2.I ABSTRACT 2.II INTRODUCTION -...

Chapter 2 : Synthesis of Nitroxide Spin Labels and Incorporation of a Nitroxide Spin- label in Oligonucleotide

2.I ABSTRACT

Hybrid nitroxide spin label and a spin label by 1-3 dipolar cycloaddition of

propargyl functionalized spin label with azide in Zidovudine molecule were

synthesized by 1,3-dipolar cycloaddition reactions. Amine functionalized spin label

was incorporated in the back bone of DNA.

2.II INTRODUCTION

The study of biomolecules like DNA and proteins has been very challenging

because of the complexicity and various specific interactions involved in these

molecules. One potential technique for probing such dynamic interactions in

biomolecules is electron spin-labeling wherein a stable nitroxide is attached to a

specific residue on the biopolymer under study. Site-directed spin labeling (SDSL)

has become an increasingly important probe of structure and dynamics in

biopolymers because of its sensitivity to motion, probe–probe interactions and local

environment. The recent development of the method of site directed spin-labeling of

proteins opens the potential to examine the local dynamical modes at or near each

labeled residue (plus the overall motions) thereby ultimately leading to a “map” of

the dynamic structure throughout the protein or other macromolecules.

Over the last fifty years , nitroxide free radicals have found applications as

diverse as redox reagents, SOD mimics (superoxide dismutase), MRI contrast

agents (Magnetic Resonance Imaging) and as reporter groups for probing the

biological systems, making them strong and versatile tool accessible to chemists and

biophysicist. Despite the vast developments in the field of spin labels, the synthesis

of spin labels possessing the desired blend of properties has always been a challenge

to synthetic chemist. In this regard the newer applications of nitroxide, in particular

their utility in the oxymetry and pH determination of biological systems, has

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intensified the efforts towards the design of novel and more suitable probes

possessing properties suitable for these applications.

Because of complexity and a variety of active sites in biomolecules it is very

difficult to directly develop nitroxide spin label on them. Usually spin labeling is

achieved by covalent anchoring of functionalized spin labeling reagents. The spin

labeled oligonucleotide has been employed for studies concerning DNA dynamics,

conformational modifications and in the detection of hybrid formation. Hence

usually attempts are made to develop spin labels in which there are electrophilic

group or groups which can easily be condensed to active sites of biomolecules.

2.III. RECENT REPORTS

Recently, Polienko et al.1 put forth a synthetic approach to access the new

nitroxides of the amidine type exhibiting pH-dependent EPR spectra through

substitution of a halide in the exo-N-halogenoalkyl chain of 1-(2-bromoethyl)-6-

oxyl- 5,5,7,7-tetramethyltetrahydroimidazo[1,5-b][1,2,4] oxadiazol-2-one. In this

approach, an oxycarbonyl moiety of the oxadiazolone heterocycle plays a role of

“protecting group” for the amidine functionality. Here a nucleophilic cleavage of the

oxadiazolone heterocycle under mild nonbasic conditions, applicable to substrates

bearing substituents vulnerable to attack by strong basic nucleophiles, have been

elaborated. The approach allows for the new amidine nitroxides bearing various

functional groups (e.g., such as CN, N3, NH2, COOEt) to be synthesized. They have

also described a series of nitroxides obtained through the Staudinger /intermolecular

aza-Wittig reaction of the azido derivative.

38


NO

NON

BrO

NO

NNH

R

NO

NN

NH

X

R= CN, NH2, N3, N(CH3)2

E+

R= N=PPh3

X=SE+ CS2=

Scheme 2.1

With the help of ESR and NMR techniques Coloumbous and Hubbel2

revealed differences in backbone motions by comparing the sequence-dependent

motions of nitroxides at structurally homologous sites. Spectral simulation

techniques and a simple line width measure were used to extract dynamical

parameters from the EPR spectra, and the results revealed that a mobility gradient

similar to that observed in NMR relaxation, further indicating that side chain

motions mirror backbone motions. With this study they predicted a model for

motion of side chain on α-Helics (Scheme 2.2).Following scheme depicts the

reaction of spin labeling reagents to generate nitroxide side chain.

39


N

S

O

SO

OProtein-SH

N

S

O

S c-c Protein

N

S

O

SO

OProtein-SH N

S

O

S c-c Protein

+

+

Scheme 2.2

Bobst et al.3 in their report demonstrated enzymatically induced sequence

specific incorporation of deoxyuridine analogous spin labels at position 5 of an

oligodeoxyribonucleotide to form a spin-labeled 26-mer. The nitroxide labeled

thymidine analogues used in the study are as depicted in Scheme 2. 3.

O

OH

RO N

NH

O

O

NH N O

O

O

OH

RO N

NH

O

O

NH N

O

O

Scheme 2.3

40


Nitroxide spin-labels have been incorporated into DNA, by conjugation to

either the nucleoside base4 or the sugar-phosphate backbone.5 However, the

currently available methods for the incorporation of spin-labels into RNA are

restricted to either an unpaired uridine6 or the 5’-end.7 Both of these strategies are

somewhat limited because the spin-label cannot be conjugated to internal, base-

paired nucleotides. A variety of molecules have been conjugated to the 2’-position

of base-paired nucleotides in RNA.8 Particularly attractive is the use of a 2’-amino-

modified RNA, which can be prepared by automated chemical synthesis using

commercially available phosphoramidites (Scheme 2.4). The 2’-amino group can be

reacted with electrophiles, such as aliphatic isocyanates9.

41


ODMTO

OP CF3

O

NNH

O

O

O

CN

NO

OP NH2

NNH

O

O

OO

O

NO

NCO

RNA Synthesis

Deprotection

O

OP

NNH

O

O

O

NH N

H

ON O

O

O

Scheme 2.4

42


Rychnovsky and colleagues10 have extended the Lai’s protocol for the

synthesis of nitroxides to the synthesis of several new chiral piperazine and

morpholine nitroxides. This strategy utilizes the Bargellini reaction as the key bond-

forming step. Several optically pure nitroxides incorporating α-aromatic and α-spiro

centers were prepared by this route. These chiral nitroxides (Figure 2.1) will be of

interest as enantioselective oxidants, as traps for prochiral radicals, and in the

preparation of new materials. One of these nitroxide compounds, compound (A),

was found to racemize under mild oxidizing conditions. They have further

investigated the mechanism for this unusual racemization reaction.

N

O

O

Ph

N

O

O

O

NO

Ph

O

NO

Ts

A

Figure 2.1

43


2.IV PRESENT WORK

A tempamine based nitroxide spin label was incorporated in back bone of

DNA. With the ground of earlier experience11 in cycloaddition reactions and

synthesis of hybrid molecules a steroid containing nitroxide spin label (7) and a spin

labeled zidovudine (8) were prepared by 1-3 dipolar cycloaddition reaction.

Attempts were made to incorporate these spin labels in DNA back bone.

2.V RESULTS AND DISCUSSION

It was first decided to incorporate a simple nitroxide spin label in back bone

of DNA. Tempamine (4-Amino-2,2,6,6-tetramethyl-piperdine-1-oxyl) was chosen

for this purpose.

Incorporation of Spin Label in DNA Back bone

H-phosphonate approach was applied for the synthesis of dimeric block of

DNA on CPG to incorporate tempamine (4-Amino-2,2,6,6-tetramethyl-piperdine-1-

oxyl) on the DNA backbone. The synthesis was performed in syringe at 1 μmol

scale. The lcaa CPG with the nucleoside loading of 44 μmol/g was used for the

synthesis and the success of the method was checked for four dimeric blocks. For

the oxidation of H-phosphonate, CCl4 was used. The dimeric blocks d(TPNA),

d(TPNT), d(TPNG), d(TPNC) were synthesized in syringe by modifying the protocol

used for the DNA synthesis by H-phosphonate approach (Table 2.1).

44


(DCA/DCM)

Detritylation

O

O

BDMTO

Pivaloyl Chloride(Py/ACN)

O

O

B'DMTO

P OH O

TEA+

O

O

BOP

O

O

B'DMTO

OH

NO

NH2

O

O

BOH

O

OH

BOP

O

O

B'DMTO

O

NH

NO

TEA/Py/CCl4NH4OH

1)

2)

- Contol pore glass

Scheme 2.5

45


Table 2.1 Standard cycle used for synthesis of dimeric block of spin labeled amidite

by using H-phosphonate chemistry

Steps

Involved Reagents and Conditions

Washing MeCN washing (3x1.5 mL)

Detritylation 3% DCA in EDC (10x0.5 mL)

Washing Pyridine:MeCN (1:4) (3x1.5 mL)

Coupling

10mg of H-phosphanate in 1 mL of Pyridine:MeCN(1:1) and 1mL Pivaloylchloride

in MeCN (72 mM) and agitate vigorously for 2 min

Chain Assembly

Washing

MeCN washing (4x1.5 mL) followed by dry MeCN

washing (4x1.5 mL) followed by washing with dry CCl4

(3x1.5 mL)

Amine-CCl4

oxidation

0.1 g Tempamine, 1 mL of CCl4, 0.75 mL Pyridine and agitate vigorously.

Washing

Dry CCl4 (3x2 mL) followed by pyridine: MeCN (1:4) (3x 1.5 mL) followed by MeCN ,

(4x1.5 mL) Detritylation 3% DCA in EDC (10x0.5 mL)

Washing

Pyridine: MeCN (1:4) (3x1.5 mL) followed by MeCN (4x1.5 mL) followed by dry MeCN

(4x1.5 mL) followed by diethyl ether,( 4x1.5 mL) and finally air dried.

Deprotection and

detachment

Concentrated aq. NH3 12 h at 55°C

46


The incorporation of spin label was evidenced by 31P NMR analysis of

aqueous solution of the oligonucleotide. The results are in support of the

incorporation of 4-Amino TEMPO spin label at DNA back bone. For example, in 31P NMR of d(TPNT) two peaks at 16.12ppm and 16.20 ppm are typical of P-N

bond.

Synthesis of New Nitroxide Spin-labels

The 1,3-dipolar cycloaddition is a classic reaction in organic chemistry

consisting of the reaction of a dipolarophile with a 1,3-dipolar compound that

allows the production of various five tempered heterocycles. This reaction

represents one of the most productive fields of modern synthetic organic chemistry.

Nitroalkanes and aldehydes through nitrile oxides can serve as useful reagents in

effecting carbon-carbon bond formation. Most of the literature data on the 1,3-

dipolar cycloaddition leading to isoxazolines refer to reactions between nitrile

oxides and alkynes. Keeping the stability of nitroalkanes in view and availability of

several efficient methods of transforming nitroalkanes into the respective nitrile

oxide13, it appeared of interest to make use of an isoxazole moiety in the 1,3-dipolar

cycloaddition reactions. We decided to take advantage of this reaction to develop

some new nitroxide spin labels.

Considering the high biological profile of steroid and use of alkynes in the

1,3-dipolar cycloaddition reactions, 16-dehydro pregnenolone acetate (16-DPA) was

chosen for the synthesis of nitrile oxide precursor and 4-Hydroxy TEMPO for the

synthesis of nitroxide alkynes as their propargyl ether derivatives.

The oxidation of 2,2,6,6-tetramethyl-piperdin-1-one (1) to its nitroxide (2) by

reported method14 was carried out. Tempol (4-Hydroxy-2,2,6,6-tetramethyl-

piperdine-1-oxyl) (3) was prepared by reduction of (2) with sodium borohydride.

47


Alcohol function of this compound (3) was propargylated with proaprgyl bromide to

give compound (4). The compound (6) was prepared by stirring 16-DPA

NH

O

N

O

O NaBH4

H2O2, NaWO4

MeOH

O

NO2

AcO

O

AcO

DBUCH3NO2

DCM

N

OH

O .

Br

NaH

THF

N O

N

O

OO

AcO

.

MeOH

PhNCOEt3NBenzene

NO

O

+

1 2

3

4

5

6

7

r.t.

.

Scheme 2.6

48


with nitromethane in dichloromethane. This reaction was carried out at room

temperature with slow addition of DBU to generate compound (6). Compound (7)

was prepared by adding compound (6) propargyl functionalized nitroxide (4) to dry

benzene and stirring the reaction mixture over night with triethyl amine and phenyl

isocyanate.

Recently 1,3- dipolar cycloaddition have generously been used in the

synthesis of a wide variety of conjugates particularly in the field of carbohydrate

chemistry.15 This has been attributed to ease of reaction and relative stability of

linker group particularly triazole as a linker employing “click chemistry”.15 The

nitroxide 8 was prepared by 1,3-cycloaddition reaction of azide function in

Zidovudine and compound 4 as indicated in Scheme 2.7.

Cu(I)

O NNH

OH

NN+

N

O

O

BuOH

Sodium Ascorbate

ON

NH

OH

O

ONN N

NO

O

.

NO

O

8

+

Zidovudine

4.

Scheme 2.7

49


However, unfortunately our attempts towards incorporation of these newly

synthesized nitroxide spin labels were hampered due to solubility problems of these

nitroxide spin labels.

2.VI EXPERIMENTAL

2.VI.1 Materials and Methods

A.R. grade 2,2,6,6-tetramethyl-piperdine-4-one was procured form Aldrich. All

other reagents were L.R. grade and procured from M/s S. D. Fine Chemicals Ltd.,

India. A.R. grade 4-amino-2,2,6,6-tetramethyl-piperdine-1-oxyl was purchased from

Aldrich. m- Chloroperbenzoic acid and other chemicals were all A.R. grade and

were procured from Spectrochem, India. The solvents used for DNA synthesis were

all A.R. grade and were dried before use. Acetonitrile (HPLC grade) and CCl4

(HPLC grade) were procured from Spectrochem, India and were dried over CaH2

and stored over 3Å molecular sieves overnight before use. Pyridine (A.R. grade)

was procured from Merck, India, dried over anhydrous KOH and stored over 3Å

molecular sieves overnight before use in the synthesis. HPLC grade ethylene

dichloride and dimethylamino pyridine and pivaloyl chloride (AR grade) were

procured from Spectrochem, India and used as received. The H-phosphonate

reagents were procured from GLEN Research, USA. The DMT-dT loaded lcaa CPG

500 was received as a gift of sample from ISIS Pharmaceuticals, USA. Protected

nucleosides and other DNA reagents were received as a gift of sample from

Innovasynth (I) Ltd., Khopoli.

50


2.V. 2 Instrumental Details and their Operational Conditions

NMR analysis

NMR analysis was performed using Bruker-Avance-II 300 MHz or Varian NMR

spectrophotometer. For NMR, CDCl3 was used as the solvent; the chemical shifts

are reported in ppm. Multiplicities are indicated by s (singlet), d (doublet), t (triplet),

q (quartet) and bs (broad singlet). The coupling constants (J) are reported in Hz. 1.5

equivalent of phenyl hydrazine (PhNHNH2) was used as quenching reagent in all

NMR spectral analysis.

GC-MS Analysis

GC-MS analysis was performed using Shimadzu, QP-5050 instrument equipped

with HP-5 column. The detector temperature was set at 300 °C. The column was

programmed initially at 60 °C for 5 min. and then with a gradient of 10 °C/min to

250 °C.

ESR Analysis

ESR spectra were recorded at room temperature on Varian E-112 spectrometer

operating in the X-band with tetracyanoethylene as internal standard (g = 2.00277).

Chloroform was used as solvent for ESR measurement and was deoxygenated by

bubbling nitrogen gas. The concentration of the nitroxide used for the experiments

were of the order of 10-4 M.

2.V. 3 General Experimental Procedure and Characterization data

Compound 3

To a well stirred solution of 2,2,6,6-tetramethyl-piperdine-4-one [1] (13 mmol, 2 g)

and sodium tungstate (150 mg) in methanol (20 ml) and acetonitrile (3 ml) was

51


added hydrogen peroxide (30 % v/v) (20 mL) drop wise from dropping funnel while

maintaining temperature at 0°C for 2 h. The reaction mixture was stirred for 5 h to

obtain red coloured solution of 2,2,6,6- tetramethyl-piperdine-4-one-1-oxyl [2]. The

solvent was evaporated under vacuo and the product obtained was extracted in

diethyl ether (30 ml). The organic layer was washed with water (2x 20 mL) to

remove soluble impurities and pure product was isolated by evaporating solvent,

with 95% yield. Thus product obtained was directly used for the next reaction. by

dissolving [2] in dry methanol with addition of 1.2 equiv. of sodium borohydride

(NaBH4) while maintaining the temperature of reaction mixture at 5-10°C. Then

reaction mixture was allowed to stirr for 2 h and methanol was evaporated under

vacuum to get product [3] in 90 % yield.

IR neat (cm-1): 3400, 2990, 1373. 1H NMR (300 MHz, CDCl3): δ 1.04 (s, 6H), 1.26 (s, 6H), 1.18-1.39 (m, 4H), 3.42

(bs, 1H), 3.9 (t, 1H). 13C NMR (75 MHz, CDCl3): δ 20.3, 32.3, 47.92, 59.3, 63.17.

MS (EI) m/z: 172 (M+), 158, 142.

ESR 10-4 M solution in chloroform gives symmetrical triplet.

Compound 4

To a well stirred solution of TEMPOL [3] (10 mmol) in dry THF (10 mL) was

added NaH (15 mmol) over a period of 15 min. The reaction mixture was allowed to

stirr for 30 minute while maintaining temperature of reaction mixture at 0°C. 3-

bromo-propyne (12 mmol) in dry THF (5 mL) was dripped into the well-stirred

reaction mixture over a period of 1 h. The solution was allowed to react overnight.

After completion of reaction THF was evaporated and the reaction mixture was

partitioned between chloroform and water to remove water soluble impurities.

52


Combined organic layer was filtered over sodium sulfated and evaporated over

rotary evaporator to get product (yield 91 %).

IR neat (cm-1): 2985, 2100, 1375. 1H NMR (300 MHz, CDCl3): δ 1.05 (s, 6H), 1.26 (s, 6H), 1.4-1.5 (m, 4H), 2.92 (s,

1H), 3.8 (t, 1H), 4.1 (s, 2H).

13C NMR (75 MHz, CDCl3): δ 20.59, 31.83, 44.17, 55.24, 59.54, 69.64, 74.11 ppm

MS (EI) m/z: 210 (M+), 154, 124, 109, 82.

ESR 10-4 M solution in chloroform gives symmetrical triplet.

Compound 6

To a stirred solution of 16-DPA (1.78 g, 5 mmol) and freshly distilled out

nitromethane (2.5 mL, 50 mmol) in dry dichloromethane (100 mL), DBU (3.7 mL,

25 mmol) was added at -15oC. The reaction mixture is then stirred at room

temperature. After 12hrs 2N HCl (100 mL) was added, the layers were partitioned,

and the acidic layer was extracted with dichloromethane (3 x 50 mL). The combined

extracts were dried over anhydrous Na2SO4 and solvent was evaporated under

reduced pressure. The product obtained was purified by recrystallization using

petroleum ether and dichloromethane affording pure nitro compound 6( yield 90%).

IR (CHCl3): 2938, 1729, 1701, 1552, 1438, 1381, 1363 cm-1. 1H NMR (300MHz, CDCl3) (Selected Signals): δ 0.68 (s, 3H), 1.02 (s, 3H), 2.04 (s,

3H), 2.16 (s, 3H), 2.48 (d, 1H, J = 9Hz), 3.37 (m, 1H), 4.28 (d, 2H, J = 6Hz), 4.58

(m, 1H), 5.37 (bs, 1H) ppm. 13C NMR (75MHz, CDCl3): δ 13.79, 19.26, 20.79, 21.44, 27.64, 28.95, 31.48,

35.34, 36.52, 36.88, 37.97, 38.67, 44.96, 49.60, 55.32, 67.36, 73.68, 79.59, 121.93,

139.66, 170.57, 206.89 ppm.

53


Compound 7

To dry benzene (25mL) containing PhNCO (1mmol) and 4 (1 mmol) was added to a

solution of nitro compound 2 (1 mmol) and Et3N (10 drops) in dry benzene (15mL).

After stirring the reaction mixture for overnight, solution was filtered. The brownish

yellow benzene solution was evaporated under reduced pressure. The residue was

dissolved in diethyl ether (25mL) and filtered and the filtrate washed with dil. HCl

and then with water and dried over anhydrous Na2SO4. The products can be purified

by column chromatography over silica gel (60-120 mesh) using a mixture of

petroleum ether and ethyl acetate to get pure products (yield 86 %).

1H NMR (300MHz, CDCl3) (Selected Signals): δ 0.71 (s, 3H), 1.05 (s, 3H), 1.29 (s,

12H), 1.57-1.67 (m, 5H), 1.88 (d, 2H), 2.07 (s, 3H), 2.20 (s, 3H), 2.37 (s, 3H), 2.50

(d, 1H), 4.30 (d, 2H), 4.67 (m, 1H), 5.41 (bs, 1H). 13C NMR (75MHz, CDCl3): δ 13.82, 19.30, 20.84, 21.47, 27.69, 29.01, 31.52,

31.66, 35.41, 36.56, 36.94, 38.02, 38.73, 44.99, 49.66, 53.88, 55.37, 67.42, 146.37,

156.9, 207.2, 210.8 ppm.

MS (EI) m/z: 619 (M+), 299, 189.

ESR : 10-4 M solution in chloroform gives symmetrical triplet.

Compound 8

Zidovudine (0.33 mmol) and compound 4 (0.33 mmol) were dissolved in a t-

BuOH/H2O mixture (2mL, 1:1). Copper acetate (0.2 equiv.) and sodium ascorbate

(0.4 equiv.) were added and the mixture was stirred at room temperature until TLC

indicated the disappearance of the starting materials. The mixture was poured into

H2O/satd. NH4Cl solution and the product was extracted with EtOAc. The organic

layer was dried with Na2SO4 and filtered, and the solvent was removed under

54


reduced pressure. The residue was purified by column chromatography to yield pure

product (yield 91 %).

1H NMR (300MHz, DMSO-d6) (Selected Signals): δ 1.09 (s, 12H), 1.76 (s, 4H),

2.15 (s, 3H), 2.15-2.29 (m, 2H), 2.34-2.36 (m, 1H), 3.58-3.61 (m, 1H), 3.77-3.80

(m, 1H), 4.38 (d, 2H), 4.98 (s, 2H), 5.23 (t, 1H), 7.66 (s, 1H), 11.32 (bs, 1H).

13C NMR (75MHz, DMSO-d6): δ 12.67, 31.91, 36.62, 53.92, 55.42, 60.60, 61.24,

83.81, 84.41, 109.95, 136.50, 149.04, 150.84, 164.15 ppm.

MS (EI) m/z: 478 (M+).


d(TPNT) 31P NMR (D2O, PhNHNH2): δ 16.12ppm and 16.20ppm


2.VI CONCLUSION

• We have incorporated the TEMPAMINE nitroxide in back bone of

DNA by oxidation of H-phosphonate using CCl4.

• We have synthesized a new TEMPO based hybrid nitroxide spin label

following a sequence of reactions and finally 1-3 dipolar cycloaddition

reaction.

• We have synthesized new TEMPO based nitroxide spin label of

Zidovudine by copper catalyzed 1-3 dipolar cycloaddition reaction.

55


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Figure 2.1 1H NMR spectrum of compound 3

58


Figure 2.2 13C NMR spectrum of Compound 3

Figure 2.3 ESR of compound 3

59


Figure 2.4 1H NMR spectrum of compound 4

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Figure 2.5 13C NMR spectrum of compound 4

Figure 2.6 ESR spectrum of compound 4

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Figure 2.7 Mass spectrum of compound 4

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Figure 2.8 1H NMR of compound 6

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Figure 2.9 13C NMR of compound 6

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Figure 2.10 1H NMR of compound 7

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Figure 2.11 13C NMR spectrum of compound 7.


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Figure2.14 1H NMR spectrumof compound 8

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Figure 2.15 13C NMR spectrum of compound 8


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Figure 2.18 31P NMR of spin labeled d(TPNT)

Figure 2.19 ESR of spin labeled d(TPNT)

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2.I ABSTRACT 2.II INTRODUCTION -...

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