Extendable stapling of unprotected peptides by crosslinking two amines with o-phthalaldehyde

Peptide modification methods that do not rely on the cysteine residue are underdeveloped, and their development could greatly expand the current toolbox for peptide chemistry. During the course of preliminary investigations into the classical ortho-phthalaldehyde (OPA)-amine-thiol condensation reaction, we found that in the absence of thiol, OPA readily condenses with two primary alkyl amines to form a class of underexplored isoindolin-1-imine compounds under mild aqueous conditions. From the intramolecular version of this OPA-2amines reaction, an efficient and selective methodology using mild reaction conditions has been developed for stapling unprotected peptides via crosslinking of two amino groups in both an end-to-side and side-to-side fashion. The stapling method is superfast and broadly applicable for various peptide substrates with the reacting amino groups separated by a wide range of different amino acid units. The macrocyclization reactions of selected substrates are completed within 10 seconds at 5 mM concentration and within 2 minutes at 50 μM concentration. Importantly, the resulting cyclized peptides with an isoindolinimine linkage can be extended in a one-pot sequential addition manner with several different electron-deficient π electrophiles, thereby generating more complex structures.

T here is increasing recognition of the importance of peptides in drug discovery 1 . However, to modulate their structures and physiochemical properties, further modification of natural peptides or those generated by the standard synthetic protocols is usually required 2,3 . In this regard, the chemoselective crosslinking or stapling of two peptide handles has proven to be highly effective for the macrocyclization of peptides into more conformationally constrained versions [4][5][6][7][8][9] . Traditional approaches, which have been successful in the past, have involved bioorthogonal coupling reactions using noncanonical amino acid (AA) units, such as alkyne-azide cycloaddition and metalcatalyzed alkene metathesis. However, an approach that would involve the stapling of native peptides in their unprotected form would be much more desirable owing to the easy accessibility of such substrates and their high compatibility with biological systems [10][11][12][13][14] . Most of the research thus far has focused on the linking of highly reactive cysteine (Cys) residues (Fig. 1a) [15][16][17][18][19][20][21] . A commonly used peptide stapling technique involves the bisalkylation or arylation of two Cys residues with linkers bearing two electrophilic sites. Recently, the groups of Li and Perrin independently reported a powerful method for the macrocyclization of native peptides between Cys and lysine (Lys) residues using a simple ortho-phthalaldehyde (OPA) reagent ( Fig. 1b) 22,23 . While Cys has proven to be a very reliable anchor for peptide modification, it is often absent from the accessible peptides of interest and needs to be incorporated by additional synthetic manipulations. The current toolbox for stapling native peptides could be greatly expanded by exploring other common amino acid residues as potential cross-linking anchors [24][25][26][27][28] . Useful methods for such noncysteine stapling are still very scarce 5,[29][30][31] . Here, we show that the primary amino groups from two AA residues can undergo a condensation reaction with OPA as the linker, forming a class of underexplored isoindolinimine structures under mild aqueous conditions (Fig. 1c). This chemoselective intramolecular OPA-2amines reaction resulted in the fast and efficient macrocyclization of native peptides in both N-end to side chain (end-to-side) and side chain to side chain (side-to-side) fashions under mild aqueous conditions with a broad substrate scope. Moreover, the resulting isoindolinimine linkage allows further extension via a one-pot process by reaction with various π electrophiles in high efficiency and selectivity, allowing secondary and even tertiary modification as well as the rapid construction of complex structures.

Results
Reinvestigation of the OPA-amine condensation reaction. OPA has been used as a color and fluorescence indicator for sensing ammonia and amine compounds, such as α-amino acids (AAs) for over a century 32,33 . In 1909, Thiele was the first to report the formation of isoindolin-1-one 1 (also called phthalimidine) by the reaction of OPA with aniline in an organic solvent at room temperature (rt) (Fig. 2a) 34 . The reaction of OPA with amines proceeds by initial formation of the cyclic hemiaminal 2, yielding iminium intermediate 3 after the loss of water. Several mechanisms, including protonation of isoindolinol intermediate 4, a [1,3]hydride shift, and the [1,3]-H sigmatropic rearrangement of 3, have been proposed for the conversion of 3 to 1 [35][36][37] . The significance of the OPA-amine reaction was recently demonstrated by Li for the selective modification of Lys in peptides and proteins 38 . In the seminal discovery by Roth from 1970, it was found that a mixture of OPA, primary amine, and 2-mercaptoethanol quickly reacted in an alkaline aqueous medium to afford a strongly fluorescent product (Fig. 2b) 39 , which was later identified by Simons as the 1-thio-isoindole 5 40 . Simons subsequent discovery that 5 reacted with dimethyl acetylenedicarboxylate (DMAC) to form a 1-thio-3-alkenylisoindole addition product with high efficiency hinted at the usefulness of this reaction type for further functionalization 41 . The three-component OPA-amine-thiol condensation reaction is believed to proceed through cyclic intermediate 6, which undergoes dehydration to form 5. Fig. 1 Stapling of unprotected peptides via non-Cys residues under mild aqueous conditions. a Common strategies for peptide stapling via two cysteine residues. b OPA-mediated peptide stapling via cysteine and lysine residues. c OPA-mediated peptide stapling via two amine groups (this work). C: cysteine, K: lysine. The thiol group of cysteine is marked in magenta. The amino group is marked in red. The linkers joining the two cysteines are marked in cayenne. The OPA reagent and isoindolinimine core are marked in green. The π electrophiles are marked in mocha. The linkages between isoindolinimine and electrophiles are marked in blue.
Owing to its rapidity and robustness, the Roth method has gained wide popularity to derivatize AA samples for chromatographic analysis. In addition, it has found usefulness to selectively modify Lys and Cys residues of proteins 42,43 . For example, Porttoghese developed an OPA-β-funaltrexamine conjugate as a fluorogenic affinity-labeling ligand of opioid receptors 42 . From these background works, Li and Perin developed the macrocyclization of unprotected peptides under biocompatible conditions by chemoselectively crosslinking Cys and Lys residues 22,23 . Furthermore, while there is much research mostly prior to 1990 describing the reaction of OPA with various partners in organic solvents, OPA chemistry under mild aqueous conditions that are potentially suitable for biomedical applications has not been vigorously investigated.
Recently, we discovered that the Roth method for HPLC analysis of AA could be repurposed for quantitative chiroptical sensing of AA using UV and circular dichroism (CD) spectroscopy 44 . As shown in Fig. 2c,d, the reaction of phenylalanine 7 (Phe, 1.0 equiv) with OPA (1.1 equiv) and p-toluenethiol 8 (1.3 equiv) in a phosphate-buffered saline (PBS) buffer (pH 8.0) and MeOH solvent mixture at room temperature (rt) quickly formed 1-thio-isoindole product 9 in high yield (see liquid chromatography (LC) trace 1). Besides MeOH, other polar organic solvents, such as EtOH, CH 3 CN, and DMSO, could also be used with success. The enantiomeric excess (ee) value could be determined from the UV and CD spectra of 9, rather than by measuring its fluorescence signal. Similar to Roth's original ARTICLE study 39 , the fluorescence signal of the reaction mixture was influenced by the addition sequence of the various reagents. If thiol 8 was incorporated a couple of minutes after the addition of OPA and Phe 7, the signal was much weaker than if thiol is first added prior to incorporation of OPA. To the best of our knowledge, a detailed explanation for this observation has still not been offered 32,33 , so we set out to further investigate the OPA condensation reactions by LC-MS analysis. As mentioned, delayed addition of thiol 8 to the mixture of OPA and 7 in a 1:1 PBS buffer (pH 8.0) and MeOH solvent mixture led to reduced 9 and formation of the new isoindolinimine adduct 10, formed from the reaction of one OPA and two Phe molecules (LC trace 2). Moreover, mixing 1.0 equiv of Phe 7 with 1.1 equiv of OPA in the absence of any thiol quickly and selectively formed 10 as the main product in 5 min (LC trace 3). A minor side product 12 with a molecular weight equivalent to the addition product of 10 and another molecule of OPA (double-OPA product) was generated in a trace amount, while no isoindolin-1-one 11 was detected. The mechanism for the formation of isoindolinimine likely starts with the reaction of a primary amine with 2, forming aminal 13 (Fig. 2e). Intermediate 13 dehydrates to generate 1-aminoisoindole 14, and then quickly tautomerizes to form isoindolin-1-imine 15. If an amine is absent, then 2 slowly dehydrates to give 1. As shown in Fig. 2f, Lys 16 (1.0 equiv) reacts with OPA (1.0 equiv) in a 1:1 PBS buffer/MeOH mixture to quickly generate cyclic product 17a in excellent yield and regioselectivity (17a/17b > 20/1), with the structure confirmed by NMR analysis. Such chemoselectivity might be attributed to the intrinsically stronger nucleophilicity of the side chain ε amine group in Lys compared to the α NH 2 group, which is deactivated by the electron-withdrawing carboxylate group. The slightly basic conditions and the MeOH co-solvent might have enhanced the formation of unprotonated Lys side chain in the reaction system 45 .
End-to-side stapling of peptides by OPA-2amines condensation. Having established the condensation reaction of Lys as a proof of concept for the intramolecular condensation between OPA and two amino groups, we next investigated whether the OPA-2amines reaction can be applied in a macrocyclic fashion to crosslink the N-terminal amino group and the Lys side chain of linear peptides (Fig. 3a), easily prepared via solid-phase peptide synthesis (SPPS). We were gratified to find that the model substrate tetrapeptide H-Ala-Trp-Gly-Lys-NH 2 18 at a 5.0 mM concentration quickly reacted with OPA (1.0 equiv) in a 1:1 mixture of PBS (pH 8.0) buffer and MeOH at rt (condition [A]), generating cyclized product 19a in 77% yield and with excellent regioselectivity (19a/19b > 20/1, see LC trace 5). To our surprise, LC-MS analysis indicated the reaction was completed in less than 10 seconds at 5 mM concentration (LC trace 6). The same reaction of 18 at 50 μM was completed within 2 min (see Supplementary Fig. 8). A small amount of side product (~10% based on LC estimation) featuring the condensation of 19a with additional OPA molecule (double OPA product) was formed when the concentration of substrate was increased to 50 mM (see Supplementary Fig. 66). Little intermolecular dimerization product was formed even when the reaction concentration was increased to 100 mM. Notably, the pH value of the medium had a strong impact on the reaction. While the reactions gave very similar results at pH values between 8.0 and 10.0, the LC yield of 19a slightly dropped to 83% at pH 7.0 and to 34% at pH 5.0 along with the significant increase of side product 20 under otherwise identical conditions. Little product was formed at pH below 3.0 (see Supplementary Fig. 68).
Interestingly, unlike in the reaction of Lys with OPA, lowering the MeOH content in the solvent mixture to a concentration such as 10% led to increased formation of the isoindolinone side product 20, along with other unidentified side products (condition [A'], LC trace 7). Other alcohol co-solvents, such as EtOH and trifluoroethanol (TFE), were also effective for promoting the desired macrocyclization. The reaction with DMSO, DMF, CH 3 CN, dioxane, or THF as cosolvents gave a considerable amount of 20 and other side products (see Supplementary Fig. 63 for the detailed evaluation of organic cosolvents). The buffer could be substituted by adding diisopropylethylamine (DIPEA, 3 equiv, for neutralization of the residual TFA in peptide substrate) to a 1:1 mixture of H 2 O and MeOH, also efficiently generating 19a (condition [B]). Alternatively, TFE could be used as an efficient cosolvent for peptide substrates with low solubility in methanol (condition [C]), although pure TFE was also a good solvent for the reaction (condition [D]). Mechanistically, we suspect that alcohols might facilitate the dehydration of aminal intermediate 13 to form 14 via solventbridged proton transfer (see Supplementary Fig. 65) for our proposed mechanistic model). In comparison to the 1-thioisoindole motif such as in 5, which is relatively prone to oxidation by air, the isoindolinimine moiety in compound 19a proved to be very robust in aqueous solvents at rt under air atmosphere over a wide pH range (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Heating 19a in PBS buffer (pH 7.3) at 60°C for 12 h gave little decomposed product. However, significant amounts of the acyclic isoindolinone side product (>30%) was formed when 19a was heated in the same buffer at 100°C for 2 h (see Supplementary Fig. 69 for details).
The OPA-2amines-mediated end-to-side macrocyclization was efficient for peptide substrates of a wide range of ring sizes and AA compositions, affording the products in excellent regioselectivity in 10 min (Fig. 3b). Most of the reactions were completed within 2 min. Two examples that demonstrate the flexibility permitted by the reaction with regards to the number of spacing AA units are seen in the formation in high yield of 21 bearing no spacing AA units and 35 bearing more than 10 AA units between the N-terminus and the Lys residue. Except for Cys, all unprotected proteinogenic AA units, such as Arg (24), His (30), Trp (33), Tyr (24), Ser (24), and Glu (31), were well tolerated. D-AA units, such as D-Phe (28), could also be incorporated. Notably, the reaction of substrates containing Cys resulted in a complex mixture of products (e.g., 28). Except for Pro, which bears a secondary amino group, a variety of proteinogenic AAs can be placed at the N-terminus. The conversion of large peptide substrates was more favorable with TFE as a cosolvent (condition [C]), owing to its superior solubilizing ability compared to MeOH.
We next set out to synthesize crosslinked peptides with potential biomimetic activity or active medical properties. For example, compound 29 was prepared in excellent yield to mimic the integrin inhibitor cyclo(RGDfK). The reaction of the 18-mer human β-melanocyte-stimulating hormone (β-MSH 5-22) afforded cyclized product 34 in 30% yield. An analog of the diabetes drug Liraglutide (without the lipophilic conjugate on the side chain of Lys20) gave 35 in 48% yield at 1 mM reaction concentration. Based on LC analysis, a slightly cleaner transformation was observed when the reaction was conducted at 5 μM (LC trace 8). Finally, the reaction of 33-mer glucagon-like peptide 2 (GLP-2), featuring 28 spacing AA units, gave 33 in 28% yield, along with acyclic isoindolinone side products. Using peptide substrate H-Ala-Val-Gly-Lys-NH 2 26, the macrocyclization reaction was also amenable to a range of OPA analogs (see 36-40, Fig. 3c). Importantly, using 41 as a model compound, we showed that OPA-2amines peptide stapling can be performed in conjunction with solid-phase peptide synthesis (Fig. 3d). Treatment of partially protected peptide substrate 41 on resin with OPA in TFE (condition [D]) followed by TFA-mediated deprotection and cleavage gave 42 bearing a free Cys residue in excellent conversion and selectivity (LC trace 9).
Side-to-side stapling of peptides by OPA-2amines condensation. As shown in Fig. 4a, the OPA-2amines reaction could also enable highly efficient side chain to side chain (side-to-side) stapling of linear peptides via two Lys side chains under the standard conditions already described in end-to-side stapling. For example, the reaction of pentapeptide 43 with an Ac-protected Nterminus with OPA (1.0 equiv) in 10 min formed the cyclic product 44 in excellent yield (LC trace 10). Notably, the product was obtained as a mixture of two regioisomers in a close ratio due to the similar reactivity of the two Lys side chains. As seen in the end-to-side stapling of 17, stapling of 43 at 5 mM can be completed within 10 seconds (LC trace 11). Stapling of 43 at 50 μM for 2 min gave almost identical results (see Supplementary  Fig. 32). Stapling of 43 also worked well at relatively high concentration, such as 50 mM, whereas a small amount of double-OPA side product (~5% based on LC analysis) was formed Reaction of substrates with free N-terminal NH 2 . Yield is based on HPLC isolated product of reactions conducted at 5.0 mM concentration and 0.01-0.05 mmol scale unless otherwise specified. All products were obtained as a mixture of regioisomers. a LC-estimated yield. b A small aliquot of the reaction mixture was taken and quenched with mixed solvents of H 2 O/MeOH/HCO 2 H before subjection to LC-MS analysis. c Adaption from an α helical sequence of RNase A. d Biotin was attached to the N-terminus. e Adaption from peptide ATSP-7041. f The structures of the major side-to-side (ss) stapled products were confirmed by comparison with the samples prepared from properly protected substrates, see Supplementary Fig. 48 for details. The minor peaks were assigned as the end-to-side (es) products according to molecular weight and their structures have not been vigorously confirmed. The ss/es ratio was estimated by LC-MS analysis. g Reaction was conducted at 1.0 mM for 10 min. h Reaction was conducted at 20.0 μM for 10 min. Nal: L-1-Naphthylalanine. PA: picolinic acid. The uncapped N-terminal AA units are marked in ocean blue, the lysine units and amino groups are marked in red, the OPA reagent and isoindolinimine core are marked in green.
when the concentration was further increased to 100 mM (see Supplementary Fig. 67). Similar to end-to-side stapling, using alcohol as a cosolvent facilitated the desired macrocyclization. A considerable amount of acyclic isoindolinone side products 45 and 46 were formed when the ratio of MeOH vs buffer was lowered from 1:1 to 1:9 (condition [A'], LC trace 12).
Substrates with a wide range of spacing units between the two Lys residues were well tolerated, and all proteinogenic AAs except for Cys are compatible (Fig. 4b). The reaction was adaptable to various unnatural AA units such as ornithine (Orn, 57) and capping groups such as biotin (54) could be incorporated. As shown by 49, the secondary amine group in N-terminal Pro remained unaffected by the reaction. The utility of the side-toside peptide stapling technique was demonstrated by the synthesis of various biomimetic analogs. For example, the reaction of a 15-mer peptide sequence derived from RNase A gave 53 in excellent yield 46 . Moreover, the macrocyclization of a 14-mer sequence derived from ATSP-7041 47 , a peptide inhibitor of the p53-MDM2 interaction, at 1 mM gave 57 in 64% yield via side-to-side stapling of two Orn side chains. As shown in LC trace 13, slightly cleaner transformation was observed when the reaction were conducted at 20 μM concentration. The macrocyclization reaction can be used to generate complex bicyclic products as exemplified by the reaction of 56, a macrolactam peptide substrate with two Lys, which undergoes cyclization to afford the bicyclic product in excellent yield and selectivity. As shown by 58-60, the stapling of substrates bearing two Lys side chains and unblocked N-terminal amine groups proceeded with high side-to-side vs end-to-side selectivity, indicating the higher reactivity of the Lys side chain than the N-terminus in our reaction system (Fig. 3c) 45 .
Further extension of isoindolinimine products. In addition to the excellent stability of the macrocyclic isoindolinimine products in an aqueous medium at rt over a wide range of pH values, they could also undergo further chemoselective reaction with certain electron-deficient π electrophiles under the same stapling conditions, enabling the highly efficient and facile construction of complex structures from simple linear precursors in a one-pot fashion (Fig. 5a). For example, when 26 was reacted with a large excess of OPA (3.0 equiv) in a H 2 O/TFE mixture (condition [C]) for 30 min, the double-OPA macrocyclization product 61 was cleanly formed in 73% yield. However, the success of the condensation reaction depends strongly on the nature of the aldehyde reaction partner, and other aldehydes, such as benzaldehyde, methylglyoxal, and glyoxylic acid, gave little 61-like product (see Supplementary Fig. 50). Similar to the reactivity of the 1-thio-isoindole product generated by the OPA-amine-thiol reaction, 26 can likewise undergo further addition reactions with appropriate electrophiles. For example, treatment of 27 with N-methyl maleimide 62 (3.0 equiv) 22 for 15 min gave the C 3alkylation product 63 in 68% yield. Alkyne DMAC 66 can act in a manner analogous to maleimide for the one-pot addition reaction, but the reactivity towards DMAC strongly depends on the reaction partner. Hence, whereas 27 reacts sluggishly giving 67 in low yield, under the same conditions the linear substrate H-Phe-Leu-Gly-Lys-Asn-NH 2 reacts to give the addition product in good yield (67%, see Supplementary Fig. 59 for details). Moreover, βnitrostyrene 64 is also an efficient acceptor for the sequential reaction using 27 as the substrate, forming 65 in high yield. It should be noted that similar results were obtained for this onepot procedure using either condition [A] or [B]. Reactions of 27 with methyl acrylate, vinyl sulfones, and 4-phenyl-3H-1,2,4triazoline-3,5(4H)-diones gave little desired products, whereas the reaction with aryl diazonium salts gave a complex mixture (see Supplementary Fig. 62).
In general, isoindoles are more reactive than indoles. The plain isoindole predominately exists in the 2H-isoindole form, which can tautomerize to 1H-isoindole at very small concentration via equilibrium (Fig. 5b) 48 . However, the ratio of the two tautomers can vary depending on the substitutions on isoindoles. In contrast with the 1-thiol-isoindole core generated by the OPA-thiol-amine reaction, the isoindole core generated by the OPA-2amines reaction predominantly exists in the isoindolin-1-imine form I, which can be viewed as a variant of 1H-isoindole. Previously studies have shown that 2H-isoindoles can undergo Diels-Alder (D-A) type of reaction with dienophiles across the C1-C3 and electrophilic substitution reaction at C1/C3 [49][50][51] . We propose that isoindolinimine I can tautomerize to 1-aminoisoindoline II, which is generated in small concentration but likely much more reactive that II. II can undergo Diels-Alder reaction with dienophile such as maleimides to generate cycloadduct III, which immediately rearranges to give the final C3 substitution product IV possibly due to the strain around the C1 aminal center of III 50 . NMR and LC-MS-based analysis of the model reaction of 68 with 62 did not identify any stable cycloadduct intermediate in our hands (see Supplementary Fig. 54 for details). Alternatively, II can undergo nucleophilic addition to alkene electrophiles to directly give IV. Nucleophilic addition of II to the aldehyde group of OPA followed by dehydration is likely responsible for the formation of the corresponding C3 condensation products (see 61) 51 .
As outlined in Fig. 5c, sequential addition of OPA and FITCconjugated maleimide 70 to the RGD-containing sequence 69 gave the fluorescently labeled product 71 in 61% yield over two steps (LC trace 16). The usefulness of our one-pot approach could even be extended to a three-step sequential addition of OPA, 4bromo-OPA 72, and methoxyamine to substrate 26, giving the triple-modified product 74 in good yield (Fig. 5d).
Through reinvestigating the classical OPA-amine-thiol condensation chemistry, we found that OPA quickly reacted with two primary alkyl amino groups to selectively form isoindolinimine products, whose chemical reactivity has been rarely explored. The OPA-2amines-mediated macrocyclization reaction provides a practical and broadly applicable method for the fast stapling of unprotected peptides via the reaction of the amino groups from two AA residues and an OPA or OPA analogs in both side-to-side and end-to-side fashions under mild conditions. Moreover, the resulting isoindolinimine linkage can undergo further reaction with electron-deficient π electrophiles under the same cyclization conditions in one pot. Overall, the reported stapling methodology and one-pot sequential addition reactions offer a powerful strategy to selectively modify unprotected peptides and construct diverse macrocyclic structures with readily accessible peptide precursors and inexpensive reagents.

Methods
Typical procedure for both end-to-side and side-to-side peptide stapling. To a solution of linear peptide precursor 18 (32.7 mg, 0.05 mmol, 1.0 equiv) in 10.0 mL of PBS buffer (pH 8.0) / MeOH (1:1), OPA was added (1.0 equiv, 50 μL of 1 M stock solution in MeOH). The reaction mixture was stirred in an air atmosphere for 10 min at rt. The reaction mixture was then purified by semipreparative HPLC in two batches with a reverse-phase C18 column (H 2 O and MeCN with 0.1% HCO 2 H as eluents) to give the product 19a as a yellowish powder after lyophilization (23.2 mg, 77%).