Tunable Electrochemical Peptide Modifications: Unlocking New Levels of Orthogonality for Side‐Chain Functionalization

Abstract

Electrochemical transformations provide enticing opportunities for programmable, residue‐specific peptide modifications. Herein, we harness the potential of amidic side‐chains as underutilized handles for late‐stage modification through the development of an electroauxiliary‐assisted oxidation of glutamine residues within unprotected peptides. Glutamine building blocks bearing electroactive side‐chain N,S‐acetals are incorporated into peptides using standard Fmoc‐SPPS. Anodic oxidation of the electroauxiliary in the presence of diverse alcohol nucleophiles enables the installation of high‐value N,O‐acetal functionalities. Proof‐of‐principle for an electrochemical peptide stapling protocol, as well as the functionalization of dynorphin B, an endogenous opioid peptide, demonstrates the applicability of the method to intricate peptide systems. Finally, the site‐selective and tunable electrochemical modification of a peptide bearing two discretely oxidizable sites is achieved.

The programmable electrochemical modification of peptides is enabled by designer electroauxiliaries which are readily compatible with solid‐phase peptide synthesis. Anodic oxidation facilitates the functionalization of “electroactive” glutamine residues to deliver high‐value peptide N,O‐acetals. By dialing in the oxidation potential of distinct electroauxiliaries, the iterative and orthogonal functionalization of peptides is accomplished.

Introduction

Residue‐specific peptide modifications serve to enhance the therapeutic capacity of peptides by enabling the fine‐tuning of their biological and pharmacokinetic properties. ^[1] The accelerating rate of discovery of distinct classes of ribosomally‐synthesized and post‐translationally modified peptides (RiPPs) ^[2] and a growing appreciation of the complexities of non‐ribosomal peptides ^[3] continues to push the boundaries of existing methodologies for peptide synthesis and modification. To meet burgeoning demand, pathways such as biocatalytic strategies, ^[4] transition‐metal catalysis, ^[5] and photochemical reactions ^[6] have received considerable attention. However, there remains a persistent demand for mild, selective, inexpensive, and sustainable ^[7] methodologies, particularly those which unlock novel structural motifs and can be employed at a late‐stage in the synthetic sequence.

Advances in modern preparative electro‐organic chemistry highlight the synergistic pairing of novel reaction development with environmentally‐friendly, sustainable and operationally simple protocols. ^[8] Despite broad appreciation of electro‐organic strategies for small molecule synthesis, ^[9] modern peptide chemistry rarely exploits this powerful technology. ^[10] Pioneering efforts in the area by Seebach and co‐workers in the 1980s applied the Hofer‐Moest reaction—an electrolytic decarboxylation leading to the generation of an N,O‐acetal—to peptide substrates to engender C‐terminally modified peptides and peptide‐based chiral building blocks. ^[11] Following extrusion of CO₂, the process involves initial formation of a reactive iminium intermediate which can be trapped as the corresponding N,O‐acetal in the presence of an alcohol‐based solvent. Inspired by this seminal work, we have recently extended the versatility of oxidative decarboxylation chemistry on peptide substrates. We have leveraged the reactivity of electrochemically‐generated N,O‐acetal intermediates to afford peptides with C‐terminal aryl groups, ^[12] diverse secondary or tertiary amides, ^[13] and N‐acylpyrrole motifs. ^[14] However, challenges remain which prevent the broader application of decarboxylative strategies. For example, the relatively high oxidation potential of the C‐terminal carboxylic acid limits the functional group compatibility of the method, and extension to side‐chain modifications (e.g., the functionalization of Asp and Glu residues) is not yet possible using this strategy.

The privileged N,O‐acetal has a storied history in electro‐organic chemistry, however, and remains an attractive synthetic target. This is perhaps best exemplified by the Shono oxidation, which involves the electrochemical α‐oxidation of amides and carbamates to form N,O‐acetals poised for subsequent Lewis acid‐mediated modification with diverse nucleophiles. ^[15] In a seminal advance on the original method, Yoshida and co‐workers incorporated “electroauxiliaries” (e.g., ‐SiR₃, ‐SR or ‐SnR₃ motifs) at the carbamate α‐position (Figure 1A), which served to lower the oxidation potential of the molecule and enable programmable oxidation with high regioselectivity. ^[16] This logic has seen valuable applications ^[17] in the synthesis of amino acid derivatives, including Chiba's access to modified N‐acylprolines via anodic oxidation of thiophenyl‐substituted proline derivatives. ^[18] In this study, a lithium perchlorate/nitromethane electrolytic system facilitates a cation pool approach which stabilizes the electro‐generated iminium intermediate for reaction with electrochemically‐sensitive nucleophiles. ^[18] Moeller and co‐workers have elegantly harnessed silyl electroauxiliaries to regioselectivity generate N‐acyl iminium ions both at proline and notably, on the peptide backbone to afford a backbone‐methoxylated tetrapeptide in excellent yield. ^[19] Anodic modification of a tripeptide bearing a 2,4,6‐trimethoxyphenyl electroauxiliary appended to a C‐terminal proline residue has also been demonstrated. ^[20]

Figure 1.

A) Electroauxiliary‐based oxidation strategies; B) Electroactive glutamine residues for tunable modification.

Despite these advances, electroauxiliary‐assisted peptide transformations to date have focused almost exclusively on proline‐bearing peptides or small peptide substrates bearing hydrophobic side‐chains. As such, there is untapped potential for leveraging a broader range of Shono‐type residue‐specific manipulations. The presence of amidic side‐chains suitable for Shono‐type oxidations and the notable lack of existing approaches to residue‐specific side‐chain functionalization at glutamine ^[1c] led us to pursue an auxiliary‐based strategy for glutamine modifications. Side‐chain arylthioether auxiliaries (e.g. N,S‐acetals) were selected as the preferred candidates for building electro‐active peptide architectures, owing to their ease of installation as well as Yoshida's early observation that the electronics of the arylsulfide group can be carefully tuned to modulate substrate oxidation potential.[ ^16a , ^16c ] Fascinatingly, this observation unveils the opportunity for sequential, tunable modification of peptides bearing two discrete electroauxiliaries[ ^16c , ²¹ ] simply by modulating the applied potential in accord with the oxidation potential of judiciously chosen arylthioether auxiliaries.

Exploiting this logic, we report herein a highly programmable electro‐synthetic platform for the modification of peptide substrates. Rationally designed glutamine building blocks adorned with arylthioether substituents serve as “electrifiable” protecting groups poised for further elaboration (see Figure 1B). These residues are amenable to Fmoc‐strategy SPPS, and their utility in anodic oxidation reactions is exemplified through the preparation of an array of functionalized peptide N,O‐acetals and through the proof‐of‐principle development of an electrochemical approach to peptide stapling. Application of the method to larger peptide substrates is highlighted by the synthesis and modification of dynorphin B, a densely‐functionalized endogenous opioid peptide. Ultimately an iterative, tunable modification protocol for the late‐stage diversification of peptide substrates is demonstrated which significantly extends the scope and utility of targeted electrochemical peptide modifications. With tunability based entirely on the disparate oxidation potentials of substituted arylthioether auxiliaries, the mild, site‐selective, and sequential electrochemical modification of a peptide bearing two discretely oxidizable sites is accomplished. Such precise modifications are unattainable using existing orthogonal protection strategies.

Results and Discussion

Initially, we designed model N,S‐acetals derived from readily available propionamide, formaldehyde, and substituted aryl thiols to establish a basis for tunable functionalization. By incorporating electronically‐distinct thiophenols, bearing both electron‐donating and electron‐withdrawing groups, we aimed to examine the relationship between the electronics of the aryl sulfide auxiliary and the oxidation potential of the amide‐derived N,S‐acetal. Importantly, given the availability of proteinogenic primary amides (e.g., glutamine and asparagine side chains; C‐terminal amides) and the broad accessibility of substituted thiophenol reagents, we hypothesized that the approach could be easily extrapolated to the context of peptides.

Accordingly, model systems were accessed by treating propionamide 1 with arylthiols A–D and formaldehyde in the presence of TFA, affording 2 a–d in good isolated yields (68–75 %, Scheme 1A). Owing to the ease of accessibility of thiophenyl derivative 2 a and the likelihood that it would display an intermediary oxidation potential relative to the substituted arylsulfides (2 b–d), we screened conditions for the Shono‐type methoxylation of 2 a to prepare N,O‐acetal 3 (Scheme 1B). The electrolysis generates an N‐acyl iminium ion through initial oxidation of the N,S‐acetal, presumably driven by loss of a high‐energy non‐bonding electron from the sulfur p‐orbital ^[16b] followed by C−S bond cleavage. The iminium subsequently engages with the solvent methanol to form N,O‐acetal 3. Methanol was preferred as the trapping nucleophile due to its relatively high oxidation potential and well‐precedented application in electro‐organic reactions.[ ^17b , ²² ] To our delight, these studies highlighted the facile nature of the reaction, which proceeded in good to excellent yields under a variety of conditions (see Supporting Information). However, we noted that carbon as the working electrode and platinum as the counter electrode represented an optimal electrode pairing, and that exploiting specific supporting electrolytes, e.g., NEt₄ClO₄ (entry 3) and NBu₄BF₄ (entry 4), positively influenced the overall yield.

Scheme 1.

Development of a tunable methoxylation strategy. A) Synthesis of model systems; B) Development of anodic methoxylation conditions; C) Competition study highlighting tunable functionalization of model N,S‐acetals. ^a  All reactions were carried out at room temperature and open to air unless otherwise specified. ^{b   1}H NMR spectra of crude reaction mixture was recorded before and after electrolysis. Selectivity was evaluated by analysis of the NH‐CH₂ ‐SAr peak associated with 2 b and 2 d.

We next conducted cyclic voltammetry studies on the model N,S‐acetals using an NBu₄PF₆/MeCN electrolyte system. A shift in the oxidation peak towards higher potential was correlated with an increase in the electron withdrawing nature (OMe<H≈Cl<NO₂) of the aryl substituent, similar to the modulation of redox potentials observed in early work by Yoshida. ^[16a] Pleasingly, the oxidation potential of N‐methyl propionamide was significantly larger than the N,S‐acetals, signifying the facile oxidation of the electroauxiliary, and underscoring the feasibility of selective oxidation of N,S‐acetals in the presence of amide functionalities, which are prevalent within the peptide backbone.

To evaluate the viability of accomplishing the selective and tunable functionalization of electron‐rich electroauxiliaries, we next explored anodic methoxylation of two N,S‐acetals (2 b and 2 d, Scheme 1C) with discrete oxidation potentials in a competition study. Among a variety of electrochemical conditions trialed (see Supporting Information, p. 9), constant potential electrolysis (2 V) using NEt₄BF₄ proved optimal, with ¹H NMR analysis of the crude reaction mixture revealing a high level of selectivity for oxidation of the more electron‐rich system (2 b) in the presence of nitro‐derivative 2 d. These results suggest that the electron‐withdrawing electroauxiliaries may be “orthogonal” to conditions that enable the functionalization of the more oxidation‐prone N,S‐acetals. Intriguingly, this selectivity is distinct from the expected reactivity profile based solely on “leaving group” ability of the arylthiol motif, in which the electron‐poor nitro auxiliary would serve as a superior leaving group.

Having established a procedure for the tunable modification of model N,S‐acetals, we next aimed to synthesize peptides incorporating an electro‐active N,S‐acetal functionality. We reasoned that peptides bearing suitable electroauxiliaries could be rapidly constructed using solid‐phase peptide synthesis (SPPS) through the preparation of orthogonally‐protected, N,S‐acetal‐containing amino acid building blocks followed by incorporation into resin‐bound peptides. To establish proof‐of‐principle for the methodology, we pursue herein a strategy for the synthesis of glutamine‐derived amino acids. Nevertheless, extension of the methodology to asparagine or C‐terminal amide functionalities is also conceivable.

Glutamine‐based building blocks were readily derived from the commercially available precursor Fmoc‐Glu‐O ^t Bu (4) (Scheme 2A). Conversion to Fmoc‐Gln‐O ^t Bu (5) by treatment with Boc anhydride and NH₄HCO₃ in the presence of pyridine was followed by reaction of compound 5 with arylthiols B or D and formaldehyde at 80 °C to forge the N,S‐acetal. Finally, acidic deprotection with TFA and ⁱ Pr₃SiH furnished building blocks 6 a and 6 b.

Scheme 2.

A) Synthesis of electroactive glutamine building blocks for Fmoc‐SPPS; B) Synthesis of electroactive peptide substrates. ^a  Yields specified are following isolation via reverse‐phase HPLC.

We envisioned that exploring the scope of anodic oxidation on peptide N,S‐acetals possessing the more readily oxidizable 4‐methoxythiophenyl group would be advantageous for future studies on tunable functionalizations. We therefore carried out the synthesis of peptide N,S‐acetals 7 a–k via Fmoc‐SPPS through the incorporation of building block 6 a (Scheme 2B). To this end, we employed commercially available Rink amide resin and standard Fmoc‐SPPS protocols (see Supporting Information) to extend the peptides. Building block 6 a was coupled using Oxyma® ^[23] and N,N′‐diisopropylcarbodiimide (DIC) in DMF. Following elongation to the target peptide sequence, the resin was treated with TFA/ ⁱ Pr₃SiH/H₂O to cleave the peptides from the resin and remove side‐chain protecting groups. Gratifyingly, peptide N,S‐acetals 7 a–k were furnished in good yields (50–79 % based on the resin loading) following purification by reverse‐phase HPLC, confirming the compatibility of building block 6 a with standard methods for iterative peptide synthesis.

Encouraged by the successful preparation of peptide N,S‐acetals, we examined the scope of electrochemical functionalizations by using tripeptide N,S‐acetal 7 a as a model substrate for Shono‐type oxidations in the presence of a variety of oxygen‐based nucleophiles (Scheme 3A). Anodic methoxylation was first performed to examine the reactivity of 7 a and confirm the ability to oxidatively activate the embedded electroauxiliary toward exogenous nucleophiles. To conduct this experiment, model peptide 7 a was dissolved in MeOH and the electrolyte NBu₄ClO₄ was added to the solution. The reaction mixture was then electrolyzed at a constant current of 1 mA (low current was applied to avoid potential off‐target oxidation at α‐positions on the peptide backbone) in an undivided chamber assembled with carbon and platinum plated electrodes (see Supporting Information). Under these conditions, the resulting N,O‐acetal 8 a was afforded in excellent yield (93 %) following HPLC purification.

Scheme 3.

A) Scope of alcohol‐based nucleophiles for anodic oxidation of peptide 7 a; B) Optimization of nucleophile stoichiometry; C) Evaluation of side‐chain compatibility. ^a  All reactions were carried out at room temperature and open to air unless otherwise specified. Yields specified are following isolation via reverse‐phase HPLC.

We next pursued the anodic oxidation of 7 a with a diverse range of alcohol nucleophiles to afford N,O‐acetals 8 b–k in moderate to excellent isolated yields (Scheme 3A). In most cases, unlike with the anodic methoxylation protocol, the alcohol nucleophile was not used in solvent quantities and the solubilizing power of the nucleophile was lower than that of MeOH. Accordingly, screening of various alcohols mandated the addition of DMSO to peptide N,S‐acetal 7 a to ensure complete dissolution of the starting material. The electrolysis of model tripeptide 7 a with excess butanol (80 vol %) afforded 8 b in 62 % yield. The reactions of peptide 7 a with readily available triethylene glycol monomethyl ether (9), cyclohexanol (10) and allyl alcohol (11) were carried out using 20 vol % of the alcohol nucleophile to deliver the corresponding N,O‐acetals in 87 % (8 c), 63 % (8 d), and 96 % (8 e), respectively. The efficiency of these transformations may be attributed to the excess of nucleophile employed as well as the relative ease with which these compounds were isolated by semi‐preparative reverse‐phase HPLC. Pleasingly, the electrochemical oxidations with ethylene glycol (12), benzyl alcohol (13), pent‐4‐yn‐1‐ol (14), 2‐chloroethan‐1‐ol (15), Boc‐Ser‐OMe (16) and 3‐hydroxypropanenitrile (17)—which were carried out with a smaller excess of nucleophile (generally 50 equiv, see Supporting Information)—successfully afforded the corresponding N,O‐acetal products 8 f–k in moderate yields (33–48 %). In most cases, we attribute slightly lower yields to the loss of material upon purification by reverse‐phase HPLC, as we did not observe considerable hydrolysis or incomplete oxidation of the electroauxiliary in these experiments. Notably, the incorporation of alcohol, alkyne, alkyl halide, nitrile and protected amino acid functionalities provide valuable handles for subsequent modification. ^[24]

Considering that the application of more precious nucleophiles in lower excesses might be desirable, we also probed the impact of nucleophile concentration on the product yield (Scheme 3B). To this end, we examined the electrochemical oxidation of peptide N,S‐acetal 7 a using triethylene glycol monomethyl ether 9 as the representative nucleophile. The highest isolated yield was obtained when 9 was used in solvent quantity (20 vol %). When the concentration of 9 was reduced to 50 equiv relative to the peptide substrate, the yield decreased to 66 %. Nevertheless, similar yields of N,O‐acetal 8 c could be achieved with both 30 and 15 equiv of 9 by incorporating molecular sieves into the reaction mixture, which presumably serves to minimize competitive hydrolysis of the iminium intermediate in cases where a smaller excess of nucleophile (e.g., <50 equiv) is employed. When the concentration of 9 was reduced to 5 equiv, the reaction proceeded in the presence of molecular sieves in 46 % yield, whereas the standard reaction conditions, in the absence of sieves, afforded product 8 c in only 19 % yield. As such, the addition of molecular sieves in the electrochemical oxidation provides a viable approach to maintaining reaction yields in cases where fewer equivalents of nucleophile are employed.

Moreover, we next investigated the side‐chain compatibility of this reaction by performing the anodic methoxylation of alternative peptide N,S‐acetals 7 b–h and 7 j–k (Scheme 3C). Pleasingly, the peptides containing unprotected Ser, Asp, Met and Lys residues (7 b, 7 c, 7 e and 7 k) proceeded to form N,O‐acetals 18 a, 18 b, 18 d and 18 i in excellent yields (67 %–84 %), a notable improvement on prior decarboxylative experiments carried out in the presence of the same unprotected side‐chain functionalities.[ ¹³ , ¹⁴ ] Oxidation of peptide 7 d, bearing an unprotected Gln residue, furnished N,O‐acetal 18 c in 30 % yield. Importantly, in this case, the electroauxiliary provides a means of differentiating between the two glutamine residues present in the peptide substrate. While conventional protecting group strategies could also conceivably be employed to enable selective targeting of a single glutamine residue within a peptide sequence, there are remarkably few side‐chain amide protection strategies available. Existing methods almost exclusively leverage acidic deprotection conditions, which are not orthogonal to the conditions required for cleavage of the peptide from Rink amide resin. ^[25] Notably, in our prior work on C‐terminal decarboxylation, ^[13] unprotected aromatic residues (e.g. His, Tyr and Trp) were incompatible with the electrolysis conditions. Herein, increased side‐chain compatibility was observed with His peptide 18 e isolated in 47 % yield and Tyr peptide 18 f isolated in 19 % yield (see Supporting Information for details). Although Trp‐containing peptide 7 h and Cys‐bearing peptide 7 j are not currently compatible with the reaction conditions, the broad chemoselectivity demonstrated herein is promising for future applications on highly functionalized peptide substrates.

We were next interested in extending the electrochemical approach to facilitate peptide stapling via reaction of the embedded N,S‐acetal with an intramolecular nucleophile present on a non‐neighboring amino acid side‐chain (Scheme 4A). To this end, substrates 7 i–k, containing the auxiliary‐functionalized Gln residue as well as an internal Ser, Cys, or Lys residue, were subjected to modified electrochemical conditions. Specifically, it was envisaged that the addition of molecular sieves would be beneficial to carry out the reaction successfully with only 1 equiv of internal nucleophile. Gratifyingly, cyclic product 19 a, containing a cyclic ether motif, was forged in good isolated yield (55 %), providing exciting proof‐of‐concept for this stapling approach. In a preliminary extension to Cys‐containing peptide 7 j, considerable starting material remained following the standard reaction time course, although the mass of the desired product 19 b could be detected by UPLC‐MS analysis (see Supporting Information). The cyclic peptide 19 c, resulting from the stapling of Lys peptide 7 k, was isolated in 11 % yield, together with isolable byproducts which presumably result from either hydrolysis or overoxidation (see Supporting Information for details and further discussion). Future work will examine the cyclization pathway in further detail in order to probe substrate scope and improve reaction efficiency with additional nucleophiles.

Scheme 4.

A) Electrochemically‐enabled peptide stapling; B) Synthesis and functionalization of dynorphin B. ^a  All reactions were carried out at room temperature and open to air unless otherwise specified. Yields specified are following isolation via reverse‐phase HPLC.

We next incorporated auxiliary‐functionalized Gln building block 6 a into a larger peptide scaffold. We identified dynorphin B, a 13‐amino acid endogenous opioid peptide containing a single glutamine residue embedded within the peptide sequence, as a suitable system (Scheme 4B). Notably, this peptide also contains several potentially oxidizable side‐chains, including Tyr, Arg, Lys and Thr, and thus represents an intricate peptide system for probing the efficiency of our electrochemical transformation. Synthesis of the dynorphin B precursor via Fmoc‐SPPS proceeded smoothly on 2‐chlorotrityl chloride resin with efficient incorporation of 6 a using Oxyma/DIC (see Supporting Information for details). Following elongation and global deprotection with an acidic cleavage cocktail of TFA/ ⁱ Pr₃SiH/H₂O the fully‐deprotected electroactive peptide 20 was isolated in 30 % yield over the 25 steps from the resin loading. Despite the presence of several unprotected side‐chains, including the highly oxidation‐prone phenolic Tyr side‐chain (cf. 18 f, Scheme 3C), electrolysis of 20 afforded N,O‐acetal 21 in 7 % isolated yield. Subjecting a fully‐protected dynorphin B precursor (accessible via HFIP cleavage of the acid‐labile resin support; see Supporting Information) to the electrolysis conditions followed by TFA deprotection afforded 22, which bears an N‐methyl glutamine residue in 18 % yield (27 steps from the resin loading). The N‐methyl group is conveniently derived from reduction of the intermediary N,O‐acetal ^[13] under the reductive cleavage conditions. This intriguing pathway to glutamine modification mimics the post‐translational methylation of glutamine residues—a process which is of broad biological significance. ^[26]

Motivated by the observed compatibility of the method with a broad array of nucleophiles and in the presence of side‐chain functional groups, we next focused on the development of a tunable and sequential anodic oxidation sequence based on the relative oxidation potentials of our electroauxiliaries. Pentapeptide 23, bearing two oxidizable sites with disparate potentials, was designed as the electro‐active precursor (Scheme 5); it was envisaged that the easily oxidizable 4‐methoxythiophenyl and less oxidizable 4‐nitrothiophenyl electroauxiliaries would provide the greatest selectivity window for sequential oxidation reactions. Accordingly, we exploited standard Fmoc‐SPPS protocols for the incorporation of building blocks 6 a and 6 b into peptide substrate 23, which was synthesized in 42 % isolated yield based on the original resin loading (see Supporting Information).

Scheme 5.

Sequential modification of electroactive peptide 23 via tunable anodic oxidation. ^a  All reactions were carried out at room temperature and open to air unless otherwise specified. Yields specified are following isolation via reverse‐phase HPLC.

Leveraging the tunable electrochemical method optimized on the model N,S‐acetals (see Scheme 1C), peptide 23 was subjected to anodic methoxylation using NEt₄BF₄/MeOH as the electrolyte‐solvent mixture at a constant potential of 2 V. Satisfyingly, the reaction gave rise to the mono‐functionalized peptide 24 in 67 % isolated yield following the delivery of 2.7 F mol⁻¹, a slight excess of charge compared to model N,S‐acetal functionalization. As anticipated, the oxidation occurred site‐selectively at the 4‐methoxythiophenyl electroauxiliary. A sequential methoxylation was also performed on 24 to afford peptide N,O‐acetal 25 under constant potential conditions at 2.5 V. Interestingly, this reaction did not proceed to completion as a result of high resistance. We therefore took advantage of constant current conditions with the intention of driving the process to completion, which resulted in superior conversion to peptide 25 (32 % isolated yield) when the reaction was conducted at 6 mA.

Having established proof‐of‐principle for the selective functionalization, we next investigated triethylene glycol monomethyl ether 9 as the initial nucleophile for tunable oxidation using the optimized method. By delivering 3.5 F mol⁻¹ charge at a constant potential of 2 V, the transformation afforded product 26 in good isolated yield (66 %). Subsequent oxidation of the second electroauxiliary using methanol resulted in lower yield (15 %) of peptide 27. We reasoned that the second oxidation, which involves the expulsion of the more oxidatively recalcitrant 4‐nitrothiophenyl group, must proceed at relatively high potential, which may serve to diminish the overall yield relative to the more facile oxidation of the 4‐methoxythiophenyl auxiliary. In a subsequent endeavor, we employed allyl alcohol as the nucleophile in the anodic oxidation of peptide 26. The product derived from this transformation (28) was furnished in 40 % isolated yield. An alternative protocol without intermediary purification also led to the successful formation of product 28 (see Supporting Information). ^[27] The incorporation of both a solubilizing triethylene glycol chain and an alkenyl handle into peptide 28 illustrates the utility of the electrochemical method for the generation of high‐value, functionalized peptide products. Moreover, the late‐stage, site‐selective installation of two distinct N,O‐acetal linkages would be difficult to accomplish with existing side‐chain protection strategies, in which three distinct levels of orthogonality (resin cleavage, followed by two discrete side‐chain deprotections) are required. In contrast to conventional “reagent‐based” chemical approaches, this method also mitigates the need to employ different deprotection agents, with sequential, selective modification achieved simply by dialling up the potential or current.

Conclusion

In summary, we report the first electrochemical approach for the tunable, residue‐specific modification of glutamine residues within a peptide sequence by exploiting the utility of electroauxiliary‐mediated Shono‐type oxidation chemistry. The electro‐active peptides were rapidly constructed via Fmoc‐SPPS employing easily accessible arylthioether‐modified glutamine building blocks. We examined the scope of electroauxiliary‐assisted electrochemical modification of glutamine residues with a wide array of alcohol nucleophiles to deliver peptide N,O‐acetals possessing valuable functional handles. Moreover, we demonstrate proof‐of‐principle for the extension of the method to the synthesis of stapled peptides and a larger bioactive peptide substrate. Galvanized by opportunities for the tunable electrochemical modification of peptides, a substrate bearing two discretely‐oxidizable sites was prepared and successfully employed in a sequential modification strategy. The most electron‐rich auxiliary, bearing a low oxidation potential, is selectively oxidized while the more electron‐deficient group, displaying a higher oxidation potential, remains intact. This powerful functionalization logic contradicts archetypal reactivity patterns based solely on “leaving group” ability and unlocks new levels of orthogonality to afford greater opportunities for the targeted modification of complex peptides. Given the operationally simple setup and mild reaction conditions, we envisage that this approach will add value to the growing repertoire of sustainable and selective peptide modification strategies.

Experimental Section

Detailed experimental procedures and characterization data is available in the Supporting Information.

Conflict of interest

The authors declare no conflict of interest.

Supporting information

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

Supporting Information

Karipal Padinjare Veedu D., Connal L. A., Malins L. R., Angew. Chem. Int. Ed. 2023, 62, e202215470; Angew. Chem. 2023, 135, e202215470.

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.