Single Atom Ring Contraction of Peptide Macrocycles Using Cornforth Rearrangement

Site-selective transformations of densely functionalized scaffolds have been a topic of intense interest in chemical synthesis. Herein we have repurposed the rarely used Cornforth rearrangement as a tool to effect a single-atom ring contraction in cyclic peptide backbones. Investigations into the kinetics of the rearrangement were carried out to understand the impact of electronic factors, ring size, and linker type on the reaction efficiency. Conformational analysis was undertaken and showed how subtle differences in the peptide backbone result in substrate-dependent reaction profiles. This methodology can now be used to perform conformation-activity studies. The chemistry also offers an opportunity to install building blocks that are not compatible with traditional C-to-N iterative synthesis of macrocycle precursors.     

Tartric acid-based linker for the solid-phase synthesis of C-terminal peptide alpha-oxo aldehydes

A novel linker, based on the anchoring of (+)-dimethyl 2,3-O-isopropylidene-D-tartrate to PEGA or PEG-PS solid supports, was developed for the solid-phase synthesis of C-terminal peptide alpha-oxo aldehydes. Peptide elongation was performed using the 9-fluorenylmethoxycarbonyl/t-Bu chemistry. The peptide and the 1,2-diol were deprotected on the solid phase. Then, a periodic oxidation of the fully deprotected peptidyl-resin led to the simultaneous cleavage of the product from the solid support and to the generation of the alpha-oxo aldehyde moiety. The methodology allowed the distance between the alpha-oxo aldehyde and the peptide to be easily modulated. The C-terminal peptide alpha-oxo aldehydes synthesized in this study were found to be useful partners in hydrazone, thiazolidine, and oxime chemical ligations.     

Synthesis of Bifunctional Azobenzene Glycoconjugates for Cysteine‐Based Photosensitive Cross‐Linking with Bioactive Peptides

Azobenzene linker molecules can be utilized to control peptide/protein function when they are ligated to appropriately spaced amino acid side chains of the peptide. This is because the photochemical E/Z isomerization of the azobenzene N?N double bond allows to switch peptide conformation between folded and unfolded. In this context, we have introduced carbohydrate‐functionalized azobenzene derivatives in order to advance the biocompatible properties of azobenzene peptide linkers. Chloroacetamide‐functionalized and O‐allylated carbohydrate derivatives were synthesized and conjugated with azobenzene to achieve new bifunctional cross‐linkers, in order to allow ligation to cysteine side chains by nucleophilic substitution or thiol‐ene reaction, respectively. The photochromic properties of the new linker glycoconjugates were determined and first ligation reactions performed.     

The development and application of a novel safety-catch linker for BOC-based assembly of libraries of cyclic peptides.

Cyclic peptides are appealing targets in the drug-discovery process. Unfortunately, there currently exist no robust solid-phase strategies that allow the synthesis of large arrays of discrete cyclic peptides. Existing strategies are complicated, when synthesizing large libraries, by the extensive workup that is required to extract the cyclic product from the deprotection/cleavage mixture. To overcome this, we have developed a new safety-catch linker. The safety-catch concept described here involves the use of a protected catechol derivative in which one of the hydroxyls is masked with a benzyl group during peptide synthesis, thus making the linker deactivated to aminolysis. This masked derivative of the linker allows BOC solid-phase peptide assembly of the linear precursor. Prior to cyclization, the linker is activated and the linear peptide deprotected using conditions commonly employed (TFMSA), resulting in deprotected peptide attached to the activated form of the linker. Scavengers and deprotection adducts are removed by simple washing and filtration. Upon neutralization of the N-terminal amine, cyclization with concomitant cleavage from the resin yields the cyclic peptide in DMF solution. Workup is simple solvent removal. To exemplify this strategy, several cyclic peptides were synthesized targeted toward the somatostatin and integrin receptors. From this initial study and to show the strength of this method, we were able to synthesize a cyclic-peptide library containing over 400 members. This linker technology provides a new solid-phase avenue to access large arrays of cyclic peptides.     

Comparative investigation of the cleavage step in the synthesis of model peptide resins: implications for Nalpha-9-fluorenylmethyloxycarbonyl-solid phase peptide synthesis

Based on our studies of the stability of model peptide-resin linkage in acid media, we previously proposed a rule for resin selection and a final cleavage protocol applicable to the Nalpha-tert-butyloxycarbonyl (Boc)-peptide synthesis strategy. We found that incorrect choices resulted in decreases in the final synthesis yield, which is highly dependent on the peptide sequence, of as high as 30%. The present paper continues along this line of research but examines the Nalpha-9-fluorenylmethyloxycarbonyl (Fmoc)-synthesis strategy. The vasoactive peptide angiotensin II (AII, DRVYIHPF) and its [Gly8]-AII analogue were selected as model peptide resins. Variations in parameters such as the type of spacer group (linker) between the peptide backbone and the resin, as well as in the final acid cleavage protocol, were evaluated. The same methodology employed for the Boc strategy was used in order to establish rules for selection of the most appropriate linker-resin conjugate or of the peptide cleavage method, depending on the sequence to be assembled. The results obtained after treatment with four cleavage solutions and with four types of linker groups indicate that, irrespective of the circumstance, it is not possible to achieve complete removal of the peptide chains from the resin. Moreover, the Phe-attaching peptide at the C-terminal yielded far less cleavage (50-60%) than that observed with the Gly-bearing sequences at the same position (70-90%). Lastly, the fastest cleavage occurred with reagent K acid treatment and when the peptide was attached to the Wang resin.     

A One‐Pot Chemically Cleavable Bis‐Linker Tether Strategy for the Synthesis of Heterodimeric Peptides

Heterodimeric peptides linked by disulfide bonds are attractive drug targets. However, their chemical assembly can be tedious, time‐consuming, and low yielding. Inspired by the cellular synthesis of pro‐insulin in which the two constituent peptide chains are expressed as a single‐chain precursor separated by a connecting C‐peptide, we have developed a novel chemically cleavable bis‐linker tether which allows the convenient assembly of two peptide chains as a single “pro”‐peptide on the same solid support. Following the peptide cleavage and post‐synthetic modifications, this bis‐linker tether can be removed in one‐step by chemical means. This method was used to synthesize a drug delivery‐cargo conjugate, TAT‐PKCi peptide, and a two‐disulfide bridged heterodimeric peptide, thionin (7‐19)‐(24‐32R), a thionin analogue. To our knowledge, this is the first report of a one‐pot chemically cleavable bis‐linker strategy for the facile synthesis of cross‐bridged two‐chain peptides.     

A novel linker methodology for the synthesis of tailored conjugate vaccines composed of complex carbohydrate antigens and specific TH-cell peptide epitopes

Pathogenic organisms or oncogenically transformed cells often express complex carbohydrate structures at their cell surface, which are viable targets for active immunotherapy. We describe here a novel, immunologically neutral, linker methodology for the efficient preparation of highly defined vaccine conjugates that combine complex saccharide antigens with specific TH-cell peptide epitopes. This novel heterobifunctional approach was employed for the conjugation of a (1-->2)-beta-mannan trisaccharide from the pathogenic fungus Candida albicans as well as the carbohydrate portion of tumor-associated ganglioside GM2 to a TH-cell peptide epitope derived from the murine 60 kDa self heat-shock protein (hsp60). Moreover, the linkage chemistry has proven well suited for the synthesis of more complex target structures such as a biotinylated glycopeptide, a three component vaccine containing an immunostimulatory peptide epitope from interleukin-1 beta (IL-1 beta), and for the conjugation of complex carbohydrates to carrier proteins such as bovine serum albumin.     

Synthesis and evaluation of 2-(2-fluoro-4-hydroxymethyl-5-methoxy-phenoxy)acetic acid as a linker in solid-phase synthesis monitored by gel-phase (19)F NMR spectroscopy

Gel-phase (19)F NMR spectroscopy is a useful monitoring technique for solid-phase organic chemistry due to the high information content it delivers and swift acquisition times, using standard NMR spectrometers. This paper describes the synthesis of the novel linker 2-(2-fluoro-4-hydroxymethyl-5-methoxy-phenoxy)acetic acid in 29% yield over seven steps, using nucleophilic aromatic substitutions on 2,4,5-trifluorobenzonitrile as key steps. Following standard solid-phase synthesis a peptide could be cleaved from the linker using 20% TFA in CH(2)Cl(2) in 30 minutes, in contrast to a previously described monoalkoxy linker that requires 90% TFA in water at elevated temperature. A resin-bound peptide could be successfully glycosylated using only two equivalents of a thioglycoside donor, activated with N-iodosuccinimide and trifluoromethanesulfonic acid, and subsequent cleavage and deprotection gave the target glycopeptide. Direct glycosylation of the linker itself followed by mild acidic cleavage gave a fully protected hemiacetal for further chemical manipulation.     

A photoinducible beta-hairpin

A photochromic azobenzene linker was incorporated as a turn element into an amino acid sequence known to fold into a beta-hairpin structure in aqueous solution. Oligomer formation when the linker was in its thermodynamically favored trans form prohibited structure determination. Light-induced conformational change of the linker to the cis form led to the formation of monomers which exhibited a well-defined beta-hairpin structure as determined by (1)H NMR. The rate of the light-induced cis-to-trans isomerization of the azobenzene-containing peptide was 30% slower compared to the unsubstituted chromophore. These results suggest that suitably substituted azobenzenes can be used as photoinducible turn elements to investigate and control the folding and stability of beta-sheets.     

A Tandem In Situ Peptide Cyclization through Trifluoroacetic Acid Cleavage

We present a new approach for peptide cyclization during solid phase synthesis under highly acidic conditions. Our approach involves simultaneous in situ deprotection, cyclization and trifluoroacetic acid (TFA) cleavage of the peptide, which is achieved by forming an amide bond between a lysine side chain and a succinic acid linker at the peptide N‐terminus. The reaction proceeds via a highly active succinimide intermediate, which was isolated and characterized. The structure of a model cyclic peptide was solved by NMR spectroscopy. Theoretical calculations support the proposed mechanism of cyclization. Our new methodology is applicable for the formation of macrocycles in solid‐phase synthesis of peptides and organic molecules.     

Protein synthesis by solid-phase chemical ligation using a safety catch linker

The native chemical ligation reaction has been used extensively for the synthesis of the large polypeptides that correspond to folded proteins and domains. The efficiency of the synthesis of the target protein is highly dependent on the number of peptide segments in the synthesis. Assembly of proteins from multiple components requires repeated purification and lyophilization steps that give rise to considerable handling losses. In principle, performing the ligation reactions on a solid support would eliminate these inefficient steps and increase the yield of the protein assembly. A new strategy is described for the assembly of large polypeptides on a solid support that utilizes a highly stable safety catch acid-labile linker. This amide generating linker is compatible with a wide range of N-terminal protecting groups and ligation chemistries. The utility of the methodology is demonstrated by a three-segment synthesis of vMIP I, a chemokine that contains all 20 natural amino acids and has two disulfide bonds. The crude polypeptide product was recovered quantitatively from the solid support and purified in 20%-recovered yield. This strategy should facilitate the synthesis of large polypeptides and should find useful applications in the assembly of protein libraries.     

A Hot‐Segment‐Based Approach for the Design of Cross‐Amyloid Interaction Surface Mimics as Inhibitors of Amyloid Self‐Assembly

The design of inhibitors of protein–protein interactions mediating amyloid self‐assembly is a major challenge mainly due to the dynamic nature of the involved structures and interfaces. Interactions of amyloidogenic polypeptides with other proteins are important modulators of self‐assembly. Here we present a hot‐segment‐linking approach to design a series of mimics of the IAPP cross‐amyloid interaction surface with Aβ (ISMs) as nanomolar inhibitors of amyloidogenesis and cytotoxicity of Aβ, IAPP, or both polypeptides. The nature of the linker determines ISM structure and inhibitory function including both potency and target selectivity. Importantly, ISMs effectively suppress both self‐ and cross‐seeded IAPP self‐assembly. Our results provide a novel class of highly potent peptide leads for targeting protein aggregation in Alzheimer’s disease, type 2 diabetes, or both diseases and a chemical approach to inhibit amyloid self‐assembly and pathogenic interactions of other proteins as well.     

Effect of linker and spacer on the design of a fibronectin-mimetic peptide evaluated via cell studies and AFM adhesion forces

The design of a fibronectin-mimetic peptide that specifically binds to the alpha 5beta 1 integrin has been widely studied because of this integrin's participation in many physiological and pathological processes. A promising design for such a peptide includes both the primary binding site RGD and the synergy site PHSRN connected by a linker and extended off of a surface by a spacer. Our original hypothesis was that the degree of hydrophobicity/hydrophilicity between the two sequences (RGD and PHSRN) in fibronectin is an important parameter in designing a fibronectin-mimetic peptide (Mardilovich, A.; Kokkoli, E. Biomacromolecules 2004, 5, 950-957). A peptide-amphiphile, PR_b, that was previously designed in our laboratory employed a hydrophobic tail connected to the N terminus of a peptide headgroup that was composed of a spacer, the synergy site sequence, a linker mimicking both the distance and hydrophobicity/hydrophilicity present in the native protein fibronectin (thus presenting an overall "neutral" linker), and finally the primary binding sequence. Even though our previous work (Mardilovich, A.; Craig, J. A.; McCammon, M. Q.; Garg, A.; Kokkoli, E. Langmuir 2006, 22, 3259-3264) demonstrated that PR_b is a promising sequence compared to fibronectin, this is the first study that tests our hypothesis by comparing PR_b to other peptides with hydrophobic or hydrophilic linkers. Furthermore, different peptide-amphiphiles were designed that could be used to study the effect of building blocks systematically, such as the peptide headgroup linker length and hydrophobicity/hydrophilicity as well as the headgroup spacer length on integrin adhesion. Circular dichroism spectroscopy was first employed, and the collected spectra demonstrated that only one peptide-amphiphile exhibited a secondary structure. Their surface topography was evaluated by taking atomic force microscopy (AFM) images of Langmuir-Blodgett peptide-amphiphile membranes supported on mica. Their adhesion was first evaluated with AFM force measurements between the different sequences and an AFM tip functionalized with purified integrins. The amphiphiles were further characterized via 1-12 h cell studies that examined human umbilical vein endothelial cell adhesion and extracellular matrix fibronectin production. The AFM studies were in good agreement with the cell studies. Overall, the adhesion studies validated our hypothesis and demonstrated for the first time that a "neutral" linker, which more closely mimics the cell adhesion domain of fibronectin, supports higher levels of adhesion compared to other peptide designs with a hydrophobic or hydrophilic linker or even fibronectin. Neutral linker lengths that were within the distance found between PHSRN and RGD in fibronectin performed equally well. However, the 10 amino acid neutral linker gave slightly better cell adhesion than did the control fibronectin at all times. Also, a short spacer was shown to give higher adhesion than other sequences with no spacer or a longer spacer, suggesting that a short spacer is necessary to extend the sequence further away from the interface. In conclusion, this work outlines a logical approach that can be applied for the rational design of any protein-mimetic peptide with two binding sites.     

Development of a fluoride-responsive amide bond cleavage device that is potentially applicable to a traceable linker

A fluoride-responsive (FR) amino acid that induces amide bond cleavage upon the addition of a fluoride was developed, and it was applied to an FR traceable linker. By the use of an alkyne-containing peptide as a model of an alkynylated target protein of a bioactive compound, introduction of the FR traceable linker onto the peptide was achieved. Subsequent fluoride-induced cleavage of the linker followed by labeling of the released peptide derivative was also conducted to examine the potential applicability of the FR traceable linker to the enrichment and labeling of alkynylated target molecules.     

Secondary Amino Alcohols: Traceless Cleavable Linkers for Use in Affinity Capture and Release

Capture and release of peptides is often a critical operation in the pathway to discovering materials with novel functions. However, the best methods for efficient capture impede facile release. To overcome this challenge, we report linkers based on secondary amino alcohols for the release of peptides after capture. These amino alcohols are based on serine (seramox) or isoserine (isoseramox) and can be incorporated into peptides during solid‐phase peptide synthesis through reductive amination. Both linkers are quantitatively cleaved within minutes under NaIO₄ treatment. Cleavage of isoseramox produced a native peptide N‐terminus. This linker also showed broad substrate compatibility; incorporation into a synthetic peptide library resulted in the identification of all sequences by nanoLC‐MS/MS. The linkers are cell compatible; a cell‐penetrating peptide that contained this linker was efficiently captured and identified after uptake into cells. These findings suggest that such secondary amino alcohol based linkers might be suitable tools for peptide‐discovery platforms.     

Targeting an Interaction Between Two Disordered Domains by Using a Designed Peptide

Intrinsically disordered regions in proteins (IDRs) mediate many disease-related protein–protein interactions. However, the unfolded character and continuous conformational changes of IDRs make them difficult to target for therapeutic purposes. Here, we show that a designed peptide based on the disordered p53 linker domain can be used to target a partner IDR from the anti-apoptotic iASPP protein, promoting apoptosis of cancer cells. The p53 linker forms a hairpin-like structure with its two termini in close proximity. We designed a peptide derived from the disordered termini without the hairpin, designated as p53 LinkTer. The LinkTer peptide binds the disordered RT loop of iASPP with the same affinity as the parent p53 linker peptide, and inhibits the p53–iASPP interaction in vitro. The LinkTer peptide shows increased stability to proteolysis, penetrates cancer cells, causes nuclei shrinkage, and compromises the viability of cells. We conclude that a designed peptide comprising only the IDR from a peptide sequence can serve as an improved inhibitor since it binds its target protein without the need for pre-folding, paving the way for therapeutic targeting of IDRs.     

A thermally-cleavable linker for solid-phase synthesis

Oxabicyclo[2.2.1]norbornenes constitute a convenient and readily cleaved linker for solid-phase organic synthesis. A simple and inexpensive furfuryl-substituted resin has been shown to capture and release maleimide dienophiles under conditions compatible with intermediate synthetic steps. The synthesis of β-amino, -thiophenoxy, and -hydrazino alcohols by epoxide ring opening, and maleimide-functionalized Leu-enkephalin by standard peptide coupling techniques, are described to illustrate the utility of the solid-phase synthesis methodology.     

Site selection in tandem arrays of metal-binding domains

The metal binding properties of peptides corresponding to metal-binding sites spanning regions that normally function as linkers in tandem arrays of metal-binding domain-containing proteins were examined. For a peptide with two His residues from one TFIIIA-like zinc finger domain, a canonical TFIIIA-like linker, and two Cys residues from an adjacent zinc domain, the dissociation constant for the 1:1 peptide to cobalt(II) was found to be 15 +/- 10 microM, compared with 60 nM for the corresponding zinc finger domains themselves. Peptides overlapping two sets of metal-binding domains from human TRAF (tumor necrosis factor receptor-associated factor) proteins were examined. In one case, the affinity of the presumed metal-binding domain and that for the linker region were comparable, while in the second case, the affinity of the linker peptide was higher than that for the corresponding presumed metal-binding domain peptide. These studies revealed that cobalt(II) affinities in the micromolar range can occur even for peptides that do not correspond to natural zinc-binding domains and that the degree of distinction between authentic metal-binding domains and the corresponding linker-spanning peptides may be modest, at least for single domain peptide models.     

Dynamics of Zn(II) binding as a key feature in the formation of amyloid fibrils by Aβ11-28

Supramolecular assembly of peptides and proteins into amyloid fibrils is of multifold interest, going from materials science to physiopathology. The binding of metal ions to amyloidogenic peptides is associated with several amyloid diseases, and amyloids with incorporated metal ions are of interest in nanotechnology. Understanding the mechanisms of amyloid formation and the role of metal ions can improve strategies toward the prevention of this process and enable potential applications in nanotechnology. Here, studies on Zn(II) binding to the amyloidogenic peptide Aβ11-28 are reported. Zn(II) modulates the Aβ11-28 aggregation, in terms of kinetics and fibril structures. Structural studies suggest that Aβ11-28 binds Zn(II) by amino acid residues Glu11 and His14 and that Zn(II) is rapidly exchanged between peptides. Structural and aggregation data indicate that Zn(II) binding induces the formation of the dimeric Zn(II)(1)(Aβ11-28)(2) species, which is the building block of fibrillar aggregates and explains why Zn(II) binding accelerates Aβ11-28 aggregation. Moreover, transient Zn(II) binding, even briefly, was enough to promote fibril formation, but the final structure resembled that of apo-Aβ11-28 amyloids. Also, seeding experiments, i.e., the addition of fibrillar Zn(II)(1)(Aβ11-28)(2) to the apo-Aβ11-28 peptide, induced aggregation but not propagation of the Zn(II)(1)(Aβ11-28)(2)-type fibrils. This can be explained by the dynamic Zn(II) binding between soluble and aggregated Aβ11-28. As a consequence, dynamic Zn(II) binding has a strong impact on the aggregation behavior of the Aβ11-28 peptide and might be a relevant and so far little regarded parameter in other systems of metal ions and amyloidogenic peptides.     

Improved Methodology for the Synthesis of a Cathepsin B Cleavable Dipeptide Linker,Widely Used in Antibody-Drug Conjugate Research

Antibody-drug conjugates (ADCs) represent an emerging class of biopharmaceutical agents that deliver highly potent anticancer agents (payloads) selectively to tumors or components associated with the tumor microenvironment. The linker, responsible for the connection between the antibody and payload, is a crucial component of ADCs. In certain examples the linker is composed of a cleavable short peptide which imparts an additional aspect of selectivity. Especially prevalent is the cathepsin B cleavable Mc-Val-Cit-PABOH linker utilized in many pre-clinical ADC candidates, as well as the FDA approved ADC ADCETRIS^® (brentuximab vedotin). An alternative route for the synthesis of the cathepsin B cleavable Mc-Val-Cit-PABOH linker is reported herein that involved six steps from l-Citrulline and proceeded with a 50% overall yield. In this modified route, the spacer (a para-aminobenzyl alcohol moiety) was incorporated via HATU coupling followed by dipeptide formation. Importantly, this route avoided undesirable epimerization and proceeded with improved overall yield. Utilizing this methodology, a drug-linker construct incorporating a potent small-molecule inhibitor of tubulin polymerization (referred to as KGP05), was synthesized as a representative example.