Richard W. Wagner

Directed Evolution of High-Affinity Antibody Mimics Using mRNA Display

We constructed a library of >10(12) unique, covalently coupled mRNA-protein molecules by randomizing three exposed loops of an immunoglobulin-like protein, the tenth fibronectin type III domain (10Fn3). The antibody mimics that bound TNF-alpha were isolated from the library using mRNA display. Ten rounds of selection produced 10Fn3 variants that bound TNF-alpha with dissociation constants (K(d)) between 1 and 24 nM. After affinity maturation, the lowest K(d) measured was 20 pM. Selected antibody mimics were shown to capture TNF-alpha when immobilized in a protein microarray. 10Fn3-based scaffold libraries and mRNA-display allow the isolation of high-affinity, specific antigen binding proteins; potential applications of such binding proteins include diagnostic protein microarrays and protein therapeutics.

Boron-dipyrromethene dyes for incorporation in synthetic multi-pigment light-harvesting arrays

The synthesis of light-harvesting arrays requires modular pigment building blocks. Boron-dipyrromethene dyes are well-suited for the photochemical sensitization of free base or metalloporphyrins. Direct access to borondipyrromethene dyes bearing reactive substituents is provided by a simple synthetic route involving two one-flask reactions. Reaction of pyrrole or 2-methylpyrrole with an aldehyde under gentle acid catalysis affords the corresponding dipyrromethane. Subsequent oxidation (DDQ or p-chloranil) and complexation affords the dye. Boron-dipyrromethene dyes bearing iodo- or ethynyl-phenyl substituents at the meso-position have been prepared as modular components for building block applications. These dyes are stable, accessible, and have suitable spectroscopic features for use as porphyrin accessory pigments in light-harvesting arrays.

Porphyrin building blocks for modular construction of bioorganic model systems

Abstract We outline a modular building block strategy for the covalent assembly of porphyrin-containing model systems. Molecular design issues for the synthesis of porphyrin dimers, dye-porphyrin dyads, and multi-porphyrin arrays have been used to guide the development of this approach. The major design constraints are to achieve directed coupling of free base and/or metalloporphyrin building blocks in dilute solution under non-acidic non-metalating conditions. A set of 24 porphyrin building blocks has been synthesized. The porphyrins are prepared by reaction of substituted benzaldehydes with pyrrole using the two-step one-flask room temperature porphyrin synthesis. Routes to 7 substituted aldehydes are described. Each porphyrin bears one or four functional groups, and many also are facially-encumbered in order to achieve increased solubility. 12 functional groups that meet the design criteria include those that can be reacted directly in coupling reactions such as active esters, α-chloroacetamido, benzoyl, and iodo groups, and others that require deprotection prior to coupling, such as phthalimidomethyl, FMOC-prolyl, trimethylsilylethynyl, dithiolane, methoxycarbonyl, and trimethylsilylethoxycarbonyl groups. The synthesis of 13 porphyrin dimers and dye-porphyrin dyads has been explored as a testbed for refining strategies for the synthesis of molecular devices containing multiple porphyrins and other components. The coupling strategies investigated have yielded dimers or dyads with subunits linked by amide, butadiyne, ethyne, or thiourea groups. This building block approach should enable rapid assembly of architecturally-defined porphyrin-based model systems.

Generating addressable protein microarrays with PROfusion covalent mRNA-protein fusion technology.

An mRNA‐protein fusion consists of a polypeptide covalently linked to its corresponding mRNA. These species, prepared individually or en masse by in vitro translation with a modified mRNA conjugate (the PROfusion™ process), link phenotype to genotype and enable powerful directed evolution schemes. We have exploited the informational content of the nucleic acid component of the mRNA‐protein fusion to create an addressable protein microarray that self‐assembles via hybridization to surface‐bound DNA capture probes. The nucleic acid component not only directs the mRNA‐protein fusion to the proper coordinate of the microarray, but also positions the protein in a uniform orientation. We demonstrate the feasibility of this protein chip concept with several mRNA‐protein fusions, each possessing a unique peptide epitope sequence. These addressable proteins could be visualized on the microarray both by autoradiography and highly specific monoclonal antibody binding. The anchoring of the protein to the chip surface is surprisingly robust, and the system is sensitive enough to detect sub‐attomole quantities of displayed protein without signal amplification. Such protein arrays should be useful for functional screening in massively parallel formats, as well as other applications involving immobilized peptides and proteins.

An improved synthesis of tetramesitylporphyrin

Abstract We report a simple two-step one-flask procedure for the synthesis of tetramesitylporphyrin in 29% yield. Pyrrole and mesitaldehyde react at room temperature to form tetramesitylporphyrinogen. The addition of an oxidant yields the porphyrin. Macrocycle formation and oxidative aromatization are thus performed separately. The reaction at higher temperature results in a lower yield of porphyrin.

Cellular penetration and antisense activity by a phenoxazine-substituted heptanucleotide

One of the major barriers to the development of antisense therapeutics has been their poor bioavailability. Numerous oligonucleotide modifications have been synthesized and evaluated for enhanced cellular permeation with limited success. Phenoxazine, a tricyclic 2´ deoxycytidine analog, was designed to improve stacking interactions between heterocycles of oligonucleotide/RNA hybrids and to enhance cellular uptake. However, the bioactivity and cellular permeation properties of phenoxazine-modified oligonucleotides were unknown. Incorporation of four phenoxazine bases into a previously optimized C-5 propyne pyrimidine modified 7-mer phosphorothioate oligonucleotide targeting SV40 large T antigen enhanced in vitro binding affinity for its RNA target and redirected RNAse H-mediated cleavage as compared with the 7-mer C-5 propynyl phosphorothioate oligonucleotide (S-ON). The phenoxazine/C-5 propynyl U 7-mer S-ON showed dose-dependent, sequence-specific, and target-selective antisense activity following microinjection into cells. Incubation of the phenoxazine/C-5 propynyl U S-ON with a variety of tissue culture cells, in the absence of any cationic lipid, revealed unaided cellular penetration, nuclear accumulation, and subsequent antisense activity. The unique permeation properties and gene-specific antisense activity of the 7-mer phenoxazine/C-5 propynyl U S-ON paves the way for developing potent, cost-effective, self-permeable antisense therapeutics.

Synthesis of porphyrins tailored with eight facially-encumbering groups. An approach to solid-state light-harvesting complexes

Abstract Synthetic models of the photosynthetic antenna complexes must achieve long-range 3-dimensional order encompassing a large number of porphyrinic pigments with limited direct contact of the pigments. In order to develop solid-state antenna complexes, we have synthesized porphyrins bearing benzyloxy groups projecting over both faces and optionally also around the periphery of the porphyrin. Routes have been established for prefunctionalizing benzaldehydes with various benzyloxy groups. Reaction of 2,6-bis, 3,5-bis, or 2,4,6-tris-(benzyloxy)benzaldehydes with pyrrole via the room temperature two-step one-flask porphyrin reaction provides direct access to the facially-encumbered porphyrins. The benzyloxybenzaldehydes react as efficiently as methoxybenzaldehydes, indicating the utility of the -OCH2- unit for introducing large substituents near the face of the porphyrin. The octakis and dodecakis(benzyloxy)porphyrins exhibit characteristic porphyrin absorption and fluorescence properties in solution. The crystal structure of meso-tetrakis[2,6-bis(2,3,4,5,6-pentafluorobenzyloxy)phenyl]porphyrin has been determined. The pentafluorobenzyloxy substituents provide a cavity on each side of the porphyrin plane which has an approximate cylindrical shape with a diameter of ~ 7.5 A and a height of ≥ 4.5 A. The porphyrin core parameters are those obtained for free base derivatives in which the inner hydrogen atoms are ordered. Crystal data: a = 14.759 (1) A, b = 25.519 (2) A, c = 13.100 (1) A, α = 100.04 (1), β = 99.83 (1), γ = 88.25 (1), V = 4767.3 (6) A3, all measurements at 127 K, triclinic, space group P 1 , Z = 2 R1(F) = 0.097, for 10020 “observed” data, and wR2(F2) = 0.275 for 17761 total unique (all) data.

Investigation of the one-flask synthesis of porphyrins bearing meso-linked straps of variable length, rigidity, and redox activity

Abstract The reactions of 18 dialdehydes have been examined in the two-step one-flask room temperature porphyrin synthesis. Efficient alkylation methods were established for the reaction of diols and diacids with m -bromomethylbenzaldehyde. Dialdehydes linked at the o,o′-or m,m′-positions were converted to strapped porphyrins in yields up to 25%, while the one p,p′-linked dialdehyde that was examined failed to give porphyrin. The resulting porphyrins bear straps joining adjacent meso -positions rather than across the face of the porphyrin. Dialdehydes incorporating rigid groups provided improved yields in some but not all cases. The yield of strapped porphyrin exhibited a maximum at 10 −2 M reactant concentrations. The o,o′-strapped porphyrins exist as atropisomers that are sufficiently stable to interconversion at room temperature to be separable chromatographically. No atropisomers of m,m′-strapped porphyrins could be separated, though some could be observed by 1 H NMR spectroscopy. For two different m,m′-strapped porphyrins, the ΔG ‡ values for interconversion of the atropisomers were found to be 66 and 68 kJ/mol. The outer rings in these strapped porphyrins range in size from 14 to 24 atoms. Five porphyrins with bridging redox-active groups (ferrocene or anthraquinone) have been prepared in one-flask reactions, including a porphyrin bearing one ferrocene and one anthraquinone in straps across adjacent meta-substituted phenyl sites.

Synthesis of facially-encumbered porphyrins. An approach to light-harvesting antenna complexes

Abstract 2,6-di- and 2,4,6-tri-benzyloxybenzaldehydes are converted to the respective octa- and dodeca-benzyloxy porphyrins in 9–52% yields via the two-step one-flask room-temperature porphyrin synthesis.

Self-assembly of molecular devices containing a ferrocene, a porphyrin and a quinone in a triple macrocyclic architecture

Porphyrinogen self-assembly is compatible with a structurally diverse set of meta-linked dialdehyde building blocks, providing rapid access under gentle conditions to porphyrins bearing redox-active units positioned in a 3-dimensional architecture.