Design and Synthesis of Crosslink-Dense Peptides by Manipulating

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Design and Synthesis of Crosslink-Dense Peptides by Manipulating Regioselective Bisthioether Crosslinking and Orthogonal Disulfide Pairing Huilei Dong, Xiaoting Meng, Xiaoli Zheng, Xueting Cheng, Yiwu Zheng, Yibing Zhao, and Chuanliu Wu J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b00164 • Publication Date (Web): 21 Mar 2019 Downloaded from http://pubs.acs.org on March 22, 2019

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The Journal of Organic Chemistry

Design and Synthesis of Crosslink-Dense Peptides by Manipulating Regioselective Bisthioether Crosslinking and Orthogonal Disulfide Pairing

Huilei Dong, Xiaoting Meng, Xiaoli Zheng, Xueting Cheng, Yiwu Zheng, Yibing Zhao, Chuanliu Wu*

Department of Chemistry, College of Chemistry and Chemical Engineering, State Key Laboratory of Physical Chemistry of Solid Surfaces, The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, Xiamen University, Xiamen, 361005, P.R. China. *To whom correspondence should be addressed, Email: [email protected]

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ABSTRACT: Existing disulfide-rich peptides, both naturally occurring and de novo designed, only represent a tiny amount of the possible sequence space, because natural evolution and de novo design only keeps sequences that are structurally approachable by correct disulfide pairings. To bypass this limitation for designing new peptide scaffolds beyond the natural sequence space, we dedicate to developing novel disulfide-rich peptides with pre-defined disulfide pairing patterns irrelevant to primary sequences. However, most of these designed peptides still suffer from disulfide rearrangements to at least 1‒3 possible isomers. Here we report a general and reliable strategy for the design and synthesis of a range of structurally diverse crosslink-dense peptide (CDP) scaffolds with two orthogonal disulfide bonds and a bisthioether bridge that are not subject to disulfide isomerizations. Altering the pattern of cysteine and penicillamine generates hundreds of different CDP scaffolds tolerant to extensive sequence manipulations. This work thus provides many useful scaffolds for the design of functional molecules such as protein binders with improved proteolytic stability (e.g., designed by epitope grafting).

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Introduction Disulfide bonds are the most prevalent crosslinks in peptides and proteins, which regulate the folding of polypeptides, enhance their thermodynamic stability and mechanical resistance, and prevent their fibrilization or oligomerization.1 Moreover, new disulfides appearing in peptides and proteins during evolution can help warp folds acquire new structures and functions, a scenario that has diversified protein folds.2 A class of well-folded peptides that has benefited from this scenario for structural and functional evolution is disulfide-rich peptides, which contain two or more disulfide bonds and have emerged as promising molecules for diagnostic and therapeutic applications.3 Among them, two peptides with three disulfides have already been approved as drugs (i.e., Ziconotide and Linaclotide),4 while even more are currently undergoing preclinical studies or are in clinical trials.5 In addition, novel disulfide-rich peptides with new functions can be designed through epitope grafting,6 computational methods,7 and directed evolution.8 However, considering that there are 2030 (~1039) sequences for even a small peptide of 30 amino acids within the entire sequence space,9 existing natural and designed disulfide-rich peptides (~103‒105, with no regard for sequence length) only occupy an infinitesimal fraction of the possible sequences.1a, 10 Natural evolution and de novo design only keeps sequences that are structurally approachable by correct disulfide connectivities. The vast majority of random sequences that are incapable of directing the correct disulfide pairing between cysteines may thus be discarded. To approach the entire sequence space for designing disulfide-rich peptides with new structures and functions, we have to break through the dogma of the “sequence determines disulfide pairing” by developing a new type of disulfide-rich peptide scaffolds that are tolerant to sequence manipulations, or in other words, peptide scaffolds with sequence-independent disulfide connectivities. Our efforts towards this direction resulted in the invention of precise disulfide pairing chemistry involving the synergistic manipulation of CXC (cysteine-any-cysteine) motifs and penicillamine (Pen, a cysteine analog), which has yielded a range of sequence-independent C/Pen-rich peptide scaffolds.11 However, except for C/Pen-rich peptides with two disulfides,11b, 11e the precise disulfide pairing to form the desired peptide scaffolds with three disulfides is still besieged by a minimum of 1‒3 possible isomers,11d which ACS Paragon Plus Environment

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limits the number of random sequences approachable to the designer scaffolds (Figure 1a). While the problem on disulfide isomerizations might be solved by replacing all disulfide bonds in peptides with stable crosslinks through a variety of orthogonal reactions,12 none of them proceeds as efficient as the disulfide formation; and moreover, introducing two or more stable crosslinks simultaneously in randomsequence peptides is technically sophisticated and is predictably of lower yield. Herein, we report a general and reliable strategy for the design and synthesis of a range of structurally diverse crosslink-dense peptide (CDP) scaffolds tolerant to extensive sequence manipulations. Our strategy takes advantage of the CXPen (or PenXC) motifs for generating two orthogonal disulfide bonds and the regioselective thiol-substitutions for generating stable bisthioether crosslinks to direct the folding of peptides into specific structures without isomers in a sequence-independent manner (Figure 1b). Altering the pattern (or framework) of cysteine, Pen, and bisthioether crosslink can generate hundreds of CDP scaffolds (Figure 2). This strategy would enable the design and discovery of novel multicyclic peptides with new functions beyond the naturally occurring sequence space for general applications. Our design on possible CDP scaffolds relies on the tunability of positions in linear peptides to place C/Pen residues, CXPen/PenXC motifs, and bisthioether crosslinks. For any linear peptide like that shown in Figure 2, there are different ways to place three cysteines, one Pen and CXPen or PenXC motif, which generates peptides with 40 different C/Pen patterns. In these C/Pen-patterned peptides, two of the three isolated cysteines can be crosslinked through selective bisthioether crosslinking, which amplifies the C/Pen pattern diversity by a factor of three (Figures 1b and 2). Accordingly, a total of 120 different peptide scaffolds with one bisthioether crosslink and two C-Pen disulfide bonds can be obtained conveniently through the orthogonal disulfide pairing (i.e., the cysteine and Pen residue in CXPen/PenXC motifs only forms the disulfide bond with the isolated Pen and cysteine, respectively). In addition, the scaffold diversity can be further increased by placing the bisthioether crosslink in the position X of the CXPen/PenXC motifs, leading to the generation of additional 48 different scaffolds


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(Figure 2). A number of reagents containing two thiol-reactive groups can be explored for generating the bisthioether crosslinks, which further increases the scaffold diversity.13


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Figure 1. a) Four expected folds formed after oxidative folding predicted based on the CXPen motif directed precise disulfide pairing rules reported previously,11d that is: 1) cysteine (C) only forms a disulfide bond with penicillamine (Pen); 2) no formation of the intra-CXPen disulfide. -x-x-x-xrepresents any peptide segments without thiol-bearing amino acids. Cysteine and Pen residues were sequentially numbered with Roman numerals. b) Design and synthesis of novel CDP scaffolds by manipulating regioselective bisthioether crosslinking and CXPen/PenXC-directed orthogonal disulfide pairing.


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Figure 2. Illustration of the diverse CDP scaffolds that can be designed by arbitrarily placing three cysteines (C), one Pen, and one CXPen or PenXC motif into linear peptides; -x-x-x-x- represents any peptide segments without thiol-bearing amino acids, purple lines represent bisthioether crosslinkers, red lines represent disulfide bonds. Additional 48 special peptide scaffolds have a bisthioether crosslinker directly conjugating onto the CXPen or PenXC motif.


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Figure 3. Amino acid sequences of peptides 1‒3 and structures of the three crosslinkers; cysteine residues in purple were deprotected first for bisthioether crosslinking; other cysteine and Pen residues were originally protected using Acm, which were deprotected for oxidation after the bisthioether crosslinking. Chromatograms showing the oxidation of the Acm-deprotected peptides in oxidized glutathione (GSSG) buffers (black lines: before oxidation; red lines: after oxidation; 1-a, 2-a and 3-a: peptides (1, 2 and 3, respectively) crosslinked by diiodomethane; 1-b, 2-b and 3-b: peptides crosslinked by 1,3-bis(bromomethyl)benzene; 1-c, 2-c and 3-c: peptides crosslinked by hexafluorobenzene). Topological drawings of the oxidative folding products were given above the corresponding chromatograms (purple and red lines denote bisthioether crosslinks and disulfide bonds, respectively).


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Results and Discussion Although stable crosslinks have been used as disulfide surrogates to regulate the folding of natural disulfide-rich peptides and proteins,12d, 14 none of them has been used in sequence-independent C/Penrich peptide scaffolds to design new and more robust multicyclic peptide scaffolds. Our work chose three

different

kinds

of

crosslinkers

diiodomethane,

1,3-bis(bromomethyl)benzene

and

hexafluorobenzene (Figure 3), and achieved regioselective bisthioether crosslinking by means of the thiol-protecting group acetamidomethyl (S-Acm).15 We first designed and synthesized two model peptides with different C/Pen patterns (1 and 2), in which lysine and tryptophan residues are strategically inserted for facilitating tryptic digestion analysis of the crosslink connectivities (Figure 3). After the bisthioether crosslinking and the deprotection of the S-Acm groups, oxidative folding of these peptides leads to exclusive formation of the desired products, as monitored by HPLC and tryptic digestion followed by LC-MS analysis (Figures 3 and S1-S6). The crosslink type does not affect the oxidative folding efficiency. We further designed and synthesized a third model peptide (3) with the crosslinks located onto the CXPen motif (Figure 3), both the crosslinking and oxidative folding are highly efficient, like the previous two peptides. As the three model peptides have a very similar sequence composed of primarily achiral glycine residues, the oxidative folding directions are certainly not driven by the primary amino acid sequence; instead they are directed by the orthogonal C/Pen disulfide pairing. To demonstrate the robustness of the strategy for manipulating random sequences, we then played on random phases by transforming keywords of the present work “CROSSLINK-DENSE PEPTIDE” and “ENTIRE SEQUENCE SPACE” into two different peptide sequences (Figure 4). Letters in these words do not code the conventional amino acids were replaced with natural ones. Lysine residues were strategically placed to facilitate the analysis of crosslink connectivity by tryptic digestion followed by LC-MS analysis. Scaffolds corresponding to the knotted crosslink connectivity (1-4, 2-5, 3-6) were selected for incorporating these sequences (4 and 5). After the crosslinking of free cysteines by diiodomethane, both peptides can be highly efficiently folded into the desired topologies after the ACS Paragon Plus Environment

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oxidation (peak-to-peak conversion observed from the HPLC chromatograms; Figures 4 and S10, S11). This exploration on random sequences indicates the feasibility of approaching the entire sequence space for discovering or designing CDPs with new structures and functions. Smaller and more constrained disulfide-rich peptides are usually more difficult to fold correctly.12d, 16 However, miniature peptide scaffolds may possess advantages over larger ones in terms of designability, applicability, production cost, and oral or deep-tissue bioavailability.17 Considering how robust the present strategy for the folding of multicyclic peptides is, we then dedicated to designing miniature CDP scaffolds consisting of two fixed mini-loops and a variable loop that can be arbitrarily grafted onto the two fixed mini-loops. We conceived that the two fixed mini-loops would provide a structurallyconstrained scaffold for displaying an additional variable loop which can further be replaced by bioactive sequences so that diverse CDPs with new biological functions can be facilely generated. This design philosophy is reminiscent of evolution of new proteins from primordial peptides.18 Our design began with an antiparallel CXPen dimer (i.e., a stable 22-membered mini-loop) and a proline-mediated β-turn motif which are terminally linked together to form a fused bicycle (6; Figure 5a).11d, 19 CD spectra indicate that the fused bicycle display a typical β-hairpin structure (Figure 5b), whereas the reduced peptide is largely disordered (Figure S49). This result is not unexpected as paired i/i+2 disulfides frequently serve as β-structure nucleators in both natural and designer peptides.20 Peptides 7‒10 were then designed and synthesized, in which a short peptide segment at the C-terminus was respectively grafted onto four different locations on the scaffold sequence segment through a thioacetal linkage formed between two cysteines (Figure 5a). All peptides can be efficiently folded into the expected tricyclic topologies after the oxidation (Figure 5b). In addition, we found that one of the four peptides (10) lost the structural characteristic of β-hairpins (Figure 5b), indicating that sequence-dependent folding of secondary structures is not the primary driven force for the precise disulfide pairing, and these tricyclic designs are applicable to design diverse structures if both the scaffold and grafted sequences are rationally manipulated.


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Figure 4. Amino acid sequences of peptides 4 and 5 and the connectivities of bisthioether crosslinks and disulfide bonds. Chromatograms showing the oxidation of the thioacetal-cyclized peptides in GSSG buffers (black lines: before oxidation; red lines: after oxidation). Topological drawings of the knotted folds were given above the corresponding chromatograms (purple and red lines denote bisthioether crosslinks and disulfide bonds, respectively).


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Figure 5. a) Amino acid sequences of peptide 6‒10 (cysteine residues in purple were inserted for crosslinking by diiodomethane) and illustration of the grafting of a C-terminal flexible loop onto a βhairpin scaffold. b) CD spectra of oxidized peptides 6‒10 (30 μM) in aqueous solutions. c) Chromatograms showing the products formed from the oxidation of the thioacetal-cyclized peptides (7‒10) in GSSG buffers.


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Finally, we explored the applicability of our arbitrarily-designed CDP scaffolds for creating protein binders through the epitope grafting strategy. In the first example, an integrin binding motif discovered from a yeast display library (PRPRGDNPPLT) was selected for the exploration,6b, 11c which was grafted into the variable loop of the tricyclic peptide 8 (Figure 6a). As expected, the epitope-bearing peptide (11) can be efficiently folded into the specific tricyclic topology (Figure S50), which can block the adhesion of U87 glioblastoma cells to cell culture plates (Figure 6b; tricyclic 8 and commercially provided cyclic RGD (cyclo-RGDyK) serve as a negative and positive control, respectively), suggesting that the binding capability of the epitope to cell-surface integrins is preserved after the grafting. Secondly, L76‒L84 (LDEETGEFL) of the nuclear factor erythroid-derived related factor 2 (Nrf2) responsible for the binding of the Kelch-like ECH-associated protein 1 (Keap1) was grafted using the tricyclic 7 and 10 as scaffolds (12 and 13; Figure 6a).21 The oxidative folding of 12 and 13 is as efficient as that of the previously designed peptides (Figure S50). Their binding affinity to the Keap1 Kelch domain was evaluated by using a previously validated fluorescence polarization competition assay (Figure 6c).21a Tricyclic 12 has a Ki of 82.8 nM, which is comparable to that of linear 12 (Ki = 65.7 nM), but significantly lower than that of tricyclic 13 (Ki = 525.8 nM; linear 13: Ki = 66.2 nM), indicating that the tricyclic structure can strongly affect the binding interaction. Moreover, tricyclic 12 and 13 are both significantly more stable towards proteolysis by chymotrypsin compared to their linear forms (Figure 6d). Epitope grafting has been one of the most promising strategies for designing bioactive peptides based on naturally occurring peptide scaffolds.22 The proof-of-concept examples presented herein demonstrate that our designed CDP scaffolds can provide an alternative for designing novel protein binders.


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Figure 6. a) Grafting of protein binding sequences into the CPD scaffolds 7, 8 and 10 to design peptides 11‒13. b) Inhibition of cell adhesion by peptides (8, 11, and c(RGDyK); 50 μM) determined by MTT assays. c) Fluorescence polarization competition assays determine the binding affinity of peptides with Keap1. Linear peptides are peptides without disulfide and thioacetal crosslinking. d) Kinetics of peptide degradation by chymotrypsin in phosphate buffer at pH 7.4. Concentration of peptides and chymotrypsin was 50 μM and 5 μg·mL-1, respectively. Data were expressed as mean ± standard deviation (n = 3).


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Conclusions In summary, this work describes a general and reliable strategy for the design and synthesis of structurally diverse CDP scaffolds tolerant to extensive sequence manipulations. This strategy relies on the use of regioselective bisthioether crosslinking and CXPen/PenXC-based orthogonal disulfide pairing in combination for directing the folding of peptides into expected multicyclic topologies without isomers in a sequence-independent manner. We evaluated the tolerance of the designed peptide scaffolds to sequence manipulations by arbitrarily tuning the pattern of the crosslinkers (thioether and disulfide bonds) and the primary sequence of peptides. All designed peptides can be highly efficiently folded into the desired multicyclic topologies with a negligible formation of possible isomers. Thus, we expect that our CDP scaffolds would enable the design and discovery of novel structurally constrained peptides beyond the naturally occurring sequence space. This would lead to the creation of novel multicyclic peptides with new functions and structures for applications in the field of drug discovery, biomaterials, and catalysis.

Experimental Section Materials. Rink amide MBHA resin (0.365 mmol/g loading), 2-chlorotrityl chloride resin (0.968 mmol/g loading) and Fmoc-protected amino acids were purchased from GL Biochem (Shanghai, China). Ethyl

cyanoglyoxylate-2-oxime

(Oxyma),

N,N'-diisopropylcarbodiimide

(DIC),

tris(2-

carboxyethyl)phosphine hydrochloride (TCEP), ethyldiisopropylamine (DIPEA), piperidine and trifluoroacetic

acid

(TFA)

were

supplied

by

Energy

Chemical

(Shanghai,

China).

4-

Mercaptophenylactic acid (MPAA) was bought from Alfa Aesar (USA). Hexafluorobenzene, 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), silver tetrafluoroborate (AgBF4), diiodomethane (CH2I2), 1,3-bis(bromomethyl)benzene and L-glutathione oxidized (GSSG) were purchased from Sigma-Aldrich (Shanghai, China). Instruments. High performance liquid chromatography (HPLC) was performed by a Shimadzu system equipped with a LC-20AD solvent delivery unit, a DGU-20A3R degassing unit, a SIL-20A autosampler, ACS Paragon Plus Environment

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a CTO-20A column oven, and a SPD-M20A photodiode array detector. Chromatograms were recorded with a HPLC InertSustain C18 5 μm (4.6×250 mm) column at a flow rate of 1.0 mL·min-1 by using gradients of ACN (0.1% TFA) and water (0.1% TFA). A Hitachi U-3900H UV-vis spectrophotometer was used for measuring peptide concentrations. A Bruker En Apex ultra 7.0T FT-MS and Bruker Esquire 3000 plus ion trap ESI mass spectrometer were used for identifying isolated peptides and fragments generated from tryptic digestion. An Olympus CKX41 inverted microscope was used for observing the ability of RGD containing peptides to block U87 cells adhesion. The solid phase peptide synthesis was performed on a CEM Liberty BlueTM automated microwave peptide synthesizer (CEM Corporation, Matthews, NC). Preparation of 2-chlorotrityl hydrazine resin 2-Chlorotrityl hydrazine resin was synthesized according to the previously described protocol.23 In brief, 2-chlorotrityl chloride resin (0.5 g) was swelled in 50% (v/v) DMF/DCM (5.0 mL) for 30 min, and then drain it. Then 4.0 mL of 5.0% (v/v) NH2NH2/DMF was added to the resin for hydrazination and gently agitate it for 30 min at 30 oC. After that, 1.0 mL of methanol was added to the solution and stirred for 10 min to cap the unreacted sites. The resulting resin was washed with DMF, H2O, DMF, MeOH and Et2O thoroughly in sequence. The resin was dried under high vacuum for 2.0 h and determined to have a loading of 0.753 mmol/g. General procedures for SPPS All peptides were synthesized at 0.025 or 0.05 mmol scales using the method of Fmoc solid-phase synthesis on the CEM Liberty BlueTM automated microwave peptide synthesizer. Deprotection was performed with 20% piperidine in DMF. Coupling reactions were performed with a 5-fold excess of Fmoc-protected amino acid, 0.5 M DIC in DMF and 1.0 M Oxyma Pure in DMF. Unless noted, coupling of the amino acid to the resin was performed within the reaction vessel using standard coupling. Each standard cycle involved a single 1.0 min/90 oC deprotection and a single 2.0 min/90 oC microwave coupling method. Cys was coupled with a special 2.0 min/RT-4min/50 °C microwave coupling method and Pen was coupled with a special 2.0 min/RT-18 min/50 °C microwave coupling method. Peptides ACS Paragon Plus Environment

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were cleaved from the resin and deprotected (except for the Acm group) by treatment with a cleavage cocktail containing TFA/phenol/TIPS/H2O in a ratio of 88/5.0/5.0/2.0 for 2.0 h at room temperature. Following cleavage, the peptide was precipitated in Et2O and purified by HPLC. Native chemical ligation The general procedure for ligation of peptides using peptide hydrazides was followed as reported previously.24 In brief, peptide hydrazides were dissolved in 6.0 M Gn·HCl containing 200 mM MPAA in a 2.0 mL Eppendorf tube to reach a concentration of 2.0 mM and adjust the pH to 3.0. Then, add 5 eq acetyl acetone (400 mM in aqueous solution) to the peptide mixture, and the mixture was allowed to stir for 4.0 h at room temperature. After 4.0 h, an equimolar amount of Cys-fragment peptide was dissolved in 6.0 M Gn·HCl with 200 mM Na2HPO4 and 50 mM TCEP to an volume equal to the thioesterification reaction mixture. Then the two solutions were mixed together and the pH was slowly adjusted to 7.0-7.4 with 2.0 M NaOH. The ligation reaction then stirred for 12 h. In this work, peptide 4 and peptide 12 were synthesized by NCL method, because this two peptides cannot be synthesized in one step. Reactions of peptides with diiodomethane To a solution of peptide (4.0 mM, 50 μL) in an Eppendorf tube, TCEP (50 mM in aqueous solution, 1.5 eq) and K2CO3 (29 mM in aqueous solution, 3.0 eq) was added. The reaction was shaken at room temperature for 15 min. Then trimethylamine (300 mM in THF, 20 eq) and diiodomethane (250 mM in THF, 20 eq) were added sequentially to the reaction mixture.25 It should be noted that the final concentration of peptide was 1.0 mM and the ratio of H2O and THF was 3:2. The reaction mixture was shaken at room temperature for 12 h. Reaction progress was monitored by HPLC (a flow rate of 1.0 mL·min−1 flow rate of H2O (+0.1 TFA) and ACN (+0.1 TFA); isocratic with 5.0% ACN (+0.1 TFA) for 5.0 min followed by a linear gradient of 5.0% to 60% ACN (+0.1 TFA) over 40 min) and ESI-MS. Reactions of peptides with hexafluorobenzene To a stock solution of peptide dissolved in DMF (3.0 mM, 20 μL) in an Eppendorf tube, hexafluorabenzene (100 mM in DMF, 40 eq) and TRIS base (50 mM in DMF, 40 eq) was added. The reaction mixture was shaken at room temperature for 3.0 h. Then, TCEP (5.0 mM in DMF, 0.5 eq) was ACS Paragon Plus Environment

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added to the reaction mixture and left at room temperature for another 3.0 h.26 The progress of the reaction was monitored by HPLC (a flow rate of 1.0 mL·min−1 flow rate of H2O (+0.1 TFA) and ACN (+0.1 TFA); isocratic with 5.0% ACN (+0.1 TFA) for 5.0 min followed by a linear gradient of 5.0% to 60% ACN (+0.1 TFA) over 40 min) and ESI-MS. Reactions of peptides with 1,3-bis(bromomethyl)benzene To a solution of peptide (3.0 mM in NH4HCO3 buffer, pH 8.0, 20 μL) in an Eppendorf tube, 1,3bis(bromomethyl)benzene (25 mM in acetonitrile, 1.1 eq) was added. The final concentration of peptide was 0.6 mM and the ratio of ACN and NH4HCO3 (100 mM) was 1:1. The reaction mixture was shaken at room temperature for 1.5 h.27 The progress of the reaction was monitored by HPLC (a flow rate of 1.0 mL·min−1 flow rate of H2O (+0.1 TFA) and ACN (+0.1 TFA); isocratic with 5.0% ACN (+0.1 TFA) for 5.0 min followed by a linear gradient of 5.0% to 60% ACN (+0.1 TFA) over 40 min) and ESI-MS. Removal of the Acm group This procedure is slightly modified from the protocol reported previously.15 To a solid sample of peptide (1.0 μmol) in a Eppendorf tube was dissolved by 200 μL of cold TFA followed by the addition of AgBF4 (20 eq/Acm group) and anisol (10 eq/Acm group) and reaction at 4 °C for 2.0 h. The peptides were recovered as the silver salt by centrifugation of precipitated form 60% (v/v) anhydrous ether. Then the precipitated was dissolved in 1.0 M acetic acid containing DTT (40 eq/Acm group) and left at room temperature for 3.0 h. The progress of the reaction was monitored by HPLC (a flow rate of 1.0 mL·min−1 flow rate of H2O (+0.1 TFA) and ACN (+0.1 TFA); isocratic with 5.0% ACN (+0.1 TFA) for 5.0 min followed by a linear gradient of 5.0% to 60% ACN (+0.1 TFA) over 40 min) and ESI-MS. Oxidative folding of peptides Fully reduced peptides (thioacetal-cyclized peptides) were dissolved in 100 mM phosphate buffer (pH 7.4) containing 0.5 mM GSSG to achieve a concentration of 50 μM. Two hours later, the reaction was quenched by adding 5.0% TFA, and the samples were then analyzed by analytical HPLC (a flow rate of 1.0 mL·min−1 flow rate of H2O (+0.1 TFA) and ACN (+0.1 TFA), isocratic with 5.0% ACN (+0.1 TFA) for 5.0 min followed by a linear gradient of 5.0% to 95% ACN (+0.1 TFA) over 40 min). ACS Paragon Plus Environment

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Tryptic digestion HPLC/MS analysis of oxidative peptides The oxidative peptides (~5.0−10 μg) were dissolved in 95 μL phosphate buffer (100 mM, pH 7.4), which was then digested by addition of 5.0 μL aqueous solution of trypsin (1.0 mg/mL) at 37°C for 2.0 h. The digested fragments were analyzed and characterized by HPLC (a flow rate of 1.0 mL·min−1 flow rate of H2O (+0.1 TFA) and ACN (+0.1 TFA); isocratic with 5.0% ACN (+0.1 TFA) for 5.0 min followed by a linear gradient of 5.0% to 50% ACN (+0.1 TFA) over 70 min) and ESI-MS. Circular Dichroism (CD) Spectroscopy CD spectra were measured at room temperature (25 °C) using a 0.1 cm path length cuvette. The spectra were recorded in wavelength range of 190−260 nm and averaged over 3 scans with a bandwidth of 2.0 nm, a resolution of 1.0 nm, and a response time of 8.0 s. The sensitivity and the speed of the spectrometer were set to 100 mdeg and 50 nm·min−1, respectively. All peptides were dissolved in aqueous to reach a concentration of 30 μM. Integrin-dependent cell adhesion assay Cell adhesion assay was evaluated by MTT assay.28 U87MG cells were incubated in a 24-well plate with an initial cell density of 80000 cells per well and grown overnight in a 37 oC incubator with 5.0% CO2 atmosphere. After 24 h, the medium was removed and the peptides (50 μM) were added to each well in 300 μL DMEM contained FBS, incubated at 37 °C, 5.0% CO2 for 3.0 h. After that, the medium was removed and cells were washed three times with DMEM to remove the detached cells. Then 300 μL fresh DMEM and 30 μL MTT (50 mg/mL) were added to each well. The cells were incubated for 4.0 h at 37 °C in culture hood. After that, removed the supernatant and added 300 µL MTT solvent (DMSO). Cover with tinfoil and agitate cells on orbital shaker for 15 min. The absorbance was recorded at 490 nm using an ELIASA reader (PerkinElmer Enspire®). The obtained absorbance was blank-corrected (blank: DMEM+MTT, no cells) and the cell viability in percent was calculated according to the following equation:

Cell viability = (OD490, sample/OD490, control) × 100% ACS Paragon Plus Environment

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where OD490, sample represents the optical density of the cells treated with peptides and OD490, control is the cells only treated with DMEM. Fluorescence polarization assay The peptide-protein interaction assays were measured by fluorescence polarization (FP) assay in 10 mM PBS (pH 7.4). Fluorescence anisotropy was recorded in a 96-well plate by Infinite® 200 PRO multimode microplate readers (TECAN). A model peptide (DEETGEF) which had been reported to effectively interact with Keap1 was labeled with fluorescein isothiocyanate (FITC) via the conjugation of residue β-Ala (FTIC-ETGE, FITC-[β-Ala]-DEETGEF).29 For a fluorescence polarization competition assay, FTIC-ETGE/Keap1 complexes (20 nM/300 nM) was prepared first and then treated with the peptides ranging from 1.0 nM to 4.0 μM. The experiments were repeated 3 times. The polarization data were fitted using Origin 8.0 based on one-site competitive model. Proteolytic stability assay Peptides were dissolved in NH4HCO3 buffer (100 mM, pH 8.0), and then treated with an aqueous solution of chymotrypsin at 37°C. The concentrations of peptides and chymotrypsin were 50 μM and 5.0 μg·mL-1, respectively. At predefined times, aliquots were taken and quenched with 5.0% aqueous TFA. The samples were then analyzed by analytical HPLC. The amount of intact peptide remained in the mixture was quantified by the peak area of the intact peptide. The digestion at each time points was repeated three times to give the average values.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website, including the HPLC, MS CD and fluorescence polarization characterization of peptides (PDF). AUTHOR INFORMATION Corresponding Author C. Wu, [email protected] Notes The authors declare no competing financial interests. ACKNOWLEDGMENT We would like to acknowledge the financial support from the National Natural Science Foundation of China (91853112, 21822404 and 21675132), the Fundamental Research Funds for the Central Universities (20720180034), the Program for Changjiang Scholars and Innovative Research Team in University (13036) and the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (21521004).


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Design and Synthesis of Crosslink-Dense Peptides by Manipulating

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