Design and validation of a frugal, automated, solid-phase peptide synthesizer.

Solid phase peptide synthesis (SPPS) has enabled widespread use of synthetic peptides in applications ranging from pharmaceuticals to materials science. The demand for synthetic peptides has driven recent efforts to produce automated SPPS synthesizers which utilize fluid-handling components common to chemistry laboratories to drive costs down to several thousand dollars. Herein, we describe the design and validation of a more 'frugal' SPPS synthesizer that uses inexpensive, consumer-grade fluid-handling components to achieve a prototype price point between US$300 and $600. We demonstrated functionality by preparing and characterizing peptides with a variety of distinct properties including binding functionality, nanoscale self-assembly, and oxidation-induced fluorescence. This system yielded micromoles of peptide at a cost of approximately $1/residue, a cost which may be further reduced by optimization and bulk purchasing.

Nanomolar Synthesis in Droplet Microarrays with UV-Triggered On-Chip Cell Screening.

Miniaturization and parallelization of combinatorial organic synthesis is important to accelerate the process of drug discovery while reducing the consumption of reagents and solvents. This work presents a miniaturized platform for on-chip solid-phase combinatorial library synthesis with UV-triggered on-chip cell screening. The platform is based on a nanoporous polymer coating on a glass slide, which is modified via photolithography to yield arrays of hydrophilic (HL) spots surrounded by superhydrophobic (SH) surface. The combination of HL spots and SH background enables confinement of nanoliter droplets, functioning as miniaturized reactors for the solid-phase synthesis. The polymer serves as support for nanomolar solid-phase synthesis, while a photocleavable linker enables the release of the synthesized compounds into the droplets containing live cells. A 588 compound library of bisamides is synthesized via a four-component Ugi reaction on the chip and products are detected via stamping of the droplet array onto a conductive substrate and subsequent matrix-assisted laser desorption ionization mass spectrometry. The light-induced cleavage shows high flexibility in screening conditions by spatial, temporal, and quantitative control.

Automated Solid-Phase Peptide Synthesis.

The development of solid-phase peptide synthesis by Bruce Merrifield paved the way for a synthesis carried out by machines. Automated peptide synthesis is a fast and convenient way of synthesizing many peptides simultaneously. This chapter tries to give a general guidance for the development of synthesis protocols for the peptide synthesizer. It also provides some suggestions for the modification of the synthesized peptides. Additionally, many examples of possible challenges during and after the synthesis are given in order to support the reader in finding the best synthesis strategy. Numerous references are given to many of the described matters.

New Developments in Microwave-Assisted Solid Phase Peptide Synthesis.

The unique combination of microwave heating with optimized carbodiimide activation has proven to be an indispensable technique for high-throughput peptide production. Here, we describe new methods in microwave-assisted solid phase peptide synthesis and optimized post-synthesis modifications that have been recently developed. These methods have drastically reduced synthesis time and solvent requirement while delivering peptides in high crude purities.

SPOT Synthesis: The Solid-Phase Peptide Synthesis on Planar Surfaces.

Peptide libraries are a highly useful tool for drug development. While most preparations of peptide libraries are laborious during either the synthesis or its screening, the SPOT synthesis offers the possibility of directly synthesizing large numbers of peptides on a planar surface. As a positionally addressable, multiple solid-phase synthesis technique, the synthesis allows a very convenient handling during the screening of that peptide library in a form of an array. This publication will provide protocols for the basic procedures of the SPOT synthesis and references to some important literature regarding that technique and its application.

Synthesis of Amide Backbone-Modified Peptides.

Solubility is a key property of peptides and of central importance to the success of solid-phase peptide synthesis and subsequent peptide purification and handling. Substitution of the backbone amide bond can dramatically increase peptide solubility. Backbone amide bond protection works by preventing the formation of interchain association and can be used both to synthesize aggregation-prone peptide sequences on solid phase and to improve solubility of a peptide post synthesis. Improving peptide solubility by judicial use of backbone protection is of growing importance, particularly for chemical protein synthesis by chemical ligation.

Regeneration of aged DMF for use in solid-phase peptide synthesis.

Dimethylformamide (DMF), which is still the most commonly used solvent for Fmoc-SPPS, has the potential for degradation over time on exposure to air (and water vapour) and storage, to give dimethylamine and formic acid impurities. In particular, dimethylamine can lead to unwanted deprotection of the fluorenylmethyloxycarbonyl (Fmoc) group during, for example, the initial loading of Fmoc amino acids in SPPS, which leads reduced calculated loading values. We have found that treatment of such aged DMF by simple sparging with an inert gas (N2 ), or vacuum sonication, can regenerate the DMF in order to restore loading levels back to those found for newer, fresh, DMF samples.

Peptide Synthesis Utilizing Micro-flow Technology.

Peptide drugs have garnered much attention in recent years. However, conventional peptide synthesis requires an excess amount of expensive reagents of low atom economy, and the large amount of waste produced by these reagents complicates the purification of desired peptides. Solid-phase approaches simplify the purification of these peptides, but these require expensive solid-phase, excess amounts of reagents, substrates, and solvents. This makes it important to develop high-yielding, cost-effective, and less wasteful synthetic approaches. Micro-flow technology (reaction space ≤1 mm) has produced many advantages over conventional batch synthesis. The advantages include precise control of short reaction time and temperature, high levels of light penetration efficiency, lowered risks of handling dangerous compounds, and ready scale-up with high reproducibility. Micro-flow peptide syntheses using these advantages have been reported in recent years. This review summarizes the solid-phase and solution-phase syntheses of α- and ß-peptides and of cyclic peptides using micro-flow technology.

Automated glycan assembly as an enabling technology.

Access to complex carbohydrates remains a limiting factor for the development of the glycosciences. Automated glycan assembly (AGA) has accelerated and simplified the synthetic process and, with the first commercially available instrument and building blocks, glycan synthesis can now be practiced by any chemist. All classes of glycans, including sulfated or sialylated carbohydrates and polysaccharides as long as 50mers are now accessible owing to optimized reaction conditions and new methodologies. These synthetic glycans have helped to understand many biological functions and to advance diagnostic and vaccine development. Establishing detailed structure-function relationships will eventually enable the production of unnatural materials with tuned properties.

Chemical Synthesis of Antimicrobial Peptides.

Solid-phase peptide synthesis (SPPS) is the method of choice for chemical synthesis of peptides. In this nonspecialist review, we describe commonly used resins, linkers, protecting groups, and coupling reagents in 9-fluorenylmethyloxycarbonyl (Fmoc) SPPS. Finally, a detailed protocol for manual Fmoc SPPS is presented.

High-molecular-weight poly(Gly-Val-Gly-Val-Pro) synthesis through microwave irradiation.

In this study, we synthesized a polypeptide from its pentapeptide unit using microwave irradiation. Effective methods for polypeptide synthesis from unit peptides have not been reported. Here, we used a key elastin peptide, H-GlyValGlyValPro-OH (GVGVP), as the monomer peptide. It is difficult to obtain poly(Gly-Val-Gly-Val-Pro) (poly(GVGVP)) from the pentapeptide unit of elastin, GVGVP, via polycondensation. Poly(GVGVP) prepared from genetically recombinant Escherichia coli is a well-known temperature-sensitive polypeptide, and this temperature sensitivity is known as the lower critical solution temperature. When microwave irradiation was performed in the presence of various additives, the pentapeptide (GVGVP) polycondensation reaction proceeded smoothly, resulting in a product with a high molecular weight in a relatively good yield. The reaction conditions, like microwave irradiation, coupling agents, and solvents, were optimized to increase the reaction efficiency. The product exhibited a molecular weight greater than Mr 7000. Further, the product could be synthesized on a gram scale. The synthesized polypeptide exhibited a temperature sensitivity that was similar to that of poly(GVGVP) prepared from genetically recombinant E. coli. Therefore, this technique offers a facile and quick approach to prepare polypeptides in large amounts. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Physical structure changes of solid medium by steam explosion sterilization.

Physical structure changes of solid medium were investigated to reveal effects of steam explosion sterilization on solid-state fermentation (SSF). Results indicated that steam explosion changed the structure of solid medium at both molecular and three-dimensional structural levels, which exposed hydrophilic groups and enlarged pores and cavities. It was interesting to find that pores where capillary water located were the active sites for SSF, due to the close relationship among capillary water relaxation time, specific surface area and fermentation performance. Therefore, steam explosion sterilization increased the effective contact area for microbial cells on solid medium, which contributed to improving SSF performance. Combined with the previous research, mechanisms of SSF improvement by steam explosion sterilization contained both chemical and physical effects.

Methods, setup and safe handling for anhydrous hydrogen fluoride cleavage in Boc solid-phase peptide synthesis.

Solid-phase peptide synthesis (SPPS) using tert-butyloxycarbonyl (Boc)/benzyl (Bzl) chemistry is an indispensable technique in many laboratories around the globe, and it provides peptides to the pharmaceutical industry and to thousands of scientists working in basic research. The Boc/Bzl strategy has several advantages, including reliability in the synthesis of long and difficult polypeptides, alternative orthogonality regarding protecting groups and ease of producing C-terminal thioesters for native chemical ligation applications. In this process, anhydrous hydrogen fluoride (HF) is used to remove the side chain protecting groups of the assembled peptide and to release the peptide from the resin, a process typically described as 'HF cleavage'. This protocol describes the general methodology, apparatus setup and safe handling of HF, with the aim of providing comprehensive information on the safe use of this valuable, well-studied and validated cleavage technique. We explain the cleavage mechanism, the physicochemical properties and risks of HF, first aid measures and the correct use of the apparatus. In addition, we provide advice on scavenger selection, as well as a troubleshooting section and video material illustrating key steps of the procedure. The protocol comprises precleavage sample preparation (30 min-2.5 h), complete HF cleavage procedure (2 h) and reaction workup (30 min).

Matrix-assisted peptide synthesis on nanoparticles.

We report a new method for multistep peptide synthesis on polymeric nanoparticles of differing sizes. Polymeric nanoparticles were functionalized via their temporary embedment into a magnetic inorganic matrix that allows multistep peptide synthesis. The matrix is removed at the end of the process for obtaining nanoparticles functionalized with peptides. The matrix-assisted synthesis on nanoparticles was proved by generating various biologically relevant peptides.

Rapid flow-based peptide synthesis.

A flow-based solid-phase peptide synthesis methodology that enables the incorporation of an amino acid residue every 1.8 min under automatic control or every 3 min under manual control is described. This is accomplished by passing a stream of reagent through a heat exchanger into a low volume, low backpressure reaction vessel, and through a UV detector. These features enable continuous delivery of heated solvents and reagents to the solid support at high flow rate, thereby maintaining maximal concentration of reagents in the reaction vessel, quickly exchanging reagents, and eliminating the need to rapidly heat reagents after they have been added to the vessel. The UV detector enables continuous monitoring of the process. To demonstrate the broad applicability and reliability of this method, it was employed in the total synthesis of a small protein, as well as dozens of peptides. The quality of the material obtained with this method is comparable to that for traditional batch methods, and, in all cases, the desired material was readily purifiable by RP-HPLC. The application of this method to the synthesis of the 113-residue Bacillus amyloliquefaciens RNase and the 130-residue DARPin pE59 is described in the accompanying manuscript.

Solid-phase synthesis of amidine-substituted phenylbenzimidazoles and incorporation of this DNA binding and recognition motif into amino acid and peptide conjugates.

Amidine-substituted phenylbenzimidazoles are well-established DNA-binding structural motifs that have contributed to the development of diverse classes of DNA-targeted agents; this ring system not only assists in increasing the overall DNA affinity of an agent, but can also influence its site selectivity. Seeking a means to conveniently exploit these attributes, a protocol for the on-resin synthesis of amino acid- and peptide-phenylbenzimidazole-amidine conjugates was developed to facilitate installation of phenylbenzimidazole-amidines into peptide chains during the course of standard solid-phase syntheses. Building from a resin-bound amino acid or peptide on Rink amide resin, 4-formyl benzoic acid was coupled to the resin-bound free amine followed by introduction of 3,4-diamino-N'-hydroxybenzimidamide (in the presence of 1,4-benzoquinone) to construct the benzimidazole heterocycle. Finally, the resin-bound N'-hydroxybenzimidamide functionality was reduced to an amidine via 1 M SnCl2·2H2O in DMF prior to resin cleavage to release final product. This procedure permits the straightforward synthesis of amino acids or peptides that are N-terminally capped by a phenylbenzimidazole-amidine ring system. Employing this protocol, a series of amino acid-phenylbenzimidazole-amidine (Xaa-R) conjugates was synthesized as well as dipeptide conjugates of the general form Xaa-Gly-R (where R is the phenylbenzimidazole-amidine and Xaa is any amino acid).

Solid-phase peptide synthesis: an introduction.

This chapter provides an introduction to and overview of peptide chemistry with a focus on solid-phase peptide synthesis. The background, the most common reagents, and some mechanisms are presented. This chapter also points to the different chapters and puts them into perspective.

Instruments for automated peptide synthesis.

This chapter provides an introduction to and an overview of current instrumentation for solid-phase peptide synthesis (SPPS). Presently, the two most common designs differ in their mode of liquid handling: the first relies on valves and valve blocks for distribution of reagents, while the second uses a robotic platform. They also differ in their mode of mixing the reactants in the reaction vessel, where the former can utilize sparging, 180° rotational shaking, or vortexing, while the latter typically uses vortexing. Valve-based instruments are often single channel (one peptide at a time), but can also be expanded to allow parallel synthesis of up to 12 and even 24 peptides, however, at the price of added complexity. Valve systems often use inert gas for their operation. The X-Y robotic platforms are ideal for parallel synthesis of large numbers of peptides up to 192 and even peptide libraries. However, although less common, the robotic platform is also very suitable for single-channel operation and can also be used for operations under inert gas. Some single-channeled synthesizers are available with UV feedback monitoring of the Fmoc removal which can be useful for some applications. Importantly, single-channel synthesizers can be equipped with fast and precise microwave heating to accelerate the synthesis and to overcome synthetic difficulties. A whole range of synthesizers with different designs are commercially available. The choice of peptide synthesizer will depend on intended application, for example on the type of chemistry, scale, and the number of peptides that are required and so on.

Microwave-assisted solid-phase peptide synthesis using the biotage Syro Wave™.

Fast and precise heating by microwave irradiation during solid-phase peptide synthesis (SPPS) can reduce reaction times as well as provide better purities and greater yields for the synthesis of difficult peptides. Microwave- assisted SPPS has proven to be a useful and reliable tool for the synthesis of peptides as well as small proteins. It is particularly well suited for sequences with a high propensity to form ß-sheet-type structures and for sterically difficult couplings. In this protocol, conditions and detailed procedures are described for performing microwave-assisted SPPS using the Syro Wave™. Here we describe the synthesis of two difficult peptide sequences: the first is derived from the C-terminus of the MuLV CTL epitope, the second is a de novo designed peptide with a C-terminal alkyne.

Solid phase synthesis of a functionalized bis-peptide using "safety catch" methodology.

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This technique quickly proved advantageous over its solution-phase predecessor in both time and labor. Improvements concerning the nature of solid support, the protecting groups employed and the coupling methods employed over the last five decades have only increased the usefulness of Merrifield's original system. Today, use of a Boc-based protection and base/nucleophile cleavable resin strategy or Fmoc-based protection and acidic cleavable resin strategy, pioneered by R.C. Sheppard, are most commonly used for the synthesis of peptides(1). Inspired by Merrifield's solid supported strategy, we have developed a Boc/tert-butyl solid-phase synthesis strategy for the assembly of functionalized bis-peptides(2), which is described herein. The use of solid-phase synthesis compared to solution-phase methodology is not only advantageous in both time and labor as described by Merrifield(1), but also allows greater ease in the synthesis of bis-peptide libraries. The synthesis that we demonstrate here incorporates a final cleavage stage that uses a two-step "safety catch" mechanism to release the functionalized bis-peptide from the resin by diketopiperazine formation. Bis-peptides are rigid, spiro-ladder oligomers of bis-amino acids that are able to position functionality in a predictable and designable way, controlled by the type and stereochemistry of the monomeric units and the connectivity between each monomer. Each bis-amino acid is a stereochemically pure, cyclic scaffold that contains two amino acids (a carboxylic acid with an α-amine)(3,4). Our laboratory is currently investigating the potential of functional bis-peptides across a wide variety of fields including catalysis, protein-protein interactions and nanomaterials.