Versatile peptide macrocyclization with Diels-Alder cycloadditions

Abstract

Macrocyclization can improve bioactive peptide ligands through preorganization of molecular topology, leading to improvement of pharmacologic properties like binding affinity, cell permeability and metabolic stability. Here we demonstrate that Diels-Alder [4+2] cycloadditions can be harnessed for peptide macrocyclization and stabilization within a range of peptide scaffolds and chemical environments. Diels-Alder cyclization of diverse diene-dienophile reactive pairs proceeds rapidly, in high yield and with tunable stereochemical preferences on solid-phase or in aqueous solution. This reaction can be applied alone or in concert with other stabilization chemistries, such as ring-closing olefin metathesis, to stabilize loop, turn, and α-helical secondary structural motifs. NMR and molecular dynamics studies of model loop peptides confirmed preferential formation of endo cycloadduct stereochemistry, imparting significant structural rigidity to the peptide backbone that resulted in augmented protease resistance and increased biological activity of a Diels-Alder cyclized (DAC) RGD peptide. Separately, we demonstrated the stabilization of DAC α-helical peptides derived from the ERα -binding protein SRC2. We solved a 2.25 Å co-crystal structure of one DAC helical peptide bound to ERα, which unequivocally corroborated endo stereochemistry of the resulting Diels-Alder adduct, and confirmed that the unique architecture of stabilizing motifs formed with this chemistry can directly contribute to target binding. These data establish Diels-Alder cyclization as a versatile approach to stabilize diverse protein structural motifs under a range of chemical environments.

Graphical Abstract

INTRODUCTION

Peptides represent powerful starting points for the development of bioactive ligands. Short linear peptide sequences mediate protein-protein interactions (PPIs) by adopting diverse secondary structures, including helix, sheet, loop and turn motifs^1–2. Removal from their native protein context often abrogates structural rigidity, leading to reduced binding affinity and pharmacologic properties. Therefore, numerous chemical strategies have been developed to synthetically stabilize peptide secondary structure and retain or augment pharmacologic utility. Most of these chemistries capitalize on alkylation or acylation reactions on naturally-occurring nucleophilic residues such as cysteine and lysine; these approaches have been applied to stabilize helix, loop, and sheet motifs^3–8. A key limitation with many of these chemistries is their incompatibility with layering multiple stabilizing chemistries. Another strategy is incorporation of non-natural amino acids that enable metal-catalyzed bond formation. The most well-studied of these strategies include olefin metathesis of terminal alkene containing groups as well as [3+2] Huisgen ligation of alkyne/azide pairs^9–14. These ‘stapled’ peptides show increased favorable properties including structural stability, binding affinity, cellular uptake, and in vivo pharmacokinetics for direct or allosteric targeting of PPIs^15–21. Notably, compounds in this class have entered clinical trials for various indications²². While these chemistries can successfully stabilize peptide secondary structure, they are often incompatible with diverse structures, other natural and non-natural functionalities and aqueous conditions necessary for proper folding of specific peptide and protein scaffolds. From a practical standpoint, the transition-metal catalyzed reactions require expensive and toxic catalysts that must be removed during purification. Therefore, new strategies to control secondary and tertiary structure, ideally with wide functional group and reaction condition tolerance, would greatly expand the synthetic arsenal for developing peptide-based chemical probes.

A notable carbon-carbon bond forming reaction that has enjoyed widespread synthetic application is the Diels-Alder [4+2] cycloaddition. First reported in 1928, this reaction is employed by nature and chemists alike to construct complex molecules^23–25. Several aspects of this transformation make it attractive for synthesis, including a wide range of diene-dienophile pairs, high regio- and chemoselectivity in many applications, simultaneous introduction of multiple stereocenters and compatibility with a range of reaction conditions²⁶. Indeed, pioneering studies demonstrated that aqueous conditions are not only tolerated, but can enhance reaction rates²⁷. While these qualities overlap with those desired for selective modification of biomolecules, beyond its limited use in intermolecular ligations^28–32, this chemistry has not been applied for site-specific stabilization of peptides or proteins. Here we report the application of Diels-Alder carbon-carbon bond forming reactions for the macrocyclization of diverse turn, loop and α-helix peptide motifs.

RESULTS AND DISCUSSION

We began our study of Diels-Alder cyclized (DAC) peptides with turn and loop motifs, including an anti-migratory RGD peptide, analogs of which are undergoing clinical testing in oncology^33–34. Diene-dienophile functional groups span a wide range of structure and reactivity, thus we chose the moderately activated diene 2,4-hexadiene and the well-characterized maleimido dienophile³². Model peptides of various length and sequence were synthesized containing orthogonally-protected cysteine (tBuS-) and lysine (Mmt-) side chains, with the former invariably in the i-position, and the latter in the i+4, i+5 and i+7 positions. Sequential on-resin cysteine deprotection and hexadiene alkylation with 1-bromo-2,4-hexadiene proceeds quantitatively (Figure 1A,B; Scheme S1). We also synthesized the corresponding diene-containing amino acid, Fmoc-Cys(2,4-hexadiene)-OH, which is compatible with direct incorporation by SPPS (Fig. 1A; Scheme S2, Supplementary Spectroscopic Data). Subsequent on-resin lysine deprotection and acylation with N-maleimido-glycine on an RGD model peptide resulted in formation of two distinct peaks on LC-MS (1 and 1a) with the same mass and ~1 minute retention time shift, suggesting formation of a cyclized product eluting earlier on the C18 column (Figure 1A,B). This product appeared rapidly, with an approximate 2:1 ratio of 1a to 1 within 5 minutes of dienophile introduction; near-complete conversion to 1a was observed upon extended incubation on resin (16 hr, Figure 1B). Kinetic reaction monitoring confirmed rapid conversion of later-eluting 1 to 1a, as well as minor product 1b, with nearly 95% formation within 1 hour (Figure 1C). This pattern was observed across diverse intervening sequences and diene-dienophile spacing (Table 1, Table S1), with high yields of ~85–95% conversion to earlier-eluting, presumably cyclized species. In each scaffold studied, additional earlier-eluting minor isomeric species (≤10% relative to major product) were observed ~4 minutes before the linear peptide (Figure 1B; Figure S1).

Figure 1.

Synthesis, stability, and activity of Diels-Alder cyclized peptides. (A) Schematic of a DAC peptide functionalization. (B) Absorbance (215 nm) and extracted ion chromatogram (EIC) LC-MS traces of crude peptide following the indicated functionalization and reaction times with the RGD compound 1 scaffold. (C) Representative reaction time course quantifying conversion of 1 to cyclized 1a/1b isomers. (D) Extended time course tracking of HPLC-purified 1a incubated with βME in aqueous buffer, verifying cycloadduct stability (βME-trapped species m/z = 914.3). (E) In vitro trypsin and chymotrypsin resistance assays for 1a versus linear analog 1wt, and 4a and linear analog 4wt, respectively. (F) Cell migration model scratch wound assay comparing reduction in wound closure by RGD peptides 1wt and 1a at 10 and 20 μM relative to DMSO control. Data represent mean ± s.d. distance measurements from images of 2 biological replicates, 5 distance measurements each. n.s. = not significant; * = P < 0.05; ** = P < 0.0005; Student’s t-test.

Table 1.

Diels-Alder cyclized loop peptides and precursors characterized in this study.

sequence	compound(s)	% conv.	product ratio*
CHexRGDKMal	1, 1a, 1b	95	>50:1
CHexVGDKMal	2, 2a, 2b, 2c	92	9:1
CHexAPVYKMal	3, 3a, 3b, 3c	89	8:1
CHexAVPAVYKMal	4, 4a, b, 4c	85	9:1

All peptides contain N-terminal acetyl and C-terminal amide groups. “Hex” indicates 2,4-hexadiene alkylation; “Mal” indicates a maleimido-glycine modified residue. Each compound number represents the non-cyclized (X) and cyclized isomers (Xa, Xb…) as determined by separation on LC-MS. *Ratios reported as major:minor product(s) were quantified by integrated LC-MS peak area. Percent conversion (% conv.) refers to the percent yield from non-cyclized to cyclized products.

A limited screen of reaction conditions revealed that mild heating on resin in DMSO was an optimal condition to maximize cyclic conversion, but also resulted in increased ratio of major to minor isomeric products (vide infra, Table S2), which may be due to de-aggregation of the protected peptide on resin. We also found that other less reactive dienophiles such as acryloyl and crotonyl groups react with hexadiene in yields comparable to maleimide (Figure S2). These viable alternative dienophiles are also compatible with direct incorporation during SPPS and offer increased diversity of cyclic structures³⁵. Similar reaction profiles were observed for peptides produced by on-resin diene incorporation or direct use of Fmoc-Cys(2,4-hexadiene)-OH, as well as upon incubation in aqueous solution following peptide cleavage (Figure S3). These data confirm the compatibility of Diels-Alder peptide cyclization in diverse organic and aqueous chemical environments.

Diels-Alder Cycloadditions Form Chemically Stable Peptide Macrocycles with Enhanced Protease Resistance and Biological Activity

To verify 1 and 1a represented non-cyclized and cyclized products, respectively, we capitalized on the maleimide dienophile uniquely present in the non-cyclized starting material. On-resin incubation of the 1 and 1a crude mixture with excess 2-mercaptoethanol (βME), which should only react with linear maleimide-containing peptide, resulted in complete consumption of 1, yielding a species with the mass of the βME conjugate addition product (Scheme S3; Figure S4). By contrast, 1a levels remained constant, consistent with the loss of the α,β-unsaturated electrophile after Diels-Alder cyclization. Likewise, prolonged exposure of purified 1a to βME did not result in any addition product, suggesting formation of a stable DAC-RGD peptide macrocycle (Figure 1D).

The RGD motif has seen considerable therapeutic development as an anti-migratory agent targeting aggressive cancers^{34, 36}. Like other peptides, in vivo use of native RGD shows limited efficacy due to cleavage by circulating proteases, whereas head-to-tail cyclized analogs demonstrate increased protease resistance and in vivo activity³⁷. To test whether DAC peptides similarly display differential protease susceptibility, we exposed cyclic 1a and linear analog 1wt to an in vitro trypsin sensitivity assay. Native peptide 1wt was rapidly degraded within two hours (Figure 1E), whereas DAC 1a showed an extended half-life, with 30% remaining intact after 4 hours. To determine if this effect was applicable to other scaffolds and proteases, we subjected the major cyclic isomer of i, i+7 DAC 4a and linear analog 4wt to chymotrypsin. As with the RGD scaffold, the linear species 4wt was degraded more rapidly than 4a (Figure 1E). Consistent with the increased stability of the cyclized RGD analog, compound 1a was significantly more effective at blocking wound closure, an integrin-mediated phenotype, relative to the linear control peptide 1wt (Figure 1F; Figure S5). Taken together, these results confirm that Diels-Adler cyclization can affect the structure and biological function of loop peptide ligands.

Diels-Alder Cycloadducts Preferentially Form with endo Stereochemistry and Stabilize Peptide Conformation

To characterize a putative Diels-Alder cycloadduct and its resulting effects on peptide conformation, 4a and precursor peptides 4wt and 4hex were subjected to a series of NMR experiments. ¹H-NMR of 4a confirmed the presence of two vinylic protons, absent in 4wt and distinct from those of the diene in 4hex (Figure 2A) or α,β-unsaturated maleimido protons. The observed doublet of triplets pattern for the vinylic protons in 4a results from small ³J and ⁴J coupling constants of the unsaturated system, indicative of near-90° dihedral angles between neighboring vinyl and allyl protons. TOCSY experiments permitted complete assignment of cyclized and non-cyclized peptides (Supplementary Spectroscopic Data). Direct through-bond coupling was observed between 4a vinylic protons and all protons within the putative cycloadduct (Figure 2B; Figure S6A), supporting the formation of a fused bicyclic adduct. The combination of TOCSY and NOESY experiments resolved the connectivity of the cycloadduct in 4a and revealed extensive through-space interactions between its protons (Figure 2B; Figure S6B). Most notably, interactions between H3/H6 allylic protons and H4/H5 succinimidyl bridgehead protons suggest the cycloadduct in 4a is the endo stereoisomer (Figure 2B).

Figure 2.

NMR spectroscopy characterization and MD simulations of DAC peptide 4a and linear precursors. (A) ¹H-NMR spectra of 4wt (black), 4hex (blue), and 4a (red) in the vinylic (~6–5 p.p.m.) region. (B) Model of 4a cycloadduct depicting through-bond and through-space interactions between cycloadduct protons as determined by TOCSY and NOESY (orange: bridgehead interactions; purple: methyl-group interactions; black: Cys-side chain interactions; red: vinylic interactions). (C) ³J_H-N-Cα-Hα coupling constant values for each residue of the three peptides studied. (D) Peptide backbone alignment over 50 ns simulations for 4wt and 4a. (E) RMSD values for 4wt and 4a peptide backbones and all atoms (excluding hydrogens).

NMR results further revealed substantial perturbation to the cyclic 4a structure relative to linear analogs. Following hexadiene incorporation, only modest changes in chemical shifts were observed for cysteine and nearby residues, with virtually no change in ³J-coupling constants between backbone amide and alpha protons (Figure 2C; Figure S6C–F). Conversely, 4a displays marked changes in chemical shifts and backbone ³J-coupling constants for several residues, most notably the tyrosine and alanine adjacent to the cyclizing residues (Figure 2C; Figure S6C–F). These alterations likely result from both cyclization-induced limits on degrees of freedom, as well as emergent intramolecular interactions such as hydrogen-bonding between amide and carbonyl oxygen atoms found on the cycloadduct and peptide.

To further investigate the structure of this i, i+7 DAC peptide, we carried out molecular dynamics (MD) studies of 4wt and 4a. Briefly, models were built in MOE and run in the NAMD2 simulation package (see Methods for additional details)^38–39. Trajectory analysis using VMD revealed linear 4wt displayed no persistent structure, while cyclized 4a displayed a highly stabilized extended loop structure over 50 ns simulations (Figure 2D)⁴⁰. RMSD measurements of the respective backbones confirm a substantial decrease in 4a conformational flexibility relative to linear 4wt (Figure 2E; average backbone RMSD = 1.07 Å and 2.69 Å, respectively), which also translated to reduced motion in the side chain dynamics of 4a (Figure 2E, Videos S1 and S2).

Diels-Alder Peptide Cycloadditions Operate in Aqueous Solution and Stabilize α-Helices

Helical peptides represent the dominant structural feature that mediates biomolecular interactions¹. We therefore asked if Diels-Alder cyclization strategies can be leveraged to stabilize α-helical peptide conformations from diverse peptide sequences. We first tested whether a conformationally-restricted and less reactive furan diene could cyclize on a p53-derived helical sequence¹⁶. Like Fmoc-Cys(2,4-hexadiene)-OH, we found that Fmoc-protected (2-furanyl)alanine (denoted as A_Fur) could be directly incorporated during SPPS (Figure 3A,B). Notably, the Diels-Alder cyclization reaction using A_Fur and K_Mal at previously established i, i+7 positions in the p53-derived sequence proceeded slowly on solid support, yielding a dominant linear species that could be purified by HPLC (Figure 3A,B). Incubation of the isolated linear peptide 5 in physiological PBS buffer, however, resulted in the rapid appearance of earlier-eluting cyclized species and a minor fraction of peptide dimers (Figure 3B). Michael-addition trapping experiments confirmed that the putatively cyclized compounds 5a, 5b and 5c lacked the reactive maleimide, which contrasted with the isolated linear peptide 5 (Figure 3C; Figure S7). Circular dichroism spectroscopy of the linear control p53 sequence and all three isolable cyclized species confirmed increased α-helical character for the dominant DAC peptide 5a relative to the minor products 5b and 5c (Figure 3D). These data are reminiscent of other studies reporting that stereochemical yield is correlated with global stability of the peptide fold⁴¹, here suggesting that the aqueous chemical environment may aid in the selection of favorable adducts that promote global helix stabilization.

Figure 3.

Synthesis, stability, and secondary structural characterization of Diels-Alder cyclized p53 peptide. (A) Schematic of DAC-p53 peptide synthesis. (B) HPLC trace and resulting aqueous solution reaction time course of conversion of 5 to cyclized isomers 5a-c. (C) Incubation of DAC-p53 peptide 5 mixture with βME in aqueous buffer, verifying Michael adduction of linear precursor 5 and stability of cyclized isomers. (D) CD spectra of linear analog 5wt and 5a-c.

Diels-Alder Cycloadduct Geometry, Alone or in Tandem with other Chemistries, Differentially Affects Peptide Helicity and Activity

To further explore the potential to stabilize diverse helical conformations, we synthesized a series of Diels-Alder stabilized, α-helical peptides derived from the co-activator protein SRC2. A short helical stretch within this protein contains a conserved LXXLL motif that engages a hydrophobic groove in estrogen receptor-α (ERα Figure 4A)^42–44. We synthesized several i, i+4 ‘DAC-stapled’ peptides, which were tolerant of direct 2-furanylalanine incorporation during SPPS. Unlike the p53-derived sequence, both SRC2 peptides 6 and 7 cyclized on resin rapidly and in high yield following maleimide incorporation (Figure 4B, Table S3). We characterized the two isolable cyclized species 6a and 6b, and the one dominant compound 7a by CD spectroscopy. The dominant species 6a and 7a were significantly more helical than either the unmodified wildtype SRC2 peptide or minor 6b compound (Figure 4C). Previous attempts to stabilize the SRC2 LXXLL motif have shown that hydrophobic residues that engage ERα can be replaced by a hydrocarbon staple without significant loss in binding affinity⁴⁵. Therefore, we sought to test whether we could synthesize a double-stapled SRC2 ligand containing both a hydrocarbon staple installed by ring closing metathesis of olefin containing amino acids in concert with Diels-Alder cyclization of a diene-dienophile pair (Figure 4A,D). Direct incorporation of olefin-containing S₅ amino acids and furan yielded a clean peptide precursor, 8pre, that was readily cyclized by Grubbs-I catalyst (8rcm; Figure 4D,E). Subsequent introduction of the maleimide dienophile resulted in immediate appearance of an earlier eluting tandem stapled peptide, which was the stoichiometric product after mild heating for 4 hr on resin (Figure 4E, Table S3). CD characterization of this bicyclic peptide 8a revealed classic α-helical minima at 208 and 222 nm (Figure 4F). ¹H-NMR analysis confirmed the presence of Diels-Alder cycloadduct vinylic protons and a single allylic proton in all major DAC SRC2 peptide products, and, in the case of 8a, peaks corresponding to the RCM alkene product were observed (Figure S8, Supplementary Spectroscopic Data).

Figure 4.

Synthesis, secondary structural characterization and activity of Diels-Alder cyclized and double-stapled SRC2 peptides. (A) Sequence of SRC2 wildtype peptide used as scaffold with conserved LXXLL motif; staple placement and structures of DAC-SRC2 peptides 6a-b and 7a and double-stapled 8a. (B) HPLC traces of pre-cursor peptide used and resulting cyclized product profile of compound 7 following 4 hr on-resin heating in DMSO. (C) CD spectra of isolated DAC-SRC2 peptides compared to unmodified wildtype sequence. (D) Synthetic scheme for layering ring-closing metathesis and Diels-Alder cyclization for the synthesis of double-stapled SRC2 peptide 8a. (E) HPLC traces of pre-cursor peptide 8pre, ring-closing metathesis product 8rcm, Diels-Alder functionalized peptide 8 and fully-DAC-SRC2 double-stapled peptide 8a. (F) CD spectra of helical compound 8a. (G) Normalized FP assay results showing a dose range of DAC-SRC2 peptides effectively competing off fluorescein-labeled wildtype SRC peptide from wildtype ERα. Each data point represents measurements from two independent experiments run in triplicate, ±s.e.m. Dose-response curves fit with a four parameter log(inhibitor) vs response equation in Prism 5 graphing software.

To probe the structure activity relationships of these DAC-SRC2 peptides we performed competitive fluorescence polarization binding assays with ERα. Both the single DAC-stapled 7a and tandem stapled 8a peptides competed a fluorescein-linked SRC2 peptide off of ERα with IC₅₀s of approximately 0.5 and 2.5 μM respectively (Figure 4G, Table S4). Intriguingly, the dominant, more helical isomer 6a demonstrated 4-fold improved activity compared to the less helical, minor product 6b (Figure 4G, Table S4). These results echo those observed for the p53 sequence, where stereochemical yield is correlated with overall peptide fold stabilization, and in the case of SRC2 improved biochemical potency.

X-ray Crystal Structure of a DAC Stapled SRC2 Peptide- ERα Complex Demonstrates Cycloadduct Stereochemistry and Contribution to Target Binding

To determine the structure of the Diels-Alder adduct in 6a and its role in promoting the interaction with ERα, we solved the X-ray structure of 6a and estradiol co-crystallized with residues 300–500 of the ERα ligand binding domain (LBD) at 2.25 Å resolution (Figure 5A; Table S5). The Y537S mutant of ERα was used as it stabilizes the agonist conformation of the receptor and aids in crystal formation. An F_O-F_c difference map using wild-type SRC2-bound ERα confirmed the unambiguous presence of estradiol bound in the core of the LBD, and DAC-stapled 6a bound in the canonical activating function 2 (AF2) cleft of ERα (Figure 5A; Figure S9A,B). The Diels-Alder adduct was highly ordered and permitted unequivocal confirmation that the more stabilizing and dominant stereochemical product in the context of SRC2 is that of the endo isomer (Figure 5B). The endo bicycle presents a convex hydrophobic surface that directly contacts ERα residues Val355, Ile358 and Leu539 that form the hydrophobic shelf adjacent to the cleft that binds the LXXLL motif (Figure 5C). Residues in 6a that are derived from Leu690 and Leu694 in SRC2 retained their canonical contacts and were deeply engaged with a network of hydrophobic residues in the AF2 groove (Figure 5C, bottom). Additional orienting contacts are mediated by His691 to the edge opposite the Diels-Alder adduct, as well as a conserved hydrogen bond between Glu542 of ERα that caps the N-terminus of the peptide helix (Figure S9C). At the C-terminus the last ordered residue present in the DAC-peptide structure, Q695, folds back on the peptide to satisfy a hydrogen bond to the i-2 amide carbonyl, effectively capping the helix (Figure S9C). Taken together, these structural data confirmed that the unique Diels-Alder chemical and stereochemical composition can stabilize an active helical conformation and directly contribute to target engagement, as evidenced here by forming a molecular “clasp” around the core and edge of the ERα AF2 cleft.

Figure 5.

X-ray crystal structure of Diels-Alder cyclized SRC2 peptide 6a bound to estrogen receptor-α (PDB: 6PIT). (A) LBD of ERα Y537S mutant protein bound with estradiol and 6a located in the AF2 cleft. (B) Two views of the structure of the DAC-SRC2 peptide 6a cycloadduct (C = yellow, N = blue, O = red) with endo stereochemistry overlaid with electron density map (grey grid) from resolved crystallographic data (2mFo-DFc map contoured to 1σ). (C) Dorsal (top) and axial (bottom) view of 6a bound in the AF2 binding pocket. Relevant residues numbered according to SRC2 sequence are highlighted, as well as the Diels-Alder cyclization moiety in yellow. Flexible portions of the structure with low electron density are omitted.

CONCLUSION

Backbone- and side chain-directed peptide cyclization strategies can effectively improve desirable properties for chemical probes or therapeutics. Our study establishes several emergent and improved capabilities offered by the incorporation of Diels-Alder cycloadditions to the toolbox of reactions used for peptide macrocyclization and stabilization. First, we demonstrated that a range of reactive diene-dienophile pairs can be applied to diverse peptide sequences in both organic and aqueous solvents. This opens up many possibilities to tailor the macrocyclization motif itself to improve not only the inherent structure of the peptide, but also physicochemical properties and target binding. This aspect was highlighted by our observation that the preferred endo DAC stapled isomer of SRC2 displays augmented helical stabilization and directly contributes to ERα binding. Specifically, the convex hydrophobic surface of the adduct presented by the endo isomer rests on a hydrophobic surface adjacent to the AF2 pocket. Future exploration of this interaction could probe whether alternative Diels-Alder adducts could be developed that contribute specific, polar contacts to the binding surface. This potential to access unique adduct structures and conformations is especially noteworthy given the physicochemical differences relative to existing, predominantly hydrophobic staples in use. Indeed, it is under-appreciated that while added hydrophobicity can improve target binding affinity, this may come at the expense of increased off-target interactions. The range of possible Diels-Alder cycloadducts may enable design of and selection for macrocycles that temper these contributions. Structural studies of both the stabilized loop and helical peptides confirmed that the expected endo Diels-Alder adduct was the major product in these contexts. Furthermore, we established that mild heating on-resin in organic solvents can drive the stereoselective formation of the endo isomer across diverse peptides. Based on the evidence here, we expect that this will be general for many peptides, however future studies are warranted to determine if there are conditions that will bias toward the formation of the exo isomer if desired.

Another compelling aspect of the DAC peptide strategy is that following installation of reactive diene and dienophile pairs, the macrocyclization reaction occurs spontaneously. This contrasts with many side-chain crosslinking approaches that require metal catalysts and the ensuing limitations on reaction conditions, cross-reactivity with other functional groups present in target peptides, and practical considerations of cost and reagent removal during synthesis and purification. Diels-Alder macrocyclizations are therefore essentially “reagentless,” and are instead only impacted by the chemical environment and reaction conditions. This suggests cyclization or intramolecular crosslinking could be performed on native protein folds, or aid in proper folding, alone or in concert with other chemistries. As was seen for DAC-p53 peptide 5, an aqueous reaction environment not only promoted spontaneous cyclization, but also preferentially formed macrocyclic isomer 5a with the greatest helicity. Indeed, this implies the context specific contributions of different Diels-Alder adducts and even stereochemical products of unique reactive pairs could be screened for inherent stabilization and extrinsic effects on the activity of stabilized peptides.

A major goal in developing new strategies to chemically modify and stabilize peptides is to permit increased access to unique and larger peptide structures, including those containing multiple secondary structural elements. Our data suggests that the current and future Diels-Alder cyclization pairs will contribute to this work in two ways. First, the stabilization of larger peptide and proteomimetics may require properly folded structures in order to template ligation reactions or other chemical modifications. In these cases, the ability to operate directly in physiological environments, which we demonstrated here for both loop and helical DAC peptides, will make the Diels-Alder cyclization an attractive choice compared to other chemistries that cannot operate on unprotected peptides and proteins, or in aqueous environments. Another avenue to stabilize larger, conformationally complex peptide structures is to incorporate multiple stabilizing elements, but finding compatible biorthogonal chemistries that can operate in concert has remained a challenge. Here we demonstrated the facile double stapling of an SRC2 helical peptide that contained RCM and DAC stapling residues adjacent to one another. Despite this proximity, these two chemistries faithfully permitted dual macrocyclization directly on-resin. We envision that future exploration of Diels-Alder stabilization of secondary and tertiary structural elements with other natural and non-natural stabilizing elements will permit improved access to hyperstable peptide and protein structures. Toward this goal, we established Diels-Alder reactions between diverse diene-dienophile pairs, and future work is warranted to explore additional combinations that are tailored for specific functional group compatibility, secondary structures, reaction conditions and ligand physicochemical properties. In summary, we believe the application of Diels-Alder as a biocompatible protein chemistry offers abundant opportunities for peptide macrocycle and protein domain construction.