Solid-phase Synthesis Protocol Research Articles

Efficient automated solid-phase synthesis of recognition-encoded melamine oligomers.

Recognition-encoded melamine oligomers (REMO) are synthetic polymers with an alternating 1,3,5-triazine-piperazine backbone and side chains equipped with either a phenol or phosphine oxide recognition unit. Here, we describe an automated method for highly efficient solid-phase synthesis (SPS) of REMO of any specified length and sequence. These SPS protocols are amongst the most robust reported to date, as demonstrated by the synthesis of a mixed-sequence 42-mer, which was obtained in excellent crude purity on a 100 mg scale. Starting from loaded Wang resin and dichlorotriazine monomer building blocks, the SPS methods were automated and optimised on a commercial peptide synthesiser. Major side products were identified using LCMS, and the undesired side reactions were suppressed by the choice of resin, solvent and coupling conditions. REMO have been shown to form high-fidelity length- and sequence-selective H-bonded duplexes, analogous to nucleic acids, and automated synthesis will facilitate exploration of related functional properties, such as molecular replication and programmable self-assembly.

Macrocyclic β‐Sheets Stabilized by Hydrogen Bond Surrogates

AbstractMimics of protein secondary and tertiary structure offer rationally‐designed inhibitors of biomolecular interactions. β‐Sheet mimics have a storied history in bioorganic chemistry and are typically designed with synthetic or natural turn segments. We hypothesized that replacement of terminal inter‐β‐strand hydrogen bonds with hydrogen bond surrogates (HBS) may lead to conformationally‐defined macrocyclic β‐sheets without the requirement for natural or synthetic β‐turns, thereby providing a minimal mimic of a protein β‐sheet. To access turn‐less antiparallel β‐sheet mimics, we developed a facile solid phase synthesis protocol. We surveyed a dataset of protein β‐sheets for naturally observed interstrand side chain interactions. This bioinformatics survey highlighted an over‐abundance of aromatic–aromatic, cation‐π and ionic interactions in β‐sheets. In correspondence with natural β‐sheets, we find that minimal HBS mimics show robust β‐sheet formation when specific amino acid residue pairings are incorporated. In isolated β‐sheets, aromatic interactions endow superior conformational stability over ionic or cation‐π interactions. Circular dichroism and NMR spectroscopies, along with high‐resolution X‐ray crystallography, support our design principles.

Macrocyclic β-Sheets Stabilized by Hydrogen Bond Surrogates.

Mimics of protein secondary and tertiary structure offer rationally-designed inhibitors of biomolecular interactions. β-Sheet mimics have a storied history in bioorganic chemistry and are typically designed with synthetic or natural turn segments. We hypothesized that replacement of terminal inter-β-strand hydrogen bonds with hydrogen bond surrogates (HBS) may lead to conformationally-defined macrocyclic β-sheets without the requirement for natural or synthetic β-turns, thereby providing a minimal mimic of a protein β-sheet. To access turn-less antiparallel β-sheet mimics, we developed a facile solid phase synthesis protocol. We surveyed a dataset of protein β-sheets for naturally observed interstrand side chain interactions. This bioinformatics survey highlighted an over-abundance of aromatic-aromatic, cation-π and ionic interactions in β-sheets. In correspondence with natural β-sheets, we find that minimal HBS mimics show robust β-sheet formation when specific amino acid residue pairings are incorporated. In isolated β-sheets, aromatic interactions endow superior conformational stability over ionic or cation-π interactions. Circular dichroism and NMR spectroscopies, along with high-resolution X-ray crystallography, support our design principles.

Optimization and Automation of Helical Aromatic Oligoamide Foldamer Solid-Phase Synthesis.

Helically folded oligoamides of 8-amino-2-quinolinecarboxylic acid composed of up to 41 units were prepared using optimized manual solid-phase synthesis (SPS). The high yield and purity of the final products places these SPS protocols among the most efficient known to date. Furthermore, analytical methods allowing for the clear identification and purity assessment of the products were validated, including 1 H NMR, a seldom used method for such large molecules. Adaption of the SPS protocols, in particular using in situ acid chloride activation under Appel's conditions, made it possible to efficiently implement SPS on a commercial peptide synthesizer, leading to a dramatic reduction of the laboratory work required to produce long sequences. Automation constitutes a breakthrough for the development of helical aromatic oligoamide foldamers.

A magnetic molecularly imprinted nanoparticle assay (MINA) for detection of pepsin

A fully abiotic Molecularly Imprinted Nanoparticle Assay (MINA) is developed for detection of the model protein pepsin. The format of the pepsin assay is based on binding of fluorescent pepsin-specific molecularly imprinted polymer nanoparticles (nanoMIPs) to the magnetic pepsin nanoparticles (MPN) immobilised on magnetic microtiter plate inserts. Competition between free pepsin and immobilised pepsin results in a decrease in nanoMIPs binding to the magnetic inserts, resulting in an increase in fluorescence. Pepsin-specific are prepared using a solid phase synthesis protocol. In order to increase the sensitivity of MINA, two labelling approaches are performed. The first approach uses a fluorescein acrylate included in the monomeric mixture during polymerisation, and the second approach is based on a post-imprinting modification of nanoparticles using the commercial fluorophore AlexaFluor® 647 NHS ester. It is observed that upon increase of free pepsin concentration from 1 μM to 100 μM, fluorescence is increased by 68%. No cross-reactivity in the presence of non-specific protein trypsin is detected. The results show that AlexaFluor-labelled nanoMIPs demonstrate higher performance towards pepsin than fluorescein-labelled nanoMIPs. The novel assay reduces the time and cost of analysis and does not uses any antibodies, eliminating the need for animal-derived reagents. The portfolio of the optimised protocols can potentially be applied for the detection of any other proteins of clinical and environmental interest.

Chemosensor Based on Molecularly Imprinted Nanoparticles for Selective Determination of Glyphosate

Glyphosate is a commonly used herbicide that can kill some weeds and grasses. Unfortunately, exposure to glyphosate is considered potentially harmful to humans. It may cause liver and kidney damage as well as can affect the endocrine system.1 Furthermore, the International Agency for Research on Cancer considers glyphosate as a probable carcinogen.2 Glyphosate determination often requires additional steps allowing quantification by commonly employed chromatographic techniques. Therefore, low-cost, rapid, and reliable ways to determine this pesticide are essential.We devised and fabricated an electrochemical chemosensor for the selective determination of the target toxin, glyphosate. As a recognition unit in this sensor, electroactive molecularly imprinted nanoparticles (nanoMIPs) with an internal redox probe were used.3 The MIP nanoparticles were synthesized using a solid-phase synthesis protocol.4 The nanoMIPs were characterized by several methods, including dynamic light scattering (DLS), scanning electron microscopy (SEM), and X-ray photoelectron spectroscopy (XPS). The obtained nanoparticles were covalently immobilized on screen-printed platinum electrodes and successfully used for electrochemical determination of glyphosate in the absence of any external redox probe. Chemosensor fabricated that way demonstrated high sensitivity and selectivity with respect to structural analogs of glyphosate and other phosphate-containing analytes. The practical application of this self-reporting MIP-based chemosensor for detecting pesticide was further verified in river water samples with satisfying results. These results prove that our sensor's detectability is sufficient for the on-site detection of glyphosate pesticide.

Molecularly Imprinted Nanoparticles (NanoMIPs) Selective for Proteins: Optimization of a Protocol for Solid-Phase Synthesis Using Automatic Chemical Reactor.

Molecularly imprinted polymer nanoparticles (nanoMIPs) are receiving broad interest as robust and highly selective synthetic receptors for a variety of molecules. Due to their stability, inexpensive synthesis and easy implementation, they are considered a promising alternative to antibodies in sensors, diagnostics and separation applications. The most challenging targets for the production of synthetic receptors are proteins due to their fragile nature and the multitude of possible binding sites in their structure. Herein, we describe the modification and optimization of the protocol for synthesis of nanoMIPs with specificity for proteins using the prototype of an automated solid-phase synthesizer. Using an automated system gives an advantage for the simple, fast and fully controlled, reproducible production of nanoMIPs. The molecular imprinting in the reactor is performed using a template covalently immobilized on a solid support, in mild conditions suitable for preserving protein native structure. The validation of the protocol was made by assessing the ability to regenerate a solid-phase, and by measuring affinity and specificity of nanoparticles. As a model protein, we have chosen trypsin since its enzymatic activity can be easily monitored by using a commercial colorimetric assay. Different protocols were tested for their ability to improve the yield of high affinity nanoparticles in the final elution.

Sequence-defined oligoampholytes using hydrolytically stable vinyl sulfonamides: design and UCST behaviour

Water soluble sequence-defined oligoampholytes with precisely positioned charges were synthesised via an iterative solid-phase synthesis protocol using vinyl sulfonamide and acrylate building blocks.

Generation of High-Affinity Molecularly Imprinted Nanoparticles for Protein Recognition via a Solid-Phase Synthesis Protocol.

Molecularly imprinted polymers are leading technology in the development of protein biomimetics. This chapter describes the protocol for the synthesis of protein imprinted nanoparticles. These materials exhibit exceptional affinity (into the nM/pM range) and selectivity for their target template. The nanoparticles can be developed for a wide range of targets, while exhibiting excellent robustness, solubility, and flexibility in use. They are finding use in the creation of drug delivery vectors and sensing and recognition assays.

Synthesis of N2 -Aryl-2'-Deoxyguanosine Modified Phosphoramidites and Oligonucleotides.

The N2 -position of 2'-deoxyguanosine (N2 -position in dG) is well known for forming carcinogenic minor groove DNA adducts, which originate from environmental pollutants, chemicals, and tobacco smoke. The N2 -dG DNA adducts have strong implications on biological processes such as DNA replication and repair and may, therefore, result in genomic instability by generating mutations or even cell death. It is crucial to know the role of DNA polymerases when they encounter the N2 -dG damaged site in DNA. To get detailed insights on the in vitro DNA damage tolerance or bypass mechanism, there is a need to synthetically access N2 -dG damaged DNAs. This article describes a detailed protocol of the synthesis of N2 -aryl-dG modified nucleotides using the Buchwald-Hartwig reaction as a main step and incorporation of the modified nucleotides into DNA. In Basic Protocol 1, we focused on the synthesis of five different N2 -dG modified phosphoramidites with varying bulkiness (benzyl to pyrenyl). Basic Protocol 2 describes the details of synthesizing N2 -dG modified oligonucleotides employing the standard solid phase synthesis protocol. This strategy provides robust synthetic access to various modifications at the N2 -position of dG; the modified dGs serve as good substrates to study translesion synthesis and repair pathways. Overall data presented in this article are based on earlier published reports. © 2019 by John Wiley & Sons, Inc.

Studies on acid stability and solid-phase block synthesis of peptide-peptoid hybrids: ligands for formyl peptide receptors.

α-Peptoids as well as peptide/α-peptoid hybrids and peptide/β-peptoid hybrids constitute major classes of proteolytically stable peptidomimetics that have been extensively investigated as mimetics of biologically active peptides. Representatives of lipidated peptide/β-peptoid hybrids have been identified as promising immunomodulatory lead compounds, and hence access to these via protocols suitable for gram-scale synthesis is warranted to enable animal in vivo studies. Recent observations indicated that several byproducts appear in crude mixtures of relatively short benzyl-based peptide/β-peptoid oligomers, and that these were most predominant when the β-peptoid units displayed an α-chiral benzyl side chain. This prompted an investigation of their stability under acidic conditions. Simultaneous deprotection and cleavage of peptidomimetics containing either α-chiral α- or β-peptoid residues required treatment with strong acid only for a short time to minimize the formation of partially debenzylated byproducts. The initial work on peptide/β-peptoid oligomers with an alternating design established that it was beneficial to form the amide bond between the carboxyl group of the α-amino acid and the congested amino functionality of the β-peptoid residue in solution. To further simplify oligomer assembly on solid phase, we now present a protocol for purification-free solid-phase synthesis of tetramericbuilding blocks. Next, syntheses of peptidomimetic ligands via manual solid-phase methodologies involving tetramericbuilding blocks were found to give more readily purified products as compared to those obtained with dimericbuilding blocks. Moreover, the tetramericbuilding blocks could be utilized in automated synthesis with microwave-assisted heating, albeit the purity of the crude products was not increased.

Preparation of Enzyme-Activated Thapsigargin Prodrugs by Solid-Phase Synthesis.

Since cells in solid tumors divide less rapidly than cells in the bone marrow or cells of the immune system, mitotic inhibitors often cause severe side effects when used for treatment of diseases like prostate cancer and breast cancer. One approach to overcome this problem involves attempts at developing drugs based on general cytotoxins, like calicheamicin and thapsigargin, which kill cells at all phases of the cell cycle. However, such toxins can only be used when efficient targeting to the malignant tissue is possible. In the case of thapsigargin, selectivity for tumor-associated cells is achieved by conjugating the drug to a peptide that is only cleaved in the vicinity of tumors to release the cytotoxic drug or an analog with retained activity. Solid-phase synthesis protocols were developed for preparation of three already validated prodrugs of thapsigargin: one prodrug cleavable by human kallikrein 2, one prodrug cleavable by prostate-specific antigen, and one prodrug cleavable by prostate-specific membrane antigen.

Synthesis, Characterization, and Function of an RNA-Based Transfection Reagent.

A synthetic 8-mer, amphipathic, trans-acting poly-2'-O-methyluridylic thiophosphate triester RNA element (2'-OMeUtaPS) can be prepared using solid-phase synthesis protocols. 2'-OMeUtaPS efficiently mediates the delivery of uncharged polyA-tailed phosphorodiamidate morpholino (PMO) sequences in HeLa pLuc 705 cells, as evidenced by flow cytometry measurements. In this cell line, 2'-OMeUtaPS-mediated transfection of an antisense polyA-tailed PMO sequence induces alternative splicing of an aberrant luciferase pre-mRNA splice site, leading to restoration of functional luciferase, as quantitatively measured using a typical luciferase assay. 2'-OMeUtaPS is also potent at delivering an uncharged antisense polyA-tailed PMO sequence in muscle cells of the mdx mouse model of muscular dystrophy; targeting the polyA-tailed PMO sequence against a splice site of the pre-mRNA encoding mutated dystrophin triggers an alternate splicing event that results in excision of the mutated exon (exon 23) from the pre-mRNA and production of functional dystrophin, as demonstrated by agarose gel electrophoresis. © 2018 by John Wiley & Sons, Inc.

Structure-Activity Relationship Study Based on Autoinducing Peptide (AIP) from Dog Pathogen S. schleiferi.

Herein, an effective protocol for solid-phase synthesis of peptide thiolactones by concomitant ring closure and cleavage from the solid support is reported. The strategy was applied for mapping the importance of the structural features in S. schleiferi AIP (5) by performing an alanine scan and truncation of this natural compound. This furnished some of the most potent inhibitors of accessory gene regulator (agr)-I in the human pathogen S. aureus reported to date.

A High-Throughput Process for the Solid-Phase Purification of Synthetic DNA Sequences.

An efficient process for the purification of synthetic phosphorothioate and native DNA sequences is presented. The process is based on the use of an aminopropylated silica gel support functionalized with aminooxyalkyl functions to enable capture of DNA sequences through an oximation reaction with the keto function of a linker conjugated to the 5'-terminus of DNA sequences. Deoxyribonucleoside phosphoramidites carrying this linker, as a 5'-hydroxyl protecting group, have been synthesized for incorporation into DNA sequences during the last coupling step of a standard solid-phase synthesis protocol executed on a controlled pore glass (CPG) support. Solid-phase capture of the nucleobase- and phosphate-deprotected DNA sequences released from the CPG support is demonstrated to proceed near quantitatively. Shorter than full-length DNA sequences are first washed away from the capture support; the solid-phase purified DNA sequences are then released from this support upon reaction with tetra-n-butylammonium fluoride in dry dimethylsulfoxide (DMSO) and precipitated in tetrahydrofuran (THF). The purity of solid-phase-purified DNA sequences exceeds 98%. The simulated high-throughput and scalability features of the solid-phase purification process are demonstrated without sacrificing purity of the DNA sequences. © 2017 by John Wiley & Sons, Inc.

Solid Phase Synthesis of Helically Folded Aromatic Oligoamides.

Aromatic amide foldamers constitute a growing class of oligomers that adopt remarkably stable folded conformations. The folded structures possess largely predictable shapes and open the way toward the design of synthetic mimics of proteins. Important examples of aromatic amide foldamers include oligomers of 7- or 8-amino-2-quinoline carboxylic acid that have been shown to exist predominantly as well-defined helices, including when they are combined with α-amino acids to which they may impose their folding behavior. To rapidly iterate their synthesis, solid phase synthesis (SPS) protocols have been developed and optimized for overcoming synthetic difficulties inherent to these backbones such as low nucleophilicity of amine groups on electron poor aromatic rings and a strong propensity of even short sequences to fold on the solid phase during synthesis. For example, acid chloride activation and the use of microwaves are required to bring coupling at aromatic amines to completion. Here, we report detailed SPS protocols for the rapid production of: (1) oligomers of 8-amino-2-quinolinecarboxylic acid; (2) oligomers containing 7-amino-8-fluoro-2-quinolinecarboxylic acid; and (3) heteromeric oligomers of 8-amino-2-quinolinecarboxylic acid and α-amino acids. SPS brings the advantage to quickly produce sequences having varied main chain or side chain components without having to purify multiple intermediates as in solution phase synthesis. With these protocols, an octamer could easily be synthesized and purified within one to two weeks from Fmoc protected amino acid monomer precursors.

Acyclic analogs of nucleosides based on tris(hydroxymethyl)phosphine oxide: synthesis and incorporation into short DNA oligomers

Abstract Tris-(hydroxymethyl)phosphine oxide (THPO) to a certain extent resembles a part of 2′-deoxyribofuranose, although it exists in an acyclic form only and the oxygen atom at the THPO phosphorus center provides additional hydration site or acceptor of hydrogen bonds. After proper protection of hydroxyl groups, THPO was functionalized with nucleobases and converted into phosphoramidite monomers suitable for incorporation into growing oligonucleotide chains within the solid phase synthesis protocol. The resultant THPO-DNA analogs show reduced affinity to complementary DNA strands, and are resistant towards snake venom and calf spleen exonucleases.

Locked 2'-Deoxy-2',4'-Difluororibo Modified Nucleic Acids: Thermal Stability, Structural Studies, and siRNA Activity.

2'-Deoxy-2',4'-difluorouridine (2',4'-diF-rU) was readily incorporated into DNA and RNA oligonucleotides via standard solid phase synthesis protocols. NMR and thermal denaturation (Tm) data of duplexes was consistent with the 2',4'-diF-rU nucleotides adopting a rigid North (RNA-like) sugar conformation, as previously observed for the nucleoside monomer. The impact of this modification on Tm is neutral when incorporated within RNA:RNA duplexes, mildly destabilizing when located in the RNA strand of a DNA:RNA duplex, and highly destabilizing when inserted in the DNA strand of DNA:RNA and DNA:DNA duplexes. Molecular dynamics calculations suggest that the destabilization effect in DNA:DNA and DNA:RNA duplexes is the result of structural distortions created by A/B junctions within the helical structures. Quantum mechanics calculations suggest that the "neutral" effect imparted to A-form duplexes is caused by alterations in charge distribution that compensate the stabilizing effect expected for a pure North-puckered furanose sugar. 2',4'-diF-RNA modified siRNAs were able to trigger RNA interference with excellent efficiency. Of note, incorporation of a few 2',4'-diF-rU residues in the middle of the guide (antisense) strand afforded siRNAs that were more potent than the corresponding siRNAs containing LNA and 2'-F-ANA modifications, and as active as the 2'-F-RNA modified siRNAs.

Automated solid-phase synthesis of high capacity oligo-dT cellulose for affinity purification of poly-A tagged biomolecules

Affinity purification of poly-adenylated biomolecules using solid supports that are derivatized with poly-thymidine oligonucleotides provides a powerful method for isolating cellular mRNA. These systems have also been used to purify mRNA–peptide fusions generated by RNA-display. However, the commercial source for high capacity oligo-dT cellulose was recently discontinued. To overcome this problem, we have developed a low cost solid-phase synthesis protocol to generate oligo-dT cellulose. Comparative binding studies indicate that chemically synthesized oligo-dT cellulose functions with superior loading capacity when compared to the discontinued product. We suggest that this method could be used to synthesize oligo-dT resin for routine purification of poly-adenylated biomolecules.

Design and application of bispecific splice-switching oligonucleotides.

Targeting of pre-mRNA by short splice-switching oligonucleotides (SSOs) is increasingly being used as a therapeutic modality, one rationale being to disrupt splicing so as to remove exons containing premature termination codons, or to restore the translation reading frame around out-of-frame deletion mutations. The aim of this study was to investigate the effect of chemically linking individual SSOs so as to ascertain equimolar cellular uptake that would provide for more defined drug formulations. In contrast to conventional bispecific SSOs generated by conjugation in solution, here we describe a protocol for synthesis of bispecific SSOs on solid phase. These SSOs comprised of either a non-cleavable hydrocarbon linker or disulfide-based cleavable linkers. To assess the efficacy of these SSOs we have utilized splice switching to bypass a disease-causing mutation in the DMD gene concurrent with disruption of the reading frame of the myostatin gene (Mstn). The premise of this approach is that disruption of myostatin expression is known to induce muscle hypertrophy and so for Duchenne muscular dystrophy (DMD) could be expected to have a better outcome than dystrophin restoration alone. All tested SSOs mediated simultaneous robust exon removal from mature Dmd and Mstn transcripts in myotubes. Our results also demonstrate that using cleavable SSOs is preferred over the non-cleavable counterparts and that these are equally efficient at inducing exon skipping as cocktails of monospecific versions. In conclusion, we have developed a protocol for solid-phase synthesis of single molecule cleavable bispecific SSOs that can be efficiently exploited for targeting of multiple RNA transcripts.