The experimental research for the present thesis was carried out from October 1, 2010 to March 31, 2011 in the laboratory of Prof. Wolfgang Pfleiderer for providing me with such an interesting topic and his valuable support and guidance throughout the project. Many thanks also go to Geeta for her very friendly working atmosphere and her much appreciated advice and encouragement.
I am especially grateful to my family and Kostas for their patience, support and continued encouragement.
Introduction
- Protective groups
- Protective groups of o-nitrobenzyl alcohol
- Protective groups of 2-(2-nitrophenylethanol)
- Solid support synthesis
A huge improvement in the photolytic blocking group strategy was recently achieved in the laboratory of Prof. In contrast to the cleavage mechanism of the 2-nitrobenzyl group, which has been extensively studied17, the photodegradation of the NPE functions follows a new pathway established as a photoinduced ß- process of elimination18. Further studies have shown that branching at the position, preferably with a methyl group, increases the photocleavage quantum yield by a factor of 10.
This led to more detailed studies of modified 2-(2-nitrophenyl)-propanol (NPP) derivatives (5)16 to further improve the photolytic characteristics. To demonstrate the anticipated project in general, the following schematics will illustrate the strategy to build up a DNA chip on a solid support. This building block is of general utility to prepare with succinic anhydride the 3'-O-succinate which will then be attached to the surface of the solid to function as a starting unit in the subsequent chain extension to form an oligonucleotide.
The second component is synthesized from the same building block by conversion to the corresponding phosphoramidite. Analogous phosphoramidites are prepared from the starting nucleosides 2'-deoxy-cytidine (dC), 2'-deoxyadenosine (dA) and 2'-deoxyguanosine (dG) which are then applied according to the predicted oligonucleotide sequence.
Research Proposal
The new approach to be investigated in this bachelor's thesis is based on special lumazine derivatives that show a strong absorption band at 360 nm. The basic molecule in this series is 1,3-dimethyl-7-phenyllumazine (10), which has been modified in its substituents (11) to meet the structural requirements for a photolabile protecting group. This means that the synthesis must start with a Traube synthesis that uses 4-ethylphenylurea and ethylcyanoacetate to form 6-amino-1-(4-ethylphenyl)uracil (12) in the first step.
Nitration of 15 should take place in the ortho position of the ethyl group forming 16 and the resulting reaction with paraformaldehyde will lead to 1-[4-(1-hydroxypropan-2-yl)phenyl]-. In order to attach compound 17 to the nucleoside thymidine, it must be reacted with phosgene to give the corresponding chloroformate, which will directly bind to thymidine to form a new photolabile building block (18). Depending on the time available, analogous reactions can be performed to increase the potential of photolabile protecting groups.
Results and Discussion
Synthesis
The subsequent reaction between 25 and 27 under mild conditions leads in high yield to the corresponding Schiff's base 28. Base-catalyzed cyclization in dilute ammonia led to the formation of 1-(4-ethylphenyl)-7-nitrophenyl-3-propylumazine (29 ) a molecule with the expected pteridine nucleus. The following modification was achieved by nitration of 29 in a mixture of nitric acid and conc.
The next step was a very difficult reaction consisting of a hydroxymethylation of 30 by paraformaldehyde in DMSO and catalyzed by DBU, leading to 31. This new building block was then reacted in a one-pot reaction first with phosgene to form the corresponding chloroformate intermediate (32), which is so reactive that without isolation, addition of the nucleoside thymidine (33) to the reaction solution led to a linkage at the 5' position of the sugar group of 33, giving thymidinyloxycarbonyloxypropan-2-yl)-3-nitrophenyl]-7-( 4 -nitrophenyl)-3-propyl}lumazine (34) in 25% yield. A second series of reactions started from 25, which was converted to the Schiff base 36 with phenylglyoxal (35)23.
The subsequent nitration of 37 in a HNO3/H2SO4 mixture led to a surprising result as a dinitro derivative was obtained. The final coupling of 39 with thymidine (33) to the second photolabile building block 40 via a carbonate bridge can be accomplished in the same way as the preparation of 34 in a yield of 25%.
Physical Data
- UV Spectra
- NMR Spectra
- Photolysis
NMR data are recorded in detail in the experimental section and are measured in CDCl3 or DMSO-d6. The structural assignments of the substituents can be determined from the splitting pattern of the aromatic protons. In contrast to the aromatic proton signals, the alkyl protons adjacent to heteroatoms and the hydroxyl groups resonate in the range of 3-4 ppm.
The methyl groups of ethyl and n-propyl substituents appear as triplets between 1-1.5 ppm due to coupling with adjacent CH2. 5'-O-substituted thymidines are little affected by the photolabile protecting group and show the normal nucleoside thymidine pattern. A singlet at 8-9 ppm that can be confused with D2O is due to the N-H proton, and the sugar proton H-C(1') appears as a pseudo-triplet at 6.3 ppm.
Shown here are the NMR spectra of compounds from the last three steps of the synthesis with NO2-feyl groups in the para and ortho positions respectively. Results from photolysis experiments show that compound 34 with the para NO2-phenyl group has a half-life of 120 sec. Compound 40 with the ortho NO2-phenyl group is less reactive as seen by its half-life of 620 sec.
Summary
Experimental section
General
Irradiation was done using an apparatus consisting of a 200W-Hg lamp and UV-DAD 8-1 filter (λ 365.6 nm) from the Schott company.
Experimental part
Methyl cyanoacetate (22 mL, 0.22 mol) was added dropwise to the mixture and refluxed for 5 hours. The resulting violet precipitate was separated by filtration and dried to give 21.9 g (80%) of the crude product (23), which was crystallized from H2O:EtOH (1:2). The crystallized precipitate was filtered off and dried in a desiccator to obtain 25.8 g (85%) of 24.
TLC still shows the presence of starting material, so the reaction mixture was stirred overnight at 70°C. The filtrate was evaporated to dryness, the residue was dissolved in CH2Cl2 (10 ml) and added slowly to an ice-cold solution. The organic layer was separated, dried over Na2SO4 and evaporated to yield a yellow foam.
After cooling, the precipitate was filtered off and dried to give 18 g (93%) of 37 Chromatographically pure product was crystallized from H2O:EtOH. TLC still showed the presence of starting material, so diphosgene (98 mg, 0.5 mmol) and NEt3 (100 mg, 1 mmol) were added again. The resulting NEt3:HCl was separated by filtration, washed with THF and the filtrate evaporated to dryness.
The residue was dissolved in CH2Cl2 (5 mL) and slowly added to an ice-cold solution.
Attachment