Vertiefungskurs Combinatorial Chemistry

Sommersemester 2000

Experiment 2:  "Detritylation"

Wednesday, May 17, 2000

Location:  You will meet for this week's experiment in room Z 744, Verfügungsgebäude, 7th floor level.

Teaching Assistant: Your teaching assistant for this experiment is Andriy Mokhir.

Information on the experiment.  Today your experiment is to determine the coupling yield of the DNA synthesis performed last week.  Since every new nucleotide in the final DNA strand is introduced as a dimethoxytrityl (DMT) or monomethoxytrityl (MMT) protected building block, its incorporation efficiency can be determined by measuring the amount of trityl cation (DMT+ or MMT+) released from the DNA-bearing solid support.  The steps releasing the trityl cations are shown in Scheme 1 on the attached sheet.  By determining the trityl yield of the first and the last deprotection reaction, the yield of the assembly of the DNA oligomer can be determined.
Last week, two syntheses were performed: synthesis A, whose oligonucleotide was detritylated already, and synthesis B, whose product is currently "trityl on".  Analysis by MALDI-TOF mass spectrometry has shown that synthesis A gave considerably higher coupling efficiency for the last, MMT-protected phosphoramidite than synthesis B.  So, if you find a low yield for the assembly of the oligonucleotide in synthesis B, don't be frustrated.  You should be able to get a reasonable yield of acylated aminonucleotide out of the DNA-bearing cpg prepared in synthesis A.

Please weigh ca. 3 mg (note down the exact amount) of DNA bearing cpg from synthesis B (will be available from your teaching assistant) into an Eppendorf polypropylene reaction vessel.  Please do not spill any of this rather precious material, if possible.  You will have to use an analytical balance for this.  Please be careful with this balance.
Treat the cpg with approximately 1 mL of deblock solution (trichloroacetic acid in dichloromethane).  Please be careful, as trichloroacetic acid is caustic and toxic.  You can use the graduation on the Eppendorf cup to reach 1 mL of solution.  Vortex briefly after addition of the reagent solution.  A yellow color should develop.  After 10 min reaction time, please aspire the supernatant (Überstand) carefully with a syringe with needle.  Transfer the solution into a 10 mL volumimetric flask (Meßkolben), wash carefully (try not to aspire any cpg!) with 2 x 0.5 mL dichloromethane.  Add 5-10 mg para-toluenesulfonic acid (tosylic acid) to the volumimetric flask and fill up to 10 mL  with dichloromethane.  The solution is now ready for UV-vis spectrophotometry, which will be performed in collaboration with your teaching assistant.
One of you should take the solution from the first step of the DNA synthesis (DMT, not MMT signal), fill the available solution up to 25 mL with CH2Cl2 and tosylic acid, and have the UV-vis spectrum of this solution determined as well.  Using the weight of your sample (the total weight of the cpg used in the synthesis was 30 mg) and the extinction coefficients given in Scheme 1, please determine the yield of the oligonucleotide synthesis.

Note: Please note that this lab has only one analytical balance in room Z 941.  Using this balance will become the "bottleneck" (i.e. rate limiting step) in this lab experiment and the next, so please try to organize your group and be a little patient, if necessary.

Procedure 2:
If you have time during or after the experiment described above, you may start preparing for next week's experiment.  The reactions to be performed next week are shown in Scheme 2.  You may begin to weigh out the reagent mixtures for the coupling reactions (HBTU or HATU, HOBT, and your carboxylic acids).  A number of the carboxylic acids ordered for you have already arrived.  We will hand you back a copy of the list you submitted, so that you can identify the acids ordered for you.  Please note that again carefully weighing out on the analytical balance can create a bottleneck.  The ratio of the reagents (90 µmol HBTU or HATU, 100 µmol HOBT, 100 µmol of your carboxylic acid) is quite important, so try to stay close to the mark and calculate the 100 µmol of your carboxylic acid carefully.  If you have the three solids for your individual coupling reaction weighed into an Eppendorf cup, please close it and label it carefully, so that there is no mix-up with other student's sample.  The general procedure for the coupling reaction is given below.  If you have a lot of time left, you can start coupling.  Good luck!

Solid-phase amide coupling, general procedure, modified from: Altman, R. K.; Schwope, I.; Sarracino, D. A.; Tetzlaff, C. N.; Bleczinski, C. F.; Richert, C. J. Combin. Chem. 1999, 1, 493-508.
 After drying by exposing the reaction column to 0.1 Torr, a sample of the support (1mg, ca. 0.03 µmol oligonucleotide) is transferred into a polypropylene reaction vessel.  A mixture of the carboxylic acid to be coupled (100 µmol), HOBT (15.3 mg, 100 µmol), and HBTU (34.1 mg, 90 µmol) is prepared separately, briefly dried at 0.1 Torr for 30 min and dissolved in DMF (600 µL).  The solution is treated with diisopropylethylamine (40 µL, 234 µmol), leading to slight darkening.  The reaction mixture is then immediately introduced into the reaction vessel containing the oligonucleotide-bearing support with a syringe.  The slurry is vortexed after addition of the coupling mixture and then every 5 min on a bench vortexer.  The reaction is allowed to proceed for 40 min.  The coupling solution is then carefully aspired and the glass support washed with CH3CN (2x3 mL), followed by drying at 0.1 Torr.