synthetic cross-linking of peptides: molecular linchpins ... · abstract: peptide-derived drugs...

$: Synthetic Cross-linking of Peptides: Molecular Linchpins ... · Abstract: Peptide-derived drugs constitute a significant fraction of therapeutic agents. In 2013, The global market$
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Protein & Peptide Letters, 2018, 25, 1-25 1 REVIEW ARTICLE

0929-8665/18 $58.00+.00 © 2018 Bentham Science Publishers

Synthetic Cross-linking of Peptides: Molecular Linchpins for Peptide Cyclization

Ratmir Derda1,* and Mohammad R. Jafari1,2

1Department of Chemistry and Alberta Glycomics Centre, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada; 2MetaSci Inc., 1 Yonge St, 1801, Toronto, M5E 1W7, Canada

A R T I C L E H I S T O R Y

Received: December 20, 2017 Revised: September 26, 2018 Accepted: October 11, 2018 DOI: 10.2174/0929866525666181120090650

Abstract: Peptide-derived drugs constitute a significant fraction of therapeutic agents. In 2013, The global market of peptide therapeutics was ca. $19 billion; this value does not include revenue from insulin derivatives of $28 million. The combined sales of insulin and non-insulin peptide drugs is estimated to exceed $70 billion by 2019. A significant fraction of peptide-derived drugs is com-posed of an amino acid sequence and additional chemical functionalities that improve biological and pharmacological properties of the drug. In this review, we focus on synthetic cross-linkers that we refer to as “linchpins”, which are commonly used to constrain the secondary structure of pep-tides and equip them with added benefits such as resistance to proteolytic degradation and confor-mational stability. The latter property leads to an increase in binding potency and increased bioavailability due to increased permeation through biological membranes. Some linchpins can even introduce properties not found in natural peptides such as light-responsiveness. Peptides cy-clized by linchpins can be viewed as a sub-class of a larger family of peptide-derived drugs with desired pharmacological performance in vivo. To understand how chemical modifications by linch-pins improve drug discovery, this review also briefly summarizes canonical examples of chemical modification used in modern peptide therapeutics.

Keywords: Peptides, peptide macrocycles, side chain cross-linking, biocompatible chemistry, chemical modification of generi-cally-encoded libraries.

1. INTRODUCTION

Peptides and peptide-derived therapeutic drugs are de-fined as molecules with less than 50 amino acids. These compounds demarcate a unique chemical space that bridges small molecule therapeutics and biological therapeutics (i.e., antibodies, recombinant proteins and protein domains) [1, 2]. By 2014, there were 60 FDA approved peptide drugs on the market, 140 peptide drugs in clinical trials and more than 500 peptide drug candidates in pre-clinical trial [3, 4]. Within the four years spanning between 2014-2017, 14 new non-insulin peptide entities were approved by the FDA [5]. The sales for the top seven selling peptide drugs (i.e., Co-paxone®, Lupron®, Sandostatin®, Zoladex®, Victoza®, Forteo®, Byetta®) were more than $9.0 billion in 2011 [3] and exceeded $11 billion in 2015 [6]. In 2013, the combined sale of peptide drugs and insulin derivatives, was ca. $47 billion and $50 billion in 2015 [6]; in turn, the sales of pharmaceuticals world-wide exceeded $1 trillion in 2014. The market of peptide drugs is smaller when compared to the combined market of small molecule therapeutics and bio-logical therapeutics. For example, antibody-based therapeu-tics is a rapidly growing class of therapeutics constituting

*Address correspondence to this author at the Department of Chemistry and Alberta Glycomics Centre, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada; E-mail: [email protected]

10% of the newly approved drugs in the past 10 years; how-ever, limited tissue penetration is a fundamental drawback of antibodies. Peptide drugs can penetrate into tissues due to their smaller size [7]. In contrast to many small molecule drugs, peptides accumulate less in the tissues and often show higher specificity [8]. Suboptimal pharmacokinetic properties of the general peptide scaffolds are a major barrier to the use of peptides as drugs. The susceptibility of the peptides to peptidases in the gastrointestinal tract reduces their bioavailability signifi-cantly through oral administration. The hydrophilic nature of many peptides dramatically reduces their delivery to the sys-temic circulation through passive diffusion. Peptides that enter the systemic circulation are subject to renal and hepatic clearance as well as degradation by peptidases. As a result, the half-life of peptides administered directly in the circula-tion can be as short as a few minutes [9-10]. Such a short half-life may have evolutionary significance: many peptides act as messenger hormones, cytokines, neural transmitters and metabolic indicators. Rapid degradation of the messen-ger peptides ensures effective turn-off mechanism and high temporal fidelity of the message. However, when peptides are adapted as drugs, in many cases the life-time of the pep-tide must be extended to avoid frequent or continuous ad-ministration.

2 Protein & Peptide Letters, 2018, Vol. 25, No. 11 Derda and Jafari

Both physical and chemical solutions have been devised to increase the bioavailability and resistance of the peptides. Examples of physical modifications include coating the pep-tide drug with polymers to protect the peptide from gastric degradation and formulation of the peptide with protease inhibitors [11]. Physical protection and peptide formulation are beyond the scope of this review and is reviewed else-where [5, 6, 11, 12]. Examples of chemical modification of peptides that improve the pharmacokinetics and pharma-codynamics include conjugation of peptides with fatty acids to increase circulation half-life via interactions with serum albumin and pegylation to increase the hydrodynamic radius. Both modifications delay renal elimination [8, 13]. An ex-ample of the first type of modification can be found in Levemir®; chemically modified insulin developed by No-voNordisk, which is an FDA approved form of insulin myristoylated at a lysine residue. A seemingly minor modifi-cation – 17 carbons added to a polypeptide with 250 carbons – yields a dramatic increase in half-life from 2.8 h to 8.8 h [14]. This increase is even more dramatic in Victoza®, a 37 amino acid glucagon-like peptide-1 receptor (GLP-1) ago-nist, also developed by NovoNordisk, which is used for the management of Type 2 Diabetes. Palmitoylation of a lysine residue in this peptide increased the half-life of the subcuta-neously injected drug from 1 h to 11-15 h [15]. Examples of the second type of modification are two FDA approved drugs Pegasys®, pegylated interferon developed by Genentech, and Pegintron®, another variant of pegylated interferon distrib-uted by Schering. These technologies highlight the benefits of introducing non-peptide structures, which equip the pep-tides with beneficial pharmacokinetic properties, while pro-viding only minor interference with their biological function. Linchpins are an emerging class of chemical modifiers that can be defined as molecules that convert a linear peptide to a cyclic peptide. Many successful natural or synthetic pep-tide-based therapeutics have cyclic structure and this cycliza-tion provides several advantages: (i) proteolytic stability (ii)

restricted conformation mobility and (iii) increased propen-sity to permeate through bio-membranes. Cyclization via linchpins can be classified as topologically orthogonal to three canonical modes of cyclization of peptides (Figure 1): (i) head to tail cyclization (or macrolactamization) (ii) head-to-side chain or tail-to-side chain cyclization, and (iii) cross-linking of functional groups in side chains, such as the con-version of thiols of cysteines to disulfide bond(s). Figure 2 depicts the structure of three FDA approved synthetic drugs and three natural peptides highlighting these three types of cyclization. Detailed reviews of these cyclizations can be found elsewhere [16]. To delineate cyclization assisted by linchpins from the canonical approaches of macrocycliza-tion, we briefly review general cyclization approaches and introduce advantages offered by linchpin cyclization.

2. ENRICHMENT OF THE CONFORMATIONAL EN-SEMBLES THROUGH MACROCYCLIZATION

Preorganization of the structure of peptides by macro-cyclization can increase the binding affinity by decreasing the entropic penalty resulting from binding [17, 18]. A linear flexible molecule exists in multiple conformational states and the contribution of the conformer necessary to achieve the most proper fit for a potent interaction is often small (Figure 3A). Constraining the skeleton of the ligand can pre-organize its structure by maximizing the fraction of con-formers that exist in the structure optimal for interaction with the receptor (Figure 3B). Therefore, macrocyclization can result in a higher binding association constant (Ka) often by increasing the on-rate of binding. Furthermore, dynamic linchpins can change the conformation in response to exter-nal stimuli. Such conformational change in a small motif of a macrocycle can dramatically change its structure, favoring a defined set of conformers over the others. When these changes are designed or selected for, they can result in a dy-namic increase or decrease in Ka. (Figure 3C).

Figure 1. Linchpin based cyclization compared to other types of cyclization.

Synthetic Cross-linking of Peptides: Molecular Linchpins for Peptide Cyclization Protein & Peptide Letters, 2018, Vol. 25, No. 11 3

3. IMPROVING THE PHARMACOKINETICS OF PEPTIDES BY MACROCYCLIZATION

Macrocyclization of the backbone can facilitate forma-tion of internal hydrogen bonds which in turn are known to facilitate masking of hydrophilic areas and exposure of hy-drophobic side chains when passing thorough hydrophobic media such as the interior of a lipid bilayer [19]. In this man-ner, peptide cyclization can overcome difficulties in active or passive intestinal absorption and/or cell penetration. In con-trast, a large exposed polar surface area is detrimental to diffusion of a peptide into the cell [20]. Cyclosporine A is a well-known example of an orally bioavailable peptide that permeates the cell membrane by this mechanism. Cy-closporine shows the same conformation when in an aqueous solvent and as a co-crystal structure complexed with its re-ceptor cyclophilin. However, upon entering the hydrophobic environment (cell membrane) cyclosporine changes its con-formation to expose its N-methylated hydrophobic backbone to the solvent by making internal hydrogen bonds [18]. Cy-clization narrows down the conformational ensemble of a peptide to two classes of conformers: one is abundant in hy-drophilic environment and the other conformer is abundant in a hydrophobic environment. Lokey and co-workers gener-alized this observation and made similar N-methylated pep-tide scaffolds that exhibit bi-stability upon transfer from po-lar to non-polar environments [21]. The structures are cur-

rently evaluated by Circle Pharma for their potential to serve as orally bioavailable pharmaceuticals [22]. An alternative solution to the cell permeability problem uses macrocyclization of peptides with simultaneous intro-duction of extended hydrophobic moieties such as a hydro-carbon side chain to yield a so-called “stapled” peptide. Since the original report by Blackwell and Grubbs [23], and later by Verdine and co-workers [24], two decades ago, the production of cell-permeable amphipathic helical peptides via “stapling” has been adopted by several pharmaceutical companies. Aileron, for example, has completed a phase I clinical trial of the stapled peptide ALRN-6924 that binds to the p53 protein for the treatment of advanced solid tumors and Acute Myeloid Leukemia (Clinicaltrials.gov ID NCT02264613 and NCT02909972). The report discloses complete responses, partial responses and evidence of stable disease [25, 26]. Interestingly, cyclization can also improve performance of a charged class of cell-penetrating sequences such as poly-arginine (Rx, where x = 4 – 9). For reasons that are not yet completely understood, cyclic variants of poly-R-peptides exhibit higher cell penetration than their linear counterparts [27, 28]. A recent summary by Pei and co-workers [29] juxtaposes five validated cell permeable bi-cyclic peptides and other known cell permeable macrocycles. In the sections below, we will describe how grafting of linchpins introduces similar promising bioavailability fea-

Figure 2. Chemical structure of selected FDA approved cyclic peptide drugs.


tures into peptides while bypassing the challenges associated with macrolactamization or cyclization via cross-linking of unnatural side-chain residues.

3. APPLICATIONS OF MOLECULAR LINCHPINS FOR MACROCYCLIZATION

A linchpin links two or more reactive groups present in amino acid side chains. Connection of n+1 anchoring amino acid will result in the production of n cycles. Mono-[30, 31], bi-[32, 33] and tricyclic [34-36] peptides have been produced using linchpins with 2, 3 and 4 anchors, respectively. The advantages of using linchpins for cyclization are: (i) or-thogonal and biocompatible functionalities can be pro-grammed in the unnatural structure of the linchpins, thereby

bypassing the use of unnatural structures in the peptide chain; (ii) linchpins can introduce extra features into the pep-tide structure such as hydrophobicity [37, 38], light-responsiveness [39, 40], conformational stability [36, 41], etc.; and (iii) a single linchpin linker can be used for cycliza-tion of diverse peptide sequences. There are three main routes for cyclization of peptides by linchpins. (i) Chemical modification using native side chain functionalities of the natural amino acids. Nucleophiles in cysteine, lysine and tyrosine [42] have been widely used for such bio-conjugation. (ii) Pre-activation of the amino acid side chains to provide new orthogonal functionalities (e.g., glyoxal [43], dehydroalanine [44, 45]) followed by bio-orthogonal oxime bond ligation [46] or Michael addition

Figure 3. The effect of cyclization by linchpins on peptide conformation. A) A linear peptide exists in multiple conformation, most of which are not optimal for interactions with the receptor. B) Macrocyclization can increase the population of the conformers that exhibit the best fit for the receptor and decrease the fraction of conformers prone to undergo degradation. C) Macrocyclization with linchpins that respond to external stimuli, such as light, can enrich the fraction of a set of productive conformer in “stimulus on” (e.g., “light-on”) condition, and de-plete it in “stimulus off” (e.g., “light-off”) condition.


[47]. (iii) Incorporation of unnatural amino acids with or-thogonally reactive functionality [48, 49]. The use of linch-pins is particularly attractive in genetically encoded libraries of peptides, such as those displayed on phage or mRNA. Introduction of an unnatural linker that is reactive towards natural amino acids is a convenient strategy to constrain 109 – 1012 peptides at once. Multiple groups have already shown the linchpin-based cyclization of peptides displayed on the coat protein of bacteriophage M13 [30, 32], T7 [50], and mRNA [51, 52]. In addition to linchpins that convert linear peptides to cyclic through covalent bonds, one can envision and implement strategies that constrain peptide structure via non-covalent interactions, such as hydrogen bonding, [53] coordination by metal such as rhodium [54-56], palladium [57], copper [58], nicker [59] or iron [60, 61] (recent reviews see [62-64]). These strategies exist but will not be discussed in this review.

4. LINCHPINS THAT FORM AMIDE BONDS

Macrolactamization is commonly used for peptide cycli-zation, since the reactive precursors – amine and carboxylic acid – are readily available in the backbone of the peptide. Amine-reactive linchpins made of bis-succinimidyl esters with different lengths are commercially available or can be readily generated. In sequences that do not contain Lys as part of the recognition sequence, such approach is very con-venient. Lys however is a common residue and its use in cyclization is possible only in a narrow class of peptide se-quences. In sequences that contain Lys as part of the func-tional sequence of the peptide, regioselective cyclization mandates the use of orthogonal protecting groups on Lys residues. Despite the need for extra synthetic steps, amide-forming linchpins are popular due to the wide availability of orthogonally protected Lys building blocks and robustness of amide bond forming reactions. We note that non-regioselective cyclization in unprotected peptides can still be a productive avenue in peptide discovery of functional mac-rocyclic structures. For example, the pioneering work of Roberts and co-workers employed bis-succinimidyl esters cross linkers to derivatize genetically-encoded peptide librar-ies displayed on mRNA [51] and used these libraries for an effective discovery of macrocyclic ligands for G-protein coupled receptors [65]. Figure 4 describes several examples of linchpins that use either one-step incorporation or multiple rounds of protec-tion/de-protection and cyclization for stepwise unmasking of the reactive amines. Pei and co-workers [33] employed a multi-step approach to incorporate trimesic acid (TA) as a linchpin that converted one-bead-two-compound peptide library with three amine residues at fixed positions into a library of bicyclic peptides (Figure 4A). First, de-protection of allyloxycarbonyl (alloc)-protected 2,3-diaminopropionic acids (Dap) revealed the free amine used to attach the TA through a single amide bond. Then, FMOC-deprotection of ε-amine of lysine and N-terminal amine exposed two other functionalities that made the other two amide bonds with the anchored TA after exposure to a PyBOP activator. Screening of the bicyclic library against tumor necrosis factor-α (TNF-α) identified two bicyclic peptides C1 and C2 with Kd = 0.45 µM and Kd = 1.6 µM, respectively. C1 exhibited no binding to TNF-α in its monocyclic form and the linear form af-

forded a Kd > 10 µM. C1 also increased the LD50 of TNF-α from 0.46 ng/mL to 1.8 ng/mL, thereby potentially protect-ing cells from TNF-α-induced cell death. Inouye and co-workers scanned 12 variants of di-carboxylic acid linchpins to study the effect of the length of the linker on stabilization of the peptides crosslinked at i, i+4, i, i+7 and i, i+11 (Figure 4B depicts peptides with lysine residue at i, i+11) [66]. They found peptides cross-linked at i, i+11 with flurorenyl- and naphtyl- dicarboxylic acid exhib-ited the highest helicity (70% and 75% helicity, respectively) at 25 °C. In a complementary example, McDowell and co-workers used alkanediamine linchpins (NH2XnNH2, n = 3-5) to cross-link between glutamic acids located at i, i+7 (Figure 4C) [67]. A 1,5-pentanediyl linker increased the helicity of the linear peptide (Ac-TNEDLAARREQQ) from 20% to 100% as measured by circular dichroism (CD). Propanediyl and butandiyl linkers increased the helicity to 84% and 63%, respectively. The last strategy mandates simultaneous pre-activation of both side chain carboxylates, making this strat-egy unreliable. Raines and co-workers developed an esterifi-cation approach that bypasses this problem by using a dia-zonium compound with exclusive reactivity towards native carboxylate nucleophile in the presence of water, amines of Lys, thiols of Cys and phenolic hydroxyl groups of Tyr [68]. But, to date, this reaction has only been employed to modify Asp and Glu side in proteins with monovalent diazonium compound [69]; bis-diazonium compounds that cross-link two carboxyl side chain have not been reported.

5. LINCHPINS THAT UNDERGO SN2 NUCLEO-PHILIC SUBSTITUTION

Thioether bond formation is a fast and selective reaction between cysteines and halo-acetamide or halo-benzyls. Rate constants of these reactions are 1-20 M-1s-1, and these values can be influenced by amino acid residues flanking the Cys residue. The reaction is conveniently accelerated at neutral pH (7-8) because the thiolate anion (pKa of cysteine is 7-9) is >10,000 times more reactive as a nucleophile than a thiol [70]. The resulting thioether can mimic disulfide bonds and is not susceptible to undesired reduction and exchange. Re-placing disulfides with more stable thioether bonds while preserving the activity of the peptides has been addressed by many research groups over the past 20 years. It was thiol-reactive linchpins that simplified chemical post-translational modifications of peptide libraries and stimulated an explo-sive development of bis-electrophiles for cyclization of pep-tides. Due to space limitations, we cannot review all pub-lished examples of thiol-reactive linchpins to-date. Rather, we summarize key examples that have historical importance and highlight specific classes of topology or reactivity of linchpins. First linchpin-mediated cyclization appears in 1985 re-port of Mosberg and Omnas who introduced S-(CH2)n-S, n=2 or 3, into deprotected [d-Pen2,L-Pen5]Enkephalin, with free C-, N-terminus and side chain of tyrosine [71]. The protocol used by the authors for site-specific alkylation of peptide in the presence of other nucleophic residues can be traced to 1936 report of de Vigneaud and Patterson, who site-selectively modified cystine (Cys-S-S-Cys) with CH2Br2 to yield djenkolic acid Cys-S-CH2-S-Cys [72]. The peptide in


1985 report, or cystine in 1936 report were dissolved in liq-uid ammonia, deprotonated by adding chunks of metal so-dium “until a blue color indicating an excess of sodium” and “stapled” by adding Br-(CH2)n-Br. As both free NH2 and COOH residues are present in cystine, the 1936 report of De Vignaurd [72] could serve as a reference to many modern bioortogonal stapling approaches. De Vignaurd’s Na-NH3 conditions have some drawbacks, such as damage to aro-matic amino acids, epimerization and cleavage of bonds with Proline [73]. First diversity-oriented “linchpin scanning” can be found in the 1992 report of Rich and co-workers, who used cycliza-tion of an analogue of pepstatin (pepsin protease inhibitor) with series allylbromide and bromomethyl benzenes (BMB) to produce new pepsin inhibitors (Figure 5A) [74]. Unfortu-nately, the new cyclized peptides did not show improved inhibition when compared to the linear one. This observation shows that simple constraint of the peptide may not lead to enhanced binding to the target. The peptide has to be con-strained in an active conformation. Another challenge is that pepsin recognizes multiple conformations and such promis-cuous recognition might be the reason why enforced deple-tion of the conformational pool made no difference in bind-ing affinity. Timmerman and co-workers reintroduced BMB in 2005 to derivatize unprotected peptides in water. They used this and other linchpins to scan the activity of an overlapping set of a 12-residue fragment of follicle-stimulating hormone (FSH-β, Figure 5B). They showed that cyclization of the sequence CRVPGAAHHADSLC with m-DBMB, o-DBMB or p-DBMB resulted in higher affinity (Kd = 2-3 µM), while the disulfide-cyclized peptide exhibited Kd = 130 µM and the

linear peptide had a Kd > 1 mM [36]. In this case, the linkers replaced a disulfide bond at i, i+13, which stabilized the shape of the loop of a β-turn. They also found o-DBMB and p-DBMB cyclize faster, most likely because the first thioether formation activates the remaining BMB. DeGrado and co-workers later expanded this approach to 21 different alkylhalides including BMBs (Figure 5C) to staple a peptide at i, i+4 positions and identified the optimal linchpin for stabilization of the helicity of the peptide Ac-IPPKYCELLC [41]. m-DBMB cyclized peptide showed the highest helix stabilization based on circular dichroism (CD) spectrometry experiments. Varying the position of cysteines in the chain (while keeping them at i, i+4) also changed the helical content of the peptide, although the authors did not quantify helicity for these peptides [41]. The m-DBMB cy-clized peptide exhibited Ki = 11 µM for Kalpain-1 protein (a calcium-regulated cysteine protease) while the unmodified linear peptide showed lower inhibition (Ki <100 µM). These results show that the structure of the linchpin is not the only factor important in stabilization of the helix, but careful posi-tioning of the cysteines is required for optimal results. Lin and co-workers used a di-bromomethylbiphenyl (Bph) and di-bromomethylbipyridine (Bpy) to staple a pep-tide with cysteines at i, i+7 (Figure 5D). The linear peptide (LTFCHYWAQLCS, a dual inhibitor of p53-Mdm2/Mdmx interactions) showed IC50 = 500 nM. After cyclization with Bph and Bpy, the IC50 decreased to 22 ± 4.0 nM and 14 ± 2.0 nM, respectively [75]. In addition, modification of the linear peptide by cyclization with Bph resulted in a 6-fold increase in cell permeability; a 5-fold increase was observed after cyclization with Bpy. To promote an α-helix structure, the linchpin should connect the amino acids located on the

Figure 4. Cyclization of peptides with amide bond forming linchpins. A) Trimesic acid (TA) reported by Pei and co-workers [33] . B) Di-carboxylic acid linchpins reported by Inouye and co-workers [66]. Alkanediamine linchpins employed by McDowell and co-workers [67].


same side of the helix, at i, i+4, i, i+7 and i, i+11 positions (Figure 7) and have appropriate end-to-end distance. For example, DeGrado found no enhanced helix stabilization by using Bph to connect cysteines at i, i+4, while Lin showed that the same linker resulted in an active peptide when the connection was at cysteines i, i+7 (Figure 5D). In another example, DeGrado reported stabilization of an α-helix when m-DBMB connects cysteines at i, i+4 (Figure 5C), while the

same linker can stabilize a β-turn, with the connection at cysteines i, i+13 (Figure 5B). Overall, the interplay of posi-tioning and distance in helix stabilization is not trivial and helicity depend on the attachment point, end-to-end distance of the linker and the composition of the linker [76]. Most of the aforementioned examples utilize resonance-activated bis-haloalkanes as linchpins. Non-activated alkyl

Figure 5. Examples of linchpins based on alkyl-halide and benzyl halides. A) Bis-alkylation of cysteine side chains in liquid ammonia in the presence of sodium used for total synthesis of djenkolic acid by De Vignaurd and co-workers [72] and linchpin geometry scan by Rich and co-workers [74]. B) Alkylation of Cys in aqueous conditions by Timmerman and co-workers. C) Scanning of 21 different by Degrado and co-workers [41]. D) Linchpin scanning to identify peptides with improved cell-permeability by Lin and co-workers [75]. B) Water insoluble [40] and B) water soluble [151] light responsive linchpins developed by Woolley and co-workers.


halides are poor electrophiles, but given extended reaction times or increased temperature, they can also be introduced into peptides and proteins. Bednares and co-workers intro-duced an oxetane pharmacophore via alkylation of cysteines in peptides and proteins by 3,3-bis(bromomethyl)oxetane [77]. Recently Cramer and co-workers [78] and Metanis and co-workers [79] revisited introduction of the “worlds-smallest linchpin”—methylene thioacetal—by cross-linking two Cys in peptides and proteins with CH2I2 and TCEP in mild biocompatible conditions in water. Earlier reports for introduction -S-CH2-S- bridge exist, including the 1987 re-ports of Mosberg [80] using Na-NH3 conditions of De Vine-guard [72] and 1947 report of De Vineguard that introduced -S-CH2-S- bridge using unprotected cystein and formalde-hyde [81]. Departing from these conditions, Ueki and coworkers produced methylene-stapled enkephaline using tetrabutulamonium fluoride (TBAF) in anhydrous dichloro-methane for concurrent removal of dimethylphosphinothioyl (Mpt) group and alkylation by Cys by CH2Cl2 solvent [82]. Hallbergand co-workers introduced -S-CH2-S- bridge into unprotected angiotensin, by dissolving the peptide in 1:1 mixture of acetonitrile-dichloromethane in the presence of tetrabutulamonium fluoride and tributylphosphine [83]. While many examples focus on stabilization of α-helices, cysteine reactive linchpins can constrain other conformations such as β-turns or even unstructured loops in larger polypep-tides. Recently Kritzer and co-workers devised a LoopFinder software to identify bioactive loops in the Protein Data Bank (Figure 8) [84]. They then used a linchpin scanning approach to identify the linchpin structure that captures the loop in an active conformation and produce inhibitors for several pro-tein targets [85-87]. In the canonical linchpin-scanning approach, it is not always known which conformation should be trapped or even what sequence is a useful binder. Heinis, Winter and co-workers used tri(bromomethyl)benzene (TBMB) to cy-clize the peptide libraries at three cysteines to form libraries of bicyclic peptides with the structure of CX6CX6C displayed on phage (Figure 9A) [32]. Screening of the library against human plasma protease kallikrein identified peptides that could inhibit the protease activity with IC50 =1.7 – 39 nM, when only in the bicyclic form. Non-modified peptides showed at least 250-fold decrease in activity. Heinis and co-workers subsequently combined library screening with a linchpin scanning approach. They observed that in a library, different linchpins synergized with different sets of sequences, and productive ligands for the specific target contained a unique combination of peptide sequence and linchpins [88]. Cysteine-reactive linchpins can accommodate reactive moieties for downstream functionalization. Dawson and co-workers used dichloroacetone (DCA) to constrain the pep-tide and simultaneously install a ketone functionality. The latter can be used for bio-orthogonal labeling of the peptide or a second cyclization (Figure 9B) [89]. For example, the authors reacted cyclized peptides with aminooxy containing substrates-such as biotin, alexafluor 647 or FLAG epitope-in the presence of 100 mM aniline at pH 4.5. In most cases, the oxime ligation proceeded in satisfactory yields (>85%) after 16 h. The authors attempted to use the DCA linchpin to form

a bicyclic product through a reaction of an N-terminal ami-nooxy group in the peptide with the ketone. However, forma-tion of a bicyclic product was modest and yielded only 24% of isolated product after 24 h. They later combined DCA and a linchpin scanning approach to show that the peptide se-quence Ac-KETAAhCKFEhCQHMDS (where hC desig-nates homocysteine) is active after cyclization with the linchpin, whereas neither the analog sequences with natural cysteines, nor its DCA conjugate were active. Ng and Derda subsequently adapted DCA to introduce glycosylated linch-pins into the phage-displayed libraries of peptides [90]. All the linchpins mentioned above have static structures. Woolley and co-workers pioneered the use of light-responsive linchpins to alter the structure of helical peptides dynamically in response to light. A classic example used chloroacetamide functionalities to conjugate light-responsive azobenzene (Azb) linkers to peptides with cysteines at i, i+4, i, i+7 and i, i+11 (Figure 5E, 5F). These light responsive linchpins (LRL) are reviewed in detail below. SN2 substitution was recently employed to construct “re-versible” linchpins that are installed on unprotected peptides in mild conditions to enhance cell-permeability of the pep-tide. The linchpins were subsequently removed from the peptide by intracellular enzymes. Building on the fundamen-tal work by Deming and co-workers; who described the scope of reversible alkylation of methionine residues in pep-tides [91], Li and co-workers employed bis-electrophiles to cross link two methionine residues [92]. The reaction occurs in a mixed water-acetonitrile solution (7:3) supplied with 1% formic acid. Among a survey of 9 cross-linkers, 16 examples of cyclization (Figure 6A) and one example of bicyclization (Figure 6C) was reported. Of note, was the selective cross-linking of two methionines present in the peptide that con-tained unprotected Cys residues (Figure 6B). Similar selec-tive alkylation of Met in the presence of Cys was reported four years earlier by Kramer and Deming [91]. The differ-ence illustrates a higher reactivity of the sulfur lone pair in R-CH2-S-CH3 of Met when compared to R-CH2-S-H in Cys and/or a higher stability of a trisubstituted vs. disubstituted sulfonium-like transition state in this reaction. A mechanistic explanation of selective reactivity of Met vs. Cys in acidic pH may be fueled by experimental measurements of a stabi-lizing effect of methyl on sulfonium cations, combined with poor solvation of S-H bonds [93].

6. LINCHPINS THAT UNDERGO SNAR SUBSTITU-TION

High reactivity of perfluorobenzene towards hydroxide, amine and thiolate nucleophiles via SNAr mechanism was noted by Tatlow as early as 1959 [94]. A few decades later, reaction of Cys in proteins with 1-chloro-2,4-dinitro-benzene via SNAr reaction was used for modification of proteins [95]. Systematic implementation of this reactions for modification of peptides was empowered by a series of publications by Pentelute and co-workers who employed perfluoarenes to design effective bidentate electrophiles for cyclization of peptides via SNAr mechanism [38]. They first used hex-afluorobenzene (HFB) and decafluorobiphenyl (DFB) to staple helical peptides at i, i+4 (Figure 10A) [38].


Figure 6. Reversibly introduced linchpins via alkylation of methionine residues used by Lin and coworkers to produce A) cyclic and B) bi-cyclic peptides which can be reversibly opened by thiol residues [92].

Figure 7. Schematic description of the location of cysteines in a peptide at i, i+4, i, i+7, i, i+11 to form a stable α-helix after cyclization with linchpins.

Figure 8. Loopfinder coupled to linchpin scanning strategy pursued by Kritzer and co-workers [84].

Cross-linking the C-CA (peptide sequence ITFCDLLCYYGKKK, an HIV-1 capsid assembly polypro-tein binder) with HFB increased the helicity from 16% to 53%, while the same stapling with the longer linchpin DFB resulted in only a 36% increase. Both stapled peptides showed increased binding to C-CA as determined by surface plasmon resonance. They also qualitatively showed that a mixture of trypsin and chymotrypsin degraded the linear

peptide in 3 h, while no significant cleavage was observed for the HFB-stapled peptide and only partial degradation was observed for the DFB-stapled peptide. Pentelute and co-workers then tested five other variants of perfluoroarenes (fAr) on peptides with cysteines posi-tioned at i, i+1 to i, i+14 (Figure 10B) [31]. The stapling of cysteines at i, i+1, i, i+2, i, i+3 with DFB occurred in modest


yield (7 – 52%), while the more flexible fAr-linchpins ex-hibited somewhat higher yields (>59%) in reactions with peptides when stapling at i, i+1 through i, i+14. The authors hypothesized that the conversions observed in these reactions reflect compatibility between end-to-end distance of the linchpin and the Cys-to-Cys distance in the peptide. Since all reactions were performed in pure DMF, the relevance of these observations to peptide conformation in H2O is not clear. Due to the limited solubility of many SNAr reagents in water, these reactions mandate the use of either pure organic solvent or a mixture of water with a high percentage of or-ganic solvent [31, 38]. To overcome this limitation, Derda and co-workers performed SAR analysis of thiol-perfluoroa-rene reactions and identified decafluoro-biphenylsulfone (DFS) as an effective reagent for cyclization of Cys-containing peptides (Figure 10D) in water [96]. The reaction yielded quantitative conversion in 5–20 min in aqueous Tris buffer containing as low as 20% acetonitrile or 5% DMF. The rate of up to 180 M-1s-1 was modulated by the sequence of the peptide: positively charged amino acids such as Arg in the vicinity of cysteine increases the rate by increasing the basicity of the thiol and by stabilizing the carbanion interme-diate. DFS effectively cyclized peptide hormones such as oxytocin, urotensin II, salmon calcitonin, melanine concen-trating hormone, somatostatin and atrial natriuretic factor in which the number of amino acids between cysteines varied between 4–15. Although SNAr between Cys and DFS was significantly faster than the reaction between DFS and the amine of Lys or N-terminal amine [96], Pentelute and co-workers subsequently demonstrated that in peptides devoid

of Cys, DFS can perform N-arylation of Lys side chains [97]. One can envision arranging multiple SNAr reactive han-dles to yield linchpins with more than two points of attach-ment. One challenge with this approach is regiochemical control of multiple reactions. A simple and elegant solution to such a problem using 2,3,5,6-tetrafluoroterephthalonitrile (4F-2CN) as a linchpin with four attachment points recently developed by Chuanliu Wu and co-workers [98]. One of the critical insights from the authors was the use of peptides that contain two Cys and two penicillamine (Pen) residues to stage a four-step reaction into two distinct steps (i) reaction of two Cys-residues 2,5-fluorines of 4F-2CN and (ii) reac-tion of thiolate of Pen with the remaining 3,6-fluorines to yield only two out of six possible regioisomers. As an alternative to perfluorenyl SNAr linchpins, Pente-lute and Buchwald developed a palladium-aryl reagent to arylate the unprotected thiols of cysteines in peptides and proteins (Figure 10C). The rate of arylation reactions was comparable to the thiol-maleimide Michael addition at pH 7.5 (>103–104 M-1s-1), although the authors did not measure the kinetics explicitly. The use of 5% organic solvent (MeCN) was sufficient for an efficient SNAr substitution with labeling agents such as biotin and fluorescein; however, the authors had to use 50% of MeCN for cross-linking the cysteine with a bidentate linchpin to cyclize the peptide. The peptide sequence IKFTNCGLLCYESLR (10 µM) was sta-pled with 4,4’-dichlorobenzopheneone-palladium complex (20 µM) resulting in the formation of an arylated macrocycle in 10 min [99]. The same groups applied a similar approach

Figure 9. Selected example of alkyl-halide and benzyl-halide linchpins that convert linear peptides to bicyclic peptides. A) Formation by bicycles in the presence of tri(bromomethyl)benzene (TBMB) [32]. B) Dichloroacetone-mediated bicyclization of peptide with N-terminal amonooxy acetic acid developed by Dawson and co-workers [89]. Bicyclization of peptide made of natural amino acids assisted by C2-symmetric light responsive linker [160].


Figure 10. Linchpins that undergo SNAr reaction with cysteines to form cyclic peptides. via SNAr mechanism. A) hexafluorobenzene [38] and B) diverse perfluoroarenes for cyclization of peptides in DMF [31]. C) Decafluoro-biphenylsulfone-mediated cyclization of peptides in water [96]. D) 2,3,5,6-tetrafluoroterephthalonitrile (4F-2CN) combined with peptides that contain two cysteine and two penicillamine residues yields only two out of six possible regioisomers [98]. Arylation of peptides using 4,4’-dichlorobenzopheneone-palladium complex [99] and o-phenylene-bridged digold(III) complex [101]. Dichlorotetrazine introduces a s-tetrazine linchpin into peptides [108]. The resulting s-tetrazine-linchpin can be used for further functionalization of the peptide via an inverse electron demand Diels–Alder reaction.


to perform arylation of unprotected Lys residues. They em-ployed preformed or in situ generated [LPd(Ar)X] com-plexes in the presence of sodium phenoxide in DMSO to cross-link two lysines in peptides using 1,2-bis(4-bromophenoxy)ethane reagent [100] Spokoyny and cowork-ers recently expanded the scope of transition metal mediated Cys-arylation using organometallic Au(III) complexes. The o-phenylene-bridged digold(III) stapling reagent [((Me-DalPhos)AuCl)2(µ2-1,4-C6H4)]2+ prepared by the authors, introduced the biphenylene “staple” into the H2N-Cys-Asp-Ala-Ala-Cys-Asp-CONH2 peptide; the reaction between pep-tide (2.8 mM) and organometallic reagent (5.6 mM) was completed in 30 min in biocompatible conditions (1:1 aceto-nitrile / aqueous Tris-buffered solution, pH 8; room tempera-ture) [101]. Other metal-catalyzed Cys arylations [102-104] and metal free [105] or photocatalytic arylation [106] of Cys residues by diazonium salts exist, but their application to-wards cross-linking of two Cys residues have not been re-ported. A notable observation by Molander and co-workers is that Ni/photoredox arylation could use peptide disulfide as starting material and bypass the need for a reducing agent [103]. The use of disulfides in S-arylation is well-known in organic methodology (e.g., see recent review [107]), but ex-amples of simultaneous reduction and modification of pep-tide disulfides by one reagent are rare. One of the most versatile yet simple linchpins introduced by SNAr reaction, described by Brown and Smith, exploits the high reactivity of dichlorotetrazine with thiols to intro-duce a s-tetrazine linchpin into peptides [108]. The resulting s-tetrazine-linchpin undergoes photochemical ring-opening and it can be used for further functionalization of the peptide via an inverse electron demand Diels–Alder reaction.

7. LINCHPINS THAT UNDERGO MICHAEL REAC-TION

Maleimides can undergo Michael addition with thiols to afford a succinimidyl adduct [109-110]. Pingoud and co-workers used an azobenzene bis-maleimide to attach two cysteines at i, i+14 in a single chain variant of PvuII (scPvuII); a restriction enzyme containing a total of 157 amino acid residues (Figure 11A) [111]. The scPvuII con-sists of two identical halves that make it possible to include an azobenzene at each half, after substitution of Tyr49 and Asn62 with cysteines. They found that the doubly stapled cis-enzyme shows up to 16-fold higher activity compared to its trans variant. The cis-azobenzene can promote the forma-tion of a β-turn while the trans azobenzene distorts it. Maleimide-based linchpins are attractive because several types of bis-maleimide cross-linkers are commercially avail-able. However the resulting product can undergo retro-Michael reaction and the succinimide moiety is prone to hy-drolysis [112]. Another drawback is the simultaneous forma-tion of two stereocenters and up to four diastereomers. Unlike classical maleimide adducts, the Michael addition to allene derivatives does not form chiral centers. Derda and co-workers employed the allenamide addition strategy de-veloped by Loh and coworkers [113] to construct LR-linchpin 3,3'-bis(sulfonato)-4,4'-bis(buta-2,3-dienoylamido) azobenzene (BSBDA) that reacts orthogonally with cysteine residues through a Michael reaction [114]. They showed that

the reaction proceeds with a rate constant k = 30 M-1s-1 at room temperature to produce the cyclic product (Figure 11B). BSBDA also effectively cyclized peptides displayed on the coat protein of M13 phage yielding 98% conversion in only 20 minutes. Allenamides have been postulated to be more stable towards undergoing retro-Micheal addition and hydrolysis (personal communication with T. Loh), but the experimental evidence for the stability of such products is limited. The Michael addition can be employed for modification of natural peptides by converting the natural cysteine resi-dues to dehydroalanine (Dha) [44]. Again, a drawback of such an approach is the formation of two stereoisomers dur-ing the Michael addition of thiols to Dha. Webb and co-workers cleverly employed this drawback to generate all possible (eight) diastereomers of a bicyclic peptide in one reaction. They used 1,3,5-benzenetrimethanethiol as a C3V symmetric tridentate linchpin to cyclize a peptide through Michael addition to a dehydroalanine produced out of cys-teines in a linear peptide (Figure 13) [115]. Cyclization of the sequence ADhaSDRFRNDhaPADEALDhaG resulted in a mixture of stereoisomers of PK15, a bicyclic peptide re-ported by Heinis et al. that binds tightly to plasma kallikrein protein (Figure 13) [32]. Binding of the mixture of peptide stereoisomers (IC50 = 238 nM) was not higher than the origi-nal PK15 (IC50 = 2.7 nM) in which all of the cysteine side chains consisted of L amino acids. Synthesis of each indi-vidual peptide isomer, showed no binding improvement (IC50 = 240 nM for the most potent binder) for any other diastereomers of PK15, but the LLL-isomer. One of the most elegant examples of linchpins that em-ploys a tandem Michael additions is the “PEG-mono sul-fone” reagent popularized by Brocchini and co-workers [116]. Its origin can be traced to 1966-1971 reports by Lawton and co-workers that pioneered the use of reagents for sequential alkylation through consecutive Michael additions (see Brocchini, Eberle and Lawton [117] and references within). The core functionality of the linchpin contains a leaving group, such as sulfone, in the alpha position to the conjugated system, such as methylacrylophenone. The first conjugate addition of thiol triggers an elimination of the sul-fone leaving group and formation of the second methylacry-lophenone moiety, followed by an addition of the second thiol. While “PEG-mono sulfone” is not symmetric, the formed linkage is C2-symmetric. In addition, modern ver-sions of cross-linking thiol residues in proteins, reported first in 1979 [118], introduces value-added moieties such as pegy-lation.

8. LINCHPINS THAT UNDERGO AZIDE-ALKYNE [3+2] CYCLOADDITIONS

The copper catalyzed [3+2] cycloaddition of azide and alkyne (CuAAC) is a popular bio-conjugation method be-cause it is orthogonal to all unprotected side chains of natural amino acids [119]. After its introduction by Sharpless and Meldal [120] in 2002, many research groups have used this method to modify peptides, proteins, multi-protein com-plexes and cells [121]. Azidoalanines, azidohomoalanine, or azidoornithine and propargyl glycines can be used in CuAAC mediated cross-linking of synthetic peptides. The


same amino acids can be also incorporated in ribosomally synthesized proteins through metabolic labeling [122]. Bong and co-workers used the CuAAC reaction to con-jugate di-alkyne linkers to peptides containing azidoalanine as orthogonal handles (Figure 12A) [123]. Placing the azides at i, i+4 resulted in helical stabilization by the linker hexa-1,5-diyne. Helical stabilization did not occur with three other rigid structures: ortho-diethynylbenzoic acid, meta-diethynylbenzoic and dipropargyl glycine linker. Spring and co-workers used poly-arginine substituted dialkynyl ben-

zenes to synthesize cyclic analogs of p53 protein, termed SP1-SP5; peptides contained azidoornithine in positions i, i+7 (Figure 12B) [48]. All of the stapled peptides showed higher binding affinity to the MDM protein (Kd = 6.7–44.3 nM) when compared to the non-stapled precursor (Kd = 483 nM); however, only SP5 with three Arg substitution could penetrate the cell. Peptides containing less than 3 arginine residues were ineffective in penetrating the cell. In vitro as-says showed that binding of SP5 to MDM resulted in a 7-fold increase in p53 activation. Thurber and co-workers used similar approach to cross-link azidohomoalanines incorpo-

Figure 11. Linchpins that undergo Michael addition to form cyclic peptides. A) maleimide-based light-responsive linchpin with two maleimide residues [111] and (B) two allenamide residues [114].

Figure 12. Linchpins that undergo CuAAC to form cyclic peptides; A) Di-alkyne linchpin introduced by Bong and co-workers [123]. B) Functional di-alkyne linchpin employed by Spring and co-workers [48]. C) Stain-promoted linchpin.


rated into glucagon-like peptide-1 receptor (GLP-1R) ligand exendin. The linker contained two propargyl groups and Al-exaFluor 680 or Cy7 dyes; its incorporation into exendin had measurable impact on stability of peptide after subcutaneous injection [124]. Other examples of double-CuAAC reactions have been recently reviewed by Spring and co-workers [125]. A copper-free, strain-promoted version of azide-alkyne cycloaddition was first reported by Wittig and Krebs in 1961 [126] and then repurposed for derivatization of biomolecules by Bertozzie and Prescher in 2004 [127]. Interestingly, a putative linchpin that contains two strained alkynes—the syn-dibenzo-1,5-cyclooctadiene-3,7-diyne (DIBOD)—had been synthesized in 2002 [128]. In 2015, Spring and co-workers employed DIBOD as a linchpin to cross-link a li-brary of p53-derived peptide segments that contained two amino acids with alkylazide size chains [129]. The unique self-accelerating reactivity of DIBOD is particularly valuable for cyclization: it exhibits slow addition to the first alkyne (k1 = 0.06 M-1s-1) followed by significantly faster addition to the second alkyne (k1 = 34 M-1s-1), as measured by Sutton and Popik [130].

9. LINCHPINS THAT USE ALDEHYDE-REACTIVE GROUPS

Horne and co-workers employed a linchpin based on reversible covalent oxime and hydrazide bonds to cyclize peptides (Figure 14). They incorporated aldehyde reactive unnatural amino acids, containing hydroxylamine and hy-drazide at i, i+4 and i, i+11 of a 17-mer peptide. They found that m-phthalaldehyde can stabilize the helix when conju-gated through an oxime bond at i, i+4 position. In contrast, stapling with three other aldehydes (p-phthalaldehyde, o-phthalaldehyde and 4,4’-biphenyldicarbaldehyde) afforded less stabilization. Conjugation through a hydrazide bond resulted in less helicity comparing to the oxime product [49]. These results are similar to the study by DeGrado which showed that only m-DBMB can stabilizes the α-helix struc-ture, while o-DBMB and p-DBMB do not show such stabilization.

The same group, in a separate publication, demonstrated that stapling the sequence Ac-WEEWDZEINZYTK LIHKLIRE (where Z is an unnatural aminooxy carrying amino acid) with 1,4-phthalaldehyde can be promoted in the presence of its receptor (HIV gp41-5 protein), suggesting that the receptor can act as a template to enhance macro-cyclization (Figure 14B) [131]. Unfortunately, this stapled peptide did not show increased helicity or improved affinity towards the protein. The binding affinity increased (4.5 fold) only after stapling with 2,5-thiophenedicarboxaldehyde, which indicates the effect of the structure of a linchpin on the binding. The cyclization did not increase the helical content of the peptide. This work, however, suggests the potential of using the target receptor as a template for cyclization and imprinting of a productive conformer. To our knowledge these are the only examples of linchpins based on dynamic reversible covalent bonds. Future elaboration of these type of approaches could potentially be used to empower de novo discovery of molecular interactions similar to technologies such as dynamic combinatorial libraries and molecular im-printing [132]. The cross-linkers that react with identical functional groups in peptides are C2V or C3V symmetric. Asymmetric linchpins for cyclization of peptides require two or more sequential reactions in orthogonal conditions. The Fasan group developed a strategy to cyclize peptides fused to N-terminal cysteine of intein GyrA, using two orthogonal reac-tive groups (Figure 15). Amber suppression was used to ge-netically encode para-acetylphenylalanine (pAcF) in the peptide sequence which provides the ketone to react with the hydroxylamine end of the linchpin. Upon oxime bond forma-tion between the peptide and the linker, splicing of the intein completes the cyclization (Figure 15). They showed disrup-tion of the interaction between p53 and HDM2 by a 12-mer peptide that was cyclized at i, i+11 position [133].

10. DYNAMIC CONTROL OF PEPTIDE CONFOR-MATION BY LIGHT-RESPONSIVE LINCHPINS

Light-responsive linchpins (LRL) mentioned in several sections above are a clear example of new functionality that

Figure 13. C3V symmetric linchpins for generation of bicyclic peptides. SN2 substitution of three thiols with bromobenzyls results in a single stereoisomer, while Michael addition of three thiols to Dha produces eight stereoisomers.


a linchpin can add into the peptide backbone. LRL-modified peptides offer a possibility to control many receptor ligand interactions dynamically with spatial and temporal resolu-tion, [134-136] and empower fields of chemical optogenetics and opto-pharmacology. LR-linchpins usually consist of substituted LR-“cores” that change conformation in response to irradiation with light. The conformational change (switch-ing) can be a result of cis/trans isomerization (e.g., azoben-zenes) [137], or formation/opening of a covalent bond (e.g., diarylethenes [138-140], spiropyranes [141, 142]). Azoben-zenes and diarylethenes are C2V symmetric and are suitable for cross-linking of two reactive groups in peptides. Asym-metric LR-linchpins with two orthogonally reactive groups are also known. While it is not difficult to take any other previously described C2V linchpins and marry them with a light-responsive core, the electronic properties of substitu-ents of the core often influence its photo-switching proper-ties (e.g., switching wavelength, thermal relaxation half-life, photostationary state, etc.). These properties have to be taken into account when designing the reactive handles for the cyclization. Reactive groups can be placed more than two bonds apart from the photo-switching core to minimize the disturbance of photochemical properties. Excessive length of linker that connects peptide and LRL, however, can dampen the transmission of structural changes from linchpin to the

peptide. Some elegant solutions yield desired electronic properties after the reaction. For example, Hopmann and co-workers incorporated a fluorinated Azb-amino acid in calmodulin (CaM) protein. This moiety can undergo an in-tramolecular SNAr reaction with a cysteine inserted in a mu-tant of the CaM. Such reaction, changed the maximum ab-sorption of the Azb from 326 nm to 340 nm and improved the performance of the photo-switch (Figure 16) [143]. Although photo-switching ligands have been known since the 1950s, the modern design of light-responsive mac-rocycles started with the pioneering report of Woolley and co-workers demonstrating the first use of an azobenzene linchpin [40]. The sequence Ac-EACARVAibAACEA AARQ (where Aib is aminobutyric acid) modified by this linchpin at i, i+7 through alkylation of thiols, increased the helical content at 11 °C from 25% to 48% after light-induced isomerization to the cis form. The helical content decreased to 12% at dark photostationary state. Notable subsequent examples by Woolley and Allemann are LRL-stapled HDH3 (a mutant of engrailed homeodomain binding peptide) with trans-BSBCA linker to increase its binding to DNA from Kd = 200 nM to Kd = 7.5 nM. After irradiation, the same cyclic peptide shows lower binding affinity (Kd = 140 nM) [144]. Using BSBCA, LR-peptide ligands have been synthesized

Figure 14. A) Oxime bond forming linchpins. B) Templated cyclization of peptides via reversible-covalent bonds.


for targets such as streptavidin, [30, 145] PDZ domain of human tyrosine-phosphatase [146], β-adaptin [147], and RNA [148]. A detailed overview of the design of LR-linchpins is be-yond the scope of this review and for an in depth review, we recommend reference [134]. Efforts that went into the design of LRL are significant. For example it was important to vary the span of LR-linchpins to identify optimal length and sub-stituents that yield the most significant change upon irradia-tion [149-151]. Scanning of various thiol-reactive groups was critical to avoid unwanted reactions with amines [151-154]. SAR studies taught the interplay of substitution posi-tion (ortho, meta, para) on switching properties and guided the design of LR-linchpins that have both optimal reactive groups and desired switching properties. The effect of amines, alkyls, amides and other substituents on the thermal relaxation half-life (0.25 ms – 43 h) [155, 156] and switching wavelengths (320 nm – 670 nm) [157, 158], is now relatively well-understood. This knowledge allowed the Woolley group and other groups to design Azb linchpins that respond to red light (635 nm) [159]. Isomerization by red light can provide a platform for photo-switchable peptide drugs, because red light exhibits a higher degree of penetration through the skin to the tissues. Although the stability of Azb in vivo is lim-ited, the fundamental knowledge acquired in the Azb-LRL field will guide future efforts in photopharmacology and

chemical optogenetics. Of note, most LR-linchpins have two attachment points to yield a mono-cyclic LR-peptide; C2-symmetric linchpins with three reactive groups that yield light-responsive bicyclic peptides have been reported re-cently (Figure 9C) [160]. Derda and co-workers [30], and then Heinis and co-workers [145] employed BSBCA linchpins to modify ge-netically encoded (GE) libraries of peptides and identify light-responsive macrocycles that bind to streptavidin only in the presence (or absence) of light. Both groups demonstrated that the LR-GE libraries of phage can be generated by con-jugating BSBCA to billions of peptides. The ligands that are identified from these LR-libraries show different binding to streptavidin in the presence and absence of light. Differences in Kd between cis and trans isomers of the ligand reached only 4-5 fold changes due to, presumably, limited yield of isomerization of the Azb scaffold. Still, these examples sug-gested that screening of a GE-library of peptides with built-in light-responsive linchpins could serve as one method for lead identification in photopharmacology.

11. OTHER CLASSES OF LINCHPINS

Baker, Caddick and co-workers pioneered the use of di-bromomaleimide [161] and dithiomaleimide [162] to pro-duce maleimide-based linchpins (Figure 18A). While dibro-momaleimide can react with both thiol and phosphine-based

Figure 15. Non-symmetrical linchpins developed by Fasan and co-workers to cyclize a peptide after intein splicing.

Figure 16. Conjugation of azobenzene linchpin induces changed in photo-switching response.


reducing agents, dithiomaleimide does not react with phosphines. Cleavage of this linchpin by reducing agents such as phosphine or thiols can be used as an advantage. If the cleavage is not desired, Baker and Caddick groups re-ported that the aryl maleimide linchpin can be hydrolyzed in controlled conditions to open the maleimide and yield an acyclic monoarylamide of maleic acids which is stable in thiol-rich media (Figure 18A) [163]. Radical reactions have also been employed to introduce linchpins into peptides. Chou and coworkers repurposed a thiol–ene reaction, promoted by irradiation by 365 nm light, to introduce high-value long alkyl chain linchpins into pep-tides [164] As in many other aforementioned reactions, the authors had to use pure organic solvents as the medium of their reaction because linear hydrocarbons are only modestly soluble in water (12 mg/L or 0.12 mM for hexane) [165]. In their subsequent report, they translated the reaction condi-tions to an aqueous acetate buffer (pH 4) supplemented with 6 M guanidinium hydrochloride [166]. To enable conjuga-tion in aqueous conditions, the authors made three changes: (i) introduction of urea as a solubilization moiety into the linker; (ii) replace the insoluble radical initiator with water soluble 2,2'-Azobis[2-(2-imidazolin-2-yl)propane] Dihydro-chloride; and (iii) identified a water soluble reducing agent (TCEP) which did not interfere with the reaction and did not promote a desulfurization of Cys residues in the peptide. The desulfurization of Cys residues is a common side-reaction in thiol-ene reactions. The authors achieved 90% conversion after 15 minutes of irradiation of a solution that contained 5 mM peptide and 15 mM linchpin, along with proper addi-tives (Figure 18B). The authors even used a linchpin-modified peptide as an inception for the next linchpin-based cyclization producing a bicycle peptide with a bridgehead linchpin (Figure 18C).

Today, there exists a wealth of reactions that selectively functionalize the aromatic chains (reviewed in [167]). In theory, any reactive group that has a reported high reaction rate and selectivity towards an aromatic side chain residue should be amenable to production of a C2-symmetric linch-pin that cross-links these side chains. Still, only a few reports incorporate the reactive groups into bidentate functionalities and show cross-linking of two aromatic residues explicitly. One example of explicit cross-linking of aromatic residues by an external linchpin is by Johannes and coworkers (Figure 19A): heating 4-bromobenzaldehyde and Camphor-10-sulfonic acid catalyst with a peptide in a sealed tube, intro-duces a linchpin that bridges two Trp residues in peptides (9 examples) [168]. A report by Albericio, Lavilla and co-workers had all functional attributes of linchpin-like cycliza-tions [169]: the authors reported that the 1,4-diiodobenzene linchpin reacts with two Trp residues, albeit the explicit demonstration was cross-linking of two cyclic peptides, each containing one Trp residue, rather than cyclization of one peptide with two Trp residues (Figure 19B).

12. LINCHPIN-BASED CYCLIZATION OF PEP-TIDES: THE BOUNDARIES OF THE DEFINITION

While most examples in this review describe side-chain-to-side-chain cyclizations, a linchpin assisted terminus-to-side-chain and head-to-tail cyclization (Figure 1) are also possible. The example of the former is a reversible macro-cyclization of peptides with linchpin derived from Meldrums acid reported by Anslyn and co-workers (Figure 19E) [170]. Yudin and co-workers reported multiple examples of head-to-tail cyclization of peptides using isocyanides and az-iridine-aldehydes. These reagents engage N- and C-termini of protected peptides in a disrupted Ugi 4-component reac-tion (Ugi-4CR) that proceeds via an imidoanhydride inter-mediate to assemble into a cyclic peptide with aziridine

Figure 17. Azobenzene-based linchpins.


linchpin [171]. Yudin and coworkers, also reported an aza-Wittig variant of an Ugi-4CR head-to-tail cyclization of pep-tide with externally added (N-isocyanimino)phosphorane and aldehydes. The two components and termini of the protected linear peptide assemble into an oxadiazole linchpin which was shown to increase passive membrane permeability (Fig-ure 19C) [172]. Based on the latter two examples, other multicomponent reactions that employ peptide-borne functionalities and ex-ternally added component(s) fall under the definition of linchpin-based cyclization. The pictorial definition of linch-pin-mediated cyclization in Figure 1, thus, can be formalized as: “the transformation of a linear peptide that links side chains, termini, or a combination of thereof, giving rise to a cyclic product that incorporates the elements of the exter-nally supplied reagents”. Recent examples that fall under this definition include a Copper-catalyzed Mannich addition (“A3 macrocyclization”) employed by Lubelle and co-workers (Figure 19D) [173], as well as the Ugi and Passerini reactions employed for peptide cyclization by the Riviera group as described in a recent Account [174]. A report by Malins et al., describing a one-pot Strecker reaction or re-ductive amination of cyclic imines formed from peptide-aldehydes could also fall under this definition because cyclic products incorporate an externally-supplied CN group (Fig-ure 19F) or hydrogen atoms, respectively [175].

13. LINCHPIN-BASED MODIFICATIONS OF GENETICALLY-ENCODED PEPTIDE LIBRARIES

Introducing linchpins into readily available peptide li-braries provided by translational machinery and encoded by DNA or RNA is an emerging general approach for late stage functionalization of millions to billions of compounds at once. Most chemical transformations applied for the modi-fication of genetically-encoded peptide libraries have been mentioned in prior sections. Figure 20A summarizes pub-lished examples of stapling of genetically-encoded libraries that use: (i) amide bond formation between two Lys residues in mRNA-encoded library [65]; and (ii) synthesis of phage-displayed cyclic libraries via SN2 alkylations of Cys side chains by bisbromoxylene [176], dichloroacetone oximes [90], chloroacetamide derivatives of azobenzene [30], and crown ether [50], Michael addition by allenamide derivatives [114], and SNAr arylation by decafluorosulfone [96]. Figure 20B describes examples of bicyclic libraries with linchpins incorporated through Michael addition to three acrylamide groups [88], SN2 reaction between 1,3,5-Tris(bromomethyl) benzene and phage displayed peptides [32], and potentially, mRNA-displayed libraries of peptides [35]. Most aforementioned examples follow the traditional canons of chemistry and strive to design reactions that intro-duce linchpin and yield a well-defined product or a mixture of a limited number of substitutional isomers or diastereo-

Figure 18. A) Actuation between reversible and irreversible thiol-crosslinking linchpins developed by Baker and Caddick and co-workers [163]. B) Linchpin installed via aqueous radical addition employed by Chou and coworkers [166]. C) The same authors reported an elegant “linchpin inside the linchpin” inception of the bicyclic peptide with a linchpin bridge [166].


mers. In contrast, Heinis and co-worker inverted the tradition and produced bicyclic peptide by stochastic cross linking of four cysteines in phage-displayed peptides by C2-symmetric electrophiles [177] (Figure 20B). Although each cross-linking produces a large number of substitutional isomers in an unpredictable ratio, the authors used selection process to find the active mixture of isomers and then deconvoluted the structure of the active isomer after the selection. Similar sto-chastic synthetic of tricyclic genetically-encoded peptide libraries was proposed a year earlier in the publication by Chuanliu Wu and co-workers (Figure 10D) [98] but the authors have not reported the implementation of this pro-posal yet.

CONCLUSION

The modification of peptides and peptide libraries with “unnatural” linchpins offers a general and versatile approach for adding new properties to peptide sequences that cannot be added with conventional macrocyclization approaches. The multi-point attachment of linchpins offers topological solutions that are distinct from those achieved by incorpora-tion of unnatural functionalities via UAA-mutagenesis and bioorthogonal reactions that target one amino acid. Examples of the application of linchpins in medium-to-large size pro-teins are currently limited, but a few published examples of light-responsive linchpins (LRL) built into the proteins high-light the potential advantages of such hybrid post-translational modification of proteins by LRL.

Figure 19. A) Linchpin that crosslinks Trp in protected peptides on solid support reported by Johannes and co-workers [168]. B) Pd-catalyzed crosslinking of Trp in unprotected peptides reported by Albericio, Lavilla and co-workers [169]. C) Multicomponent head-to-tail cyclization installation of the oxazole linchpin by Yudin and co-workers [172]. D) Copper-catalyzed Mannich addition (“A3 macrocycliza-tion”) reported by Lubelle and coworkers introduces methylene linchpin into protected peptides on the solid support [173]. E) Reversible tail-to-side-chain macrocyclization of unprotected peptides with linchpin derived from Meldrums acid reported by Anslyn and co-workers [170]. F) Head-to-tail cyclization with concurrent incorporation of exocyclic CN linchpin by Malins, Baran and co-workers [175].


The field is fueled by an ever-expanding palette of un-natural amino acids that can be introduced in peptides via synthetic or biological approaches. Despite the increasing availability of peptides and peptide libraries with UAA, there will always remain a need for introduction of linchpin-type structures into “natural” translationally-made peptides and proteins. We envision that new challenges in this area could be: the identification of uniquely reactive linchpins that cross-link non-traditional residues, i.e., not Cys, Lys and Asp/Glu, in natural peptides and proteins. Linchpins that cross-link Tyr, Trp, His, or Arg residues can already be en-gineered based on known reactions that target these residues. Even a difficult chemical problem such as design of a linch-pin that can cross-link primary or secondary alcohols (side chains of Ser/Thr) in water in the presence of other nucleo-philes and electrophiles could be potentially solved via prox-imity-directed reactions and multi-dentate reactions with boron derivatives. As in classical organic synthesis, the most interesting and practical solutions could emerge from multi-step reactions that capitalize on intra-molecular events at a later stage of the multi-step manifold. For example, a robust inter-molecular reaction between the linchpin and the peptide can be used to introduce the first 1-2 attachment points. Sub-sequent intra-molecular reactions can drive regiospecific reactions between linchpin and other side chains within the peptide. The DCA linchpin introduced by Dawson can be

used as a platform for a “thought experiment” in design of such staged, multi-step cyclizations: for example, robust alkylation of Cys docks the linchpin and places the ketone group in proximity to other side-chain residues. The ketone moiety exhibits low reactivity to all natural side chain resi-dues. But a careful design can build on intra-molecular ar-rangement of ketone and side chains of Lys, Tyr, Trp or other residues and trigger their reaction in appropriate condi-tions. Linchpins could be thought of as co-factors that equip peptides with functions that side chain residues of the pep-tides alone cannot provide. This analogy helps to illustrate the future impact of linchpins on peptide science. The evolu-tion of life on Earth reached a critical irreversible decision to keep only ~20-amino acid oligomers and five-nucleotide oligomers and add further upgrades to the “oligomer world” via introduction of cofactors and post-translational modifica-tions. These co-factors allowed proteins to perform new tasks without the need to produce and encode new amino acids. Oligomers of a limited set of monomers combined with versatile cofactors and PTMs are the only molecular solution to “life” as we know it. It allows organisms across all domains of life to perform all critical biochemical func-tions and evolve new functions. Similarly, future molecular solutions in the fields of therapeutics, materials, and bioma-

Figure 20. Application of linchpins to modify phage displayed peptides and genetically-encoded peptide libraries displayed on M13 phage, T4 phage and mRNA.


terials, could be empowered by the combination of peptide oligomers and linchpins.

CONSENT FOR PUBLICATION

Not applicable.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

Declared none.

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