INTRODUCTION
DNA (deoxyribonucleic acid) consists of double helix structure formed by spiral binding of two strands of nucleotides. Nucleotides contain deoxyribose sugar composed of a phosphate and a sugar molecule making it to form deoxyribonucleic acid. Out of four different nitrogenous bases which are ,adenine (A) ,thymine (T), guanine (G) and cytosine (C),one of them is attached to neucleotides forming a complete structure of DNA.The complete understanding of DNA structure,its binding with molecule and morphology of strands is shown in Fig 1.
Fig 1: Structure of DNA
The initial discovery in the field of DNA chemistry was done by Watson and Crick by illustrating the purine-pyrimidine pairs in 1953.The further research in mid 1990s brought in focus the paradigmatic rule of purine-pyrimidine for non- Adeinine-Thymine and Gunaine -Cytosine pairs which are the design pairs of Benner and Rappaport with the nautral pairs (fig 1). The basic knowledge about the functioning of genetic material in natural DNA and the understanding of the chemical structure of DNA is important for the research and studies in medicines.DNA consist of backbone called phosphodiesters which stores complete genetic information and of the different bases to encode the biological instructions.The base pairs A-T and G-C provides the flow of genetic information from DNA to RNA and then to protein. Leonard brought the concept of size expansion in DNA by synthesizing adenine ribonucleoside analogue (2) by benzofusion.This analogue was then used as substrate to study cAMP but it was not possible to synthesize oligonuctleotide durinf Leonard decade.However Matsuda synthesiezed base pairs of DNA having extra ring to change the number of hydrogen bond to four in each pair without causing any change to original structure of DNth
Benzo-fused Moleculer Designs
Benzo fused moleculer designs was used to expand the base pairs of DNA. The design of xDNA analogues is similar to that of natural DNA but different in sequencing and positioning of bases.In natural DNA the four different bases are on either side causing the purines to pair with pyrimidine bases where as in benzo fusion the benzo pyrimidine paired with purine consist four types of ring system with eight base components of xDNA as shown in the Figure 2.The selevtivity of the bases in xDNA is not complementary to hydrogen bonding only but it also depend on the DNA size. In this case the adenine paired with thymine in xDNA is a mismatch because it is not possible for such a small analogue to form double helix and because of this reason the xDNA is not expected to have its interection with the natural DNA.However the xDNA formed by benzene ring extension are more stable than the natural DNA and also shows fluorescent properties in aqueous buffers.
The research being carried out for the size-expanded DNA is with a goal to achieve the non-Natural DNA which can function physiological, biochemical and genetically similar to natural genetic system. The ability of non-natural DNA to initiate its own synthesis and then catalyzed by polymerase enzyme. The research on all these aspects and its success is not always guaranted with xDNA and yDNA as the natural DNA functions are more specialized.DNA polymerase enzyme function accurately and with high specifity for nucleotides. The specifity of the enzyme depends largely on the type of base pair attached to it with steric properties. The nucleotide with a little difference of sterric and kinetic properties is rejected and this is the danger in selection of xDNA in place on natural DNA.
Figure 2 .Four base-pairing schemes of xDNA which consist of eight bases in comparison to the natural DNA,which consist of four bases with two pairs
The xDNA bases which are synthesized, aimed to be used further for alteration of gene expression by incorporating them in to oligonucleotides to form a triplex helical structure called Triplex forming oligonucleotides (TFO) in order improve the specifity, efficiency and gene targeting for the genetic disease management.
Oligonucleotides and Triplex Forming Oligonucleotides.
The stretched bases in xDNA are synthesized in order to incorporate in oligonucleotides to form a triplex helical structure. Oligonucleotides bind to purine-pyrimidine sites to form triplex forming oligonucleotides (TFO). Triplex forming oligonucleotides are formed in order to increase the gene specifity and gene therapy as biological agents. It was discovered in 1957 Felsen feld at al, synthesized this triplex complex. However the binding of the third helix was found weak in comparison to the double helix, but it showed gene specific sequence and by using divalent cations they were stabilized as well.IN 1968, a research paper was published by Morgan and Wells ,showing the ability of triplex RNA strand of inhibiting the transcription. This research was a breakthrough in the field of gene specific targeting through triplex helical structure.TFO structure consists of double stranded DNA with a single strand of messenger RNA. The binding of third strand in oligonucleotide is achieved by natural and expanded DNA oligonucleotides.TFO in DNA binds to spefic sites having purine strand on the one and the pyrimidine on the second strand.The size of the targets of TFO is 10-30 nt in length.TFO binds by following a code which is specific to purine-rich strand targets forming either polypurine and polypyrimidine analougue.Size-expanded DNA bases incoroporated in oligonucleotide to form TFO have been research in approach of alteration of gene expression.The ability of TFO to change a gene expression depends on gene specific code,mutagenesis and reverse transcription.The specific gene targeting and binding with other DNA analogues has made them potent gene alteration oligonucleotides.They can cause heritable genetic changes by the regulation of RNA transcription permenantly.
In Fig 4,the change or alteration in gene expression by using TFO where in FigA shows how the transcriptional process can be stopped by following two different strategies.These strategies followes the similar path by blocking the normal cellular path when the TFO binds to the target site.Whereas in FigB the TFO ability to form a tethered mutagen as well as mutagenesis when the a reactive molecule of DNA is not conjugated at the binding site. Fig C shows the TFO to be used as homologous recombination technology between the target genome site which is used after homologous recombination having a donor fragment induced by TFO.
Fig 5: Steps of Gene expression Alteration (A) Blocking the transcription after competing the transcriptional factors or their interception. (B) Mutagenesis caused by direct site-specific TFO (C) Homologous Recombination.
The advancement in size-expanded DNA with synthesis of new TFO,there are still major issues regarding the intracellular delivery of TFO and the proper treatment of abnormal gene expression casuing genetic diseases.In order to achieve the goal of to use TFO as spicific gene binding molecules and to have modification in the genome structure ,their delivery ,genome availiblity and effieciency must be analysed and confirmed.
Aim
The aim of the research is to synthesize pyridine-stretched adenine nucleoside phosphoramidite in sugar protected form for its incorporation into oligomers for the formation of size-expanded DNA by oligomerization.
Objective
To synthesize Starting material and intermediate to syntheisize pyridine-stretched adenine (strA).
To synthesize strA by cyclisation with ionexchange resin and Acetonitrile.
To convert the strA in to protected strA with diethoxyethylacetate.
To synthesize the deprotected strA from protected strA with methanol,deionized water and hydrogen oxide pallets.
To convert the deprotected strA in to an amidine-protected phosphoramidite and to incorporate that in to oligonucleotide to synthesize synthetic DNA oligonuctleotide by solid phase synthesis.
Instrumental Techniques and Methods.
The techniques,instruments and methods which are used throughout the research project have been followed by previous work in these field.The identification of the synthesized product and the characterization of the purity of the product depends on the analysis done by these methods.At every stage of synthesis, Thin layer chormoatograpy (TLC) analysis has been done, as it's the most simplesest,cheapest and a fast way for the analysis of the product for further steps.TLC consist of a silica gel bed on aluminium plate.The surface is smooth
1 - TLC
At every stage of synthesis, Thin layer chormoatograpy (TLC) analysis has been done, as it's the most simplesest,cheapest and a fast way for the analysis of the product for further steps.TLC consist of a silica gel bed which is adsorbed on aluminium plate.The surface is smooth and it provides quick result by spotting the reaction mixture on plate.TLC is used to compare and identify the product from the reaction mixture.The TLC analysis from the analysis of starting material to the end of identifying the product is done.The presence of other material with product is visible by the fluorescent spot.Different solvents according to the polarity of the reaction mixture are used in TLC analysis.
Fig 7 : A model of a TLC tank ,TLC plate and solvent according to time zero and after ten minutes.
2 - NMR
NMR (Nuclear Magnetic Resonance) in the field of chemistry is one of major spectroscopic technique being used on large scale .The NMR is used for the determination of organic and inorganic stereochemistry,purity,structure and formation of molecules when sample is placed in a magnetic field.There are two possible spectrum from NRM,the 1H proton and 13C carbon spectrum.The most commonly used is the H proton NMR because of the presence of H protons in all types of molecules.The active nuclei (I > 0) is used for all the substance present in periodic table but there is a distinction in active nuclei of 1/2 of proton and the molecule having high spin.NMR has also created new ways of spectroscopic methods for determining the protein and nucleic acid with three dimentional structures which is not present in other techniques like X-ray diffraction.In drug discovery NMR is the most fundamental technique tobe used as it can determine the binding of putative ligands in proteins and the target site at which binding is taking place.This can help by using small molecules and binding them to adjacent sites in order to increase the stability and effinity which leads discover new compounds by using different small molecules on different sites.
In this research project the NMR scpectroscpy hase been used in every step to determine the structure and purity of the synthesized products.The 1H proton spectra are taken on every stage where as 13C spectrum have been taken where necessary.
3 - IR
Infrared spectroscopy ( IR) is a technique which identifies different peaks at IR frequencies when the sample is place in front the IR beam and after absorption ,the measurement of peaks at different frequencies is done.IR spectroscopy determines the functional group present with in the sample as each functional gropu absorbs to diferrent IR frequencies.IR is used to elucidate structure of different compounds , solid,liquid and gases and their functional group.The electromagnet range of IR is from wavenumbers 13,000 to 10 cm-1 and possibly the wavelength range consist of 0.78 to 1000µm.
The solid products synthesized during the research were characterized by making a KB (Potassium Bromide) disc.The solid is first mixed with KBr and then it forms a disc under pressure and then it is placed under IR spectra beam.The spectra obtained by IR is by detecting transmittance change according to the intensity of IR frequency
4 - Mass Spectroscopy.
Mass Spectroscopy is a technique used to determine unknown moleculer structures,components in mixture and probing the chemistry of compounds.It fundamental application is to determine the weight of the molecule in compound.This technique works by detection the ions generating from compound when it is bombarded with electrons.The generated ions are follow a circuler pathby the deflection from the magnetic field.The circuler path runs under a radius depending on the mass charge ratio (m/z).Deflection of the ions depends on (m/z),the ions having (m/z) value low are deflected more compared to the ions having (m/z) high value.The vm/z value of ions is either determined by single focusing through magnetic field scan or by double focusing which is done by scanning both magnetic and electric field.The scientific method is define by following figure for mass spectrometry.
Fig 9: Schematic diagram of mass spectroscopy
5- Column Chromatography
Column Chormatography is a separation technique which works on mobile and the stationary phase.The stationary phase consist of a solid adsorbant (silica gel) which is filled in a glass tube vertically to form a columns and the mobile phase which is the solvent is dropped inside the vertical tube from the top and it flows down to the bottom to form a complete column.The reaction mixture which is to be separated or either to be collected in pure form is added from the top and then the solvent eluent is added with a use of air vaccum in order to increase the flow of the column which is therefor called as flash column chormatography.Maxiumum number of fractions are taken of the seprated product.The change in the colour shows the progess of the reaction.The size of the column depends on the quantity of the reaction mixture to be used.Specific range is used depending upon the amount of reaction mixture.This technique is widely used as it is most convient and easy way to separate the product on small scale extraction.
6-Dry Vacuum Chromatography
This technique was first used by L.M Harwood to be used as an alternative for flash chromatography for large scale separation in laboratory.It consist of parallel-sided vacuum filter funnel.silica gel of TLC grade is added in to the funnel according to need of sepration and is tapped well.It s tapped until it the silica gel is well settled from top to bottom.Then water is added and a suction pump is used to to press the silica and make it compact.It is pressed hard until the whole bed is compact for the mixture to be separated.After forming a firm bed,the column is pre-eluted with the solvent covering the whole silica column and then it is sucked to make the column dry.Then the reaction mixture which is to be separated is added in to solvent used for pre-eluting the columns.The fractions are collected by adding the solvent as described in table according to required.Each time adding the solvent the column is sucked to dry and then the next amount of solvent is used.
Funnel
Diameter
Funnel
Length
Silica
Weight
Sample
Weight
Fraction
Size
30mm
45mm
15g
15-500mg
10-15ml
40mm
50mm
30g
0.5-2g
15-30ml
70mm
55mm
100g
1-5 g
20-50ml
Synthesis of 2-deoxychlorosugar
HCl gass was used at 0 0C to saturate acetic acid ( 200 mL) for 2 hours. 1'-O-methyl 2-deoxy-3',5'- di-O-p-toluoyl-α/ï¢-D-ribofuranoside ( 28.8 g) solution in acetic acid (45 mL) was added to the HCL saturated solution with continuous stirring over a period of 10 minutes.The mixture was then filtered to get solid and was washed with diethyl ether to recover all the remaining solid.The collected solid was then placed under high vaccum overnight to get dry solid compound. The product, 2-deoxy-3',5'- di-O-p-toluoyl-α-D-ribofuranosyl chloride ( 18 g) was obtained.The NMR characterization showed that the synthesis is complete and the product is pure.
TLC Analysis = TLC (EtOAc) Rf = 0.60
1H NMR (CDCl3): ï¤ 2.39 (6H, s, 2 x 4-tol-CH3), 2.72 (1H, m, 2′-CH), 2.83 (1H, m, 2′-CH), 4.58 (1H, m, 5′-CH2), 4.61 (m, 1H, 5′-CH), 4.80 (1H, m, 4′-CH), 5.52 (1H, m, 3′-CH), 6.51 (1H, d, J 4.8 Hz, 1′-CH), 7.24 (4H, q, 3,5-tol-CH), 7.93 (4H, q, 2,6-tol-CH) .
Synthesis of 4-Nitroimidazole Caesium Salt
4-Nitroimidazole (15.00 g, 132.6 mmol) added in methanol (150 mL) with stirring at a room temperature and caesium hydroxide (50 % w/v) (23.4 mL, 133.1 mmol) was added in to the solution.It was allowed to stir for one hour and pale yellow oil was formed after removing the solvent.iso-propanol was used to recrystallize the residue and dried under vaccum at 90 oC to give a bright yellow the crystals ( 32.20 g , 99% yield) ; m.p : 157-160 oC.
IR (KBr) : = 867,984,1257,1303,1379,1626 cm-1
1H NMR (d6- DMSO): ∞ 7.07 (1H, br s, imidazole(5)-H), 7.69 (1H, d, J = 1.0 Hz)
13C NMR (d6-DMSO): ∞ 132.26 (C4), 146.42 (C5), 148.29 (C2).
MS (ES/LR) (C3H2N3O2) ; m/z = 122 (M , 100 %),82 (99%) . 66(12 %).
MS (ES/LR) (Cs+) : m/z = 133 (M+ , 100 %).
Synthesis of 1-5-nitroimidazole
4-Nitroimidazole caesium salt (12.4 g , 20.3 mmol) was added to dry THF (700 mL) and stirred at 67oC under argon. Chlorosugar (18 g , 13.6) was added in three separate solution of cholorosugar (6 g) in 200 mL of dry THF for 2 h. The temperature was increased to (70 oC ) after addition of cholorosugar and THF was distilled to recycle it. Rotary evaporation was donr to rtered emove the remaining THF and chloroform (100 mL) was added to the residue to dissolve the solids.The mixture was filtered by celite filtration and washed with 20 mL for 2 times .Choloroform was removed by rotary evaporator to give the crystals of 4β and 5β isomers confirmed by TLC analysis and NMR. Separation of the product was done by dry vacuum chromatography. For filtration (100 mL x 2) was used to pack the silica and to make the column wet. The crude product was dissolved in 100 mL of chloroform. Chloroform (100 mL) again used to was the remaining material.The product was separated by using 11 concentrations of solvent eluent. Each eluent used was 100 mL with 5%,10%,15% and 20% ether in chloroform. After collecting 11 fractions of the product,TLC analysis was done and the fractions 4,5 and 6 showed fluorescent spot of β-isomers and a negligible amount of α-isomers. The fraction 7, 8 and 9 also showed fluorescent spot with some isomers of β and α. Fraction 4, 5 and 6 was combined together and evaporated by rotary evaporator to get dry solid crystal . It was characterized by NMR after which showed the compound is pure and contains α and β isomers. It was vaccumed to get dry crystals (11.78 g) and was characterized by NMR showing a 50% yield. The fraction 7, 8 and 9 was also combined and evaporated.NMR was done which showed some impurities along with the product.
Treatment with Ether and Filtration
Ether (50 mL) was added to solid (11.78 g) and was sonified to separate the 5-β-crystals from the remaining isomers. Mixture was the filtered by scinter funnel filtration. The product was washed (10 mL x 3) to get the total amount of product. The filtered solid was collected from the top of funnel and was vacuumed. The weight of the dry crystals of 5-β-isomer separated by dry vacuum chromatography obtained was (8.2 g) .TLC analysis showed single fluorescent spot of the product. NMR showed that the product is completely separated from other isomers and is 97% pure, ready to be used in the next stage of reaction.
The fractions 7,8 and 9 was then combined with 50 mL of ether to dissolve the remaining impurities and was brought to sonicator. The mixture was then filtered with scinter funnel filtration. The mivture was washed (10 mL x 3) to obtain the remaining material.The filterd product was collected from the top of the funnel and NMR was done to check the product purity.NMR showed that 5-β isomers are present with α-isomers.
Synthesis of 5-Amino-4-imidazole
The synthesized 1-5-nitoimidazole (8.2 g, 10.8 mmol), was added with 5% palladium on carbon catalyst (8.3 g) and dry THF (100 mL).Hydrogen gas was used to remove air from the mixture flask and hydrogenated temperature was maintained with a hydrogen gass filled ballon.The reaction mixture was brought to automated shaker and was allowed to shake for 3 h under atmospheric pressure.After 3 h ,TLC analysis was done which showed fluorescent spot for the product and no starting material..Filtration of the product was done through dry celite under argon pressure. Round bottom flask containing EMMN collected the solvent.THF was used for filtration with (3 x 30mL and then (3 x 25mL).After filtration it was stirred overnight at 50 oC. It was mixed with ethyl acetate and allowed to chill in freezer for 1 h .The crystals were separated by scinter funnel filtration under vacuum and was left to dry under vacuum to get solid crystals and the weight of the product was (4.40 g).NMR and IR showed that the product is 98% pure and is ready to be used in the next stage of synthesis.
Yield = 53.65%
TLC Analysis : TLC (EtOAc-MeOH-NH3 10:1:1) Rf = 0.76
1H NMR (d6-DMSO): δ 2.38 (3H, s, 4-tol-CH3), 2.41 (3H, s, 4-tol-CH3), 2.73 (1H, m, 2′-CH), 2.85 (1H, m, 2′-CH), 4.51 (3H, m, 5′-CH2 and 4′-CH), 5.62 (1H, d, 3′-CH), 6.09 (1H, dd, 1′-CH), 7.34 (2H, d, 3,5-tol-CH3), 7.37 (2H, d, 3,5-tol-CH3), 7.69 (2H, br s, NH2), 7.81 (1H, s, C=CH), 7.83 (1H, s, 2-H), 7.84 (2H, d, 2,6-tol-CH), 7.92 ppm (2H, d, 2,6-tol-CH).
RESEARCH PROJECT
METHODS FOR THE SYNTHESIS OF DEPROTECTED strA
Method A
Synthesis of 5-Amino-6-cyano-3-(2'-deoxy-3', 5'-di-O-p-tolouyl-β-D-erythro-ribofuanosyl)-imidazole|4,5-b| pyridine (6)
5-Amino-6-cyano-3-(2'-deoxy-3', 5'-di-O-p-tolouyl-β-D-erythro-ribofuanosyl)-imidazole (1.0 g) was added to acetonitrile (25 mL) and an ion-exchange resin (302.7 mg).This mixture was then heated under reflux (70 OC) for 3 h. After 3 hours of refluxing, TLC analysis showed a single fluorescent spot. Single spot was distinct from the starting material, confirming the conversion of the starting material to the desired dinitrile product. The product mixture was then filtered into a round bottom flask to remove the resin that was used as a catalyst for the reaction. After filtration, the solvent was removed by rotatory evaporation under vacuum yielding yellow crystals. These crystals were then left to dry overnight under a vacuum to give the product (0.52 g) yellow colour solid. To prepare the sample for NMR, the small amount of product was added to deuteriated DMSO. The proton NMR showed only peaks corresponding to the desired product and solvent, and was thus judged to be of acceptable purity to be used in the next stage of synthesis.
Yield (%) = 52%
TLC Analysis: TLC (ethyl acetate: MeOH 90:1) : Rf = 0.64
1H-NMR (d6-DMSO) = 2.38 (3H, s, -CH3), 2.48 (3H, s, -CH3), 2.52 (DMSO), 2.77 (1H,m,3'-CH), 3.26 (2H, m, 2' -CH2), 4.61 (2H, m,5' -CH), 5.78 (1H,d,4' -CH), 6.47 (1H, t,1' -CH), 6.90 (2H, s,NH2,D2O exch.), 7.36 (4H,dd,Ar-H), 7.94 (4H,dd,Ar-H), 8.30 (1H,s,Ar-H), 8.44 (1H,s,Ar-H).
13C-NMR APT (d6-DMSO)= 21.15 (s, -, 2 x CH3), 35.37 (s, +, 2' -CH2), 64.24 (s, +, 2), 64.24 (s, +, 5' -CH2), 75.13 (s, -, 3' -CH), 81.71 (s, -, 4' -CH), 83.38 (s, -, 1' -CH), 129.30 (s, -, 8 x Ar -CH on toluoyl groups), 165.29 (d, +, 2 x C=O)
Synthesis of 8- Amino-3-(2'-deoxy-β-D-ribo furanosyl)-imidazo |4', 5':5, 6| pyrido |2, 3-dipyrimidine (7)
The synthesized 5-Amino-6-cyano-3-(2'-deoxy-3', 5'-di-O-p-tolouyl-β-D-erythro-ribofuanosyl)-imidazole|4,5-b| pyridine (0.52 g) was added to diethoxymethyl acetate (5 mL). After mixing it, TLC analysis (EtOAc-MeOH-NH3 10:1:1) was done on the reaction mixture. It was then set to reflux (160 0C) for 4 h. After the reflux, TLC analysis showed a slight decrease in the Rf value of the reaction product compared with the starting material, indicating that the reaction is complete. High vacuum was used to remove the solvent from the reaction mixture. Brown oil suspension was produced which was added to 2.0M ammonia in methyl alcohol (20 mL) and was then left to stir overnight. Hazel brown suspension was left behind and its TLC analysis showed two fluorescent spot but there was some negligible starting material. It was afforded to heat with gaseous vapours and was placed in an ice bath for 1h in order to make the reaction mixture cool and crystallize. It was then filtered through scinter funnel under vacuum leaving behind a crystalline precipitate that was left to dry overnight under vacuum. The brownish coloured crystals of strA (0.45 g) were collected as a product and their TLC analysis showed a single fluorescent spot. The remaining filtrate was mixed with methanol and was left overnight to crystallize in order to extract the remainder of the product from the filtrate. The crystals were isolated by filtration through scinter funnel and giving a further crop of crystalline material (0.04 g). TLC analysis of these crystals along with the filtered solvent was carried out in order to confirm that the product gave one fluorescent spot. TLC analyses showed that some negligible amount of the starting material was still present with the one fluorescent spot. NMR characterization showed good purity and that the product could be used in the next stage of the synthesis without purification.
Yield (%) = 81%
TLC Analysis: TLC (EtOAc-MeOH-NH3 10:1:1) : Rf = 0.63 (After 4 hours)
TLC (EtOAc-MeOH-NH3 10:1:1) : Rf = 0 and 0.25 ( Two fluorescent spot)
1H-NMR (d6-DMSO) = 2.34 (3H, s, -CH3), 2.44 (3H, s, -CH3), 2.51 (DMSO), 2.87 (1H,m,3'-CH), 3.42 (2H, m, 2' -CH2), 4.64 (2H, m,5'-CH), 5.93 (1H, d, 4' -CH), 6.76 (1H, t,1' -CH), 6.89 (2H, s, NH2, D2O exch.), 7.94 (4H, dd, Ar-H), 7.94 (4H, dd, Ar-H), 8.30 (1H, s, Ar-H), 8.44 (1H, s, Ar -CH), 9.23 (1H, s, Ar-H).
13C-NMR APT (d6-DMSO)= 21.13 (s, -, 2 x CH3), 35.41 (s, +, 2' -CH2), 74.24 (s, +, 2), 63.24 (s, +, 5'-CH2), 74.13 (s, -, 3' -CH), 81.71 (s, -, 4' -CH), 83.38 (s, -, 1' -CH), 129.30 (s, -, 8 x Ar -CH on toluoyl groups), 165.29 (d, +, 2 x C=O)
Synthesis of 8- Amino-3-(2'-deoxy-β-D-ribo furanosyl )-imidazo |4', 5':5,6| pyrido |2,3-dipyrimidine (8)
Method A
The synthesized protected strA (0.45 g) was added to methanol (35 mL), deionised water (5 mL) and 2 pallets (0.10 g) of sodium hydroxide. The mixture was then brought under reflux for 20 min and after that its TLC analysis (EtOAc-MeOH-NH3 10:1:1) showed only one florescent spot visible at the bottom near the baseline which confirmed the completion of reaction. The pH of the reaction mixture was adjusted to pH 6 by using citric acid 20% solution.The solvent from the reaction mixture was then removed by rotatory evaporation under pressure liberating methanol and leaving behind water. The remaining solvent was then set to reflux (room temperature) for 30 min and then was placed in an ice bath in order to crystallize. After several hours, crystals were collected from the top of scinter funnel by filtration and the filtrate which was collected in the round bottom flask was retained in order to check if any remainder of product is still present. The filtrate was left overnight to precipitate and was collected (0.05 g).The two solids collected were then mixed together with ethanol and were gently heated with the help of heat gun to dissolve them. The mixture was placed inside the fridge for 30 min. The solids were then filtered and dried under vacuum. The total weight of product was (0.20 g) and then proton NMR characterization was done.
Yield % = 38%
TLC Analysis: TLC (EtOAc-MeOH-NH3 10:1:1) : Rf = 0.06
1H-NMR (d6-DMSO) = 2.44 (DMSO), 2.84 (1H, m, 3' -CH), 3.39 (2H, m, 2' -CH2), 4.54 (2H, m, 5'-CH), 6.76 (1H, t, 1' -CH), 8.09 (2H, br, s, NH2, D2O exch), 8.30 (1H, s, Ar-H), 8.44 (1H, s, Ar -CH), 9.23 (1H, s, Ar-H).
As the quantity of the synthesized deprotected strA was not enough for the next stage of reaction, the synthesis was again repeated to synthesize more deprotected strA.
5-Amino-6-cyano-3-(2'-deoxy-3', 5'-di-O-p-tolouyl-β-D-erythro-ribofuanosyl)-imidazole (1.0 g) was added to acetonitrile (25 mL) and an ion-exchange resin (302.7 mg). After all the steps of synthesis of protected strA, the amount of product synthesized was (0.61g). Some brown filtrate was also present which was retained to crop the remainder of the product. The product was de-protected with a different method on this occasion along with the brown filtrate.
Method B
The synthesized product (0.61 g) was added to resin (0.2 g) and MeOH (20 mL) and the mixture was stirred at room temperature for 3 h. The brown filtrate was also added with resin (0.1 g) and was stirred at room temperature.TLC analysis showed a single fluorescent spot but the starting material was still present. Resin (0.2 g) and MeOH (15 mL) again added to reaction mixture and (0.1 g) of resin was added to filtrate.TLC analysis still showed the starting material with the product. Resin (0.2 g) was again added to the reaction mixture and (0.1 g) to the filtrate. They were left for stirring for another 1 h. TLC analysis again showed some negligible amount of starting material. They were again refluxed for 3.5 h and then left overnight. The TLC analysis still showed enough starting material and it was again left to reflux for 1 h. The TLC analysis on this occasion showed single fluorescent spot for the reaction mixture. In case of the filtrate (0.1 g) resin was added and refluxed for 3 h. Ammonia (conc.) (2 mL) was added to dissolve the solid of reaction mixture and then it was filtered through scinter funnel. Solvent from the filtration was then evaporated by rotatory evaporation. The deprotected strA (0.21 g) was collected from the solvent. The proton NMR showed that the product is pure and ready to be used in the next stage of the synthesis.
Yield % = 45%
TLC Analysis: TLC (EtOAc-MeOH-NH3 10:1:1) : Rf = 0.56
1H-NMR (d6-DMSO) = 2.38 (1H, m , 2' -CH), 2.52 (1H, m, 2' -CH), 2.52 (DMSO), 2.77 (1H, m ,5'- CH2), 3.26 (2H, m, 2' -CH2), 3.78 (1H, d, 4'-CH), 4.32 (1H, s, 3' -CH), 6.22 (1H, t, 1' -CH), 6.79 (2H, s, NH2, D2O exch.), 8.21 (1H, s ,7' -CH), 8.21 (1H, s ,2 -CH).
The remaining filtrate was added to methanol and filtered. The solvent was collected 3 times in order to collect complete product of protected strA. The total weight of the product was (0.17 g).
The brown filtrate which was left in vacuum to gain the remaining material was treated with methanol and warmed with a heat gun. It was left in fridge for 30 min and the solid material was collected from filtration. The collected solid were placed under vacuum overnight to dry and was ready to be used in the next stage of the synthesis.
Treatment of deprotected strA with N, N-dimethylactamide dimethyl acetal.
Method A
The synthesized deprotected strA (0.1 g) was treated with N, N-dimethylactamide dimethyl acetal (2 mL, 34.2 m mol) and heated (90 oC) for 66 h. The crystals from the filtrate (0.17 g) were mixed with the initial crystals collected from first filtration (0.21 g) and were mixed with ethanol. Vacuumed under rotatory evaporation to remove methanol and gain crystals of strA. After the evaporation it was vacuumed. Water (0.2 mL) was then added and stirred for 10 min, dark brown viscous liquid was obtained by removing the solvent under reduced pressure. In the obtained residue, minimum quantity of dichloromethane was added to dissolve it. Column chromatography used to purify by eluting it with 9:1 (CH2Cl2: MeOH). Set of fraction was obtained and combined in a flask. Solvent was evaporated to give the product.TLC analysis showed that the yield was not successful to show single fluorescent spot of the product. This reaction was done again with another method.
TLC Analysis: TLC (CH2Cl2: MeOH 9:1) : Rf = 0.10
Method B
The second Sample of strA was treated with N, N-dimethylactamide dimethyl acetal (0.15 mL, 34.2 m mol) and was stirred for 72 hrs (90 oC).Water (0.3 mL) was added to mixture and was stirred for 10 mins.It was then subjected to remove water under reduced pressure.Dicholoromethane (3 mL) was added to dissolve the solid.Now the solvent to be used Column chromatography as a eluent is 8:1:1(CH2Cl2 : MeOH)).11 Fractions from eluent was taken and TLC analysis was done for the product fluorescent spots.The single spots were not visible for the product as it also had starting material spots as well.This attempt was also not successful and all the astarting material was used in during this stage of synthesis so the experiment was stopped here.
TLC Analysis: TLC (CH2Cl2 : MeOH) 8:1:1) : Rf = 0.8
RESULTS AND DISCUSSION
Synthesis of strating material 5-amino -4- imidazole for strA
Fig : 2-deoxycholorosugar (1) ,4-Nitroimidazole Caesium salt (2) , 1-5,Nitroimidazole (3) 5-Amino- 4-imidazole (4)
Felsenfeld, G., Davies, D.R. and Rich, A. (1957) Formation of a three-stranded polynucleotide molecule. J. Am. Chem. Soc., 79, 2023-2024.
2 Morgan, A.R. and Wells, R.D. (1968) Specificity of the three-stranded complex formation between double-stranded DNA and single-stranded RNA containing repeating nucleotide sequences. J. Mol. Biol., 37, 63-80
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