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Leveraging the Mukaiyama oxidation–reduction condensation reaction for on-resin aryl thio-esterification for bio-conjugation†

Andrew Siow and Paul W. R. Harris*
School of Biological Sciences, School of Chemical Sciences and Maurice Wilkins Centre for Molecular Biodiversity. The University of Auckland, The University of Auckland, 3b and 23 Symonds Street, Auckland 1010, New Zealand. E-mail: asio616@aucklanduni.ac.nz; paul.harris@auckland.ac.nz

First published on 20th August 2024

Originally, synthetic methods to form C-terminal α-thioesters peptides utilised Boc-solid phase peptide synthesis (Boc-SPPS) using mercaptoalkyl acid linkers, cleaved via hazardous reagent HF.⁶ However, the susceptibility of thioesters to secondary amines such as piperidine utilised in 9-fluorenyl-methoxycarbonyl (Fmoc)-SPPS renders this approach unsuitable for direct preparation of peptide thioesters via Fmoc-SPPS. This has set precedence for the development of chemical technologies to facilitate synthesis of peptide thioesters bearing the thiol auxiliary or reactive intermediates that can be post modified to aryl or alklythioester precursors. Such methodologies developed, but not limited to are listed in (Fig. 1A–I).^3,7–18 This demonstrates that no one protocol can be considered as universal for Fmoc-based thioester synthesis.²⁹ We envisioned a new methodology to directly install both aryl and alkyl thioesters on-resin would be attractive as C^α-terminal arylthioesterification is highly sought after for the chemical synthesis of both peptides and proteins (Fig. 1J).

	Fig. 1 (A–I) Overview of selected current thioesterification methodologies. (J) Our presented work.

To enable arylthioesterification on-resin we took inspiration from the named Mukaiyama oxidation–reduction condensation reaction. Mechanistically this approach follows a Mukaiyama oxidation–reduction condensation procedure in which the carboxylic acid 1 is activated with intermediate 2 generated by 2,2′-dipyridyldisulfide (DPS) and triphenylphosphine (PPh₃) to form 3 and triphenylphosphine oxide 4 by-product, followed by formation in situ to the reactive 2-pyridyl thioester species 5 (Scheme 1A).^19,20 This activated thioester derivative 5 has been utilised in various chemical reactions such as the Corey–Nicolaou macrolactonization reaction or aminolysis of 2-pyridyl thioesters for peptide condensation reactions.^21,22 However, currently there are no reports of the Mukaiyama oxidation–reduction condensation being used in Fmoc-SPPS to directly afford aryl(or alkyl)-thioesters on-resin, and it was proposed that this procedure could be repurposed to furnish C^α-terminal thioesters from protected peptides containing unprotected C^α-carboxylic acids 6 (Scheme 1B). This resulting 2-pyridylthiol ester 7 should undergo rapid thiolysis via nucleophilic acyl thiol substitution.

	Scheme 1 (A) Mukaiyama oxidation–reduction condensation reaction mechanism.21,26 (B) Our modified Mukaiyama oxidation–reduction condensation thioesterification reaction on-resin and application.

Herein, we report a modified Mukaiyama oxidation–reduction condensation reaction demonstrating nucleophilic aryl-thiol substitution on the corresponding in situ 2-pyridylthiol ester 7 on-resin, which synthetically furnishes a wide variety of peptide thioesters on-resin 8. This would facilitate NCL between the proposed synthesised C^α-terminal thioester 9 and corresponding N-terminal cysteine 10b resulting in a S-to-N internal acyl shift affording the desired ligated polypeptide 11b (Scheme 1B). Our approach circumvents the requirement of excess exogenous thiol during NCL to drive the in situ formation of more active C^α-arylthioesters from the less reactive C^α alkylthioesters. This can invariably lead to unwanted mixed disulphides, and hamper reaction monitoring and purification. The work developed herein also provides a more straightforward entry into the convergent kinetic chemical ligation (KCL) chemistry,²³ allowing a direct synthesis of C^α-arylthioesters on-resin circumventing often used solution-phase conversion of C^α-alkythiolester peptides to C^α-arylthioester peptides that is governed by equilibrium processes and which requires a tedious HPLC purification prior to KCL.

Our initial investigation employed a C^α-terminally allyl protected resin-bound peptide 12 (Scheme 2) orthogonally loaded on 2-chlorotrityl chloride polystyrene resin (2-CTC) (0.1 mmol, 0.64 mmol g⁻¹ loading). This would provide a platform for orthogonal allyl removal exposing the C-terminal acid 13 for thioesterification. This C-terminal expansion approach has been exploited to facilitate side anchoring as reported by Barany and co-workers via a backbone reductive amination on the α-amine of the prospective C-terminal amino acid followed by secondary amine protecting group strategies/peptide extension and further arylthioester couplings; or Payne and co-workers who synthesised alkyl thioesters via direct coupling of alkylthioester derived building block couplings to extend the C-terminus. To our knowledge the above methods have not been shown to directly install C^α-terminal arylthioesters at the desired C-terminal amino acid by direct aryl-thioesterification via a Mukaiyama oxidation–reduction condensation type reaction procedure in a streamlined approach.^15,24,25 With precursor 13 in hand, we applied our Mukaiyama oxidation–reduction condensation modification employing 4-MPAA for nucleohpilic insertion.

	Scheme 2 Synthesis of 15. Reaction conditions: (i) Pd(PPh3)4 (0.2 mmol), PhSiH3 (0.9 mmol), DMF/CH2Cl2, r.t, 3 h; (ii) DPS (0.4 mmol), PPh3 (0.4 mmol) anhy. CH2Cl2, r.t, 1 h; (iii) anhy. CH2Cl2, 2, r.t, 1 h; (iv) 4-MPAA (0.4 mmol), i-Pr2NEt (0.4 mmol), anhy. DMF, 16 h, r.t; (v) TFA/H2O/TIPS/EDT, r.t, 2 h.

Thus activation of DPS (4 equiv.) with PPh₃ (4 equiv.) in anhydrous CH₂Cl₂ afforded 2 which is cannulated to 13 affording 14 in situ. To intermediate 14 was added 4-MPAA/base in anhydrous DMF, in a one-pot reaction followed by acidolysis affording 15. This methodology demonstrated a reasonable degree of scope (Table 1) (see the ESI† for details). Retrospectively, it should be noted that attempts to isolate the 2-pyridinethiol intermediate 14 were unsuccessful as owing to the intrinsic nature of 14, ester hydrolysis can likely occur on 14 after resin cleavage. Additionally, to establish that the in situ 14 was indeed formed on-resin, parallel to our attempt i-Pr₂NEt and 4-MPAA were directly added to C-terminal carboxylate 13 to observe if thioesterification occurred without the presence of 2-pyridinethiol ester. No thioesterification was observed by LC-MS and RP-HPLC.

However, initial studies identified the formation of diasteroisomers occuring during the Mukaiyama oxidation–reduction condensation step for entries 7–9, 11–12 as evidenced by RP-HPLC/LC-MS (see the ESI Fig. S30, S31, S44, S45 and S25† respectively). It was theorised that prolonged exposure of C^α-terminal residues existing as the transitional 2-pyridylthiol construct 7 would promote epimerisation from H⁺ abstraction at the labile C^α-H site due to resonance stabilization via the 2-pyridylthiolate moiety. This limitation could be addressed by incorporation of 1-hydroxy-6-chlorobenzotriazole (6-Cl-HOBt) to suppress proton abstraction during the activation step of the corresponding C^α carboxylic acid site. Analysis confirmed that this modification suppressed epimerisation as evidenced by RP-HPLC analysis allowing for the synthesis of the C^α-arylthioester peptides (entries 10, 13–19, see ESI Fig. S19, S20, S32, S33, S46–S49, S21, S23, S25, S51, S52, S54 and S55† respectively). To ensure epimerisation was not occurring at the C-terminal activation step both (L) and (D) diastereomers of Glu-23 and 24 and snakin-1 (12-33) 26 and 27 were synthesised separately and co-injected to confirm the absolute configuration of separate diastereomers (see ESI Fig. S25, S56 and S57†), thereby confirming retention of sterochemistry during thioesterifcation. We subsequently screened NCL reactions to validate our C^α-arylthioester peptides synthetic strategy (see ESI,† pages 61–82). We determined the scope by several practical NCL synthetic applications (Scheme 3A–F). Our protocol was appied to the synthesis of a truncated cysteine rich natural product snakin-1 isolated from Solanum tuberosum.²⁷ A convergent NCL synthesis could be applied by direct C^α-thioarylesterification on the appropriate fragments of snakin-1 furnishing the reactive thioester branch point for NCL (Scheme 3E). Through analysis it was confirmed that our optimised Mukaiyama oxidation–reduction condensation C^α-thioarylesterification reaction suppressed epimerisation as evidenced by RP-HPLC allowing for the synthesis of C^α-arylthiol 4-MPAA (26) and thiophenol (27) fragments (see ESI Fig. S25†). This facilitated direct NCL between arylthiol (26) and cysteinyl fragment (35) affording ¹²Arg-⁴⁶Glu 36 (Scheme 3E, see ESI Fig. S66 and S67†).

	Scheme 3 (A) NCL between 15 and 30. (B) NCL between 17 and 30. (C) NCL between 23 and 30. (D) NCL between 22 and 30. (E) Synthesis of truncated snaking-1 analogue 36. (F) NCL between 28 and 37 and NCL between 29 and 37 for synthesis of vascular endothelial growth factor peptide analogue 38. Reaction conditions: (i) 2 mM peptide concentrations, 6 M Gdn·HCl, 0.2 M Na2HPO4, 50 mM TCEP. HCl, r.t, 3 h.

We further elucidated our methodology by applying it to the synthesis of an analogue of the endothelial cell-specific mitogen protein vascular endothelial growth factor (VEGF) (Scheme 3F).²⁸ C^α-arylthioesterifications on fragment ¹⁹Cys-⁵⁹Glu could be furnished affording 28/29 (Scheme 3F, see ESI Fig. S50–S55†). NCL ligations with the corresponding cysteinyl fragment 37 (see ESI Fig. S80–S83†) afforded the crude 82-residue polypeptide 38, with only by-product resulting from N–C intramolecular cyclisation of thioester intermediates 28 and 29 which could be avoided with Thz masked terminal cysteines.

This approach demonstrates the first example of C^α-thioarylesterification on-resin via Fmoc-SPPS using a modified Mukaiyama oxidation–reduction condensation inspired thioesterification step affording C-terminal arylthioesters on a diverse range of resin/linkers/amino acids and thiols. Importantly this methodology provides C^α-4-MPAA peptide auxiliaries on and off resin as relevant intermediates which were successfully employed in NCL reactions and rapidly gave the expected ligation products. We consider our presented methodology offers a facile, direct synthetic route in a straightforward manner via arylthiolesterification on-resin providing an alternative strategy to this critical chemical scaffold expanding the chemical toolbox for C^α-thioarylester synthesis and therefore both polypeptide and chemical protein synthesis.

Data availability

Conflicts of interest

Acknowledgements

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