Indian Journ al of Biotechnology Vo l 1, April 2002, pp 158-163
New Curcumin - Bioconjugate: Synthesis and DNA Binding
Sanjay Kumar, Vibha S hukla , Arvind Misra, S ne hl ata Tripathi and Krishna Mi s ra *
Nucleic Ac ids Research Laborato l'y , Department of Chem istry, University of All ahabad , All ahabad 211 002, India.
Received I May 200 1; revised 8 Febrl/at)' 2002
A triglycyl derivative of curcumin, 1,7-bis (4-0-glycinoyl-3-methoxy phenyl)-1,6-heptadine-C-4-glycinoyl-3, 5-dione, was synthesized and characterized by UV, elemental analysis and IH NMR. Interaction studies of curcumin and curcumin-bioconjugate with calf thymus DNA were carried out using UV -absorbance, gel electmphoresis and viscometric studies. Curcumin-bioconjugate was found to be A-T specific minOI- groove bindeL
Keywords: curcumin, glycine, bioconjugate, viscosity, interaction, gmove binding
Introduction One of the most important approaches of drug de
velopment and of current chemotherapy against some vi ral and parasitic di seases including cancer and AIDS invo lve drugs, which interact reversibly with nucleic ac ids. Natural antibiotics e.g. adri amycin and synthetic drug such as amsacrine interac t with DNA and are widely used in clinical treatment of a variety of neoplastic diseases . Duplex structure of nucleic ac ids is not exclusive requirement for its reversible interaction. Single stranded RNA can form extensive intramolecul ar duplex region that arises from fo lding of RN A strands fro m biological systems such as ribosomes, t-RNA or the genomic RNA of some vi ruses. These perturbed duplex conformati ons can undergo very specifi c interactions and in pathogenic RNA viruses, such as H1V -I offer an exciting potenti al target in drug design.
Molecules interact with duplex nucleic acids in three significantly di fferent primary ways, electrostatic interaction, groove binding and intercalation (Wil son, 1996, Dougherty, 1984). Curcumin, the main colouring component of turmeric, 1,7-bis-(4-hydroxy-3-methoxy phenyl)-1,6-heptadiene-3, 5-dione/diferuloylmethane (Fig. la), offers excellent molecular dimension havi ng a flexible C-C chain, which is stable in trans-position with two phenyl rings at both the ends (Govindarajan, 1979) . Turmeric, a vulnerary agent, is being used for centuries as traditional medicine for external/internal wounds, li ver di seases
* Author for correspondence: Tel: (9 I )-0532-46 I 236; Fax: +9 1-(0532)-623221
E-mai l: kllli sraI23@ red iffma il. colll.krishnam isra @hotmail.co m
(particularly jaundice), blood purification and inflamed joi nts (Am mon & Wahl , 199 1; Srimal & Dhawan, 1973; Stoskar et af, 1986; Sharma, 1976; Toda et af, 1985) . Authors have studied curcumin and its bioconjugates fo r their antibacterial and antifungal properties (Kumar et af, 2000). By covalently linking amino acids through their carboxyl function to the phenolic hydroxyl in the two phenyl rings of curcumin , free amino groups can be generated at two sites. The active methylene is an additional site at which a third glycine molecule could be linked; thus making three am ino groups avai lable for binding to different enzymatic substrates. A number of diamidines, viz. Berenil and Pentamidine (Fig. I c & d) have similar curved structure (Turner et ai, 1998; Kielkopf et a f, 2000), having two phenyl rings and bind in the minor groove in AT specific fashion (J enkins, 1993; Edwards et ai, 1992; Neidle & Abraham, 1984; Brown, 1990). Berenil have an ti-trypanosomal activity and pentamidine is used clinically against Pneumocystis carinii pneumonas, a common opportu nistic infection reported in AIDS patients. In both the cases, the two planar units are twi sted by 35°C with respect to each other as they follow the curvature of grooves and the phenyl rings are in close contact with adenine C2 protons. In the pentamidine complex, each phenylamidinium group is an approximately planar unit, which is inserted deep into the minor groove and is ali gned parallel to the walls of groove. On parallel lines, it can be reasoned that groove binding ability of the curcumin bioconjugate could be a combination of positive (hydrogen bonding, van der Waal's and electrostatic) and negative (steric repulsion) effects. The carbon chain fits snugly into the minor groove and
KUMAR et al: CU RCUMIN-BIOCONJUGATE 159
o 0
HO (a) CCOCI-lJ
I d' OB
H'NyO/"~"'-OyNH' -INH2 (d) "'NB
2
Fig. I - (a) Curcumin ; (b) Triglycinoy l-curcumin (Protonated); (c) Berenil; (d) Pentamidine.
assumes a conformation to allow hydrogen bonding of the amino with adenine N3 group and 02 of thymine at the flow of groove.
Keeping thi s rationale in mind bioconjugate of curcumin, 1,7-bi s (4-0-glycinoyl-3-methoxyphenyI)-1,6-heptadiene-C-4-glycinoyl-3,5 -di one, has been synthesised. Interaction studies of curcumin (a) and curcumin-bi oconjugate (Fig. 1 b) have been carried out with CT-DNA and sy ntheti c polynucleotides .
Materials and Methods UV absorption and emission spectra were recorded
on Hitachi 220S spectrophotometer, 'H-NMR spectra (chemical shi ft in 8 ppm) on Brooker AMX 500. Curcumin and g lyc ine were purchased from MerckSchuchardt, Germany. The purification was done on sili ca gel column chromatography. CT-D NA was purchased from Genei, Bangalore, InJia and polynucleotides fro m Perseptive Biosys tems, Framingham, MA, USA. Water used was triple distilled and autoclaved. All the glasswares were also autoclaved pri or to their use. The stock so lution of CT-DNA and polynuc\eotides were prepared in 6X Tris-EDT A buffer (PH 8.0) and co ncentrat ions were determined spectroscopically using the following ex tincti on coefficient (M-' , cm-') ,
£260 nm = 6600 for CT-DNA , po ly [d (A-T).d(A-T)] £26(J nm = 8100 for poly [d (G-C).d (G-C)]. Gel elec-
Table 1- Viscometric properti es of binding of Curcumin (a) with various natural and sy nthetic DN A
Polymer ~ i\\ (nm) % heli x length enhancement
at r m"
CT-DNA 1.1±0.05 0.3±0.0 15 0.1 Poly[d(G-C)]
[d(G.C)] 0.00 0.00 0.00 Poly[d(A-T)]
[d(A-T)] 1.3±0.05 0.012±0.02 0.013
trophores is was done on Pharmacia Horizontal type electrophoresis unit using 1 % agarose gel in TrisEDT A buffer (PH 8.0) and bromophenol blue and xylene cyanol FF as dyes. Mobility on gel, the visualisation and photograph of spot was done using a trans-illuminator. The buffer containing 10 mM Tris and 1 mM EDT A was prepared by dissolving 0 .24g Tri s in 200 ml water containing 400 ).lml (0.5 M stock) EDT A solution (PH was adjusted to 8.0 with glacial acetic acid before making up the final volume). BPES-DMSO buffer was prepared with 1.5 mM Na2HP04, 0.5 mM NaH2P04, 0.25 mM EDTA, 240
mM OM SO pH 7.0 ± 0.05 . Absorbance measurements were performed using Hitachi 220 S spectrophotometer at room temperature in quartz ce ll of Icm path length. Concentration of (a) and (b) in thi s study was kept less than 30 ).lM as it confirms Beer's law in this concentration range. Changes in absorption characteristics of (a) and (b) when bound to natural DN A was determined at varied nucleotide/drug ratio . The concen tration of curcumin itself and curcuminbioconjugate were kept constant at J mM in all the experimental sets. Observations were made by varying the concentration of CT -ON A with the ligands/ DNA ratio of 1: 0 .5, 1: I , 1: 1.5 and 1:2, respective ly . Studies have been carried out in presence of di ffe rent sa lts, viz. NaCI, ZnCh, MgS0 4 and also in different buffers, BPES-DMSO, potass ium- di-hydrogen orthophosphate, Tris-EDTA and ammonium acetate. Viscosity measurements were done in Ostwald type capillary vi scometer at 15°,35" and 45°C (Tab le I ).
Synthesis of 1,7-Bis( 4-0-glycinoyl-3-methoxy-phenyl)-1,6-heptadiene-C'-glycilloyl-3,S-diolle
Curcumin (368 mg; Im11101 ) was taken in C2HsOH and the NaOC2Hs (containing 83 mg of metallic sodium; 3.6 mmol) was added drop wise for 10 min and the reac tion mi xture was stilTed at room temperature for 30 min . The resulting sodium salt was concentrated under vacuo and thoroughly washed with
160 INDIAN J BIOTECHNOL, APRIL 2002
HO
° °
(al
1. C2HSOJ V C211 50 Nal3 0 rnin .lr.t. 2. Pyridint: I N· Phthn loylgl}'cinoylchIOridc / 611 I r. t. J. Nil} :Pyridinc(9 :1)v/v
Fig. 2 - Synthesis of 1,7-bis(4-0-glyc inoyl-3-methoxyphenyl)I ,6-heptadiene-C4 -g lyci noy 1-3,5-d ione.
C2HsOH and taken in dry pyridine. N-Phthaloyl glycinoyl chloride (804 mg; 3.6 mmol) was added to the reaction mixture and stirred at room temperature for 6 hrs. After completion of the reaction, the mixture was poured into crushed ice and thoroughly extracted with EtOAc. The organic layer was concentrated and treated with ammonia: pyridine (9:1 v/v) for 1 min at room temperature. The reaction mixture was poured into crushed ice and extracted again with EtOAc. The organic layer was concentrated and purified by silica gel column chromatography using dichloromethane: methanol gradient, yield 38% (205 mg) Am"X 330 nm (Fig. 2b) . The pure product was characterized by elemental data and 'H NMR. Anal. found: C, 59.88; H, 5.60; N, 7.5 I % calcd For C27H2<)09N3; C, 60.04; H, 5.48; N, 7.78%. 'H NMR (CDCl]) 8 = 3.73 (S, 6H, -OCH3), 4.05 (S, I H, C4-H), 4.43-4.79 (M, 6H, -CH2-NH2), 6.58 (d, 2H,C2-H & C6-H), 6.81-7.03 (M, 6H, Ar-H), 7.56 (d, 2H, C,-H & CrH).
Results
Interaction of Curcumin and Curcumin Bioconjugate with Calf-Thymus DNA, Poly [d(A-T)'(A-T)] and Poly [d(G-C) -(G-C)]
The binding experiments were performed in BPESDMSO buffer (PH 7±0.05) and in presence of different Na+, ZnH and MgH molarity obtained by addition of required volume of NaCl, ZnCl2 and MgS04 for a known concentration stock. Studies were performed at 15°,35° and 45°C using the reported procedure (Chakraborty et aI, 1989).
The absorbance spectra lacks (Fig. 3) a common isosbestic point and, therefore, the data could not be used for calculating the free and bound form of the ligand. Similar pattern was observed in case of (b) also (Fig. 4). As the ligand / DNA ratio increases, hyperchromism with a red shift of about 5 nm was observed, which reaches saturation at li gand/DNA ratio greater than 10. Visible absorption changes do not indicate any difference between (a) and (b) with respect to their binding with this natural DNA. Presence of metal ions has significant effect on the mode of binding (Muller & Crothers, 1968). The effect is more pronounced for (b) as compared to (a). The amine function on the ring in (b) is expected to have more affinity for ionic environment and at higher ionic strength this might be leading upto a greater loss of interaction with the DNA helix. Zn2+ show the best result among the ions selected-Na+, Zn2+ and Mg2+. The effect of progressive increment In the
0.4
0.3 1" v u
0.2 ta .D
~ .D ..: 0.1
Wavelength - )
Fig. 3 - Absorbance spectra of clirclimin (a) in the presence of varying concentration of CT-DNA wilh the ligand-DNA ratio of 1= 1.05, II=I: 1, 111=1.5 , IV=l:2 and V= control (clirclIlllin) at 420 nm.
0.4
0.3
1" 0,) 0.2 ~
.D <; ~ 0.1
Wavelength -4
Fig. 4 - Absorbance spectra of triglycinoyl clIrclimin (b) in the presence of varying concentration of CT-DNA with the ligandDNA ratio of 1= 1.05, 1I=1:1 , III= 1.5, IV=l:2 and V= control (triglycinoyl-clirclimin) at 333 nm.
KUMAR ef al: eUReUMTN-BIOeONJUGATE 161
O. 14 rr---r---,--;-----r---,--.,..------,
A
0.12
0.10
0.08
0.14
B
§ on 0.12 -rrl rrl ....... ro (1) (.)
@ ..0 ....
0.10 0 Ul
..0
-<
0.08
0.14
C
0.12
0.10
. 0.08
o 8 16 48
[DNA / Curcumin conjugate]
Fig. 5 - Spectrophotometric titration data on binding of triglycinoyl curcumin (b) to calf thymus DNA in BPES-DMSO buffer in presence of Zn ++, 0.02 M (d) 0.05 M ("'), 0.1 M (0), and 0.2 M (e), AT 15° (A), d3SO (B) and 45°e (e), respectively .
concentration of the CT-DNA on the absorbance spectra of conjugate was studied in three different molarities of Zn++ at pH 7.0 (Fig. 5). The spectrophotometric measurement in buffer of particular Zn++ molarity was observed at 15", 35° and 45°C. The spectrophotometric changes involve essentially a red shift and hypochromacity in complex until saturation is reached . The fact that the observed hypochromacity of the complex of the conjugate and CT-DNA is also significantly effected by the presence of ions, specifically on their concentration.
Synthetic polynucleotides provide a homogeneous lattice to study the interaction of ligands with DNA as a function of base pairs . Authors have studied the interaction of (a) and (b) with poly [d(A-T) 'd(A-T)] and poly [d(G-C)·d(G-C)]. Changes in the visible absorbance of (a) and (b) were studies with both. The pattern shows that the changes are analogous to those observed in the case of calf-thymus DNA. The gel pattern of the same studies with polynucleotides shows better quenching in case of poly [d(A-T) d(A-T)] then poly [d(G-C)' d(G-C)].
Assessment of Binding by Viscometric Method Experiments designed to measure the change in
specific viscosity produced by binding of curcumin (a) and curcumin bioconjugate (b) were performed (Cohn & Eisenberg, 1966, 1969). For viscometric experiments, a sample of linear duplex calf thymus DNA was vortexed by using a needle probe of 4 mm diameter as described earlier (Maiti et ai, 1982, 1984). The vortexed DNA sample used had a molecular weight of the order of 2.0-3.5x105 (Chakraborty et ai, 1989). Synthetic polynucleotides were used as such without further purification. Viscometric experiments were performed in an Ostwald type capillary viscometer, mounted vertically in a constant temperature water bath maintained at 25±0.05°C. Flow time of DNA alone and DNA-Curcumin complexes were measured by an electronic stopwatch with an accuracy of 0.01 s. The increase in the helix length of sheared DNA and synthetic polynucleotides were calculated from the experimental data, which were transformed directly from flow times to by using the expression:
UL" = [tc-tjt,rt,,} 1I3
Where L is the contour length in presence of curcumin bioconjugates, Lo is the counter length of free DNA, tc is the flow time for complex, td is the flow time for pure DNA, to is the flow time of the buffer at a given volume in the viscometer and B is the slop
162 INDIAN J BIOTECHNOL, APRIL 2002
when LILa is plotted against y. The expression was derived directly from the theory of Cohn and Eisenberg with the added assumption that the extrinsic viscosity approximated the reduced viscosity for the complex (Maiti et ai, 1982). Results obtained with (a) and (b) showed no change in the length on adding each of these compounds to vortexed DNA solution at pH 8.0. In order to subtract the electrostatic interaction between the positively charged amidinium group and DNA, same experiments performed at pH 7.0, leading to the same observation (Table I).
Discussion Study of the structure, binding specificity and dy
namics of drug-DNA complexes has dual objectives: to elucidate the properties of the drug, and to probe the interaction capability of the host nucleic acids in this host-guest interaction . Structure of the drug is of vital importance, since it is on this basis the spectroscopic properties, binding specificity, hydrodynamics and dynamic characteristics may be explained. In particular, the studies of variations of hydrodynamics properties of the DNA in the presence of a drug are believed to be essential in determining the reality of interaction. Intercalation into DNA makes the molecule rigid and thus significantly increases the viscosity of DNA solution. The reason for this is that intercalation results in greater distance between base pairs leading to an apparent increase in length of the molecule (Wi lson, 1996). Viscosity is, therefore, a useful measure of intercalation. Since, in the present communication, in viscometric studies there is no change in the length of free DNA and bound DNA, therefore, it may be precisely assumed that it is specifically interacting as groove binder.
Absorbance is particularly convenient method for characterization of DNA binding ligands. Its use requires that there should be a detectable difference between absorption properties of the ligands in free form and in bound form. The spectra of ethidium bromide in presence of increasing amount of DNA shows a progressive red shift and also a reduction in the peak absorbance (Waring, 1968). The same pattern is shown by (a) and (b) in presence of increasing amount of CT-DNA but it lacks isosbestic point. The isosbestic point is obtained when there are only two forms of drug, but the lack of it shows that there must be more than two physical states bound in that form, with the remainder free in solution. However, at low DNA concentration , the drug becomes crowded on the polymer and the isosbestic point disappears. This
effect may be due to the binding of the drug by a different mechanism, namely stacking on the outside of the helix, mediated by electrostatic forces and tendency of the drug to self associate. Large changes in the absorbance are often observed when drug stacks together on the surface of DNA, for example, a nonintercalating derivative of proflavine shows a red shift of the absorption spectrum upon binding (Muller et ai, 1973). The spectra (Figs 3 & 4) can be explained on the same pattern. There is a red shift of 5 nm on increasing the concentration of DNA and it reaches saturation as the ligand/DNA ratio reaches 10. This shows that on increasing the concentration of DNA, free drug gets stacked on the double helix and results in the decrease in the peak intensity .
Since, the presence of ions has a significant effect of ions on the extent of binding therefore absorbance studies have been carried out in presence of three ions -Na+, Zn2+ and Mg2+. Although, all of these are biologically important and are assumed to be present in in vivo systems but Zn2+ shows the best result, therefore, data with Zn2+ ions interaction are included as Fig 5. The red shift is more prominent as the molarity and the temperature of the solution increases. This is again observed with electrophoretic studies while running the gel. The maximum quenching is found in the case of interaction with zinc ions.
Electrophoretic studies with polynucleotide sequences support that (a) and (b) are interacting with the helix. Since, the interaction is more in the case of A-T rich sequences, therefore, it may be assumed that it is specifically A-T specific minor groove binder. This may be explained as hydrogen bonds can be accepted by A-T pairs from the bound molecules to the C2 carbonyl oxygen of the thymine or N3 nitrogen of the adenine (Kielkopf et ai, 1998). Although, similar groups are present on G-C base pairs, the amino group of guanine presents a steric block to hydrogen bond formation at N3 of guanine and at the C2 carbonyl of cytosine and thus, the sterically inhibits penetration of molecules. Thus, the aromatic rings of many groove binding molecules form close contacts with adenine C2 protons in the minor groove of DNA and there is no room for the added steric bulk of the guanine -NH2 function in G-C base pairs.
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