it does have a GpT step in the middle of
the duplex. Again, this highlights the importance of sequence composition. The trends seen here
for the 4-substituted naphthalimides with Duplexes E-G are the same trends observed for the 3-
substituted naphthalimides.16 Namely, a stabilizing effect by the compounds for a poly (GC) and
GC rich sequence with up to two A-T pairs and a loss of effect in a GC rich sequence with three
A-T pairs. By performing structural studies on some of these complexes, more information may
be gained as to the stability afforded by the intercalating compound.
Duplexes H and I are the RNA equivalents of Duplexes F and E, respectively. Both of
the RNA duplexes had only one intercalator (NN or BN, respectively) that showed any
stabilizing effect. The same substituents at the 3-position on the naphthalimide also showed little

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to no stabilizing effect when complexed to RNA duplexes.16 The explanation for the difference
in stabilizing effect when comparing DNA and RNA highlighted by Johnson et al.16 can also
explain the differences seen here. Whether a substituent is at the 3-position or the 4-position, the
inherent differences in helical structure between DNA and RNA (namely, the 2’-OH group on
the ribose sugar, the sugar conformation, and the helical rise and twist) may dictate the altered
ability to accommodate an intercalating moiety. Also, the base pairs are more easily accessible
from the major groove of DNA than RNA49, which has been shown previously to be the
preferred binding mode.27
3.2.2 Substituent Effects
A systematic study on the effects of different functional groups as substituents at the 3-
position on a naphthalimide core has previously been investigated.16 The work presented here
supplements the previous study by investigating the same substituents at the 4-position on a
naphthalimide core. The results shown in Table 1 highlight the effect of changing a substituent
at the 4-position on the stability of the complex it forms when intercalating into DNA. These
results compared to previous work by Johnson et al.16 also highlight the effect the position of the
substituent on the core molecule has on the stability of the DNA-intercalator complex. In
general, the 3-substituted naphthalimides had a larger effect on the stability of the DNA-
intercalator complexes formed, thus showing a difference based on position on the naphthalimide
core.
The largest ΔTm value for a substituent at the 4-position is seen for IPN with Duplex C.
This result is somewhat surprising considering the bulkiness of the substituent. In fact, both IPN
and NAN are the bulkiest substituents and exhibit some of the largest average ΔTm values, 8.4
and 7.6°C, respectively. A study by Zhou et al.33 also resulted in larger binding abilities for
bulky substituents at the 4-position of a naphthalimide intercalator when complexed to calf
thymus DNA. The larger binding ability of the bulky guanidinoethyl substituent was attributed
to the group’s ability to have stronger hydrogen bonding ability and a pKa such that the group is
fully protonated.33 These arguments help to explain the results seen here. The bulkiness of a
substituent at the 4-position provides stability that is not accessible to bulky substituents at the 3
position. Indeed, the IPN and NAN substituents at the 3-position do not exhibit large ΔTm
values, 5.6 and 5.4°C, respectively.33 Other studies have reported slight differences in binding
for substituents at the 3 position and 4 position.30,33 Previous work by Stevenson et al.30
explained these differences due to enhanced stacking and less steric hindrance for 3-nitro-1,8-
naphthalimides when compared to 4-nitro-1,8-naphthalmides. The nitro group was assumed to
adopt a coplanar orientation when at the 3-position that could not be achieved at the 4-position.30
Both the IPN and NAN groups are more bulky substituents than the nitro group described in the
previous study. The bulkiness at the 3-position, which likely causes steric hindrance with the
bases, potentially is in an orientation at the 4-position that interacts favorably with the
neighboring atoms. Also, the bulky IPN and NAN groups are activating to the aromatic ring,
while the nitro group is deactivating. Structural studies of IPN with a duplex whose sequence
composition is similar to that of Duplex C could explain the source of stabilization seen for such
a large group and aid in the design of more efficient therapeutic agents.

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The smaller substituted naphthalimides, or NN, AN, CN, BN, and IN, had average ΔTm
values that range from 4.7°C to 5.5°C. Interestingly, the BN naphthalimide had one of the
lowest average ΔTm values, yet exhibited binding to the largest number of duplexes (seven out of
nine). It may be that a more ubiquitous binding intercalator is more desirable as an effective
therapeutic agent than an intercalator that binds more efficiently to one specific binding site. For
example, Zhou et al.33 tested cytotoxicity of a series of naphthalimide derivatives. The results of
that study show that those derivatives with the higher binding ability exhibited the weaker
cytotoxicity.
4. CONCLUSION
The results of this study show the differing effects on the stability of DNA-intercalator
complexes. A naphthalimide intercalator can stabilize a DNA duplex to a varying degree
depending on the substituent attached to the naphthalimide, the position of the substituent, and
the sequence of the DNA. Varying substituent groups, position of the group on the
naphthalimide core, as well as nucleotide sequence of the target DNA all play a role in the
stability of the complex. In some cases, 4-substitutents exhibited similar trends as the 3-
substituents (higher stabilizing with DNA than RNA, similar sequence preferences, etc.).
Differences were observed, however, based on substituent position. For example, IPN had the
largest ∆TM value of all of the 4-substituents but fell in the middle of the ∆TM range at the 3-
position. We also observed that placing substituents at the 3-position provided much greater
stabilization than placing substituents at the 4-position. We feel that these insights for
substituents at the 4-position as well as comparisons between 3- and 4-substituents are important
as we (and others) continue to investigate naphthalimide derivatives as anti-cancer agents.
FUNDING
Funding was provided by Saint Louis University and Saint Louis University’s President’s
Research Fund.
ACKNOWLEDGEMENTS
The researchers would like to acknowledge Dr. Michael Nichols at the University of Missouri-
St. Louis for the use of the Jasco spectropolarimeter. We would also like to thank the National
Science Foundation for the Major Research Instrumentation Award (#1337638) that funded
acquisition of the Jasco 1500 circular dichroism spectrometer at the University of Missouri-St.
Louis. In addition, Joseph Kramer at the University of Missouri-St. Louis is acknowledged for
obtaining the high-resolution mass spectra of the 4-substituted naphthalimides.

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Graphical abstract

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Highlights


4-substituted naphthalimide intercalators were synthesized.
A systematic thermodynamic study of 4-substituted naphthalimide-DNA complexes was
completed.

A naphthalimide intercalator can stabilize a DNA duplex to a varying degree depending on the
substituent attached to the naphthalimide, the position of the substituent, and the sequence of the
DNA.


Varying substituent groups, position of the group on the naphthalimide core, as well as nucleotide
sequence of the target DNA all play a role in the stability of the complex.
In some cases, 4-substitutents exhibited similar trends as the 3-substituents (higher stabilizing
with DNA than RNA, similar sequence preferences, etc.).
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
Differences were observed, however, based on substituent position.
Placing substituents at the 3-position provided much greater stabilization than placing
substituents at the 4-position.