Postassembly Modifications of Peptides via Metal-Catalyzed C–H Functionalization

Abstract

Open AccessCCS ChemistryMINI REVIEW1 Mar 2021Postassembly Modifications of Peptides via Metal-Catalyzed C–H Functionalization Hua-Rong Tong, Bo Li, Guoxing Li, Gang He and Gong Chen Hua-Rong Tong State Key Laboratory and Institute of Elemento-Organic Chemistry, College of Chemistry, Nankai University, Tianjin 300071 , Bo Li State Key Laboratory and Institute of Elemento-Organic Chemistry, College of Chemistry, Nankai University, Tianjin 300071 , Guoxing Li State Key Laboratory and Institute of Elemento-Organic Chemistry, College of Chemistry, Nankai University, Tianjin 300071 , Gang He State Key Laboratory and Institute of Elemento-Organic Chemistry, College of Chemistry, Nankai University, Tianjin 300071 and Gong Chen *Corresponding author: E-mail Address: [email protected] State Key Laboratory and Institute of Elemento-Organic Chemistry, College of Chemistry, Nankai University, Tianjin 300071 https://doi.org/10.31635/ccschem.020.202000426 SectionsAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail The growing importance of peptides and proteins in therapeutic and biomedical applications has provided immense motivation toward the development of new ways to construct and transform peptide molecules. As in other areas of organic synthesis, C–H functionalization (CHF) chemistry could potentially exemplify disruptive technologies for peptide engineering. Over the past decade, the field has witnessed an exciting surge of reports of various metal-catalyzed CHF chemistry for postassembly modification of peptides and proteins. This review chronicles present advances in this research area up to June 2020. The content is organized based on the location of CHF on peptides: amino acid side chains (aromatic and nonaromatic), backbone, and appendant groups on peptide terminus. In addition to the reaction mechanisms of the metal-catalyzed CHF chemistry used in these peptide modification protocols, brief comments on the corresponding nonmetal-mediated strategies are included to provide readers a broad view of the current status of CHF-enabled peptide modification. Download figure Download PowerPoint Introduction Via simple iterative amide linkages, a small set of α-amino acid (αAA) building blocks can be assembled by biological or chemical approaches to form peptides and proteins of almost infinite structural diversity. Peptides of varied length, composition, and sequence occupy a vast chemical space between the small molecules and biological macromolecules, playing many irreplaceable biological roles, and provide an increasingly valuable platform for therapeutic and biomedical applications.1–4 In nature, peptides and proteins often need to be modified after the assembly of primary sequence to adopt proper structural, biophysical, and functional properties.5,6 In biomedical engineering, peptides and proteins also need to be modified to improve their therapeutic efficacy, enable new functionality, or be repurposed for new tasks.7–13 Despite significant development, the existing methods for postassembly modification of peptides are limited mainly to reactions involving polar nucleophilic residues such as cysteine and lysine. In comparison, practical methods for modifying hydrophobic aliphatic and aromatic residues, which are essential in engaging in biological targets, are much less developed.12,14–16 In this context, methods based on functionalization of C–H bonds of hydrophobic residues could offer a new set of tools to enable unprecedented peptide modification, thus, dramatically expanding the arsenal for peptide engineering.17–32 As shown in Scheme 1a and 1b, nature has shown that C–H functionalization (CHF) could provide a powerful means to modify and transform peptides and proteins.33 As exemplified by C2-mannosylation of tryptophan (Trp) in specific membrane proteins, C5-alkylation of histidine (His) in elongation factor 2 protein (EF2), and C3-hydroxylation of proline (Pro) in collagen enzyme-catalyzed CHF reactions enable unique post-translational modification (PTM) of proteins.34–36 Enzyme-catalyzed CHF also promotes the biosynthesis of natural peptide products with highly complex structures such as penicillin, celogentin C, and subtilosin A.37–39 However, useful biological methods based on enzyme-mediated CHF for peptide modification remain elusive. Chemical CHF methods have also long been used for peptide and protein modifications. For example, photoaffinity labeling reactions that are widely used for identifying protein target of small-molecule ligand typically proceed via photoactivated insertion of carbenes into C–H bonds of aliphatic and aromatic side chains or radical-mediated alkylation of peptide backbone C–H bonds with benzophenone.40,41 Electrophilic aromatic substitution (SEAr) of electron-rich aromatic side chains of peptides such as C2-thiolation of Trp and ortho-iodination of tyrosine (Tyr) could proceed in high efficiency and residue selectivity under mild conditions.42,43 Dimethyldioxirane (DMDO)-mediated hydrogen atom transfer (HAT) abstraction chemistry has also been used for the hydroxylation of weaker tertiary C–H bonds of aliphatic side chains of simple peptides.44 However, these classic CHF reactions mostly rely on the reactivity of highly reactive organic intermediates, which lack tunable mechanisms to control their reactivity and selectivity. In comparison, metal-catalyzed CHF reactions could potentially not only allow a broader range of modification modes but also exert control of reactivity through the innovation and fine-tuning of reaction pathways, reagents, and ligands to achieve more discriminative peptide modifications (Scheme 2). Over the past three decades, metal-catalyzed CHF chemistry has been advanced markedly, offering increasingly powerful methods for synthesis and late-stage modification of small molecules.17–32 Various metal-mediated C–H cleavage pathways such as concerted oxidative addition (OA), concerted metalation–deprotonation (CMD), carbene and nitrene C–H insertion, electrophilic addition to a π system, and HAT, have been identified (Scheme 2b). Many of these reactions have been used in the synthesis and modification of single protected AA units. The field is poised to tackle the more difficult challenges of modifying and transforming large peptides and even proteins. Besides the common issues associated with CHF reactions, peptide substrates present multitudes of additional problems, including a complex landscape of C–H bonds displayed by various AAs, interference of metal-coordinating side chains and backbones, the presence of sensitive side chains (e.g., thiol group), maintaining the integrity of chiral centers (e.g., Cα position of AAs), along with issues of solubility and purification. Ideally, these reactions would operate under near-physiological conditions for native peptides and proteins. Despite these issues, encouraging progress has been made to advance CHF strategies for selective modification of peptides. C–H bonds in AAs with high innate or inducible reactivities undergo residue and regioselective modification of peptides with or without the assistance of directing groups (DGs) (Scheme 2a). Metal-catalytic reactions via either inner-sphere or outer-sphere mechanisms have been utilized. For instance, the innate reactivity of C2–H bond of indole allows undirected C2-selective labeling of Trp in complex peptides under the catalysis of palladium (Pd) and gold (Au), respectively. In peptides, the β methyl C–H bond of alanine (Ala) could be activated selectively and converted to a variety of functional groups via the induction of either endogenous or exogenous DGs under Pd catalysis. Even the typically inert aryl C–H bonds of phenylalanine (Phe) of peptides could be borylated selectively under the control of the iridium (Ir) catalyst. The weak 3° C–H bonds of valine (Val) and leucine (Leu) in short peptides are oxygenated selectively via the HAT-mediated outer-sphere mechanism under iron (Fe) catalysis. Scheme 1 | (a and b) C–H functionalization of proteins and peptides in nature. Download figure Download PowerPoint This review intends to briefly chronicle the representative advances in the postassembly modification of peptides via the metal-catalyzed CHF chemistry strategies utilized up to June 2020. The discovery and basic reaction mechanisms of the original CHF chemistry used in these peptide modification protocols are discussed concisely. General comments on the C–H reactivities of various αAA units via nonmetal-mediated strategies are included to complement the state-of-the-art of metal-catalyzed CHF strategies for peptide modification. Since studies on the CHF-enabled synthesis and modification of AA substrates have been covered by several recent reviews, this review focuses mainly on the reports of metal-catalyzed CHF of peptides containing more than two AA residues.45–50 Examples of single AA, and occasionally, dipeptide substrates are included for specific CHF reactions. The content is organized based on the location of CHF on the peptides: AA side chains (aromatic and nonaromatic), backbone, and appendant groups on peptide terminus. Typically, one representative example with detailed reaction conditions would be presented from each study. Scheme 2 | (a and b) General information on metal-catalyzed CHF of AA in peptides. Download figure Download PowerPoint CHF of the side chains of aromatic AAs in peptides There are four proteinogenic aromatic αAAs: Trp, Tyr, Phe, and His. While Trp and Tyr carry an electron-rich aryl side chain, His possesses an electron-deficient imidazole ring.51 Trp Trp, bearing an indole side chain, is the second-lowest abundance αAA with ∼1% frequency (Scheme 3a). Approximately 90% of proteins contain at least one Trp residue. Trp has unique photoelectronic properties and often engages in noncovalent binding interactions through its π orbitals. The C2 = C3 region of the indole ring is very electron-rich and could react with a variety of electrophiles under mild conditions. C2–H is the most acidic C(sp2)–H bond.52 With the C3 position occupied, the C2–H bond of Trp provides a target for CHF modification via various pathways (Scheme 3b). Historically, the SEAr reaction with chlorosulfonic reagents provided an efficient method for C2-selective thiolation of Trp in peptides.42 However, these reagents are not selective and could also react with Tyr and cysteine. Trp of peptides could undergo regioselective C2–H alkylation with electrophilic alkyl radicals via radical-mediated pathways.53,54 Besides C2, the benzylic C–H bonds at Cβ of Trp provide a different kind of target for HAT-mediated reactions. Recently, a research team at the Merck Company (Rahway, NJ, USA) showed that the benzylic β C–H of Trp in peptides could be alkylated selectively with electron-deficient olefins to give β-alkylated products under photoredox catalysis.55 Scheme 3 | (a–d) Undirected CHF of indole and Trp. Download figure Download PowerPoint In 2004, Antos and Francis56 reported the first metal-catalyzed CHF of Trp in proteins via rhodium (Rh)-catalyzed carbenoid insertion reaction pathway with stabilized vinyl diazo reagents (Scheme 3b). However, while the proteins could be labeled in excellent residue selectivity and with good conversion in aqueous media, the modification of Trp took place at both N1 and C2 positions, forming a regiomeric mixture of N1- and C2-alkylation products. The formal C2-alkylation product, which was slightly favorable over the N-alkylation, was formed presumably via the rearrangement of a cyclopropane intermediate. Later, Popp and Ball57 modified this Rh-catalyzed carbenoid insertion strategy by introducing a molecular recognition element via coiled-coil interaction between the substrate and the catalyst. The new design enabled a more controllable selectivity for Trp in peptide substrates and could be extended to the modification of Tyr and Phe with slightly lower efficiency. However, the main modification mode of Tyr is likely an esterification process through O–H insertion; that of Phe is undetermined and could be through aryl, benzylic C–H insertion, or dearomatization cyclopropanation. Ir-catalyzed C–H borylation reactions developed by the laboratories of Hartwig and Smith have emerged as one of the most broadly applicable methods for CHF of arenes and heteroarenes without the use of DGs (Scheme 3c).58–60 While the site selectivity for arenes is predominantly controlled by steric effects, that for heteroarenes is strongly influenced by electronic effects. C–H activation of heteroarenes is generally favored at the most acidic C–H bond in the absence of steric effects. In 2009, Kallepalli et al.61 first demonstrated that the Ir-catalyzed C–H borylation of Boc-Trp-OMe could take place at the most acidic C2 position to give compound 1 in exclusive selectivity. By installing a bulky Triisopropylsiyl (TIPS) group on the N1 of Trp, Feng et al.62 showed that the regioselectivity of C–H borylation could be switched to C6 and C5 positions in an 8:1 ratio under slightly modified conditions (see 2). Loach et al.63 showed that the reaction of the same substrate using excess pinacolborane (HBpin) could give a diborylated product 3 at C2 and C7 positions in high yield. Interestingly, the C2-boronate group of 3 could be removed selectively by treatment with Pd(OAc)2 in AcOH in a single-pot fashion to give the mono C7-borylated product 4 in high yield. However, the application of these reactions to more complex peptide substrates has not been reported. Pd-catalyzed undirected C2-selective C–H arylation of Trp in peptides and proteins Pd-catalyst has proven to be the most versatile among the catalytic CHF of small molecules.17–32 Pioneering studies by several investigators have shown that simple indole compounds could undergo undirect C–H arylation at C2 position with various aryl coupling partners via different catalytic cycles (Scheme 3d).64–69 In most of these reaction systems, the catalytic cycle involves two critical steps of the C–H palladation of indole to generate an indolyl PdII species and the reductive elimination (RE) of PdII or PdIV intermediate to form the arylation product. The aryl group could be added onto Pd before or after the C–H palladation step via OA to Pd0 or transmetalation to PdII. The binding interactions between the C2 = C3 region of indole and the cationic PdII likely facilitate the initial indole/Pd contact. The CMD mechanism, featuring carboxylate or carbonate ligand as an internal base, has been invoked predominantly for the palladation step.22 The high acidity of the C2–H bond plays a crucial role in controlling the regioselectivity. Lane and Sames64 reported the first C2–H arylation of indole with aryl iodide via a Pd0/II catalytic cycle using a strong base and high reaction temperature. Later, Lebrasseur and Larrosa65 showed that the use of a proper combination of carboxylic acid and silver salt additives could allow the reaction to proceed at room temperature (rt). It was proposed that the silver salt removes the iodide from PdII after OA to generate a more electrophilic PdII species to accelerate the C–H palladation step via CMD. Deprez et al.66 showed that the reaction via a PdII/IV catalytic cycle using diaryl-iodonium (III) salts, premade or in situ generated by mixing ArB(OH)2 with PhI(OAc)2, can also proceed at rt. More recently, Gemoets et al.68 showed that the C–H arylation of indole with aryldiazonium salts ArN2BF4 could proceed efficiently with low loading of Pd(OAc)2 catalyst (<1 vs 5–10 mol %) under mild conditions at rt. In comparison to the previous methods, this reaction generated much less waste. ArN2BF4 is electrophilic and could react rapidly with Pd0 to generate Ar-PdII species. A mechanistic study suggested that the arylation of indole in this system proceeded through a Heck-type carbopalladation mechanism via a different Pd0/II cycle. Fairlamb69 also investigated independently the Pd-catalyzed C–H arylation of indole with aryldiazonium salt. In 2010, Ruiz-Rodríguez et al.70 reported the first Pd-catalyzed undirected C2–H arylation of Trp in short peptides (mostly tri- and tetramers) with aryl iodides using a modified protocol of Sames and Larrosa's method (Scheme 4a). For example, tetrapeptide 5 was obtained in 95% yield under the conditions of 5 mol % of Pd(OAc)2, 10 equiv of Ar-I, 2-nitro-benzoic acid (2-NO2-BzOH), and AgBF4 additive in an aqueous phosphate buffer at 80 °C for 10 min under microwave irradiation (MW). A high temperature was required to achieve sufficient reactivity. Conventional heating in dimethylformamide (DMF) at 150 °C resulted in undesired racemization of AA units. Notably, unprotected Arg, Lys, Tyr, Gln, and Asp were tolerated. Met was incompatible as it promoted the Pd-catalyzed hydrolysis of the peptide backbone. Unprotected His residues caused reduced reaction yield. Scheme 4 | (a and b) Pd-catalyzed undirected C2–H arylation of Trp in peptides and proteins. Download figure Download PowerPoint In 2014, Williams et al.71 reported the C2–H arylation of Trp in short peptides with aryl boronic acids in the presence of Cu(OAc)2 as cocatalyst and air oxidant at 40 °C. The method worked well for simple dipeptides but required more vigorous conditions for longer peptides. For example, hexapeptide 6 containing a free Lys was obtained in >86% conversion with 30 mol% of Pd(OAc)2 and 60 mol% of Cu(OAc)2 in AcOH solvent. Notably, impurities arising from the use of Ar-B(OH)2/Cu(OAc)2 included hydroxylation (major) and arylation (minor) of the newly installed aryl group. In 2015, Reay et al.72 reported C2–H arylation of Trp in short peptides with premade mesityl substituted diaryl-iodonium reagent [MesArI]OTf using a modified protocol of Sanford's method. The reaction could take place in EtOAc or iPrOH solvent at 25 °C. For example, tetrapeptide 7 bearing a free Ser and unprotected terminal CO2H was obtained in >50% conversion with 10 mol % of Pd(OAc)2 and 2 equiv of [MesPhI]OTf. Around the same time, Zhu et al.73 reported a similar protocol with symmetric diaryliodonium salts [Ar2I]OTs in AcOH, AcOH/1,2-dichloroethane (DCE), or even water solvent at rt. For example, tripeptide 8 was obtained in 60% yield with 5 mol % of Pd(OAc)2 and 1 equiv of [Ph2I]OTs in H2O at rt. Interestingly, Zhu et al.74 also showed that the indole moiety of 3-indoleacetamide that appended on the N-terminus of the protected short peptides could undergo C2–H arylation with [Ar2I]OTs at 100 °C in DMF in the absence of any metal catalyst. Notably, the Trp residue in the peptide substrates was untouched under the reaction conditions. In 2017, Reay et al.75 reported a C2–H arylation of Trp in short peptides with aryldiazonium salts under mild conditions. For example, tetrapeptide 5 bearing a free Ser and terminal CO2H was obtained in 45% yield using 1.1 equiv of PhN2BF4 and 20 mol % of Pd(OAc)2 in MeOH at 37 °C. Notably, the terminal CO2H was converted to methyl ester under the reaction conditions. The catalytic cycle presumably starts with the OA of ArN2+ to a Pd0 species, generated via in situ reductions of Pd(OAc)2 to form Ar-PdII. It was found that the aggregated Pd0 nanoparticulate species were formed in the reaction, and Pd0 nanoparticle (PdNPs) stabilized by polyvinyl pyrrolidinone (PVP) as a viable catalyst. Mechanistic studies showed that such heterogenous Pd0 species might serve primarily as a catalyst reservoir, and the reaction mainly proceeded homogenously. Building on the works of Noel and Fairlamb, Perez-Rizquez et al.76 recently demonstrated the first application of Pd-catalyzed C2–H arylation of Trp for site-selective modification of proteins with aryldiazonium salt in pure aqueous solution at rt. A heterogeneous Pd nanoparticle biohybrid catalyst (PdNPs-E), prepared by dispersion of PdNPs in a protein network, was used. The reaction of Candida antarctica lipase protein (Cal-B, 33 kDa) with an excess amount of p-MeO-C6H4N2BF4 and PdNPs-E catalyst in H2O at for proceeded with high at residue or at both and residues was formed selectively on the amount of Pd catalyst The excellent selectivity and made this method a for labeling of proteins in complex biological As shown in Scheme Pd-catalyzed C2–H arylation has also been in an fashion to construct peptide In et first reported a peptide via Pd-catalyzed C2–H arylation of Trp with aromatic AA side chains or aryl For example, compound bearing a between Trp and Phe was obtained in yield under the conditions of 5 mol % of Pd(OAc)2, and AgBF4 additive in at °C for 30 min under Notably, the reaction could proceed at a without significant and aryl iodides could be used. The products of ring between and 25 were obtained in good yield. Unprotected Tyr was tolerated. Notably, most peptide substrates used or favorable units. The nucleophilic functional groups were mostly In 2015, et extended this strategy to more peptides between Trp and Phe or Tyr under conditions. This could be in both solution and Notably, several unprotected AA units such as Arg, Gln, and Ser were tolerated. As exemplified by product complex could be via C–H arylation of an residue. undirect C2–H of Trp in peptides and proteins In 2009, et reported that C–H of with reagent could proceed efficiently at (Scheme While the reaction of indole place selectively at the C3 give product in high yield. The reaction mechanism it could proceed via a sequence of C–H of indole and the addition of indole to the bond of could form a vinyl gold complex by elimination to give the product. Later, and demonstrated that the thiol groups of peptides and even proteins could be efficiently with the treatment of in aqueous in the absence of catalysis. Scheme 5 | (a and b) C2–H of Trp in peptides and proteins. Download figure Download PowerPoint In et first demonstrated that C–H reaction could be for modification of peptides and proteins under slightly modified conditions (Scheme For example, was modified in yield and exclusive Trp selectivity under the conditions of equiv of catalyst and 3 equiv of the in with acid at rt. The group of could be removed by to give bearing a free which could be used for the via with The AA including free and were tolerated. a reaction of of with 5 equiv of catalyst and 10 equiv of in of and water proceeded with excellent conversion and Trp selectivity to give a mixture of and products and the method is only for peptides and proteins that organic this study the of a metal-catalyzed C–H arylation reaction for selective modification of native proteins under mild conditions. et also reported their of C–H of Trp in simple peptides to using 5 mol % of in at 40 °C. CHF of Trp in peptides using exogenous DGs While the C2 position of Trp provides a for various undirected CHF reactions, DGs could this innate reactivity and enable CHF at other A variety of DGs installed on the and CO2H groups have been utilized to facilitate different CHF of Trp (Scheme The of most metal-catalyzed reactions is controlled by the favored intermediate formed via a CMD the reactions could proceed through occasionally, intermediate. For instance, DGs on could aryl CHF at C2 and DGs on CO2H were to functionalization of one of the β C–H DGs installed on N1 strongly favored CHF at C2 to the high acidity of C2–H and of the DGs on N1 could be to CHF at Recently, CHF at the more C6 position has been using longer DGs on N1 under Pd CHF at C5 Scheme 6 | C–H functionalization of Trp in peptides. Download figure Download PowerPoint Recently, the use of strong metal or DGs on N1 has enabled several C2-selective CHF of Trp in peptides using metal such as and (Scheme In 2017, et showed that Trp in short protected peptide substrates could be modified selectively at C2 via C–H arylation with aryl at an temperature (see Notably, the Trp of was In 2017, et reported that Trp in short peptides could be modified selectively via C2–H with under the conditions of 5 mol % of and base in at 80 °C (see C–H activation mechanism involving a was The on simple indole substrate could be removed with good but required conditions in at 80 Notably, C–H with via a in good yield under modified conditions with mol % of More recently, et showed that an existing in protected peptides could be modified via C2–H alkylation with methyl carbonate reagents bearing protected As exemplified by bearing various could be obtained in good yield under the conditions of 20 mol % of and 30 mol % base in at °C. A group could be removed from simple peptides using a with by treatment with in MeOH at 60 or catalytic transfer with and in at 60 °C. In addition to et also showed in protected peptides could be modified via with For example, the tetrapeptide 20 was obtained in yield under the conditions of 5 mol % of and additive in at 80 °C. A reaction pathway of C–H insertion, and was As exemplified by et showed that in protected peptides could be modified via alkylation with electron-deficient terminal under conditions of 10 mol % of in at 80 °C. Notably, H2O is

Topics & Concepts