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.

References Ammon H P T & Wahl M A, 1991. Pharmaco logy of Curcuma

longa. Planta Med, 57, 1-7. Brown D G, 1990. Crystal structure of a berenil-dodeca nucleo­

tide complex: the role of water in sequence specific ligand binding. EM 801. 1329-1334.

KUMAR el al: CURCUM IN-BI OCONJUGATE 163

Chakraborty S el ai, 1989. Ari stolac tam-0-D-g lucoside: a ne w DNA binding monofun ctio nal - intercalating alkal oid. Bio­chem Pharmacal, 38, 3683-3687.

Cohn G & Ei senberg H, 1966. Studies of the bindi ng of ac tino­myc in and related compounds to DNA. BiopolYIII.er, 4, 429-440.

Cohn G & Eisenberg H, 1969. Vi scos ity and sedimentation study of sonicated DNA- proflavine complexes. Biopolymer, 8,45-55.

Dougherty G, 1984. Phys ico-chem ical probes o f intercalation. In l J Biochem, 16, 11 79-1192.

Edwards J K et ai, 1992. Crystal structure of pentamidine­o li gonuc leotide complex: implication fo r DNA binding prop­erties. Biochelllistry, 31, 7103-7109.

Gov indraj an V S, 1979. Turmeric chemi stry, techno logy and quality. CRC Cril Rev Food Sci, 12, 199-30 I.

Jenkins C T, 1993. NMR and molecular modeling studies of inter­ac ti on of berenil and pentamidine with d (CGC AAA TIT GCG)z. Eur J Biochem, 213, 11 75- 11 84.

Kielkopf C L eI ai, 1998. A structural basis for recognition of A T and T A base pairs in the minor groove o f 0-DNA. Science, 282, 111-115.

Kielkopf C L et ai, 2000. Structural effects of DNA sequence on T .A recognition by hydroxypyrro le / pyrro le pairs in the mi­nor groove. J Mol Bioi, 295, 557-567.

Kumar S et ai, 2000. Synthesis of Curcumin-bioconjugates and study of their antibacterial activities against 0-lactamase pro­ducing microorgani sm. Bioconjugate Chern, 12, 464-469.

Maiti M el ai, 1982. Sanguanarine: A monofunctional intercalat­ing alkaloid. FEBS Lell, 142, 280-284.

Maiti M el ai, 1984 . Interacti on of Sanguanarine with natural and sy nthe ti c deoxyribonucle ic acids. Indian J Biochem Biophys, 21 ,58- 165.

MUlier W & Crothers D M, 1968. Studi es of binding of actinomy­cin and related compounds to DNA. J Mol Bioi, 35, 25 1-290.

MUller W et ai, 1973. A non-intercalat ing proflavin e derivative. Eur J Biochem, 39, 223-234.

Ne idle S & Abraham Z, 1984. Structural sequence dependent aspects of drugs: intercalation into nucle ic ac ids. CRC Cril Rev Biochell1, 17, 73- I 2 1.

Sharma 0 P, 1976. Antioxidant activity of curcumin and related compounds. Biochem Biopharmacol, 25, 18 11-1 8 12.

Sri mal R C & Dhawan B N, 1973. Pharmacology of di feruloy l methane (curcumin) , a non-steroidal anti -inflammatory agent. J Pharrn Pharmacol, 25,447-452 .

Stoskar R R et ai, 1986. Evaluation of anti-inflammatory property of curcumin (d iferuloy l methane) in patients with postopera­tive inflammation. Int J Clin Phannacol Th er Toxicol, 24 , 65 1 - 654.

Toda S el ai, 1985. Natural anti oxidant III. Antioxidant compo­nents isolated from rhizome of Curcuma longa L. Chern Pharm Bull, 331, 1725-1728.

Turner J M et ai, 1998. Aliphatic/aromat ic amino ac id pairing for polyamide recognition in the minor groove of DNA. J Am Chem Soc, 120,62 19-6226.

Waring M J, 1968. Drugs, which affect the structure and function of DNA. Nature (Lond), 219, 1320-1325.

Wil son W D, 1996. Reversible interaction of nucleic acids with small molecules. in Nucleic Acids in Chemistry and Biology, edited by G M Blackburn & M J Gait, 2nd edn. Oxford Uni ­vers ity Press, New York. Pp 329-374.