# FOLDED SYNTHETIC PEPTIDES FOR BIOMEDICAL APPLICATIONS

EDITED BY : Alessandro Contini and Jutta Eichler PUBLISHED IN : Frontiers in Chemistry

#### Frontiers Copyright Statement

© Copyright 2007-2019 Frontiers Media SA. All rights reserved. All content included on this site, such as text, graphics, logos, button icons, images, video/audio clips, downloads, data compilations and software, is the property of or is licensed to Frontiers Media SA ("Frontiers") or its licensees and/or subcontractors. The copyright in the text of individual articles is the property of their respective authors, subject to a license granted to Frontiers.

The compilation of articles constituting this e-book, wherever published, as well as the compilation of all other content on this site, is the exclusive property of Frontiers. For the conditions for downloading and copying of e-books from Frontiers' website, please see the Terms for Website Use. If purchasing Frontiers e-books from other websites or sources, the conditions of the website concerned apply.

Images and graphics not forming part of user-contributed materials may not be downloaded or copied without permission.

Individual articles may be downloaded and reproduced in accordance with the principles of the CC-BY licence subject to any copyright or other notices. They may not be re-sold as an e-book.

As author or other contributor you grant a CC-BY licence to others to reproduce your articles, including any graphics and third-party materials supplied by you, in accordance with the Conditions for Website Use and subject to any copyright notices which you include in connection with your articles and materials.

All copyright, and all rights therein, are protected by national and international copyright laws.

The above represents a summary only. For the full conditions see the Conditions for Authors and the Conditions for Website Use. ISSN 1664-8714 ISBN 978-2-88963-045-5 DOI 10.3389/978-2-88963-045-5

#### About Frontiers

Frontiers is more than just an open-access publisher of scholarly articles: it is a pioneering approach to the world of academia, radically improving the way scholarly research is managed. The grand vision of Frontiers is a world where all people have an equal opportunity to seek, share and generate knowledge. Frontiers provides immediate and permanent online open access to all its publications, but this alone is not enough to realize our grand goals.

#### Frontiers Journal Series

The Frontiers Journal Series is a multi-tier and interdisciplinary set of open-access, online journals, promising a paradigm shift from the current review, selection and dissemination processes in academic publishing. All Frontiers journals are driven by researchers for researchers; therefore, they constitute a service to the scholarly community. At the same time, the Frontiers Journal Series operates on a revolutionary invention, the tiered publishing system, initially addressing specific communities of scholars, and gradually climbing up to broader public understanding, thus serving the interests of the lay society, too.

#### Dedication to Quality

Each Frontiers article is a landmark of the highest quality, thanks to genuinely collaborative interactions between authors and review editors, who include some of the world's best academicians. Research must be certified by peers before entering a stream of knowledge that may eventually reach the public - and shape society; therefore, Frontiers only applies the most rigorous and unbiased reviews.

Frontiers revolutionizes research publishing by freely delivering the most outstanding research, evaluated with no bias from both the academic and social point of view. By applying the most advanced information technologies, Frontiers is catapulting scholarly publishing into a new generation.

#### What are Frontiers Research Topics?

Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office<EMAIL_ADDRESS>
# FOLDED SYNTHETIC PEPTIDES FOR BIOMEDICAL APPLICATIONS

Topic Editors: Alessandro Contini, Università degli Studi di Milano, Italy Jutta Eichler, University of Erlangen–Nurnberg, Germany

Image: Folded Synthetic Peptides in Biomedicine. by Alessandro Contini and Jutta Eichler

Folded peptides - and peptide motifs within proteins - are abundant in living organisms, where they are essential for the biological activities of the peptides and proteins. During the past decades, much research has been dedicated to understanding the rules that govern peptide folding. Simultaneously, a range of strategies have been established for the conformational stabilization of bioactive peptides, as well as for the de novo design of peptides with defined secondary structures. These methods are either based on the chemical modification of the peptide backbone, such as cyclization and stapled peptides, or on the use of a range of non-proteinogenic amino acids that, in a defined sequential arrangement, induce secondary structures peptides. Such building blocks include D- and other non-proteinogenic amino acids, as well as beta- and gamma-amino acids.

This Research Topic comprises a collection of papers by an international group of 77 scientists with a background in synthetic, analytical, computational and medicinal chemistry, as well as in biochemistry and pharmacology. Their research is presented here in a total of 11 papers (8 original research reports and 3 reviews), covering diverse aspects of folded synthetic peptides. These studies include the preparation and characterization of new peptide monomers with interesting folding properties, the synthesis and conformational analysis of non-natural peptides, as well as the use of folded peptidomimetics as molecular switches. Additionally, a range of biomedical applications, such as antimicrobial, anti-inflammatory, antiangiogenic and immune-stimulating activities, are also reported.

We hope this eBook will be a source of inspiration and knowledge for scientist in various disciplines related to folded peptides and their many applications, as well as for those who want to learn more about this fascinating field of research.

Citation: Contini, A., Eichler, J., eds. (2019). Folded Synthetic Peptides for Biomedical Applications. Lausanne: Frontiers Media. doi: 10.3389/978-2-88963-045-5

# Table of Contents

*06 Editorial: Folded Synthetic Peptides for Biomedical Applications* Jutta Eichler and Alessandro Contini

#### CHAPTER 1

#### NON-NATURAL AMINO ACIDS AND PEPTIDOMIMETICS FOR PEPTIDE FOLDING

## CHAPTER 2

#### BIOPHARMACEUTICAL APPLICATIONS OF FOLDED SYNTHETIC PEPTIDES

Anne C. Conibear, Karine Thewes, Nadja Groysbeck and Christian F. W. Becker

*93 Probing Anti-inflammatory Properties Independent of NF-*κ*B Through Conformational Constraint of Peptide-Based Interleukin-1 Receptor Biased Ligands*

Azade Geranurimi, Colin W. H. Cheng, Christiane Quiniou, Tang Zhu, Xin Hou, José Carlos Rivera, Daniel J. St-Cyr, Kim Beauregard, Vadim Bernard-Gauthier, Sylvain Chemtob and William D. Lubell

#### CHAPTER 3

FOLDED SYNTHETIC PEPTIDES FOR THE DESIGN OF FUNCTIONAL BIOMATERIALS

*113 Self-Assembling Peptides as Extracellular Matrix Mimics to Influence Stem Cell's Fate*

Katharina S. Hellmund and Beate Koksch

*121 Novel Materials From the Supramolecular Self-Assembly of Short Helical ß3-Peptide Foldamers*

Ketav Kulkarni, Nathan Habila, Mark P. Del Borgo and Marie-Isabel Aguilar

*133 Enhancement of the Stability and Anti-DPPIV Activity of Hempseed Hydrolysates Through Self-Assembling Peptide-Based Hydrogels* Carmen Lammi, Carlotta Bollati, Fabrizio Gelain, Anna Arnoldi and Raffaele Pugliese

# Editorial: Folded Synthetic Peptides for Biomedical Applications

Jutta Eichler 1 and Alessandro Contini 2 \*

1 Department of Chemistry and Pharmacy, University of Erlangen–Nurnberg, Erlangen, Germany, 2 Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Generale e Organica "Alessandro Marchesini", Università Degli Studi di Milano, Milan, Italy

Keywords: foldamer, bioactive peptide, conformational analysis, molecular switches, hydrogels

**Editorial on the Research Topic**

#### **Folded Synthetic Peptides for Biomedical Applications**

Folded peptides—and peptide motifs within proteins—are abundant in living organisms, where they are essential for the biological activities of the peptides and proteins. During the past decades, numerous research programs have been dedicated to understanding the rules that govern peptide folding. Simultaneously, a range of strategies have been established for the conformational stabilization of bioactive peptides, as well as for the de novo design of peptides with defined secondary structures. These methods are either based on the chemical modification of the peptide backbone, such as cyclization and side chain stapling, or on the use of a range of non-proteinogenic amino acids that, in a defined sequential arrangement, induce secondary structures in peptides. Such building blocks include D- and other non-proteinogenic amino acids, as well as β- and γ-amino acids.

This Research Topic comprises a collection of papers by an international group of 77 scientists with a background in synthetic, analytical, computational, and medicinal chemistry, as well as in biochemistry and pharmacology. Their research is presented here in a total of 11 papers (8 original research reports and 3 reviews), covering diverse aspects of folded synthetic peptides. These studies include the preparation and characterization of new peptide monomers with interesting folding properties, the synthesis and conformational analysis of non-natural peptides, as well as the use of folded peptidomimetics as molecular switches. Additionally, a range of biomedical applications, such as antimicrobial, anti-inflammatory, antiangiogenic, and immune-stimulating activities, are also reported.

Examples for the use of non-peptidic chemical moieties or non-natural amino acids for the generation of folded synthetic peptides include the use of 1,4-disubstituted 1H-1,2,3-triazoles (Schröder et al.) to mimic the amide bond. This bioisosteric replacement led to peptidodimetics whose foldamer properties were confirmed by both instrumental analysis and computational simulations. Similarly, two enantiomers of a bicyclic pyrrolidine-isoxazoline γ-amino acid were used to prepare diastereoisomeric model peptides (Oliva et al.). NMR, FT-IR, circular dichroism and molecular modeling studies confirmed that the (+) enantiomer was able to stabilize an α-turn conformation. Conversely, Bucci et al. used syn-β2,3-diarylamino acids in conjunction with S-alanine for the generation of foldameric antiparallel β-sheets. As in the study by Oliva et al., foldamer properties were confirmed here as well by combining instrumental analysis and theoretical calculations. Applications of synthetic peptides/peptidomimetics include their use as molecular switches, utilizing the light-triggered reversible shift from one conformation to another. In this context, Nuti et al. investigated the ability of a new photochromic azobenzene amino acid derivative to act as a conformational switch, when inserted into a model peptide.

Edited and reviewed by: Tony D. James, University of Bath, United Kingdom

> \*Correspondence: Alessandro Contini<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Supramolecular Chemistry, a section of the journal Frontiers in Chemistry

Received: 14 May 2019 Accepted: 03 June 2019 Published: 20 June 2019

#### Citation:

Eichler J and Contini A (2019) Editorial: Folded Synthetic Peptides for Biomedical Applications. Front. Chem. 7:448. doi: 10.3389/fchem.2019.00448

A broad range of biopharmaceutical applications of synthetic peptides are also reported in this Research Topic. Recent applications of folded synthetic peptides targeting proteins of the outer membrane of gram-negative bacteria are reviewed by Robinson. In particular, synthetic β-hairpin mimetic peptides were found to interact with β-barrel and β-jellyroll domains in bacterial lipopolysaccharide transport and β-barrel folding machine complexes, thus representing a new frontier in the discovery of novel antimicrobial agents. Folded peptides are also reported to target tumor angiogenesis. Zanella et al. present oligopeptides, designed by structural analysis and computational calculations, where Cα,α-disubstituted amino acids are exploited to stabilize the helical conformation that is essential to bind to the VEGF receptor at nanomolar concentration. Synthetic peptides as cancer-targeting immune system engagers (ISErs) are reported by Conibear et al. These molecule are generated using a range of chemoselective ligations, including copper-catalyzed azide-alkyne "click," oxime, maleimide, and native chemical ligations. Furthermore, anti-inflammatory peptides targeting the interleukin-1 receptor (IL-1R) are reported by Geranurimi et al. The authors describe the structure-activity relationships of 12 peptides, in which different configurations of the α-amino-γlactam and β-hydroxy-α-amino-γ-lactam moieties were used to conformationally constrain an IL-1R peptide ligand.

This Research Topic also presents articles on the use of folded synthetic peptides for the design of functional biomaterials. Hellmund and Koksch review the recent literature on the use of self-assembling peptides as mimics of the extracellular matrix. This is illustrated by peptide- and protein-based biomaterials that are able to support proliferation and differentiation of stem cells, demonstrating great potential of these peptides as tools in regenerative medicine. Applications of N-acetyl-β3-peptides to obtain innovative bio- and nanomaterials by supramolecular self-assembly are reviewed by Kulkarni et al. Compared to other organic and inorganic self-assembled systems, these foldamers show advantages in terms of biocompatibility, toxicity and functionalization potential. Last but not least, Lammi et al. report a strategy to increase the stability and antidiabetic properties of hempseed protein hydrolysates. This was achieved through encapsulation of hempseed hydrolysates into ionic self-complementary RADA16 peptide-based hydrogels. This study also evaluated the synergistic activity of RADA16-hemp hydrogels and sitagliptin, an orally available DPPIV inhibitor.

As a collection, the papers of this Research Topic demonstrate the broad impact of folded synthetic peptides on various fields of biological and biomedical research. It can be expected that research on folded synthetic peptides will continue to result in applications in medicinal chemistry and drug design, as well as the design of novel biomaterials.

## AUTHOR CONTRIBUTIONS

AC and JE edited the articles of this research topic and wrote the editorial.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Eichler and Contini. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# 1,4-Disubstituted 1H-1,2,3-Triazole Containing Peptidotriazolamers: A New Class of Peptidomimetics With Interesting Foldamer Properties

David C. Schröder 1 , Oliver Kracker 1 , Tanja Fröhr 1 , Jerzy Góra1,2, Michał Jewginski 2 , Anke Nieß1 , Iris Antes 3 , Rafał Latajka2 , Antoine Marion3,4 \* and Norbert Sewald1 \*

1 Organic and Bioorganic Chemistry, Department of Chemistry, Bielefeld University, Bielefeld, Germany, 2 Department of Bioorganic Chemistry, Wrocław University of Science and Technology, Wrocław, Poland, 3 Center for Integrated Protein Science, TUM School of Life Sciences, TU Munich, Freising, Germany, 4 Department of Chemistry, Middle East Technical University, Ankara, Turkey

#### Edited by:

Jutta Eichler, University of Erlangen Nuremberg, Germany

#### Reviewed by:

Nicolas Inguimbert, Université de Perpignan Via Domitia, France Yuji Nishiuchi, GlyTech, Inc., Japan Norbert Schaschke, Hochschule Aalen, Germany

#### \*Correspondence:

Norbert Sewald<EMAIL_ADDRESS>Antoine Marion<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Chemical Biology, a section of the journal Frontiers in Chemistry

Received: 30 November 2018 Accepted: 01 March 2019 Published: 26 March 2019

#### Citation:

Schröder DC, Kracker O, Fröhr T, Góra J, Jewginski M, Nieß A, Antes I, Latajka R, Marion A and Sewald N (2019) 1,4-Disubstituted 1H-1,2,3-Triazole Containing Peptidotriazolamers: A New Class of Peptidomimetics With Interesting Foldamer Properties. Front. Chem. 7:155. doi: 10.3389/fchem.2019.00155

Peptidotriazolamers are hybrid foldamers with features of peptides and triazolamers, containing alternation of amide bonds and 1,4-disubstituted 1H-1,2,3-triazoles with conservation of the amino acid side chains. We report on the synthesis of a new class of peptidomimetics, containing 1,4-disubstituted 1H-1,2,3-triazoles in alternation with amide bonds and the elucidation of their conformational properties in solution. Based on enantiomerically pure propargylamines bearing the stereogenic center in the propargylic position and α-azido esters, building blocks were obtained by copper-catalyzed azide-alkyne cycloaddition. With these building blocks the peptidotriazolamers were readily available by solution phase synthesis. A panel of homo- and heterochiral tetramers, hexamers, and heptamers was synthesized and the heptamer Boc-Ala-Val-Ψ [4Tz]Phe-LeuΨ[4Tz]Phe-LeuΨ[4Tz]Val-OAll as well as an heterochiral and a Gly-containing equivalent were structurally characterized by NMR-based molecular dynamics simulations using a specifically tailored force field to determine their conformational and solvation properties. All three variants adopt a compact folded conformation in DMSO as well as in water. In addition to the heptamers we predicted the conformational behavior of similar longer oligomers i.e., Boc-Ala-(AlaΨ[4Tz]Ala)6-OAll as well as Boc-Ala-(D-AlaΨ[4Tz]Ala)6-OAll and Boc-Ala-(GlyΨ[4Tz]Ala)6-OAll. Our calculations predict a clear secondary structure of the first two molecules in DMSO that collapses in water due to the hydrophobic character of the side chains. The homochiral compound folds into a regular helical structure and the heterochiral one shows a twisted "S"-shape, while the Gly variant exhibits no clear secondary structure.

Keywords: peptidotriazolamers, 1, 4-disubstituted 1H-1, 2, 3-triazole, peptide bond isoster, foldamer, molecular dynamic simulations

## INTRODUCTION

Since the discovery of the CuI catalyzed azide-alkyne cycloaddition (CuAAC) by the groups of Meldal (Tornøe et al., 2002) and Sharpless (Rostovtsev et al., 2002), this so-called "click"-reaction proved to be suitable for a broad number of applications in bioorganic and pharmaceutical chemistry (Kolb and Sharpless, 2003; Bock et al., 2006a; Angell and Burgess, 2007; Gil et al., 2007; Moses and Moorhouse, 2007; Hein et al., 2008; Tron et al., 2008; Nwe and Brechbiel, 2009) The CuAAC is compatible with most functional groups present in biomolecules and the 1,4-disubstituted 1H-1,2,3 triazole obtained as reaction product is metabolically inert (Kolb and Sharpless, 2003). Size and dipole moment of the 1,4 disubstituted triazole ring are larger compared to a trans-amide bond (Kolb and Sharpless, 2003), but overall physicochemical properties are similar enough to enable these triazoles to act as trans-amide mimetics (**Figure 1**; Brik et al., 2003, 2005; Horne et al., 2004, 2009; Angell and Burgess, 2005, 2007; van Maarseveen et al., 2005; Bock et al., 2006b, 2007; Wang et al., 2006; Appendino et al., 2007; Kim et al., 2007; Lee et al., 2007; Beierle et al., 2009).

In 2005 the counterpart of the CuAAC, the ruthenium(II) catalyzed azide-alkyne-cycloaddition (RuAAC), yielding selectively 1,5-disubstitued 1H-1,2,3-triazoles, was presented by Zhang et al. (2005). The latter can act as cis-amide mimetic (**Figure 1**; Zhang et al., 2005; Pedersen and Abell, 2011).

Several amide bonds within biologically active substances have already been replaced by triazoles. The bio-isosterism has been exemplified by triazole analogs of a matrix metalloprotease inhibitor (Wang et al., 2006), of the immunestimulating natural compound α-galactosylceramide (Kim et al., 2007; Lee et al., 2007), and of capsaicin in its role as agonist of the vanilloid-receptor TRPV1 (Appendino et al., 2007). Small bivalent peptidomimetics based on triazole formation from amino acid-derived propargylamines and amino acid-derived α-azido acids were shown to adopt turnlike conformations and serve as ligands for the receptor TrkC (Chen et al., 2009).

Further examples are triazole analogs of the peptidic tyrosinase inhibitor cyclo-[Pro-Tyr-Pro-Val] (Bock et al., 2006b, 2007), of the histone deacetylase inhibitor apicidin (Horne et al., 2009), the antimitotic cyclodepsipeptide cryptophycin (Nahrwold et al., 2010) and of peptides containing the pharmacophoric residues of somatostatin (Beierle et al., 2009). Moreover, X-ray crystal structure analysis revealed that the triazole ring within an analog of the HIV-1-protease inhibitor amprenavir interacts with the enzyme in the same way as an amide bond in the parent compound (Brik et al., 2003, 2005). Recently Ben Haj Salah et al. gave a good overview on the topic of substituting single or isolated amide moieties by 1H-1,2,3 triazoles and showed the influence of isolated triazole-amidebond substitution as well as two cases with multiple substitutions containing a motif of two triazole replacements separated by one amide bond in two antimicrobial peptides with helical conformations (Ben Haj Salah et al., 2018).

Triazolamers, oligomers of 1,4-disubstituted triazoles connect a series of triazole rings by single C1 unit that, at the same time, is a stereogenic center (Angelo and Arora, 2005, 2007; Jochim et al., 2009). The CuAAC can be performed either in solution or on solid phase to provide the 1,4-disubsituted triazoles. Best results are obtained in a sequential protocol when using triflic azide and Cu(II) for the diazo transfer, where the copper was subsequently reduced by ascorbate to bring about the CuAAC (**Figure 2**; Angelo and Arora, 2005; Beckmann and Wittmann, 2007).

Since the triazole ring is characterized by a large dipole moment it might stabilize discrete conformations by dipole-dipole interactions. The Arora group investigated relatively short oligomers and found out that they are likely to adopt a zigzag conformation reminiscent of β-strands found in peptides. Indeed, the dipole-dipole-interaction of neighboring triazole moieties seems to crucially influence the overall conformation in polar solvents (Angelo and Arora, 2005).

amide bond isoster.

The zigzag triazolamer structure also resembles the oligopyrrolinones described by Smith and Hirschmann (Smith et al., 1992). The axial Cβ to Cβ distance between the i and i+2 side chain residues in peptide β-strands is 7.2 Å; this distance is roughly 7.9 Å in the zigzag triazolamer. The Cβ to Cβ distances in adjacent residues are 5.5 Å in peptide β-strands and a little longer (6.8 Å) in the triazolamer (Angelo and Arora, 2005).

In addition, the group of L. Mindt investigated besides the substitution of isolated peptide bonds by 1,4-disubstituted triazoles also the replacement of multiple adjacent peptide bonds attempting to improve the tumor targeting qualities of bombesin, an amphibious analog of the gastrin releasing peptide (Valverde et al., 2015).

Peptidotriazole foldamers represent repetitive hybrid structures of peptides, where e.g., every second amide bond is being replaced by a triazole. Such short triazole-based peptide analogs have been obtained from achiral propargylamines and α-amino acid derived α-azido carboxylic acids (**Figure 3**, left). They were found to self-dimerize and act as organogelators (Ke et al., 2012).

In a similar fashion, the Hecht group synthesized bifunctional monomers from amino acid-derived α-azido acids and propargylamine for "click"-polyaddition (**Figure 3**, right; Hartwig and Hecht, 2010).

FIGURE 3 | Triazole-based peptidomimetic with unsubstituted propargylamines (left) and "Click"-polyaddition oligomer; bifunctional monomers from amino acid-derived α-azido acids and unsubstituted propargylamine (right).

In accordance with recommendations for nomenclature of amide bond replacements we will use Ψ[4Tz] in this publication as a placeholder of a 1,4-disubstituted 1H-1,2,3-triazole–i.e., a dipeptide H-Xaa-Yaa-OH is replaced by H-XaaΨ [4Tz]Yaa-OH. In a similar fashion Ψ[5Tz] stands for a 1,5-disubstituted 1H-1,2,3-triazole replacement in the same orientation.

Recently we published on 1,5-disubstituted 1,2,3-triazole containing peptidotriazolamers (Kracker et al., 2018). Beside the robust synthetic route it was shown that the homochiral and heterochiral oligomers adopt versatile foldamer conformations. Furthermore, the solid state conformation of the homochiral tetramer Boc-ValΨ[5Tz]Ala-LeuΨ[5Tz]Val-OBzl was in an excellent agreement with an in silico conformational analysis, which was performed using a specifically truncated TZLff molecular mechanics force-field parametrization for 1,4- and 1,5 disubstituted peptidotriazolamers that we recently established (Marion et al., 2018).

For the synthesis of the chiral propargylamines, we used the methodology previously described by Ellman et al. using (R)- or (S)-tert-butylsulfinamide as a chiral auxiliary (Patterson and Ellman, 2006). According to the protocol of Ellman et al. the chiral tert-butylsulfinamide is prepared by enantioselective oxidation of di-tert-butyl disulfide with a chiral catalyst, followed by reaction with lithiumamide (Weix and Ellman, 2005). After formation of the imine using the auxiliary and an aldehyde as starting material, the diastereoselective synthesis of propargylamines is possible by reaction of the N-sulfinyl imine with lithium trimethylsilylacetylide (Wünsch et al., 2017). Cleavage of the trimethylsilyl protecting group is achieved with TBAF or AgNO3 to provide the N-sulfinylpropargylamines with a terminal alkyne moiety. Numerous tert-butylsulfinyl protected propargylamines [(S)- or (R)-Bus-Xaa≡CH] have been prepared in our group, bearing natural amino acid side chains as well as non-amino acid like side chains **(Figure 4**; Wünsch et al., 2017).

## RESULTS AND DISCUSSION

#### Synthesis

Inspired by the Arora triazolamers, we embarked on a project directed toward the synthesis and characterization of peptidotriazole foldamers, where both components, the propargylamine and the α-azido carboxylic acids are chiral. The chiral propargylamines were synthesized via the Ellman strategy and the α-azidocarboxylic acids from commercially available α-amino acids by diazotransfer (Yan et al., 2005). Several αazidocarboxylic acids as well as the corresponding amino acid allyl esters can be synthesized under mild reaction conditions with good to very good yields.

In principle, there are two strategies for the synthesis of peptidotriazole foldamers: the building block approach (**Figure 5**), where the triazole is formed from the propargylamine and the azide by CuAAC and then coupled by amide bond formation, and the submonomer approach (**Figure 5**), where first

FIGURE 4 | Synthesis of diasteromerically pure N-sulfinyl propargylamines, used for the oligomers of this publication.

the α-azido carboxylic acid is coupled and then the N-protected propargylamine is reacted by CuAAC.

In the case of activation of the free acid of the triazolebuilding block **4** (Val side chain), epimerization was observed even upon using TBTU/HOBt/DIPEA or HATU/HOAt/DIPEA as the coupling reagents. Therefore, the submonomer approach was initially chosen for the synthesis of the peptidotriazole foldamers. In this case the α-azido carboxylic acid can be activated with e.g., HATU/HOAt without any epimerization. Eventually it has been shown, that a carbodiimide-mediated preactivation in DCM, without the use of a base, and subsequent coupling with the use of 2,4,6-collidine, effectively prevents epimerization during the coupling of the free acid of the triazole-building block **4** (Kracker et al., 2018). K Ben Haj Salah et al. also used the building block approach for their synthesis of peptaibols analogs containing AibΨ[4Tz]-Xaa dipeptides (Ben Haj Salah et al., 2015).

The C-terminal component was protected as allyl ester and the propargylamines were employed as sulfinamide as well as Boc protected derivatives. The tert-butylsulfinyl group (Bus) is acid-labile and can be cleaved e.g., by treatment with 4 M HCl in dioxane at room temperature. In addition, it was reported to be cleavable with 50% trifluoroacetic acid in CH2Cl2 (Lee and Silverman, 2000).

An example of the peptidotriazole foldamer synthesis is given in **Figure 6**. Triazole formation was achieved by CuAAC in the presence of 0.5 eq. of CuSO4·5 H2O and 1 eq. sodium ascorbate in DMF/water mixtures. Lower amounts of copper salt and reductant led to incomplete conversion, as had been previously observed by others (Angelo and Arora, 2005).

Several homo- (**7a**, **9a**, **10a**) and heterochiral (**7b**, **9b**, **10b**) peptidotriazole foldamers as well as one where every second amino acid side chain is exchanged by a hydrogen atom (**7c**, **9c**, **10c**), have been obtained (**Figure 7**). Some peptidotriazole foldamers with Nterminal Bus group that had been purified by HPLC with an acetonitrile/water/TFA gradient slowly decomposed in DMSO solution upon standing. Notably, this instability also depends on the sequence. After cleavage of the Bus group and acylation e.g., with Boc-Ala the resulting heptamers were completely stable. The tert-butylsulfinyl group had been suggested as a substitute for the Boc group. However, its stability, in particular at elevated temperatures, is not sufficient to qualify Bus as a versatile protecting group. According to the rearrangement suggested for the Ellman auxiliary by Arava et al. (2011) we found out that a fragment (S) or (R)-BusXaaΨ[4Tz]Yaa-OAll rearranges with a second equivalent to form (S) or (R)- H-XaaΨ[4Tz]Yaa-OAll and (tert-Bu-SO2)(tert-Bu-S)Xaa Ψ[4Tz]Yaa-OAll.

#### Conformational Analysis

We analyzed the conformational preferences of 1,4-disubstituted peptidotriazolamers in solution by means of CD spectroscopy and molecular dynamics (MD) simulations based on the specific molecular mechanics force-field parameterization TZLff (Marion et al., 2018). As representative molecules, we selected compounds **10a**, **10b,** and **10c** (further referred to as homochiral, heterochiral, and homochiral∗ , respectively) to investigate the effect of chirality and of the introduction of glycine-like moieties on the conformational preferences of the peptide mimetics. Schematic representations of the three molecules are given in **Figure 8**. Therein, we additionally indicate our definition for the backbone conformational degrees of freedom for peptidotriazolamers as the φ and ψ torsion angles. In the following, φ and ψ notations are used indifferently whether it refers to a standard amino acid or to a triazole-based derivative (Marion et al., 2018).

ascorbate, H2O/DMF, RT, 15 h; e) 4 M HCl in dioxane, MeOH, RT, 2 h; f) N2=Phe-OH, HATU, HOAt, DIPEA, DMF, RT, 15 h; g) (S)-Bus-Val-CH, CuSO4·5H2O, sodium ascorbate, H2O/DMF, RT, 15 h; h) 4 M HCl in dioxane, MeOH, RT, 2 h; i) Boc-Ala-OH, HATU, HOAt, DIPEA, DMF, RT, 15 h.

#### CD Spectroscopy

CD spectra were recorded to get a first experimental insight into the conformational properties of the three oligomer types. It should be emphasized that the solvent used for the measurements is acetonitrile since DMSO is not applicable due to its strong absorbance below 270 nm. It becomes clear (**Figure 9**) that the different types of peptidotriazole heptamers have characteristic CD signatures. The heterochiral compound **10b** has a positive CD below 210 nm, while the CD remains negative for the homochiral all-(S)-configured **10a**. The CD spectrum of **10c** is somewhat intermediate, with a sign inversion at about 202 nm.

#### Molecular Dynamics

For each of the three representative molecules (**10a-c**), we performed one simulation in DMSO (i.e., the solvent used for NMR measurements) and one simulation in water, as representative of a more biologically relevant environment. The simulations in solution were started using an initial structure derived from an extensive, simulated annealing-based sampling in gas phase during which NMR-derived interproton distance restraints were applied (see section Methodology for details).

Monitoring of the backbone root mean square deviations (RMSD) along the trajectories of each molecule showed that most compounds adopt one main stable conformation in both solvents, with standard deviations ranging from 0.18 to 0.79 Å over the whole trajectory for all six MD trajectories (**Supplementary Figure 10**). Repetitive bumps in the RMSD time series of **10c** in water show that the molecules adopt at least two stable conformations in water and that our MD simulations sampled them appropriately. Radius of gyration (ROG) time series indicate similar results for all compounds with mean values ranging from 3.5 to 4.0 Å with minor amplitudes of fluctuations after 100 ns (**Supplementary Figure 11**). Such small values for the ROG of the 1,4-disubstituted peptidotriazolamers are indicative for a compact folded conformation for all three variants. All the time series indicate that the simulations reached convergence after 100 ns at most. We therefore considered the last 100 ns as converged and used this part in each simulation for the following analysis.

The distribution of φ/ψ backbone torsional degrees of freedom is depicted in **Figure 10** for each molecule in both solvents. Although the plots resemble Ramachandran distributions, a direct comparison with known patterns for standard peptides is not straightforward due to the introduction of the triazole moieties in the sequence. Nevertheless, the plots give a qualitative picture of the differences and similarities

between the three variants considered in this analysis and of the effect of the solvent polarity on their conformational preferences. Overall, all three molecules appear to have their own unique signature in terms of distribution of backbone torsion angles. While distribution maxima depict distinct patterns for homo- and heterochiral, the glycine-containing derivative (homochiral∗ ) shows features of both parent oligomers. The polarity of the solvent appears to have little to no effect on the φ/ψ distribution for all three molecules.

Clustering based on the backbone RMSD of the oligomers for the second half of the simulations in DMSO and in water resulted in the following populations for the main structural clusters: 94 and 52% for the homochiral molecule in DMSO and in water, respectively, 71 and 59% for the heterochiral, and 60 and 76% for the homochiral∗ compound. For the heterochiral compound in water, a second structural cluster was found with a population of 40% that differed from the main representative structure with a backbone RMSD value of 0.54 Å. The differences were localized on the flexible terminal residues and both structures can, therefore, be formally assigned to the same conformation with a total population of 99%, as confirmed by the rather flat RMSD and radius of gyration time-series (**Supplementary Figures 10, 11**). Similarly, the homochiral∗ variant in DMSO results in a second main cluster with a trajectory fraction of 21% and a backbone RMSD value of 0.81 Å, primarily caused by both flexible ends pointing in orthogonal direction. Thus, the two main clusters can be joined with a combined trajectory fraction of 81%. Such large populations are consistent with the rigid character of the peptidomimetics already observed from the RMSD time series. To a given extent, the homochiral∗ molecule appears to be slightly more flexible than the others when plunged in water. The representative structure for the main cluster of each simulation is depicted in the upper panel of **Figure 11**. In general, all oligomers considered in this analysis fold with a helically twisted conformation. Consistently with our observations thus far, the solvent has little to no effect on the conformation of the oligomers. The values of backbone torsional angles for the

FIGURE 8 | Homo- and heterochiral peptidotriazolamers as representatives for in silico structure determination. The definition for the torsion angles φ and ψ for these peptidomimetic torsions is annotated by the colored bonds.

representative structure in DMSO and in water are listed in **Table 1**. The lower panel of **Figure 11** shows a direct comparison of the backbone of the homochiral and homochiral∗ variants, the heterochiral and homochiral∗ ones, as well as a comparison between homo- and heterochiral ones. The figure illustrates that the homochiral and homochiral∗ compounds fold into a nearly identical conformation in DMSO and in water, while the heterochiral molecule adopts a slightly different orientation of the triazole rings in DMSO and a significantly yet still compact conformation in water. Taking the homochiral molecule as reference, the homochiral∗ and heterochiral compounds have a backbone RMSD value of 0.45 and 1.97 in DMSO, respectively, and of 1.27 and 3.61 Å in water.

We further analyzed the potential solvation properties of the three oligomers in water by calculating the radial distribution functions (RDF) around key chemical groups. The complete results are available as supporting information (**Supplementary Figures 12–14**). In general, the RDFs show that the molecules are poorly solvated in water, which is expected regarding the highly hydrophobic character of the side chains. Only specific groups such as the backbone carbonyl moiety show stable interactions with one water molecule over the whole simulation with distances characteristic of hydrogen bonds. Such interactions are conserved over all three molecules, as illustrated by the RDF for the carbonyl group of the first alanine amino acid in **Figure 12**. Therein, we observe a sharp peak at about 1.8 Å, but no further structure formation at larger distances

due to the hydrophobic character of the rest of the molecule. Among all the potential hydrogen bond donor/acceptor groups in the peptidomimetic considered in this work, we noted only few significant differences. (i) A shallow peak is present at short distances in the RDF of the nitrogen atom (N3) of the first triazole ring for both the homochiral and the homochiral∗ molecules. The integration of this peak returns a value corresponding to a sporadic interaction with a water molecule occurring for about a third of the simulation time only. This weak interaction, however, is totally absent in the case of the heterochiral compound. (ii) Similar observations can be made regarding the hydrogen of that same triazole ring, which is involved in a weak interaction with water in two of the three molecules. As opposed to (i), however, this interaction is absent for the homochiral molecule and present for the two others. We also notice that, considering the sharpness of the peak, this interaction is tighter in the case of the heterochiral molecules than it is for the homochiral∗ one. (iii) Comparison of the RDF around the amide nitrogen of the 8 th residue (i.e., Leu or Gly) indicates that this site is slightly more solvent accessible in the case of the heterochiral compound than it is for the two other molecules. These differences are due to a slightly better orientation of the corresponding chemical groups favoring interactions with the solvent and potentially with other polar compounds in different environments. Although these interactions with water are somewhat weak for these oligomers, the differences that we observed are indicative of potential interaction sites that could discriminate one molecule from another in a hypothetical protein environment.

Overall our conformational analysis revealed only little differences between the three variants of the oligomers under investigation. All three adopt a compact folded conformation in DMSO. The homochiral compound (**10a**) and the glycine containing analog (**10c**) adopt nearly identical conformations in DMSO, which tends to correlate with the observation of a similar pattern on the CD spectra for these two molecules. Considering the high hydrophobic character of the side chains, it is not surprising that the molecules remain folded in water in order to minimize the interaction surface area with the polar environment. This observation that the chirality of the different stereocenters has no significant impact on the conformation of the 1,4-disubstituted peptidotriazolamers is in contrast with our results for the 1,5-disubstituted counterpart (Kracker et al., 2018). There we identified that changes in the chirality of the α-carbons can induce drastic changes in the conformation of the molecules, i.e., while the homochiral hexapeptidotriazolamer Boc-AlaΨ[5Tz]Phe-ValΨ[5Tz]Ala-Leu-Ψ[5Tz]Val-OBzl formed

a compact β-turn-like structure, the heterochiral equivalent was observed and predicted in an extended polyproline I-like conformation (Ke et al., 2012).

homochiral\* in orange (DMSO) and purple (Water), respectively.

To further characterize the conformational properties of 1,4-disubstituted peptidotriazolamers, we analyzed a longer, hypothetical oligomer bearing only methyl side chains to mimic a poly-alanine peptide **Figure 8**). Similarly, to the case of shorter molecules, we considered three variants labeled as homochiral, heterochiral, and homochiral∗ . Due to the significantly larger conformational space of these oligomers, we performed four parallel 200 ns simulations for each molecule in each of the two solvents (i.e., in DMSO and in water, for a total of 24 simulations) in order to reach a statistically significant sampling.

The RMSD time series for the whole trajectory of each replicate simulation are shown in **Figure 13**. As expected, the simulations took longer to reach their local equilibrium compared to the case of the smaller molecules. After 150 ns, all trajectories reach a stable value of RMSD or oscillate between two conformational basins in a balanced manner. Compared to the case of shorter oligomers in which we had NMR information to produce relevant initial structures, the lack of experimental data in the present case translates into higher RMSD values since our initial geometries are likely to be far from a minimum conformation. Nevertheless, all simulations reached equilibrium and yield a distribution of stable conformers.

In contrast to the case of short oligomers, the homo- and heterochiral polyalanine derivatives suffer from a significant decrease of flexibility when moving from DMSO to water. This is illustrated in **Figure 13** by the fact that all four simulations systematically converge toward the same value of

RMSD in water only. Once this stable plateau is reached, the amplitude of fluctuations also decreases, showing the tight conformational restraints exerted by the solvent on the solute. Such a drastic effect of the solvent was, however, not observed in the case of the glycine-containing variant (homochiral∗ ), which is most likely due to the significantly reduced hydrophobic character of that molecule. ROG time series are available as supporting information (**Supplementary Figure 15**) for the whole trajectories and show that even significant fluctuations in RMSD do not result in large changes in the compactness of the conformations. The φ/ψ torsion angle distributions (**Supplementary Figure 16**) are in a good agreement with the RMSD fluctuations, showing that all three molecules have a unique conformational signature.

We performed the clustering analysis over the last 50 ns of the trajectories of all four replica. The main structural clusters in DMSO and water showed a population of 55 and 95%, respectively for the homochiral, 72 and 91% for the heterochiral, and 52 and 40% for the homochiral∗ oligomer. For the homochiral∗ molecule in water three other clusters with significant population (i.e., 32 and 27%) were identified, but showing a high similarity with the main cluster as indicated by backbone RMSD values as low as 1.4 and 0.8 Å.

The representative structures are shown in **Figure 14**. The homochiral molecule shows a clear helical conformation in DMSO, which collapses in water. The heterochiral molecule shows a compactness in DMSO similar to that of the homochiral one. However, the conformation of the former describes a twisted "S"-shape, which also collapses in water. The Gly-substituted oligomer shows no clear secondary structure in either of the solvents, which is in agreement with the more flexible character of this molecule as interpreted from the RMSD time series.

homochiral, the heterochiral, and the homochiral\* oligomers (10a-c, homochiral, heterochiral, homochiral\*) in TIP4Pew water.

## CONCLUSION

Herein we present a new class of peptidomimetics containing 1,4-disubstituted 1H-1,2,3-triazoles and amide bonds in an alternating fashion. The synthesis is based on chiral propargylamines and chiral α-azido acids. Beside the introduction of a versatile and robust synthetic strategy we elucidated their foldamer properties by means of molecular dynamics simulations in explicit solvent. Homo- and heterochiral tetra-, hexa-, and heptamers based on two different sequences as well as a homochiral oligomer, in which every second sidechain is replaced by a proton, have been synthesized based on a submonomer approach. Molecular modeling of the Boc-Ala-ValΨ[4Tz]Phe-LeuΨ[4Tz]Phe-LeuΨ[4Tz]Val-OAll sequence (homo- and heterochiral) as well as a Gly-substituted derivative revealed a compact folded conformation in DMSO, similar for all three variants. The conformation of these molecules remains compactly folded in water as well, due to the hydrophobic character of the non-polar side chains. In addition, we predicted the conformations of homo-, heterochiral, and glycine-containing poly-alanine peptidotriazole oligomers. In contrast to the shorter oligomers, our calculations suggest a well-defined and significantly different secondary structure

for the homo- (helix) and heterochiral (twisted S) variants in DMSO, which collapses in water. The glycine-substituted oligomers, however, shows no clear secondary structure and a significantly higher flexibility compared to the two other variants, with a conformational space that is little-affected by the solvent's polarity.

## EXPERIMENTAL SECTION

#### Synthesis

All chemicals were purchased from Sigma Aldrich (Taufkirchen, Germany), Acros (Geel, Belgium), Alfa Aesar (Ward Hill, USA), VWR (Darmstadt, Germany), Chempur (Karlsruhe, Germany), Iris Biotech (Marktredwitz, Germany), Bachem (Bubendorf, Switzerland), and were employed without additional purification. Moisture- and air-sensitive reaction steps were conducted in flame-dried glassware and under argon atmosphere. Dichloromethane and toluene were freshly distilled from CaH2 and Na, respectively. THF was pre-dried over KOH followed by drying over sodium/benzophenone under reflux conditions and was distilled freshly prior usage.

Analytical RP-HPLC was performed on a Thermo Separation Products system equipped with a UV-6000 LP detector, a P-4000 pump, a Hypersil Gold 3µm (C18; 150 × 2.1 mm) column. A flow rate of 0.7 mL min−1 using Eluent A: H2O/CH3CN/TFA (95/5/0.1) and Eluent B: CH3CN/H2O/TFA (95/5/0.1) was employed. Method A1: 0–3 min (100% A 0% A), 3–4 min (0% A), 4–5 min (0% A 100% A).

Preparative RP-HPLC was performed on a Hitachi MERCK LaChrom system equipped with a UV-Vis L-7420 detector, a L-7150 pump and a Phenomenex Jupiter 10µm column (C18; 300 Å, 250 × 21.1 mm). A flow rate of 10.0 mL min−1 using Eluent A: H2O/CH3CN/TFA (95/5/0.1) and Eluent B: CH3CN/H2O/TFA (95/5/0.1) was employed.   Method P3: 0–5 min (100% A), 5–35 min (100% A 0% A), 35–40 min (0% A), 40–45 min (0% A 100% A).

In addition, a Thermo Separation Products system equipped with a UV-1000 detector, a P-4000 pump, was used utilizing the same methods.

NMR spectra were recorded at 298 K on a DRX 500 (1H: 500 MHz, 13C: 126 MHz), an Avance III 500 (1H: 500 MHz, 13C: 126 MHz), and an Avance 600 spectrometer (1H: 600 MHz, 13C: 151 MHz) (Bruker Biospin, Rheinstetten, Germany). Chemical shifts are reported relative to residual solvent peaks (DMSOd6: 1H: 2.50 ppm, 13C: 39.52 ppm; CDCl3: 1H: 7.26 ppm, 13C: 77.16 ppm) (Gottlieb et al., 1997). 1H- and 13C-NMR spectra (**Supplementary Figures 1–9**) as well as 1H-1H-COSY-, 1H-1H-TOCSY, 1H-13C-HMBC, and 1H-13C-HMQC-NMR spectra were used for the signal assignments. 1H-1H-ROESY-NMR spectra were only taken for the restraints list.

ESI/APCI mass spectra were recorded using an Esquire 3000 ion trap mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) equipped with a standard ESI/APCI source. Samples were introduced directly with a syringe pump. Nitrogen served both as the nebulizer gas and the dry gas. Nitrogen was generated using a Bruker nitrogen generator NGM 11. Helium served as cooling gas for the ion trap and collision gas for MS experiments.

MALDI TOF mass spectra were recorded with a Ultraflex MALDI-TOF/TOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) operated in reflectron mode. 2,5- Dihydroxybenzoic acid was used as the matrix. Ionization was achieved using a LTB nitrogen laser MNL 200 (337 nm beam wavelength, 50 Hz repetition rate).

High-resolution masses were recorded using a MicroToF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) with sample-loop injection. Mass calibration was performed immediately prior measurement with sodium formate cluster and quasi-internal calibration.

CD-spectra were recorded on a JASCO J-810 spectrometer in acetonitrile (HPLC grade) at 20◦C.

#### General Procedures

Bus-protected propargylamines were synthesized according to Wünsch et al. (2017) as well as the α-azido acids in the style of Kracker et al. (2018).

**GP-1:** The azide (1.1 equivalent) and the alkyne (1.0 equivalent) were dissolved in DMF/H20 (10:1, 0.1 M solution). Afterwards, CuSO4·5H2O (0.5 equivalents) and sodium ascorbate (1.0 equivalent) are added. The mixture was stirred 15 h at room temperature. After completion of the reaction the solvent was evaporated. The residue was dissolved with saturated NaCl-solution. After extraction with DCM (3×), drying over Na2SO4, and evaporation of the solvent, the pure triazole was delivered after purification via preparative HPLC or column chromatography.

**GP-2:** The free amine was dissolved in DMF (0.1 M solution). After addition of HATU (1.5 equivalents), HOAt (1.5 equivalents), DIPEA (9.0 equivalents) and an azidocarboxylic acid (1.1 equivalents) the reaction mixture is stirred 15 h at rt. After evaporation of the solvent purification via preparative HPLC or column chromatography yields the pure amide.

#### Explicit Experimental Procedures

**(S)-Bus-Leu**Ψ **[4Tz]Val-OAll (3a):** N2=Val-OAll (1a, 132 mg, 0.720 mmol), (S)-Bus-Leu-CH (2a, 104 mg, 0.483 mmol), CuSO4·5H2O (60 mg, 0.24 mmol) and sodium ascorbate (96 mg, 0.48 mmol) were stirred according to **GP-1**. Purification via column chromatography (DCM/MeOH 80:1, Rf = 0.13) yielded the product as yellow oil. Yield: 180 mg (0.452 mmol, 94%); 1H-NMR (500 MHz, CDCl3): δ [ppm] = 7.88 (s, 1H); 5.88 (m, 1H); 5.31 (dddd, 4 J = 1.3 Hz, 1.3 Hz, 3 J = 17.1 Hz, 2 J = 1.1 Hz, 1H); 5.25 (dddd, 4 J = 1.3 Hz, 1.3 Hz, 3 J = 10.5 Hz, 2 J = 1.1 Hz, 1H); 5.10 (d, 3 J = 8.8 Hz, 1H); 4.68–4.60 (m, 2H); 4.53 (m, 1H); 3.69 (d, 3 J = 8.1 Hz, 1H); 2.47 (m, 1H); 1.90–1.77 (m, 2H); 1.68 (m, 1H); 1.19 (s, 9H); 1.00 (d, 3 J = 6.7 Hz, 3H); 0.91 (d, 3 J = 6.7 Hz, 3H); 0.89 (d, 3 J = 6.6 Hz, 3H); 0.83 (d, 3 J = 6.8 Hz, 3H); 13C-NMR (126 MHz, CDCl3): δ [ppm] = 168.5; 150.2; 131.0; 121.3; 119.8; 68.9; 66.6; 56.4; 51.4; 45.5; 32.5; 24.6; 22.9; 22.8; 21.9; 19.3; 18.8; MS (ESI): m/z = 421.3 ([M+Na]+); HRMS (ESI): m/z calcd. for C19H34N4O3SNa+ ([M+Na]+): 421.2244, found: 421.2253.

**N2**=**Phe-Leu**Ψ **[4Tz]Val-OAll (6a):** (S)-Bus-LeuΨ[4Tz]Val-OAll (3a, 171 mg, 0.429 mmol) was dissolved in MeOH (4 mL). After addition of 4 M HCl in dioxane (2.15 mL, 8.6 mmol) the mixture was stirred for 2 h at rt. After evaporation of the solvent the product was used for the next step without further purification. 1H-NMR (500 MHz, CDCl3): δ [ppm] = 8.53 (s, 3H); 8.09 (s, 1H); 5.89 (m, 1H); 5.33 (dddd, 4 J = 1.4 Hz, 1.4 Hz, 3 J = 17.2 Hz, 2 J = 1.2 Hz, 1H); 5.27 (dddd, 4 J = 1.2 Hz, 1.2 Hz, 3 J = 10.3 Hz, 2 J = 1.0 Hz, 1H); 5.14 (d, 3 J = 8.5 Hz, 1H); 4.71–4.60 (m, 3H); 2.49 (m, 1H); 2.00 (m, 1H); 1.90 (m, 1H); 1.48 (m, 1H); 0.99 (d, 3 J = 6.7 Hz, 3H); 0.90 (d, 3 J = 6.5 Hz, 3H); 0.87 (d, 3 J = 6.6 Hz, 3H); 0.81 (d, 3 J = 6.7 Hz, 3H).

H-LeuΨ[4Tz]Val-OAll·HCl was stirred with the azido carboxylic acid N2=Phe-OH (1b, 90 mg, 0.47 mmol), HATU (247 mg, 0.650 mmol), HOAt (88 mg, 0.65 mmol) and DIPEA (0.65 mL, 3.87 mmol) according to **GP-2**. Purification via column chromatography (DCM/MeOH 50:1, Rf = 0.27) yielded the product as colorless solid. Yield: 181 mg (0.387 mmol, 86%); 1H-NMR (500 MHz, CDCl3): δ [ppm] = 7.75 (s, 1H); 7.32–7.25 (m, 5H); 6.80 (d, 3 J = 8.7 Hz, 1H); 5.89 (m, 1H); 5.33 (dddd, 4 J = 1.5 Hz, 1.5 Hz, 3 J = 17.1 Hz, 2 J = 1.1 Hz, 1H); 5.28 (dddd, 4 J = 1.5 Hz, 1.5 Hz, 3 J = 10.2 Hz, 2 J = 1.0 Hz, 1H); 5.21 (m, 1H); 5.13 (d, 3 J = 8.7 Hz, 1H); 4.71–4.62 (m, 2H); 4.18 (dd, 3 J = 7.8 Hz, 4.2 Hz, 1H); 3.32 (dd, 3 J = 4.3 Hz, 2 J = 14.0 Hz, 1H); 3.06 (dd, 3 J = 8.1 Hz, 2 J = 14.2 Hz, 1H); 2.47 (m, 1H); 1.70–1.67 (m, 2H); 1.34 (m, 1H); 1.02 (d, 3 J = 6.7 Hz, 3H), 0.89 (d, 3 J = 6.6 Hz, 3H); 0.88 (d, 3 J = 6.5 Hz, 3H); 0.82 (d, 3 J = 6.7 Hz, 3H); 13C-NMR (126 MHz, CDCl3): δ [ppm] = 168.5; 167.7; 148.2; 136.1; 131.0; 129.7; 128.7; 127.3; 120.8; 119.9; 68.9; 66.6; 65.5; 44.1; 44.0; 38.5; 32.5; 24.7; 22.7; 22.3; 19.3; 18.8; MS (ESI): m/z = 490.2 ([M+Na]+'); HRMS (ESI): m/z calcd. for C24H33N7O3Na+ ([M+Na]+): 490.2537, found: 490.2542.

**(S)-Bus-Leu**ψ**[4Tz]Phe-Leu**Ψ **[4Tz]Val-OAll (7a):** N2=Phe-LeuΨ[4Tz]Val-OAll (6a, 181 mg, 0.387 mmol) was stirred with the alkyne (S)-Bus-Leu-CH (2a, 92 mg, 0.43 mmol), CuSO4·5H2O (50 mg, 0.20 mmol) and sodium ascorbate (77 mg, 0.39 mmol) according to **GP-1**. Purification via preparative RP-HPLC yielded the product as colorless solid. Yield: 155 mg (0.227 mmol, 59%); Analytical RP-HPLC(Method A1): tR = 3.49 min; Preparative RP-HPLC(Method P2, 220 nm): tR = 42.0 min; 1H-NMR (500 MHz, CDCl3): δ [ppm] = 7.67 (s, 1H); 7.59 (s, 1H); 7.15–7.14 (m, 3H); 7.06 (d, 3 J = 8.4 Hz, 1H); 6.99–6.97 (m, 2H); 5.90 (m, 1H); 5.32 (dddd, 4 J = 1.2 Hz, 1.2 Hz, 3 J = 17.1 Hz, 2 J = 1.6 Hz, 1H); 5.28 (dddd, 4 J = 1.2 Hz, 1.2 Hz, 3 J = 10.3 Hz, 2 J = 1.2 Hz, 1H); 5.26–5.19 (m, 2H) 5.10 (d, 3 J = 8.8 Hz, l1H); 4.71– 4.61 (m, 2H); 4.46 (m, 1H); 3.88 (m, 1H); 3.56 (dd, 3 J = 6.5 Hz, 2 J = 14.1 Hz, 1H); 3.38 (dd, 3 J = 9.2 Hz, 2 J = 14.1 Hz, 1H); 2.47 (m, 1H); 1.86–1.77 (m, 2H); 1.74–1.70 (m, 2H); 1.58 (m, 1H); 1.45 (m, 1H); 1.19 (s, 9H); 1.01 (d, 3 J = 6.7 Hz, 3H), 0.91 (m, 12 H); 0.81 (d, 3 J = 6.7 Hz, 3H); 13C-NMR (126 MHz, CDCl3): δ [ppm] = 168.4; 166.8; 149.7; 148.2; 135.7; 131.0; 129.1; 128.8; 127.3; 122.7; 121.1; 119.9; 69.0; 66.7; 66.1; 56.5; 51.4; 45.1; 44.6; 43.8; 39.1; 32.5; 24.9; 24.6; 22.8; 22.7; 22.7; 22.2; 21.2; 19.3; 18.8; MS (ESI): m/z = 705.4 ([M+Na]+); HRMS(ESI): m/z calcd. for C35H54N8O4SNa+ ([M+Na]+): 705.3881, found: 705.3871.

**N2**=**Phe-Leu**Ψ **[4Tz]Phe-Leu**Ψ **[4Tz]Val-OAll (8a):** (S)-Bus-LeuΨ[4Tz]Phe-LeuΨ[4Tz]Val-OAll (7a, 33 mg, 0.048 mmol) was dissolved in MeOH (2 mL) and stirred at rt with 4 M HCl in dioxane (24 µL solution). After 2 h the solvent was evaporated. The free amine was dissolved in DMF and stirred with the azido carboxylic N2=Phe-OH (1b, 10 mg, 0.052 mmol), HATU (28 mg, 0.073 mmol), HOAt (10 mg, 0.073 mmol) and DIPEA (74 µL, 0.44 mmol) according to **GP-2**. Purification via preparative RP-HPLC yielded the product as colorless solid. Yield: 10 mg (13 µmol, 27%); Preparative RP-HPLC: tR = 27.2 min (Method P1, 220 nm); 1H-NMR (500 MHz, DMSO-d6): δ [ppm] = 8.94 (d, 3 J = 8.4 Hz, 1H); 8.56 (d, 3 J = 8.6 Hz, 1H); 7.98 (s, 1H); 7.93 (s, 1H); 7.29–7.26 (m, 2H); 7.22–7.21 (m, 3H); 7.17–7.11 (m, 5H); 5.90 (m, 1H); 5.61 (dd, 3 J = 9.5 Hz, 6.4 Hz, 1H); 5.29–5.20 (m, 3H); 5.06–5.01 (m, 2H); 4.70–4.63 (m, 2H); 4.00 (dd, 3 J = 9.0 Hz, 5.5 Hz, 1H); 3.35 (dd, 3 J = 6.4 Hz, 2 J = 14.2 Hz, 1H); 3.30 (dd, 3 J = 9.4 Hz, 2 J = 14.0 Hz, 1H); 3.02 (dd, 3 J = 5.4 Hz, 2 J = 13.9 Hz, 1H); 2.90 (dd, 3 J = 9.0 Hz, 2 J = 13.9 Hz, 1H); 2.57 (m, 1H); 1.73– 1.60 (m, 4H); 1.47–1.37 (m, 2H); 0.96 (d, 3 J = 6.7 Hz, 3H), 0.88 (m, 6H); 0.83 (d, 3 J = 6.6 Hz, 3H); 0.72 (m, 6H); 13C-NMR (126 MHz, DMSO-d6): δ [ppm] = 168.2; 167.9; 166.7; 148.1; 147.9; 136.8; 136.1; 133.8; 129.0; 128.9; 128.4; 128.2; 126.7; 126.7; 122.1; 121.3; 118.4; 67.6; 65.7; 63.7; 62.1; 43.7; 43.6; 43.4; 43.4; 37.4; 36.9; 30.6; 24.3; 24.2; 22.6; 22.6; 21.9; 21.8; 18.8; 18.2; MS (ESI): m/z = 774.4 ([M+Na]+).

**(S)-Bus-Val**Ψ **[4Tz]Phe-Leu**Ψ **[4Tz]Phe-Leu**Ψ **[4Tz]Val-OAll (9a):** N2=Phe-LeuΨ[4Tz]Phe-LeuΨ[4Tz]Val-OAll (8a, 10 mg, 13 µmol) was stirred with the (S)-Bus-Val-CH (2b, 4.0 mg, 20 µmol), CuSO4·5H2O (2.5 mg, 10 µmol) and sodium ascorbate (2.6 mg, 13 µmol) according to **GP-1**. Purification via preparative RP-HPLC yielded the product as colorless solid.

#### **Boc-Ala-Val**Ψ **[4Tz]Phe-Leu**Ψ **[4Tz]Phe-Leu**Ψ **[4Tz]Val-**

**OAll (10a):** (S)-Bus-ValΨ[4Tz]Phe-LeuΨ[4Tz]Phe-LeuΨ[4Tz]Val-OAll (9a, 5.0 mg, 5.2 µmol) was dissolved in MeOH (0.5 mL) and 4 M HCl in dioxane (26 µL solution) was added. After 2 h stirring at rt the solvent was evaporated and the free amine was dissolved in DMF. It was stirred with Boc-Ala-OH (1.5 mg, 7.9 µmol), HATU (3.0 mg, 7.9 µmol), HOAt (1.0 mg, 7.3 µmol) and DIPEA (8.0 µL, 47 µmol) according to **GP-2**. Purification via preparative RP-HPLC yielded the product as colorless solid.

**(R)-Bus-D-Leu**Ψ **[4Tz]Val-OAll (3b):** The azide N2=Val-OAll (1a, 70 mg, 0.38 mmol) was stirred with the alkyne (R)- Bus-D-Leu-CH (2c, 41 mg, 0.19 mmol), CuSO4·5H2O (24 mg, 96 µmol) and sodium ascorbate (38 mg, 0.19 mmol) according to **GP-1**. Purification via column chromatography (DCM/MeOH 80:1, Rf = 0.18) yielded the product as yellow oil. Yield: 74 mg (0.19 mmol, 98%); 1H-NMR (500 MHz, CDCl3): δ [ppm] = 7.90 (s, 1H); 5.84 (m, 1H); 5.26 (dddd, 4 J = 1.4 Hz, 1.4 Hz, 3 J = 17.3 Hz, 2 J = 1.3 Hz, 1H); 5.20 (dddd, 4 J = 1.2 Hz, 1.2 Hz, 3 J = 10.4 Hz, 2 J = 1.1 Hz, 1H); 5.06 (d, 3 J = 8.6 Hz, 1H); 4.74– 4.64 (m, 2H); 4.55 (m, 1H); 3.61 (d, 3 J = 6.8 Hz, 1H); 2.43 (m, 1H); 1.85 (m, 1H); 1.77 (m, 1H); 1.70 (m, 1H); 1.19 (s, 9H); 0.94 (d, 3 J = 6.7 Hz, 3H); 0.91 (d, 3 J = 6.5 Hz, 3H); 0.90 (d, 3 J = 6.5 Hz, 3H); 0.78 (d, 3 J = 6.7 Hz, 3H); 13C-NMR (126 MHz, CDCl3): δ [ppm] = 168.4; 150.7; 131.0; 121.6; 119.7; 68.8; 66.5; 56.4; 51.7; 45.7; 32.4; 24.5; 23.0; 22.7; 21.7; 19.2; 18.6; MS (ESI): m/z = 421.2 ([M+Na]+); HRMS(ESI): m/z calcd. for C19H34N4O3SNa+ ([M+Na]+): 421.2244, found: 421.2239.

**N2**=**Phe-D-Leu**Ψ **[4Tz]Val-OAll (6b):** (R)-Bus-D-Leu Ψ[4Tz]Val-OAll (3b, 74 mg, 0.19 mol) was dissolved in MeOH (2 mL). After addition of 4 M HCl in dioxane (0.14 mL) the mixture was stirred 2 h at rt. After evaporation of the solvent the product was used for the next step without further purification. **1H-NMR** (500 MHz, CDCl3): δ [ppm] = 8.45 (s, 3H); 8.13 (s, 1H); 5.89 (m, 1H); 5.33 (dddd, 4 J = 1.3 Hz, 1.3 Hz, 3 J = 17.2 Hz, 2 J = 1.2 Hz, 1H); 5.28 (dddd, 4 J = 1.2 Hz, 1.2 Hz, 3 J = 10.3 Hz, 2 J = 1.0 Hz, 1H); 5.14 (d, 3 J = 8.4 Hz, 1H); 4.72–4.61 (m, 3H); 2.50 (m, 1H); 2.00 (m, 1H); 1.92 (m, 1H); 1.50 (m, 1H); 0.99 (d, 3 J = 6.7 Hz, 3H); 0.90 (d, 3 J = 6.5 Hz, 3H); 0.89 (d, 3 J = 6.5 Hz, 3H); 0.81 (d, 3 J = 6.7 Hz, 3H). H-D-Leu[4Tz]Val-OAll·HCl (81 mg, 0.19 mmol) was stirred with the N2=Phe-OH (1b, 39 mg 0.21 mmol), HATU (106 mg, 0.279 mmol), HOAt (38 mg, 0.28 mmol), and DIPEA (282 µL, 1.66 mmol) according to **GP-2**. Purification via column chromatography (DCM/MeOH 50:1, Rf = 0.35) yielded the product as colorless solid. **Yield:** 34 mg (73 µmol, 39%); **1H-NMR** (500 MHz, CDCl3): δ [ppm] = 7.68 (s, 1H); 7.24–7.18 (m, 5H); 6.67 (d, 3 J = 8.7 Hz, 1H); 5.90 (m, 1H); 5.34 (dddd, 4 J = 1.4 Hz, 1.4 Hz, 3 J = 17.2 Hz, 2 J = 1.1 Hz, 1H); 5.29 (dddd, 4 J = 1.1 Hz, 1.1 Hz, 3 J = 10.4 Hz, 2 J = 1.0 Hz, 1H); 5.21 (m, 1H); 5.12 (d, 3 J = 8.8 Hz, 1H); 4.72–4.62 (m, 2H); 4.15 (dd, 3 J = 8.1 Hz, 4.2 Hz, 1H); 3.29 (dd, 3 J = 4.7 Hz, 2 J = 14.0 Hz, 1H); 2.96 (dd, 3 J = 8.1 Hz, 2 J = 14.0 Hz, 1H); 2.46 (m, 1H); 1.83–1.73 (m, 2H); 1.45 (m, 1H); 1.03 (d, 3 J = 6.7 Hz, 3H), 0.93 (d, 3 J = 6.6 Hz, 3H); 0.92 (d, 3 J = 6.4 Hz, 3H); 0.82 (d, 3 J = 6.7 Hz, 3H); 13C-NMR (126 MHz, CDCl3): δ [ppm] = 168.4; 168.0; 148.1; 136.2; 131.0; 129.5; 128.8; 127.3; 120.8; 120.0; 69.0; 66.7; 65.5; 44.1; 44.0; 38.7; 32.6; 25.0; 22.7; 22.4; 19.3; 18.7; MS(ESI): m/z = 490.2 ([M+Na]+); HRMS(ESI): m/z calcd. for (C24H33N7O3)2Na+ ([2M+Na]+): 957.5182, found: 957.5158.

**(R)-Bus-D-Leu**Ψ **[4Tz]Phe-D-Leu**Ψ **[4Tz]Val-OAll (7b):** N2=Phe-D-LeuΨ[4Tz]Val-OAll (6b, 34 mg, 73 µmol), the alkyne **(R)-Bus-D-Leu-CH** (2c, 17 mg, 79 µmol), CuSO4·5H2O (9.0 mg, 36 µmol) and sodium ascorbate (14 mg, 71 µmol) were stirred according to **GP-1**. Purification via preparative RP-HPLC yielded the product as colorless solid. Yield: 39 mg (56 µmol, 77%); Analytical RP-HPLC: tR = 3.60 min (Method A1); Preparative RP-HPLC: tR = 42.6 min (Method P2, 220 nm); 1H-NMR (500 MHz, CDCl3): δ [ppm] = 7.72 (s, 1H); 7.69 (s, 1H); 7.22–7.20 (m, 3H); 7.09–7.08 (m, 2H); 6.84 (d, 3 J = 8.1 Hz, 1H); 5.90 (m, 1H); 5.34 (dd, 3 J = 8.4 Hz, 6.9 Hz, 1H) 5.33 (dddd, 4 J = 1.4 Hz, 1.4 Hz, 3 J = 17.1 Hz, 2 J = 1.3 Hz, 1H); 5.28 (dddd, 4 J = 1.2 Hz, 1.2 Hz, 3 J = 10.5 Hz, 2 J = 1.2 Hz, 1H); 5.19 (m, 1H); 5.10 (d, 3 J = 8.8 Hz, 1H); 4.71–4.61 (m, 2H); 4.79 (m, 1H); 3.62 (s (br), 1H); 3.58 (dd, 3 J = 6.9 Hz, 2 J = 14.0 Hz, 1H); 2.96 (dd, 3 J = 8.7 Hz, 2 J = 13.9 Hz, 1H); 2.47 (m, 1H); 1.84 (m, 1H); 1.75 (m, 1H); 1.69–1.58 (m, 2H); 1.32–1.24 (m, 2H); 1.19 (s, 9H); 1.01 (d, 3 J = 6.7 Hz, 3H), 0.91 (d, 3 J = 7.0 Hz, 3H); 0.90 (d, 3 J = 6.7 Hz, 3H); 0.85 (d, 3 J = 6.5 Hz, 3H); 0.84 (d, 3 J = 6.6 Hz, 3H); 0.80 (d, 3 J = 6.7 Hz, 3H); 13C-NMR (126 MHz, CDCl3): δ [ppm] = 168.5; 166.8; 150.4; 148.3; 135.6; 131.1; 129.2; 128.9; 127.4; 122.3; 120.9; 119.9; 68.9; 66.7, 66.0; 56.4; 51.8; 45.2; 44.8; 43.6; 39.2; 32.6; 24.8; 24.6; 22.9; 22.8; 22.7; 22.3; 21.9; 19.3; 18.7; MS (ESI): m/z = 705.4 ([M+Na]+); HRMS (ESI): m/z calcd. for C35H54N8O4SNa+ ([M+Na]+): 705.38809, found: 705.38774.

**N2**=**Phe-D-Leu**Ψ **[4Tz]Phe-D-Leu**Ψ **[4Tz]Val-OAll (8b):** (R)-Bus-D-LeuΨ [4Tz]Phe-D-LeuΨ[4Tz]Val-OAll (15 mg, 22 µmol) was dissolved in MeOH (2 mL). After addition of 4 M HCl in dioxane (11 µL solution) the mixture was stirred 2 h at rt. After evaporation of the solvent the free amine was dissolved in DMF and stirred with the azido N2=Phe-OH (1b, 5.0 mg, 26 µmol), HATU (13 mg, 34 µmol), HOAt (4.0 mg, 29 µmol), and DIPEA (34 µl, 0.20 mmol) according to **GP-2**. Purification via preparative RP-HPLC yielded the product as colorless solid. Yield: 9.0 mg (12 µmol, 54%); Preparative RP-HPLC: tR = 28.2 min (Method P1, 220 nm); 1H-NMR (500 MHz, DMSO-d6): δ [ppm] = 8.96 (d, 3 J = 8.6 Hz, 1H); 8.53 (d, 3 J = 8.7 Hz, 1H); 8.16 (s, 1H); 8.01 (s, 1H); 7.32–7.18 (m, 10H); 5.87 (m, 1H); 5.61 (dd, 3 J = 8.6 Hz, 6.7 Hz, 1H); 5.27 (d, 3 J = 8.4 Hz, 1H); 5.23 (dddd, 4 J = 1.4 Hz, 1.4 Hz, 3 J = 17.3 Hz, 2 J = 1.5 Hz, 1H); 5.19 (dddd, 4 J = 1.3 Hz, 1.3 Hz, 3 J = 10.6 Hz, 2 J = 1.5 Hz, 1H); 5.03–4.96 (m, 2H); 4.67–4.60 (m, 2H); 3.95 (dd, 3 J = 7.9 Hz, 6.7 Hz, 1H); 3.39 (dd, 3 J = 6.7 Hz, 2 J = 13.5 Hz, 1H); 3.34 (dd, 3 J = 8.2 Hz, 2 J = 13.5 Hz, 1H); 3.07 (dd, 3 J = 6.8 Hz, 2 J = 13.5 Hz, 1H); 2.98 (dd, 3 J = 8.2 Hz, 2 J = 13.5 Hz, 1H); 2.54 (m, 1H); 1.57 (m, 2H); 1.48–1.45 (m, 2H); 1.25 (m, 1H); 1.10 (m, 1H); 0.92 (d, 3 J = 6.7 Hz, 3H), 0.82–0.78 (m, 9H); 0.75 (d, 3 J = 6.5 Hz, 3H); 0.72 (d, 3 J = 6.7 Hz, 3H); 13C-NMR (126 MHz, DMSO-d6): δ [ppm] = 168.1; 167.9; 165.5; 148.1; 147.8; 136.6; 135.8; 133.7; 129.1; 129.1; 128.3; 128.2; 126.; 126.7; 122.0; 121.3; 118.5; 67.5; 65.7; 63.7; 62.1; 43.7; 43.6; 43.5; 43.3; 39.5; 37.0; 30.6; 24.1; 23.9; 22.7; 22.5; 21.9; 21.8; 18.8; 18.2; MS(ESI): m/z = 774.4 ([M+Na]+).

#### **(R)-Bus-D-Val**Ψ **[4Tz]Phe-D-Leu**Ψ **[4Tz]Phe-D-**

**Leu**Ψ **[4Tz]Val-OAll (9b):** N2=Phe-D-LeuΨ[4Tz]Phe-D-LeuΨ[4Tz]Val-OAll (8b, 24 mg, 32 µmol), the alkyne (R)-Bus-D-Val-CH (9.0 mg, 45 µmol) CuSO4·5H2O (4.0 mg, 16 µmol) and sodium ascorbate (6.0 mg, 30 µmol) were stirred according to **GP-1**. Purification via preparative RP-HPLC yielded the product as colorless solid. Yield: 17 mg (18 µmol, 56%); Analytical RP-HPLC: tR = 3.74 min (Method A1); Preparative RP-HPLC: tR = 46.2 min (Method P2, 220 nm); 1H-NMR (600 MHz, DMSO-d6): δ [ppm] = 8.90 (d, 3 J = 8.6 Hz, 1H); 8.85 (d, 3 J = 8.6 Hz, 1H); 8.21 (s, 1H); 8.11 (s, 1H); 7.98 (s, 1H); 7.23–7.15 (m, 10H); 5.87 (m, 1H); 5.62–5.56 (m, 2H); 5.26 (d, 3 J = 8.4 Hz, 1H); 5.23 (dddd, 4 J = 1.5 Hz, 1.5 Hz, 3 J = 17.2 Hz, 2 J = 1.6 Hz, 1H); 5.20 (s (br), 1H); 5.18 (dddd, 4 J = 1.5 Hz, 1.5 Hz, 3 J = 10.6 Hz, 2 J = 1.2 Hz, 1H); 5.01–4.96 (m, 2H); 4.67–4.60 (m, 2H); 4.11 (dd, 3 J = 6.5 Hz, 1H); 3.39–3.27 (m, 4H); 2.52 (m, 1H); 2.05 (m, 1H); 1.57–1.54 (m, 2H); 1.54–1.47 (m, 2H); 1.24 (m, 1H); 1.17 (m, 1H); 1.12 (s, 9H); 0.91 (d, 3 J = 6.7 Hz, 3H); 0.82–0.78 (m, 15H); 0.75 (d, 3 J = 6.7 Hz, 3H); 0.72 (d, 3 J = 6.7 Hz, 3H); 13C-NMR (151 MHz, DMSO-d6): δ [ppm] = 167.9; 166.5; 166.5; 148.3; 148.1; 147.6; 135.8; 135.8; 131.7; 129.1; 129.0; 128.2; 128.2; 126.7; 126.7; 122.2; 122.0; 121.3; 118.; 67.5; 65.7; 63.7; 63.7; 58.3; 55.7; 43.8; 43.6; 43.5; 43.4; 38.4; 37.9; 33.3; 30.6; 24.1; 24.0; 22.7; 22.6; 22.5; 21.9; 21.8; 19.0; 18.8; 18.3; 18.2; MS(ESI): m/z = 975.5 ([M+Na]+); HRMS(ESI): m/z calcd. for C35H54N8O4SNa+ ([M+Na]+): 975.5362, found: 975.5332.

#### **Boc-Ala-D-Val**Ψ **[4Tz]Phe-D-Leu**Ψ **[4Tz]Phe-D-**

**Leu**Ψ **[4Tz]Val-OAll (10b):** (R)-Bus-D-ValΨ [4Tz]Phe-D-LeuΨ[4Tz]Phe-D-LeuΨ[4Tz]Val-OAll (9b, 23.3 mg, 25.0 µmol) is dissolved in 1 mL of a 1:1 mixture of DCM and HCl (4 M in dioxane) and stirred for 2 h at rt. After complete cleavage of the Boc-group, the free amine is evaporated to dryness. In a second flask, Boc-Ala-OH (9.5 mg, 50 µmol) and HOAt (7.5 mg, 55 µmol) are dissolved in DCM (2 mL, dry). After addition of DIC (8.5 µL, 55 µmol), the mixture is stirred 5 min at RT for preactivation and is then added to the free amine, followed by TMP (16.5 µL; 125 µmol). This solution is left stirring over night at rt. After evaporation of the solvent, the crude product is purified by preparative HPLC. Yield: 15.4 mg (15.1 µmol, 60%); Analytical RP-HPLC: tR = 6.60 min (Method A2); 1H-NMR (600 MHz, DMSO-d6): δ [ppm] = 8.92 (d, J = 8.6 Hz, 1H), 8.89 (d, J = 8.7 Hz) 8.19 (s, 1H), 8.14 (s, 1H), 8.00 (s, 1H), 7.88 (d, J = 9.2 Hz), 7.23–7.14 (m, 10H) 6.89 (d, J = 7.6 Hz, 1H), 5.86 (ddt, J = 17.2, 10.6, 5.5 Hz, 1H), 5.63–5.58 (m, 2H), 5.26 (d, J = 8.4 Hz, 1H), 5.23 (dd, J = 17.2, 1.7 Hz, 1H), 5.18 (dd, J = 10.6, 1.5 Hz, 1H), 5.01–4.96 (m, 2H), 4.81 (dd, J = 9.2, 6.4 Hz, 1H), 4.64 (m, 2H), 4.03 (dq, J = 7.6, 7.2 Hz, 1H), 3.40–3.26 (m, 4H), 2.50 (m, 1H), 2.05–1.96 (m, 1H), 1.58–1.50 (m, 4H), 1.36 (s, 9H), 1.28–1.14 (m, 2H), 1.18 (d, J = 7.2 Hz), 0.91 (d, J = 6.7 Hz, 3H), 0.82–0.78 (m, 12H), 0.75 (d, J = 6.7 Hz, 3H), 0.72 (d, J = 6.8 Hz, 3H), 0.70 (d, J = 6.8 Hz, 3H); 13C-NMR (151 MHz, DMSO-d6): δ [ppm] = 172.77, 168.32, 166.96, 166.94, 155.42, 148.53, 148.04, 147.33, 136.27, 136.22, 132.16, 129.50, 129.48, 128.62, 127.20, 122.46, 122.28, 121.82, 118.91, 78.49, 67.96, 66.14, 64.17, 50.82, 50.38, 44.21, 44.05, 43.90, 38.81, 38.41, 32.80, 31.08, 28.63, 24.56, 24.46, 23.09, 22.97, 22.37, 22.26, 19.34,19.24, 18.84, 18.68, 18.41; HRMS (ESI): m/z calcd. for C54H78N13O + 7 ([M+H]+): 1020.6142, found: 1020.6126.

**Boc-Gly**Ψ **[4Tz]Val-OAll (3c):** N2=Val-OAll (104 mg, 0.568 mmol), the alkyne Boc-Gly-CH (2e, 100 mg, 0.644 mmol), CuSO4·5H2O (71 mg, 0.29 mmol) and sodium ascorbate (112 mg, 0.565 mmol) were stirred according to **GP-1**. Purification via column chromatography (PE/EE 2:1, Rf = 0.54) yielded the product as colorless solid. Yield: 138 mg (0.408 mmol, 72%);[α] 20 D = 40.8 (c = 0.7, CHCl3); 1H-NMR (500 MHz, CDCl3): δ [ppm] = 7.86 (s, 1H); 5.90 (m, 1H,); 5.34 (dddd, 4 J = 1.5 Hz, 1.5 Hz, 3 J = 17.2 Hz, 2 J = 1.2 Hz, 1H); 5.29 (dddd, 4 J = 1.5 Hz, 1.5 Hz, 3 J = 10.4 Hz, 2 J = 1.0 Hz, 1H); 5.23 (s (br), 1H); 5.12 (d, 3 J = 8.6 Hz, 1H); 4.71–4.64 (m, 2H); 4.42 (d, 3 J = 5.3 Hz, 2H); 2.49 (m, 1H); 1.43 (s, 9H); 1.02 (d, 3 J = 6.7 Hz, 3H); 0.83 (d, 3 J = 6.7 Hz, 3H); 13C-NMR (126 MHz, CDCl3): δ [ppm] = 168.3; 156.0; 145.5; 131.0; 122.0; 120.0; 80.0; 69.2; 66.8; 36.0; 32.5; 28.5; 19.3; 18.6; MS (ESI): m/z = 361.2 ([M+Na]+).

**N2**=**Phe-Gly**Ψ **[4Tz]Val-OAll (6c):** Boc-GlyΨ[4Tz]Val-OAll (3c, 63 mg, 0.19 mmol) was dissolved in TFA (2 mL) and stirred for 12 h. After evaporation of TFA in vacuo the crude product was used for the next step without further purification. 1H-NMR (500 MHz, CDCl3): δ [ppm] = 8.62 (s, 3H); 8.16 (s, 1H); 5.89 (m, 1H); 5.33 (dddd, 4 J = 1.4 Hz, 1.4 Hz, 3 J = 17.2 Hz, 2 J = 1.3 Hz, 1H); 5.27 (dddd, 4 J = 1.3 Hz, 1.3 Hz, 3 J = 10.4 Hz, 2 J = 1.0 Hz, 1H); 5.14 (d, 3 J = 8.5 Hz, 1H); 4.71–4.60 (m, 2H); 4.37 (s (br), 2H); 2.50 (m, 1H); 0.99 (d, 3 J = 6.7 Hz, 3H); 0.81 (d, 3 J = 6.7 Hz, 3H); 13C-NMR (126 MHz, CDCl3): δ [ppm] = 168.2; 161.9 (q); 140.3; 131.0; 124.2; 120.0; 116.3 (q); 69.2; 66.9; 34.8; 32.3; 19.1; 18.3. H-GlyΨ [4Tz]Val-OAll·TFA (55 mg, 0.16 mmol) and the azido acid N2=Phe-OH (1b, 34 mg, 0.18 mmol) were stirred with HATU (93 mg, 0.24 mmol), HOAt (33 mg, 0.24 mmol) and DIPEA (0.25 mL, 1.5 mmol) according to **GP-2**. Purification via preparative RP-HPLC yielded the product as colorless solid. Yield: 34 mg (83 µmol, 52% over two steps); Preparative RP-HPLC: tR = 23.8 min (Method P1, 220 nm); 1H-NMR (500 MHz, CDCl3): δ [ppm] = 7.82 (s, 1H); 7.29–7.21 (m, 5H); 7.01 (dd, 3 J = 5.2 Hz, 5.2 Hz, 1H); 5.91 (m, 1H); 5.35 (dddd, 4 J = 1.3 Hz, 1.3 Hz, 3 J = 17.2 Hz, 2 J = 1.2 Hz, 1H); 5.30 (dddd, 4 J = 1.2 Hz, 1.2 Hz, 3 J = 10.4 Hz, 2 J = 1.1 Hz, 1H); 5.13 (d, 3 J = 8.6 Hz, 1H); 4.72–4.64 (m, 2H); 4.56–4.48 (m, 2H); 4.19 (dd, 3 J = 8.3 Hz, 4.4 Hz, 1H); 3.32 (dd, 3 J = 4.4 Hz, 2 J = 14.0 Hz, 1H); 3.00 (dd, 3 J = 8.3 Hz, 2 J = 14.0 Hz, 1H); 2.49 (m, 1H); 1.03 (d, 3 J = 6.7 Hz, 3H); 0.85 (d, 3 J = 6.7 Hz, 3H); 13C-NMR (126 MHz, CDCl3): δ [ppm] = 169.0; 168.2; 144.0; 136.1; 130.9; 129.6; 128.8; 127.4; 122.3; 120.1; 69.2; 66.8; 65.5; 38.7; 34.8; 32.5; 19.3; 18.7; MS (ESI): m/z = 434.2 ([M+Na]+).

**Boc-Gly**Ψ **[4Tz]Phe-Gly**Ψ **[4Tz]Val-OAll (7c):** N2=Phe-GlyΨ[4Tz]Val-OAll (6c, 34 mg, 83 µmol) was stirred with Boc-Gly-CH (2e, 19 mg, 0.12 mmol), CuSO4·5H2O (10 mg, 0.040 mmol) and sodium ascorbate (16 mg, 81 µmmol) according to **GP-1**. Purification via preparative RP-HPLC yielded the product as colorless solid. Yield: 19 mg (34 µmol, 41%); Analytical RP-HPLC: tR = 3.04 min (Method A1); Preparative RP-HPLC: tR = 32.9 min (Method P2, 220 nm); 1H-NMR (600 MHz, CDCl3): δ [ppm] = 7.74 (s, 1H); 7.59 (s, 1H); 7.22–7.19 (m, 3H); 7.03–7.02 (m, 2H); 6.99 (dd, 3 J = 5.8 Hz, 5.8 Hz, 1H); 5.89 (m, 1H); 5.34 (dddd, 4 J = 1.5 Hz, 1.5 Hz, 3 J = 17.2 Hz, 2 J = 1.4 Hz, 1H); 5.29 (dddd, 4 J = 1.3 Hz, 1.3 Hz, 3 J = 10.4 Hz, 2 J = 1.3 Hz, 1H); 5.27 (dd, 3 J = 8.6 Hz, 6.9 Hz, 1H); 5.11 (d, 3 J = 8.6 Hz, 1H); 5.11 (s (br), 1H); 4.70 (dddd, 4 J = 1.3 Hz, 1.3 Hz, 3 J = 6.0 Hz, 2 J = 13.0 Hz, 1H); 4.65 (dddd, 4 J = 1.3 Hz, 1.3 Hz, 3 J = 6.0 Hz, 2 J = 13.0 Hz, 1H); 4.74–4.64 (m, 2H); 4.35 (m, 2H); 3.56 (dd, 3 J = 6.5 Hz, 2 J = 13.9 Hz, 1H); 3.38 (dd, 3 J = 8.9 Hz, 2 J = 13.9 Hz, 1H); 2.47 (m, 1H); 1.43 (s, 9H); 1.03 (d, 3 J = 6.7 Hz, 3H); 0.83 (d, 3 J = 6.7 Hz, 3H); 13C-NMR (151 MHz, CDCl3): δ [ppm] = 168.4; 167.4; 155.9; 145.7; 143.9; 135.4; 131.0; 129.0; 128.9; 127.6; 122.4; 122.0; 120.1; 79.9; 69.1; 66.8; 66.3; 39.5; 36.2; 35.5; 32.6; 28.5; 19.3; 18.8; MS (ESI): m/z = 589.3 ([M+Na]+); HRMS (ESI): m/z calcd. for C19H34N4O3SNa+ ([M+Na]+): 589.2857, found: 589.2852.

**N2**=**Phe-Gly**Ψ **[4Tz]Phe-Gly**Ψ **[4Tz]Val-OAll (8c):** Boc-GlyΨ[4Tz]Phe-GlyΨ[4Tz]Val-OAll (7c, 78 mg, 0.138 mmol) was dissolved in TFA (2 mL) and stirred for 18 h. After evaporation of TFA in vacuo the free amine, the azido carboxylic acid N2=Phe-OH (1b, 39 mg, 0.21 mmol), HATU (79 mg, 0.20 mmol), HOAt (28 mg, 0.21 mmol) and DIPEA (0.21 mL, 1.2 mmol) were stirred according to **GP-2**. Purification via preparative RP-HPLC yielded the product as colorless solid. Yield: 38 mg (59 µmol, 43%); Preparative RP-HPLC: tR = 34.2 min (Method P3, 254 nm); 1H-NMR (500 MHz, CDCl3): δ [ppm] = 7.75 (s, 1H); 7.58 (s, 1H); 7.48 (dd, 3 J = 5.6 Hz, 5.6 Hz, 1H); 7.28–7.16 (m, 8H); 7.08 (dd, 3 J = 5.1 Hz, 5.1 Hz, 1H); 7.03–7.01 (m, 2H); 5.88 (m, 1H); 5.38 (dd, 3 J = 8.4 Hz, 7.1 Hz, 1H); 5.32 (dddd, 4 J = 1.5 Hz, 1.5 Hz, 3 J = 17.1 Hz, 2 J = 1.5 Hz, 1H); 5.28 (dddd, 4 J = 1.3 Hz, 1.3 Hz, 3 J = 10.4 Hz, 2 J = 1.3 Hz, 1H); 5.09 (d, 3 J = 8.6 Hz, 1H); 4.70–4.60 (m, 2H); 4.74–4.46 (m, 2H); 4.43–4.38 (m, 2H); 4.16 (dd, 3 J = 8.4 Hz, 4.4 Hz, 1H); 3.55 (dd, 3 J = 6.8 Hz, 2 J = 13.9 Hz, 1H); 3.35 (dd, 3 J = 8.7 Hz, 2 J = 13.9 Hz, 1H); 3.29 (dd, 3 J = 4.4 Hz, 2 J = 14.0 Hz, 1H); 2.97 (dd, 3 J = 8.4 Hz, 2 J = 14.0 Hz, 1H); 2.47 (m, 1H); 1.01 (d, 3 J = 6.7 Hz, 3H); 0.81 (d, 3 J = 6.7 Hz, 3H); 13C-NMR (126 MHz, CDCl3): δ [ppm] = 169.0; 168.3; 167.4; 144.2; 143.9; 136.3; 135.3; 131.0; 129.6; 129.0; 128.9; 128.8; 127.5; 127.3; 122.8; 122.2; 120.0; 69.1; 66.8; 65.9; 65.3; 39.4; 38.6; 35.3; 34.9; 32.5; 19.3; 18.7; MS(MALDI): m/z = 640.3 ([M+H]+); 662.3 ([M+Na]+ ; 678.3 ([M+K]+).

#### ) **Boc-Gly**Ψ **[4Tz]Phe-Gly**Ψ **[4Tz]Phe-Gly**Ψ **[4Tz]Val-OAll**

**(9c):** N2=Phe-GlyΨ[4Tz]Phe-GlyΨ[4Tz]Val-OAll (8c, 38 mg, 59 µmol) was stirred with the alkyne Boc-Gly-CH (2e, 14 mg, 88 µmol), CuSO4·5H2O (7.4 mg, 30 µmol) and sodium ascorbate (11.7 mg, 59.0 µmol) according to **GP-1**. Purification via preparative RP-HPLC yielded the product as colorless solid. Yield: 7.0 mg (9 µmol, 15%); Analytical RP-HPLC: tR = 3.12 min (Method A1); Preparative RP-HPLC: tR = 34.0 min (Method P3, 220 nm); 1H-NMR (500 MHz, DMSO-d6): δ [ppm] = 9.05 (dd, 3 J = 5.5 Hz, 5.5 Hz, 1H); 8.99 (dd, 3 J = 5.6 Hz, 5.6 Hz, 1H); 8.07 (s, 1H); 8.05 (s, 1H); 7.94 (s, 1H); 7.28 (dd, 3 J = 6.0 Hz, 6.0 Hz, 1H); 7.22–7.14 (m, 10H); 5.90 (m, 1H); 5.67–5.57 (m, 2H); 5.31 (d, 3 J = 8.2 Hz, 1H); 5.28 (dddd, 4 J = 1.5 Hz, 1.5 Hz, 3 J = 17.2 Hz, 2 J = 1.5 Hz, 1H); 5.23 (dddd, 4 J = 1.4 Hz, 1.4 Hz, 3 J = 10.5 Hz, 2 J = 1.4 Hz, 1H); 4.70–4.62 (m, 2H); 4.41–4.34 (m, 2H); 4.33–4.26 (m, 2H); 4.14 (m, 2H); 3.41–3.30 (m, 4H); 2.55 (m, 1H); 1.30 (s, 9H); 0.95 (d, 3 J = 6.7 Hz, 3H); 0.77 (d, 3 J = 6.7 Hz, 3H); 13C-NMR (126 MHz, DMSO-d6): δ [ppm] = 167.9; 167.4; 167.3; 155.6; 145.3; 143.8; 143.6; 136.2; 136.0; 131.8; 128.9; 128.9; 128.3; 128.3; 126.8; 126.8; 123.1; 122.2; 121.9; 118.6; 77.9; 67.5; 65.8; 63.7; 63.7; 37.8; 37.6; 35.6; 34.4; 34.4; 30.7; 28.2; 18.8; 18.3; MS(ESI): m/z = 817.5 ([M+Na]+); HRMS(ESI): m/z calcd. for C40H50N12O6Na+ ([M+Na]+): 817.3868, found: 817.3874.

**Boc-Ala-Gly**Ψ **[4Tz]Phe-Gly**Ψ **[4Tz]Phe-Gly**Ψ **[4Tz]Val-OAll (10c):** Boc-GlyΨ[4Tz]Phe-GlyΨ[4Tz]Phe-GlyΨ[4Tz]Val-OAll (9c, 50 mg, 63 µmol) is dissolved in 2 mL of a 1:1 mixture of DCM and HCl (4 M in dioxane) and stirred for 2 h at rt. After complete cleavage of the Boc-group, the free amine is evaporated to dryness. In a second flask, Boc-Ala-OH (24 mg, 0.13 mmol) and HOAt (19 mg, 0.14 mmol) are dissolved in DCM (5 mL, dry). After addition of DIC (22 µL, 0.14 mmol), the mixture is stirred 5 min at RT for preactivation and is then added to the free amine, followed by TMP (42 µL, 0.32 mmol). This solution is left stirring over night at rt. After evaporation of the solvent, the crude product is purified by preparative HPLC. Yield: 41 mg (47 µmol, 75%); Analytic RP-HPLC: tR = 5.3 min (Method A2); 1H-NMR (600 MHz, DMSO-d6): δ [ppm] = 9.04 (dd, J = 5.61, 5.57 Hz, 1H), 9.00 (dd, J = 5.56, 5.43 Hz, 1H), 8.26 (dd, J = 5.99, 5.93 Hz, 1H), 8.08 (s, 1H), 8.04 (s, 1H), 7.94 (s, 1H), 7.25–7.08 (m, 10H), 6.91 (d, J = 7.4 Hz, 1H), 5.94–5.85 (m, 1H), 5.62–5.57 (m, 2H), 5.30 (d, J = 8.4 Hz, 1H), 5.28 (dd, J = 17.2, 1.7 Hz, 1H), 5.23 (dd, J = 10.5, 1.6 Hz, 1H), 4.70–4.62 (m, 2H), 4.41–4.24 (m, 6H), 3.97 (dq, 1H, J = 7.4, 7.2 Hz), 3.42–3.27 (m, 4H) 2.54 (dqq, J = 8.4, 6.7, 6.5 Hz, 1H), 1.38 (s, 9H), 1.17 (d, J = 7.2 Hz, 3H), 0.94 (d, J = 6.5 Hz, 3H), 0.77 (d, J = 6.7 Hz, 3H); 13C-NMR (151 MHz, DMSO-d6): δ [ppm] = 173.15, 168.33, 167.73, 155.51, 145.26, 144.25, 144.03, 136.52, 136.49, 132.21, 129.25, 128.24, 127.24, 123.49, 122.66, 122.40, 119.06, 78.44, 67.98, 66.23, 64.16, 50.13, 38.29, 38.08, 34.84, 34.83, 34.80, 31.12, 28.68, 19.26, 18.71; HRMS (ESI): m/z calcd. for C43H56N13O + 7 ([M+H]+): 866.4420, found: 866.4413.

## Conformational Analysis

Molecular dynamics (MD) simulations were performed on a compute cluster located at the Center for Biotechnology (CeBiTec), Bielefeld University, using the CUDA accelerated version of the pmemd program as implemented in AMBER (Amber 16, AmberTools 17) (Case et al., 2017). All simulations were based on the additional force-field TZLff (Marion et al., 2018) parametrized for 1,4- and 1,5 disubstituted peptidotriazolamers.

Identical simulation strategies were applied for the conformational analysis of all molecules. First, 100 parallel simulated annealing (SA) runs were performed in the gas phase by applying NMR-derived inter-proton restraints (**Supplementary Tables 1–3**). No cutoff was applied on nonbonded interactions and the temperature ramp was set to gradually heat up the molecule from 0 to 600 K and finally cool it down to 0 K over a 1 ns-long MD run(10 K, 5 ps; 100 K, 5 ps; 200 K, 90 ps; 600 K, 400 ps; 400 K, 100 ps; 200 K, 200 ps; 100 K, 75 ps; 10 K, 20 ps; 0 K, 5 ps). Interproton distances were calculated from ROESY-NMR spectra using equation 3 by Ämmälahti et al. (1996). The harmonic restraints were applied only to the upper bound, with a force constant of 32 kcal·mol−1 ·Å −2 (as set in the input of AMBER). After an initial series of 100 runs, NMR restraint restraints causing penalties <5 kcal·mol−1 , were regarded as potential artifacts from the spectrum interpretation and removed from the set of restraints. The procedure was then repeated until no restraint passed the penalty ceiling.

The representative structure of the main cluster (based on backbone atoms only) among the SA replica was further selected as a starting structure for MD simulations in solvent. Each molecule was solvated in a truncated octaedron box of (i) TIP4Pew water (Horn et al., 2004) molecules and (ii) DMSO (Fox and Kollman, 1998) molecules, the solvent used for NMR, with a 15 Å buffer region. After minimization, the systems were gradually heated up, first in the NVT ensemble from 0 to 100 K, and then in the NPT ensemble from 100 to 300 K over a total of 890 ps (10 K, 10 ps; 50 K, 10 ps; 100 K, 20 ps; 100 K, 50 ps; 200 K, 100 ps; 300 K, 200 ps; 300 K, 500 ps). Production runs were then performed in the NPT ensemble for 200 ns. All simulations of solvated systems used a time step of 2 fs, periodic boundary conditions with particle mesh Ewald and a non-bonded cutoff of 12 Å, and the SHAKE algorithm to constrain the length of all bonds involving a hydrogen atom. Temperature and pressure were controlled with Langevin dynamics (collision frequency of 2.0 ps−1 ) and isotropic position scaling, respectively. For each system, one simulation in water and one simulation in DMSO were performed without applying any NMR restraint.

The simulations for the longer poly-alanine mimics (poly-Ala) followed the same protocol and were performed without restraints in water and in DMSO. For each poly-Ala system, four parallel simulations were ran starting from the same coordinates after heatup but with different initial velocities in order to increase the sampling of the conformational space and ensure convergence of our analysis.

Analysis of the trajectories were performed using cpptraj module of AmberTools17 (Roe and Cheatham, 2013). The output data was post processed using python 2 including NumPy and matplotlib modules (Hunter, 2007; Oliphant, 2007). Cluster analysis was performed using cpptraj's hierarchical agglomerative approach with a total of 10 clusters based on backbone RMSD (N, CO, Cα , Ψ[4Tz]). Root mean square deviation (RMSD) and radius of gyration (RoG) analysis were performed based on backbone atoms only (N, CO, Cα , Ψ[4Tz]) using the heatup structure as reference point in the case of RMSD. Dihedral analysis was done using the φ- and ψ- torsion angles defined in **Figure 4**. The torsion angle distribution was plotted in a Ramachandran like style using matplotlib. Radial distribution functions (RDFs) were calculated using a bin spacing of 0.1 Å and normalized using the default density of water as implemented in cpptraj. The representative structures of

#### REFERENCES

Ämmälahti, E., Bardet, M., and Molko, D., Cadet, J. (1996). Evaluation of distances from ROESY experiments with the intensity-ratiomethod. J. Magn. Reson. Ser. A. 122, 230–232.

the main clusters were prepared using the VMD software (Humphrey et al., 1996).

## DATA AVAILABILITY

This manuscript contains previously unpublished data. The name of the repository and accession number are not available.

## AUTHOR CONTRIBUTIONS

The project was designed, coordinated and supervised by NS. Synthesis of the peptidotriazolamers was performed by TF, OK and AN as well as the measurement of spectroscopic data by TF, OK, AN, and DS. Analysis of the NMR data for the use as distance restraints was done by DS, MJ, JG, under supervision of RL. The molecular modeling study was designed by AM and IA, supervised by AM, and executed mainly by DS, with assistance of MJ and JG. DS analyzed and compiled the data and prepared the manuscript with support of AM, NS, RL, and IA. The final manuscript was read and approved by all authors.

## FUNDING

The authors gratefully acknowledge financial support from Deutsche Forschungsgemeinschaft (DFG, Project SE 609/10– 1, SFB 749, project C8, CIPSM), Deutscher Akademischer Austauschdienst (DAAD, Project ID 57333947) and the Leading National Research Centre (KNOW, Poland), the BioNam: Bionanomaterials project. This work was supported by the BMBF-funded de.NBI Cloud within the German Network for Bioinformatics Infrastructure (de.NBI) (031A537B, 031A533A, 031A538A, 031A533B, 031A535A, 031A537C, 031A534A, 031A532B). We acknowledge support for the Article Processing Charge by the Deutsche Forschungsgemeinschaft and the Open Access Publication Fund of Bielefeld University.

## ACKNOWLEDGMENTS

The authors gratefully thank the de.NBI Cloud Team for the access to the de.NBI Cloud as major computational resource for this project. The authors thank the NMR spectroscopy as well as the Mass spectrometry department of Bielefeld University.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem. 2019.00155/full#supplementary-material

Supplementary Material as mentioned above as well as NMR data and PDB files of the representative structures are available online.

Angell, Y. L., and Burgess, K. (2007). Peptidomimetics via copper-catalyzed azidealkynecycloadditions. Chem. Soc. Rev. 36, 1674–1689. doi: 10.1039/b701444a

Angell, Y., and Burgess, K. (2005). Ring closure to β-turn mimics via copper-catalyzed azide/alkynecycloadditions. J. Org. Chem. 70, 9595–9598. doi: 10.1021/jo0516180

application to the design of novel inhibitors of proteolytic enzymes. J. Am. Chem. Soc. 114, 10672–10674. doi: 10.1021/ja00052a093

surrogates in peptidomimetics. Beilstein J. Org. Chem. 13, 2428–2441. doi: 10.3762/bjoc.13.240

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Schröder, Kracker, Fröhr, Góra, Jewginski, Nieß, Antes, Latajka, Marion and Sewald. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Bicyclic Pyrrolidine-Isoxazoline γ Amino Acid: A Constrained Scaffold for Stabilizing α-Turn Conformation in Isolated Peptides

Francesco Oliva1 , Raffaella Bucci 2 , Lucia Tamborini 2 , Stefano Pieraccini 1 \*, Andrea Pinto3 \* and Sara Pellegrino2 \*

#### Edited by:

Jutta Eichler, Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany

#### Reviewed by:

Lihua Yuan, Sichuan University, China Cheng Yang, Sichuan University, China

#### \*Correspondence:

Stefano Pieraccini<EMAIL_ADDRESS>Andrea Pinto<EMAIL_ADDRESS>Sara Pellegrino<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Supramolecular Chemistry, a section of the journal Frontiers in Chemistry

Received: 29 November 2018 Accepted: 20 February 2019 Published: 18 March 2019

#### Citation:

Oliva F, Bucci R, Tamborini L, Pieraccini S, Pinto A and Pellegrino S (2019) Bicyclic Pyrrolidine-Isoxazoline γ Amino Acid: A Constrained Scaffold for Stabilizing α-Turn Conformation in Isolated Peptides. Front. Chem. 7:133. doi: 10.3389/fchem.2019.00133 1 Department of Chemistry, University of Milan, Milan, Italy, 2 Department of Pharmaceutical Sciences, University of Milan, Milan, Italy, 3 Department of Food, Environmental and Nutritional Sciences, University of Milan, Milan, Italy

Unnatural amino acids have tremendously expanded the folding possibilities of peptides and peptide mimics. While α,α-disubstituted and β-amino acids are widely studied, γ-derivatives have been less exploited. Here we report the conformational study on the bicyclic unnatural γ amino acid, 4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-3-carboxylic acid 1. In model peptides, the (+)-(3aR6aS)-enantiomer is able to stabilize α-turn conformation when associated to glycine, as showed by 1H-NMR, FT-IR, and circular dichroism experiments, and molecular modeling studies. α-turn is a structural motif occurring in many biologically active protein sites, although its stabilization on isolated peptides is quite uncommon. Our results make the unnatural γ-amino acid 1 of particular interest for the development of bioactive peptidomimetics.

Keywords: unnatural γ-amino acids, peptidomimetic, isoxazoline, α-turn, metadynamic studies, conformational analysis

## INTRODUCTION

Amino acids are the key building blocks of proteins and biomolecules and are widely exploited in different applications, from pharmaceutical chemistry and biomedicine to material science, optoelectronics and catalysis (Zhang et al., 2012; Mikhalevich et al., 2017; Solomon et al., 2017; López-Andarias et al., 2018; Raymond and Nilsson, 2018). The insertion of unnatural amino acids (UAAs) in peptides and peptide mimics could add specific features to these molecules, such as proteolytic stability, active functional groups and new reactivity (Clerici et al., 2016; Pellegrino et al., 2016, 2017; Bucci et al., 2017). α,α disubstituted and β- homologs of natural amino acids have been particularly studied during the years for their ability to introduce conformational constrains in peptides and to stabilize specific secondary structures (Bonetti et al., 2015; Pellegrino et al., 2015; Fanelli et al., 2017; Kobayashi et al., 2017). On the other hand, γ UAAs provide a further opportunity to engineer the available backbone through the incorporation of an additional methylene group (Vasudev et al., 2011; Sohora et al., 2018). Recent studies report that γ-peptides are able to form helices, sheets and turns, whose conformational stability is increased by introducing substituents on the backbone chain (Bouillère et al., 2011). Moreover, cyclic γ UAAs, as gabapentin (Gpn) (Chatterjee et al., 2009; Konda et al., 2018) and γ-cyclohexyl amino acid (Guo et al., 2012) are able to stabilize both turn and helix conformation in oligomers and in mixed α-γ and β-γ sequences. On the other hand, the use of bicyclic γ amino acids for the stabilization of the peptide structure is much less investigated (Machetti et al., 2000).

In this work, we investigated the conformational behavior of both the enantiomers of the bicyclic unnatural γ amino acid 4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-3-carboxylic acid **1**, obtained starting from the corresponding ethyl esters recently described by us (Tamborini et al., 2015). Compound **1** is a conformationally constrained dipeptide analog and, in principle, it could substitute two amino acids in a peptide chain. The presence of the constrained bicyclic system could induce specific secondary structure allowing a proper orientation of the peptide arms at C- and N- termini. Furthermore, the presence of the isoxazoline ring, a core often found in biologically active compounds, could be particularly useful for future applications in the pharmaceutical field. In fact, isoxazoline derivatives are important scaffolds found in many naturally occurring and biologically active compounds possessing a wide range of bioactivities, such as antibacterial, antifungal, antiparasitic (Conti et al., 2011; Bruno et al., 2014; Pinto et al., 2016a), anticancer (Castellano et al., 2011; Kaur et al., 2014), anti-inflammatory and anticonvulsant activity (Sperry and Wright, 2005; Pinto et al., 2011, 2016b). Isoxazolines are also considered to be important precursors for the synthesis of β-hydroxyketones (Kozikowski and Park, 1990; Tsantali et al., 2007), β-aminoalcohols (Fuller et al., 2005), isoxazolidines (Itoh et al., 2002), and many other valuable compounds. Recently, peptidomimetics containing the isoxazoline ring have been reported as β-turn mimics (Bucci et al., 2018; Memeo et al., 2018).

Starting from the (–)-(3aS6aR)-**1** and (+)-(3aR6aS)-**1** enantiomers, model peptides containing Leu-Val dipeptide at C-terminus and variable sequences at N-terminus were prepared. Their conformational behavior was investigated by NMR spectroscopy, FT-IR, circular dichroism, and molecular modeling. Our results indicated that (+)-(3aR6aS)-**1** enantiomer, in combination with glycine, is effective in stabilizing the α-turn conformation in peptides (**Figure 1**). This structural motif occurs quite often in many key sites of proteins, such as enzyme active site, and metal binding domains (Wintjens et al., 1996), although few molecules are known to mimic or stabilize it on isolated peptides (Kelso et al., 2004; Hoang et al., 2011; Krishna et al., 2014; Wang et al., 2018.). Our results make thus the unnatural γ-amino acid **1** of particular interest for future development of bioactive peptidomimetics.

## MATERIALS AND METHODS

## Materials

Chemicals were obtained from Zentek (Italy) and used without further purification. Melting points were determined in a Stuart Scientific melting point apparatus in open capillary tubes and are certified. ESI mass spectra were recorded on an LCQESI. MS was recorded on a LCQ Advantage spectrometer from Thermo Finningan and a LCQ Fleet spectrometer from Thermo Scientific. CD experiments were carried out on a Jasco J-810 instrument. Spectra were obtained from 195 to 250 nm with a 0.1 nm step and 1 s collection time per step, taking three averages. The CD spectra were plotted as mean residue ellipticity θ (degree × cm2 × dmol−1 ) vs. wave length λ (nm). Noise-reduction was obtained using a Fourier-transform filter program from Jasco. The NMR spectroscopic experiments were carried out on a Varian OXFORD 300 MHz (300 and 75 MHz for 1H and 13C, respectively). Chemical shifts, δ, are given in ppm relative to the CHCl3 internal standard, and the coupling constants, J, are reported in hertz (Hz).

## Methods

#### Synthesis of (–)-(3aS,6aR)-1 and (+)-(3aR,6aS)-1

Compound (**–**)-**2** or (+)-**2** (200 mg, 0.70 mmol) was dissolved in MeOH (3.0 mL) and treated with 1N NaOH aqueous solution (1.4 mL). The mixture was stirred at room temperature for 1 h and the disappearance of the starting material was monitored by TLC (Cyclohexane/EtOAc 7:3). The mixture was diluted in water (20 mL), made acidic with 2N aqueous HCl and extracted with EtOAc. The organic phase was dried over anhydrous Na2SO4 and after evaporation of the solvent, the acid derivate (–)-(3aS,6aR)**-1** or (+)-(3aR,6aS)**-1** (170 mg, 95%) was obtained as a white solid.

**(–)-(3aS,6aR)-1:** Rf (CH2Cl2/MeOH 9:1 + 1% AcOH): 0.40; [α] 20 D : (–) 192.2 (c: 0.55 in MeOH); mp = dec. T > 137◦C; 1H NMR (300 MHz, CD3OD): δ 5.35 (dd, J = 4.8, 9.2, 1H); 4.10 (ddd, J = 2.2, 9.2, 9.2, 1H); 3.86–3.96 (m, 2H); 3.38–3.50 (m, 2H); 1.44 (s, 9H) ppm; 13C NMR (75 MHz, CD3OD): δ 161.28, 154.77, 152.95, 87.62, 80.33, 53.06, 50.93, 49.10, 27.12 (3C) ppm.

**HRMS** (ESI) [M + Na]+ calculated for C11H16N2O5Na: 279.0957, found: 279.0950.

**(**+**)-(3aR,6aS)-1:** [α] 20 D : (+) 192.5 (c: 0.57 in MeOH).

## General Procedure for Coupling Reaction

Free carboxylic acid (1 equiv.) was dissolved in CH2Cl2 (0.01M solution) and the mixture was cooled to 0◦C. HOBt (1.1 equiv.) and EDC (1.1 equiv.) were added. After 1 h, free amino compound (1 equiv.) was added, followed by the addition of DIEA (2 equiv.). The reaction mixture was stirred at room temperature for 24 overnight (TLC analysis). The organic layer was washed with a 5% solution of KHSO4 (3 times), with a saturated solution of NaHCO3 (3 times) and with brine (1 time). The organic phase was dried over Na2SO4 and the solvent was removed under vacuum. The products were purified by column flash chromatography on silica gel (hexane/ethyl acetate gradient) followed by a crystallization from a mixture of ethyl acetate/hexane.

**Abbreviations:** ACN, Acetonitrile; Boc, tert-butyloxycarbonyl; DIEA, N,N-Diisopropylethylamine; DMF, Dimethylformamide; EDC, 1-Ethyl-3-(3 dimethylaminopropyl)carbodiimide; Gly, Glycine; HOBt, Hydroxybenzotriazole; Leu, Leucine; Phe, Phenylalanine; TFA, Trifluoroacetic acid; Val, Valine.

a see Steinke and Kula (1990).

#### **N-Boc-(–)-**∆2 **-isox-Leu-Val-CONH**2 **(4)**

1H NMR (300 MHz, CDCl3): δ 7.11 (bs, 1H), 6.88 (d, J = 8.8 Hz, 1H), 6.19 (bs, 1H), 5.72 (bs, 1H), 5.34–5.29 (m, 1H), 4.56–4.48 (m, 1H), 4.31 (dd, J = 8.2; 6.9 Hz, 1H), 4.17–3.83 (m, 3H), 3.56 – 3.42 (m, 2H), 2.23–2.07 (m, 1H), 1.85–1.61(m, 3H), 1.43 (s, 9H), 0.99 – 0.92 (m, 12H) ppm.

13C NMR (75 MHz, CDCl3): δ 173.04, 171.32, 159.44, 154.28, 153.93, 87.40, 80.43, 58.24, 53.26, 52.30, 50.53, 49.32, 40.87, 30.53, 28.31, 24.82, 22.91, 21.92, 19.27, 17.93 ppm.

HRMS (ESI) [M + Na]+ calculated for C22H37N5O6Na: 490.2642, found: 490.2635.

#### **N-Boc- (**+**)-**∆2 **-isox-Leu-Val-CONH**2 **(5)**

1H NMR (300 MHz, CDCl3): δ 7.05 (bs, 1H), 6.73 (d, J = 7.9 Hz, 1H), 6.13 (bs, 1H), 5.69 (bs, 1H), 5.37–5.28 (m, 1H), 4.56–4.47 (m, 1H), 4.36–4.27 (m, 1H), 4.18–4.07 (m, 1H), 3.99–3.77 (m, 2H), 3.61–3.41 (m, 2H), 2.28–2.10 (m, 1H), 1.78–1.57(m, 3H), 1.49 (s, 9H), 1.01 – 0.90 (m, 12H) ppm.

13C NMR (75 MHz, CDCl3): δ 173.16, 171.54, 159.33, 154.16, 153.96, 80.34, 60.38, 58.16, 53.27, 52.25, 49.41, 41.02, 30.67, 28.32, 24.83, 22.87, 21.99, 19.20, 18.02 ppm.

HRMS (ESI) [M + Na]+ calculated for C22H37N5O6Na: 490.2642, found: 490.2635.

HRMS (ESI) [M + Na]+ calculated for C31H46N6O7Na: 637.3326, found: 637.3332.

HRMS (ESI) [M + Na]+ calculated for C31H46N6O7Na: 637.3326, found: 637.3332.

HRMS (ESI) [M + Na]+ calculated for C33H49N7O8Na: 694.3540, found: 694.3539.

#### General Procedure for N-Boc-Deprotection

N-Boc protected compound (1 equiv.) was dissolved in CH2Cl2 (0.01M) and the solution cooled at 0◦C. TFA (50% v/v) was added dropwise, the solution was warmed up at room temperature and was stirred for 2 h. The solvent was removed under vacuum with the obtainment of the trifluoroacetate salt that was directly used in the next coupling step.

a see Smith and Spackman (1955).

#### **NH-(–)-**12 **-isox-Leu-Val-CONH2 (6)**

1H NMR (300 MHz, CD3OD): δ 7.90 (d, J = 8.7 Hz, 1H), 5.53 (dd, J = 9.5, 4.9 Hz, 1H), 4.54 (dd, J = 9.8, 4.9 Hz, 1H), 4.42 (t, J = 8.8 Hz, 1H), 4.26 – 4.17 (m, 1H), 3.90 – 3.84 (m, 1H), 3.89 – 3.75 (m, 2H), 3.58 – 3.49 (m, 2H), 3.22 (dd, J = 5.9, 4.1 Hz, 1H), 2.05 (dq, J = 13.6, 6.8 Hz, 1H), 1.80 – 1.59 (m, 3H), 1.05 – 0.82 (m, 12H) ppm.

13C NMR (75 MHz, CD3OD): δ 147.47, 172.82, 159.52, 152.51, 85.48, 58.22, 52.96, 52.21, 50.55, 49.27, 40.31, 30.68, 24.54, 21.98, 20.47, 18.32, 17.08 ppm.

HRMS (ESI) [M + Na]+ calculated for C17H29N5O4Na: 390.2117, found: 390.2111.

#### **NH-(**+**)-**12 **-isox-Leu-Val-CONH2 (7)**

1H NMR (300 MHz, CD3OD); δ 7.97 (d, J = 8.6 Hz, 1H), 5.53 (dd, J = 9.5, 4.9 Hz, 1H), 4.55 (m, 1H), 4.39 (m, 1H), 4.26 – 4.16 (m, 1H), 3.90 – 3.72 (m, 2H), 3.58 – 3.43 (m, 2H), 3.24 – 3.16 (m, 1H), 2.05 (m, 1H), 1.75 – 1.58 (m, 3H), 1.02 – 0.81 (m, 12H) ppm.

13C NMR (75 MHz, CD3OD): δ 147.47, 172.99, 159.40, 152.40, 128.72, 85.21, 58.05, 53.12, 52.17, 50.66, 49.14, 40.21, 30.91, 29.36, 24.84, 21.99, 20.25, 18.26, 16.90 ppm.

HRMS (ESI) [M + Na]+ calculated for C17H29N5O4Na: 390.2117, found: 390.2111.

## Molecular Modeling

Compound **9** and **10** were modeled in explicit solvent with periodic boundary conditions using metadynamics and restrained molecular dynamics simulations. For each of the compounds two isomers (cis and trans with respect to the amide bond) were considered, resulting in a set of four peptides. Such molecules were solvated in a cubic box of 4 nm with chloroform (compound **8**) and acetonitrile (compound **10**) to reproduce the NMR experiment conditions. Every system has been submitted to geometry optimization with the steepest descent algorithm with a convergence of 100 kJ mol−1 nm−1 . Then we performed a 1 ns equilibration in NVT conditions at 300 K, followed by a 1 ns NPT equilibration at the 1 bar and at the same temperature. After the equilibration phase, we ran a 50 ns Well Tempered Metadynamics (WTMD) with a 8.0 kJ mol−1 bias-factor, at a reference temperature of 300 K. To study the open and closed state of both peptides, we selected as collective variable (CV) the distance between CβVAL and the N-tert-butoxy carbonyl (N-Boc) quaternary carbon. A Gaussian hill with σ = 0.1 Å and an initial height of 1 kJ mol−1 was added once every 100 steps. The simulation was sped up by saving the Gaussian hills on a grid that ranged from 0 to 16 Å and spaced 0.02 Å. Compound **10** was also simulated using a 50 ns restrained molecular dynamics. Special potentials are used for imposing restraints on the motion of the system, to include knowledge from experimental data such as the NMR derived interatomic distances. Distance restraints add a penalty to the potential when the distance between specified pairs of atoms exceeds a threshold value (Abraham et al., 2014). The threshold values used in our simulation are reported in the **Supplementary Materials**.

The restraints are applied in a progressive way to give time to the peptide to relax, using a τ equal to 500 ps. Since we are using multiple distance restraints, they are not necessary satisfied at each simulation step but as a time average. The atoms restrained and the relative distances are shown in **Table S1**. Compound **9** and **10** have been described using the Amber99SB-ILDN Force Field (Hornak et al., 2006). 4,5,6,6a-Tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-3-carboxylic acid residues were parameterized according to standard procedures as explained in reference (Gandini et al., 2018). During molecular dynamics (MD) and metadynamics simulations, temperature was held constant with the v-rescale algorithm (Bussi et al., 2007), while pressure was kept constant through the Berendsen barostat (Berendsen et al., 1984). MD simulations were performed using the leap-frog algorithm with 2 fs time-step, with holonomic constraints on every bond enforced using the LINCS algorithm. Simulations and subsequent analysis were performed with the GROMACS 5.0.4 (Van Der Spoel et al., 2005) program suite, while metadynamics was run along with PLUMED version 2.2.2 (Tribello et al., 2014).

## RESULTS AND DISCUSSION

The two enantiomers of compound **1** were obtained starting from the corresponding ethyl esters recently described by us (Tamborini et al., 2015). Racemate (±)-**2** (Conti et al., 2006) was synthesized in a flow chemistry reactor exploiting the 1,3-dipolar cycloaddition reaction of N-Boc-3-pyrroline with ethoxycarbonyl formonitrile oxide. An excellent enantiomeric separation (e.e. >99%) of the racemate was achieved by semi-preparative chiral HPLC. Alkaline hydrolysis of (–)-(3aS6aR)-**2** and (+)-(3aR6aS)- **2** gave the desired (–)-(3aS6aR)-**1** and (+)-(3aR6aS)-**1**, respectively (**Scheme 1**).

The ability of compound **1** to stabilize secondary structures was evaluated in model peptides containing (L)-Phe at N-terminus and (L)-Leu-(L)-Val sequence at C-terminus and (**Schemes 2**, **3**). This last dipeptide was chosen as it normally adopts extended conformation in solution. Compound **1** was used in both the 3aS6aR and 3aR6aS stereochemistries [(–)-**1** and (+)-**1**, respectively], as a different effect on peptide conformations could be expected depending on the stereochemistry of the unnatural amino acids (Pellegrino et al., 2012).

Firstly, Leu-Val dipeptide **3** was prepared starting from (L)-Valinamide and NH-Boc-(L)-Leu, according to the general coupling procedure, followed by N-terminus deprotection in TFA (91% overall yield). Diastereoisomeric dipeptides **4** and **5** were then achieved in good yields (65% and 63%, respectively) by a coupling reaction of (–)-**1** or (+)-**1** with dipeptide **3** (**Scheme 2**).

Both **4** and **5** compounds were completely characterized by NMR spectroscopy (CDCl3, see SI for complete proton assignment). In both tripeptides, no significant Noesy effects between Leu-Val dipeptide and the scaffold were detected. Furthermore, in variable temperature experiments, the obtained 1δ/1T is higher than 5 ppb/K for all the amide protons, indicating the absence of H-bonds stabilizing a particular

conformation. The JNH−CHα value for NH-Val is of 8.8 Hz and 7.9 Hz, for **4** and **5**, respectively (NH-Leu appears as a broad signal for each compound). From these findings, we hypothesized that both **4** and **5** adopt an extended conformation and that the isoxazoline scaffold is not interacting with Leu-Val dipeptide.

The peptide chain was then elongated at N-terminus, through Boc-deprotection and coupling with (L)-Phe. Diastereoisomeric dipeptides **8** and **9** were achieved in good overall yields (73% and 76%, respectively) (**Scheme 3**). From NMR studies, it was found that both model peptides **8** and **9** are present as a mixture of conformers in 2:1 ratio in CDCl3 solution. The presence of these two conformers is probably due to the low-energy barrier cis/trans isomerization of the tertiary amide on the pyrrolidine as it is frequently observed on the acylated proline and in the case of tertiary cyclic amides (Laursen et al., 2013; Pellegrino et al., 2014). Regarding compound **8**, this rotation led to a splitting of the (L)-Phe proton signals only. Furthermore, no significative Noesy effects were detected, and, in variable temperature experiments, the obtained 1δ/1T coefficient is high for all the amide protons. Taking all these data together, we can assume that Phe and Leu-Val dipeptide are oriented in opposite directions and that the 3aS6aR stereochemistry of the scaffold is not effective in inducing specific secondary structures when used in combination with (L)-α-amino acids.

A different scenario was observed for compound **9**. Its two rotamers are indeed characterized by different chemical shifts, suggesting that the isomerization of the tertiary amide leads to two different structures conformations influencing the entire molecule. Unfortunately, due to several overlapping signals, it was not possible to assessing significative Noesy proximities. Variable temperature experiments were thus done, in order to investigate if the NH were involved in hydrogen bonds and, as a consequence, if the two isomers of **9** were characterized by a stable conformation in solution. In the case of the major isomer, the obtained 1δ/1T is around 4 ppb/K for NH-Phe, indicating that this proton could be involved in a weak/medium hydrogen bond. All the other amide protons had higher 1δ/1T. On the

SCHEME 3 | Reagents and conditions: a) TFA, CH2Cl2 from 0◦C to r.t, 2 h; b) EDC/HOBt/DIEA, N-Boc-(L)-Phe, CH2Cl2 from 0◦C to r.t, 24 h; c) EDC/HOBt/DIEA, N-Boc-(L)-Phe-Gly-OH, CH2Cl2 from 0◦C to r.t, 24 h.

other hand, in the minor conformer the obtained 1δ/1T is of around 2 ppb/K for NH-Val, indicating that this proton is involved in a strong hydrogen bond. Metadynamic studies were then performed on the cis/trans tertiary amide conformers of compound **9** (**Figure 2**).

In particular, we performed two 50 ns long metadynamics simulations, using the distance between Boc quaternary carbon and CβVal as collective variable (CV). This geometric parameter was selected as a suitable CV because it discriminates well between closed and extended states of the peptide. In this way, it could be possible to evaluate if the two conformers had an intrinsic tendency toward turn conformation. The cis isomer showed a broad free energy minimum corresponding to CV values between 5 and 8 Å, while the trans isomer showed much higher free energy values in this region, exhibiting a shallow minimum around 10–15 Å (**Figure 3**).

This different behavior could be indicative of a preference of cis-**9** toward a more closed conformation, although from these data it was not possible to make any further assumptions. For this reason, we envisaged that the introduction of a Gly residue between Phe and isoxazoline scaffold could increase the conformational flexibility of the N-terminus peptide arm favoring its interaction with the C-teminus part of the molecule. Compound **7** was thus elongated at N-terminus, through coupling with N-Boc-(L)-Phe-Gly-OH dipeptide using general coupling reaction conditions to obtain peptide **10**

(65%, **Scheme 3**). The NMR study on **10** (CD3CN, see SI for complete assignment), showed that it is present in solution as a single stable conformer (only trace amounts of a second isomer were detected). A complete set of NHi-NHi+1 Noesy proximities, whose calculated distances ranged from 2.68 to 2.96 Å, were observed (**Figure 4** on the top right). Furthermore, long range Noesy effects involving HαGly and NHVal (3.03 Å) and HβLeu (2.73 Å) were found (**Figure 4** on the bottom).

In variable temperature experiments (**Figure 5**), the obtained 1δ/1T is of around 2 ppb/K for NH-Val, while NH-Leu and one of the C-terminus amide protons possess 1δ/1T of around 3 and 4 ppb/K, respectively. The presence of hydrogen bonds was also confirmed by FT-IR analysis. N-H stretching bands A and B (3,500–3,000 cm−1 region, **Figure S20**) are indeed downshifted as frequently observed in intramolecular hydrogen bonded conformations (Tonan and Ikawa, 1996; Barth, 2007). Furthermore, the deconvolution of the amide I band (1,700– 1,600 cm−1 ) showed the presence of a band at 1,655 cm−1 (**Figure S20**), typical of α structures (Kong and Yu, 2007).

In order to gain more information on the conformation of compound **10**, far-UV circular dichroism (CD) analysis in CH3CN (0.1 mM solution) was then performed. In **Figure 6** the obtained CD spectrum is reported.

It showed a maximum at around 250 nm due to the strong absorption of the isoxazoline ring, as observed by Memeo et al. (2018) on similar compounds (see also the UV spectrum in the SI). In the amide bond region, two negative minima at 225 and 208 nm, with the same intensity, and a slightly positive band at 200 nm were also observed. The presence of the exciton splitting of the π→ π ∗ transition band suggested that a α-turn conformation was present (Wang et al., 2018), although in short

peptides it is difficult to correctly evaluate the intensity of Cotton effects with respect to secondary structure motifs (Chin et al., 2002; Wang et al., 2018).

In order to completely elucidate the conformation of peptide **10**, a restrained molecular dynamic was finally performed. The restraints were based on the obtained NOESY values. When the hydrogen was not uniquely defined, like in HαGly and HβLeu, we used the closest carbon, e.g., CαGly and CβLeu to implement distance restrains (**Figure S3**). The analysis showed that CαGly-CβLeu (A) and CαGly-HNHVal (B) are mutually exclusive in the trans isomer but not in the cis one as shown in **Figure S1.** Furthermore, H-bond analysis resulted in the observation that a H-bond is between CO-Gly and NH-Val. In **Figure 7** the most representative structure is reported.

Taking together both experimental and molecular modeling data, we can assume that, in solution, compound **10** effectively adopts a α-turn conformation. This motif is normally formed

by 5 α-amino acids and is characterized by a H-bond between i and i+4 residues. In our case, the isoxazoline scaffold replaces two of the three core amino acids (**Figure 1**) and the H-bond is formed by the CO-Gly and NH-Val, leading to the formation of a 12 member pseudo-cycle. Furthermore, the overall structure of the peptidomimetic is reinforced by medium-weak H-bonds involving NH-Leu and one C-terminus-NH2, as evicted from NMR data. The presence of a α-turn conformation is also confirmed by the computational analysis of the dihedral angles of the residue i+3 and of the distances between residues i and i+4 (**Table S9**), as their values rely within the α-turn parameters (Pavone et al., 1996).

In conclusion, as α-turns are often found on biologically active sites and few molecules have been reported to mimic or stabilize them, the ability of compound (+)-**1** to stabilize α-turn conformation on isolated peptide is particularly important in view of future biological applications.

## DATA AVAILABILITY

All datasets generated for this study are included in the manuscript and/or the supplementary files.

## AUTHOR CONTRIBUTIONS

FO and RB equally contributed to this work. The project was conceived by SaP, LT, and AP. SaP designed and coordinated the research. LT and RB chemically synthesized and analyzed the materials, RB performed NMR, FT-IR and CD experiments, FO and StP performed molecular modeling. SaP, StP, LT, and AP analyzed and compiled the data and co-wrote the manuscript. The final manuscript was read and approved by all the authors.

#### ACKNOWLEDGMENTS

We thank Donatella Nava for the NMR analysis and Michele Consonni and Sabrina Giofrè for practical help in the synthesis.

#### REFERENCES

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem. 2019.00133/full#supplementary-material

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Oliva, Bucci, Tamborini, Pieraccini, Pinto and Pellegrino. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Fluoro-Aryl Substituted α,β 2,3-Peptides in the Development of Foldameric Antiparallel β-Sheets: A Conformational Study

Raffaella Bucci 1 , Alessandro Contini 1 , Francesca Clerici 1 , Egle Maria Beccalli 1 , Fernando Formaggio2 , Irene Maffucci 3,4, Sara Pellegrino1 and Maria Luisa Gelmi 1 \*

1 Department of Pharmaceutical Sciences (DISFARM), University of Milan, Milan, Italy, 2 Department of Chemistry, University of Padova, Padova, Italy, 3 CNRS UMR 7025, Génie Enzymatique et Cellulaire, Centre de Recherche de Royallieu, Compiègne, France, 4 Génie Enzymatique et Cellulaire, Centre de Recherche de Royallieu, Sorbonne Universités, Université de Technologie de Compiègne, Compiègne, France

#### Edited by:

Assaf Friedler, Hebrew University of Jerusalem, Israel

#### Reviewed by:

Vladimir Torbeev, Université de Strasbourg, France James Gardiner, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australia

> \*Correspondence: Maria Luisa Gelmi<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Chemical Biology, a section of the journal Frontiers in Chemistry

Received: 31 January 2019 Accepted: 13 March 2019 Published: 02 April 2019

#### Citation:

Bucci R, Contini A, Clerici F, Beccalli EM, Formaggio F, Maffucci I, Pellegrino S and Gelmi ML (2019) Fluoro-Aryl Substituted α,β 2,3-Peptides in the Development of Foldameric Antiparallel β-Sheets: A Conformational Study. Front. Chem. 7:192. doi: 10.3389/fchem.2019.00192 α,β 2,3-Disteroisomeric foldamers of general formula Boc(S-Ala-β-2R,3R-Fpg)nOMe or Boc(S-Ala-β-2S,3S-Fpg)nOMe were prepared from both enantiomers of syn H-2-(2-F-Phe)-h-PheGly-OH (named β-Fpg) and S-alanine. Our peptides show two appealing features for biomedical applications: the presence of fluorine, attractive for non-covalent interactions, and aryl groups, crucial for π-stacking. A conformational study was performed, using IR, NMR and computational studies of diastereoisomeric tetra- and hexapeptides containing the β 2,3-amino acid in the R,R- and S,S-stereochemistry, respectively. We found that the stability of peptide conformation is dependent on the stereochemistry of the β-amino acid. Combining S-Ala with β-2R,3R-Fpg, a stable extended β-strand conformation was obtained. Furthermore, β-2R,3R-Fpg containing hexapeptide self-assembles to form antiparallel β-sheet structure stabilized by intermolecular H-bonds and π,π-interactions. These features make peptides containing the β 2,3-fluoro amino acid very appealing for the development of bioactive proteolytically stable foldameric β-sheets as modulators of protein-protein interaction (PPI).

Keywords: β 2,3-diaryl-amino acid, α, β 2,3-peptide, extended peptide, antiparallel β-sheet, conformational analyses, foldamers

## INTRODUCTION

Amino acids are small molecules able to induce high molecular complexity, promoting discrete three-dimensional folded structures in peptides. α-Amino acid-based peptides show some drawbacks such as the low stability to proteases, and, in the case of short peptides, the lack of a stable secondary structure. The use of oligomers with unnatural backbones is a powerful strategy to overcome those issues, while maintaining the desired fragment structure.

In particular, β-peptide foldamers (Seebach et al., 1996, 2004; Gellman, 1998) have been extensively studied in the last two decades. These molecules can adopt specific compact conformations despite the increased conformational space due to the additional methylene group of the β-amino acid. Furthermore, their substitution pattern can influence the conformation of

**37**

β-peptides, as reported in several experimental (Cheng et al., 2001; Seebach et al., 2006; Seebach and Gardiner, 2008; Pilsl and Reiser, 2011; Vasudev et al., 2011; Berlicki et al., 2012; Johnson and Gellman, 2013; Basuroy et al., 2014; Lee et al., 2014; Wang and Schepartz, 2016; Bucci et al., 2017a) and molecular modeling studies (Wu et al., 2008; Zhu et al., 2010; Baldauf and Hofmann, 2012). An even larger pool of secondary structure motifs could be obtained, by the combination of α- and both acyclic and cyclic β-amino acids (αβ; ααβ; αββ; etc). Inspired by the diversity of folded structures and functions manifested by peptides and proteins, β-peptide mimics and foldamers have been thus exploited for different applications ranging from biomedicine (Horne, 2011; Cabrele et al., 2014; Checco and Gellman, 2016) to material science (Gopalan et al., 2015; Clerici et al., 2016; Del Borgo et al., 2017).

Only few examples are reported related to α,β-repeating sequences containing acyclic amino acids (Sharma et al., 2005; Srinivasulu et al., 2006; Angelici et al., 2007; Balamurugan and Muraleedharan, 2012; Basuroy et al., 2014), the majority of them giving helix constructs.

Our interest toward the preparation of non-natural amino acids (Pellegrino et al., 2008; Penso et al., 2012; Ruffoni et al., 2015) and their use for peptidomimetic synthesis for different applications (Pellegrino et al., 2015, 2016; Ruffoni et al., 2016; Bucci et al., 2018, 2019; Tonali et al., 2018) is well-documented. Recently, our research group reported on a diastreoselective synthesis of a new class of β-amino acids, syn-S ∗ , S∗ -β 2,3 diarylamino acids, differently substituted on the aromatic ring (Bonetti et al., 2014).

Due to the lack of information related to α,β 2,3-amino acids repeating sequences, here we report on the preparation and conformational studies of α,β 2,3-peptides, containing syn H-2-(2-F-Phe)-h-PheGly-OH, named β-Fpg, and helicogenic Salanine (**Figure 1**). This scaffold shows two features that can be appealing for biomedical applications: the presence of a fluorine, useful for non-covalent interactions, and two aryl groups, crucial for π-stacking and hydrophobic interactions. Taking advantage of the use of fluorine-substituted β-2S,3S-Fpg (**Figure 1**), in combination with S-Ala or S-Arg-S-Ala, we recently prepared short peptides able to generate proteolytically stable nanotubes (Bonetti et al., 2015) and spherical aggregates (Bucci et al., 2017b) as drug delivery systems.

Here we report on a conformational study on diastereoisomeric tetra- and hexapeptides α,β 2,3-foldamers

of general formula Boc(S-Ala-β-2R,3R-Fpg)nOMe and Boc(S-Ala-β-2S,3S-Fpg)nOMe (**Figure 1**).

Our results showed that the stability of peptide conformation is dependent on the stereochemistry of the β-amino acid. Combining S-Ala with β-2R,3R-Fpg, a stable extended βstrand conformation was obtained. Moreover, it was found that hexapeptide **11** (**Figure 1**) self-assembles to form antiparallel βsheet structure stabilized by intermolecular H-bonds and π,πinteractions. This result is important since, to the best of our knowledge, no examples of extended α,β 2,3-foldamers are reported. Furthermore, there is a need of the development of bioactive proteolytically stable foldameric β-sheets that might be useful as modulators of protein-protein interactions (PPI) (Hegedüs et al., 2016). The design of this secondary structure is indeed not trivial, although β-sheet interfaces frequently occurred in PPI.

## MATERIALS AND METHODS

#### Chemistry

Experimental procedures, compound characterization data for newly synthesized peptides (**3-5** and **7-12**; for 1H, 13C, and 19F NMR spectra see **Figures S10–S39**) are reported in the **Supplementary Material**. Dipeptide **1**, **2** (Bonetti et al., 2015) and **6** (Bucci et al., 2017b) are known compounds.

Melting points were determined with a Stuart Scientific melting point apparatus in open capillary tubes and are uncorrected. Chemicals were purchased from Sigma Aldrich and were used without further purification. ESI MS were recorded on a LCQ Advantage spectrometer from Thermo Finningan and a LCQ Fleet spectrometer from Thermo Scientific. The FT-IR absorption spectra were recorded with a Perkin-Elmer 1720X spectrophotometer, nitrogen flushed, equipped with a sampleshuttle device, at 2 cm−1 nominal resolution, averaging 100 scans. Solvent (base-line) spectra were recorded under the same conditions. Cells with path lengths of 1.0 and 10 mm (with CaF2 windows) were used. Spectrograde CDCl3 (99.8% D) was purchased from Fluka. The NMR spectroscopic experiments were carried out either on a Varian MERCURY 200 MHz (200 and 50 MHz for 1H and 13C, respectively), Varian MERCURY 300 MHz (300, 75, 282 MHz for 1H, 13C, and 19F, respectively), or Bruker Avance I 500 MHz spectrometers (500 and 125 MHz for 1H and 13C, respectively). Chemical shifts (δ) are given in ppm relative to the CHCl3 internal standard, and the coupling constants J are reported in Hertz (Hz). Optical rotations were measured on a Perkin–Elmer 343 polarimeter at 20◦C (concentration in g/100 mL).

## Computational Methods

The non-natural β-amino acids, capped respectively with an acetyl (Ac) and an OMe group at the N- and C-termini, were designed using MOE (Molecular Operating Environment, 2013) and submitted to a "low mode" conformational search (MMFF94x force field, Born solvation, iteration limit = 40 000, MM iteration limit = 2,500, rejection limit = 500). The two lowest energy conformations having φ, θ, and ψ dihedrals corresponding to two different secondary structures, namely the extended one and a β-turn, were selected to derive partial charges with the R.E.D.III.52 software (Dupradeau et al., 2010). Each geometry was optimized at the HF/6-31G(d) level, and two different spatial orientations were used to derive orientation- and conformation-independent RESP-A1 charges.