Vertiefungskurs Combinatorial Chemistry

Sommersemester 2000

Experiment 3:  Coupling of Acids

Wednesday, May 24, 2000

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

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

Information on the experiment.  Today, you will perform the only combinatorial step in your synthesis.  It will only be combinatorial, if you choose to make it so, i.e. if you perform a mixed coupling with more than one carboxylic acid building block in one solution.  To do so may be risky, as it is known that the reactivity of carboxylic acids depends on their structure.  More sterically hindered carboxylic acids, carboxylic acids that tend to aggregate, and carboxylic acids deactivated for electronic reasons will react more slowly than their competitors not suffering from these disadvantages.  For amino acid building blocks, particularly those of Boc- and Fmoc-protected proteinogenic amino acids, we have good data on relative reactivites (see e.g. Ostresh, J.M., Winkle, J.M., Hamashin, V.T., and Houghten, R.A. Peptide libraries: Determination of relative reaction rates of amino acids in competitive couplings. Biopolymers 1994, 34, 1681-1689), for other acids, one will have to guess.  If you do not want to risk a failure in a competitive coupling reaction, couple your acids individually.
The coupling conditions chosen are taken from established solid phase peptide coupling protocols that we have previously adapted for the synthesis of peptide-DNA hybrids (C.N. Tetzlaff, I. Schwope, C.F. Bleczinski, J.A. Steinberg, C. Richert, A convenient synthesis of 5'-amino-5'-deoxythymidine and preparation of peptide-DNA hybrids. Tetrahedron Lett. 1998, 39, 4215-4218.).  Scheme 1 gives an overview over the reactions for this week's experiment and structural formulae for the reagents.  Please think about how HBTU and HOBT can induce coupling.  A hint: HBTU is ultimately converted into tetramethylurea and HOBT.
As mentioned last week, the weighing of the coupling reagents on the analytical balance is the "bottleneck" in this experiment.  Please organize yourself as well as possible to avoid a long line at the balance.  Please also treat the balance carefully.  It appears as if the balance was already damaged during last week's experiment.  Also, we have decided to provide a stock solution of HOBT in DMF, so that you have to weigh out only the HBTU and your carboxylic acid.  A revised protocol is given below.

Solid-phase amide coupling, revised 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.
 A sample of the support (1 mg, ca. 0.03 µmol oligonucleotide) is transferred into a polypropylene reaction vessel (Eppendorf cup).  A mixture of the carboxylic acid to be coupled (100 µmol), and HBTU (34.1 mg, 90 µmol) is prepared separately, and dissolved in a solution of HOBT (15.3 mg, 100 µmol) in DMF (600 µL).  The solution of HOBT in DMF will be provided by your teaching assistant.  You may have to vortex the solution to get the components to dissolve fully.  Please make sure you close the reaction vessels when you vortex them, so that none of your solution is spilled.  In some cases, the carboxylic acid will only dissolve upon addition of the diisopropylethylamine (DIEA).  The solution is treated with DIEA (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 once after addition of the coupling mixture and then every 5 min during the coupling time with 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.  Please be careful with the acetonitrile.  It is rather toxic for an organic solvent.
If you do not have time to do the drying of your cpg, please mention this to your teaching assistant, so that he can take care of this.

At the end of your coupling reactions, please label your reaction vessels carefully, so that they cannot be mixed up.  It is recommend that you use colored tape for this.  Your teaching assistant will start the deprotection reaction for you next Tuesday.  For this, you have to leave the reaction vessels clearly visible and labeled in the laboratory (room Z 744).  On Tuesday, Andriy will add an aliquot of ammonium hydroxide solution, so that next Wednesday by the time you enter the lab the deprotection will be completed.  You will then be able to proceed by removing excess ammonia and drawing samples for mass spectrometric analysis.  The general deprotection protocol is given below.  We may decide on a change in the removal of excess ammonia, to make this more feasible for your situation.  Please note that the treatment with ammonium hydroxide cleaves the linker to the cpg, removes the cyanoethyl protecting groups, and the protecting groups on the exocyclic amino groups of the nucleobases in one step.

Deprotection and release from support, 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.   The hybrid-bearing controlled pore glass (1 mg, ca. 0.03 µmol oligonucleotide) in a polypropylene cup is treated with ammonium hydroxide (30% NH3 in water, 0.25 mL) for 16 h.  The supernatant is aspired, the residue washed with water (0.5 mL) and excess NH3 is removed from the combined solutions with an air stream.  The resulting solution is lyophilized to dryness, taken up in water and filtered (0.2 µm) for HPLC purification and/or MALDI analysis.  It is also possible to draw a sample for MALDI analysis prior to lyophilization.

A Note at the End.  Most probably, this week's experiment will be the most time consuming.  Please do not panic, if you cannot finish all five couplings in the allotted time.  Rather, I recommend that you either make an appointment with Andriy during the next few days and finish your work outside the time slot for the Praktikum, or that you finish your experiments next week.  It is better to extend the schedule a little (and we are not in a squeeze, right now) than to work in too hectic a fashion, risking the success of your synthesis and the well being of the instruments and your neighbors.
If everything goes well, you will have made several new compounds.  Good luck!