Wednesday, May 10, 2000
Vertiefungskurs Combinatorial Chemistry

Sommersemester 2000,

Selection of Acid Building Blocks for your Library of 5'-Modified Oligonucleotides

Selection of carboxylic acids.
The selection of the carboxylic acid building blocks for your individual library is an important step, as it is in this selection that you "bet on a horse" (or several ones) to win the race for the tightest binder to the target DNA strand.  As mentioned in last week's lecture, ideally you want to find a carboxylic acid of a molecular weight between 200 and 600 that can be coupled to the amino-terminal DNA via amide-bond formation.  The carboxylic acid thus linked is expected to bind to the terminus of the duplex of the oligonucleotide you are synthesizing and a complementary DNA strand (the target).  A good carboxylic acid substituent on your DNA will bind tightly and specifically to the terminal base pair through interactions such as p-stacking, hydrogen bonding, van der Waals interactions, hydrophobic effect, and possibly ion pair formation.
Just so you know, the coupling reaction must be specific, i.e. the carboxylic acid you use should have only one carboxylic acid or should be symmetrical (the former is preferred).  Further, it should not have amino or other highly nucleophilic groups (unless they are protected), so that it does not react with itself rather than with the amino group on the DNA that you want it to couple to.  You should also bear in mind that the final deprotection step will be performed with a saturated solution of ammonia in water (NH4OH), a reagent that cleaves esters, labile amides, and a number of other groups, including b-elimination protecting groups, such as Fmoc (fluorenylmethoxycarbonyl) or CNE (cyanoethyl) groups.  If you use an ester or another base-labile group, cleavage will occur, leaving only the portion that is connected to the carboxylic acid (now an amide) connected to the DNA.

What should you choose?  I recommend that you choose at least on each of the following:

-  One of the acids available in our (currently small) collection here in our new lab at U. Constance.  A hand-written sheet with these carboxylic acids is available (prepared by Andriy Mokhir).  Please talk to the other members of your lab group, so that you can avoid choosing the same acid.

-  One Fmoc- or Boc-protected amino acid or an amino acid derivative protected on N-a with an Fmoc- or Boc-group.  The Fmoc-group will be removed during NH4OH treatment, whereas the Boc-group (tert-butyloxycarbonyl) will stay intact.  A large number of these amino-protected amino acids is commercially available (Acros, Fluka, Sigma, Novabiochem, Bachem, etc.)  Please note the limitations in terms of reactivity stated above.  For example, if you want to use aspartic acid, you will need to have a derivative that has its side chain carboxylic acid group protected (e.g. Boc-Asp(OBn)-OH; in amino acid/ peptide short hand, the substituent on the Na-group precedes the three letter name of the amino acid residue, the substituent on the side chain is in parentheses, and the substituent on the carbonyl group follows; -OH means free acid, of course).

-  Two or three carboxylic acids that you select from a catalog, find in the list of the Chemikalienausgabe, or in the set of compounds that Prof. Jochims makes available.

Please submit a list of the proposed carboxylic acid building blocks you plan to use on the attached sheet no later than Thursday, May 11, 3:00 PM (15:00 Uhr) to Andriy Mokhir, Room Z 937.  If Andriy is not in his lab, just tape your list to his door.  We will then screen the proposals and edit the list.  Feel free to submit more than five suggestions, so that deletion of candidates judged poor by us leaves some compounds on the list.  Good luck!

Recommended Literature:

Bleczinski, C. F.; Richert, C. Steroid-DNA interactions increasing stability, sequence-selectivity, DNA/RNA discrimination, and hypochromicity of oligonucleotide duplexes. J. Am. Chem. Soc. 1999, 121, 10889-10894.

Altman, R. K.; Schwope, I.; Sarracino, D. A.; Tetzlaff, C. N.; Bleczinski, C. F.; Richert, C. Selection of modified oligonucleotides with increased target affinity via MALDI-monitored nuclease survival assays. J. Combin. Chem. 1999, 1, 493-508.

Schwope, I.; Bleczinski, C.F.; Richert, C. Synthesis of 3',5'-dipeptidyl oligonucleotides, J. Org. Chem. 1999, 64, 4749-4761.

Tetzlaff, C. N.; Schwope, I.; Bleczinski, C. F.; Steinberg, J. A.; Richert, C. A convenient synthesis of 5'-amino-5'-deoxythymidine and preparation of peptide-DNA hybrids. Tetrahedron Lett. 1998, 39, 4215-4218.