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Synthesis and Conformational Analysis of Macrocyclic Peptides Consisting of Both A-Helix and Polyproline Helix Segments

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Synthesis and Conformational Analysis of Macrocyclic Peptides Consisting of Both A-Helix and Polyproline Helix Segments Synthesis and Conformational Analysis of Macrocyclic Peptides Consisting of Both a-Helix and Polyproline Helix Segments Sung-ju Choi,1 Soo hyun Kwon,1 Tae-Hyun Kim,2 Yong-beom Lim1 1 Translational Research Center for Protein Function Control and Department of Materials Science & Engineering, Yonsei University, Seoul 120-749, Korea 2 Department of Chemistry, Incheon National University, Incheon 406-840, Korea Received 29 April 2013; revised 18 June 2013; accepted 9 July 2013 Published online 19 July 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22356 INTRODUCTION ABSTRACT: eptides have been used, as isolated monomeric mol- ecules or as basic building blocks for self-assembly Macrocycles are interesting molecules because their topo- into nanostructures, to mimic the functions of natu- logical features and constrained properties significantly ral proteins. Peptides have several advantages in that affect their chemical, physical, biological, and self- they can be relatively easily synthesized and have Pbroader chemical diversity than natural proteins.1,2 However, assembling properties. In this report, we synthesized unique macrocyclic peptides composed of both an a-helix peptides typically have unstable molecular conformations due to their lack of the multiple stabilizing interactions pro- and a polyproline segment and analyzed their conforma- vided by a folded protein environment.3,4 Such conforma- tional properties. We found that the molecular stiffness of tional instability can significantly limit the affinity and the rod-like polyproline segment and the relative orienta- selectivity of peptide epitopes for target receptors. Therefore, tion of the two different helical segments strongly affect the the maintenance and stabilization of the actively folded form of peptides are important for increasing their biomacromo- efficiency of the macrocyclization reaction. Conformational lecular recognition capabilities. analyses showed that both the a-helix and the polyproline The a-helix is a secondary structure element of proteins II helix coexisted within the macrocyclic peptides and that and is well known to play an important role in specific bioma- the polyproline segment exerts significant effect on the cromolecular recognition processes, such as protein–protein, protein–RNA, and protein–DNA interactions. a-Helical struc- overall helical stability and conformation of the a-helical tures are stabilized within the context of well-folded protein segment. VC 2013 Wiley Periodicals, Inc. Biopolymers 101: structures; however, when isolated from the intact protein, the 279–286, 2014. stability of the helical motif is compromised and is rarely heli- 3 Keywords: a-helix; polyproline; macrocycles; peptides cal in solution. Because the stabilization of the active molecu- lar conformation of a peptide is a crucial factor for maintaining the unique functions of a protein, extensive This article was originally published online as an accepted pre- research into stabilized a-helical peptides has been conducted. print. The “Published Online” date corresponds to the preprint Such studies include side chain crosslinking, hydrogen-bond version. You can request a copy of the preprint by emailing the surrogates, metal coordination, salt bridge formation, and syn- Biopolymers editorial office at [email protected] thetic a-helix receptor approaches.5–8 Macrocycles, which have a cyclic structure, are interest- ing molecules because their topological features signifi- Additional Supporting Information may be found in the online version of this article. cantly affect their chemical, physical, biological, and self- 9–13 Correspondence to: Yong-beom Lim; e-mail: [email protected] or Tae-Hyun Kim; assembling properties. Macrocycles have superior pro- e-mail: [email protected] teolytic stability and often display enhanced biological Contract grant sponsor: Research Institute of Industry-Academic Cooperation Foundation Incheon National University Research Grant (2010) activity because of their increased affinity toward target VC 2013 Wiley Periodicals, Inc. receptors. Macrocycles can maintain more rigid and Biopolymers Volume 101 / Number 3 279 280 Choi et al. conformationally constrained structures than their linear RESULTS AND DISCUSSION counterparts. It has been shown that that macrocyclization of the N- and C-termini of an a-helical peptide with a rigid Design and Synthesis of Macrocyclic Peptides 14 crosslinker helps stabilize an otherwise unstable a-helix. The macrocyclic peptides designed for this study consist of a One notable example of the stabilization of an a-helical segment with high a-helix propensity, a PPII helix, and two peptide by macrocyclization is the peptide stapling linker segments that connect the two different helical segments 5,15 method. In this approach, side chains at the same face (Figure 1). The a-helix segment is derived from the HIV-1 Rev of an a-helix are crosslinked by covalent bonds, and the protein. A short stretch of amino acids within Rev, called the formation of this intramolecular covalent bridge can arginine-rich motif (ARM; approximately 14–17 amino acids), decrease the conformational entropy of the unfolded state. is known to mediate specific binding to the cognate Rev- This approach yields a stabilized a-helical peptide in response element (RRE) RNA in an a-helical conforma- monomeric form. In addition to this single molecule tion.25,26 Although Rev-ARM has a high propensity to form approach, there are multimeric approaches in which supra- a-helical structures, it is not structured under ambient condi- 16 molecular peptide assemblies are constructed. It has been tions, similarly to most short peptides.27 Because both helical reported that nanostructures with multiple stabilized a hel- segments are rigid and are contained in the constrained cyclic 17,18 ices can be fabricated by using macrocyclic peptides. structure, it was of interest to investigate how they mutually The formation of multiple stabilized helices was made pos- influence each other. Moreover, we envisioned that the rigid sible by the self-assembly-mediated coil-to-rod transition PPII helix might stabilize an otherwise unstructured Rev-ARM in the self-assembling b-sheet segment within the macrocy- segment by further constraining and thereby lowering the con- clic peptide, which constrains and subsequently stabilizes formational entropy of the unfolded state of Rev-ARM. the a-helices. Linear peptides were synthesized on Rink amide MBHA The polyproline helix is one of the common secondary resin. A cysteine protected with highly acid labile methoxytrityl structures found in natural proteins. Proline oligomers (Mmt) was placed at the C-terminus to enable an orthogonal tend to adopt the polyproline II (PPII) conformation in deprotection scheme. Because we aimed to obtain macrocyclic 19,20 aqueous solution. The PPII conformation is character- peptides with very large rings of 32–34 amino acid residues, ized by a left-handed helix with three residues per turn, the cyclization reaction was a challenging one (Figure 1A). As i.e., every third residue is stacked on top of one another. an initial attempt, we synthesized the a-helical segment first Amide bonds in the PPII conformation exist in a trans con- from the resin and then subsequently elongated the linker and formation. The PPII conformation has been recognized to polyproline segments. Then, bromoacetic acid was coupled to 21–23 form rigid rod-like structures in aqueous solutions. In the N-terminal portion of the peptide chain. To achieve a organic solvents, polyproline adopts the polyproline I pseudo-dilution effect, the cyclization reaction was performed (PPI) conformation, a right-handed helix with all amide while the protected peptide was still bound to the resin.28 Fol- bonds in a cis conformation. lowing selective deprotection of the Mmt group using 1% tri- Here, we report the synthesis of macrocyclic peptides con- fluoroacetic acid (TFA) while side chains for other amino sisting of both an a-helix and a polyproline segment and the acids were protected, an intramolecular SN2 reaction (S-alkyla- analysis of their conformational properties. Syntheses of mac- tion of a cysteine thiol for cyclic thioether formation) was rocycles from large peptides are often extremely difficult commenced by adding a base, N,N-diisopropylethylamine because of the entropic penalty associated with intramolecular (DIPEA), in N-methyl-2-pyrrolidone (NMP). A small portion cyclization. We found that rigid polyproline-containing pep- of the resin was sampled occasionally and treated with cleavage tides display an orientation-dependent structural effect during cocktail. Monitoring the progress of the reaction by mass spec- the head-to-tail cyclization reaction. Macrocyclic peptides con- trometry revealed that the cyclization reaction did not occur taining both an a-helix and a polyproline helix represent a under these conditions, even after several days of extensive unique class of cyclic molecules because two different types of reaction. peptide helices are contained within a single constrained cyclic The N- and C-termini must meet for effective intramolecu- scaffold. We analyzed whether the rigid PPII helix could assist lar cyclization to take place. We speculated that the long per- the stabilization of the potential a-helix segment and examined sistence length of the rigid polyproline rod might reduce the how these two different helices affect each other (Figure 1C). total possible degrees of freedom for reactive
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