Volume 29, Issue 70 e202302689
Research Article
Open Access

Cysteine-Cysteine Cross-Conjugation of both Peptides and Proteins with a Bifunctional Hypervalent Iodine-Electrophilic Reagent

Ilias Koutsopetras

Ilias Koutsopetras

UMR 7199 CNRS-UdS, Chime Bio-Fonctionnelle, Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch cedex, France

These authors contributed equally to this work.

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Dr. Abhaya Kumar Mishra

Dr. Abhaya Kumar Mishra

Laboratory of Catalysis and Organic Synthesis, Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédéralede de Lausanne, 1015 Lausanne, Switzerland

These authors contributed equally to this work.

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Rania Benazza

Rania Benazza

Laboratoire de Spectrométrie de Masse BioOrganique, IPHC UMR 7178, Université de Strasbourg CNRS, 67087 Strasbourg, France

Infrastructure Nationale de Protéomique ProFI-FR2048, 67087 Strasbourg, France

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Oscar Hernandez-Alba

Oscar Hernandez-Alba

Laboratoire de Spectrométrie de Masse BioOrganique, IPHC UMR 7178, Université de Strasbourg CNRS, 67087 Strasbourg, France

Infrastructure Nationale de Protéomique ProFI-FR2048, 67087 Strasbourg, France

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Sarah Cianférani

Sarah Cianférani

Laboratoire de Spectrométrie de Masse BioOrganique, IPHC UMR 7178, Université de Strasbourg CNRS, 67087 Strasbourg, France

Infrastructure Nationale de Protéomique ProFI-FR2048, 67087 Strasbourg, France

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Dr. Guilhem Chaubet

Corresponding Author

Dr. Guilhem Chaubet

UMR 7199 CNRS-UdS, Chime Bio-Fonctionnelle, Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch cedex, France

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Dr. Stefano Nicolai

Corresponding Author

Dr. Stefano Nicolai

Laboratory of Catalysis and Organic Synthesis, Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédéralede de Lausanne, 1015 Lausanne, Switzerland

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Prof. Dr. Jérôme Waser

Corresponding Author

Prof. Dr. Jérôme Waser

Laboratory of Catalysis and Organic Synthesis, Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédéralede de Lausanne, 1015 Lausanne, Switzerland

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First published: 15 September 2023

Graphical Abstract

Easily accessible crosslinking ethynylbenziodazolone (EBZ) JW-AM-005 enables the conjugation of peptides and proteins through the selective linkage of cysteine residues. Peptide dimers or stapled peptides were obtained under mild and tuneable conditions. The rebridging of antibody fragments was also performed in a one-pot three-reaction process with high regioselectivity, outperforming the standard reagents commonly used for this transformation.

Abstract

Peptide and protein bioconjugation sees ever-growing applications in the pharmaceutical sector. Novel strategies and reagents that can address the chemo- and regioselectivity issues inherent to these biomolecules, while delivering stable and functionalizable conjugates, are therefore needed. Herein, we introduce the crosslinking ethynylbenziodazolone (EBZ) reagent JW-AM-005 for the conjugation of peptides and proteins through the selective linkage of cysteine residues. This easily accessed compound gives access to peptide dimers or stapled peptides under mild and tuneable conditions. Applied to the antibody fragment of antigen binding (Fab) species, JW-AM-005 delivered rebridged proteins in a one-pot three-reaction process with high regioselectivity, outperforming the standard reagents commonly used for this transformation.

Introduction

The bioconjugation of peptides and proteins with natural or synthetic molecules stands at the forefront or modern drug discovery and biomaterial sciences.1-5 Chemical modifications allow optimizing the pharmacological properties and bioactivity of peptides, proteins and oligonucleotides.6-8 The development of new bioconjugation techniques is essential for continuing progress in this area, yet the multiple functional groups present in biomolecules constitute a formidable challenge for selective transformations.1-10 The most successful approaches to perform bioconjugation are based on either the installation and modification of non-natural amino acids with unique reactivity, or the functionalization of more reactive natural amino acids present in low abundance.6-12 The latter has the advantage of not requiring time-consuming and technically difficult modifications of peptides and proteins with non-natural amino acids, but represents a challenge of selectivity, as most reactive natural amino acids are inherently nucleophilic.13-16 Cysteine bioconjugation has been most successful due to the unique nucleophilicity of the sulfur atom combined with its low abundance.16

When considering the preponderance of nucleophilic functionalities in biomolecules, the use of bis-electrophilic linkers appears especially attractive for bioconjugation (Figure 1a). Linkers based on alkylation (1),8, 16 acylation (2),17 Michael addition (3,4),18 nucleophilic aromatic substitution (6),19 and metal mediated coupling (7)20 have been therefore developed for cysteine-cysteine and, to a lesser measure, lysine-lysine cross-conjugation. The use of these linkers with two identical electrophilic groups limits potential applications to either intramolecular processes (macrocyclization, stapling, disulfide re-bridging) or the formation of homodimers.1, 9, 19 However, the intermolecular coupling of two different biomolecules is highly desirable for the formation of peptide-peptide, protein-peptide, peptide-drug,21, 22 protein-drug, antibody-drugs conjugates (ADC)23 and antibody-oligo conjugates (AOC),24 all of which are extensively used in vaccines, immunotherapy, drug delivery and cancer therapy.

Details are in the caption following the image

Bis-electrophilic linkers for bioconjugation, a) homo-bifunctional, b) hetero-bifunctional, and c) our previous work: hypervalent iodine based linkers.

In order to realize such selective cross-conjugation, the use of non-symmetrical linkers with sufficient difference in reaction rates between the two electrophilic groups is needed. Maleimide-succinimidyl esters 9 are currently among the most popular hetero-bifunctional cross linkers used in bioconjugation.1 However, maleimide conjugates can also present issues of stability in biological systems and the activated ester of 9 get rapidly hydrolyzed in alkaline aqueous media and reacts with both thiols and amines.26 The issue of the ester reactivity can be solved by replacing it with an azide (10 a) to perform a biorthogonal reaction, but this does not allow any longer the use of natural amino acids as conjugation partners.27 Next generation maleimide (NGM) dibromide cross-linkers (10 b) have also been recently found particularly attractive for their high cysteine selectivity as well as for their convenient use in the generation of ADCs through disulfide rebridging.28, 29 Nevetheless, there is still a urgent need for crosslinkers that are stable under physiological conditions and easily further functionalized after rebridging. In a recent work, Pentelute, Buchwald and co-workers reported the successful use of palladium complex 11 for protein-protein cysteine bioconjugation leading to stable products with high selectivity.30 However, this complex requires arduous and costly synthesis to be accessed, thus limiting its application.

In the Waser group, we have introduced ethynylbenziodoxolones (EBXs) hypervalent iodine reagents for cysteine bioconjugation. These compounds present higher thiol selectivity than alkylation reagents, with reaction rates comparable to Michael addition to maleimides, and lead to the formation of stable thioalkynes or vinylbenziodoxolones (VBXs) as conjugates.31, 32 The use of EBXs such as 12 was first reported, which could be used in water for the generation of VBX adducts without by-product formation, and enable further conjugation via biorthogonal azide cycloaddition reactions.31 More recently, we developed reagents with two electrophilic reactive sites, such as 13 – bearing two EBX groups – or 14, which has one EBX and one activated ester.32 These reagents could be respectively used for cysteine-cysteine or cysteine-lysine conjugation, albeit exclusively in organic solvents, as the use of water often led to a mixture of products and degradation.

Herein, we report the design and application of the new crosslinking hypervalent iodine reagent JW-AM-005 (15), a nitrogen analogue of EBXs coined ethynylbenziodazolone (EBZ) (Figure 2). In the Waser group, we have used EBZs as electrophilic alkynylation reagents for the enantioselective copper-catalyzed oxyalkynylation of diazo compounds,33 but they had never been used for bioconjugation so far. Compared to EBXs, they have the unique advantage of possessing a nitrogen substituent poised for further diversification. Inspired by the work of the Pentelute group, who have extensively studied the reactivity of perfluoro aryl groups in bioconjugation via SNAr reactions,19 we designed water compatible JW-AM-005 (15), which is easily accessible in two steps. Similarly to EBX reagent 12, JW-AM-005 (15) undergoes fast addition to thiols (<10 minutes at RT) at low concentrations (5–20 mM range) to give the corresponding S-VBZ derivatives. The electron-poor perfluorinated aryl group on the sulfonamide can then be reacted in a second step with another thiol-containing molecule. This second reaction is orders of magnitude slower (3–5 h at 37 °C) than the first one, allowing smooth and selective crosslinking of two different thiols without the use of a large excess of reagent or reaction partner. Added advantages of this new EBZ crosslinking reagent is that only fluoride is generated as a by-product and the hypervalent bond can be easily cleaved using a copper(I) salt, offering promising perspectives for the controlled release of the conjugated thiols.

Details are in the caption following the image

This work: JW-AM-005 (15) – a bifunctional EBZ reagent with high rate difference for Cys-Cys cross-linking.

Results and Discussion

The synthesis of reagent JW-AM-005 (15) was carried out in just two steps starting from the commercially available 2-iodobenzamide (16) (Scheme 1a). The latter was first treated with sulfonyl chloride 17 in the presence of sodium hydride to give sulfonamide 18 in 73 % yield. Subsequently, oxidation of 18 with mCPBA followed by reaction with silyl alkyne 19, furnished the desirable compound 15 in 42 % yield, whose structure was also confirmed by X-ray single crystal analysis (Scheme 1b).34

Details are in the caption following the image

a) Synthesis of reagent JW-AM-005 (15). b) X-Ray crystal structure of 15.

Unprotected hexapeptide amide 20 a bearing a cysteine, a threonine and a free N-terminus was then synthesized using standard Fmoc-solid phase synthesis (See Supporting Information: page S20; the syntheses of the peptides used in this work are reported in pages S20-25) (Table 1). This peptide was selected to optimize the reaction for the first Cys bioconjugation, as it would allow assessing selective sulfur functionalization over oxygen or nitrogen nucleophiles. When 20 a was treated with only 1.2 equivalents of 15 in a mixture of 50 mM Tris buffer pH 8.0 : DMF (1 : 1), vinylbenziodazolone (VBZ) product 21 a was obtained in 65 % HPLC calibrated yield (for the calibration, see Supporting Information: pages S25–26) (entry 1). The reaction profile was very clean, with complete conversion of the starting peptide 20 a. Only two by-products could be observed: a small variable amount of disulfide dimer (2–14 %), and alkyne 21 a’, which was formed in 8 % yield. Thioalkynes are the major products obtained, when the reaction with EBX or EBZ reagents is carried out in organic solvents.35, 36 Neither reaction on threonine or the N-terminus nor degradation of product 21 a′ occurred.

Table 1. Optimization of the first Cys bioconjugation.

image

Entry

Reaction conditions

21a [%][a]

21a’ [%][a]

1

50 mM Tris : DMF, pH 8.0

65

8

2

50 mM HEPES : DMF, pH 8.0

77

11

3

50 mM PB : DMF, pH 8.0

74

13

4

50 mM PB : DMSO, pH 8.0

47

14

5

50 mM PB : ACN, pH 8.0

75

6

6

50 mM PB : ACN, pH 7.4

73

7

7

50 mM PB : ACN, pH 6.0

79

8

8

H2O : ACN

75

7

9[b]

50 mM PB : ACN, pH 8.0

76

4

10[c]

50 mM PB, pH 8.0; DMF 2 % v/v

53 %

11[d]

50 mM PB : ACN, pH 8.0

82[f]

7

12(d)[e]

50 mM PB : ACN, pH 8.0

77[f]

6

  • Reaction conditions: 0.0010 mmol of 20 a, 0.0012 mmol of 15, shaken in the indicated 1 : 1 mixture of buffer and organic solvent for 20 minutes. The overall concentration was 10 mM with respect to 20 a, unless specified otherwise. [a] Yields of 21 a were determined by HPLC-MS and are calibrated; yields of 21 a’, were estimated without calibration. [b] The reaction was performed at a 2 mM concentration, for 80 minutes. [c] The reaction was performed at a 2 mM concentration in 2 % DMF/50 mM PB pH 8.0 (v/v), for 40 min. The yield was estimated by uncalibrated HPLC-MS. [d] The reaction was performed using 0.0080 mmol of 20 a, 0.0096 mmol of 15. [e] The reaction was performed at 20 mM concentration. [f] Isolated yield upon preparative RP-HPLC. ACN: acetonitrile, PB: phosphate buffer.

Using a HEPES buffer resulted in a higher 77 % yield of 21 a, with no significant relative increase in the formation of alkyne 21 a’ (entry 2). A very similar result was obtained in phosphate buffer (PB; 50 mM, pH=8.0) (entry 3), and prompted us to further investigate the reaction in this more physiologically compatible buffer. Other organic co-solvents were tested at first. While isopropanol had to be excluded because of the limited solubility of the reacting species, the transformation worked in DMSO, although only in 47 % yield, and with a less favorable 3 : 1 ratio between 21 a and 21 a’ (entry 4). The reaction in acetonitrile (ACN) provided comparable results to DMF (75 % yield of 21 a; entry 5) and it was therefore adopted as the optimal co-solvent for the following experiments. Interestingly, lowering the pH from 8.0 to 7.4 or even 6 had no impact on the yield (entries 6 and 7), which remained unaffected even when no buffer at all was used (reaction in water-ACN, entry 8). The reaction worked as well under much more diluted conditions (2 mM), providing 21 a in 76 % yield after 80 minutes (entry 9) with minimal amounts of alkyne by-product. These evidences highlight that the Cys-conjugation of 15 is very robust and highly tolerant to variations in buffer composition, solvent, pH and concentration. In order to be closer to physiological conditions, we then run the transformation in the presence of smaller amounts of organic co-solvent. At 2 mM and with only 2 % of DMF in a 50 mM PB buffer, 21 a was still obtained in 53 % yield after 40 min, alongside with 5 % of 21 a’ and disulfide (entry 10). Under these conditions, precipitation of 15 was observed, explaining the lower yield. Selecting the 50 mM PB pH 8.0/ACN (1 : 1) mixture as our model solvent system, we were also pleased to observe that the reaction could be effectively scaled up (81 % yield; entry 11) and successfully carried out at higher concentration (20 mM) with no significant diminution of yield (77 % yield; entry 12).

Interested in how broadly applicable our method was (Scheme 2), we tested it first on unprotected L-cysteine, which was efficiently converted into the corresponding VBZ 21 b in 79 % yield. With glutathione, no complete conversion was obtained even after a longer reaction time (40 minutes). The reason for the lower reactivity shown by this substrate remains unclear. Moreover, while the yield of product 21 c was estimated around 70 % based on HPLC-MS analysis, only 36 % of it could be isolated. This could indicate a poor stability of 21 c on preparative HPLC.

Details are in the caption following the image

[a] 0.020 mmol 20, 0.022-0.024 mmol 15, PB (50 mM, pH=8.0) : ACN, 1 : 1, 20 mM with respect to 20, at room temperature; (indicative HPLC-MS-based yields are reported in parentheses), isolated yields. [b] Reaction performed starting from 0.040 mmol L-cysteine. [c] Reaction performed at a 10 mM overall concentration. [d] Reaction performed starting from 0.0040 mmol 22, 0.0080 mmol 15, 0.53 mL PB (5.0 mM, pH=8.0), 0.27 mL EDTA 6.7 mM in PB (50 mM, pH=8.0), 0.80 mL DMF, at room temperature; (indicative HPLC-MS-based yields are reported in parentheses), isolated yields.

Better results were obtained with the Cys-conjugation of N-acylated tetrapeptides 21 d and 21 e. The former, derived from SARS-COV 2 (Ala211-Thr214) protein and containing both serine and threonine residues gave the corresponding VBZ 21 d in 68 % yield, with full conversion within 20 minutes. Similarly, 21 e was isolated in 62 % yield starting from histidine-containing 20 e. As shown with model substrate 20 a, peptides with unprotected N-termini also worked effectively, with complete cysteine selectivity observed in all cases. In particular, the reaction tolerated the presence of nucleophilic side chains, with both basic (product 21 f, isolated yield: 49 %) and acidic character (product 21 g, isolated yield 61 %). Hexapeptide 20 h, mostly containing lipophilic residues, provided VBZ derivative 21 h in 41 % yield. We then turned our attention to larger peptides, and in particular to fragments with biological significance. With these more complex substrates, issues with solubility of the peptides and competing oxidative homodimerization, through disulfide bond formation, became more pronounced. An adjustment of the reaction conditions was necessary to address these problems: the Cys-conjugation was carried out at higher dilution (2.5 mM), with replacement of ACN with DMF, while the addition of EDTA helped to limit oxidative dimerization. Under such re-optimized conditions, we could isolate VBZ 23 a in 36 % yield starting from fragment 22 a, derived from hepatitis C virus envelope glycoprotein E2.37 Endorphine, one of the most important natural peptide hormone, was modified on its C-terminus to add a cysteine residue,38 resulting in modified peptide 22 b that gave its corresponding VBZ conjugate 23 b in 41 % yield. Finally, we synthesized peptide 22 c containing the H6F7R8W9 sequence, which is present in human adrenocorticotropic hormone (ACTH) and acting as binding site to the melanocortin receptor 2 (MC2R).39 The corresponding product 23 c was also isolated in 41 % yield. Overall, in all these larger peptides, the presence of potentially reactive amino acids such as histidine, tyrosine, arginine and methionine was tolerated, although the corresponding VBZ derivatives 24 were obtained in lower yields and the formation of several (unidentified) by-products was observed by HPLC-MS analysis.

Having developed an efficient method for the monoconjugation of reagent 15 with cysteine-containing substrates, we then investigated the possibility to add a second thiol peptide to the so obtained conjugates 21 via a SNAr reaction on the polyfluorinated aromatic group. As a proof of concept, we started our study on the homoconjugation of our model hexapeptide 21 a. A stepwise protocol was considered at first, consisting in the treatment of the preformed VBZ-derivative 21 a with a small excess (1.5 equivalents) of 20 a. After a short reoptimization of the reaction conditions (see Supporting Information for details: pages S47–50), we found that an effective procedure relied on the reaction of the two species in a 1 : 1 mixture of PB buffer (50 mM, pH 8.0) and DMF at 37 °C for 4 hours (Scheme 3a). While the transformation also occurred at room temperature within the same reaction time, mild heating led to higher yield and cleaner HPLC-MS profiles. Under these adjusted conditions, homodimer 24 aa was isolated by preparative RP-HPLC in 69 % yield (Scheme 3d). In order to avoid the isolation and purification of VBZ intermediate 21, we then developed a one-pot protocol (one-pot protocol 1; Scheme 3b). Accordingly, upon reacting 20 a with 1.2 equiv. 15 at room temperature for 30 minutes, adding an excess of 20 a to the untreated reaction mixture resulted in the formation of 24 aa in 51 % yield after 4 hours at 37 °C. An even more straightforward approach implied the direct treatment of 15 with 2.2 equivalents of the starting peptide (one-pot protocol 2; Scheme 3c). In this case, homodimer 24 aa was obtained in 41 % yield.

Details are in the caption following the image

[a] 0.0040 mmol 21, 0.0044-0.0048 mmol 15, PB (50 mM, pH=8.0) : DMF, 1 : 1, 10 mM with respect to 21, at 37 °C; (indicative HPLC-MS-based yields are reported in parentheses), isolated yields. [b] 0.0040 mmol 20, 0.0048 mmol 15, PB (50 mM, pH=8.0) : DMF, 1 : 1, 10 mM with respect to 20 at room temperature for 20 minutes; then 0.0044-0.0048 mmol 20 at 37 °C; (indicative HPLC-MS-based yields are reported in parentheses), isolated yields. [c] 0.0072 mmol 20, 0.0032 mmol 15, PB (5.0 mM, pH=8.0) : DMF, 1 : 1, 10 mM with respect to 20, at 37 °C; (indicative HPLC-MS-based yields are reported in parentheses), isolated yield. [d] Same conditions as in [a] but at room temperature.

At this point, we wondered whether our stepwise and one-pot methods might be applied to the cross-conjugation of two different thiol-containing molecules (Scheme 3e). The use of the stepwise protocol was successful in all the examined examples, with heterodimers 24 ae, 24 fe, 24 ge and 24 gf isolated in good to excellent yields (62-89 %). The application of the one-pot procedure, while viable, proved less effective. Much lower yields were obtained, and the formation of the homoconjugation products corresponding to both reacting peptides was also observed. In particular, under such conditions 24 ae and 24 fe could be only isolated in 39 % and 18 % yields respectively. While this homodimerization is due to an SNAr process occurring on the cross conjugates, it remains unclear why it was not observed in the stepwise experiments. The specific reactivity of the used second peptide must certainly play a significant role, as suggested by the fact that no homoconjugate was generated together with 24 ge when the one-pot protocol was utilized.

Importantly, all obtained VBZ cross-linked conjugates were shown to be stable under the conditions classically used for their synthesis or purification. In addition, treating conjugate 24 ae with an excess of glutathione in a 10 mM DMF : PB 1 : 1 mixture for 24 hours did not lead to any significant degradation (see Supporting Information: pages S56-58). Nevertheless, the weak hypervalent iodine bond should be still cleavable using stronger reductants. This has the potential to make our method even more attractive, as it could be used to release selectively the two individual biomolecules, each labelled with a fragment of the initial EBZ reagent. In a preliminary approach, a modified version of the L-proline-promoted Rosenmund-von Braun reaction first reported by Yoshikai and co-workers on VBX compounds40 was successfully applied to cross-conjugate 24 gf to generate two main fragments 25 and 26 in 24 % and 38 % yield, respectively (Scheme 4). Interestingly, two other analogues of 26 were also detected by HPLC-MS, the reduced arene 26’ and nitrile derivative 26’’, presumably resulting from the reductive elimination of a Cu(III)CN intermediate. While these results are promising, they also indicate that further investigations will be needed to obtain a clean and selective linker cleavage.

Details are in the caption following the image

Reaction conditions: 0.0025 mmol 24 af, 0.025 mmol L-proline, 0.050 mmol CuCN, ACN : PB (50 mM, pH=8.0)=1 : 1, 2.5 mM with respect to 24 gf, at 37 °C for 18 hours; isolated yields.

Having shown the utility JW-AM-005 (15) for the crosslinking of different peptides, we wondered if this compound might also be used for the cysteine-cysteine rebridging of peptidic fragments resulting from the reduction of disulfide bonds. Such an operation would provide a convenient access to VBZ conjugates easily further modified thanks to the azide group. A large number of bioactive peptides in nature are in fact characterized by one or multiple intramolecular disulfide bridges. As a notable example, oxytocin (27) is a hormone and neuropeptide containing a single S−S bond that plays a role in processes such as reproduction, childbirth and lactation (Scheme 5).41

Details are in the caption following the image

Reaction conditions [a] 0.0090 mmol Oxytocin (27, as acetate salt), 0.0180 mmol TCEP, 5 mM in PB (50 mM, pH=8.0), at 37 °C for 2 hours; then EDTA in PB (50 mM, pH=8.0), ACN (2 mM overall concentration), 0.0085 mmol 15, at room temperature for 24 hours; isolated yield. [b] 0.0021 mmol 28, 0.025 mmol CuI, PB (50 mM, pH=8.0) : DMF, 1 : 1, 5 mM with respect to 28, at 37 °C for 30 hours; isolated yield.

Native oxytocin was treated with the reductant TCEP in a PB buffer. The subsequent addition of JW-AM-005 (15) in the presence of EDTA (to minimize intramolecular reoxidation) and ACN as the organic co-solvent provided the desired macrocycle 28 in 23 % isolated yield. Surprisingly, only one of the two possible isomers was observed. It cannot be excluded that the second one was not stable under the reaction conditions. The structure of 28 was later on confirmed by the MS-MS analysis of product 29 resulting from the reductive cleavage of the hypervalent bond in 28, which was efficiently accomplished in 70 % yield using copper(I) iodide under aqueous conditions. In this reaction, the formation of multiple coupling derivatives of the VBZ core could be avoided by using CuI instead of the aforementioned combination of CuCN and L-proline: the 2-iodobenzamide 29 was cleanly obtained in 70 % yield as the sole product.

In an effort to evaluate the potential of JW-AM-005 (15) as disulfide crosslinking reagent on more complex substrates, we investigated the rebridging of fragment antigen-binding (Fab) species. Fabs are ~50 kDa proteins classically produced by the enzymatic digestion of a parent monoclonal antibody (mAb). With the retained targeting properties of whole mAbs but a smaller size, Fab conjugates are attractive due to their enhanced tissue penetration.42 Structurally speaking, they consist of a light chain (LC) covalently connected through a single interchain disulfide bond to a Fd chain, the digested remnant of the mAb's heavy chain (HC). For our optimization studies, we opted for the Fab fragment 30 of trastuzumab – an FDA-approved monoclonal antibody (mAb) used against HER2+ breast cancer cells43 – which we easily obtained after two consecutive pepsin- and papain-mediated digestion steps.44 Reduction of the single interchain disulfide bond to free the cysteines’ thiols was carried out following known procedures, using 5 equivalents of tris(2-carboxyethyl)phosphine (TCEP) at 37 °C, followed by gel filtration chromatography to eliminate the excess of reagent. Rebridging efficacy was determined by SEC-MS in denaturing conditions (dSEC-MS), an approach specifically developed for our study,45 according to two parameters: the amount of Fab detected (corresponding to the percentage of both Fab species – i. e., native disulfide-bonded Fab 30 and VBZ-rebridged Fab 31 – versus that of LC and Fd sub-species, including fragmented adducts 32 and 33) and its average degree of conjugation (avDoC), used as a direct indicator of the rebridging efficiency.

We began our investigations by applying to our reduced trastuzumab-Fab similar conditions as those previously optimized for the production of dimers 22 (see Supporting Information, Table S8, page S73). However, to our dismay, this led to poor rebridging due to two main factors: the generation of LC and Fd side species 32 and 33, respectively, bearing only fragments of the expected payload; and the incomplete conjugation of 30 (avDoC <0.3). A plausible explanation behind the formation of 32 and 33 is the fragmentation of the JW-AM-005 (15) payload upon thiolate addition through elimination; the reasons behind the observed selectivity of this side reaction, with the alkynyl and sulfonamide fragments being only detected on the Fd and the LC, respectively, remain poorly understood.

Adding EDTA in an attempt to minimize the reoxidation of cysteines and playing on both the reaction time and number of equivalents of JW-AM-005 (15) helped to increase rebridging, whilst maintaining the same amount of side species 32 and 33 (see Supporting Information, Table S8, page S73).45 Interestingly, a stark improvement was noticed when switching to borate buffered saline (BBS; entry 1; Table 2), with 70 % of Fab being detected with an avDoC of 0.80, whilst decreasing the reaction time to 6 h led to improved amounts of Fab but to a less efficient rebridging, as evidenced by its halved avDoC (entry 2).

Table 2. Optimisation of rebridging of Fab-trastuzumab 30.

image

Entry

Reaction conditions

Fab [%][a]

avDoC

1

15 (5 equiv. in DMSO), BBS, 2 mM EDTA, pH 8, 37 °C, 16 h

70

0.80

2

15 (5 equiv. in DMSO), BBS, 2 mM EDTA, pH 8, 37 °C, 6 h

79

0.41

3[b]

15 (5 equiv. in DMSO), BBS, 2 mM EDTA, pH 8, 37 °C, 6 h

70

1.0

4[b]

15 (5 equiv. in DMSO), PBS 1X, 2 mM EDTA, pH 8, 37 °C, 6 h

71

0.91

5[b]

15 (5 equiv. in DMSO), Tris, 2 mM EDTA, pH 8, 37 °C, 6 h

82

0.27

6[b]

15 (5 equiv. in MeCN), BBS, 2 mM EDTA, pH 8, 37 °C, 6 h

58

0.95

7[b]

15 (5 equiv. in DMF), BBS, 2 mM EDTA, pH 8, 37 °C, 6 h

70

1.0

8[b]

15 (5 equiv. in DMSO), BBS, 2 mM EDTA, pH 8.5, 37 °C, 6 h

46

0.82

9[b]

15 (5 equiv. in DMSO), BBS, 2 mM EDTA, pH 7.0, 37 °C, 6 h

61

1.0

10[b]

15 (5 equiv. in DMSO), BBS, 2 mM EDTA, pH 8, 37 °C, 5 h

72

1.0

11[b]

15 (5 equiv. in DMSO), BBS, 2 mM EDTA, pH 8, 37 °C, 2 h

82

0.85

12[b]

15 (5 equiv. in DMSO), BBS, 2 mM EDTA, pH 8, 37 °C, 0.5 h

80

0.69

13[b]

15 (5 equiv. in DMSO), BBS, 2 mM EDTA, pH 8, 25 °C, 6 h

76

0.97

14[b]

15 (5 equiv. in DMSO), BBS, 2 mM EDTA, pH 8, 4 °C, 6 h

69

0.58

15[b]

15 (7.5 equiv. in DMSO), BBS, 2 mM EDTA, pH 8, 37 °C, 6 h

84

1.06

  • [a]: Relative quantification determined by integration of peak areas from UV chromatograms – the remaining fraction of protein species are LC and Fd (see Supporting Information, page S4 for more details); [b]: Reduction and rebridging conducted in one pot.

At this stage, we hypothesized that the gel purification conducted after the TCEP-mediated reduction step might favor the reoxidation of the thiols, and therefore we evaluated the possibility of conducting both the reduction and the rebridging steps in one pot (see Supporting Information for procedure, page S72). Gratifyingly, this led to full rebridging of Fab 30 (i. e., avDoC=1.0), the sole double-chain species detected by denaturing LC-MS (entry 3). This excellent stability of the iodine(III) bond toward these reductive conditions is remarkable, and far superior to that of more standard reagents classically used for protein rebridging (v. infra), and is further highlighted by a perfect stability of the rebridged Fab in plasma for 5 days at 37 °C (see Supporting Information, page S93). All our subsequent efforts were thus dedicated to improving the percentage of fully rebridged Fab obtained, by minimizing the side reactions leading to partially conjugated LC 32 and Fd 33. Varying buffer and co-solvent led systematically to a decrease in DoC, highlighting the profound influence of solvent effects on the efficacy of the conjugation (entries 4–7). Increasing pH had a detrimental impact on Fab's percentage and DoC, whilst decreasing it had little effect on both terms (entries 8–9). We also noted that similar results could be obtained after just 5 h (entry 10), but that decreasing the reaction time further led to an erosion of the DoC values and hence the rebridging efficiency (entries 11–12). Lowering the reaction temperature to 25 °C led to an improved amount of Fab with a minimal decrease in avDoC (entry 13). Interestingly, formation of side species 32 and 33 could be completely suppressed by working at 4 °C, albeit at the expense of both avDoC and amount of Fab (entry 14). Any attempt at improving the latter – notably by increasing the number of JW-AM-005 15 equivalents (entry 15) – led to a parallel increase of the former, due to the appearance of doubly conjugated Fab species, indicating chemoselectivity issues.

Having developed optimal conditions on trastuzumab Fab, we were keen to apply our protocol to Fab from other mAb sources. Enzymatic digestion of bevacizumab, avelumab and rituximab furnished the desired Fab species in high yield and purity, which were also successfully rebridged (Scheme 6). Gratifyingly, we also demonstrated that JW-AM-005 (15) could be applied to whole mAbs: under slightly tweaked conditions, trastuzumab led to 82 % of rebridged mAb (avDoC=2.5), with only 11 % of half mAb and 7 % of LC and HC subspecies, demonstrating the broader applicability of our strategy. For the rebridged mAb, the incorporation of two VBZs was most frequent (D2), but insertion of 1, 3 and even 4 VBZs was also observed (D1, D3 and D4).

Details are in the caption following the image

Reaction conditions: [a] Fab (1.5 mg/mL in BBS, 2 mM EDTA, pH 8), TCEP (15 mM in water, 5 equiv.), 15 (10 mM solution in DMSO, 5 equiv.) at 37 °C for 5 hours. [b] trastuzumab (10 mg/mL in BBS, 6 mM EDTA, pH 8), TCEP (15 mM in H2O, 10 equiv.), 15 (10 mM solution in DMSO, 10 equiv.) at 37 °C for 5 hours. Chromatogram and mass spectrum excerpts from the dSEC-MS analysis of the rebridging of trastuzumab are provided.

Having validated the rebridging step, we next focused on the functionalization of rebridged Fab 31 through bioorthogonal reaction, taking advantage of the azide group on the VBZ payload. Performing this stepwise rebridging/functionalization process on Fab trastuzumab with strained alkyne 34 led to fully rebdriged iminobiotin-containing Fab species 35, albeit in a lower proportion than before (Scheme 7). Suspecting that this was caused by the two successive purification steps, we also evaluated the concomitant one-pot reduction/rebridging/functionalization sequence by mixing trastuzumab Fab with a mixture of TCEP, JW-AM-005 (15) and BCN 34 under our optimized conjugation conditions at 37 °C. Pleasingly, this led to an increase in rebridged Fab proportion (72 %), the only double-chain species detected. To the best of our knowledge, this intricate chemoselective ballet between four reactive species has never been reported before and is key in improving both the efficacy and the efficiency of our conjugation sequence. More importantly, any attempt at performing the same one pot three-step reaction with a classical rebridging dibromomaleimide reagent 36 led to mediocre rebridging (i. e., 92 % Fab, avDoC=0.10), presumably because of TCEP-mediated decomposition of the maleimide motif.28, 44, 47-49

Details are in the caption following the image

Reactions conditions: [a] 1. Fab 30 (1.5 mg/mL in BBS, 2 mM EDTA, pH 8), TCEP (15 mM in water, 5 equiv.) 15 (10 mM solution in DMSO, 5 equiv.) at 37 °C for 5 hours; 2. 34 (10 mM in DMSO, 30 equiv.) at 25 °C for 24 hours. [b] Fab 30 (1.5 mg/mL in BBS, 2 mM EDTA, pH 8), TCEP (15 mM in H2O, 5 equiv.), 15 (10 mM solution in DMSO, 5 equiv.), 34 (10 mM solution in DMSO, 20 equiv.) at 37 °C for 5 hours.

Finally, we wanted to investigate whether the controlled reductive cleavage of the hypervalent iodine bond could also be applied to rebridged Fab 31. Using a mixture of copper(I) iodide and tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) in the presence of sodium ascorbate and aminoguanidine at 37 °C for 16 h, nearly quantitative cleavage of Fab 31 was observed, leading to fragmented species 37 – the sole Fd fragmentation species detected, which tends to suggest a regioselective rebridging –, and 38 – the main LC fragmentation species detected –, as determined by denaturing LC-MS (Scheme 8).

Details are in the caption following the image

Reaction conditions: 31 (1.5 mg/mL in PBS 1X, pH 7.4), 20 equiv. CuI, 40 equiv. THPTA, 100 equiv., aminoguanidine ⋅ HCl, 300 equiv. NaAsc,at 37 °C for 16 hours.

Conclusions

In conclusion, we reported the synthesis of a novel bifunctional cross-linker incorporating EBZ and pentafluorophenyl motifs. This platform was applied to the single or double conjugation of cysteine-containing peptides and proteins, offering access to stapled peptides, homo- and hetero-dimers. We showed that the method was tolerant to a wide range of functionalities and that excellent chemo- and regioselectivity could be attained. In particular, the rebridging of the Fab fragment of several antibodies could be achieved with high efficiency and an incorporated azide group enabled further diversification of the payload. A further advantage of incorporating a hypervalent iodine bond into the cross linker is high stability under physiological conditions, even in plasma, but still inherent lability towards copper(I) species, allowing a controlled cleavage of bioconjugates. These properties further highlight the advantages of hypervalent iodine(III) reagents for the selective conjugation of cysteines and open promising perspectives in the development of new hetero-crosslinking reagents.

Supporting Information

The authors have cited one additional reference within the Supporting Information.50

Acknowledgments

This work was supported by the European Research Council (ERC Consolidator Grant SeleCHEM, No. 771170), the European Union's Horizon 2020 Research and Innovation Programme Marie Sklodowska Curie Action ITN under Grant Agreement No 859458 (IK, RB), the CNRS, the University of Strasbourg, the “Agence Nationale de la Recherche” (Conformabs; ANR-21-CE29-0009-01), the French Proteomic Infrastructure (ProFI; ANR-10-INBS-08-03) and the Interdisciplinary Thematic Institute IMS (Institut du Médicament Strasbourg), as part of the ITI 2021–2028 supported by IdEx Unistra (ANR-10-IDEX-0002), SFRI-STRAT'US project (ANR-20-SFRI-0012). Dr. Farzaneh Fadaei Tirani and Dr. Rosario Scopelliti (ISIC, EPFL) are acknowledged for the X-ray study. Dr. Daniel Ortiz, Francisco Sepulveda and Dr. Laure Menin (ISIC, EPFL) are acknowledged for HR-MS analyses. S.N. would like to express his gratitude to Dr. Tobias Milzarek (LCSO, EPFL) and Xingyu Liu (LCSO, EPFL) for the fruitful discussions. Additionally, we thank Dr. Tobias Milzarek for reviewing the Supporting Information. IK and GC would like to thank Valentine Vaur for providing avelumab and bevacizumab Fabs, and Louis Moreira da Silva for providing next-generation maleimide. Open Access funding provided by École Polytechnique Fédérale de Lausanne.

    Conflict of interest

    The authors declare no conflict of interest.

    Data Availability Statement

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