Vertiefungskurs Combinatorial Chemistry

Sommersemester 2000

Experiment 5:  Pre-Purification or diversification

Wednesday, June 7, 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.  This week, the crude products obtained from syntheses will be pre-purified via HPLC.  Those of you who did not get at least four of your products in detectable quantities also will have to do a mixed coupling during today's lab session in order to get the required number of compounds for your library.
The goal of the HPLC pre-purification is to remove failure products from the DNA synthesis and any fragmented DNA that could have resulted from nucleophilic attack of functional groups of the substituent you introduced on the DNA during the deprotection procedure.  If the product was the predominant molecular species in your crude products, we could skip this prepurification (as we have in the past when performing nuclease selection experiments), but inspection of the MALDI spectra of the crudes you obtained indicated that in the majority of all cases too high a level of shorter failure sequences was present.  These shorter failures also have a measurable affinity for the target strand that we will use in the nuclease selection experiments.  If their concentration is high, they can "swamp out", i.e. displace in the equilibrium, the binding of the compounds of interest, thus making it difficult to perform a meaningful selection among them.
The HPLC technique we are using is a standard technique for oligonucleotides.  It uses a reverse phase (RP) HPLC column material that in our case bears butyl chains on its surface ("C4 column").  The compounds are being retained on the column based on their lipophilicity.  Since DNA itself is a polyanion that is well solvated and would thus usually not be retained on the RP column, one uses a buffer salt in the aqueous solvent that contains lipophilic counterions to the phosphodiester anions of the DNA.  We will use triethylammonium acetate, conveniently prepared from equimolar quantities of triethylamine and acetic acid.  The triethylammonium cations associate with the phosphodiester groups in a dynamic equilibrium.  Since the counterions are now lipophilic and thus "sticky" for the RP column, the DNA is being retained.  A gradient of acetonitrile (CH3CN) is used to elute the DNA.  The organic solvent competes with the hydrophobic interactions that retain the DNA on the column, and at the percentage of acetonitrile at which this competition is won by the solvent, the compound elutes.
Experience shows that the longer the DNA strand, the later it elutes in the acetonitrile gradient.  Further, appending a lipophilic appendage to one of the termini (such as the carboxylic acid residues you have appended to the 5'-terminus of your oligonucleotides) also leads to a greater retention of the DNA on the column,  As a result, we expect your modified oligonucleotides to elute last.  The compounds can conveniently be obtained in pure form by lyophilizing (freeze drying).  This removes the solvents and the buffer (triethylammonium acetate is volatile, when pumped on for long enough).
A typical HPLC run will take approximately one hour, so it would be inefficient to pre-purify all your library components individually.  Therefore, it is planned that you prepare a mixture of the compounds prepared (a true library) and inject the entire library.  In order to prepare for the HPLC purification, you have to prepare an injection solution that is rather concentrated (so you get a narrow band on the column) and that is free of particles.  For this, the solution with the pooled compounds has to be centrifuged and filtered through a filter with submicrometer pore size.  After injection, the chromatogram has to be monitored carefully, since the eluting solutions have to be collected by hand.  If you miss a peak, you will lose the compound.  It is therefore suggested that all peaks are being collected (though one fraction per peak should suffice) and MALDI spectra are being acquired of the fractions, starting from the latest fraction forward, until all library components have been found.  We are planning to do a single injection with a gradient that should elute all your compounds.  There is no need to get your library compounds really pure.  As long as the bulk of the failure sequences is removed, we should be able to do meaningful selection experiments, hence the term "pre-purification".

Prepurification of libraries via HPLC.  The conditions chosen are standard conditions for oligonucleotides, see e.g. Eckstein, F. (ed.) Oligonucleotides and analogues, a practical approach. IRL Press at Oxford University Press, Oxford, 1991.
Please note that this step can only be performed after having obtained at least four crude products with a substantial amount of the desired modified oligonucleotide product in them. 
When you enter the lab, your teaching assistant will have lyophilized the solutions of those of your crude products that contain a measurable amount of your product.  Those, that (according to MALDI analysis) do not contain a substantial amount of product will not be pursued at the moment.   Please take the residue up in 100 µL of deionized water.  Please vortex well to ensure that the material is fully dissolved (lipophilically modified oligonucleotides are notorious for sticking to surfaces).  Of the 100 µL solution obtained for every crude product, 50 µL will be used for the HPLC injection.  The rest is being kept for possible later use.  Please pool 50 µL aliquots from each of your solutions into one Eppendorf reaction vessel.  Centrifuge the resulting mixed solution for 2 min in a fast microcentrifuge to spin down larger particles.  The supernatant will now be aspired into a 1 mL syringe and filtered through a small disc filter attached to the tip of the syringe.  Please rinse the syringe and the filter with 100 µL of 0.5 M ammonium acetate solution (not triethylammonium acetate solution, but ammonium acetate).  It is important that the Eppendorf cup into which you are filtering your solution has previously been washed with filtered water, so that it is as dust-free as possible.  Please vortex briefly and carefully to ensure full mixing of the solutions.
Of the resulting filtered solution, 80% should be injected into the HPLC injector.  This and the handling of the HPLC instrument will be performed by your teaching assistant.  Please be ready with numbered Eppendorf cups to collect the eluting fractions.  Please keep notes, based on which you can correlate the numbers of the cups with the elution time in the chromatogram.  The main gradient will go from 0 to 50 % acetonitrile in 0.1 M triethylammonium acetate (TEAA).  After the "washing step" of the gradient is completed, the fractions obtained may be analyzed via MALDI.  Again, this will be performed by your teaching assistant.  Please do not pool product-containing fractions yet.  These will have to be pooled in the correct ratios in the next two lab experiments.

After you have seen one HPLC run, those of you who have to perform a mixed coupling to obtained the required minimum of four library components will have to proceed to room Z 744 to do their couplings.  We have picked mixtures of carboxylic acids that are expected to give you the additional library components.  Your teaching assistants (Andriy Mokhir and Jason Mayo) will have prepared coupling mixtures that are ready to be taken up in 600 µL of DMF.  The resulting solutions should be treated with the diisopropylethylamine quantity given (not more!), and transferred into the Eppendorf cup containing the DNA-bearing cpg.  Please use 1 mg of cpg per component in your mixed coupling, i.e., if you need two additional compounds, you should use 2 mg of DNA-bearing cpg, if you need an additional 4 components (and therefore have a coupling mixture with 4 building blocks prepared), you should use 4 mg of cpg.  A reminder of how the coupling works is given below.

Reminder, coupling reaction.  A sample of the support (1 mg, ca. 0.03 µmol oligonucleotide per component in your mixed coupling) is transferred into a polypropylene reaction vessel (Eppendorf cup).  A mixture of the carboxylic acids to be coupled (a total of 100 µmol), HBTU (34.1 mg, 90 µmol), and HOBT (15.3 mg, 100 µmol) will be ready for use for you.  Please take these up in DMF (600 µL).  You may have to vortex the solution to get the components to dissolve.  The solution is treated with diisopropylethylamine, DIEA (40 µL, 234 µmol), leading to slight darkening.  The reaction mixture is then immediately transferred to 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.  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 brief drying.  The crude oligonucleotide may then be deprotected and released from the cpg with a concentrated solution of ammonium hydroxide (30% NH3 in water, 0.25 mL).  Your teaching assistant will terminate the deprotection and will acquire MALDI spectra of the crudes.

A Note at the End.  Just a reminder - Please label everything carefully.  The more advanced you are in this lab, the more important is this advice.  As everywhere in combinatorial chemistry, managing the compounds and the information is critical.  This means careful labeling of all vessels and careful documentation of your steps in the lab notebook.  Please also communicate your labeling scheme to your teaching assistant.  Thank you and good luck!