Total Stepwise Solid-Phase Peptide-Oligonucleotide Conjugate Synthesis on Macroporous Polystyrene

Abstract

An efficient total stepwise solid-phase synthesis of oligonucleotide-peptide conjugates on a macroporous polystyrene is described. Extending our homoserine linker approach, we prepared a range of fluorescein-labelled conjugates containing one of two different peptides together with oligonucleotides containing 2′-deoxynucleoside or 2′-O-methylribonucleoside phosphodiesters, or gapmers containing 2′-deoxyphosphorothioate sequences flanked by 2′-O-methyl wings.     

The Stepwise Solid-Phase Synthesis Methodology is Suitable for the Preparation of a Great Variety of Nucleopeptides

Abstract

The preparation of nucleopeptides containing tryptophan and basic residues (lysine, arginine) is described. Different solid supports and the necessity of primary carboxamide protection have also been evaluated.     

Solid-Phase Synthesis of Modified Oligonucleotides

Synthetic oligonucleotides are ubiquitously found in most laboratories since solid-phase synthesis protocols have become highly optimized. These protocols make it possible to synthesize a large variety of modified oligonucleotides. As one example, we will review some of the developments regarding oligonucleotide synthesis from our own group. In particular, we will describe the synthesis of oligonucleotides carrying non-natural bases, of oligonucleotide–peptide conjugates, and of modified oligonucleotides used in the assembly of nanomaterials. This work is dedicated to the memory of Bruce Merrifield.     

A Rapid and Effective Solid-Phase Synthesis of DNA-Sequence-Specific Polyamides

A rapid and effective variant of solid-phase synthesis of DNA-sequence-specific polyamides on the basis of 4-amino-1-methylpyrrole-2-carboxylic acid, 4-amino-1-methylimidazole-2-carboxylic acid, -alanine, and -aminobutyric acid was suggested. The method is based on the use of di- and trimeric oligocarboxamide building blocks, which help reduce the time of synthesis, increase the yield and purity of products, and enables efficient use of manual synthesis for long oligocarboxamides. The yields of hairpin ligands with up to 10 units are 35–50% and the synthesis takes no more than 6 h.     

Further Optimization of Detritylation in Solid-Phase Oligodeoxyribonucleotide Synthesis

Various conditions for optimum detritylation (i.e., the removal of 5′-O-trityl protecting groups) during solid-phase synthesis of oligodeoxyribonucleotides were investigated. Di- and tri-chloroacetic acids of variable concentrations were used to study the removal of the 4,4′-dimethoxytrityl (DMTr) group. It was found that the DMTr group could be completely removed under much milder acidic conditions than what are currently used for automated solid-phase synthesis. The 2,7-dimethylpixyl (DMPx) is proposed as an alternative and more readily removable group for the protection of the 5′-OH functions both in solid- and solution-phase synthesis. The improved detritylation conditions are expected to minimize the waste and offer a protocol for incorporation of acid sensitive building-blocks in oligonucleotides.     

固相法合成多肽AN-M的工艺研究

目的:探讨生物活性肽AN-M的固相合成工艺,并为工业化合成目的肽提供理论依据。方法采用固相法,以Fmoc-保护基保护的α-氨基酸和Wang树脂为原料,经1—氧—3—双二甲胺羧基苯骈三氮唑四氟化硼盐(TBTU)、1—羟基苯并三氮唑(HoBt)、二异丙基乙胺(DIEA)缩合,20%哌啶的DMF溶液脱保护,用切割试剂将AN-M粗品从Wang树脂上切割下来。结果经反相高效液相色谱分析纯化,可得目的肽的得率大于67．00%,最终成品的纯度在98．78%以上,经质谱鉴定其分子量与理论值一致。结论该合成方法步骤简便、便于操作、产品得率高,可用于大规模合成目的肽。     

Ribo-, Xylo-, and Arabino-Configured Adenine-Based Nucleoside Phosphonates: Synthesis of Monomers for Solid-Phase Oligonucleotide Assembly

Abstract

Adenine-based, regioisomeric nucleoside phosphonates with ribo, xylo and arabino configuration were synthesized in the protected form suitable for the phosphotriester-like, solid-phase synthesis of oligonucleotides. Phosphonate moiety was protected by 4-methoxy-1-oxido-2-picolyl group and the furanose hydroxyl by the dimethoxytrityl group.     

Solid-Phase Synthesis of Heterobivalent Ligands Targeted to Melanocortin and Cholecystokinin Receptors

Heteromultivalency provides a route to increase binding avidity and to high specificity when compared to monovalent ligands. The enhanced specificity can potentially serve as a unique platform to develop diagnostics and therapeutics. To develop new imaging agents based upon multivalency, we employed heterobivalent constructs of optimized ligands. In this report, we describe synthetic methods we have developed for the preparation of heterobivalent constructs consisting of ligands targeted simultaneously to the melanocortin receptor, hMC4R, and the cholecystokinin receptors, CCK-2R. Modeling data suggest that a linker distance span of 20–50 Å is needed to crosslink these two G-protein coupled receptors (GPCRs). The two ligands were tethered with linkers of varying rigidity and length, and flexible polyethylene glycol based PEGO chain or semi-rigid [poly(Pro-Gly)] linkers were employed for this purpose. The described synthetic strategy provides a modular way to assemble ligands and linkers on solid-phase supports. Examples of heterobivalent ligands are provided to illustrate the increased binding avidity to cells that express the complementary receptors.     

Solid-Phase Capture of Proteins, Spores, and Bacteria

Current methods for the detection of pathogens in food and water samples generally require a preenrichment step that allows selective enrichment of the test organism. The objective of this research was to eliminate an enrichment step to allow detection of bacteria directly in food and water samples in 30 min. A high-flow-rate, fluidized bed to capture and concentrate large (bacteria and spores) and small (protein) molecules was developed. This format, ImmunoFlow, is volume independent and uses large beads (greater than 3 mm in diameter) when capturing bacteria to prevent sample clogging when testing food samples. Detection of bound targets was done using existing enzyme-linked immunosorbent assay (ELISA) protocols. Four antibodies (anti-Escherichia coli O157:H7, -Bacillus globigii, -bovine serum albumin [BSA], and -ovalbumin [OVA]) were covalently coupled to various glass and ceramic beads. Very small amounts of BSA (<1 ng) and OVA (0.2 to 4.0 μg) were detected. Various industrial and environmental samples were used to observe the effect of the sample composition on the capture of anti-B. globigii and anti-E. coli O157:H7 modified beads. The lower limit of detection for both E. coli O157:H7 and B. globigii was 1 spore/cell independent of the sample size. The activity of anti-B. globigii modified beads declined after 3 days. Anti-E. coli O157:H7 modified beads declined in their capture ability after 2 days in various storage buffers. Storage temperature (4 and 25°C) did not influence the stability. The ImmunoFlow technology is capable of capturing bacteria and spores directly from samples, with subsequent detection in an ELISA format in 30 min.     

Solid-Phase Synthesis of Core 8 O-Glycan-Linked MUC5AC Glycopeptide

The benzyl-protected disaccharide building blocks of core 8 O-glycan (15a/15b) for glycopeptide were stereoselectively synthesized by two glycosidation reactions with the glycosyl fluoride method. The building blocks were utilized in the solid-phase synthesis of a glycopeptide carrying two O-glycans with the consensus sequence of the tandem-repeat domain of MUC5AC. The synthetic glycopeptide was detached from the resin with reagent K, and subsequent debenzylation under conditions of low-acidity TfOH afforded glycopeptide 2. The synthetic sample will be used as a suitable standard in studies of the physicochemical or immunochemical characterization of mucin glycoforms.     

Solid-Phase Synthesis of Biaryl Cyclic Peptides Containing a Histidine-Phenylalanine Linkage

The feasibility of the solid-phase intramolecular 4(5)-arylation of a histidine residue to obtain biaryl cyclic peptides bearing a His-Phe linkage was esta     

Magnetic CLEAR Supports for Solid-Phase Synthesis of Peptides and Small Organic Molecules

Paramagnetic versions of the CLEAR supports were prepared by entrapment of magnetite particles during suspension polymerization of allylamine, trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate, and trimethylolpropane trimethacrylate. Swelling studies in a broad range of solvents showed comparable swelling of magnetic beads and regular CLEAR beads. The application of the magnetic beads as the support in solid-phase synthesis of peptides (Leu-enkephalinamide, substance P, acyl carrier protein (65–74) amide, and amyloid β-protein (34–42) amide) and small organic affinity ligands based on the s-triazine scaffold was demonstrated. Adequate stability of the beads and retention of their magnetic properties were observed throughout the synthesis steps. Abbreviations used for amino acids follow the IUPAC-IUB Commission of Biochemical Nomenclature (Jones, J. Pept. Sci., 12 (2006) 1). All amino acids used were of the L-configuration unless otherwise stated. All solvent ratios are volume/volume. A preliminary report of portions of this work was presented at the 19th American Peptide Symposium in San Diego, CA, 2005 (Sasikumar and Kempe, 2006). Dedicated to the memory of Bruce Merrifield (July 15, 1921–May 14, 2006).     

Multilayered Magnetic Nanoparticles as a Support in Solid-Phase Peptide Synthesis

The synthesis of multilayered magnetic nanoparticles (MNPs) for use as a support in solid-phase peptide synthesis (SPPS) is described. Silanization of magnetite (Fe₃O₄) nanoparticles with 3-(trimethoxysilyl)propyl methacrylate introduced polymerizable groups on the surface. Polymerization with allylamine, trimethylolpropane trimethacrylate, and trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate provided a polymeric coating and amino groups to serve as starting points for the synthesis. After coupling of an internal reference amino acid and a cleavable linker, the coated MNPs were applied as the solid phase during synthesis of Leu-enkephalinamide and acyl carrier protein (65-74) by Fmoc chemistry. A “high-load” version of the MNP support (0.32 mmol/g) was prepared by four consecutive cycles of Fmoc-Lys(Fmoc)-OH coupling and Fmoc deprotection. Successful synthesis of Leu-enkephalin was demonstrated on the “high-load” MNPs. Chemical stability studies proved the particles to be stable under SPPS conditions and magnetization measurements showed that the magnetic properties of the particles were maintained throughout derivatizations and SPPS. The MNPs were further characterized by high-resolution transmission electron microscopy, inductively coupled plasma atomic emission spectrometry, elemental analysis, and nitrogen gas adsorption measurements.     

Development of a Method for the Solid-Phase Peptide Synthesis in Water

Various organic solvents are routinely used in peptide synthesis, safe disposal of which are now an important environmental problem. To circumvent this problem, during the last few years we focused on developing an organic solvent-free SPPS method using aqueous solvents. For the SPPS in water, we designed protected amino acids that could be used in the aqueous media. Here we described development of several types of water-soluble protected amino acids and their application to the SPPS in water, and a novel technology that uses water-dispersible protected amino acids for in-water peptide synthesis.     

Solid-Phase Synthesis and Antibacterial Activity of an Artificial Cyclic Peptide Containing Two Disulfide Bridges

International Journal of Peptide Research and Therapeutics - Synthesis of artificial antibacterial peptides containing multiple disulfide bridges is of particular interest in bioorganic chemistry....     

Serine-71 phosphorylation of rac1 modulates downstream signaling

The Rho GTPases Rac1 and Cdc42 regulate a variety of cellular functions by signaling to different signal pathways. It is believed that the presence of a specific effector at the location of GTPase activation determines the route of downstream signaling. We previously reported about EGF-induced Ser-71 phosphorylation of Rac1/Cdc42. By using the phosphomimetic S71E-mutants of Rac1 and Cdc42 we investigated the impact of Ser-71 phosphorylation on binding to selected effector proteins. Binding of the constitutively active (Q61L) variants of Rac1 and Cdc42 to their specific interaction partners Sra-1 and N-WASP, respectively, as well as to their common effector protein PAK was abrogated when Ser-71 was exchanged to glutamate as phosphomimetic substitution. Interaction with their common effector proteins IQGAP1/2/3 or MRCK alpha was, however, hardly affected. This ambivalent behaviour was obvious in functional assays. In contrast to Rac1 Q61L, phosphomimetic Rac1 Q61L/S71E was not able to induce increased membrane ruffling. Instead, Rac1 Q61L/S71E allowed filopodia formation, which is in accordance with abrogation of the dominant Sra-1/Wave signalling pathway. In addition, in contrast to Rac1 transfected cells Rac1 S71E failed to activate PAK1/2. On the other hand, Rac1 Q61L/S71E was as effective in activation of NF-kappaB as Rac1 Q61L, illustrating positive signal transduction of phosphorylated Rac1. Together, these data suggest that phosphorylation of Rac1 and Cdc42 at serine-71 represents a reversible mechanism to shift specificity of GTPase/effector coupling, and to preferentially address selected downstream pathways.     

A Solid-Phase Method for the Synthesis of Small to Medium-Sized Cyclic Oligonucleotides

Abstract

Cyclic oligonucleotides (2- to 30-mer) are synthesized by a solid-phase method, for both chain elongation and cyclization, employing a new linker and standard phosphoramidite chemistry. Fairly pure crude products (>90% by HPLC) are obtained.     

L-丝氨酸高产菌株的选育和摇瓶发酵条件优化

采用B revibacterium flavmC-11A为出发菌株,经紫外线照射和亚硝基胍诱变处理,选育出一株L-丝氨酸高产菌株C32为目的突变株,使摇瓶产酸率由12.1 g.L-1增加到16.4 g.L-1,然后对其进行摇瓶发酵条件优化,使菌株C32的L-丝氨酸产率提高到30.1 g.L-1。