Sequence-specific Synthesis Research Articles

Two-Component Redox Organocatalyst for Peptide Bond Formation.

Peptides are fundamental therapeutic modalities whose sequence-specific synthesis can be automated. Yet, modern peptide synthesis remains atom uneconomical and requires an excess of coupling agents and protected amino acids for efficient amide bond formation. We recently described the rational design of an organocatalyst that can operate on Fmoc amino acids─the standard monomers in automated peptide synthesis (J. Am. Chem. Soc. 2019, 141, 15977). The catalytic cycle centered on the conversion of the carboxylic acid to selenoester, which was activated by a hydrogen bonding scaffold for amine coupling. The selenoester was generated in situ from a diselenide catalyst and stoichiometric amounts of phosphine. Although the prior system catalyzed oligopeptide synthesis on solid phase, it had two significant requirements that limited its utility as an alternative to coupling agents─it depended on stoichiometric amounts of phosphine and required molecular sieves as dehydrating agent. Here, we address these limitations with an optimized method that requires only catalytic amounts of phosphine and no dehydrating agent. The new method utilizes a two-component organoreductant/organooxidant-recycling strategy to catalyze amide bond formation.

Peptoid microsphere coatings to improve performance in sandwich ELISA microarrays

Enzyme-linked immunosorbent assay (ELISA) microarray performance is limited by low assay sensitivity and dynamic range. Increasing the surface area for reagent binding can help to improve performance, but standard techniques such as roughening the surface or adding a polymer coating lead to increased non-specific fluorescence and do not have reproducibly improved performance. Another approach to increase surface area is adding a microsphere coating on the surface. Poly-N-substituted glycine (peptoid) microspheres are ideal for this application due to low immunogenicity, protease-resistance, and biocompatibility. Peptoids are polymers with a backbone similar to peptides, but with the side chains appended to nitrogen rather than the alpha carbon. A variety of side chain chemistries can be incorporated into peptoids through a solid-phase, sequence-specific synthesis protocol. Here we report the development of sandwich ELISA microarray on peptoid microsphere coated glass slides. Coating morphology was evaluated via SEM and efficacy was assessed by ELISA microarray performance. Peptoid microsphere coated glass slides exhibit an increase in signal intensity and dynamic range as compared to commercially available microarray slides. These studies show the potential for peptoid microspheres as coatings for ELISA microarray slides, as well as for use in other biosensor applications.

Diverging from Nature: A New Reactivity for Molecular Synthesizers

Download : Download high-res image (475KB) Download : Download full-size image Dr. Charlie McTernan is currently a Leverhulme Early Career Research Fellow and a Research Fellow at Sidney Sussex College, Cambridge, where he works in the group of Prof. Jonathan Nitschke on the design and synthesis of asymmetric molecular capsules. In 2013, he received his MChem from the University of Oxford, where he worked in Prof. Tim Donohoe’s group for his fourth year. He then moved to work with Prof. David Leigh FRS in Manchester for his PhD, during which he investigated the synthesis of artificial molecular machines capable of unidirectional motion and sequence-specific synthesis, before moving to Cambridge in 2017.

Stereodivergent synthesis with a programmable molecular machine.

It has been convincingly argued that molecular machines that manipulate individual atoms, or highly reactive clusters of atoms, with Ångström precision are unlikely to be realized. However, biological molecular machines routinely position rather less reactive substrates in order to direct chemical reaction sequences, from sequence-specific synthesis by the ribosome to polyketide synthases, where tethered molecules are passed from active site to active site in multi-enzyme complexes. Artificial molecular machines have been developed for tasks that include sequence-specific oligomer synthesis and the switching of product chirality, a photo-responsive host molecule has been described that is able to mechanically twist a bound molecular guest, and molecular fragments have been selectively transported in either direction between sites on a molecular platform through a ratchet mechanism. Here we detail an artificial molecular machine that moves a substrate between different activating sites to achieve different product outcomes from chemical synthesis. This molecular robot can be programmed to stereoselectively produce, in a sequential one-pot operation, an excess of any one of four possible diastereoisomers from the addition of a thiol and an alkene to an α,β-unsaturated aldehyde in a tandem reaction process. The stereodivergent synthesis includes diastereoisomers that cannot be selectively synthesized through conventional iminium-enamine organocatalysis. We anticipate that future generations of programmable molecular machines may have significant roles in chemical synthesis and molecular manufacturing.

Efficient Assembly of Threaded Molecular Machines for Sequence-Specific Synthesis

We report on an improved strategy for the preparation of artificial molecular machines that can pick up and assemble reactive groups in sequence by traveling along a track. In the new approach a preformed rotaxane synthon is attached to the end of an otherwise fully formed strand of building blocks. This "rotaxane-capping" protocol is significantly more efficient than the "final-step-threading" method employed previously and enables the synthesis of threaded molecular machines that operate on extended oligomer, and potentially polymer, tracks. The methodology is exemplified through the preparation of a machine that adds four amino acid building blocks from a strand in sequence, featuring up to 20-membered ring native chemical ligation transition states.

Fully Automated Sequence-Specific Synthesis of α-Peptides Using Flow Chemistry

The efficient and convenient synthesis and manipulation of the peptides beyond what is normally feasible to achieve in a living cell offers a powerful strategy for investigating the complex processes that occur in living organisms and may lead to elucidation of mechanisms that are of direct relevance to new drug discovery initiatives. We report an efficient and sequence-specific stop flow strategy for the synthesis of α-peptides using natural amino acids as well as synthetic amino acid analogues, activated as N-carboxyanhydrides with traceless protection groups. The coupling step is carried out by a robotic liquid handler and by maintaining the product in the liquid phase we have overcome several challenges posed by traditional peptide synthesis. The removal of the protection group uses a continuous flow hydrogenolysis method for highly efficient deprotection. We demonstrate that high yields and purities are obtainable via this new automated solution-phase approach that runs under a dedicated software con...

Sequence-Specific Synthesis of Platinum-Conjugated Trichromophoric Energy Cascades of Anthracene, Tetracene, and Pentacene and Fluorescent “Black Chromophores”

A sequence-specific synthesis of trichromophoric energy cascades containing anthracenyl (A), tetracenyl (T), or pentacenyl (P) rings is achieved by coupling PtII ions and singly desilylated bis(triisopropylsilylethynyl)acenes (acene = anthracene, tetracene, or pentacene). Four sequences, T-P-T, P-T-P, A-P-T, and A-T-P, are generated. Absorption spectra of the triads show intense bands due to the S0 → S1 transitions of the acenes. The A-P-T and A-T-P, being able to absorb strongly throughout the entire visible region, are rare examples of “black chromophores”. Different sequences are shown to have different emission fingerprints. Emission spectra of A-P-T and A-T-P show that excitation at the S0 → S1 band of A gives rise to emissions from the S1 excited states of A, T, and P, implying the presence of energy transfer from the excited state of A to those of T and P.

Multiscale modeling of metabolism and macromolecular synthesis in E. coli and its application to the evolution of codon usage.

Biological systems are inherently hierarchal and multiscale in time and space. A major challenge of systems biology is to describe biological systems as a computational model, which can be used to derive novel hypothesis and drive experiments leading to new knowledge. The constraint-based reconstruction and analysis approach has been successfully applied to metabolism and to the macromolecular synthesis machinery assembly. Here, we present the first integrated stoichiometric multiscale model of metabolism and macromolecular synthesis for Escherichia coli K12 MG1655, which describes the sequence-specific synthesis and function of almost 2000 gene products at molecular detail. We added linear constraints, which couple enzyme synthesis and catalysis reactions. Comparison with experimental data showed improvement of growth phenotype prediction with the multiscale model over E. coli’s metabolic model alone. Many of the genes covered by this integrated model are well conserved across enterobacters and other, less related bacteria. We addressed the question of whether the bias in synonymous codon usage could affect the growth phenotype and environmental niches that an organism can occupy. We created two classes of in silico strains, one with more biased codon usage and one with more equilibrated codon usage than the wildtype. The reduced growth phenotype in biased strains was caused by tRNA supply shortage, indicating that expansion of tRNA gene content or tRNA codon recognition allow E. coli to respond to changes in codon usage bias. Our analysis suggests that in order to maximize growth and to adapt to new environmental niches, codon usage and tRNA content must co-evolve. These results provide further evidence for the mutation-selection-drift balance theory of codon usage bias. This integrated multiscale reconstruction successfully demonstrates that the constraint-based modeling approach is well suited to whole-cell modeling endeavors.

Biophysical mimicry of lung surfactant protein B by random nylon-3 copolymers.

Non-natural oligomers have recently shown promise as functional analogues of lung surfactant proteins B and C (SP-B and SP-C), two helical and amphiphilic proteins that are critical for normal respiration. The generation of non-natural mimics of SP-B and SP-C has previously been restricted to step-by-step, sequence-specific synthesis, which results in discrete oligomers that are intended to manifest specific structural attributes. Here we present an alternative approach to SP-B mimicry that is based on sequence-random copolymers containing cationic and lipophilic subunits. These materials, members of the nylon-3 family, are prepared by ring-opening polymerization of beta-lactams. The best of the nylon-3 polymers display promising in vitro surfactant activities in a mixed lipid film. Pulsating bubble surfactometry data indicate that films containing the most surface-active polymers attain adsorptive and dynamic-cycling properties that surpass those of discrete peptides intended to mimic SP-B. Attachment of an N-terminal octadecanoyl unit to the nylon-3 copolymers, inspired by the post-translational modifications found in SP-C, affords further improvements by reducing the percent surface area compression to reach low minimum surface tension. Cytotoxic effects of the copolymers are diminished relative to that of an SP-B-derived peptide and a peptoid-based mimic. The current study provides evidence that sequence-random copolymers can mimic the in vitro surface-active behavior of lung surfactant proteins in a mixed lipid film. These findings raise the possibility that random copolymers might be useful for developing a lung surfactant replacement, which is an attractive prospect given that such polymers are easier to prepare than are sequence-specific oligomers.

Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7

DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5′-TGGTC-3′) than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain.

DNA polymerase kappa produces interrupted mutations and displays polar pausing within mononucleotide microsatellite sequences

Microsatellites are ubiquitously present in eukaryotic genomes and are implicated as positive factors in evolution. At the nucleotide level, microsatellites undergo slippage events that alter allele length and base changes that interrupt the repetitive tract. We examined DNA polymerase errors within a [T]11 microsatellite using an in vitro assay that preferentially detects mutations other than unit changes. We observed that human DNA polymerase kappa (Pol κ) inserts dGMP and dCMP within the [T]11 mononucleotide repeat, producing an interrupted 12-bp allele. Polymerase β produced such interruptions at a lower frequency. These data demonstrate that DNA polymerases are capable of directly producing base interruptions within microsatellites. At the molecular level, expanded microsatellites have been implicated in DNA replication fork stalling. Using an in vitro primer extension assay, we observed sequence-specific synthesis termination by DNA polymerases within mononucleotides. Quantitatively, intense, polar pausing was observed for both pol κ and polymerase α-primase within a [T]11 allele. A mechanism is proposed in which pausing results from DNA bending within the duplex stem of the nascent DNA. Our data support the concept of a microsatellite life-cycle, and are consistent with the models in which DNA sequence or secondary structures contributes to non-uniform rates of replication fork progression.

Synthesis of Oligonucleotides Containing the N2-Deoxyguanosine Adduct of the Dietary Carcinogen 2-Amino-3-methylimidazo[4,5-f]quinoline

2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is a highly mutagenic heterocyclic amine formed in all cooked meats. IQ has been found to be a potent inducer of frameshift mutations in bacteria and carcinogenic in laboratory animals. Upon metabolic activation, IQ forms covalent adducts at the C8- and N2-positions of deoxyguanosine with a relative ratio of up to approximately 4:1. We have previously incorporated the major dGuo-C8-IQ adduct into oligonucleotides through the corresponding phosphoramidite reagent. We report here the sequence-specific synthesis of oligonucleotides containing the minor dGuo-N2-IQ adduct. Thermal melting analysis revealed that the dGuo-N2-IQ adduct significantly destabilizes duplex DNA.