Secondary-Amine Relay Enables Lysine-Directed Multicomponent Functionalization of Peptides

Abstract

Multicomponent reactions provide powerful modularity for peptide functionalization, but their application to lysine residues remains limited by the low efficiency of direct primary-amine-based activation pathways under peptide-compatible conditions. Here we report a secondary-amine relay strategy for lysine-directed multicomponent functionalization of peptides. In this approach, salicylaldehyde derivatives, arylboronic acids, and a cyclic secondary amine are combined to generate a transient electrophilic intermediate, which is subsequently intercepted by the ε-amine of lysine-containing peptides. By separating electrophile generation from direct lysine activation, this relay protocol improves the efficiency of lysine modification while retaining the modularity of multicomponent chemistry. Mechanistic experiments, isolated intermediate studies, kinetic comparisons, and density functional theory calculations support a pathway involving secondary-amine-mediated intermediate formation followed by lysine capture. The method enables modification of diverse bioactive peptides, incorporation of drug-derived and fluorescent aldehyde or boronic acid components, and lysine–lysine stapling using bifunctional aldehydes. The reaction is further compatible with automated and parallel solid-phase peptide modification workflows, providing rapid access to libraries of lysine-functionalized peptide conjugates. This study provides a practical secondary-amine relay platform for modular peptide functionalization and expands the utility of multicomponent chemistry in automated peptide synthesis.

1. Introduction

Site-selective modification of peptides and proteins is central to the preparation of peptide-drug conjugates, probe-labeled biomolecules, and chemically diversified bioactive scaffolds. − Among native functional groups, lysine ε-amines are particularly attractive because of their abundance, nucleophilicity, and frequent presence in bioactive peptides and proteins. − However, the same abundance and high basicity that make lysine synthetically accessible also complicate selective and modular functionalization, especially under aqueous or peptide-compatible conditions. ,

A variety of chemical and enzymatic strategies have been developed for lysine modification. − These methods have greatly expanded the toolbox for amine-directed bioconjugation, yet many rely on electrophilic reagents or activation modes in which productive bond formation is closely linked to direct reaction with the target lysine ε-amine. , For multicomponent reactions involving primary amines, this requirement can be particularly limiting, because direct formation of lysine-derived imines or related intermediates is often inefficient, reversible, or hydrolytically sensitive in aqueous and dilute environments. − As a result, achieving efficient lysine modification while simultaneously preserving the modularity of multicomponent chemistry remains a significant challenge in peptide functionalization. ,

Borono-Mannich multicomponent chemistry has been widely used to assemble structurally diverse amine-containing products from aldehyde, amine, and boronic acid components. − In peptide settings, related transformations have enabled modification of secondary amines, N-terminal residues, and macrocyclization through iminium-based pathways (Figure a). , However, direct application of this chemistry to lysine ε-amines in unprotected or minimally protected peptides remains less straightforward. Classical primary-amine-based pathways require the target amine to participate in imine formation before C–N bond construction, , and lysine-derived imines are generally less favorable under peptide-compatible conditions, leading to reduced efficiency and limited practical scope in aqueous media (Figure b).

1.

Secondary-amine relay strategy for lysine-directed multicomponent functionalization. (a) Established iminium-based multicomponent reactions involving secondary amines for peptide modification and macrocyclization. (b) Direct primary-amine-based pathways for lysine modification are often inefficient under aqueous and peptide-compatible conditions because lysine-derived imines are reversible and hydrolytically sensitive. (c) In the secondary-amine relay design, a cyclic secondary amine mediates formation of a transient electrophilic intermediate, which is subsequently captured by the lysine ε-amine without requiring direct lysine-imine formation.

We envisioned that these limitations could be addressed by separating electrophile generation from direct lysine activation. In contrast to classical designs in which the lysine ε-amine participates directly in the formation of the reactive imine or iminium intermediate, , our strategy assigns this upstream activation role to a cyclic secondary amine. The secondary amine engages salicylaldehyde derivatives and arylboronic acids to generate a transient salicylaldehyde–boronic acid-derived electrophilic intermediate, which can then be intercepted by the lysine ε-amine as a downstream nucleophile (Figure c). In this secondary-amine relay design, lysine is no longer required to serve simultaneously as the site of electrophile generation and nucleophilic capture, thereby providing a practical route to lysine-directed multicomponent peptide functionalization.

Here we report the development of this secondary-amine relay strategy for lysine-directed functionalization of peptides. Mechanistic experiments, isolated-intermediate studies, kinetic comparisons, and density functional theory calculations support a pathway involving secondary-amine-mediated intermediate formation followed by lysine capture. The method enables modification of diverse bioactive peptides, incorporation of drug-derived and fluorescent aldehyde or boronic acid components, and lysine–lysine stapling using bifunctional aldehydes. In addition, the reaction is compatible with automated and parallel solid-phase peptide modification workflows, providing rapid access to libraries of lysine-functionalized peptide conjugates. These results establish a practical multicomponent platform for modular peptide functionalization and highlight the value of relay-based activation strategies in automated peptide synthesis.

2. Results and Discussion

2.1. Relay-Mediated Electrophile Generation Enables Efficient Lysine Functionalization

We first examined whether a lysine-containing peptide could be functionalized under direct primary-amine-based multicomponent conditions. Treatment of peptide 3a with salicylaldehyde (1a) and 4-methoxyphenylboronic acid (2a) in acetonitrile/water at 80 °C afforded the desired lysine-modified product 4a in only 19% conversion, as determined by LC-MS analysis (Figure a, entry 1). This low efficiency is consistent with the limited reactivity of primary amines in peptide-compatible borono-Mannich processes and highlights the need for an alternative activation design.

2.

Relay-mediated electrophile generation enables lysine-directed functionalization. Reaction conditions: salicylaldehyde 1a (2.0 equiv), 4-methoxyphenylboronic acid 2a (3.0 equiv), and 2-methylpyrrolidine (3.0 equiv) were combined at 80 °C for 2 h prior to peptide addition. Yields were determined by HPLC-UV, and isolated yields are shown in parentheses. ^a Product 4ax was monitored as the target product (see Supporting Information, Section 2.2).

To evaluate whether electrophile generation could be promoted independently of direct lysine activation, a series of secondary amines were introduced into the reaction system. Cyclic secondary amines substantially altered the reaction outcome, although they were not incorporated into the final lysine-modified product. Among those examined, 2-methylpyrrolidine increased the conversion of 3a to 4a to 51% (Figure a, entry 2), whereas pyrrolidine, piperidine, and l-proline showed more modest effects (Figure a, entries 3–5). LC-MS monitoring revealed new species derived from salicylaldehyde, the arylboronic acid, and the secondary amine, suggesting the formation of reactive intermediates capable of promoting lysine capture. In contrast, replacement of the arylboronic acid with (E)-phenylethenylboronic acid led to only 8% yield of the target product (4ax) (Figure a, entry 6), consistent with the lower stability of the corresponding intermediates and products. ,

These observations led us to develop a one-pot, two-step protocol in which electrophile generation and lysine capture were temporally separated. Salicylaldehyde (1a), 4-methoxyphenylboronic acid (2a), and 2-methylpyrrolidine were combined to generate the reactive intermediate in situ, followed by addition of peptide 3a. Under these conditions, product 4a was obtained in 89% yield and 77% isolated yield (Figure a, entry 7), representing a substantial improvement over the direct reaction. Comparable efficiency was maintained at elevated temperature (Figure a, entry 9), whereas room-temperature conditions resulted in slower but still productive conversion (Figure a, entry 8). Solvent screening further showed that DMF/water provided alternative performance, while methanol and DCE gave diminished yields, likely due to reduced solubility of the peptide substrate (Figure a, entries 10–12).

Kinetic analysis revealed that the rate of lysine functionalization was strongly influenced by the identity of the secondary amine (Figure a). Intermediates generated from 2-methylpyrrolidine promoted rapid formation of 4b, reaching high conversion within 20 min, whereas those derived from pyrrolidine, piperidine, and l-prolinamide required longer reaction times to achieve comparable yields. Preformed or in situ generated intermediates Int-ii-a–d all afforded efficient lysine modification after extended reaction times (>85% yield of 4b, Figure b), indicating that secondary amines play an active and structure-dependent role in upstream intermediate formation rather than serving as simple additives.

To further define the structural requirements for productive relay-mediated functionalization, a series of designed intermediates were evaluated (Figure b). Intermediate Int-ii-e, which retains the ortho-hydroxy group but contains a modified amine moiety, afforded 4c in moderate yield with minimal bis-addition. In contrast, non-hydroxylated analogues Int-ii-f and Int-ii-g failed to produce detectable lysine-modified products (4d and 4e). These results indicate that the ortho-phenolic hydroxyl group is critical for productive reactivity and electrophile generation. Control experiments using non-ortho-hydroxybenzaldehyde derivatives failed to afford the desired product, and the corresponding intermediates were not detected (Figure S1). This observation highlights the importance of the ortho-hydroxyl group in facilitating boron coordination and promoting formation of Int-ii, which is proposed to undergo subsequent conversion into Int-iii. This requirement also represents a current limitation of the transformation, as productive reactivity is largely restricted to ortho-hydroxyl-containing aldehydes. These findings are consistent with the proposed relay pathway, in which secondary-amine-derived intermediates undergo proton-transfer-assisted elimination to generate a proposed transient electrophilic species that can be captured by lysine residues without requiring direct lysine-imine formation from the peptide substrate (Figure ).

3.

Computational analysis of the secondary-amine relay pathway for lysine functionalization.

2.2. Mechanistic Origin of Relay-Mediated Lysine Functionalization

To gain insight into the mechanistic basis of the relay-mediated pathway, density functional theory (DFT) calculations were performed to compare direct lysine-based activation with secondary-amine-mediated relay activation (Figure ). We first examined a classical primary-amine-based pathway involving imine formation between the lysine ε-amine and salicylaldehyde. Consistent with the low conversion observed experimentally under direct reaction conditions, this lysine-derived imine showed limited thermodynamic driving force. Subsequent C–N bond formation through addition of the arylboronic acid to the lysine-derived imine was calculated to require a high activation barrier (ΔG‡ = 63.0 kcal mol^–1), suggesting that this direct pathway is kinetically unfavorable under the examined peptide-compatible conditions (Figure ).

In contrast, secondary amines more readily participate in salicylaldehyde activation to generate iminium intermediates, providing a favorable entry point for relay-mediated intermediate formation. The calculated free energies for iminium formation were −22.3 kcal mol^–1 for 2-methylpyrrolidine, −22.4 kcal mol^–1 for pyrrolidine, −18.9 kcal mol^–1 for piperidine, and −20.4 kcal mol^–1 for l-prolinamide (Figure ). Subsequent addition of the arylboronic acid affords intermediates Int-ii, which can serve as precursors for downstream electrophile generation.

Proton-transfer-assisted elimination of the secondary amine from Int-ii is proposed to generate a transient ortho-quinone methide intermediate (Int-iii), which can subsequently be captured by lysine residues. − The calculated free energy changes for the Int-ii → Int-iii conversion were +1.0 kcal mol^–1 for 2-methylpyrrolidine, +7.9 kcal mol^–1 for pyrrolidine, +6.8 kcal mol^–1 for piperidine, and +1.9 kcal mol^–1 for l-prolinamide, revealing a clear dependence on the identity of the secondary amine (Figure ). These energetic trends are consistent with the experimentally observed differences in reaction kinetics and help rationalize the higher reactivity observed for intermediates derived from 2-methylpyrrolidine.

Capture of the relay-generated electrophilic species by lysine residues was calculated to proceed through a lower-energy transition state, with an activation barrier of ΔG‡ = 12.2 kcal mol^–1 (Figure ). This barrier is substantially lower than that calculated for direct C–N bond formation through the lysine-derived imine pathway, supporting the kinetic advantage of the relay-mediated process. These calculations, supported by the intermediate studies and kinetic experiments, are consistent with a mechanism in which secondary-amine-mediated electrophile generation is separated from lysine capture.

2.3. Scope of Peptide Functionalization

With the optimized relay conditions established, we evaluated the scope of lysine-directed functionalization across structurally diverse bioactive peptides and functional components (Figure ). Sixteen lysine-containing peptides, ranging from dipeptides to nonapeptides, were converted to the corresponding modified products in 36–77% isolated yields. Short peptides, including the dipeptide vilon (3c), underwent lysine modification to afford 4f in 52% isolated yield. Tri- and tetrapeptides bearing free carboxyl, hydroxyl, or guanidino groups also reacted efficiently, delivering products 4g–4k in 41-76% isolated yields. The cell-penetrating peptide CPPP-2, bearing a free C-terminal carboxyl group, afforded 4l in 67% isolated yield. Pentapeptide substrates, including the Bax inhibitor peptide V5 (3j) and antagonist tcY-NH₂ (3k), were also compatible with the relay protocol. Peptides containing oxidation- or nucleophile-sensitive residues, such as tryptophan, methionine, and glutamine, furnished hexa- and heptapeptide conjugates 4o–4s in 36-66% isolated yields. Longer nonapeptides, including β-neo-endorphin (3q) and melanostatine-5 (3r), afforded the corresponding lysine-modified products in 59% and 77% isolated yields, respectively. Peptides containing Cys, His, or a free N-terminal amine together with Lys were further evaluated (Table S1). These substrates exhibited residue-dependent reactivity, with preferential Lys modification accompanied by competitive modification at other unprotected nucleophilic sites in certain cases. These results further define the modification preference and limitations of the relay strategy and demonstrate its capability for lysine-directed functionalization across diverse peptide sequences under peptide-compatible conditions.

4.

Scope of relay-mediated lysine functionalization across bioactive peptides and functional components. Reaction conditions: salicylaldehyde derivatives 1 (2.0 equiv), arylboronic acid derivatives 2 (3.0 equiv), and 2-methylpyrrolidine (3.0 equiv) were combined at 80 °C for 2 h before peptide addition. The reaction mixture was then stirred at 80 °C for an additional 2 h. Yields were determined by HPLC-UV, with isolated yields shown in parentheses.

We next examined the modularity of the reaction using CPPP-2 (3b) as a representative peptide scaffold. Diverse salicylaldehyde and arylboronic acid components were incorporated without reoptimization, enabling installation of structurally varied functional groups. Salicylaldehyde derivatives introduced aldehyde-containing and heterocyclic motifs, affording products such as 4v in 60% isolated yield, while thiazole and polyaromatic substituents gave modified peptides 4w and 4x. Drug-derived aldehydes and boronic acids, including ibuprofen, naproxen, fenbufen, indomethacin, estrone, and diclofenac derivatives, were directly incorporated to afford peptide-drug conjugates 4y-4ab and 4ai-4aj in 40-68% isolated yields. Additional arylboronic acids bearing click handles, PEGylated groups, heteroaromatic frameworks, and a fluorescent dansyl group delivered products 4ac-4ah and 4ak in synthetically useful yields. The peptide and component scope highlights the utility of the secondary-amine relay protocol for modular access to lysine-functionalized peptide conjugates with both sequence variation and functional-group diversity.

2.4. Peptide Stapling and Exploratory Protein Modification

To examine whether the secondary-amine relay strategy could be extended beyond linear peptide modification, bifunctional aldehydes were used to promote lysine–lysine crosslinking (Figure a). Treatment of lysine-containing peptides with 2,6-diformylphenol enabled intramolecular stapling, converting tetrapeptide-30 (3s) and the Ca²⁺ channel blocker CALP3 (3t) into cyclic peptides 5a and 5b in 33% and 28% isolated yields, respectively.

5.

Extension of relay-mediated lysine functionalization to peptide stapling and exploratory protein modification. (a) Peptide stapling. Reaction conditions: 2,6-diformylphenol 1i (2.0 equiv), 4-methoxyphenylboronic acid 2a (6.0 equiv), and 2-methylpyrrolidine (6.0 equiv) were combined at 80 °C for 2 h before peptide addition. Yields were determined by HPLC-UV, with isolated yields shown in parentheses. (b) Exploratory protein modification. Reaction conditions: salicylaldehyde derivatives 1 (20 equiv. per lysine residue), 4-methoxyphenylboronic acid 2a (30 equiv. per lysine residue), and 2-methylpyrrolidine (30 equiv. per lysine residue) were combined in MeCN at 80 °C for 2 h. After cooling to room temperature, protein (1 mM) and PBS (pH ∼7) were added. The reaction mixture was stirred at room temperature for the indicated time and analyzed by HPLC/MS.

We also evaluated the applicability of this strategy to small protein substrates (Figure b). Aprotinin, a 58-amino-acid protein containing four lysine residues, afforded lysine-modified species as detected by intact protein LC–MS analysis, including conjugation with a naproxen-derived motif. Lysozyme (∼14.3 kDa) gave mono- and di-modified products under aqueous conditions. These preliminary protein results indicate that relay-generated electrophilic intermediates can be captured by accessible lysine residues in folded protein substrates, while also highlighting the need for further site-mapping studies for applications requiring defined protein conjugates.

2.5. Compatibility with Automated and Parallel Solid-Phase Synthesis

We next examined the compatibility of the secondary-amine relay strategy with solid-phase peptide synthesis workflows (Figure ). In an initial demonstration, resin-bound CPPP-2 prepared by conventional solid-phase peptide synthesis was subjected to on-resin lysine functionalization under the relay conditions (Figure a). Installation of a dansyl boronic acid derivative together with salicylaldehyde derivatives bearing anti-inflammatory drug motifs afforded peptide–drug–probe conjugates 4al-4ao in 40-47% isolated yields after cleavage, showing that the relay-mediated lysine functionalization can be performed on solid support.

6.

Integration of relay-mediated lysine functionalization with solid-phase and automated peptide synthesis workflows. (a) On-resin synthesis of peptide-drug-probe conjugates. (b) Automated workflow for peptide assembly and lysine modification, and parallel workflow for peptide library modification.

Encouraged by this solid-phase compatibility, we integrated the reaction into an automated solid-phase flow synthesis platform for peptide assembly and modification (Figure b). − Resin-bound peptides were assembled under automated flow conditions and deprotected at lysine residues by Dde removal, followed by relay-mediated lysine functionalization at elevated temperature. The recirculating flow design allowed the modification solution to be reused during the reaction while maintaining efficient contact with the resin-bound peptide before final cleavage from the solid support.

We further constructed an automated parallel solid-phase flow platform containing 20 reaction columns to enable higher-throughput peptide modification (Figure b). Each column was loaded with a distinct immobilized bioactive peptide substrate, and a relay-activated modification solution was sequentially passed through all columns in a continuous workflow. After cleavage and analysis, the modified peptides were obtained in 33-87% isolated yields. LC-MS analysis confirmed the formation of the expected lysine-modified products across the peptide set, demonstrating the compatibility of this relay strategy with automated and parallel peptide modification workflows.

3. Conclusion

This study describes a secondary-amine relay strategy for lysine-directed multicomponent functionalization of peptides. By separating electrophile generation from direct lysine activation, the method improves the efficiency of lysine modification under peptide-compatible conditions while preserving the modularity of aldehyde and boronic acid components. Mechanistic experiments, isolated-intermediate studies, kinetic comparisons, and DFT calculations support a pathway in which secondary amines mediate upstream electrophile formation, followed by capture of the transient intermediate by lysine ε-amines. The reaction was applied to diverse bioactive peptides, enabled installation of drug-derived and fluorescent functional groups, and supported lysine–lysine stapling with bifunctional aldehydes. Preliminary experiments with small proteins further showed that accessible lysine residues can participate in this relay-mediated process. The compatibility of the reaction with automated and parallel solid-phase workflows provides a practical route to libraries of lysine-functionalized peptide conjugates. This secondary-amine relay design offers a useful platform for modular peptide modification and expands the application of multicomponent chemistry in automated peptide synthesis.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.6c00945.

• General experimental information; experimental procedures; compound characterization data; DFT computational details; protein modification experiments; and automated and parallel solid-phase synthesis workflows (PDF)

C.R. and C.L. conceived and designed the experiments; C.R., M.Z., T.J., F.W., and Y.L. performed and analyzed the experiments; C.R. and C.L. wrote the manuscript; C.L. and L.Y. supervised and guided the entire project.

The authors declare no competing financial interest.