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Cyanosulfurylides as protecting groups for carboxylic acids. a Cyanosulfurylide 1 undergoes a rapid reaction with electrophilic halogen species, leading to the release of the unprotected carboxylic acid 2. A postulated mechanism proceeds via electrophilic halogenation followed by hydration and elimination of cyanohalogenylide. b Reaction screening was performed using cyanosulfurylide (5 mM) and electrophilic halogen species (10 mM) in aqueous solvent (CH 3 CN/H 2 O, 1:1); NCS = N-Chlorosuccinimide, NCSa = N-Chlorosaccharine, NBS = N-Bromosuccinimide, NIS = N-Iodosuccinimide. Reactions were analyzed by LC-MS after 1 min. c LC-MS traces of the reaction between sulfurylide 1 and NCS. d Fmoc-Asp(CSY)-OH 3 is readily synthesized from commercially available Fmoc-Asp(OH)-O t Bu 4 in two steps. e Reaction between aspartic acid monomer 5 and NCS was performed on a larger scale to determine the isolated yield (80%). Chiral HPLC traces showed that the stereoinformation is retained upon CSY removal. f Incorporation of cyanosulfurylide-protected aspartic acid into model peptide 6b and comparison with conventional O t Bu ester 6a upon incubation in 20% piperidine in DMF (12 h, room temperature).
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Although peptide chemistry has made great progress, the frequent occurrence of aspartimide formation during peptide synthesis remains a formidable challenge. Aspartimide formation leads to low yields in addition to costly purification or even inaccessible peptide sequences. Here, we report an alternative approach to address this longstanding challe...
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... mechanism for the conversion of CSY and the related phosphorus ylides, originally developed by Wasserman to vicinal dicarbonyls 27 , as well as the documented addition of halogens to sulfurylides 28 , we anticipated that we would be able to oxidize CSYs to a species susceptible to C-C bond cleavage in a manner similar to haloform reactions (Fig. 2a) 29 . With this working hypothesis, we screened various oxidizing agents and observed that electrophilic halogen species rapidly react with CSY 1 to yield the free carboxylic acid 2 under aqueous conditions (Fig. 2b). Among the halogen reagents evaluated, N-chlorosuccinimide (NCS) was particularly promising as it had already been ...
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... that we would be able to oxidize CSYs to a species susceptible to C-C bond cleavage in a manner similar to haloform reactions (Fig. 2a) 29 . With this working hypothesis, we screened various oxidizing agents and observed that electrophilic halogen species rapidly react with CSY 1 to yield the free carboxylic acid 2 under aqueous conditions (Fig. 2b). Among the halogen reagents evaluated, N-chlorosuccinimide (NCS) was particularly promising as it had already been reported to be compatible with all amino acids apart from methionine-which is, however, relatively rare and commonly substituted with norleucine in SPPS 30,31 . The deprotection of CSY 1 proceeded rapidly to full ...
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... acid 2 (Fig. 2c). These observations are consistent with a mechanism featuring chlorination of the ylide followed by hydration of the carbonyl and loss of the electron deficient ylide species ( Fig. 2a). The carboxylic acids masked as CSYs showed remarkable stability to strongly acidic and basic milieu, as well as to oxidative conditions other than ...
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... acid 2 (Fig. 2c). These observations are consistent with a mechanism featuring chlorination of the ylide followed by hydration of the carbonyl and loss of the electron deficient ylide species ( Fig. 2a). The carboxylic acids masked as CSYs showed remarkable stability to strongly acidic and basic milieu, as well as to oxidative conditions other than halogenation (e.g., NaNO 2 in AcOH), and in the presence of radicals; no major degradation being observed after 1 hour (Supplementary Figs. 1-5, Supplementary Table 1). Having identified ...
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... SPPS. Fmoc-Asp(CSY)-OH 3 was readily prepared from commercially available Fmoc-Asp(OH)-O t Bu 4 in two steps, in 80% overall yield, to provide the benchstable amino-acid derivative. When Fmoc-Asp(CSY)-O t Bu 5 was treated with an stochiometric amount of NCS, the enantiomerically pure carboxylic acid 4 was obtained in excellent isolated yield ( Fig. 2e and Supplementary Fig. 6). We incorporated 3 into the resin-bound pentamer H-FDGLA-OH. Two variants were synthesized; pentamer 6a was synthesized using traditional Fmoc-Asp (O t Bu)-OH and pentamer 6b was prepared with Fmoc-Asp(CSY)-OH 3. With 6b, we were pleased to observe that no aspartimide formation occurred even after incubation ...
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... prepared with Fmoc-Asp(CSY)-OH 3. With 6b, we were pleased to observe that no aspartimide formation occurred even after incubation in 20 vol% piperidine in DMF for 12 h at room temperature. In contrast, pentamer 6a-containing the conventional Asp(O t Bu) monomer-showed a high degree of aspartimide formation and piperidine substituted products ( Fig. 2f, for Asp(OMpe) see Supplementary Fig. 7). These results confirmed our hypothesis that masking the carboxylic acid with a C-C bond instead of a C-O ester bond could overcome the problem of aspartimide ...
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... were deprotected using TCEP under aqueous conditions (pH 7.0, 50 °C, 12 h), delivering unfolded LDLa 14 on a multi-milligram scale. This result demonstrates that CSY-masked aspartic acids can enable the synthesis of otherwise inaccessible peptides. The resulting synthetic LDLa 14 was folded using reported conditions and purified by HPLC ( Fig. 5c and Supplementary Fig. ...
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Citations
... Asi is formed most rapidly when the (i + 1) residue with respect to amino acid (i), which is Asp (or Asn), is flexible and has no side chain, making Asp-Gly the most susceptible subunit for isomerization [11][12][13][14][15][16]. Moreover, the catalytic effect of the side chain of the (i + 1) residue has also been proposed, suggesting that Ser and Thr, both residues with short side chains, -Asp-Ser-and -Asp-Thr-, are ready for isomerization within days under synthetic conditions [17]. ...
... Therefore, Asi formation and isomerization could be prevented by masking the latter two residues as Ser(ΨPro) or Thr(ΨPro). Due to difficulties in acylating the sterically hindered secondary amine of Xxx(ΨPro) on the resin, ΨPro derivatives Asi is formed most rapidly when the (i + 1) residue with respect to amino acid (i), which is Asp (or Asn), is flexible and has no side chain, making Asp-Gly the most susceptible subunit for isomerization [11][12][13][14][15][16]. Moreover, the catalytic effect of the side chain of the (i + 1) residue has also been proposed, suggesting that Ser and Thr, both residues with short side chains, -Asp-Ser-and -Asp-Thr-, are ready for isomerization within days under synthetic conditions [17]. ...
... Throughout our work, all syntheses were performed on our continuous flow peptide synthesizer, using previously developed coupling protocols. Asi is formed most rapidly when the (i + 1) residue with respect to amino acid (i), which is Asp (or Asn), is flexible and has no side chain, making Asp-Gly the most susceptible subunit for isomerization [11][12][13][14][15][16]. Moreover, the catalytic effect of the side chain of the (i + 1) residue has also been proposed, suggesting that Ser and Thr, both residues with short side chains, -Asp-Ser-and -Asp-Thr-, are ready for isomerization within days under synthetic conditions [17]. ...
Pseudoproline derivatives such as Thr(ΨPro)-OH are commonly used in peptide synthesis to reduce the likelihood of peptide aggregation and to prevent aspartimide (Asi) formation during the synthesis process. In this study, we investigate notable by-products such as aspartimide formation and an imine derivative of the Thr(ΨPro) moiety observed in flow peptide chemistry synthesis. To gain insight into the formation of these unexpected by-products, we design a series of experiments. Furthermore, we demonstrate the oxazolidine character of the pseudoproline moiety and provide plausible mechanisms for the two-way ring opening of oxazolidine leading to these by-products. In addition, we present evidence that Asi formation appears to be catalyzed by the presence of the pseudoproline moiety. These observed side reactions are attributed to elevated temperature and pressure; therefore, caution is advised when using ΨPro derivatives under such harsh conditions. In addition, we propose a solution whereby thermodynamically controlled Asi formation can be kinetically prevented.
... Because target Uln-3d lacks a Ser9 or Thr9 residue needed to use the same pseudoproline dipeptide approach, we applied the cyanosulfurylide (CSY) strategy recently developed by Bode and coworkers. 22 This strategy temporarily masks the requisite carboxylic acid functionality at Asp8, and unlike conventionally used sterically encumbered ester derivatives (e.g., Asp(Ompe)), avoids the potential for aspartimide formation all together. As described, we found that Fmoc-Asp(CSY)-OH S-3 is readily prepared (SI Scheme S-1). ...
Lasso peptides are a structurally distinct class of biologically active natural products, defined by their short sequences with impressively interlocked tertiary structures. Their characteristic peptide [1]rotaxane motif confers marked proteolytic and thermal resiliency, and reports on their diverse biological functions have been credited to their exceptional sequence variability. Because of these unique properties, taken together with improved technologies for their biosynthetic production, lasso peptides are emerging as a designable scaffold for peptide-based therapeutic discovery and development. Although the defined structure of lasso peptides is recognized for its remarkable properties, the role of the motif for imparting bioactivity is less understood. For example, sungsanpin and ulleungdin are natural lasso peptides that similarly exhibit encouraging cell migration inhibitory activities in A549 lung carcinoma epithelial cells despite sharing only one-third sequence homology. We hypothesized that the shape of the lasso motif is beneficial for the preorganization of the conserved residues, which might be partially retained in variants lacking the threaded structure. Herein, we describe solid-phase peptide synthesis strategies to prepare acyclic, head-to-sidechain (branched), and head-to-tail (macrocyclic) cyclic variants based on the sungsanpin (Sun) and ulleungdin (Uln) sequences. Proliferation assays and timelapse cell motility imaging studies were used to evaluate the cell inhibitory properties of natural Sun compared alongside the synthetic Sun and Uln isomers. These studies demonstrate that the lasso motif is not a required feature to slow cancer cell migration, and more generally show that these non-threaded isomers can retain similar activity to the natural lasso peptide despite the differences in their overall structures.
... These remarkably stable zwitterionic protecting groups effectively prevented aspartimide formation and enabled the synthesis of otherwise inaccessible peptides but required stochiometric amounts of electrophilic halogen species for final deprotection of the Asp residues. 10,11 This oxidative reaction necessitated a separate step after standard resin cleavage and global deprotection and was incompatible with methionine residues and the most common cysteine protecting groups. Therefore, although this solution to aspartimide formation is appropriate for many challenging peptides, it is not suitable for the synthesis of complex dimeric LA modules by convergent assembly using native chemical ligation (NCL) and/or the alpha-ketoacidhydroxylamine (KAHA) ligation. ...
Low-density lipoprotein receptor class A domains (LA modules) are common ligand-binding domains of transmembrane receptors of approximately 40 amino acids that are involved in several cellular processes including endocytosis of extracellular targets. Due to their wide-ranging function and distribution among different transmembrane receptors, LA modules are of high interest for therapeutic applications. However, the efficient chemical synthesis of LA modules and derivatives is hindered by complications, many arising from the high abundance of aspartic acid and consequent aspartimide formation. Here, we report a robust, efficient and general applicable chemical synthesis route for accessing such LA modules, demonstrated by the synthesis and folding of the LA3 and LA4 modules of the low-density lipoprotein receptor, as well as a heterodimeric LA3-LA4 constructed by chemical ligation. The synthesis of the aspartic acid-rich LA domain peptides is made possible by the use of cyanopyridiniumylides (CyPY) - reported here for the first time - as a masking group for carboxylic acids. We show that cyanopyridiniumylide masked aspartic acid monomers are readily available building blocks for solid phase peptide synthesis and successfully suppress aspartimide formation. Unlike previously reported ylide-based carboxylic acid protecting groups, CyPY protected aspartic acids are converted to the free carboxylic acid by acidic hydrolysis and are compatible with all common residues and protecting groups. The chemical synthesis of Cys- and Asp-rich LA modules enables new access to a class of difficult to provide, but promising protein domains.
... The aspartimide installed by O-methyltransferase ThfM differentiates fuscimiditide from other characterized graspetides. Normally, aspartimide functions as either an intermediate in protein repair or as a nuisance product in solid-phase peptide synthesis or protein pharmaceutical formulation 44,45,65 . Our data strongly support the idea that, at least in cellulo, the aspartimidylated product fuscimiditide is the final, intended product of the BGC. ...
Microviridins and other ω-ester-linked peptides, collectively known as graspetides, are characterized by side-chain–side-chain linkages installed by ATP-grasp enzymes. Here we report the discovery of a family of graspetides, the gene clusters of which also encode an O-methyltransferase with homology to the protein repair catalyst protein l-isoaspartyl methyltransferase. Using heterologous expression, we produced fuscimiditide, a ribosomally synthesized and post-translationally modified peptide (RiPP). NMR analysis of fuscimiditide revealed that the peptide contains two ester cross-links forming a stem–loop macrocycle. Furthermore, an unusually stable aspartimide moiety is found within the loop macrocycle. We fully reconstituted fuscimiditide biosynthesis in vitro including formation of the ester and aspartimide moieties. The aspartimide moiety embedded in fuscimiditide hydrolyses regioselectively to isoaspartate. Surprisingly, this isoaspartate-containing peptide is also a substrate for the l-isoaspartyl methyltransferase homologue, thus driving any hydrolysis products back to the aspartimide form. Whereas an aspartimide is often considered a nuisance product in protein formulations, our data suggest that some RiPPs have aspartimide residues intentionally installed via enzymatic activity.
... We first chose ubiquitin (1-76), which is a protein-size PTM (ubiquitination) regulating other proteins' degradation, location, and activity and has been used as a model substrate in protein chemistry. 8,11,28,[30][31][32][33] The ubiquitin A46C mutant 41 obtained via NCL was smoothly desulfurized to 41 in the add-and-done manner with 52% isolated yield. ...
Highly effective yet chemoselective chemical transformation strategies enable the facile access and precise modification of complicated biomacromolecules. In particular, the application of desulfurization chemistry expands the dimension of chemical protein synthesis with the cysteine-based peptide ligation. Considering the existing peptide desulfurization methods, a milder, faster, and easier strategy is still required for the increasing complexity of proteins by chemical synthesis. Herein, we report a superfast desulfurization strategy based on tetraethylborate for effectively and chemoselectively desulfurizing peptides/proteins containing cysteine or penicillamine in an add-and-done manner. This strategy can be simply applied under ambient conditions without requirement of inert atmosphere protection, irradiation, heating, or exogenous thiol additives. Such desulfurization can even overcome a certain amount of radical scavengers. Various peptide and protein substrates were examined, and a practical one-pot native chemical ligation (NCL)-desulfurization was developed for the synthesis of leukocyte-associated immunoglobulin-like receptor 1 (LAIR1) cytoplasmic domain and semisynthesis of serotonylated histone H3 (H3Q5ser) protein.
... However, like PEDOT:PSS, PEDOTS lacks functional groups that will enable post-functionalization [3]. The carboxyl group is widely used in coupling chemistry for modifying biomaterials with biomolecules and proteins containing, for instance, amine groups [22]. Having this group tethered to the conjugated polymer backbone could pave the way to potentially modifying these materials with other chemicals to achieve the desired properties [23]. ...
Poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) is the most researched conjugated polymer in the field of organic bioelectronics. The conjugated PEDOT backbone features good redox stability in aqueous electrolyte, and low oxidation potential. However, PEDOT:PSS has two major drawbacks. The PEDOT backbone lacks biofunctionality, limiting the fine tuning of its interface with the biological environment. The dopant PSS is insulating, resulting in a decrease in the capacitance of the polymer. Here, we describe the design of a random copolymer, P(EDOTCOOH-co-EDOTS), based on EDOT monomers functionalized with sulfonic and carboxylic acid groups. The copolymer was successfully synthesized by electropolymerization as confirmed by X-ray photoelectron spectroscopy. Contact angle measurements illustrated the high hydrophilicity of the P(EDOTCOOH-co-EDOTS) (28 ± 6º), attributed to the sulfonate group in the side chains. This in turn led to a higher water penetration into the copolymer film, enhancing significantly its volumetric capacitance (69 ± 4 F cm⁻³), and thereby, its performance when used as an active channel in an organic electrochemical transistor. Of note, we incorporated the sulfonate group in its sodium salt form retaining its highly ionized properties. This is the first instance of utilizing an uncapped, ionized sulfonate group covalently bound to the backbone of a polymer, where the resultant polymer is oxidized at very low potentials, as well as stable and electroactive in aqueous electrolytes. Furthermore, our molecular design to incorporate carboxylic acid groups paves the way for the development of conjugated polymers that can be tailored for bioelectronic applications.
... The SPPS process is holistic and, therefore, its success depends on several factors, such as the resin [5][6][7], solvent (taking into consideration its polarity and viscosity) [8,9], coupling reagents [10][11][12], and protection scheme [13][14][15][16][17]. Despite the immense amount of work done over the years to improve these factors and thus facilitate the development of any kind of sequence, some "difficult peptide sequences" remain challenging [18,19]. ...
Amino-Li-resin is a new and unique polyacrylamide resin presented in the form of fibers and is found to be well suited for solid-phase peptide chemistry. Although amino-Li-resin swells much better in polar solvents, it is also compatible with some non-polar solvents. It comes with a high loading of functional amino groups, thus maximizing its productivity in terms of the amount of peptide per gram of resin. In addition to its mechanical stability, this resin shows excellent stability in basic and acidic reagents; thus, allowing its broad applicability for the synthesis of a wide range of biomolecules. Finally, the appropriateness of amino-Li-resin for solid-phase peptide synthesis (SPPS) has been demonstrated for the synthesis of several model peptides, including difficult sequences and those containing hindered amino acids, all of which afforded excellent crude purity, as shown by high-performance liquid chromatography (HPLC) analysis.
... Area % [4] can be circumvented by simple alterations: oxidation of Cys by disulfide protective groups (e.g. StBu or SIT) and oxidation of Met by substitution by norleucine. ...
... Area % [4] can be circumvented by simple alterations: oxidation of Cys by disulfide protective groups (e.g. StBu or SIT) and oxidation of Met by substitution by norleucine. ...
... 53 Approaches to mitigate aspartimide formation include backbone amide protecting groups 54−57 or masking the side-chain carboxylate as a cyanosulfurylide (CSY). 58 Another approach is using bulky side-chain ester protecting groups such as 3-methylpent-3-yl (OMpe) ester for Asp, 59 and we have recently demonstrated that using this protecting group in combination with binary solvents significantly reduces aspartimide formation compared to DMF. 21 To assess aspartimide formation during Fmocremoval in different solvents using pyrrolidine as the base, we used the aspartimide-prone scorpion toxin II peptide (Fmoc-Val-Lys(Boc)-Asp(OtBu)-Gly-Tyr(OtBu)-Ile on a Wang-PS resin) 53,60,61 as a model. ...
Green binary solvent mixtures with a polarity and viscosity close to that of DMF perform similarly in solid-phase peptide synthesis (SPPS). However, while coupling reactions readily proceed in solvents of significantly lower polarity than that of DMF, a high solvent polarity is essential for Fmoc-removal using piperidine, which limits the options for green SPPS solvents. Herein, we report our efforts to expand the available solvent polarity space for green SPPS. We identified pyrrolidine as an efficient base to enable Fmoc-removal in less polar solvent mixtures that also favor coupling reactions, such as dimethyl sulfoxide/ethyl acetate (1:9) and N-butylpyrrolidone/1,3-dioxolane (2:8 and 4:6). Employing less polar binary solvent mixtures in combination with pyrrolidine gave crude peptide purities comparable to or better than for DMF with piperidine in the SPPS of challenging peptide targets. An evaluation of base-dependent side reactions such as diketopiperazine (DKP) and aspartimide formation showed increased side-product formation when using pyrrolidine on DKP- and aspartimide-prone sequences. However, the scaled-up syntheses (5 and 7.5 mmol, respectively) of the peptide therapeutics dasiglucagon (29-mer) and bivalirudin (20-mer) gave good crude peptide purities and purity profiles amenable to SPPS optimization. Pyrrolidine therefore represents a useful alternative to piperidine for Fmoc-removal in an expanded solvent space for green SPPS.
