A Reactivity Test for HBTU-Activated Carboxylic Acids with Low Reactivity and Competitive Coupling of N-Methylpyrrole Derivatives

Abstract

N-Methylpyrrole carboxylic acids are building blocks for oligopyrroleamides that bind DNA duplexes via the minor groove. The reactivity of HBTU-based active esters of four ­methylpyrroles in amide-forming reactions was determined. When assayed against HBTU-activated N-acetylleucine, a 6-250-fold lower reactivity was found. When assayed against the NHS ester of Boc-valine, the reactivity was up to 4-fold lower. Despite large differences in reactivity, mixed couplings were successfully performed with all four pyrroles, generating small libraries of modified oligonucleotides suitable for spectrometrically monitored selection experiments. ­Microwave irradiation accelerated coupling of an Fmoc-protected pyrrole to an amine on solid support.

Key words

DNA - pyrroles - amides - combinatorial chemistry - ­solid-phase synthesis

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18

Reactivity Assay.
Aliquots (9.2 µL each) of stock solutions of HBTU and HOBT monohydrate (each 476 mM in DMF) were mixed with an aliquot (35 µL) of a stock solution of the test acid (127 mM in DMF) in a reaction cup, followed by addition of 1.6 µL (9.7 µmol) of diisopropylethylamine (DIEA) and vortexing. N-Acetyl-l-leucine was activated analogously. When N-Boc-l-valine N-hydroxysuccinimide ester was used as reference acid, DIEA was mixed with the stock solution. After 1 h, aliquots from the activation mixtures (23 µL each) were combined and treated with an aliquot (1.7 µL) of a stock solution of 6 (3.05 mM in DMF), followed by vortexing. After 1 h, a sample (0.5 µL) of the assay solution was spotted on a stainless steel MALDI target, followed by drying at 0.1 Torr for 10 min. Then, 1.0 µL of the matrix-co-matrix mixture (2,4,6-trihydroxyacetophenone 0.3 M in EtOH; diammonium citrate, 0.1 M in H₂O; 2:1, v/v) was added, followed by drying and acquisition of the mass spectrum on a Bruker REFLEX IV spectrometer in positive mode with delayed extraction at 20 kV total acceleration voltage. Equireactive acids give a peak pattern of 1:4:6:4:1 for the porphyrin products. Deviations from this ratio of peak intensities were used to calculate relative reactivities, as described earlier (ref. [6] ).

21

Mixed Coupling.
Acids 1-4 were activated individually with HBTU, HOBT, and DIEA, as described for the reactivity assay above. An aliquot of a stock solution of 13 (35 µL, 127 mM in DMF) was treated with DMF (18.4 µL) and DIEA (1.6 µL). Then, aliquots from solutions of activated acids corresponding to the amount of the building block given in Table [2] were pooled and the resulting mixture was added to a sample of 19 (1.0 mg, approx. 33 nmol DNA) in a reaction cup. The coupling was allowed to proceed for 6 h with occasional gentle vortexing. The supernatant was aspired and the support washed with DMF, MeOH, and CH₂Cl₂, followed by drying in an air stream. Chemset 1 thus prepared was treated with sat. aq NH₃ (NH₄OH, 150 µL) for 16 h at r.t. to give a solution of chemset 2 that was freed from excess NH₃ by exposure to air for 4 h. The solution was aspired and analyzed by MALDI-TOF mass spectrometry, as previously described (ref. [6] ).

23

A slurry of pyrrole 4 (19.3 mg, 53 µmol) in DMF (420 µL) was activated with HBTU, HOBT and DIEA in DMF for 1 h, as described for the reactivity assays above. The activation solution was added to a conical glass vial for small scale microwave-assisted reactions containing 19 (1.0 mg, approx. 33 nmol DNA), followed by irradiation in a CEM Discover chemical microwave apparatus using the settings for DMF at up to 200 W, a ceiling temperature of 80 °C, and a hold time of 10 min. After cooling, the support was washed with DMF, MeOH, and CH₂Cl₂, followed by air drying and deprotection with sat. aq NH₃.