Peptides are of continued interest for therapeutic applications, from soluble and immobilized ligands that promote desired binding or uptake to self-assembled supramolecular structures that serve as scaffolds in vitro and in vivo. These applications require efficient and scalable synthetic approaches because of the large amounts of material that often are needed for studies of bulk material properties and their translation. In this work, we establish new methods for the synthesis, purification, and visualization of assembling peptides, with a focus on multifunctional collagen mimetic peptides (mfCMPs) relevant for formation and integration within hydrogel-based biomaterials. First, a methodical approach useful for the microwave-assisted synthesis of assembling peptide sequences prone to deletions was established, beginning with the identification of the deleted residues and their locations and followed by targeted use of dual chemistry couplings for those specific residues. Second, purification techniques that integrate the principles of heating and ion displacement with traditional chromatography and dialysis were implemented to improve separation and isolation of the desired multifunctional peptide product, which contained blocks for thermoresponsiveness and ionic interactions. Third, an approach for fluorescent labeling of these mfCMPs, which is orthogonal to their assembly and their covalent incorporation into a bulk hydrogel material, was established, allowing visualization of the resulting hierarchical fibrillar structures in three dimensions within hydrogels using confocal microscopy. The methods presented in this work allow the production of multifunctional peptides in scalable quantities and with minimal deletions, enabling future studies for better understanding of composition–structure–property relationships and for translating these biomaterials into a range of applications. Although mfCMPs are the focus of this work, the methods demonstrated could prove useful for other assembling peptide systems and for the production of peptides more broadly for therapeutic applications.

中文翻译：

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快速生产用于水凝胶生物材料中并入和可视化的多功能自组装肽

从促进所需结合或摄取的可溶性和固定化配体到在体外和体内用作支架的自组装超分子结构，肽在治疗应用中一直受到关注. 这些应用需要高效且可扩展的合成方法，因为研究散装材料特性及其转化通常需要大量材料。在这项工作中，我们建立了组装肽的合成、纯化和可视化的新方法，重点是与水凝胶生物材料中的形成和整合相关的多功能胶原模拟肽 (mfCMP)。首先，建立了一种可用于微波辅助合成易于缺失的肽序列组装的有条理的方法，首先是识别缺失的残基及其位置，然后针对这些特定残基有针对性地使用双重化学偶联。第二，实施了将加热和离子置换原理与传统色谱和透析相结合的纯化技术，以改善所需多功能肽产品的分离和分离，其中包含热响应性和离子相互作用的模块。第三，建立了一种对这些 mfCMPs 进行荧光标记的方法，该方法与它们的组装和共价结合到块状水凝胶材料中是正交的，从而允许使用共聚焦显微镜在水凝胶内的三个维度上可视化所得的分层纤维状结构。这项工作中提出的方法允许以可扩展的数量和最少的缺失生产多功能肽，使未来的研究能够更好地理解成分-结构-性能关系并将这些生物材料转化为一系列应用。尽管 mfCMPs 是这项工作的重点，但所证明的方法可能对其他组装肽系统和更广泛地生产用于治疗应用的肽有用。