In the Laboratory
An Undergraduate Organic Chemistry Laboratory Experiment: The Multistep Synthesis of a Modified Nucleoside Joseph Howell and Peter deLannoy* Department of Chemistry, Black Hills State University, Spearfish, SD 57799
Recent advances in the biotechnology and pharmaceutical industries have created an increased interest in nucleic acid chemistry among students, yet experimentation using biomolecules is typically of such a level that this area of chemistry is generally unavailable to undergraduates. We report here the design of a multistep organic synthesis of a modified nucleoside with biological significance and have recently updated our laboratory curriculum in organic chemistry to include this experiment. We designed the synthesis of 5′-O-dimethoxytrityl-2′-O-methyluridine, and 5′-Odimethoxytrityl-3′-O-methyuridine directly from the literature and over the last five years have developed a protocol that is appropriate in a small-school setting. This particular synthesis was chosen for several reasons. First, modified nucleosides have been used extensively by the pharmaceutical industry for the design of certain types of new drugs (1). 3′-Azido-2′,3′-dideoxythymidine (AZT), for example, is a modified nucleoside used to treat AIDS, and the target molecules of interest herein have been used to synthesize antisense compounds that may have therapeutic potential against a variety of diseases and biological functions (2, 3). Secondly, the synthesis required the students to master many important bench techniques commonly used in the multistep synthesis of complex target molecules, including thin-layer chromatography, flash chromatography, use of the Schlenk line, and use of the rotovap. Thirdly, the relevant intermediates and products have been extensively characterized in the literature and NMR, mass spectrum, and other important data were available (4, 5). Additionally, the products were well characterized by TLC, which made the synthesis ideal for a small-school setting where no NMR or mass spectrometer was available. This experiment worked best with groups of two students and in our experience usually took about six laboratory periods to complete. This was an excellent special project for the latter half of a second-semester undergraduate organic chemistry laboratory course. The synthesis of the methylated nucleosides was relatively straightforward and required synthesis steps (Fig. 1). Students began with the unmodified uridine nucleoside, chosen because it does not require the use of protecting groups and has few of the purification problems that sometimes occur with the more complex purines (4). Uridine was first converted to the dibutylstannylene derivative (2) to activate the 2′ and 3′ hydroxyls of the nucleoside. Next, 2 was combined with iodomethane to methylate the 2′ and 3′ positions of the nucleoside (3 and 4). Finally, the methylated nucleosides were tritylated at the 5′ position to facilitate separation and purification by flash chromatography (5 and 6). It should be noted that the length and expense of the project can be reduced by eliminating the last step of the synthesis. Although more difficult, the untritylated molecules can be separated by flash chromatography using the *Corresponding author.
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conditions described below. Also noteworthy was that although methyl iodide was chosen for the alkylating agent in this synthesis, other unbranched alkyl halides may be substituted and we have successfully nonylated uridine by the same procedure. Finally, the products at each of the three steps could be stored without detectable decomposition for several days between laboratory periods. Experimental Procedure
Materials All reagents and solvents were purchased from either Aldrich or Sigma Chemicals. 2′ ,3′ -O-(Dibutylstannylene)uridine (2) (4) Uridine (2 mmol, 0.488 g) and dibutyltin oxide (2 mmol, 0.500 g) were carefully added to 100 mL of anhydrous methanol that had been cannulated into a 250-mL roundbottom flask. The clear solution was refluxed with a Friedrich condenser for one hour and evaporated to dryness in vacuo using the rotovap. The white powdery residue had a melting point between 232 and 234 °C. The residue was stored overnight under nitrogen at room temperature. Uridine and the dibutylstannylene derivative could not be resolved by TLC. However, we assumed that this reaction was quantitative, based on our melting point data and the reHO HO
U O
U O
1) Dibutyltin oxide
Step 1 methanol O
O
OH OH Sn Bu (1)
HO
Bu (2) HO
HO
U O
U
U
O
O
2) CH3I
+
Step 2 dimethylformamide OH OCH3
O
O
OH
H3CO
Sn Bu
Bu (2)
HO
HO
(4)
(3)
U
U O
O
DMTO
DMTO 3) DMTCl
Step 3
U
U
O
O
+
+ pyridine
OH OCH3 (3)
H3CO
OH (4)
OH OCH3 (5)
H3CO
OH
(6)
Figure 1. The three-step synthesis of 5′- O -dimethoxy-2′- O methyluridine and 5′- O-dimethoxy-3′-O -methyluridine. U = uridine, Bu = butyl, DMTCl = dimethoxytritylchloride, DMT = dimethoxytrityl protecting group.
Journal of Chemical Education • Vol. 74 No. 8 August 1997
In the Laboratory sults demonstrated by Wagner et al. (4). The residue was used directly in the next step with no other manipulations.
2′ -O-Methyluridine and 3′-O -Methyluridine (3 and 4) (4) Thirty milliliters of anhydrous dimethylformamide was cannulated into the 250-mL round-bottom flask from above. Iodomethane (64 mmol, 4 mL) was added to the anhydrous mixture with a syringe (students should be wearing gloves). The reaction mixture was maintained at 40 °C under nitrogen overnight. The solution was evaporated to a gum in vacuo and the residue stored under nitrogen. The products were characterized by TLC in ethyl acetate/methanol (4:1) on silica gel 60 F254 plates. Rf values for 1, 3, and 4 were .60, .68, and .56, respectively. The products may also be characterized by TLC using dichloromethane/methanol/triethylamine (96:3:1) on silica gel 60 F254 plates (Rf values for 1, 3, and 4 were .05, .17, and .14, respectively). The overall yield of 80% was estimated based on the TLC results.
Flash Chromatography (7) The products from above were separated and purified by flash chromatography in a 55 × 2.5-cm column with silica gel 60 (230–400 mesh; Aldrich) equilibrated in 99:1 dicloromethane/triethylamine. The products were loaded onto a 7-inch bed of silica gel and washed with 5 column volumes of dichloromethane with 1% triethylamine to remove unwanted reaction products and solvent. The products of interest were then eluted with several column volumes of 2% methanol in 99:1 dichloromethane/triethylamine. Fractions were monitored by TLC as described above and the products of interest were pooled, evaporated to dryness, and stored under nitrogen. The best results using flash chromatography were obtained after extensive TLC of the product mixtures. We also found that our results varied depending on the batch of silica gel and recommend predrying the silica gel at 110 °C. Acknowledgments
5′ -O-Dimethoxytrityl-2′-O -methyluridine and 5′-ODimethoxytrityl-3 ′-O-methyluridine (5 and 6) (6) Fifty milliliters of anhydrous pyridine was cannulated into the residue from the previous step (~2 mmol, 0.52 g). The solution was evaporated to dryness in vacuo and 50 mL of fresh anhydrous pyridine was cannulated into the flask. Dimethoxytritylchloride (2.2 mmol, 0.75 g) was added and the mixture and stirred at room temperature for two hours under nitrogen. The reaction’s progress was monitored by TLC. However, because of the pyridine, it is recommended that the plates first be run in diethyl ether and then in ethyl acetate/triethylamine (99:1). Under these TLC conditions 3 and 4 were not resolved (one spot, Rf .06) and 5 and 6 have Rf’s of .48 and .36, respectively. After prerunning the plates in diethyl ether the products may also be fractionated in dichloromethane/methanol/triethylamine (96:3:1) (Rf’s for 3, 4, 5, and 6 were .17, .14, .27, and .24, respectively). After the reaction was complete the solution was evaporated to a gum. Yields at this step were based on TLC and were estimated as better than 80%.
This work was supported in part by a grant from the Black Hills State University Faculty Research and Instructional Improvement committees and an award from the Center for Innovation and Economic Development at Black Hills State University for undergraduate research and laboratory facility improvement. Literature Cited 1. Wagner, R. W. Nature 1994, 372, 333–335. 2. Blackburn, G. M.; Gait, M. J. Nucleic Acids in Chemistry and Biology, 1st ed.; Oxford University: New York, 1990; p 160. 3. Johansson, H.; Belsham, G. J.; Sproat, B. S.; Hentze, M. W. Nucleic Acids Res. 1994, 22, 4591–4598. 4. Wagner, D.; Verheyden, J. P. H.; Moffatt, J. G. J. Org. Chem. 1974, 39, 24–30. 5. Sproat, B. S.; Lamond, A. I.; Beijer, B.; Neuner, P.; Ryder, U. Nucleic Acid Res. 1989, 17, 3373–3386. 6. Smith M.; Rammler, D. H.; Goldberg, I. H.; Khorana, H. G. J. Am. Chem. Soc. 1962, 84, 430. 7. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923–2925.
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