File Name: solid-phase organic synthesis concepts strategies and applications .zip
Editor-in-Chief: P. Seeberger Beilstein J.
- Automated solid-phase peptide synthesis to obtain therapeutic peptides
- Peptide synthesis
- Linker Strategies in Solid-Phase Organic Synthesis
In organic chemistry , peptide synthesis is the production of peptides , compounds where multiple amino acids are linked via amide bonds, also known as peptide bonds. Peptides are chemically synthesized by the condensation reaction of the carboxyl group of one amino acid to the amino group of another. Protecting group strategies are usually necessary to prevent undesirable side reactions with the various amino acid side chains.
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Automated solid-phase peptide synthesis to obtain therapeutic peptides
Editor-in-Chief: P. Seeberger Beilstein J. The great versatility and the inherent high affinities of peptides for their respective targets have led to tremendous progress for therapeutic applications in the last years. In order to increase the drugability of these frequently unstable and rapidly cleared molecules, chemical modifications are of great interest. Automated solid-phase peptide synthesis SPPS offers a suitable technology to produce chemically engineered peptides.
Critical issues and suggestions for the synthesis are covered. The development of automated methods from conventional to essentially improved microwave-assisted instruments is discussed. In order to improve pharmacokinetic properties of peptides, lipidation and PEGylation are described as covalent conjugation methods, which can be applied by a combination of automated and manual synthesis approaches.
The synthesis and application of SPPS is described for neuropeptide Y receptor analogs as an example for bioactive hormones. The applied strategies represent innovative and potent methods for the development of novel peptide drug candidates that can be manufactured with optimized automated synthesis technologies.
Keywords: automated synthesis; automation; lipidation; PEGylation; peptide drugs; solid-phase peptide synthesis; therapeutic peptides. Peptides and proteins are involved in a large variety of biochemical processes and physiological functions. Peptides can consist of up to 50 amino acids and have generally no tertiary, three-dimensional structure compared to proteins . In nature, the oligomers or polymers are assembled at ribosomes by aminoacyl-tRNAs transfer ribonucleic acid .
The individual building blocks occur as L-enantiomers throughout living organisms in case of ribosomal synthesis and only 20 monomers are generally found in peptides and proteins with few rare exceptions. Those canonical amino acids vary in their side-chain functionality and possess different polarities that are important for their biological function. Scheme 1: Formation of a dipeptide 3.
Reaction of the amino group of amino acid 2 with the carboxylic acid moiety of amino acid 1 leads to a mesomeric peptide bond highlighted in red. Reaction of the amino group of amino acid 2 with the carboxylic acid mo Peptides can be biologically active hormones, neurotransmitters and neuropeptides, growth factors, signaling molecules and antibiotics.
These diverse functions make peptides an interesting target on the pharmaceutical market. Diseases such as cancer, diabetes, obesity but also osteoporosis, cardiovascular diseases and inflammation can be treated by peptide-based drugs [4,5]. Within the last decades, the fast development of omics technologies such as genomics, proteomics and transcriptomics led to the identification of a great number of target peptides or proteins .
This trend successively offers new targets for peptide drugs that classical small organic molecules cannot cover . Although small synthetic drugs are in general orally applicable owing to their high metabolic stability, capable to cross cell membranes and small in size, which simplifies their production and costs, they reveal considerable shortcomings.
They show, for example, often moderate target potency and selectivity, which manifest in side-effects. In contrast, the strong and specific binding of peptides and proteins to their molecular targets can reduce the drug dose. This high selectivity leads to fewer side effects, which is considered as the greatest benefit of peptides and proteins over small molecules [7,8]. Peptides share all superiorities of proteins but are significantly smaller in size and hence, easier and cheaper to synthesize using chemical strategies .
Thereby, they provide a vast perspective for novel drug design. The high potency and selectivity of peptides are of great advantage for drug development . The metabolization leads to non-toxic degradation products, which, combined with their high specificity, goes along with low adverse effects.
Furthermore, peptides do not tend to interact with other drugs and exhibit a more predictable in vivo behavior owed to their biochemical nature . The extended size and the tremendous biological and chemical diversity of peptides opposed to small organic drugs opens targets for multiple applications .
In the last decades, the production of therapeutic peptides has been revolutionized by new methods and strategies for automated approaches, which simplifies peptide manufacturing. Combined with the mentioned advantages of peptide-based drugs, their application as novel biopharmaceuticals is pushed forward. Within the last years, the global market for peptide therapeutics expanded nearly twice as fast as overall drugs .
In , the market for peptide drugs covered 8 billion EUR and was estimated to reach Notably, their low bioavailability owing to proteolytic degradation by enzymes of the intestine, blood and cell plasma leads to short circulating half-lives . Depending on their size, peptides are excreted by kidneys renal clearance or liver hepatic clearance within minutes [5,9].
Nevertheless, their ability to pass through membranes and the urgent need of alternative, more comfortable administration routes as the commonly used parenteral subcutaneous, intramuscular and intravenous application, have prompted further research in this field .
Therefore, methods to prolong peptide stability are of great interest. Here, we highlight the importance of automated solid-phase peptide synthesis SPPS in the process of peptide modification. Recent advances in automation devices are described, with attention to the comparison between conservative SPPS robots and microwave-assisted automated SPPS.
Moreover, strategies for modulating peptide stability with an emphasis on lipidation and PEGylation are characterized. Last, the syntheses of selected peptide hormones are presented exemplarily. In the past, pioneering of Emil Fischer at the beginning of the 20 th century  and du Vigneaud in  have made the synthesis of peptides possible, as at that time, they were relatively unknown biomolecules.
Fifty years later, du Vigneaud developed a strategy for the production of a polypeptide. For the synthesis of the polypeptide hormone oxytocin, organic protecting groups were introduced to trifunctional amino acids  in order to ensure specific amide-bond formation . The principle of peptide synthesis in homogenous solution is based on the reversible blocking of the carboxylic acid function of the C-terminal amino acid and the amino group of the N-terminal amino acid.
In addition, activation of the free carboxy group of the N-terminal amino acid is necessary to obtain the peptide bond. For this approach, all peptide intermediates have to be isolated and purified before they can be used for further reaction steps. Although this assures a good quality control, it is a very time-consuming and a technical-demanding process .
This manifests especially at larger and more complex peptides, for which the protected fragments often tend to be rigid and insoluble . These disadvantages in the synthesis of peptides led to the revolutionary inception of a completely different strategy. The method was named solid-phase peptide synthesis and accounts for a peptide construction between two phases, an insoluble solid support and liquid soluble reagents .
Here, the first amino acid is coupled for the time of the synthesis with its carboxylic acid terminus to a resin that consists of polymer particles and protects the C-terminus from side reactions.
In order to overcome aggregate formation, a distinct short organic linker is interposed between the amino acid and the solid support, which also determines the C-terminal modification of the synthetic peptide .
Moreover, the relatively inert carboxy group has to be activated by a special auxiliary to increase the electrophilicity . After loading of the resin, the N-terminal protecting group of the first amino acid can be removed and the next activated building block can be coupled. The last step of SPPS should be performed in the presence of scavengers to trap highly reactive carbocations that are formed during the cleavage procedure and that might react with the peptide to form unwanted byproducts .
The crude product can be easily separated from the resin and purified by standard analytical methods such as the diverse chromatographic techniques. Their strong development with excellent improvement in separation of similar components was a major prerequisite for the success of SPPS, both with respect to analytics and preparative purification .
Furthermore, high-quality mass spectrometry MS with soft ionization techniques such as MALDI—TOF matrix-assisted laser desorption ionization — time of flight and ESI electrospray ionization MS allows nowadays rapid and clear identification of the respective product and all byproducts .
First, the C-terminal amino acid is coupled to the linker. The peptide chain will be elongated by repeating a cycle of 1 deprotection of NPG, 2 activation of the carboxy group and 3 coupling. At the end of the synthesis, the protecting groups will be cleaved and the desired peptide obtained.
First, the C-terminal amino acid is This heterogeneous synthesis technique offers great advantages. Certainly, the most important benefit of SPPS is the feasibility of carrying out all reactions in a single vessel. Following a coupling step, unreacted reagents and byproducts can be easily removed by washing, which makes purification of intermediates redundant.
Based on the use of excess amounts of reactants, high coupling yields can be obtained and the incorporation of difficult sequences and modifications to the polymer are enabled. Moreover, the reaction cycles are very short compared to solution synthesis, which allows faster manufacturing . Additionally, the solid-phase concept is not only an elegant way to build up peptides but also other oligomers such as polyamides  , polynucleotides  and polysaccharides .
This method simplified the chemical synthesis of peptides and allowed the automation of the process  , which has led to a breakthrough of SPPS and the establishment as one major technique for therapeutic peptide production [8,19]. Requirements for appropriate protecting groups are the simple incorporation into the desired molecule, a high stability against various conditions as well as easy and safe removal . The initial method applied by Merrifield was based on the use of the Boc group as temporary protecting group for the amino function and Bn benzyl or related protecting groups for the side chains of trifunctional amino acids.
Usually, Boc can be removed by treatment with TFA trifluoroacetic acid , whereas Bn deprotection requires strong acids such as HF . Whilst the Boc group has been used exclusively during the first years of SPPS, the introduction of the Fmoc-group  opened the path for a novel, more variable synthesis concept. Nowadays, both protecting group strategies are used for the synthesis of peptides and both methods can be applied for automated synthesis.
Figure 1: Five issues that have to be resolved prior to peptide synthesis. Removal reactions of the respective protecting groups are illustrated. Dashed boxes stress the COOH-side-chain protecting group of glutamic acid exemplarily, used in each strategy. A The Fmoc-group is rem This concept  enables the selective removal of the protecting groups using completely different chemical conditions and cleavage mechanisms, which ensures milder overall reactions .
The orthogonality is the main benefit of the Fmoc-based concept allowing a higher flexibility for complex strategies during synthesis. Moreover, the Fmoc strategy does not require the use of special vessels that have to be stable towards the corrosive and toxic HF and in some cases, the repetitive TFA acidolysis for Boc deprotection could have an impact on sensitive peptide bonds and acid-catalyzed side reactions .
And, since it is no orthogonal strategy, the Bn removal always leads to Boc deprotection. A tremendous diversity of side-chain protection groups for trifunctional amino acids has been evolved since the development of SPPS more than 50 years ago. Proteinogenic amino acids contain different functional groups: amino, carboxyl, hydroxy, thio, pyrrolidinyl, imidazolyl, guanidinyl, amido and indolyl.
Basically, every amino acid containing chemically reactive side chains has to be equipped with a protecting group during peptide assembly by SPPS in order to prevent side reactions and the formation of byproducts.
These protecting groups are orthogonal to the base-labile Fmoc-group and can be cleaved by highly concentrated TFA solutions. In addition to these examples there is a number of diverse orthogonal protecting groups commercially available. They will have to be used, if peptides are modified additionally and they are cleaved under specific conditions as, e. For a precise overview, the review of Isidro-Llobet and detailed manuals of major companies are recommended .
Trt: trityl, Pbf: pentamethyl-2,3-dihydrobenzofuransulfonyl. Trt: trityl, Optimal resins and linkers for peptide synthesis: The solid phase has to meet a number of requirements to be suitable for peptide synthesis. It has to be insoluble in all solvents, chemically and physically resistant and mechanically stable to allow filtration.
A large number of organic, inorganic, or organic-inorganic hybrid materials have been employed as polymeric solid supports to promote or catalyze various organic reactions. The reaction parameters, scopes, and limitations, particularly in the context of green chemistry, have been highlighted with pertinent approaches by other groups. The concept of solid-phase organic synthesis SPOS dates back mids, and the solid-phase peptide synthesis in s developed by Merrifield has been a pioneering work [ 1 ]. Over the last two decades, there has been a surge generating tremendous interest in expanding this field of solid-phase synthesis [ 2 — 10 ]. There is a clear emphasis in synthetic chemistry towards developing environmentally friendly and sustainable routes to a myriad of materials.
Presents both the fundamental concepts and the most recent applications in solid-phase organic synthesis With its emphasis on basic concepts, Solid-Phase.
Linker Strategies in Solid-Phase Organic Synthesis
Practical and theoretical aspects of instrumentation in chemistry. Other topics may also be presented. This course will focus on 1 the meaning and practice of writing organic reaction mechanisms and 2 standard synthetic reactions, their mechanisms, and modern refinements.
Linker Strategies in Solid-Phase Organic Synthesis guides the reader through the evolution of linker units from their genesis in solid-supported peptide chemistry to the cutting edge diversity linker units that are defining a new era of solid phase synthesis. Individual linker classes are covered in easy to follow chapters written by international experts in their respective fields and offer a comprehensive guide to linker technology whilst simultaneously serving as a handbook of synthetic transformations now possible on solid supports. Linker Strategies in Solid-Phase Organic Synthesis is an essential guide to the diversity of linker units for organic chemists in academia and industry working in the broad areas of solid-phase organic synthesis and diversity oriented synthesis, medicinal chemists in the pharmaceutical industry who routinely employ solid-phase chemistry in the drug discovery business, and advanced undergraduates, postgraduates, and organic chemists with an interest in leading-edge developments in their field.
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Presents both the fundamental concepts and the most recent applications in solid-phase organic synthesis With its emphasis on basic concepts, Solid-Phase Organic Synthesis guides readers through all the steps needed to design and perform successful solid-phase organic syntheses. The authors focus on the fundamentals of heterogeneous supports in the synthesis of organic molecules, explaining the use of a solid material to facilitate organic synthesis. This comprehensive text not only presents the fundamentals, but also reviews the most recent research findings and applications, offering readers everything needed to conduct their own state-of-the-art science experiments. Featuring chapters written by leading researchers in the field, Solid-Phase Organic Synthesis is divided into two parts: Part One, Concepts and Strategies, discusses the linker groups used to attach the synthesis substrate to the solid support, colorimetric tests to identify the presence of functional groups, combinatorial synthesis, and diversity-oriented synthesis. Readers will discover how solid-phase synthesis is currently used to facilitate the discovery of new molecular functionality.