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A Multicomponent Stapling Approach to Exocyclic Functionalized Helical Pep-tides: Adding Lipids, Sugar, PEGs, Labels and Handles to the Lactam Bridge Aldrin V. Vasco, Yanira Mendez, Andrea Porzel, Jochen Balbach, Ludger A. Wessjohann, and Daniel G. Rivera Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00906 • Publication Date (Web): 21 Dec 2018 Downloaded from http://pubs.acs.org on December 24, 2018

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Bioconjugate Chemistry

A Multicomponent Stapling Approach to Exocyclic Functionalized Helical Peptides: Adding Lipids, Sugars, PEGs, Labels and Handles to the Lactam Bridge Aldrin V. Vasco,† Yanira Méndez,†,‡ Andrea Porzel,† Jochen Balbach,§ Ludger A. Wessjohann†,* and Daniel G. Rivera†,‡,* †Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, D-06120,

Halle (Saale), Germany ‡Center

for Natural Products Research, Faculty of Chemistry, University of Havana, Zapata y G,

Havana 10400, Cuba §Institute

of Physics/Biophysics and Center for Structural and Dynamics of Proteins, Martin Luther

University Halle-Wittenberg, D-06120, Halle (Saale), Germany.

Corresponding Authors *E-mails: [email protected], [email protected]

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ABSTRACT: Peptide stapling is traditionally used to lock peptide conformations into α-helical structures using a variety of macrocyclization chemistries. In an endeavor to add a diversitygenerating tool to this repertoire, we introduce a multicomponent stapling approach enabling the simultaneous stabilization of helical secondary structures and the exocyclic N-functionalization of the side chain-tethering lactam bridge. This is accomplished by means of a novel solid-phase methodology comprising, for the first time, the on-resin Ugi reaction-based macrocyclization of peptide side chains bearing amino and carboxylic acid groups. The exocyclic diversity elements arise from the isocyanide component used in the Ugi multicomponent stapling protocol, which allows for the incorporation of relevant fragments such as lipids, sugars, polyethylene glycol, fluorescent labels and reactive handles. We prove the utility of such exocyclic reactive groups in the bioconjugation of a maleimide-armed lactam-bridged peptide to a carrier protein. The on-resin multicomponent stapling proved efficient for the installation of not only one, but also two consecutive lactam bridges having either identical or dissimilar N-functionalities. The easy access to helical peptides with a diverse set of exocyclic functionalities shows prospect for applications in peptide drug discovery and chemical biology. Graphical Abstract


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Introduction Multicomponent reactions (MCRs) have recently experienced a renaissance as powerful peptide cyclization tools enabling the generation of high levels of molecular diversity and complexity during the ring-closing step.1-3 Recent examples of MCRs employed in peptide macrocyclization are the Strecker reaction,4 the metal-catalyzed amine/alkyne/aldehyde coupling, 5 and a wide repertoire of isocyanide-based MCRs. These latter include the Passerini reaction,6 the Ugi-Smiles reaction7 and a novel MCR developed by Yudin and co-workers comprising the use of an amphoteric aziridinealdehyde component,8,9 among others.10 Moreover, the Ugi reaction11,12 was the first one to be employed in peptide cyclization13 and it is also the MCR providing the highest complexity and diversity-generating characters due to its four component nature.14,15 However, despite the recognized value of MCRs in macrocyclization strategies,1-15 a successful input of MCRs in stapling methodologies enabling the combined stabilization and diversity-oriented derivatization of helical peptides has remained elusive. The term peptide stapling was coined for the synthesis of all-hydrocarbon bridged peptides by ringclosing metathesis.16-18 Even before, the stabilization of helical peptide sequences by the introduction of lactam bridges proved to be effective.19-23 Since then, both macrocyclization techniques have furnished a variety of helical synthetic peptides with remarkable biological and medicinal applications.24-28 Currently, the stapling chemistry used for side chain-to-side chain tethering has greatly diversified.29,30 This includes the CuI-catalyzed alkyne-azide cycloaddition (click),31,32 Lys Nε- and Cys S-arylations,33-35 Cys alkylation,36,37 and Pd-catalyzed C-H activation.38,39 In addition, there is a special type of ‘two-component’ stapling approach,31 in which the side chain-bridging moiety is designed to bear an additional handle suitable for the modulating bioactivity, labeling or conjugation. Thus, Spring and co-workers have introduced a double-click, two-component stapling 3 ACS Paragon Plus Environment


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with functionalized dialkyne linkages.40-42 Similarly, Dawson and co-workers described an acetonelinked side-chain bridge capable of stabilizing a helical structure and providing the carbonyl functional group for further labeling and conjugation through oxime ligation.43 Herein we describe a general and versatile solid-phase methodology enabling the stabilization of peptide helical structures and the simultaneous functionalization of the lactam bridge with diverse exocyclic appendages. Never before has the helix inducing lactam-bridge strategy been combined with the synchronized installation of biologically relevant exocyclic functionalities such as lipid, polyethylene glycol (PEG), carbohydrate and fluorescent label. Of note, this is actually unfeasible by the classic lactamization approach leading to a lactam-bridge with a secondary amide. Thus, we aimed at proving a multicomponent macrocyclization that could do the double purpose of locking a helical peptide conformation and providing a varied set of exocyclic moieties at the lactam bridge, all in one step. Most intriguingly, we expected that some of the appended moieties may facilitate helix formation in water, e.g. by mimicking lipid surfaces, where simple lactam bridges fail to do so. Results and Discussion Over the past several years, our laboratories have employed isocyanide-based MCRs in peptide cyclization,6,7 with special emphasis on exploiting the scope of Ugi-type multicomponent reactions.44,45 This class of four component reaction is ideal for peptide modification because it can include the amino and carboxylic acid groups present in the peptide side chains and termini without any modification or activation.6,14,15 However, such Ugi cyclization methods only involved solutionphase procedures and short peptide sequences,

44,45

and they were not designed to access helical

secondary structures.44 Besides the current interest on helical peptides promoted us to focus on this type of secondary structures, we also realized that the scope of the solution-phase Ugi macrocyclization was very limited, as this reaction is not bioorthogonal like, e.g., click chemistry. In 4 ACS Paragon Plus Environment

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consequence, the peptide sequences should contain only one (Ugi reactive) amino and carboxylic acid group in deprotected form, thus restraining applications to some bioactive peptide sequences. Accordingly, to demonstrate the potential of Ugi multicomponent stapling in a more general sense, the development of a solid-phase protocol that enables the growth of longer peptides and the multicomponent cyclization/functionalization was required. So far, there are no previous reports of Ugi reaction-based peptide macrocyclization on solid-phase. On-resin isocyanide-based MCRs with resin-linked peptides are efficient46-48 – including cyclization48 – but only with reactive aliphatic or aromatic aldehydes. However, due to the poor stereoselectivity of the classic Ugi reaction,11 the use of paraformaldehyde is preferred when applied to macrocyclization chemistry, thus leaving the diversity-generating task to the other components.49,50 Unfortunately, imine formation with resin-linked peptides and paraformaldehyde has recurrently proven to be inefficient, thus limiting an effective translation from solution to solid-phase Ugi macrocyclizations. Recently, our group described aminocatalysis-mediated transimination protocols enabling the easy formation of paraformaldehyde-derived methylene-imines at the peptide Nterminus, and their subsequent participation in on-resin Ugi reactions.51,52 Thus, our endeavor was to translate that protocol to Lys side chains to allow the on-resin Ugi multicomponent stapling with the side chain tethering combinations Lys/Glu or Lys/Asp in the presence of isocyanides. Synthesis and structural analysis of helical peptides with diverse exocyclic N-functionalization A solid-phase method based on three dimensions of orthogonality was employed for the preparation of peptide 1 (see the Supplementary Information, SI). As depicted in Scheme 1, after Alloc/Allyl deprotection of the Lys and Glu side chains chosen to be cyclized, the imine formation at the Lys NH2 was undertaken by transimination for 30 min with fourfold excess of pyrrolidinium ion arising 5 ACS Paragon Plus Environment


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from the reaction of paraformaldehyde and pyrrolidine. This step is crucial to ensure the complete conversion of the free amine into the imine, as it is the washing of the resin prior to the addition of the isocyanide component to avoid basic conditions during the Ugi cyclization. TentaGel S RAM resin with low loading (0.24 mmol/g) was used, while a careful screening of solvents proved that the mixture DCM/trifluoroethanol (TFE) 1:1 (v/v) is the best option for this multicomponent macrocyclization step. Intriguingly, DCM/TFE showed better results than the mixtures THF/MeOH – used in the transamination step – and DCM/MeOH, both previously employed for on-resin Ugi reaction at the peptide N-terminus.51,52 We hypothesize that the helical-inducing effect of TFE facilitates the engagement of the reacting carboxylic (Glu) and imine groups (Lys) by placing them in spatial proximity (see wheel diagram in scheme 1). Mini-cleavages and HPLC/ESI-MS analyses after 12 h of reaction often showed complete consumption of the linear peptide. In cases of incomplete on-resin macrocyclization after 12h, a second cycle of imine formation and reaction with isocyanide for additional 12 h was required. Thus, the criterion for ending the process after either one or two cycles was the disappearance of the linear peptide in HPLC. The isolated yields reported in scheme 1 can be considered as good to excellent, as they correspond to the overall solid-phase process including all peptide couplings, deprotections and the final on-resin multicomponent macrocyclization, with the latter alone featuring low yields in standard solution-phase chemistry without pseudo-dilution. We consider that the overall efficiency of this method is comparable to that of standard solid-phase approaches using macrolactamization as stapling protocol. The rationale for this lies at the fact that similar protecting groups tactics are used, while the efficiency of the Ugi multicomponent ring closure is as high as that of a lactamization, although of slower kinetics. However, the value of the multicomponent method should be assessed in terms of the creation of a higher complexity at the lactam bridge. 6 ACS Paragon Plus Environment

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Scheme 1. Multicomponent stapling approach to lactam-bridged N-functionalized helical peptides. A) On-resin Ugi macrocyclization enabling the lactam bridge formation and N-functionalization with exocyclic appendages. B) Ugi-derived helical peptides with diversely N-functionalized lactam bridges. C) CD spectra of the stapled peptides in water and aqueous trifluoroethanol solution. In order to address the influence of the exocyclic N-functionality on the helical stability of the peptide, we initially chose an amino acid sequence with helical propensity, and bearing two Lys alternating with a Trp at i, i+4 positions in order to favor further helical stabilization by cation-π interactions. Despite this side chain support, linear peptide 1 (in deprotected form) does not yet possess a purely α-helical structure, as is evident from its CD spectrum (see the SI). As illustrated in Scheme 1, the lactam bridge N-functionality can have an influence on the helical type and stability of the stapled 7 ACS Paragon Plus Environment


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peptides. For example, lipidated peptides 2 and 3 – obtained using a lipidic isocyanide53 – show typical -helical CD spectra in aqueous solution (10 mM, phosphate buffer, pH 7.4), evidenced by the maximum at 191 nm and double minima at 207 and 222 nm. Alternatively, the CD spectrum of stapled peptide 4 – which bears a hydrophilic carboxylate as exocyclic appendage – in phosphate buffer shows a slight shift of the absorption minima to lower wavelengths compared to 2 and 3 (i.e., 222 nm218 nm, 207 nm202 nm). For peptide 4, a marked decrease in the intensity of the maximum and a slight shift to 187 nm is also observed. These features are commonly found in 310helical peptides, showing the change from an -helix when the lactam N-substituent is a lipid to a 310-helix when it is a carboxylate group. A more drastic change is observed in the CD spectra of stapled peptides 5 and 6, bearing peracetylated D-glucose and a PEG chain, respectively, as exocyclic N-substituents of the lactam bridge. For both compounds, there is a significant loss of helical content in aqueous solution compared to the stapled lipopeptides 2 and 3. To address the further capacity of stapled peptides 2-6 for helical stabilization, the CD spectra were measured in 50% aqueous TFE. As depicted in Scheme C, a notable improvement in the -helicity was found for the stapled peptides 4, 5 and 6, which are those bearing more polar exocyclic lactam N-functionalities. The rationale for the excellent -helicity of peptides 2 and 3, even in water, could be the reinforcement of their amphiphilic character due to the presence of the lipid tail in the face opposite to the cationic side chains. Based on these results, we were interested in addressing the influence of both the exocyclic lactam N-functionality and the amino acid sequence on the helical stability of this novel system. As depicted in Figure 1, the change of the two Lys by two Arg leading to stapled peptide 7 does not affect the -helical character of this type of amphiphilic structure. However, the replacement of Trp by Lys to produce peptide 8 – also having the exocyclic lipid tail in the face opposite to the three Lys 8 ACS Paragon Plus Environment

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– gives rise to a 310-helix CD spectrum in phosphate buffer. This latter might be due to the loss of cation-π interations and subsequent appearance of repulsive electrostatic interactions between the three cationic side chains of peptide 8. Similarly, the installation of a positive charge at the lactam Nsubstitution in both stapled peptides 9 and 10 also provokes a shift towards 310-helix. Analysis of the CD spectra of the four peptides in 50% aqueous TFE shows peptides 7, 8 and 9 displaying a marked -helicity, while peptide 10 shows a much lower -helical character.

Figure 1. A) Structures of Ugi-derived stapled peptides with lactam bridge N-functionalities of lipidic and cationic nature. B) CD spectra of stapled peptides 7-10. To get a deeper insight into the three-dimensional structure in water, we turned to study a model compound by means of NMR and molecular dynamics (MD) simulation. While the lipidic stapled peptides show the higher -helical character according to CD, their analysis by NMR proved difficult 9 ACS Paragon Plus Environment


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due to the presence of broad resonance signals in the 1H NMR spectra. As a result, we prepared peptide 11 having a shorter aliphatic chain, and therefore, a lower tendency to aggregation and micelle formation. As depicted in Figure 2, the CD spectrum of this peptide also shows a typical -helical behavior in phosphate buffer. Thus, 11 can serve as model compound to study the -helical conformation by NMR. Full assignment of the NMR resonances was achieved, confirming the presence of a major conformer in more than 90% (see the SI). Due to the tertiary nature of the Ugiderived amide at the lactam bridge, it was important to know whether the major conformer occurs in the s-cis or s-trans amide configuration. The NOE data unequivocally proved the presence of the scis rotamer at the bridge amide bond, with the consequent positioning of both the Glu and Lys side chains toward the same side. The 3JNHCH coupling constants of most residues were lower than 6 Hz, which is characteristic of -helices. However, the expected NOEs between i,i+3 and i,i+4 residues were not detected, probably due to low signal-to-noise ratio derived from the low concentration required to avoid peptide aggregation. Despite of this, the combined CD and NMR information enabled the building of an -helical model as starting point for studying the helical stability by MD simulation. Figure 2C illustrates the evolution in time of the -helical content calculated as the percentage of residues having their  and  angles in the -helical region. Snapshots at 50 ns and 96 ns are representative conformations populated during the MD simulation time, showing a wellconserved helical structure up to 50 ns and a tendency towards a partial unfolding at the C-terminus at later simulation times.


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Figure 2. A) Ugi-derived stapled peptide 11. B) CD spectrum of peptide 11 in phosphate buffer. C) Analysis of the -helical population by Molecular Dynamics simulation (program: YASARA, force field: AMBER 14, solvent: explicit water at pH 7.4, temp.: 298 K, simulation time: 100 ns). Synthesis of doubly functionalized bicyclic helical peptides After proving the capacity of the Ugi multicomponent stapling to improve formation of helical peptides and to introduce at the same time exocyclic fragments of biological (e.g., lipid, sugar), pharmacological (e.g., PEG) or diagnostic (e.g., fluorescent dyes) relevance, we looked at demonstrating that even more complex multicyclic peptides are available through this solid-phase methodology. In this sense, a traditional approach for the stabilization of longer helical structures is the installation of more than one lactam bridges along the sequence.26-28 Bicyclic helical peptides are commonly produced on solid-phase by implementing a first on-resin lactamization, followed by 11 ACS Paragon Plus Environment


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peptide growth and a second lactamization. As shown in Scheme 2, a solid-phase protocol comprising two subsequent on-resin Ugi multicomponent macrocyclizations was implemented to incorporate the exocyclic lactam N-substituents R1 and R2. Again, three degrees of orthogonality were required to enable the first on-resin Ugi macrocyclization at one Lys side chain, while having other amino groups protected as Fmoc (N-terminus) and Boc (Lys). The sequence was designed to construct the lactam bridges by a suitable positioning two Asp (Glu)/Lys pairs placed at i, i+4 residues. This concept provides a unique type of bicyclic peptides with either identical or different exocyclic Nfunctionalities. For example, peptide 12 features a bilipidated bicyclic structure resulting from the employment of lipidic isocyanides in both cyclization steps. Alternatively, peptides 13 and 14 were produced using the lipid/PEG and fluorescent label/sugar combinations, respectively, derived from using different isocyanides for the consecutive Ugi macrocyclizations.

Scheme 2. Solid-phase approach to bicyclic lactam-bridged peptides with exocyclic N-functionalities derived from sequential Ugi multicomponent macrocyclizations Analysis of the CD spectra of peptides 12-14 in phosphate buffer reinforces the idea that the amphiphilic nature of the Ugi-stapled peptide favors the -helical character. Thus, peptide 12, bearing 12 ACS Paragon Plus Environment

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three cationic Lys side chains at the counter-face of the lipidated lactam bridges, shows a strong helical character (Scheme 2), overriding a suspected coulomb repulsion as observed for peptide 8. On the other hand, the incorporation of a PEG chain (peptide 13) reduces slightly the -helical character. A similar bicyclic skeleton was assembled for peptide 14, but using Glu instead of Asp and incorporating a fluorescent tag and a sugar at each lactam bridge. The CD spectrum of bicyclic peptide 14 also shows -helicity, but again in a lesser extent than in peptide 12. The success of this consecutive stapling approach shows great promise for applications in the development of inhibitors of protein-protein interactions. For example, the exocyclic functionalities may be tailor-made to enhance target affinity, to enable imaging or simply to improve pharmacological properties. Bioconjugation of a lactam bridge-functionalized helical peptide To extend the potential of the multicomponent stapling concept even further, we sought to combine the stabilization an -helix with a reactive handle installed as exocyclic appendage for subsequent reactions. The previously introduced exocyclic functional handles like hydroxyl, amino or carboxylate groups do not allow for further derivatization, e.g., conjugation, without interference or competition of similar side chains. As a result, we sought to incorporate a bioorthogonal reactive group suitable for conjugation to other biomolecules. Thus, peptide 15 was prepared by means of the on-resin Ugi macrocyclization using a maleimide-functionalized isocyanide (see the SI). As depicted in scheme 3, this type of -helical structure (helical CD spectrum of 15 shown in Scheme 3C) can be readily conjugated to a thiol-containing protein like bovine serum albumin (BSA), which has a Cys at position 34. MALDI-MS analysis proves the formation of helical peptide-BSA conjugate 16 (see the SI). This result shows great prospect in chemical biology and immunology, as it may enable, for example, the Ugi multicomponent assembly of N-functionalized helical epitopes and their subsequent conjugation to carrier proteins or immunogenic peptides. Importantly, this type of conjugation of 13 ACS Paragon Plus Environment


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stapled peptides can be executed without affecting other amino acids, besides the Lys and Glu/Asp used to introduce the lactam bridge. Once more, it is worth noting that this is impossible to do with the ring-closing metathesis and lactamization approaches, which do not introduce additional reactive handles during the stapling process.

Scheme 3. Bioconjugation of a lactam-bridged helical peptide bearing an exocyclic reactive handle derived from an initial Ugi macrocyclization Conclusions We have developed a solid-phase multicomponent methodology that enables, for the first time, the simultaneous stabilization of peptide helical structures and a diversity-oriented functionalization of the resulting lactam bridge. This novel peptide stapling technique, herein referred to as multicomponent stapling, makes use of an on-resin Ugi reaction-based macrocyclization to lock the peptide conformation into a helical structure, and at the same time, it allows to introduce lipids, carbohydrates, PEGs, fluorescent labels and reactive handles as exocyclic functionalities. The αhelical character of the Ugi-derived stapled peptides was proven by CD analysis, while a deeper insight into their three-dimensional structure was achieved by means of NMR and MD simulation. The versatility of the Ugi reaction in the realm of peptide chemistry could be extended to solid-phase peptide stapling purposes. As a demonstration of the synthetic scope, we proved that on-resin Ugi macrocyclization can be used for peptide-protein bioorthogonal conjugation without affecting other 14 ACS Paragon Plus Environment

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peptide side chains. In addition, the methodology allows for the solid-phase construction of bicyclic, stapled peptides having two consecutive lactam bridges. The feasibility of the solid-phase protocol, including the efficient Ugi multicomponent macrocyclization, shows promise for applications in structure and chemical biology, immunology and drug discovery. Acknowledgments A.V.V. and Y.M. are grateful to DAAD for PhD fellowships. D.G.R is grateful to the Alexander von Humboldt Foundation for an Experienced Researcher fellowship. We thank Stefan Gröger for his support during NMR data acquisition and interpretation. Supporting Information Available. Experimental procedures, HPLC chromatograms, NMR, CD and HR-MS analyses of final compounds. References 1

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