Investigation on interaction of L-methionine dipeptide with ct-DNA by ultraviolet spectroscopy

Article abstract of DOI:10.1080/10426500701793170

In this article, L-methionine peptides were synthesized with the assistance of phosphorus oxychloride. L-methionine dipeptide was purified by HPLC and characterized by the means of 1H-NMR, 13C-NMR and ESI-MS. The interaction of L-Methionine dipeptide with ct-DNA was studied by ultraviolet spectra. The results showed that L-Methionine dipeptide could interact with phosphorous groups of ct-DNA. The influences of interaction time, ionic strength, and phosphate anion on the interaction between L-methionine dipeptide and ct-DNA were also investigated. Copyright Taylor & Francis Group, LLC.

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Phosphorus, Sulfur, and Silicon
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Investigation on Interaction
of L-Methionine Dipeptide
with ct-DNA by Ultraviolet
Spectroscopy
Kui Lu a , Rui Li a & Li Ma a
a School of Chemistry and Chemical Engineering ,
Henan University of Technology , Zhengzhou, P. R.
China
Published online: 20 Jun 2008.
To cite this article: Kui Lu , Rui Li & Li Ma (2008) Investigation on Interaction of L-
Methionine Dipeptide with ct-DNA by Ultraviolet Spectroscopy, Phosphorus, Sulfur, and
Silicon and the Related Elements, 183:2-3, 596-602, DOI: 10.1080/10426500701793170
To link to this article: http://dx.doi.org/10.1080/10426500701793170
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Phosphorus, Sulfur, and Silicon, 183:596–602, 2008
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500701793170
Investigation on Interaction of L-Methionine Dipeptide
with ct-DNA by Ultraviolet Spectroscopy
Kui Lu, Rui Li, and Li Ma
School of Chemistry and Chemical Engineering, Henan University
of Technology, Zhengzhou, P. R. China
In this article, L-methionine peptides were synthesized with the assistance of
phosphorus oxychloride. L-methionine dipeptide was purified by HPLC and char-
1
13
acterized by the means of H-NMR, C-NMR and ESI-MS. The interaction of
L-Methionine dipeptide with ct-DNA was studied by ultraviolet spectra. The re-
sults showed that L-Methionine dipeptide could interact with phosphorous groups
of ct-DNA. The influences of interaction time, ionic strength, and phosphate anion on
the interaction between L-methionine dipeptide and ct-DNA were also investigated.
Keywords ct-DNA; interaction; L-methionine dipeptide; Phosphorus oxychloride;
ultraviolet spectra
INTRODUCTION
The study of small molecules that specific sites along a DNA helix has
become a subject of considerable interest.1−7 Several small molecules
have been shown to interact with DNA at the molecular level by specific
binding modes. These binding studies were driven partly by the need to
understand the mechanism of anticancer drug action,8 to decipher the
chemical basis for the carcinogenicity of environmental pollutants and
toxic chemicals9 and to serve as analogues in studies of protein-nucleic
acid recognition,10 provide site specific reagents for molecular biology,
and yield rationales for new drug design. Many small molecules based
chemical reagents have already been proved to be useful as sensitive
probes of local nucleic acid structure.
The authors would like to thank the financial supports from the Chinese National
Science Foundation (No. 20272055, 20572016), the Henan Province Science Foundation
for Prominent Youth (No. 0312000900), and the Office of Education of Henan Province
(No. 2006KYCX017).
Address correspondence to Kui Lu, School of Chemistry and Chemical Engineering,
Henan University of Technology, Zhengzhou 450052, P. R. China. E-mail: luckyluke@
haut.edu.cn
596

Interaction of L-Methionine Dipepteide
597
FIGURE 1 Illustration of the static interaction, intercalation and groove bind-
ing of small molecules to DNA.
Small molecules interact with the DNA double helix by three domi-
nant modes, namely static interaction, intercalation and groove binding
(Figure 1). It would be valuable to understand quantitatively the con-
tributions from these different modes to the stabilization of bound DNA
complex.
Phosphorus plays an important role in the origin of life and control of
life; we studied the peptide formation reactions with the assistance of
inorganic phosphorous compound.11 The investigation on interactions
between biological macromolecules and small molecules by ultraviolet
spectroscopy is common, sensitive and convenient.12 In this article, we
prepared L-methionine dipeptide (Met-Met) by assembled reaction with
the assistance of phosphorus oxychloride, and explored the interaction
of Met-Met with calf thymus DNA (ct-DNA) by ultraviolet spectroscopy.
The result of ultraviolet absorption spectra suggested the static inter-
action between Met-Met and ct-DNA.
EXPERIMENTAL
Reagents and Chemicals
L-Methionine was purchased from Yuanju Biochemical Co. (Shanghai,
China). Phosphorus oxychloride, acetonitrile and THF (HPLC grade)
were purchased from Tianjin Chemical Reagent Co. (Tianjin, China).
Calf thymus DNA (ct-DNA) (Sigma Co., America) was commercially
purchased and accepted for use without further purification. The stock
solutions were prepared by dissolving the solid DNA in doubly distilled
water and stored at 4◦C. The concentration of DNA was determined

598
K. Lu et al.
according to the absorbance at 260 nm after establishing that the ab-
sorbance ratio A260/A280 was in the range of 1.80–1.90. The molarities
of DNA were calculated using εDNA = 6600 M−1cm−1 13
.
The stock solu-
tion of 5.6 × 10−3 mol L−1 Met-Met (Prepared and purified by HPLC)
was prepared by directly dissolving its crystal into water. A sodium
acetate-acetic acid buffer solution (pH 4.8) was used to control the
pH value of the interacting system. Deionized water was generated
from Milli-Q water purifying system purchased from Millipore (MA,
America). Other common chemicals used were of the highest purity
commercially available.
Apparatus
A Bruker Esquire 3000 plus ion trap mass spectrometer interfaced to
electrospray ionization (ESI) source was used for mass analysis and de-
tection. Agilent 1200 HPLC system consisted of a UV detector, a Ternary
HPLC pump with a SB C18 prepared column (10 μm, 250 mm × 10 mm).
Peaks were monitored by UV absorbance at 214 nm. A mixture of ace-
tonitrile (A) and water (B) was used as the mobile phase. The gradient
elution was programmed as follows: 0–5 min, 70% B; 5–15 min, 70–80%
B; 15–20 min, 85% B; 20–25 min, 85–70% B, and 25–30 min, 70% B. The
column temperature was maintained at room temperature (25 ◦C). The
flow rate was 1.0 mL/min. The 1H and 13C NMR spectra were recorded
on Bruker DPX-400 spectrometer, using CDCl3 as solvent and TMS as
internal standard. IR spectra were taken from Shimadzu IR-Prestige
21 spectrophotometer. The ultraviolet absorption spectra were recorded
on a TU-1900 spectrophotometer.
Preparation of L-Methionine Dipeptide
L-Methionine oligopeptide libraries were prepared according to the
literature,11 and the mixture of oligopeptides was separated and pre-
pared on SB C18 prepared column (10 μm, 250 mm × 10 mm) using
a mixture of acetonitrile (A) and water (B) as elute. Then, the collec-
tion was concentrated under reduced pressure. L-Methionine dipeptide
was obtained as white power in 4.01% yield (225 mg). m.p.:148–150◦C;
IR (KBr pellet, cm−1): 3440, 3330, 2950, 2920,1660, 1580, 1450, 1380,
1420, 1340; 1H-NMR (CDCl3, ppm): δ1.97 (s, 3H, CH3), 1.98 (s, 3H, CH3),
1.87–2.07 (m, 4H, 2CH2), 2.40–2.54 (m, 4H, 2CH2), 4.03 (t, J = 6.2 Hz,
1H, CH), 4.32 (t, J = 6.1Hz, 1H, CH); 13C-NMR (CDCl3, ppm): δ 13.88
(SCH3), 27.81 (CH2S), 29.97 (CHCH2CH2), 52.07 (HNCHCO), 53.01
(H2NCHCO), 168.92 (HNCOCH), 175.98 (CHCOOH); MS2, m/z (RA%):
264.2 (19), 233.3 (7), 187.4 (3), 150.5 (100), 132.6 (<1), 104.8 (52).

Interaction of L-Methionine Dipepteide
599
Spectral Measurements
The ultraviolet absorption titrations with DNA were conducted by keep-
ing the concentration of nucleic acid constant, and varying the Met-Met
concentrations. This was done by dissolving an appropriate amount of
Met-Met in the DNA stock solution and mixing various proportions of
Met-Met and the DNA stock solutions, while maintaining the total vol-
ume of the solution constant. This resulted in a series of solutions with
varying concentrations of Met-Met but with a constant concentration of
DNA. The ultraviolet absorbance of the samples was monitored at 260
nm. The temperature (37◦C) of the solution was continuous monitored
with a thermo-couple attached to the sample holder.
The intrinsic binding constants of Met-Met with calf thymus DNA
was determined by ultraviolet absorption. In the case of the former, the
absorbance at 260 nm was recorded after each addition of Met-Met. The
intrinsic binding constant K was determined from Eq. (1):14
A0/A = −(1 − A/A0) cK + npK + 1,
(1)
where K is the intrinsic binding constant; n is the binding site size;
p is the concentration of DNA determined by the absorbance at 260
nm; c is the concentration of Met-Met. A; and A0 are the absorbance
values of DNA-Met-Met and DNA at 260 nm, respectively. The binding
data were treated by constructive methods and values of K and n were
obtained.
The effects of interaction time, ion strength, and phosphate ion on
the interaction of Met-Met and DNA were investigated by controlling
interaction time, and varying ion strength, phosphate and ion concen-
tration of the interaction system.
RESULTS AND DISCUSSION
Study of Ultraviolet Absorption Spectra
The ultraviolet absorption spectra of DNA was decreased with in-
creasing amounts of Met-Met (hypochromicity), but there is no obvi-
ous shift at the peak intensities (Figure 2). The absorbance of DNA in
the peak (260 nm) was decreased by ca. 20%. The addition of further
amounts of Met-Met to the DNA solution did not cause any more ab-
sorption changes, due to the binding saturation of the Met-Met with
DNA. We considered the electrostatic interactions between the Met-
Met and DNA because of the dipeptide with positive charge and DNA
with negative polyphosphate skeleton. Hypochromism was suggested
to be due to weak interactions between the electronic states of the

600
K. Lu et al.
FIGURE 2 The change of ultraviolet spectra of dipeptide-DNA solutions with
dipeptide concentration R = [Met-Met]/[DNA]; a: R = 0; b: R = 1; c: R = 1.5;
d: R = 3.0; e: R = 5.0; f: [DNA] = 0.0.
dipeptide and that of the DNA phosphate ion. The observed spectral
changes (hypochromicity) are consistent with the static interaction be-
tween dipeptide and DNA phosphate ion.
Absorbance data of DNA at 260 nm with different concentration of
Met-Met were used to obtain the intrinsic binding constant by Equa-
tion (1) (Figure 3). According to Rosenthan plot, the best-fit linear re-
gression equation was: A0/A = −0.711 + 0.722 (1−A/A0) c (R = 0.9982).
Then, the intrinsic binding constant of Met-Met with DNA (Ka) of
1.39 × 102 L/mol was deduced.
FIGURE 3 Rosenthan plot of DNA (5.6 × 10−5 mol/L) with Met-Met dipeptide
as determined from the electronic absorption spectra.

Interaction of L-Methionine Dipepteide
601
The Effects of Interaction Time, Ion Strength and Phosphate
Anion on the Interaction System
It was expected that the longer the interaction time of L-methionine
dipeptide with ct-DNA, the more the hypochromic effect. It was found,
however, that there was an energetic equilibrium after 3 h.
Moreover, in order to test whether the electrostatic interaction be-
tween Met-Met and DNA exists, the strong electrolyte, sodium chloride
(NaCl), was used. The electrostatic interaction would be weakened by
the addition of counter ion because of the conversion of the electro-
static atmosphere of DNA periphery. Some reports showed that very
high concentration of NaCl would hinder the binding of small molecule
with DNA.15 If there did exist electrostatic interaction between Met-
Met and DNA, by increasing the ionic strength in the solution of Met-
Met and DNA, the absorbance of solutions would increase when NaCl
was added, but it would never exceed the absorbance of the pure DNA
solution. However, the effect was not as great as expected. The ultra-
violet absorption were decreased greatly from 0.360 to 0.310 when the
concentration of NaCl (i.e., ionic strength) increased from 0.00 to 0.10,
but there was no obvious change for ultraviolet absorption when the
concentration of NaCl increased further (Figure 4), which indicated
that there was competition of interaction between Met-Met and NaCl
with DNA. It is suggested that the additional sodium cations interacted
with DNA, and hindered the interaction of Met-Met with DNA. So the
probable intercalation process was that Met-Met was attracted to the
periphery of DNA by electrostatic attraction. If other cations occupied
the effective range of electrostatic interaction, the affinity of Met-Met
with DNA would be greatly weakened. Anyway, the salt effect could not
inhibit the binding of Met-Met with DNA completely.
Furthermore, the electrostatic interaction between Met-Met and
DNA was tested by the influence of phosphate anion (Table I). As shown
in Table I, the observed spectral changes (hypochromicity) were weak-
ened with adding phosphate anion, which indicated that Met-Met was
attracted to the periphery of DNA by electrostatic attraction.
TABLE I Influence of Phosphate Anion on the Binding of Met-Met
with DNA
KH2PO4
Concentration
0
0.005
0.01
0.02
0.05
0.1
0.2
0.5
A260
δA260
0.360
0
0.332
0.028
0.335
0.025
0.337
0.023
0.341
0.019
0.333
0.027
0.323
0.037
0.313
0.047

602
K. Lu et al.
FIGURE 4 Influence of ionic strength on the binding of Met-Met with DNA.
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