DNA (deoxyribonucleic acid) is the genetic material of eve living organisms including some viruses. It is a dimer consists of two strands that immerse upon each other and appear as a double helix that are linked together covalently with each other. Each strand is made up of similar repeating units called nucleotides. Each nucleotide composed of three different moieties,a 2-deoxyribose sugar,a phosphate group and a nitrogenous base.
1.1.1 2-Deoxyribose sugar
The 2-deoxyribose sugar, a major structural component of DNA is a cyclic molecule .
The sugars are joined together by phosphate groups that form phosphodiester bonds between third and fifth carbon atoms of adjacent sugar rings.The 5′ carbon of deoxyribose sugar is attached to the 3′ carbon of the next, and make a network of 3′ carbon and 5′ carbon.5’end of a DNA molecule is characterized by a free phosphate (P) group and the 3′ end is characterized by a free hydroxyl (OH) group. It lacks an hydroxyl group at the 2 position as in a ribose therefore a sugar moiety is a 2-deoxyribose.
Two free hydroxyl groups are also located on the 5 carbon and 3-carbon of 2-deoxyribose sugar.These hydroxyl groups give a DNA oligomer its designation of 5 and the 3 end(usually accent as “three prime end” and “five prime end”).
1.1.2 Sugar-Phosphate backbone
The 2-deoxyribose sugar and a phosphate group forms the backbone in the DNA which are highly polar and defines directionality of the molecule. The polar hydrophilic back- bone is surrounded by a core of hydrophobic bases and is important for the stability and structure of DNA. The phosphate groups have a negative charge that gives a concentra- tion of negative charge on the backbone of DNA and also makes DNA,a negatively charge
molecule. The charge is also neutralised by DNA-binding proteins that contain the pos- itively charged amino acids lysine and arginine, which are attracted to the negatively charged phosphate backbone. See Fig. 1.1.
Figure 1.1: DNA backbone
1.1.3 Nucleic acid bases
DNA contain four different nitrogenous bases that make monomer of one nucleotide different from other. These bases are adenine (A), thymine (T), cytosine (C), and gua- nine(G). The bases come in two categories pyrimidines and purines. Larger nucleic acids adenine and guanine are members of a class of doubly ringed structures called purines while the smaller nucleic acids cytosine and thymine are members of a class of singly- ringed chemical structures called pyrimidines .A six-membered ring with two-nitrogen molecule formed a pyrimidine structure whereas purine is produced by a nine-membered, ring with four- nitrogen molecule. Each unit of the ring constructing the base is numbered to for specific identification. They are arranged in a particular order along the backbone of DNA to make a long chain of varying sequence that contains the code for proteins.The sequence specifies the exact genetic instructions required to create a particular organism with its own unique traits.
1.1.4 Base Pairing in DNA
The nitrogenous bases are responsible to form double-strand of DNA in consequence of weak hydrogen bonds and have specific shapes and hydrogen bond properties. The three hydrogen bonds form between guanine and cytosine and then denoted as G.C or C.G,depending on which is associated with the first strand. Similarly adenine and thymine also bond exclusively by pairing of two hydrogen bonds and then denoted as A.T or T.A. This coupling up of nitrogen bases termed as complementarity.,A hydrogen bond donor need an equivalent hydrogen bond acceptor to form a hydrogen bond in the base across from it. Purines are only complementary with pyrimidines because molecules in pyrimidine-pyrimidine pairings are very far from each other that doesn’t makes the hydrogen bonding to be established. Purine-purine pairing are energetically unfavourable because the molecules are too close and create an electrostatic repulsion. The only possible pairings are GT and AC. Primary and secondary amine groups or hydroxyl groups are common hydrogen bond donar while carbonyl and tertiary amines are common hydrogen bond acceptor groups. There are two hydrogen bonds between an A:T base pair. One hydrogen bond lie between the 6′ primary amine of adenine and the 4′ carbonyl of thymine. The other hydrogen bond form between the 1′ tertiary amine of adenine and the 2′ secondary amine of thymine. On the other hand,G:C base pair has three hydrogen bonds. One hydrogen bond lie between guanine with its 6′ hydrogen bond accepting carbonyl and cytosine having 4′ hydrogen bond accepting primary amine. The second hydrogen bond also formed between guanine on 1′ secondary amine and cytosine 3′ tertiary amine and the third formed between the 2′ primary amine on guanine and the 2′ carbonyl on cytosine.
The directionality of DNA is vitally important to many cellular processes. since,double helices are necessarily directional(a strand running 5 to 3 pairs with strand running 3 to 5 )and processes such as DNA replication occur in only one direction. The two DNA strands in a duplex are anti parallel and form a chemically stable structure. That is, one strand running from the 5-phosphate to 3-OH is paired with the other strand arranged with its 3-OH opposite the 5-phosphate of the first strand, and its 5-phosphate opposite the 3- OH of the first strand.
1.1.6 3 end and 5 en
DNA strand is inherently directional.The “3 prime end” has a free hydroxyl (or phos- phate) on a 3′ carbon and is called as the tail end. New nucleic acid molecules are formed by one end of 3-hydroxyl as it is ligated to the other end of 5-phosphate of a different nucleotide that make it possible to form strands of connected nucleotides.Molecular biologists can use nucleotides that has a deficiency of 3-hydroxyl(dideoxyribonucleotides) to stop DNA replication .The “5 prime end” has a free hydroxyl (or phosphate) on a 5′ carbon in the sugar-ring and this end is called as the tail end . If a phosphate group bind with the 5 end, ligation of two nucleotides can form, with a phosphodiester bond from the 5-phosphate group to the 3-hydroxyl end of other nucleotide. ligation can also stop if the above process is eliminated. Molecular biologists have an advantage of the above phenomenon to stop ligation of any unnecessary nucleic acid by removing the 5-phosphate with a phosphatase.
1.2 DNA-Ligand Binding
The structure of DNA represents a variety of sites where ligands may interact and bind
with DNA.The binding interaction between a drug and DNA often leads to a signi_-
cant modi_cation of the structure of the DNA and may have an important inuence on
their physiological functions associated with several biological e_ects including antivi-
ral,antibacterial,antipotozoal and antitumor.
Modes of Binding
Because of the complex double-helical structure of DNA,drug molecule interact with
DNA in a number of modes. A number of forces of varying strength involved in each
interaction. Electrostatic forces with the phosphate backbone,sequence sensitive van der
Waals interaction and hydrogen bonding interactions that occur between polar atom of
bases and hydrogen molecules are incorporated singly or in combination.To understand
the mechanism of interaction of each mode,it is best to discuss di_erent binding modes
that can act on DNA. (a) External Binding (b) Intercalators (c) Groove binding (i)
Major groove binders (ii)Minor groove binders
This type of binding results due to electrostatic forces applied to the negatively charge
phosphodiester group along the backbone of DNA for cationic molecule.Ligand charge,
hydrophobicity and size a_ect on electrostatic interactions.External binding may also be
due to either covalent or non-covalent interactions.This mode of binding is characteristics for major groocould potentially be sampled during simulations where the charge and shape of helical molecules are both changed.
An important class of molecules that binds to DNA are intercalators,which have been
extensively used as a anti-cancer drug.Intercalation occurs due to immersion of a at
aromatic drug molecule between nucleic bases contributes to unwind DNA helix(67).The
interaction between a positively charged intercalator and a negatively charged DNA
can be quite strong and form complex through electrostatic forces.Energy consumed to
unstacked the nucleic acid bases which forms a gap between neighbouring base pairs
into which the intercalator can _t easily.Because of small binding site,they have a little
sequence selectivity and many known intercalators shows limited selectivity for GC base
pairs such as ethidium bromide which has a high a_nity towards GC site.Several other
drugs such as propidium,proavin, anti-tumor drugs adriamycin and actinomycin D
intercalate with DNA.
Smaller ligands preferentially binds to minor groove region whereas proteins and other
large molecules speci_cally _ts into the major groove region of DNA. They have crescent -shaped conformation due to presence of two or more than two aromatic rings that gives a conformational exibility to the molecule and makes it perfect to _t in the groove. They also possess some functional group that forms hydrogen bonds at lower most part of DNA bases.They perfectly accommodate in the AT rich regions but some known groove binders show little preference towards GC site.
Major Groove binders
Presence of number of hydrogen bonds on the DNA major groove enhance its recognition potential. Major groove speci_c compounds are alkylating and methylating agents and and N 7 position of guanine in the major groove take part in interaction.one of the most common example is Cis platin which is a well known anti cancer drug.
Minor Groove binders
The most widely studied DNA interacting agents are minor groove binders that occurs
naturally and also synthesize according to their sequence speci_c properties as they have
pronounced binding a_nity towards AT rich groove.AT binding site is more thinner
and deeper than GC so that all heteroaromatic rings such as furan,pyrole,benzene and
Imidazole of minor groove binders twisted and _t better into AT site by applying van
der waals force.Hydrogen bonds of bound molecule attached to the AT base pairs to
the C-2 carbonyl oxygen of thymine or N-3 nitrogen of adenine.GC base pairs also
contain same functional groups but a steric block form by amino group of guanine in
GC locations which causes hinderence to the formation of hydrogen bond on guanine at
N-3 position and on cytosine at O-2 cabonyl position,prohibiting vad derWaal forces and
inhibit penetration of small molecules at GC sites of minor groove.AT site selectivity for
positively charged minor groove binders also enhanced due to high negative electrostatic
potential as compared to GC site. A number of experimental studies shows that minor
groove of B type of DNA duplexes more suitable for binding of small molecules most
often with Dickerson-Drew sequence d(CGCGAATTCGCG) and also similar such as
X-ray crystallography proof complex formation of berenil with dodecanucleotides,i.e.
d(CGCGAATTCGCG) and d(CGCAAATTTGCG)which in turn shows its preference
of binding with AT rich site of DNA minor groove and reside between three (AAT) or
four(AATT) base pairs. A number of research on berenil also con_rm its weak interac-
tion and intercalating behavior.Hydrogen bonds are also formed between the amidinium
groups and adenine N-3 or thymine O2 atoms on reverse strands of a double helical DNA
oligonucleotide.Berenil is a curve shape drug which match the helical structure of DNA
One of the most clinically important drug,pentamidine is a synthetic antimicrobial com-
pound also known aspentamidine (1,5-bis(4-amidinophenoxy)pentane,among all the mi-
nor groove binders.It has been use as a secondary drug for treating aids related P.carinii
pneumonia.Foot printing and X-ray crystallography shows its pronounced attachment
to DNA sites which has minimum four to _ve successive AT base pairs with the charged
amidinium group shows hydrogen bonding to O2 of thymine or N3 of adenine on oppo-
site DNA strands. It contains two phenyl rings that are twisted after binding with the
minor groove by 35Â° with respect to each other by van der Waals forces.
DAPI also called 4,6-diamidino-2-phenylindole(DAPI) is a synthetic,unfused aromatic
compound is widely used in molecular biology as a uorochrome on binding upon AT
site of minor groove binder as well as an intercalating drug.upon binding to GC rich
sequence without showing any property of uorescence.X-ray structure of DAPI with
d(CGCGAATTCGCG)exhibited that the drug span three base pairs and also give a
clear picture of parallel attachment of phenyl and indole rings to the minor groove walls
of DNA. ||||||||
1.4 UV-Visible Spectroscopy
Spectroscopy is a valuable tool in the study of intermolecular interactions. It is a well
developed routine technique and plays an important role in analytical chemistry as well
as it has widespread application in physics and life sciences. It deals with the mea-
surement of the absorption of radiations in the ultraviolet and visible region of spec-
trum.Spectroscopic techniques form the largest and the most important single group of
techniques used in analytical chemistry,and provide a wide range of quantitative and
qualitative information. All spectroscopic techniques depend on the emission or ab-
sorption of electromagnetic radiations and used to determine the electronic structure of
atoms and molecules. In order to understand these techniques,it is necessary to have
some knowledge about properties of electromagnetic radiations and the nature of atomic
and molecular energy. The ultraviolet region extends from 10 to 400nm.It is subdivided
into near ultraviolet region (200 to 400nm) and the far or vacuum ultraviolet region(10
to 200 nm).The visible region extends from 400 to 800 nm.
1.4.1 Electromagnetic radiations
Electromagnetic radiations are produced by the oscillation of electric charge and mag-
netic _eld residing on the atom and has its origins in atomic and molecular processes. It
vibrates perpendicular to the direction of propagation with a wave motion and can travel
in space and does not need a medium like air or water to travel through. There are various
forms of electromagnetic radiations e.g. visible,ultraviolet,infra-red, X-rays,microwaves
and cosmic rays. They are characterised by frequencies,wavelength or wave numbers.
The most familiar form of electromagnetic radiations is visible light which forms only a
small portion of full electromagnetic spectrum.
A plot which shows a number of absorption bands with respect to energy versus wave-
length has some properties yield various information and is broken into several regions
called as Electromagnetic Spectrum.Di_erent regions of the electromagnetic spectrum
provide di_erent kinds of information as a result of interactions. Electromagnetic spec-
trum covers a very wide range of electromagnetic radiation that starts from gamma
rays and ends on to radio waves. The boundaries between the regions are approximate
and the molecular process associated with each region are quite di_erent.The regions in
increasing order of frequency are
1/ Radio frequency region ;Nuclear magnetic resonance and electron spin resonance
spectroscopy.The energy changes with change in direction of spin of a nucleus and elec-
2/ Micro wave region:Rotational spectroscopy .Change in energy arise from transi-
tions to higher energy associated with change in the rotational quantum number of the
molecule. 3/Infra-red region:Vibrational spectroscopy The energy changes associated
with transitions between vibrational levels of molecules.
4/Vis- ible and Ultraviolet region:Electronic spectroscopy The energy changes accom-
pained with valence electrons of molecules.
5/X-ray region: inner electrons of an atom or a molecule invole in order to change
energy of molecule.
6/ X-ray region: nuclear excitations necessary for an enegy change.
1.4.2 Law of molecular Absorption:Beer-Lambert law
All spectrophotometric methods that measure concentration in terms of absorbance,including
detection of proteins and nucleic acids,determine molar absorptivity of metal com-
plex,various enzyme essay,describe attenuation of solar or stellar radiation and di_er-
ent metabolites based upon two basic rules,which combinely spoken as Beer-Lambert
law.This law was basically originate by a French mathematician Lambert,which states
that the function of light absorbed by a transparent medium s independent of the inci-
dent light assing through it.This shows that logarithm of the decrease in light intensity
along the light path with respect to thickness of medium which can be written as follow
log10(I0/I) = kl
where IÂ° is incident light intensity,I is light path length,k is a medium constant which is
further interpret by a Beer,a German Physicist in the same year states that the amountof
light absorbed is proportional to the number of molecules of the chromophore through
which the light passes.One can also says that constant K is directly proportional to
the chromophore concentration i.e. K=eC,e is the molar absorptivity of chromophore
and is equal to absorption of 1M of solution at a path length of 1 cm and their unit is
M-1cm-1.Now,combinely Lambert-Beer law presented as
A = lC,
whereby,the term log10(I0/I) is re_ered as absorbance(A),l is the thickness of solution
and E is the molar absorption coe_cient.
1.4.3 Electonic transitions in Nucleic Acids
Absorption or emission of radiations in nucleic acid causes di_erent types of transitions
in UV-visible spectral regions and appear from n-pi* and pi -pi* transitions of purine
and pyramidine bases.
Large amount of energy required for the shifting of an electron from a bonding molec-
ular orbital to a * antibonding molecular orbital in the UV region.Unsaturated hydro-
carbons shows this type of transition and being transprent in the near UV such as
methane,heptane and cyclohexane that shows maximum absorbance below 200 nm due
to the fact that absorbance is equal to 1 for a thickness of 1 cm below 200nm. Similarly,
water in the near UV(A=0.01 for 1cm ,at lambda =190nm)is transparent due to the
presence of -* and n-* transitions.
This type of transition usually occur in compounds having lone pair of electrons and
required energy lower than -* transition for the promotion of an n electron from an
atom to an * molecular orbital.Moderate wavelength range for this transition is 150 to
250 nm as 180nm for alcohols,near 190nm for ethers or halogen derivatives and in the
region of 220nm for amines.
Most of the organic compounds have a – conjugate system and shows -* transitions
with an intense strong absorption band occuring anywhere in the near UV region which
depends upon the presence of heteroatoms substituents.These compounds also shows a slightly blue and red shift with respect to its polarity.
These bands are called forbidden bands having a low molar absorptivity less than 100
and originate from promotion of electron from a non bonding molecular orbital to an
anti-bonding *orbital.This transition is more pronounced in molecules having a hetero
atom with a lone pair of electron i.e.carbonyl which requires low energy and occur in
the regions from 270 to 300 nm. d-d transition
electrons placed in incompletely _lled d orbitals of most of the inorganic salts are re-
sponsible for transitions of weak absorption and also color and located in the visible
region..That is why the solutions of metallic salts of titanium or copper are blue,while
potassium permeganate yeilds violet solutions, and so on.
1.4.4 Chemical shift
change in max to longer wavelength(lower frequency)also change absorption,reectance
transmittance or emission spectrum of a molecule mostly due to substitution or solvent
e_ect i.e change in polarity of solvent called as bathochromic shift or red shift.Solvent
e_ect is weak in less polar compounds as compared to polar one which can stabilise
excited form,favours transition and causes a change in wavelength towards longer side. Hypsochromic shift
The opposite e_ect of bathochromic shift also called as blue shift as max shift towards
the blue end of spectrum.Unbonded electron pair lowers the energy of the n-orbital
and increased solvation causes hysochromic shift.Mostly polar solvents such as water
and alcohol have pronounce e_ect of hypsochromism due to broad hydrogen bonding
between protons and the non-bonded electron pair during solvation.
reduction in the intensity of uv light without any change in wavelength called as hypochormic
e_ect which caused by the entry of an auxochrome which distrots the chromophore.For
example ,biphenyl shows lAMDAmax 252nm,Emax19,000,whereas 2,2-dimethylbiphenyl
shows Lambda max 270nm,Emax 800.
This e_ect leads to an increase in absorption of UV light at same wavelength due to
appearance of an au that causes hyperchromic shift.For example,benzene shows B-band
at 256nm,Emax 200,whereas aniline shows B-band AT 280nm,Emax 1430.The increase of
1230 in the value Emax of aniline compared to that of benzene is due to the hyperchromic
e_ect of the auxochrome NH2.
1.4.5 Chromophore groups
Organic compound mostly containing double bond is responsible to produce color and
absorption of ultraviolet or visible radiations as single bond is not enough to do that
but if many are present in conjugations,sharp color can produce. A single functional
group or a collection of functional groups also capable for absorption and they also act
as a chromophore. A complex molecule can contain more than one chromophore so the
e_ect of conjugation on the chromophore is to shift the maximum absorption to a longer
wavelength .i.e. a bathochromic shift or red shift appear with an increase in absorption
intensity and the spectrum is strongly upset with respect to the superimposing e_ects of
random chromophores. The more the number of carbon atoms on which the conjugated
system is spreaded,the more the decrement in the di_erence between energy levels.and
accounts large bathchromic e_ect. A very simple spectrum of a compound having one
main peak absorbing below 300nm possibly contains a very simple conjugated system
Instrumentation in UV-Visible Spectrophotometer
UV-Visible spectrophotometer is a very simple to operate and able to perform quick
qualitative as well as quantitative analysis.It is usuallay designed around _ve funda-
mentals parts i.e. a radiation source,a monochromater(wavelength selector),a samplecell(cuvette),detector and a signal processor (readout device) for measuring the absorp-
tion of uv or visible radiations.These components are typically integrated in a unique
frame work to make spectrometers for chemical analysis.Two types of UV-Visible spec-
trophotometers are generally in use:a _xed spectrophotometer with a single beam and
a scanning spectrophotometer with double beams.Single beam spectrophotometers are
highly sensitive devices and obtaining a spectrum requires measuring the transmittance
of the sample and the blank at each wavelength separately.In the double beam spec-
trophotometer,the light split into two parallel beams,each of which passes through a
cell;one cell contains the sample dissolved in a solvent and the other cell contains the
solvent alone.The detector measures the intensity of light transmitted through the sam-
The intensity of radiation coming from the light source varies over the entire UV-Vis
range.More than one type of source can be used in UV-Vis spectrophotmeter which au-
tomatically swap lamps when scanning between the UV and visible range .A deutrium
lamp is used for the wavelengths in the UV range,a tungsten lamp is used for the wave-
lengths in the visible range and alternatively for the entire UV-Visible region,a xenon
lamp can be used.
Its role is to spread the beam of light into its component wavelengths and a system of
slits focuses the desired wavelength on the sample cell.The most widely used dispersing
device is a prism or a grating made p of quartz because quartz is transparent throughout
the UV range.
The detector converts the intensity of light reaching it to an electrical signal.It is by
nature a single channel device.Two types of detector are used,either a photomultiplier
tube or a semiconductor.For both of which the sensitivity depends upon the wavelength.
QSAR and Drug design
Quantitative structure-activity relationship (QSAR) (sometimes QSPR: quantitative
structure-property relationship) is the process by which chemical structure is quanti-
tatively correlated with a well de_ned process, such as biological activity or chemical
For example, biological activity can be expressed quantitatively as in the concentra-
tion of a substance required to give a certain biological response. Additionally, when
physicochemical properties or structures are expressed by numbers, one can form a math-
ematical relationship, or quantitative structure-activity relationship, between the two.
The mathematical expression can then be used to predict the biological response of other
QSAR’s most general mathematical form is:
* Activity = f(physiochemical properties and/or structural properties)
Quantitative structure-activity relationships (QSAR) represent an attempt to corre-
late structural or property descriptors of compounds with activities. These physico-
chemical descriptors, which include parameters to account for hydrophobicity, topology,
electronic properties, and steric e_ects, are determined empirically or, more recently, by
computational methods. Activities used in QSAR include chemical measurements and
biological assays. QSAR currently are being applied in many disciplines, with many
pertaining to drug design and environmental risk assessment.
Organic compound mostly containing double bond is responsible to produce color and absorption of ultraviolet or visible radiations as single bond is not enough to do that but if many are present in conjugations,sharp color can produce. A single functional group or a collection of functional groups also capable for absorption and they also act as a chromophore. A complex molecule can contain more than one chromophore so the effect of conjugation on the chromophore is to shift the maximum absorption to a longer wavelength .i.e. a bathochromic shift or red shift appear with an increase in absorption intensity and the spectrum is strongly upset with respect to the superimposing effects of random chromophores. The more the number of carbon atoms on which the conjugated system is spreaded,the more the decrement in the difference between energy levels.and accounts large bathchromic effect. A very simple spectrum of a compound having one main peak absorbing below 300nm possibly contains a very simple conjugated system such as diene or an enone whereas, if the spectrum is much mixed and also allocated in a visible region,then the molecule must contain chromophore having large red shift such as polyene ,polycyclic aromatic system etc.
Selection of solvent used in UV-visible spectroscopy is very important. The prime requirement for a solvent is that it should be transparent to radiation over full UV range and also not absorb UV radiations in the region of substance whose spectrum is actually analysed .Most of the organic solvents successfully meet that criteria and solvents without having any conjugtion are very convenient for this purpose.Among the solvents ,the water ,95% ethanol and hexane are most commonly used and are transparent in the full uv spectrum. Another valuable requirement for selecting a solvent is that it gives a nice spectrum of a set a absorption bands because polar solvent form hydrogen bonds with solute and the fine spectrum of the complex may vanish but this is not the case for non polar solvents where a fine spectrum often easily appears because of the absence of hydrogen bonding.Polar solvents also shows bathochromic effect which causes a decrease in electronic state.
Asecond criteria for agood solvent is its effect on the fine strusture of an absorption band.Ano polar solvent doesnot hydrogen bond with the solute,and the spectrum of the solute closely approximate s the spectrum that would be produced in the gaseous state ,in which fine structure is often observed.In a polar solvent the hydrogen bonding forms a solute solvent comlex and the fine structure may disappear.
Athird criteria for a good solvent is its ability to influence th