Biotage^® Initiator+ Alstra^™

Read the feedback from Dr. Longhi and his team in Milan about Biotage^® Initiator+ Alstra^™

Professor Roger Strömberg is Head of the Bioorganic Chemistry Unit at the Department of Biosciences and Nutrition, Karolinska Institutet in Huddinge, Sweden. He leads a number of research projects in the area of nucleic acids and peptides in biotechnology and therapy, including tools for molecular biology, treatment of multi-resistant bacterial infections and prevention of Alzheimer’s Disease.

Dr. Andrew Jamieson leads a team of peptide chemists at the University of Glasgow, where they synthesize complex molecules for chemical biology applications. An important tool that contributes to their success has been Biotage® Initiator+ Alstra™ microwave peptide synthesizer, and they are even breaking the dogma that only HPLC can be used for peptide purification, by using cutting edge reversed phase flash purification techniques provided by Biotage.

Dr. Aaron Muth from St. John’s University, Queens, New York speaks about his work in cancer research with Biotage lab equipment and specifically Biotage® Initiator+ Alstra™ and the SNAP Bio cartridges. As an Assistant Professor of Medicinal Chemistry, Dr. Muth and the members of his research group use this equipment every day. 

Red Glead Discovery is a pre-clinical drug discovery CRO offering a broad range of services to Life Science clients. With a focus on small molecules and peptides, their drug discovery platform ranges from medicinal chemistry and synthesis to ADME and biology. In addition to the CRO business, they also perform research collaborations with various biotech and academic partners. We met with Richard E. Johnsson, Ph.D., Head of Peptide Chemistry at Red Glead Discovery, Lund, Sweden, where he told us about using Biotage products in his workflow.

Tokyo based PeptiDream Inc. is a company specializing in non-standard peptide therapeutics containing non-standard amino acids, and conducts research and development of new drug candidates. PeptiDream uses the peptide synthesizers Syro I and Biotage® Initiator+ Alstra™ for the synthesis of various kinds of peptides to support drug discovery and development.

The study of peptides, especially for therapeutic use, has grown significantly in recent years. However, producing peptides for such research is challenging, not just in the synthesis. The molecules must be visualized to allow synthetic approaches to be designed, and after synthesis the purification and isolation of peptides is not straightforward. In order to address these challenges, Biotage has developed a holistic approach to the entire peptide workflow via an automated solution, designed for dedicated peptide researchers and those new to the field.

Peptides have garnered much attention in a variety of applications over the last few years, and interest only continues to grow. With improved synthetic strategies, peptides are now being synthesized in greater length and complexity than ever before. However, very few changes have been made when it comes to peptide purification, leading to a bottleneck in the overall peptide workflow. Peptide purification via flash chromatography has recently been demonstrated as a viable alternative to the more standard HPLC methods currently utilized. Flash chromatography offers peptide chemists the advantage of significantly greater loading capacity reducing the overall purification time but with a compromise of decreased peak resolution. Herein we present several strategies that, when implemented, allow for very high purity peptide samples purified by flash chromatography.

The interest in naturally occurring and designed bioactive cyclic peptides is ever-increasing. Cyclic peptides often have an increased stability against proteolysis compared with linear peptides, which can be used in the design of potential peptide drugs. The decreased flexibility can provide more specific interactions with receptors. The fast and convenient synthesis of cyclic peptides is thus very appealing. The cyclic peptide mupain-1 (CPAYSRYLDC) is an inhibitor of the cancer-related urokinase-type plasminogen activator. Here we present two methods to assemble cyclic peptide analogues of mupain-1.

Synthesis of branched peptides is very challenging. Here we show the synthesis of a complex multi-branched peptide and how this process can be simplified using the innovative Branches™.

A branched peptidoglycan mimic and a tetra-branched antimicrobial peptide analogue were synthesized on a lysine scaffold using Biotage® Initiator+ Alstra™ microwave peptide synthesizer. These peptide modifications are challenging to synthesize and automate, however, the procedure was operationally simplified using Branches™.

Labeled peptides are important tools to study molecular interactions and are increasingly utilized with the growing number of peptide-based therapeutics. Biological assays frequently require imaging agents to study drug molecules and fluorescent labels are often employed for this purpose, but can be expensive and thus present a cost barrier for synthesis. In an effort to demonstrate the cost savings by employing small μmol scale, automated solid phase peptide synthesis through robot liquid handling of reagents with accurate dispensing of small volumes, a 10-mer antimicrobial peptide3 1 was synthesized and labeled with 5(6)-carboxyfluorescein (FAM) at the N -terminus.

Structural and functional studies of membrane proteins remain a challenge due to difficulties in their handling. In the last decade, several lipoprotein based methods have been proposed for solving exactly this problem in biophysical and functional studies.1 The lipoproteins used for this purpose, were derived from naturally occurring protein analogues and in particular, from the amphipathic 243 amino acid long Apolipoprotein A1 (ApoA1), which is the main constituent in high density lipoproteins (HDLs), the carriers of so-called “good” cholesterol.

Despite the improvements in automated solid phase peptide synthesis, such as the use of elevated temperatures, whereby peptides of greater complexity and purity can be synthesized routinely, the purification step is one of the main bottlenecks in the peptide synthesis workflow. Preparative RP-HPLC is normally the method of choice but is limited by small loading amounts, long separation times, poor recoveries and high costs. In addition, crude synthetic peptides contain impurities with retention characteristics very similar to the target peptide which can present additional purification challenges. Although there are a number of examples in the literature, flash chromatography or medium pressure liquid chromatography (MPLC) is almost never considered as a technique for purification of synthetic peptides, as it is not perceived to be suitable for this application. However, with recent advances in flash using 20–25 micron spherical particles, flash chromatography can be considered an efficient technique for synthetic peptide purification.

The purification step is one of the main bottlenecks in the peptide synthesis workflow. Preparative RP-HPLC is normally the method of choice but is limited by small loading amounts, long separation times, poor recoveries and high costs. In addition, crude synthetic peptides contain impurities with retention characteristics very similar to the target peptide which can present additional purification challenges. Although there are a number of examples in the literature, flash chromatography or medium pressure liquid chromatography (MPLC) is almost never considered as a technique for purification of synthetic peptides, as it is not perceived to be suitable for this application. However, with recent advances in flash using 20-25 micron spherical particles, flash chromatography can be considered an efficient technique for synthetic peptide purification.

Peptide drug discovery efforts increase as the rules governing cytosolic delivery and improved pharmacokinetic stability are further elucidated. Progress has certainly been aided by advances in coupling reagents and synthetic methodologies, thus enabling chemical synthesis of peptides with greater lengths and higher crude purities. Despite these improvements, crude peptides are still subjected to laborious purification protocols. Reversed-phase flash chromatography presents itself as an attractive tool for peptide purification due to its high sample loading capacities and expedient chromatographic methods. Many questions arise when considering a new technique for such an important component of the peptide drug discovery process though. Herein we present purification efficiency and recovery results to address some of these critical concerns.

This application note describes optimization for a fully automated synthesis and on-resin cyclization of oxytocin enabled by Branches™, a unique software feature of the Biotage® Initiator+ Alstra™ peptide synthesizer. The optimized protocol is then applied to the synthesis of isotopically labeled oxytocin. Also identified during this process is a strategy for an efficient separation of oxidized from reduced oxytocin via flash chromatography by modulating the pH of the mobile phase.

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