1.1 Synthesis of Oligonucleotides 1.1.1...

Chapter 1: Synthesis of Oligonucleotides and Spin-labeling Techniques

1.1 Synthesis of Oligonucleotides

1.1.1 INTRODUCTION

Deoxyribonucleic acid (DNA) is a nucleic acid that encodes entire hereditary

information and controls the growth and division of cells of all known living

organisms and some viruses.1,2 It contains the information needed to construct other

components of cell such as RNA molecules and proteins. DNA was first isolated in

1869 from the nuclei of white blood cells. As this material was found in the nucleus

and was acidic, it was called nucleic acid. Eventually, scientist found that nuclei of

all cells contain DNA and it acts as carrier of genetic information. In 1953, James

Watson and Francis Crick described the three dimensional structure of DNA - the

famed Double helix, for which they were awarded 1962 Nobel price in medicine.3

This led to many important developments in the field of DNA. One such important

milestone which received great deal of interest was chemical synthesis of DNA or

oligonucleotide. The intension of artificially synthesizing DNA by H. G. Khorana

and coworkers in 1967 raised many doubts regarding practical applications of such

artificial DNA. However, the numerous uses developed for synthetic DNA in recent

years have dispelled all such doubts and the ability to synthesize DNA fragments

rapidly has become an important asset in many laboratories. This chapter gives a

brief account of different structural aspects of DNA, chemical strategies used for

synthesis of oligonucleotides, and spin labeling techniques.

1


1.I.2 Structure of DNA

Primary Structure of DNA

Chemically DNA consists of two long polymeric strands of simple units called

nucleotides. Each nucleotide in turn is made up of nucleoside and phosphate

molecule. Nucleoside further consists of a pentose sugar attached to nitrogen atom

of heterocyclic molecules called as nitrogen bases. The pentose sugar observed in

DNA is 2-deoxy-D-ribose, which discriminates it from another type of nucleic acid,

ribonucleic acid (RNA) containing D-ribose sugar. The nitrogen bases observed in

DNA are either substituted purines (adenine and guanine) or pyrimidines (thymine

and cytosine) (Figure 1.1). In case of RNA thymine is replaced by uracil. The

nitrogen bases are bonded to the anomeric carbon of sugar via β-glycosidic linkage

forming the corresponding nucleoside (Figure 1.2). Nucleotides are the phosphate

ester of nucleosides (Figure 1.3).2

NH

N

N

N

NH2

NH

N

N

NH

NH2

O

NH

N

O

NH2

NH

NH

O

OCH3

NH

NH

O

O

adenine guanine cytosine thymine uracil

1

2

34

5 67

8

9 1

2

345

6

Figure 1.1

2


N

N

O

OH

OHN

N

NH2

N

N

O

OH

OHN

NH

O

NH2N

N

O

OH

OH

NH2

O N

NH

O

OH

OH

O

O

CH3

2' - deoxyadenosine 2' - deoxyguanosine 2' - deoxycytosine thymidine

1'

2'3'

4'5'

Figure 1.2

N

N

O

OH

ON

N

NH2

PO

O

O

N

N

O

OH

ON

NH

O

NH2PO

O

O

N

N

O

OH

O

NH2

OPO

O

ON

NH

O

OH

O

O

O

CH3

PO

O

O

2' - deoxyadenosine- 5' - monophosphate (dA)

2' - deoxyguanosine- 5' - monophosphate (dG)

2' - deoxycytosine- 5' - monophosphate (dC)

thymidine- 5' - monophosphate (dT)

--

--

--

--

Figure 1.3

Two nucleotide units attached to each other forms a dinucleotide. An

oligonucleotide consists of three to ten nucleotide subunits and polynucleotide

contains many nucleotide subunits. A single strand of DNA is a polynucleotide

consisting of either of four nucleosides attached to each other via phosphodiester

linkage connecting 5'-hydroxyl group of one nucleotide to 3'-hydroxyl group of

another nucleoside. This forms the primary structure of DNA, uniqueness of which

solely resides in sequence of nitrogen bases. The nitrogen bases in DNA are capable

of existing in tautomeric forms (either amino - imine or keto - enol or both) which

are alternative structures differing in the location of hydrogen atom. It has been

established by UV, NMR and IR studies that all nitrogen bases exist

overwhelmingly (99.99 %) in amino - and keto - tautomeric forms at physiological

pH. The primary structure of oligonucleotide containing four nucleotides with

3


nitrogen bases adenine (A), thymine (T), guanine (G), cytosine (C) can be shown as

in (Figure 1.4).

N

N

O

O

NH2

O

N

N

O

O

N

NH

O

NH2

N

NH

O

O

O

O

CH3

N

N

O

O

ON

N

NH2

PO

PO

OO

PO O

O

PO

O

O

OO -

-

-

-

3'

5'

A

T

G

C

Figure 1.4

Watson and Crick Model of DNA

James D. Watson and Francis Crick who, using x-ray diffraction data collected by

Rosalind Franklin, proposed the double helix or spiral staircase structure of the

DNA molecule in 1953. According to Watson and Crick, the DNA molecule consist

of two very long single polynucleotide strands wrapped around each other in the

form of a double helix.3 The width of the double stranded helix is 20 Å. Each turn of

helix is 34 Å and contains 10 nucleotides pairs. The twisting of strands results in

4

http://en.wikipedia.org/wiki/James_D._Watson

http://en.wikipedia.org/wiki/Francis_Crick

http://en.wikipedia.org/wiki/Rosalind_Franklin

http://en.wikipedia.org/wiki/Double_helix

http://en.wikipedia.org/wiki/DNA

http://en.wikipedia.org/wiki/1953


formation of deep and shallow spiral grooves. The double helix structure is

analogous to a coiled ladder, outsides of which are formed by the pentose-phosphate

backbones. The purine and pyrimidine bases of each strand are facing inward

towards each other and interacting thus forming the rungs of the ladder and holding

the strands together. Each base on one chain interacts with complementary base on

the second chain via hydrogen bonds. This interaction was found to be specific as

adenine (A) interacts with thymine (T) via two hydrogen bonds and vice versa.

Whereas, guanine (G) interacts with cytosine (C) via three hydrogen bonds and vice

versa (Figure 1.5).

Figure 1.5

5


Different Conformers of DNA

The DNA duplex can exist in three major conformations namely A-DNA, B-DNA

and Z-DNA depending upon the sequence and the environment (Figure 1.6).4,5 The

A and B - DNA are both right-handed helices, whereas Z-DNA is a left handed

helix. The A-helix is the predominant form in nonpolar solvents, while B-helix is

the predominant form in aqueous solution. Nearly all the DNA in living organisms

is in B-helix form. Z-DNA is observed in regions where there is high content of G-C

base pairs. The A-helix is shorter (for a given number of base pairs) and about 3%

broader than B-helix, which is shorter and broader than Z-helix. A-DNA has 11 base

pairs per turn with rise (distance between adjacent base pairs) of 2.3 Å. B-DNA has

10 base pairs per turn and rise of 3.4 Å. Z-DNA has 12 base pairs per turn with rise

of 3.8 Å.

Figure 1.6

6


In the late 1950s and early 1960s it was shown that double stranded DNA or

RNA containing purines (Pu) in one strand and pyrimidines (Py) in another, forms

triple stranded structure containing either one polypurine strand and two

polypyrimidine strands (Py*Pu-Py) or one polypyrimidine strand and two

polypurine strands (Pu*Py-Pu). Watson-Crick interaction between two strands of

duplex is represented by (-) and the interaction between duplex and third strand

called as Hoogstein H-bonding is represented by (*) (Figure 1.7). The

oligonucleotides that invade dulplex DNA and form triple helix are termed as

Triplex Forming Oligonucleotides (TFOs).6

N

NN

N

NH

HOH

R

N

NN

N

R NH

H

H

O NN

O R

CH3NH

H

C - G * G

G

G

C

N

NN

N

NH

R

H

H

N

NN

N

R NH

H

H

O NN

O R

CH3NH

H

C - G * A

A

G

C

N

N

R

OO

CH3

H

NNN

N

R

NH

H

NN

O

H

O R

CH3

T - A * T

T

A T

N

N

N

N

NH

H

R

NNN

N

R

NH

H

NN

O

H

O R

CH3

T - A * A

A T

T

Figure 1.7

7


1.I.3 Chemical Synthesis of Oligonucleotides

Discovery of DNA, understanding of its structure and functions was followed by its

applications in variety of areas such as site-directed mutagenesis, recombinant DNA

technology, cloning, chip based DNA-microarrays, PCR technologies, etc. This led

to the exponential increase in the demand for synthetic oligonucleotides. Most of the

applications mentioned above require smaller quantities (< 1 μg) of synthetic

oligonucleotides. However, much larger quantities of highly pure DNA are required

for structural studies involving NMR-spectroscopy or X-ray crystallography.7

Oligonucleotides can be synthesized via solid phase synthesis or classical

solution phase synthesis. Solid phase synthetic route is useful, practicable, and

reasonably economical for preparation of oligonucleotides up to about 50 mg.

Beyond this amount, classical solution phase synthesis is more appropriate but it is

relatively laborious. Isolation of pure oligonucleotides in both the routes is highly

time consuming.

The key step in DNA synthesis is the specific and sequential formation of

phosphate linkage between 3' - O of one nucleoside and 5' - O of another nucleoside.

Since a deoxyribonucleoside monomer contains two hydroxyl groups (primary

hydroxyl at 5' and secondary hydroxyl at 3' position) one must be chemically

protected, while the other is specifically phosphorylated or phosphitylated and then

coupled to the next deoxyribonucleoside unit. In the process other reactive

functional groups present must be protected. Various strategies developed for the

synthesis of oligonucleotides involve two types of protecting groups.

• Permanent protecting groups, which remain attached to the oligonucleotide

throughout the synthesis and are removed at the end of the synthesis.

8


• Temporary protecting groups, which are used to obtain specificity in a single

reaction and are removed immediately.

Permanent protection is required for the heterocyclic bases in the nucleosides

during DNA synthesis. The exocyclic amino group needs to be protected due to its

reactive nature. The most commonly used amino protecting group is benzoyl for

adenine and cytosine and isobutyryl for guanine.8 Thymine usually requires no

protecting group. Recently, it was observed that during extended synthesis side

reactions take place at 6 - O position of guanine. Thus various protecting groups are

proposed for 6 - O position of guanine such as, phosphinothioyl9, 2-nitrophenyl10, β-

cyanoethyl11, nitrophenylethyl11, etc.

Most of the synthesis strategies for oligonucleotides involve selective

phosphorylation or phosphitylation of 3' - OH group thus 5' - OH group needs to be

protected temporarily. The 4,4'-dimethoxytrityl chloride (DMT-Cl) remains the best

choice for this purpose.12 Due to the bulky nature of DMT- group, it can be

introduced selectively at 5'-position of sugar unit and can be removed easily under

very mild acidic conditions as strong acidic conditions results in depurination.

Further efficiency of deprotection step can be monitored easily by

spectrophotometer due to strong visible absorption of the cationic species produced

upon detritylation.

The phosphate group needs to be permanently protected as it may compete

with sugar hydroxyl group during chain extension in oligonucleotide synthesis. Thus

development of permanent phosphate protecting groups selectively removable at the

end of the synthesis has received a lot of importance. Earlier chlorophenyl13 and

methyl14 groups were used as phosphate protecting groups. Chlorophenyl group can

be cleaved selectively by use of mild nucleophile, an oximate anion and the methyl

9


group by the thiophenate anion. Recently these protecting groups were replaced by

β-cyanoethyl group15, which can be easily removed by β-elimination with mild

ammonia solution. The other phosphate protecting groups such as (9-

fluorenyl)methyl16, 2,2,2-trichloroethyl17, 2,2,2-trichloro-1,1-dimethylethyl18.19,

2,2,2-tribromoethyl20, 2-(p-nitrophenyl)ethyl21, 2-(2-pyridyl)ethyl22, 2-

(methylsulfonyl)ethyl23, 2-(phenylsulfonyl)ethyl24, o-nitrobenzyl25, o-(t-

butyl)phenyl26, o-or p-chlorophenyl27, 8-quinolyl28 and allyl29 have been found to be

functional in place of β-cyanoethyl group.

Michelson and Todd Synthesis

The first chemical synthesis of dinucleotide phosphate ([d(TpT)] thymidylyl-

(3'→5')-thymidine) and a dinucleotide ([d(pTpT)] 5'-O-phosphorylthymidylyl-

(3'→5') thymidine) with natural 3' to 5' internucleotide linkages was reported by

Michelson and Todd in 1955.30 It involves phosphorylation of 3'-O-acetylthymidine

with 3'-O-phosphorochloridate of 5'-O-acetylthymidine (Scheme 1.1). Phosphate

functionality was protected by benzyl group. However the approach did not received

much attention and no further work was carried out on this approach.

10


O

O

TRO

POOBn

Cl

O

AcO

TOH

O

O

AcO

TOPO

RO T

O OBn

O

O

OH

TOPO

R'O T

O O

OPOOBn

OBn

OPOOH

O

2,6-lutidineMeCN

Deprotection

-

+

R = -OAc or

R' = -H or-

i. H2SO4, EtOH, Waterii. Ba(OH)2, H2O

Scheme 1.1 Michelson and Todd synthesis

The Phosphodiester Approach

The phosphodiester approach for oligonucleotide synthesis was introduced by H. G.

Khorana in 1956. It involved the condensation of appropriate nucleoside with

nucleotide derivative in presence of dicyclohexylcarbodiimide (DCC) or

arylsulfonyl chloride as the activating agent.31 The approach was simple and

dominated the field of oligonucleotide synthesis for almost 20 years. However it

suffers from disadvantage offered by the presence of anionic phosphodiester

11


function in the backbone of DNA fragment used in the synthesis at which side

reaction occurs, leading to complications in isolation of products. Moreover it

requires large excess of nucleoside reagents and coupling yields are usually low.

Thus the approach is now only of historical and developmental interest.

O

OH

B1RO O

AcO

B2OPOH

O

O

O

O

AcO

B2OPO

RO B1

O O

O

O

OH

B2OPO

OH B1

O O

Coupling agent

Deprotection

-

--

B1 and B2 are A Bz, C Bz, C Ibu or T

Coupling agent is DCC or triisopropylbenzenesulfonyl chloride or mesitylsulfonyl chloride

+

Scheme 1.2 Phosphodiester Approach

The Phosphotriester Approach

The phosphotriester approach involves protection of the anionic site in the

phosphate backbone during oligonucleotide synthesis.32 In this approach, a

phosphotriester is generated by condensation of phosphotriester intermediate of one

12


nucleoside with another protected nucleoside or oligonucleotide. The 5'- O and 3'- O

protecting groups can be selectively removed to extend the synthesis in either

direction. After oligonucleotide chain has formed, all the P - O protecting groups are

removed in one step.

O B1RO

OH

PO

ClCl

OR'O B1RO

OP OR'OO

O B2OH

R''O

O

O B1RO

OP OR'OO B2

R''O

O

O B1OH

OP OOO B2

OH

i)

ii) Hydrolysis

Base

Methylsulfonylchloride / base

Deprotection

R' is 2-cyanoethyl, phenyl or o-chlorophenyl

B1 and B2 are A Bz, C Bz, C Ibu or TR is trityl or substituted trityl

-

Scheme 1.3 The Phosphotriester Approach

The phosphitetriester Approach

R. L. Letsinger has introduced the phosphotriester approach for the synthesis of

oligonucleotides in 1975.33 It involves use of highly reactive phosphate reagent for

coupling two deoxyribonucleosides. Suitably protected nucleoside is reacted with

bifunctioal phosphodichloridite to form nucleoside-3'-phosphomonochloridite which

13


is then reacted with protected nucleoside in which 5'-hydroxyl group is free to react.

Upon coupling the trivalent phosphorous is converted into more stable pentavalent

phosphorous via mild oxidation method. The procedure is summarized in Scheme

1.4. Later on this approach was used in solid phase synthesis of oligonucleotides

using deoxyribonucleoside-3'-phosphomonochloridite or monotetrazolides and

deoxyribonucleoside derivatised silica gel as the insoluble support. Although

monochloridite and monotetrazolide had the rapid and efficient coupling potential,

they were not ideal reagents for oligonucleotide synthesis. These reagents were

exceptionally sensitive to hydrolysis and air oxidation, difficult to prepare and

isolate, required special handling techniques, had limited life time in solution and

often contains appreciable amount of inert 3'-3'dinucleoside phosphine.34

O B1RO

OH

PClCl

OR'O B1RO

OP

R'O Cl

O B2OH

AcO

O

O B1RO

OP OR'O B2

R''O

O

O B1OH

OP OOO B2

OH

Base

Base

ii) Deprotection

R' is 2-cyanoethyl, phenyl or o-chlorophenyl

B1 and B2 are A Bz, C Bz, C Ibu or TR is trityl or substituted trityl

i) Oxidation, I2/H2O

-

Scheme 1.4 The Phosphitetriester Approach

14


The Phosphoramidite Approach

The problems faced by monochloridite (Figure 1.8, X = Cl) and monotetrazolide

(Figure 1.8, X = tetrazole) trivalent phosphorous reagents in phosphitetriester

approach were resolved by Beaucage and Caruthers by introducing a new class of

deoxyribonucleoside phosphites called as deoxyribonucleoside-3'-O(N,N-

dimehylamino) phosphoramidites35 (Figure 1.8, X = NMe2). These reagents had

improved stability and were resistant to hydrolysis by water or oxidation to air.

Further they were much easier to prepare and use.

O BDMTO

OP

MeO X

NN

NN

ON

X

= N Me2

= N Et2

= N iPr2

= Cl

=

=

Figure 1.8

Unlike their phosphomonochloridite and monotetrazolide counterparts

phosphoramidite reagents were less reactive and could not react directly with

another 5'-hydroxyl containing deoxyribonucleoside. They need to be activated by

treatment with weak acid such as tetrazole. Adams et al. observed that increase in

the steric hindrance around the nitrogen in phosphoramidites leads to increase in

their stability.36 McBride and Caruthers found that morpholino and diisopropyl

phosphoramidites are the reagents of choice as they are nonhygroscopic, stable at

room temperatures as dry powders as well as in anhydrous acetonitrile solution for

at least a week, and can be purified easily on a silica gel column.37 With the

15


commercial availability of these reagents synthesis of oligonucleotides in laboratory

has become accessible even to non-chemists particularly via solid phase method.

O B1DMTO

O

O B2DMTO

OP

RO N

O

O B2DMTO

OP ORO B1

O

O

O B2OH

OP OOO B1

OH

O B1OH

O

CPG

ii) Deprotection

R is 2-cyanoethyl or methyl


i) Oxidation, I2 / H2O

-

Trichloroaceticacid / CH2Cl2

CPG CPG

CPG is Controlled pore glass

1H-tetrazole / ACN

Scheme 1.5 The Phosphoramidite Approach

The H-phosphonate Approach

The H-phosphonate approach for oligoncleotide synthesis involves reaction of

suitably protected nucleoside-3'-H-phosphonate with 5'-hydroxyl group of protected

nucleoside. As the H-phosphonate is less reactive, it is first activated by treatment

with acyl chloride. The acyl chlorides used are pivaloyl chloride or 1-admantoyl

chloride. Use of these activating agent may lead to side reactions involving

modification of heterocyclic bases.38 A new activating reagent, dipentafluorophenyl

16


carbonate was introduced by Efimov et al. which gives high coupling efficienty with

considerable decrease in the side reactions.39 In H-phosphonate chemistry H-

phosphonate moity acts as protecting group for phosphorus through out the

synthesis. After completion of the synthesis all H-phosphonate moieties are

simultaneously oxidized by iodine solution to the corresponding phosphates.

O B1DMTO

O

O B2DMTO

OPOO

H

O

O B2DMTO

OPO B1

O

O H

O

O B2OH

OP OOO B1

OH

O B1OH

O

CPG

ii) Deprotection


i) Oxidation, I2 / H2O

-

Trichloroaceticacid / CH2Cl2

CPG CPG

CPG is Controlled pore glass Pivaloyl chloridebase

- TEA+

Scheme 1.6 The H-phosphonate Approach

17


1.I.4 Purification of Synthetic Oligonucleotide

All the protecting groups used in the oligonucleotide synthesis needs to be removed

in correct order. Temporary protecting groups are removed at each cycle during

chain elongation process, whereas permanent protecting groups are removed at the

end of synthesis. Various techniques are used for the purification of synthesized

oligonucleotide sequence such as polyacrylamide gel electrophoresis (PAGE) and

chromatographic techniques. The PAGE technique is useful for isolating the product

directly from the small scale reactions or at the end of chromatography. Various

chromatography techniques are also useful for purifying the oligonucleotides. High

performance liquid chromatography technique proves to be much useful for

oligonucleotide purifications. Rapid and efficient separations can be obtained with

the anion exchanger pellinox AL WAX,40 Permaphase AAX,41 Pellionex SAX,42

reverse phase column μ-Bondapak,43 Partsil ODS-244 and RP C-18 column.45

1.II Spin labeling Technique

1.II.1 Introduction

The concept of labelling a biological system with some external probe was

first introduced by Burr and Koshland. The technique involves the introduction of

the probe at specific site of the system to be studies. By monitoring the alternation

in the spectroscopic properties of the group, inferences can be drawn about the

molecular architecture in the vicinity of the probe, also commonly known as a

“reporter group” and the technique involves referred to as reporter group techniques.

Such external probes can be in the form of a fluorescent dye such as biotin,

radioactive labels such as 2H, 13C, 31P and 19F etc or a paramagnetic species called as

spin label and variety of spectroscopic methods have been used in the past to study

18


the dynamics, conformational mobility and other structural properties of

biomolecules.

Requirement of a Reporter Group:

The probe used in such methods should fulfill the following requirements:

1. It should be sensitive to changes in environment of the site of intrest and must

subsequently ‘report’ the changes to the external detector.

2. It should have well characterized spectroscopic or physical properties which are

either unique or distinct from the properties of the system under investigation.

3. The introduction of the probe should not alter the structure and function of the

system under study.

1.II.2 Spin Labeling Technique

McConnell46 first introduced spin labeling in 1965 and since then it has been

rapidly emerged as one of the most relible and extensively used tool for research in

biophysics. Owing to the relatively low content of stable paramagnetic species in

biological systems, a paramagnetic reporter group which is ESR-sensitive

constitutes a physically distinct moiety from the rest of system. A paramagnetic

reporter group is termed as a spin label. The technique which involves the

introduction of spin labels as a paramagnetic probe into a system followed by the

monitoring of the changes in its ESR spectra is called spin labeling. This method has

distinct advantages over other methods due to the fact that extremely low

concentration of spin labels can be used in ESR experiments. Paramagnetic species

which have been used for spin labeling studies are nitric oxides, lanthanide ions and

a few organic radicals for specific experiments. The great versatility, sensitivity,

immense stability and the variety of information available from nitroxide radicals

19


has made it synonymous with spin labeling. It is necessary at this point to make a

distinction between the terms spin labels and spin probes. The term spin label is

used to describe a molecule to which a nitroxide is covalently attached. On the

other hand a spin probe is a nitroxide containing molecules which is not attached to

molecules of the system under study. A spin probe can be a paramagnetic metal ion

almost always of transition or lanthanide series. The distinction is hard and fast, as

some times a spin label may be used as a spin probe to study more complex systems.

Nitroxides in spin labeling

Nitroxide or nitroxyl radicals are compounds containing the >N-O group.

This has one unpaired electron. Although the inorganic nitroxide Fremy’s salt47 A

had know since 1845, it was one in 1901 that the first organic nitroxide was isolated

and characterised by Piloty and Schwerin.48 This heterocyclic free radical,

porphyrexide B was formulated at that time as C a derivative of “quadrivalent

nitrogen”.

O NSO3K

SO3K

. NH

N NH

NH

O

NH

N NH

NH

O

A B C Figure 1.9

Electronic Structure of nitroxide

Nitroxides may be considered as derivatives of the stable inorganic radical

nitric oxide. The possible resonating forms of nitric oxide are shown below:

20


N O+ -

: :... ..

N O: :. .. +-

N O:.. .

.. N O. . .: ,

1 2 3 4

Figure 1.10 Resonance hybrid of nitroxide

Another mode of representation is by a structure with five bonding electrons where

there is a sigma bond and a three electron bond between nitrogen and oxygen. By

analogy, nitroxides can also be represented as a resonance hybrid of the forms a and

b by c with a three electron bond.

-N O

R

RN O

R

R

+

N OR

R.

. . .

a b c Figure 1.11 Resonating forms of nitroxides

The contributions of the above resonating forms differ in the ground state depending

on the conjugation and polarity of the medium. In the present thesis, for the purpose

of convenience in representation and familiarity with representations from literature,

nitroxides will be represented as 5a, regardless of their actual electronic structure.

Stability of Nitroxides

Johnson and coworkers49 have suggested that nitroxides are rendered stable

by the presence of tertiary carbon atom flanking the >N-O groups. Such nitroxides,

unlike most other free radicals, are extremely stable towards purification, handling

and storage. The instability of nitroxides owing to the presence of hydrogen on α-

carbon atom leads to a disproportionation reaction50 producing a nitrone A and

hydroxylamine B via the formation of a dimer.

21


H

O

H

O

H

O N

HO

ON

H

N O OH NH

+

A B Figure 1.12 Disproportionation reaction of nitroxides with hydrogen on α-carbon

atom.

The rate of disproportionation depends on the degree of substitution on the carbon

atom and the nature of the solvent. The structure of the dimer is uncertain.

Basic building block nitroxide

To date essentially all biological nitroxide spin-labeling studies have utilized

derivatives of three simple stable nitroxides. These are 2,2,6,6-

tetramethylpiperidine-N-oxyl (sometimes referred to as TEMPO), 2,2,5,5-

tetramethylpyrroline-N-oxyl (sometimes referred to as PROXYL) and 4,4-

dimethyloxazolidine-N-oxyl (sometimes referred to as DOXYL).

N

O

R

N

O

R

NO

R R

O

Tempol Proxyl Doxyl

Figure 1.13

22


Typically these structures bear functional group at the postion 4 and/ or 3

capable of undergoing chemical reaction with some functional group of the

biomolecules to be spin labeled. Some of the examples51 of nitroxides are

NX

O

XN O

NO

O

NO

I NO

CH2Br

NO

ONO

OO N

O

O

NO

NHCOCH2X

NO

COOH

NO

COOHNH2

X = OSO2CH3, I etc

NO

OPX

NN

X = O,S

N O

N

O

OP

O

CH2ClO

N

O

NHCOCH2Br

NO

O

O

NO

Br

NO

CHO

Figure 1.14

23


Characterization of Nitroxides:

Nitroxides are deeply coloured solids or liquids. However, bis-

[trifuloromethyl]-nitroxyl is a gas under normal condition.

1. Infrared Spectra: Generally all nitroxides shows characteristic IR band in the

range of 1310 to 1370 cm-1. This is attributed to the stretching of N-O..

However it has been proposed that the infrared stretching frequency of several

piperidine notroxides to be in the range of 1373 ± 7 cm-1.

2. UV-Visible Spectra: Nitroxides are orange-yellow in colour owing to an

absorption band in the region 410-450 nm (ε~ 20). A much more intense

absorption band is observed at about 230 (ε~ 3000). The visible band exhibits a

hypsochromic shift with highly polarity of the solvent and hence, is ascribed to

n→π* transition, whereas the absorption in the UV region is due to π→π*

transition. Other than the yellow and orange colours observed in some

nitroxides, α-nitrosonitroxides are blue in colour and has absorption bands at

238, 263, 360 nm. Resonance between the nitroxides group and the aromatiaac

ring, expectedly leads to bathochromic shifts up to the extent of 335 to 340 nm

and 490 to 510 nm.

3. Circular Dichroism: The circular dichroism spectra of several optically active

nitroxides are reported. The octant rule as used for ketones is also applicable in

the case.

4. NMR Spectra: The presence of unpaired electron in the nitroxide group

leads to the broadening of signals in the NMR spectra. Interaction of the

unpaired electron with nuclear spins (both intermolecular as well as

intramolecular), shortens the lifetime of spins in excited state and hence, leads to

line broadening. To overcome this difficultly NMR spectra are run at high

concentration viz. of the order of 3M, so that the electron spin relaxation time is

24


considerably shortened compared to the inverse of proton hyperfine coupling

constant. Alternatively though less conveniently, one may employ paramagnetic

solvent such as di-tert-butly nitroxide or 2-doxyl propane or corresponding

completely duterated analogues as spin-relaxer solvents for NMR studies of

nitroxide free radicals.

NO O N

O

Figure 1.15

5. Mass spectra: The mass spectra of several types of nitroxides have been

reported. The five membered pyrrolidine nitroxide substituted with –CH2OH,

OH or –NH2 exhibits (M+ 1) peak along with peaks corresponding to the loss of

methyl, isobutylene and nitric oxide. Six membered nitroxide shows a (M+1)

molecular ion peak along with a peak corresponding to the loss of methyl group.

Nitroxides of the doxyl type fragments producing a protonated ketone which

constitutes the base peak in the mass spectra.

25


N

O

R

N

O

R

NO O N+

O OO

HCH2O

H

NO

++

Figure 1.16 Mass spectral fragmentation pattern of nitroxide

6. Electron Spin Resonance Spectra: The electron spin resonance (ESR) spectra,

also known as electron paramagnetic resonance (EPR) spectra, of nitroxides

have been studied extensively. It constitutes the subject of numerous books and

review articles and continuous to be a subject of immense interest till today. A

detailed account of the ESR spectra features will be presented in the later

section. The ESR spectrum of nitroxide, which is employed to detect the present

of nitroxides radicals, consists of a three line spectrum in solution. (Figure 1.17)

Figure 1.17 Typical nitroxide ESR spectra

The three lines appears owing to the coupling of the unpaired electron spin in the

free radicals with the nuclear spin (J = 1) of 14N. the (2 J + 1) component which

26


results in a three line spectrum of nitroxide with 14N, collapses into a doublet on

replacement of 14N by 15N having J=1/2. Presence of atoms having non-zero J values

in the vicinity of the >N-O group results in finer splitting of the three line spectrum.

Some complex spectra cannot be unambiguously interpreted, though it provides

information regarding the structure of the radical. At this point it is worthwhile to

mention the salient features of the ESR spectra of biracials or polyradicals. As

expected, nonconjugatead biradicals do not give a nitroxide triplet. On the contrary,

a five line spectrum is obtained indicating the interaction of both unpaired electron

with both nitrogen atoms. This phenomenon is termed as ‘exchange interaction’ and

is observed in di- or polynitroxides wherein the >N-O group lies in close proximity

with each other. The structure of the intervening framework between the >N-O

groups in polynitroxdes or binitroxides determine the shape of the ESR spectra. This

phenomenon was explored to determine the stereochemistry of organic molecules.

This involved the reaction of cyclopropane-1,2-cis- and the corresponding trans-

dicarboxylic acid dichlorides. The resulting compound, a dinitroxide diester, in

each case exhibited different ESR spectra. The cis compounds had a five line ESR

spectra where as the trans isomer exhibit a triplet. This reflected the presence of the

>N-O group in close proximity in the former isomer, compared to the trans

compounds where the greater distance inhibits the desired exchange phenomenon.

Applications of Spin labeling technique

The spin labeling field blossoms even more as we head into the next

millenium. It has been applied to systems in food chemistry, nucleic acid and

nucleotide biochemistry, to cell application, in vivo applications with small animals

and most recently as a tool for specifically incorporating spin labels at unique amino

acid residues position in mutated proteins.

27


Spin Labelling In Oligonucleotides

Magnetic resonance spectroscopy is well adapted to the study of molecular

structure and dynamics. It’s application to biological molecules fetches information

about the chemical structural and biochemical aspects of the biomolecules. The

electron spin resonance calls for the presence of an unpaired electron on the

biomolecule to make it suitable for the above investigations. The method is

recognized as an extremely specific tool, since it is most unlikely to have unpaired

electron in the biological systems. The range of applications can be extended by

attaching synthetic free radicals (spin labels) in the precise chemical and structural

manner. The most important feature is the anisotropy of the spectrum with respect

to the magnetic field orientation, which can yield detailed structural information.

The line splitting in spectra, fetches information about the quantitative aspects of the

chemical bond. The detection of DNA sequence by using DNA biochip is a, rapidly

developing technology. In its present form, DNA sequences are attached to a chip,

which is then exposed to the solution containing DNA. If the complementary

sequence is present in the solution, it anneals to the chip, the chip is then treated

with a fluorescent probe which detects the double stranded DNA. The technique is

very sensitive mainly due to the method of detection. However, there are some

negative aspects of the method. For example, hybridization may be incomplete, or

may be difficult to optimize. The hybridization condition or the detection of

hybridization may be non selective. It is highly desirable to device a method which

shall provide an insight into the interaction of oligonucleotide on the chip. The spin

labeled DNAs can be used to differentiate the DNAs which are annealed to the solid

bound nucleotide versus the oligonucleotide which are in the solution. This is

because the spin labels are uniquely sensitive to their environment. Spin labelling

also helps in understanding the optimal set of condition (temperature, time and

buffer) under which the annealing process should be conducted.30 In the discussion

that follows we have emphasized on the reports which proved the applicability of

28


the DNA spin labelling as applied to the determination of structure, conformation

and detection of hybrid formation. An enormous growth is witnessed by the area of

development of novel Nitroxides, which can be easily used for the labeling of bin

molecules, and at equally high peace the novel method for labeling and detection are

also developed.

Gannett et. al. showed the application of spin probe labeled oligonucleotides

(Figure 1.18) for detecting the oligonucleotide binding and its extent by using EPR.

The authors further demonstrated the utility of same probes for optimizing the

conditions required for annealing of DNA. The method can also serve as a selective

method for detection of oligonucleotide hybridization under the conditions that

model DNA biochip.52

N

NH

N

O

O

O

O

O ODNA

N

NH

N

O

O

O

O O

O

DNA

..

Figure 1.18

In order to achieve an EPR sensitive probe for DNA, Giordanol et. al. linkded 3-

carboxy-Proxy1 free radical to O6of dG through a five-atoms-tether. The modified

base was incorporated into a 30-mer ODN, and then annealed to its complementary

DNA strand ODN in order to verify the possibility to monitor changes in DNA by

EPR spectroscopy.53 Hydrodinamic parameters show only a slight destabilization

with respect to the equivalent unlabeled hybrid. EPR could monitor the hybrid

29


formation showing a progressive enlargement of the upfield signal in passing from

the labeled ss- to the ds-30-mer. Several non-isotopic DNA probing techniques

have been developed by means of the insertion of a modified base carrying a group

selected to be easily detectable by spectroscopic methods. Since the probe bound to

O6 of dG protrudes into the central space of the major groove, it is expected to cause

only a minor perturbation of the duplex DNA structure. (Figure 1.19)

N

N

N

ODMTrO

O

N

N

PN O CN

NHR

O NH

O

Figure 1.19

Cekan et. al. reported a rigid spin-labeled nucleoside C*, an analog of deoxycytidine

that base-pairs with deoxyguanosine, was incorporated into DNA oligomers by

chemical synthesis.54 Thermal denaturation experiments and circular dichroism

(CD) measurements showed that C*¸ has a negligible effect on DNA duplex

stability and conformation. Nucleoside C*¸ was incorporated into several positions

within single-stranded DNA oligomers that can adopt two hairpin conformations of

similar energy, each of which contains a fourbase loop. The relative mobility of

nucleotides in the alternating C/G hairpin loops, 5’-d(GCGC) and 5’-d(CGCG), was

determined by electron paramagnetic resonance (EPR) spectroscopy. The most

mobile nucleotide in the loop is the second one from the 5’-end, followed by the

third, first and fourth nucleotides, consistent with previous NMR studies of DNA

hairpin loops of different sequences. The EPR hairpin data were also corroborated

30


by fluorescence spectroscopy using oligomers containing reduced Ç (Ç f), which

is fluorescent. Furthermore, EPR spectra of duplex DNAs that contained Ç at the

end of the helix showed features that indicated dipolar coupling between two spins.

These data are consistent with end-to-end duplex stacking in solution, which was

only observed when G was paired to C*, but not when Ç was paired with A, C or T.

(Figure 1.20)

N

N

N

O

N

O

O

H

N N

N

N

NH H

H

ON

N

NHO

N

O

O

ODMTrO

O

P

N

O

NC

2 C*G

Figure 1.20

In site-directed spin labelling (SDSL), Grant et. al. described a nitroxide moiety

containing a stable, unpaired electron is covalently attached to a specific site within

a macromolecule, and structural and dynamic information at the labeling site is

obtained via electron paramagnetic resonance (EPR) spectroscopy. Successful

SDSL requires efficient site-specific incorporation of nitroxides. Work reported here

presents a new method for facile nitroxide labeling at the 50 terminus of nucleic

acids of arbitrary sizes. T4-polynucleotide kinase was used to enzymatically

substitute a phosphorothioate group at the 50 terminus of a nucleic acid, and the

resulting phosphorothioate was then reacted with an iodomethyl derivative of a

nitroxide. The method was successfully demonstrated on both chemically

synthesized and naturally occurring nucleic acids. The attached nitroxides reported

31


duplex formation as well as tertiary folding of nucleic acids, indicating that they

serve as a valid probe in nucleic acid studies.55 (Figure 1.21)

N

OOP-O

O

S

H (or OH)O

B

O

Figure 1.21

Obeid et. al. described the synthesis of two modified 2’-deoxyuridine triphophates

bearing a spin label linked to the base by a rigid linker to ensure a tight coupling of

spin label dynamics. The incorporation of both spin-labeled nucleotides could be

shown in primer extensions reactions in presences of DNA polymerases from

eukaryotic, prokaryotic and archaic origin are competent in the employment of spin

labeled nucleotides as surrogate of natural building blocks in enzymatic DNA

synthesis. This finding opens new opportunities for further advanced applications

e.g. in vivo spin labelling or the generations of complex multi spin systems which

might be useful for DNA-based nanobiotechnology.56 (Figure A.1.22)

N

N

NH

OO

OH

O4P

OO

.

1

NO

N

NH

OO

OH

O4P

O

2

.

Figure A.1.22

32


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