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A prominent target of monoclonal antibodies as targeted therapies for cancer is the epidermal growth factor receptor, which is overexpressed on the surface of various cancer cell types. Its natural binder, the epidermal growth factor (EGF), is a 53 amino acid polypeptide. Anticancer synthetic targeted immune system engagers (ISErs) comprising two binder' peptides, which are attached to a scaffold conveying immune stimulating effector' properties, via monodisperse polyethylene glycol chains. So far, preparation of ISErs has been limited to the use of small peptides (8-20 amino acids) as binding functionalities, and they have been entirely synthesized by solid phase peptide synthesis. Here, we describe a synthetic and a semisynthetic approach for the preparation of an ISEr bearing two murine EGF molecules as binding entities (ISEr-EGF(2)). EGF was either synthesized in segments by solid phase peptide synthesis or expressed recombinantly and ligated to the scaffold by native chemical ligation. We report the successful generation of synthetic and semisynthetic ISEr-EGF(2) as well as several challenges encountered during the synthesis and ligations. We demonstrate the application of native chemical ligation for the design of larger ISEr constructs, facilitating new objectives for the coupling of small binder peptides and larger proteins to multivalent ISEr scaffolds. Copyright (c) 2017 European Peptide Society and John Wiley & Sons, Ltd.

Deciphering the pathophysiologic events in prion diseases is challenging, and the role of posttranslational modifications (PTMs) such as glypidation and glycosylation remains elusive due to the lack of homogeneous protein preparations. So far, experimental studies have been limited in directly analyzing the earliest events of the conformational change of cellular prion protein (PrPC) into scrapie prion protein (PrPSc) that further propagates PrPC misfolding and aggregation at the cellular membrane, the initial site of prion infection, and PrP misfolding, by a lack of suitably modified PrP variants. PTMs of PrP, especially attachment of the glycosylphosphatidylinositol (GPI) anchor, have been shown to be crucially involved in the PrPSc formation. To this end, semisynthesis offers a unique possibility to understand PrP behavior in vitro and in vivo as it provides access to defined site‐selectively modified PrP variants. This approach relies on the production and chemoselective linkage of peptide segments, amenable to chemical modifications, with recombinantly produced protein segments. In this article, advances in understanding PrP conversion using semisynthesis as a tool to obtain homogeneous posttranslationally modified PrP will be discussed., The key pathophysiologic event in prion diseases is based on a conformational change of cellular (PrPC) into scrapie prion protein (PrPSc) and is closely linked to posttranslational modifications (PTMs). Semisynthesis offers a unique opportunity to study the impact of PTMs on prion conversion, transmission, and pathogenicity, which is the major focus of this review.

The properties of elastin-like polypeptides (ELPs), specifically the fact that they are soluble in aqueous buffers below and aggregate reversibly above a well-defined transition temperature, are extensively used for protein purification, enzyme recycling, and more recently, for in vivo applications such as drug delivery and tissue engineering. ELPs are artificial but biocompatible polypeptides composed of pentameric repeats (Val-Pro-Gly-Xaa-Gly) containing different guest residues Xaa, derived from mammalian elastin. The temperature-dependent aggregation and desaggregation of ELPs is controlled by composition of the pentameric repeats as well as the number of repetitive units within the ELP. External parameters such as ELP concentration, pH, and most importantly, salt effects heavily influence the transition temperature. Here, we explore the chemical synthesis of a series of 51mer peptides consisting of 10 pentameric ELP repeats with hydrophobic as well as charged guest residues such as isoleucine, leucine, alanine, lysine, and/or glutamate all prepared by Boc-based solid phase peptide synthesis. These guest residues expand the available toolbox of synthetic ELPs and provide ELPs that can be chemically modified and tuned to specific environments. An N-terminal cysteine is added allowing disulfide-based crosslinking of ELPs and to link synthetic ELPs to a recombinantly produced protein using native chemical ligation. Transition temperatures of all synthetic ELPs and the fusion construct were determined by measuring turbidity in solution and spanned a large temperature range between 25 and 70 °C, providing synthetically accessible ELPs with transition temperatures suitable for in vitro and in vivo applications. Cycling between their soluble and aggregate state has been observed at least 6 times without significant loss of material for all synthetic ELPs. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Peptide hydrazides are valuable building blocks in peptide and protein chemistry, e.g. as precursors of peptide thioesters that allow the preparation of these important intermediates under mild conditions. Additional robust and versatile methods for the generation of peptide hydrazides from standard solid supports are therefore highly desired in order to facilitate access to peptide thioester via Fmoc-based SPPS. Here, the efficient generation of peptide hydrazides from conventional 4-hydroxymethyl phenol Wang-TentalGel peptidyl resins is described. Direct hydrazinolysis of a 19mer mucin1 peptide gives the protected peptide hydrazide in excellent yields. Testing a series of octapeptides carrying the 20 common proteinogenic amino acids at their C-terminus led to preparation of all corresponding peptide hydrazides in very good to excellent yields and purities. The available set of octapeptides allowed analyzing the influence of the nature of the C-terminal amino acid and of the solvent on the hydrazinolysis reaction. Furthermore, the compatibility of the method with posttranslational modifications (here glycosylation) and with potentially sensitive functional groups in amino acid side chains makes this approach a viable alternative for obtaining peptide hydrazides. It combines the advantages of a straightforward synthesis with stereochemical stability and flexibility, as it provides easy access to the peptide acid and the peptide thioester (via the hydrazide) from the same solid support.

The R5 peptide is derived from silaffin peptides naturally occurring in the diatom Cylindrotheca fusiformis and exhibits outstanding activity in silica precipitation. Because of its ability to cause silicification under mild conditions, several biotechnological applications based on R5-mediated biomimetic silica formation have already been reported. Yet a more detailed understanding of the R5 peptide and its intrinsic silica precipitation activity will help the rational design of R5 peptide variants as efficient agents for defined silica precipitation. The herein presented analysis of the relationship between the R5 amino acid sequence and its activity in silica precipitation emphasizes the essential role of the lysine residues in mediating silica polycondensation. Furthermore, a tetra amino acid motif (RRIL) has to be present within the R5 sequence, but in contrast to previous reports, we demonstrate that localization of the RRIL motif shows minor impact on silica precipitation activity but rather on morphology of the resulting silica material. The amino acid sequence of silaffin peptides is a well-balanced arrangement in terms of charges, functional groups and distances. The impact of this pattern of charges and functionalities was highlighted by the disturbed morphology of silica spheres resulting from R5 variants with scrambled sequences. A detailed understanding of the highly evolved silaffin sequence(s) will contribute to unravel the intriguing process of silica biomineralization in diatoms.