Biomod/2013/Aarhus/Results And Discussion/Chemical Modification: Difference between revisions
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==DNA conjugation== | ==DNA conjugation== | ||
The labeling of DNA strands with cholesterol was carried out over two steps. The first step was a reaction | The labeling of DNA strands with cholesterol was carried out over two steps. The first step was a labeling reaction using the enzyme terminal deoxynucleotidyl transferase (TdT), where the nucleotide 5-propargylamino-ddUTP was added to the 3’-end of the DNA strand. The second step was the amide bond formation where the free amine will react with the NHS-ester. The outline of the two steps can be seen in Scheme 20 below. | ||
The procedure for the first step was inspired by the article by Jahn et al. | The procedure for the first step was inspired by the article by Jahn et al. | ||
Revision as of 11:12, 20 October 2013
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Preview
Cholesterol
Introduction to cholesterol
Cholesterol (Figure 1) is a natural and important steroid in many living organisms. The steroid structure of cholesterol is the precursor for a number of important steroid hormones. Besides being the precursor for other compounds, cholesterol has important functions as a lipid. In cells cholesterol is imbedded in the lipid bilayer to control the permeability and the fluidity.
When cholesterol is imbedded in the membrane, the alcohol group is oriented in order to interact with the head group of the phospholipids while the rigid Quattro cyclic system and the tail are parallel to the fatty acid chains. This ability can be exploited by using cholesterol to direct functionalities to the cell surface. The idea has been proven viable in experiments by Langecker et al. where a lipid membrane channel is bound to the cell surface using cholesterols affinity for the lipid membrane (Figure 2).
The ability to attach to cell membranes is also exploited in this project in order to bind the origami plate to the cell surface. Thereby insuring a high local concentration of the photosensitizer at the cell surface. In order to conjugate cholesterol to DNA staple strands, the cholesterol is modified with an NHS-ester handle. This NHS-ester can be used in an amide coupling reaction with a beforehand amine modified DNA strand. In total 19 staple strands are modified with cholesterol for use in the origami plate.
Synthesis of cholesterol derivative
The cholesterol derivative 5 is synthesized in a three step procedure (Scheme 2). The linker and cholesterol are envisioned to react in a simple SN2 reaction. The oxyanion, from the deprotonated alcohol, performs a nucleophilic attack on the α-carbon with bromide as the leaving group. The second step is an acidic deprotection of the ester and the third step is an activation of the acid with N-hydroxysuccinimide (NHS).
The synthesis of 3 resulted in a low yield (8%), when using the procedure described by Simeone et al. and an optimization was as a consequence tried. However the optimization did not result in a significant improvement as the highest obtained yield was 12% in a time-consuming reaction procedure.
The synthesis of 4 is a deprotection of the tert-butyl ester with acid. Compound 3 was dissolved and left to react in formic acid at room temperature. After the reaction was judged to be finished by TLC, the formic acid was evaporated under reduced pressure after which the product could be extracted from the organic phase with aq. saturated solution of sodium hydrogen carbonate. Hereafter the water phase was made acidic with 2 M hydrochloric acid and the product was back-extracted using dichloromethane (DCM). This method of purification results in the pure product in 76% yield after evaporation in vacuo.
To heighten the reactivity of the carboxylic acid it is converted into an NHS-ester, compound 5. The acid 4 was mixed with the coupling reagent N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC) and NHS after which the reaction was left to stir for 3 h. An NHS-ester is easily hydrolyzed as NHS is a very good leaving group. The inherent reactivity, which makes it useful as a coupling intermediate, also renders it hard to handle without the functionality decomposes. Therefore, the work-up had to be performed quickly. The desired product 5 was synthesized in 80% yield, which was deemed successful.
Synthesis of the Photosensitizer
The photosensitizer used in the project is indium(pyropheophorbide a)chloride (5), which can be synthesized in five steps starting from pheophytin a, a natural compound that can be extracted from spirulina powder. The first four steps were performed according to literature. By activating indium(pyropheophorbide a)chloride as an NHS ester it can be conjugated with an amine modified DNA strand.
After the photosensitizer was synthesised and conjugated with DNA. The photosensitizers ability to induce apoptosis in cells was analysed in a test setup.
In order to extract pheophytin a (1) from the spirulina powder it was necessary to lyse the cells, which was obtained by performing a cryogenic extraction. The extracted amount of pheophytin a corresponded with the 1 %w/w of spirulina powder reported in the literature.The melting point interval measured was lower and broader than reported in the literature, thus indicating a contaminated product. Impurities were also found in the NMR spectra. When performing the extraction again, further purification of the product unfortunately proved to be unsuccessful. Since it was possible to obtain a pure product at a later point in the synthesis of In(PPa)Cl, these impurities were concluded to be of less importance.
The first synthetic step was a trans-esterification of pheophytin a (1). Compound 1 was dissolved in allyl alcohol before conc. sulphuric acid was added. This reaction afforded the desired product in 79% yield.
The second step was performed to remove the methyl ester group. In order to accomplish this, compound 2 was dissolved in 2,4,6-collidine and heated to 170 °C. Compound 3 was obtained in a high yield (91%).
The subsequent reaction was insertion of indium in the porphyrin ring. When inserting indium in the porphyrin ring, the compound changed color from black to dark blue/green, and the insertion was confirmed by a UV-Vis spectrophotometry. The UV-Vis spectrum of In(PPa allyl ester) Cl (appendix) was compared with a UV-Vis spectrum of PPa allyl ester (appendix). Pyropheophorbide a allyl ester had a maximum absorbance at 274 nm and moderate absorbance at 410 nm. In the UV-Vis spectrum for In(PPa allyl ester)Cl no significant absorbance below the maximum absorbance at 423 nm is observed. In the 1H-NMR spectrum the amine protons were no longer present, and a downfield shift for two of the methine bridge protons was observed, as expected upon metalation of the porphyrin ring. This reaction afforded the indium compound in 45% yield.
The next step was a deallylation, better known as the Tsuji Trost reaction. The yield of the deallylation was much lower (21%) than reported in the literature (95%).
The last step was a formation of an activated acid in form of an NHS-ester. This reaction was performed using a general procedure.
This reaction afforded compound 6 in 91% yield without purification, as this treatment would decompose the labile NHS-ester. The NHS ester was used to conjugate the photosensitizer with a DNA strand. After conjugation the product was purified. The NHS ester was used in such excess that impurities were of less importance.
Synthesis of 5-propargylamino-ddUTP
For the chemical modification of DNA staple strands the chosen method involved the enzyme terminal deoxynucleotidyl transferase, which was used to add a modified nucleotide to the 3′-end of the DNA strand. This chosen nucleotide was modified with a terminal amine, which could subsequently be used in an amide bond formation. In order to only add one nucleotide to the DNA strand a dideoxynucleotide was used. The nucleotide, which had already proved viable with the TdT method, was 5-propargylamino ddUTP (13). As compound 13 is ideal when a large number of different DNA strands need to be labeled with a terminal amine it was decided to synthesize the compound instead of buying it commercial as it is very expensive (598 euro/mg). The synthesis is carried out over seven steps, starting from 2′-deoxyuridine (6) (5.6 euro/g). The first part of the strategy has previously been reported by McGuigan et al., however, modifications of the procedures were necessary.
The overall strategy (Scheme XX) is to remove the 3′-hydroxy group, functionalize the 5-position with propargylamine and functionalize the 5′-alcohol with a triphosphate chain. This is accomplished by converting the hydroxy groups into good leaving groups followed by two SN2 reactions, giving the bicyclic system in 8. An elimination reaction affords 9, where the oxetane ring is opened and a double bond is formed. A hydrogenation of the double bond followed by an iodination of the 5-position in uridine leads to compound 11. A Sonogashira cross coupling reaction is used to incorporate a protected amine, which after the triphosphate synthesis can be deprotected.
The two first steps were carried out without purification of 7 as described in the literature. In the first reaction it was important to control the temperature because the sulfene species is unstable at higher temperature. So the solution of 6 in pyridine was cooled to 0 °C before MsCl was added. The first reaction resulted in brown sticky oil, which was subsequently dissolved in MeOH and NaOH, heated to 80 °C and left for the oxetane ring to be formed. These two reactions yielded 8 in 48% over two steps.
The next step was a ring opening by an elimination reaction giving the double bond and homoallylic alcohol in compound 9. After purification the desired compound was obtained in an acceptable yield 58%.
The subsequent step was a hydrogenation of 9 resulting in 2′,3′-dideoxyuridine (10). This was accomplished with a catalytic amount of palladium over carbon and hydrogen gas. This reaction resulted in a quantitative yield.
The iodination of the base moiety was performed using iodine and ceric ammonium nitrate (CAN) as in the procedure by Asakura et al.The nucleoside, iodine and CAN were dissolved in glacial acetic acid and then heated to 80 °C. After 37 min the reaction was complete and the solvent was evaporated. This reaction afforded the product in a good yield 72%.
The Sonogashira cross coupling was inspired by literature and performed under inert conditions with pre-dried reagents, however, the product was only obtained in 41% yield after purification.
The triphosphate synthesis was performed using a procedure by Caton-Williams et al.The reagents were dried overnight before they were dissolved in dry DMF. The tributylammonium pyrophosphate reagent was mixed with salicyl phosphorochloridite for half an hour to make the reactive species (Scheme XX), after which the mixture was added to the nucleoside for further reaction.
After 3 h an oxidative iodine solution was added followed by addition of water in order to hydrolyze the cyclic intermediate (Figure XX).
Crude 1H- and 31P-NMR were acquired which showed peaks corresponding to the desired product. However, the product could not be obtained after purification by preparative TLC. Because of time constrains the last reaction could not be performed again and another purification technique could not be tried.
DNA conjugation
The labeling of DNA strands with cholesterol was carried out over two steps. The first step was a labeling reaction using the enzyme terminal deoxynucleotidyl transferase (TdT), where the nucleotide 5-propargylamino-ddUTP was added to the 3’-end of the DNA strand. The second step was the amide bond formation where the free amine will react with the NHS-ester. The outline of the two steps can be seen in Scheme 20 below. The procedure for the first step was inspired by the article by Jahn et al.
picture
The experiment was at first performed on 6 of the 19 DNA staple strands, from the origami baseplate, targeted for modification. 0.5 nmol of each strand were mixed together and to the solution were added the co-factor CoCl2, the reaction buffer containing the potassium cacodylate, the 5-propargylamino-ddUTP (13) and the enzyme. The volume was diluted to 100 μL and incubated at 37 °C for 15 min. The reaction was stopped using EDTA and an ethanol precipitation was performed to isolate the DNA. After the liquid was discarded the residue was dissolved in H2O and a solution of 5 in DMF was added. MeCN was added together with TEA and the reaction was incubated overnight at room temperature. After another ethanol precipitation the product was purified by RP-HPLC using a gradient (10% to 70% MeCN in TEAA buffer over 30 min). Even though there were 6 different strands it was not a problem separating the cholesterol modified from the ones without. The yield was calculated to 17% by UV absorbance.
Conclusion
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</style> </head> <body> <div id="indexing"> <div id="sitemap"> <p id="sitemapTitle">SITEMAP | BIOMOD 2013 NANO CREATORS | Aarhus University</p> <div id="footer-contents"> <div class="footer-section"> <p class="footer-section-title">INTRODUCTION</p> <ul> <li><a href="/wiki/Biomod/2013/Aarhus">Home, abstract, animation and video</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Introduction">Introduction</a></li </ul> </div> <div class="footer-section"> <p class="footer-section-title">RESULTS AND DISCUSSION</p> <ul> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/Origami">Origami</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/Peptide_lock">Peptide lock</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/Chemical_Modification">Chemical modification</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/sisiRNA">sisiRNA</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/System_In_Action">System in action</a></li> </ul> </div> <div class="footer-section"> <p class="footer-section-title">MATERIALS AND METHODS</p> <ul> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/Origami">Origami</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/Peptide_lock">Peptide lock</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/Chemical_Modification">Chemical modification</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/sisiRNA">sisiRNA</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/System_In_Action">System in action</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/Methods">Methods</a></li> </ul> </div> <div class="footer-section"> <p class="footer-section-title">SUPPLEMENTARY</p> <ul> <li><a href="/wiki/Biomod/2013/Aarhus/Supplementary/Team_And_Acknowledgments">Team and acknowledgments</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Supplementary/Optimizations">Optimizations</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Supplementary/Supplementary_Data">Supplementary data</a></li>
<li><a
href="/wiki/Biomod/2013/Aarhus/Supplementary/Supplementary_Informations">Supplementary informations</a> <li><a href="/wiki/Biomod/2013/Aarhus/Supplementary/References">References</a></li> </ul> </div> </div> <div> <p id="copyright">Copyright (C) 2013 | BIOMOD Team Nano Creators @ Aarhus University | Programming by: <a href="mailto:pvskaarup@gmail.com?Subject=BIOMOD 2013:">Peter Vium Skaarup</a>.</p> </div> </div>
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