A Reactivity Test for HBTU-Activated Carboxylic Acids with Low Reactivity and Competitive Coupling ofN-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.Combinatorial searches for new structural motives that bind tightly to a given target can be performed in a number of different ways. 1 For modified oligonucleotides the synthesis of small combinatorial chemsets (libraries) that are then subjected to mass spectrometrically monitored selection experiments (SMOSEs) 2 has been one successful approach. 3 Other approaches involving combinatorial steps and spectrometric or spectroscopic techniques are known. 4,5 The small chemsets employed in SMOSE assays are usually prepared via mixed coupling of carboxylic acids and amine-terminated DNA on solid support. 3 After high yielding mixed couplings, crude libraries can be assayed directly, without purification or pooling steps, greatly enhancing the throughput. SMOSE assays require the unambiguous detection of all components of a given chemset. Since the reactivity of building blocks varies with structure, equireactive mixtures of the building blocks have to be generated for mixed couplings by adjusting the concentration of building blocks such that increased reactivity is compensated by a lower concentration and decreased reactivity by a higher concentration. This demands a knowledge of the relative reactivity of the active esters of the carboxylic acids. Since conventional methods of determining rate constants are time consuming we developed a rapid assay for determining relative reactivities, 6 building on earlier work on combinatorial libraries of porphyrins. 7 The assay involves competitive coupling reactions between test and reference acid with a porphyrin tetraamine under pseudo-first order conditions and product analysis via MALDI-TOF mass spectra. 6One class of carboxylic acids, namely the N-methylpyrrole carboxylic acids, has not yet been explored by SMOSE. Methylpyrroles are building blocks for oligopyrroleamides that bind sequence-specifically in the minor groove of DNA duplexes, 8,9 and conjugates of oligonucleotides and oligopyrroleamides show promising binding properties. 10 Our recent synthesis of stilbenelinked DNA-oligopyrroleamide hybrids required separate assembly of oligopyrroles in solution, 11 as coupling to amino-terminated DNA on controlled pore glass (cpg) was extremely low yielding. Attempts to terminate stilbene caps 12 in pyrrole amides using mixed coupling were also...