[2.2.1]Heterobicyclic Bromovinyl Sulfones for Thiol-Triggered Strategies in Linker Chemistry: Aza- vs Oxa-Norbornadienic Systems

Abstract

Azanorbornadienes (ANDs) containing a bromovinyl sulfone are able to accept a first thiol and, in a further stage, fragment upon reaction with a second thiol. This fragmentation has been studied in a collection of differently substituted ANDs. The substitution pattern of the AND influences the rate of the first thiolation and, specially, the further fragmentation. N-pyramidalization of selected ANDs was demonstrated via X-ray diffraction. This structural feature attenuates the resonance effect of N-substituents in the further reactivity of ANDs. A comparison with related oxanorbornadienes is also reported. The installation of a fluorogenic AND onto a single domain Antibody against PD-L1 (PD-L1 sdAb) resulted in a conjugate capable of releasing the corresponding fluorogenic pyrrole in the presence of glutathione (GSH) under physiological conditions. Overall, these scaffolds demonstrate potential to be implemented as new drug delivery systems.

1. Introduction

Conjugation of a drug to a targeting vector moiety (e.g., antibody or receptor ligand) is a common strategy for enhancing the selectivity of a cytotoxic compound, ensuring efficacy and avoiding off-target effects. The resulting conjugate would enrich the desired site due to the action of the vector and release/activate the drug there. This latest aspect highlights the importance of linker chemistry, as the linker moiety between the vector and the drug should avoid premature drug release and response specifically to a stimulus at the desired site. Several triggering mechanisms can operate for this response. One of the most exploited mechanisms is the thiol-triggered fragmentation of disulfide moieties that has been broadly used in the development of antibody-drug-conjugates (ADCs). This mechanism takes advantage of the high concentration of tripeptide glutathione (GSH) inside cells (1–10 mM, primarily in the cytoplasm) with respect to the extracellular environment (2–20 μM), moreover the intracellular [GSH] is even higher in tumor cells. GSH promotes the reduction of the disulfide moiety of the linker via thiol–disulfide exchange. Although disulfide-based linkers constitute one of the primary linkers employed for drug conjugation, they are not exempt from problems related with stability of the disulfide-based conjugates in serum: it is problematic to reach a good balance between the stability of the conjugate and the ability to release the payload. , Given that GSH has proven to be an effective intracellular trigger for linker fragmentation, the design of novel chemical systems capable of efficiently fragmenting in response to this thiolthrough mechanisms other than disulfide bond reductionremains a significant challenge. The reactivity of electrophilic oxa- and azanorbornadiene (OND and AND) systems toward thiols makes them excellent candidates to be used as thiol-sensitive linkers in targeted drug delivery strategies. (Hetero)­norbornadienes able to spontaneously fragment by accepting one thiol molecule were previously described by Finn and co-workers. These systems upon fragmentation were able to deliver a pyrrole/furan moiety anchored to a biologically relevant molecule (cargo). Our group recently reported that AND vinyl and bromovinyl sulfones 1 (Scheme ) react selectively with the side chain of cysteine 2 via thio-Michael addition (for R = H) or concerted nucleophilic vinylic substitution (S_NV_σ, for R = Br) in the presence of other nucleophilic amino acids, being excellent tools for site-selective modification of proteins. ,

1. Preferential Reactivity of Azanorbornadienic (bromo)­vinyl Sulfones towards Thiol-Based Nucleophiles.

Inspired by these results, we have also explored the thiol-triggered fragmentation of 2-halo-3-tosyl-oxanorbornadienes (Scheme , ZO) as a means to design new thiol-responsive chemical systems. We have shown that these compounds can sequentially react with two thiol molecules in distinct stages. The first step involves a nucleophilic vinylic substitution, while the second implies a thiol-triggered two-step fragmentation, initiated by a conjugate addition followed by a retro-Diels–Alder (rDA) reaction. In this study, we explore the impact of incorporating a nitrogen atom into the bridge of ANDs on their thiol-promoted fragmentation, enhancing their utility in biomedical applications. The presence of this nitrogen provides an additional site for the attachment of biologically relevant moieties, such as drugs, fluorophores, or affinity probes. We hypothesized that the introduction of an N-substituent could influence both the rate of the initial nucleophilic substitution and the subsequent fragmentation steps, compared to their oxa-analogues. To address this, we first performed a comparative analysis of the thiol-triggered substitution and fragmentation sequences in a series of differently substituted ANDs (and related analogues), using N-acetylcysteamine as a model thiol. Finally, we achieved the Cys-selective labeling of a nanobody with a fluorogenic AND, and evaluated the stability of the resulting bioconjugate in plasma, as well as its capacity to undergo GSH-mediated fragmentation.

2. Reaction of (hetero)­norbornadienes with Two Thiols at Different Stages.

2. Results and Discussion

2.1. Synthesis and Structural Studies (Pyramidalization) of 2-bromo­(aza)­norbornadienic Systems

To investigate how structural modifications influence the reactivity of (aza)­norbornadienes toward thiols, we prepared a series of AND derivatives incorporating different N-bridge substituents, electron-withdrawing groups at C-3 and substitution patterns on the bicyclic scaffold. Thus, we carried out the synthesis of 2-bromo-(aza)­norbornadienes 4a-18a and (carba)­analogues 19a-22a (Scheme ).

3. (Hetero)­norbornadienic and Related Systems for This Study .

^a Reaction conditions: (i) N-acetylcysteamine, DMF-Phosphate buffer pH 8.0.

These compounds were synthesized via Diels–Alder (DA) reactions between the appropriate cyclic dienes and electron-deficient alkynes (see Supporting Information for details). DA reactions involving pyrroles required higher temperatures than those with furans, consistent with the lower dienic character of pyrroles due to their significant aromatic stabilization. Introduction of electron-withdrawing groups on the nitrogen atom (e.g., via amidation, alkoxycarbonylation, or sulfonylation) is a well-established strategy to reduce aromaticity by delocalizing the nitrogen lone pair, thereby enhancing their reactivity in DA cycloadditions. However, this electronic modulation must be carefully balanced, as overly electron-deficient pyrroles become inefficient dienes in normal electron-demand DA reactions. The Boc group offers an optimal balance of electron-withdrawing character, and most N-substituted ANDs were obtained from AND 4a through a deprotection/acylation sequence (see Supporting Information).

Amides of bicyclic 7-azanorbornanes are known to exhibit intrinsic nitrogen pyramidalization, which reduces the electron delocalization from the nitrogen lone pair (nN) to the amide π*CO orbital. Motivated by this, we decided to investigate the extent of N-pyramidalization in selected N-substituted 7-azanorbornadienes as a potential factor influencing their reactivity toward nucleophiles. Crystals of ANDs 4a, 6a, 8a, 12a, 15a, and 23a (Table ) were obtained and analyzed by single-crystal X-ray diffraction. The pyramidalization of the bridging nitrogen correlates directly with the angular parameters α and θ (Table ). Additionally, deviation from planarity at the nitrogen center can result from torsion around the N-substituent bond, quantified by the twist angle τ in amides or carbamates, which corresponds to the rotation about the N–(CO) bond. For an ideal sp²-hybridized nitrogen, the angles α, θ, and τ would be 180°, 360°, and 0°, respectively.

1. Selected Angular Parameters from Crystal Structural Data of ANDs 4a, 6a, 8a, 12a, 15a and 23a .

AND	G	X	R	α (deg)	θ (deg)	τ (deg)
4a	Boc	Br	H	136.7	333.9	12
				134.8	331.7	4
				136.4	333.5	6.5
6a	Boc	Br	Me	145.2	342.9	1.3
8a	Moc	Br	H	135.2	332.2	1.7
12a	Bz	Br	H	141.3	338.4	15.1
15a	Ts	Br	H	142.1	339.4
				142.0	339.4
23a	Boc	H	H	136.5	333.8	9.2

^aAngular parameters related with pyramidalization (α,θ) and twist (τ).

^bTwo/three molecules are involved in the unit cell.

^cCompound previously synthesized by us, see ref .

The crystallographic data for carbamates 4a, 6a, 8a, 23a, amide 12a, and sulfonamide 15a reveal significant pyramidalization of the nitrogen atom in these compounds. In acyclic systems, the nitrogen atom in N-sulfonylamides typically exhibit greater pyramidalization than the corresponding in N-acyl amides. − This behavior is also observed in N-substituted pyrrolidines and pyrrolines. For instance, N-(p-nitrobenzoyl)-3-pyrroline and N-benzoylpyrrolidine are essentially planar, whereas N-tosylpyrrolidine and N-tosyl-3-pyrroline show remarkable pyramidalization. , In contrast, our azanorbornadienic compounds display pronounced nitrogen pyramidalization regardless of the functional group (carbamate, amide, or sulfonamide), with carbamates 4a, 8a, and 23a exhibiting slightly higher pyramidalization than amide 12a and sulfonamide 15a. Substitution with methyl groups at C1 and C4 of the bicyclic scaffold reduces pyramidalization (4a vs. 6a), whereas bromine substitution does not significantly affect the pyramidalization degree (4a vs. 23a). Due to N-pyramidalization, a syn-tilt of the N-substituent relative to the C2 and C3 substituents of the azanorbornadienic core is observed in all cases (Figure S1). Moreover, a notable twist around the N–C­(=O) bond is evident in 4a, 23a and, particularly, in amide 12a.

2.2. Reactivity of Azanorbornadienic Bromovinyl Sulfones toward Thiol Nucleophiles

The nucleophilic vinylic substitution reaction of a set of bromovinyl bicyclic systems toward thiol nucleophiles was explored. The reaction of 4a-22a with N-acetylcysteamine under buffered (pH 8.0) aqueous/DMF conditions originated the expected [2.2.1/2]­bicyclic thiovinyl derivatives (4b-22b) in a fast (less than 30 min, Scheme ) and clean reaction (no byproducts were identified). Then, competition reactions by pairing selected (hetero)­norbornadienic systems against N-acetylcysteamine in DMF-phosphate buffer were accomplished (Table ). The previously studied oxanorbornadiene (OND) 3a was chosen as reference for comparative studies in some of the competition experiments.

2. Competition Experiments: Reaction of a Mixture of Two Bromo-(Hetero)­norbornadienes (Br-HNDs) with N-Acetylcysteamine .

entry	Br-HNDs (A/B)	structural variation	% Conversion into A′/B′
1	3a:4a	X/Y: O/NBoc	61:39
2	3a:9a	X/Y: O/NAc	37:63
3	4a:8a	X/Y: NBoc/NMoc	40:60
4	4a:10a	X/Y: NBoc/NC(=O)Pr	28:72
5	4a:11a	X/Y: NBoc/NC(=O)CF3	0:100
6	4a:12a	X/Y: NBoc/NBz	27:73
7	13a:14a	X/Y: NC(=O)PMP/PNP	36:64
8	13a:15a	X/Y: NC(=O)PMP/NTs	0:100
9	3a:19a	X/Y: O/CH2	66:34
10	3a:20a	X/Y: O/C(=CMe2)	82:18
11	3a:21a	X/Y: O/CH2CH2	73:27
12	3a:22a	X/Y: O/-CHCH-	92:8
13	6a:7a	EWG: Ts/COOMe	35:65

^aReaction conditions: To an equimolar solution of (hetero)­norbornadienes A/B (50 mM each, 1.0 equiv. each) and N-acetylcysteamine (0.9 equiv) in DMF, a similar volume of phosphate buffer solution (pH 8.0, 50 mM) was added. The reaction mixture was stirred for 30 min at room temperature. After workup, the ratio A′:B′ was analyzed by ¹H NMR of the unreacted starting material. PMP: p-methoxyphenyl; PNP: p-nitrophenyl.

The electronegativity of the atom or group in the bridge was found to significantly influence the electrophilic character of these (hetero)­norbornadienes. Specifically, OND 3a exhibited higher reactivity compared to its N-Boc azanorbornadiene analogue 4a, as well as norbornadienes 19a and 20a (entries 1, 9, 10). Among the N-substituted derivatives, N-acyl compounds demonstrated greater reactivity than N-Boc carbamates (entries 4–6), with the trifluoroacetamide derivative 11a showing a remarkable reactivity (entry 5). The reduced reactivity of 4a may be attributed to steric hindrance from the bulky Boc group, as evidenced by increased reactivity upon substitution with the less hindered Moc group (4a vs. 8a, entry 3). This effect could be rationalized by the syn-tilting of the N-substituent relative to the C2 and C3 substituents of the bicyclic framework. Within the series 13a–15a, which share similar steric profiles, thiol reactivity follows the trend 15a > 14a > 13a (entries 7 and 8). As we have demonstrated, our ANDs exhibit significant N-pyramidalization regardless of the bridge substituent (carbamate, acyl, or sulfonamide), which increases the predominance of inductive effects over resonance. The sulfonamide substituent in 15a, being more electronegative than the amide groups in 14a and 13a, contributes to the observed reactivity. On the other hand, the presence of a methoxycarbonyl group instead of a tosyl as electron-withdrawing (EWG) group (6a vs 7a, entry 13) accelerated the substitution reaction. Finally, we also observed that the extension of the bridge (from [2.2.1] to [2.2.2]­bicyclic skeleton) decreased the reactivity of the corresponding compounds (entries 11 and 12).

At this stage, we explored the thiol-promoted fragmentation of selected thio-substituted AND systems (Scheme ). Compounds 4b-19b were reacted with N-acetylcysteamine in CD₃OD in the presence of Et₃N. For comparative purposes, data from previously studied fragmentations of selected ONDs (3b, 24b–26b) were also included. The reaction time (t) required to achieve 50% conversion of the heteronorbornadienes into the expected heterocycle (furan or pyrrole) and ketene S,S-acetal products (27 or 28) was monitored. This parameter (t) was determined by ¹H NMR spectroscopy through plotting the % conversion of the (hetero)­norbornadiene versus reaction time. The direct comparison between oxabicyclic and azabicyclic systems (3b vs 4b, 24b vs 5b, 25b vs 6b, 26b vs 7b) shows that the fragmentation of the aza-analogues is in general much faster than for the oxa-derivatives. Unlike ONDs, where the thioketal intermediate was quantitatively and immediately formed (detected by ¹H NMR), and the rDA was the rate-limiting step, in the case of ANDs we have found three different situations. In the first one (Scheme , ANDs in blue), compounds 4b, 6b, 7b and 17b were transformed into the corresponding pyrroles without detection of the thioketal intermediate by ¹H NMR. This fact indicates that the rate-limiting step was the thio-Michael addition, in accordance with the second-order reaction determined for these fragmentations (Figures S15, S17, S18 and S27). The second situation encompasses ANDs 11b and 15b, where the rDA of the resulting thioketal intermediate was the rate-limiting step and a first-order reaction kinetic was determined in both cases (Figures S21 and S26). A third situation was observed for several AND derivatives (5b, 9b, 10b, 12b–14b, and 16b; ANDs in red, Scheme ), where a mixture of starting material, thioketal intermediate and final pyrrole derivative are detected by ¹H NMR during the fragmentation process. This indicates that the rates of conjugate addition (k ₁) and retro-Diels–Alder (k ₂) reactions are comparable. Compound 18b (green) showed exceptionally fast fragmentation, precluding its classification into any of the previous situations.

4. Thiol-Promoted Fragmentation of ANDs and Time (t) Required for the Conversion of (hetero)­norbornadienes Into 50% of the Expected Heterocycle (Furan or Pyrrole) .

^a Reaction conditions: To a solution of thio-substituted (hetero)­norbornadiene in CD₃OD (70 mM), 1.1 equiv of N-acetylcysteamine in CD₃OD and Et₃N (1.1 equiv) were added. The mixture was heated at 30 °C and ¹H NMR spectra were registered at different intervals. % Conversion of heteronorbornadienes into the expected heterocycle (furan or pyrrole) and ketene S,S-acetal vs time was plotted. ^bData from ref . ^cThe resulting pyrrole was not stable under reaction conditions. ^dThis experiment was accomplished in DMSO-d ₆ (13b and 14b were not soluble in CD₃OD). ^eThio-Michael addition (3.8 h for completion) is slow although much faster than rDA. ^fStarting material mostly remains after 24 h. In blue (2nd order kinetics): only starting material + final product is detected; in black (1st order kinetics): only intermediate + final product is detected; in red: intermediate + starting material + final product is detected in the experiment; in green: the transformation was so fast that the identification of the species in solution was not possible during the experiment; in lilac: fragmentation products were not observed.

The reactivity of compounds 4b, 6b, 7b, and 17b, all of them in situation 1 (thio-Michael addition as rate-limiting step), can be attributed to two main factors: (1) steric hindrance from the N-Boc group, whose syn-tilt relative to C2/C3 may interfere with nucleophilic attack; and (2) the lower electronegativity of the carbamate moiety compared to the bridge oxygen in ONDs, which reduces the electrophilicity of the azabicyclic system in the thio-Michael addition. AND 11b behaved similarly to ONDs, with rapid and quantitative formation of the thioketal intermediate as confirmed by ¹H NMR, followed by a slow rDA fragmentation (t = 13.5 h). In the case of 15b (t > 80 h) both, the thio-Michael addition and the rDA were slow, with the rDA being markedly slower than the initial addition. Both compounds, 11b and 15b present a notably slow fragmentation kinetic. In both cases, the rate-limiting step corresponds to the retro-Diels–Alder (rDA) reaction, reflecting the low dienic character of the resulting pyrrole which would difficult both, the direct and the retro Diels–Alder reaction. Concerning amides 10b and 12b-14b no significant differences in t (78–119 min) were observed, despite the different electron-withdrawing character of the acyl group of the nitrogen. Although the type of substitution of the N atom should influence both the conjugate addition and the rDA reaction, the notable pyramidalization of the N in the azanorbornadienic skeleton avoids, in part, the delocalization of the lone electron pair on the N by resonance effect, and therefore, reduces the expected electronic effect of the amide substituents.

The introduction of substituents at C1 and/or C4 of ANDs did not significantly affect the fragmentation rate (compounds 6b, 16b, and 17b vs 4b). In contrast, the incorporation of a formyl group at the bridgehead carbon (compound 18b) led to a dramatic increase in the fragmentation rate (t < 2 min). This could be due to the electronic repulsion between nearby CHO and Ts groups in the thioketal intermediate, resulting in destabilization that would be the driving force for the extremely fast rDA reaction. The influence of the electron-withdrawing group at the thiovinyl moiety (C3) was also examined. Replacing the tosyl group with an ester proved to be detrimental to the fragmentation process (5b vs 4b, 7b vs 6b), likely due to the superior thio-Michael acceptor ability of vinyl sulfones over acrylates. In the case of carba-bicyclic systems 19b and 20b, no thio-Michael addition was observed even after 24 h.

2.2.1. Azabicyclic Bromovinyl Sulfones for Protein Labeling. GSH-Triggered Degradation of the Bioconjugate

To evaluate the feasibility of our ANDs as cleavable linkers for drug delivery system design, we conjugated dansylated AND 29 to a PD-L1 single-domain antibody (sdAb), following the same experimental protocol previously optimized for the bioconjugation of ubiquitin with a related bromo-AND. We monitored the fragmentation of the resulting bioconjugate A in the presence of GSH at various time points (Scheme ). The fluorophore in 29 was incorporated through a glycine spacer as N-acyl ANDs showed higher fragmentation rates than their N-sulfonyl analogues (see Scheme , compounds 15b and 10b).

5. (a) GSH-Promoted Fragmentation Reaction between Bioconjugate A and GSH; (b) Bioconjugate A Incubated with GSH at different Times on a NuPAGE SDS Gel. Lane 1 Bioconjugate A; Lanes 2 to 7: A + GSH after 5, 15, 30, 45, 60, 120, and 240 min Incubation at 37 °C; (c) MS Trace of the Reaction Crude between GSH and Bioconjugate A after 240 min at 37 °C.

Bioconjugate A presented attenuated fluorescence compared to that shown by dansyl pyrrole 30, presumably due to photoinduced electron transfer (PET) effect between the conjugated double bond and the fluorophore. This effect was also observed for a model of bioconjugate A (Supporting Information, Figure S29). Since this PET is not completely quenching dansyl fluorescence, residual emission can still be observed in bioconjugate A (Scheme b, lane 1) and, therefore, can be monitored over time as an indicative of pyrrole release when the bioconjugate is incubated in GSH solution (Scheme b, lanes 2 to 7). As shown in Scheme b, dansyl residual fluorescence quickly decreases over time and after 240 min, emission was virtually undetectable- consistent with the conversion of A into B. LCMS studies carried out on the reaction mixture after 4 h incubation (Scheme c) confirmed the formation of product B as the main species identified (expected 16,723 Da, found 16,721 Da). Interestingly, intermediate C (GSH-adduct) was also detected by MS (expected 17,080, found 17,076). However, its concentration in the sample must be very low as almost no fluorescence was observed at that time.

Finally, we decided to investigate the stability of bioconjugate A in the presence of other biologically relevant thiols, selecting albumin as a representative example. Albumin is the most abundant circulating protein in blood and its potential interaction with AND-derived conjugate may result in an undesirable cargo release thereby, limiting the utility of AND scaffolds as new cleavable linkers. For that reason, bioconjugate A was incubated in both Human Serum Albumin (HAS) and human plasma at 37 °C for 4 days and the reactivity of the conjugate was assessed as it was previously described for GSH (Supporting Information, Figure S34). To our delight, no fluorescence decrease was observed over the curse of the incubation, which may indicate that cysteine 34 of the albumin is unable to attack the thiovinyl sulfone function on A -most likely due to steric hindrance- thus confirming not only the stability of the bioconjugate in plasma, but also the applicability of these ANDs as cleavable linkers.

3. Conclusions

ANDs incorporating a bromovinyl sulfone moiety exhibit a unique sequential reactivity, undergoing initial thiol–Michael addition followed by a second thiol-triggered fragmentation of the bicyclic scaffold. This dual-thiol responsiveness makes them promising candidates as cleavable linkers for targeted drug delivery, where the first thiol could mimic a targeting vector and the second be supplied by an intracellular trigger such GSH. Unlike previously reported ANDs and ONDs, which accept only one thiol, these systems support a two-step, stimulus-responsive fragmentation. Structural effects play a key role in modulating this reactivity. The pyramidalization of the N-bridge confirmed in selected cases partially attenuates the resonance contribution of N-substituents, resulting in generally consistent fragmentation rates across most ANDs studied (t = 56–255 min). However, strong electron-withdrawing substituents such as sulfonamides can significantly reduce reactivity (15b, t > 80 h), making this linkage unsuitable for N-decoration with biologically relevant fragments. In contrast, amide and carbamate linkages proved more effective for such purposes. The steric consequences of nitrogen pyramidalization probably contribute to the distinct fragmentation behavior observed in ANDs compared to previously studied ONDs. Specifically, the initial thiol–Michael addition tends to proceed more rapidly in ONDs; nevertheless, the overall fragmentation process is generally faster in AND analogues. Finally, conjugation of AND 29 to a single-domain antibody (sdAb) produced a plasma-stable bioconjugate that underwent efficient GSH-triggered cleavage. Taken together, these results highlight the utility of AND-based scaffolds as tunable, cleavable linkers with potential applications in drug delivery and targeted bioconjugation.

4. Experimental Procedures

4.1. General Methods

¹H- and ¹³C NMR spectra were recorded with a Bruker AVIII300, NEO300, NEO400 and NEO500 spectrometer for solutions in CDCl₃, CD₃OD, (CD₃)₂CO, (CD₃)₂SO, and C₆D₆. δ are given in ppm and J in Hz. Chemical shifts are calibrated using residual solvent signals. All the assignments were confirmed by 2D spectra (COSY and HSCQ). High resolution mass spectra were recorded on a Q-Exactive-quadrupole mass spectrometer. TLC was performed on silica gel 60 F₂₅₄ (Merck), with detection by UV light charring with KMnO₄, ninhydrin, or with reagent [(NH₄)₆MoO₄, Ce­(SO₄)₂, H₂SO₄, H₂O]. Purification by silica gel chromatography was carried out using either hand-packed glass columns (Silica gel 60 Merck, 40–60 and 63–200 μm) or Puriflash XS520 Plus Interchim system with prepacked cartridges.

4.2. General Procedure for the Preparation of Bromo-ANDs 3a–8a, 15a–17a

To a solution of activated alkyne S1 or S2 (x mmol) in toluene (2 mL/mmol), the corresponding commercial or synthetic pyrrole derivative S3–S8 (z mmol) was added, and the reaction mixture was stirred at 50–90 °C. After the reaction was completed, the solvent was evaporated, and the resulting residue was purified by a chromatography column on silica gel to give the corresponding bromo-AND.

4.2.1. General Procedure for the Synthesis of Thio-(hetero)­Norbornadienes 4b–20b.

A solution of the corresponding bromo-AND (x mmol) in DMF or MeCN (10 mL/mmol), a solution of N-acetylcysteamine (z mmol) in DMF or MeCN (5 mL/mmol) and phosphate buffer solution (pH 8.0, 50 mM, 10 mL/mmol), were added simultaneously and the mixture was stirred at r.t. for 30 min. Then, solvents were evaporated, and the residue was dissolved in EtOAc and washed with water. The organic phase was dried with Na₂SO₄ anh., filtered and concentrated. Purification by silica gel column chromatography afforded the corresponding thio-AND.

4.2.2. (rac) tert-Butyl-2-((2-acetamidoethyl)­thio)-3-tosyl-7-azabicyclo­[2.2.1]­hepta-2,5-diene-7-carboxylate (4b)

Starting from 4a (412 mg, 0.95 mmol) and N-acetylcysteamine (95 mg, 0.80 mmol) in MeCN following the general procedure, afforded after chromatographic purification (EtOAc/CyHex 2:1 → 10:1) compound 4b (258 mg, 0.55 mmol, 69%) as a brown oil. ¹H NMR (300 MHz, CDCl₃, δ ppm): 7.76 (d, 2H, J = 7.9 Hz, Ar–H), 7.32 (d, 2H, J = 7.7 Hz, Ar–H), 6.82 (br s, 2H, H-5, H-6), 6.36 (br s, 1H, NH), 5.54 (br s, 1H, H-1 or H-4), 5.31 (br s, 1H, H-1 or H-4), 3.55–3.51 (m, 2H, H-2′), 3.19–3.09 (m, 2H, H-1′), 2.42 (s, 3H, CH₃ of Ts), 1.98 (s, 3H, CH₃CO), 1.28 (s, 9H, CH₃ of Boc). ¹³C NMR (75 MHz, CDCl₃, δ ppm): 170.7, 166.9, 154.2, 144.6, 142.6, 141.1, 138.2, 137.2, 129.9, 127.3, 81.9, 70.8, 69.4, 40.3, 32.2, 27.3, 23.1, 21.6. HRMS (ESI) m/z; found, 487.1330 calcd for C₂₂H₂₈N₂NaO₅S₂ [M + Na]⁺: 487.1337.

4.2.3. (rac) 7-(tert-Butyl)-2-methyl-3-((2-acetamidoethyl)­thio)-7-azabicyclo­[2.2.1]­hepta-2,5-diene-2,7-dicarboxylate (5b)

Starting from 5a (250 mg, 0.76 mmol) and N-acetylcysteamine (75 mg, 0.63 mmol) in DMF following the general procedure, afforded after chromatographic purification (EtOAc/CyHex 1:1 → EtOAc) compound 5b (159 mg, 0.43 mmol, 68%) as a yellow oil. ¹H NMR (300 MHz, CD₃OD, δ ppm): 7.08 (s, 2H, H-5, H-6), 5.74–5.64 (m, 1H, H-1 or H-4), 5.41 (ap q, J = 1.6 Hz, H-1 or H-4), 3.72 (s, 3H, COOCH₃), 3.54–3.29 (m, 2H, H-2′), 3.19–3.09 (m, 2H, H-1′), 1.96 (s, 3H, COCH₃), 1.40 (s, 9H, CH₃ of Boc). ¹³C NMR (75 MHz, CD₃OD, δ ppm): 172.3, 164.3, 142.9, 139.9, 81.2, 69.7, 68.3, 67.6, 60.2, 50.8, 40.4, 30.7, 27.0, 21.1. HRMS (ESI) m/z; found, 391.1289 calcd for C₁₇H₂₄N₂NaO₅S [M + Na]⁺: 391.1281.

4.2.4. (rac) tert-Butyl-2-((2-acetamidoethyl)­thio)-1,4-dimethyl-3-tosyl-7-azabicyclo­[2.2.1]­hepta-2,5-diene-7-carboxylate (6b)

Starting from 6a (206 mg, 0.85 mmol) and N-acetylcysteamine (84 mg, 0.71 mmol) in MeCN following the general procedure, afforded after chromatographic purification (EtOAc/CyHex 10:1) compound 6b (152 mg, 0.31 mmol, 44%) as a brown oil. ¹H NMR (300 MHz, CD₃OD, δ ppm): 7.76 (d, 2H, J = 8.4 Hz, Ar–H), 7.42 (d, 2H, J = 8.0 Hz, Ar–H), 6.62 (d, 1H, J = 5.4 Hz, H-5 or H-6), 6.55 (d, 1H, J = 5.2 Hz, H-5 or H-6), 3.26–3.19 (m, 2H, H-2′), 3.14–3.11 (m, 2H, H-1′), 2.44 (s, 3H, CH₃ of Ts), 1.98 (s, 3H, CH₃CO), 1.94 (s, 3H, CH₃), 1.92 (s, 3H, CH₃), 1.31 (s, 9H, CH₃ of Boc). ¹³C NMR (75 MHz, CD₃OD, δ ppm): 173.3 (CO), 170.9 (CO), 156.4, 153.6 (C-2, C-3), 148.5 (C-5 or C-6), 146.3 (C_qAr), 146.2 (C-5 or C-6), 139.3 (C_qAr), 131.0, 128.7 (CH-Ar), 83.0, 82.8, 79.7 (C-1, C-4, C_q of Boc), 40.8 (C-2′), 34.4 (C-1′), 28.4 (CH₃ of Boc), 22.3 (CH₃CO), 21.6 (CH₃ of Ts), 18.5 (CH₃), 18.0 (CH₃). HRMS (ESI) m/z; found, 515.1642 calcd for C₂₄H₃₂N₂NaO₅S₂ [M + Na]⁺: 515.1650.

4.2.5. (rac) 7-(tert-Butyl)-2-methyl-3-((2-acetamidoethyl)­thio)-1,4-dimethyl-7-azabicyclo­[2.2.1]­hepta-2,5-diene-2,7-dicarboxylate (7b)

Starting from 7a (190 mg, 0.53 mmol) and N-acetylcysteamine (58 mg, 0.48 mmol) in DMF following the general procedure, afforded after chromatographic purification (EtOAc/CyHex 10:1 → EtOAc) compound 7b (85 mg, 0.21 mmol, 44%) as a dark brown oil. ¹H NMR (300 MHz, CD₃OD, δ ppm): 6.72 (d, 1H, J = 5.8 Hz, H-5 or H-6), 6.67 (d, 1H, J = 5.3 Hz, H-5 or H-6), 3.78 (s, 3H, COOCH ₃), 3.25–3.19 (m, 2H, H-2′), 3.09–3.01 (m, 2H, H-1′), 1.94 (s, 3H, CH₃CO), 1.93 (s, 3H, CH₃), 1.89 (s, 3H, CH₃), 1.42 (s, 9H, CH₃ of Boc). ¹³C NMR (75 MHz, CD₃OD, δ ppm): 171.9 (CO), 165.9 (CO), 165.7 (CO), 155.2 (C-2 or C-3), 147.4, 145.2 (C-5, C-6), 141.3 (C-2 or C-3), 80.9 (C_q of Boc), 79.7, 77.6 (C-1, C-4), 50.6 (COOCH₃), 38.4 (C-2′), 31.8 (C-1′), 27.2 (CH₃ of Boc), 21.1 (CH₃CO), 16.8 (CH₃), 15.4 (CH₃). HRMS (ESI) m/z; found, 419.1610 calcd for C₁₉H₂₈N₂NaO₅S [M + Na]⁺: 419.1617.

4.2.6. (rac) Methyl-2-((2-acetamidoethyl)­thio)-3-Tosyl-7-azabicyclo[2.2.1]­hepta-2,5-diene-7-carboxylate (8b)

Starting from 8a (61 mg, 0.16 mmol) and N-acetylcysteamine (17 mg, 0.15 mmol) in MeCN following the general procedure, afforded after chromatographic purification (EtOAc) compound 8b (40 mg, 0.094 mmol, 63%) as a brown solid. ¹H NMR (300 MHz, DMSO-d ₆, 353 K, δ ppm): 7.88 (br s, 1H, NH), 7.69 (d, 2H, J = 8.4 Hz, Ar–H), 8.16 (d, 2H, J = 8.1 Hz, Ar–H), 7.00 (ddd, 1H, J = 5.3, 2.8, 0.7 Hz, H-5 or H-6), 6.93 (ddd, 1H, J = 5.6, 2.3, 0.5 Hz, H-5 or H-6), 5.72 (t, 1H, J = 2.2 Hz, H-1 or H-4), 5.30 (t, 1H, J = 2.2 Hz, H-1 or H-4), 3.43 (s, 3H, COOCH ₃), 3.40–3.09 (m, 4H, H-1′, H-2′), 2.42 (s, 3H, CH₃ of Ts), 1.85 (s, 3H, CH₃CO). ¹³C NMR (75 MHz, DMSO-d ₆, 353 K, δ ppm): 169.1 (CO), 154.0 (CO), 143.8 (C-2 or C-3), 142.0 (C-5 or C-6), 138.8 (C_qAr), 138.4 (C-5 or C-6), 136.8 (C-2 or C-3), 133.5 (C_qAr), 129.3 (CH-Ar), 70.2, 68.2 (C-1, C-4), 52.2 (COOCH₃), 40.2–38.0 (C-1′ or C-2′, under solvent signal), 30.9 (C-1′ or C-2′), 21.9 (CH₃ of Ts), 20.5 (CH₃CO). HRMS (ESI) m/z; found, 445.0868 calcd for C₁₉H₂₂N₂NaO₅S₂ [M + Na]⁺: 445.0859.

4.2.7. N-(2-(((rac)-7-Acetyl-3-Tosyl-7-azabicyclo­[2.2.1]­hepta-2,5-dien-2-yl)­thio)­ethyl)­acetamide (9b)

Starting from 9a (135 mg, 0.37 mmol) and N-acetylcysteamine (37 mg, 0.31 mmol) in MeCN following the general procedure, afforded after chromatographic purification (EtOAc/CyHex 10:1 → EtOAc) compound 9b (77 mg, 0.19 mmol, 61%) as a brown oil. ¹H NMR (300 MHz, CD₃OD, δ ppm, major rotamer): 7.76 (d, 2H, J = 8.3 Hz, Ar–H), 7.45–7.39 (m, 2H, Ar–H), 7.05–6.88 (m, 2H, H-5, H-6), 6.01 (t, 1H, J = 2.3 Hz, H-1 or H-4), 5.58 (t, 1H, J = 2.5 Hz, H-1 or H-4), 3.56–3.35 (m, 2H, H-2′) 3.26–3.11 (m, 2H, H-1′), 2.44 (s, 3H, CH₃ of Ts), 2.15, (s, 3H, CH ₃CO), 1.96 (s, 3H, CH ₃CO). ¹³C NMR (75 MHz, CD₃OD, δ ppm, major rotamer): 172.5 (CO), 169.3 (CO), 166.6, 145.2 (C-2, C-3), 142.5 (C-5 or C-6), 140.3 (C_qAr), 138.1 (C-5 or C-6), 136.8 (C_qAr), 129.7 (CH-Ar), 126.9 (CH-Ar), 69.9, 68.1 (C-1, C-4), 40.6 (C-2′), 31.1 (C-1′), 21.11 (CH₃ of Ts), 21.07 (CH₃CO), 19.7 (CH₃CO). HRMS (ESI) m/z; found, 429.0905 calcd for C₁₉H₂₂N₂NaO₄S₂ [M + Na]⁺: 429.0919.

4.2.8. N-(2-(((rac)-7-Butyryl-3-Tosyl-7-azabicyclo­[2.2.1]­hepta-2,5-dien-2-yl)­thio)­ethyl)­acetamide (10b)

Starting from 10a (195 mg, 0.49 mmol) and N-acetylcysteamine (53 mg, 0.45 mmol) in MeCN following the general procedure, afforded after chromatographic purification (EtOAc → EtOAc/Acetone 10:1) compound 10b (96 mg, 0.22 mmol, 49%) as a yellow oil. ¹H NMR (300 MHz, CD₃OD, δ ppm, major rotamer): 7.78 (d, 2H, J = 8.4 Hz, Ar–H), 7.47–7.41 (m, 2H, Ar–H), 7.07–6.93 (m, 2H, H-5, H-6), 6.04 (t, 1H, J = 2.3 Hz, H-1 or H-4), 5.63 (t, 1H, J = 2.2 Hz, H-1 or H-4), 3.61–3.36 (m, 2H, H-2′), 3.28–3.11 (m, 2H, H-1′), 2.46 (s, 3H, CH₃ of Ts), 2.26–2.03 (m, 2H, CH ₂–CH₂–CH₃), 1.99 (s, 3H, CH₃CO), 1.59–1.27 (m, 2H, CH₂–CH ₂–CH₃), 0.90–0.79 (m, 3H, CH₂–CH₂–CH ₃). ¹³C NMR (75 MHz, CD₃OD, δ ppm, major rotamer): 172.3 (CO), 169.4 (CO), 145.2, 143.4 (C-2, C-3), 142.6 (C-5 or C-6), 139.2 (C_qAr), 138.2 (C-5 or C-6), 137.0 (C_qAr), 129.8 (CH-Ar), 126.9 (CH-Ar), 68.0, 67.8 (C-1, C-4), 40.0 (C-2′), 34.8 (CH ₂–CH₂–CH₃) 30.5 (C-1′), 21.1 (CH₃CO), 20.2 (CH₃ of Ts), 17.9 (CH₂–CH ₂–CH₃), 12.5 (CH₂–CH₂–CH ₃). HRMS (ESI) m/z; found, 457.1221 calcd for C₂₁H₂₆N₂NaO₄S₂ [M + Na]⁺: 457.1232.

4.2.9. N-(2-(((rac)-3-Tosyl-7-(2,2,2-Trifluoroacetyl)-7-azabicyclo­[2.2.1]­hepta-2,5-dien-2-yl)­thio)­ethyl)­acetamide (11b)

Starting from 11a (75 mg, 0.23 mmol) and N-acetylcysteamine (25 mg, 0.21 mmol) in MeCN following the general procedure, afforded after chromatographic purification (EtOAC/CyHex 10:1 → EtOAc) compound 11b (25 mg, 0.07 mmol, 33%) as a yellow oil. ¹H NMR (300 MHz, CD₃OD, δ ppm, mixture of rotamers): δ 7.65 (d, 2H, J = 8.5 Hz, Ar–H), 7.32 (d, 2H, J = 7.9 Hz, Ar–H), 7.01 (dd, 1H, J = 5.2, 2.9 Hz, H-5 or H-6), 6.91 (dd, 1H, J = 5.6, 2.4 Hz, H-5 or H-6), 6.14–6.11 (m, 1H, H-1 or H-4), 5.64 (br s, 1H, H-1 or H-4), 3.45–3.22 (m, 4H, H-1′ or H-2′), 2.34 (s, 3H, CH₃ of Ts), 1.86 (s, 3H, CH ₃CO). ¹³C NMR (75 MHz, CD₃OD, δ ppm, mixture of rotamers): δ 172.4, 167.6, 165.3, 143.8, 139.0, 138.4, 136.5, 129.8, 127.0, 67.9, 65.8, 40.0, 31.1, 21.0, 20.2. HRMS (ESI) m/z; found, 483.0631 calcd for C₁₉H₁₉N₂F₃NaO₄S₂ [M + Na]⁺: 483.0631.

4.2.10. N-(2-(((rac)-7-Benzoyl-3-Tosyl-7-azabicyclo­[2.2.1]­hepta-2,5-dien-2-yl)­thio)­ethyl)­acetamide (12b)

Starting from 12a (175 mg, 0.41 mmol) and N-acetylcysteamine (44 mg, 0.37 mmol) in MeCN following the general procedure, afforded after chromatographic purification (EtOAC/CyHex 10:1 → EtOAc) compound 12b (116 mg, 0.25 mmol, 68%) as a yellow solid. ¹H NMR (300 MHz, DMSO-d ₆, δ ppm, mixture of rotamers): 7.58–7.26 (m, 9H, Ar–H), 7.18–7.01 (m, 2H, H-5, H-6), 6.07 (br s, 1H, H-1 or H-4), 5.36 (br s, 1H, H-1 or H-4), 3.23–2.87 (m, 4H, H-1′, H-2′), 2.38 (s, 3H, CH₃ of Ts), 1.90 (s, 3H, CH ₃CO). ¹³C NMR (75 MHz, DMSO-d ₆, δ ppm, mixture of rotamers): 170.2, 168.7, 144.8, 143.5, 139.5, 137.1, 132.2, 130.4, 130.2, 129.0, 128.9, 128.3, 127.8, 126.8, 71.0, 69.4, 60.2, 38.7, 31.8, 23.0, 21.6. HRMS (ESI) m/z; found, 491.1075 calcd for C₂₄H₂₄N₂NaO₄S₂ [M + Na]⁺: 491.1070.

4.2.11. N-(2-(((rac)-7-(4-Methoxybenzoyl)-3-Tosyl-7-azabicyclo­[2.2.1]­hepta-2,5-dien-2-yl)­thio)­ethyl)­acetamide (13b)

Starting from 13a (187 mg, 0.43 mmol) and N-acetylcysteamine (47 mg, 0.39 mmol) in MeCN following the general procedure, afforded after chromatographic purification (EtOAc/CyHex 10:1 → EtOAc) compound 13b (93 mg, 0.19 mmol, 49%) as a pale yellow solid. ¹H NMR (300 MHz, DMSO-d ₆, δ ppm, mixture of rotamers): 7.58–7.50 (m, 2H, Ar–H), 7.40–7.26 (m, 4H, Ar–H), 7.18–7.10 (m, 2H, Ar–H), 7.00–6.94 (m, 2H, H-5, H-6), 6.06 (br s, 1H, H-1 or H-4), 5.45 (br s, 1H, H-1 or H-4), 3.88 (s, 3H, OCH₃), 3.58–3.20 (m, 4H, H-1′, H-2′), 2.43 (s, 3H, CH₃ of Ts), 1.91 (s, 3H, CH₃CO). ¹³C NMR (75 MHz, DMSO-d ₆, δ ppm, mixture of rotamers): 170.2 (CO), 162.4 (CO), 144.7, 143.5, (C-2, C-3), 139.3, 137.1, 130.5, 130.4, 126.8 (CH-Ar), 114.2 (C-5, C-6), 71.5, 69.7 (C-1, C-4), 55.9 (OCH₃), 40.0, 31.8 (C-1′, C-2′), 23.O (CH₃ of Ts), 21.6 (CH₃CO). HRMS (ESI) m/z; found, 521.1166 calcd for C₂₅H₂₆N₂NaO₅S₂ [M + Na]⁺: 521.1175.

4.2.12. N-(2-(((rac)-7-(4-Nitrobenzoyl)-3-Tosyl-7-azabicyclo­[2.2.1]­hepta-2,5-dien-2-yl)­thio)­ethyl)­acetamide (14b)

Starting from 14a (165 mg, 0.37 mmol) and N-acetylcysteamine (40 mg, 0.34 mmol) in MeCN following the general procedure affored 14b (71 mg, 0.14 mmol, 41%) as a pale-yellow solid after precipitation with AcOEt. ¹H NMR (300 MHz, DMSO-d ₆, δ ppm, mixture of rotamers): 8.27–8.12 (m, 2H, Ar–H), 7.51 (m, 5H, Ar–H), 7.24–7.16 (m, 4H, Ar–H, H-5, H-6), 6.16 (br s, 1H, H-1 or H-4), 5.37 (br s, 1H, H-1 or H-4), 3.49–3.09 (m, 4H, H-1′, H-2′), 2.36 (s, 3H, CH₃ of Ts), 1.87 (s, 3H, CH₃CO). ¹³C NMR (75 MHz, DMSO-d ₆, δ ppm, mixture of rotamers): 170.2 (CO), 166.9 (CO), 149.4, 144.9 (C-5, C-6), 143.4, 138.2, 138.0, 137.6, 137.1, (C_qAr), 130.5, 129.7, 126.9, 124.2 (CH-Ar), 70.6, 69.1 (C-1, C-4), 40.0, 31.8 (C-1′, C-2′), 23.0 (CH₃ of Ts), 21.5 (CH₃CO). HRMS (ESI) m/z; found, 536.0913 calcd for C₂₄H₂₃N₃NaO₆S₂ [M + Na]⁺: 536.0920.

4.2.13. N-(2-(((rac)-3,7-Ditosyl-7-azabicyclo­[2.2.1]­hepta-2,5-dien-2-yl)­thio)­ethyl)­acetamide (15b)

Starting from 15a (80 mg, 0.17 mmol) and N-acetylcysteamine (18 mg, 0.15 mmol) in MeCN following the general procedure, afforded after chromatographic purification (EtOAc/CyHex 10:1 →EtOAc) compound 15b (51 mg, 0.098 mmol, 65%) as a yellow solid. ¹H NMR (300 MHz, CD₃OD, δ ppm): 7.69–7.61 (m, 4H, Ar–H), 7.43–7.34 (m, 4H, Ar–H), 6.84 (dd, 1H, J = 5.5, 3.2 Hz, H-5 or H-6), 6.70 (dd, 1H, J = 5.5, 2.6 Hz, H-5 or H-6), 5.75 (t, 1H, J = 2.4 Hz, H-1 or H-4), 5.30 (t, 1H, J = 2.5 Hz, H-1 or H-4), 3.28–3.09 (m, 4H, H-1′, H-2′), 2.44 (s, 6H, CH₃ of Ts), 2.04 (s, 3H, CH ₃CO). ¹³C NMR (75 MHz, CD₃OD, δ ppm): 165.6 (CO), 145.0, 144.5 (C-2, C-3), 143.1, 138.7 (C-5, C-6), 137.6, 134.7 (C_qAr), 129.7, 129.6 (CH-Ar), 129.4 (C_qAr), 128.3 (CH-Ar), 128.2 (C_qAr), 127.0 (C_qAr), 71.4, 68.5 (C-1, C-4), 40.2, 30.8 (C-1′, C-2′), 21.1 (CH₃CO), 20.1 (CH₃ of Ts). HRMS (ESI) m/z; found, 541.0903 calcd for C₂₄H₂₆N₂NaO₅S₃ [M + Na]⁺: 541.0896.

4.2.14. tert-Butyl (rac)-3-((2-acetamidoethyl)­thio)-1-(hydroxymethyl)-2-Tosyl-7-azabicyclo[2.2.1]­hepta-2,5-diene-7-carboxylate (16b)

Starting from 16a (240 mg, 0.53 mmol) and N-acetylcysteamine (57 mg, 0.48 mmol) in MeCN following the general procedure, afforded after chromatographic purification (EtOAc/Acetone 15:1) compound 16b (71 mg, 0.14 mmol, 29%) as a yellow solid. ¹H NMR (300 MHz, CD₃OD, δ ppm): 7.62 (d, 2H, J = 8.4 Hz, Ar–H), 7.28 (d, 2H, J = 8.2 Hz, Ar–H), 6.79 (dd, 1H, J = 5.5, 3.0 Hz, H-5), 6.72 (d, 1H, J = 5.4 Hz, H-6), 5.70 (d, 1H, J = 2.9 Hz, H-4), 4.40–4.24 (m, 2H, CH₂), 3.46–3.28 (m, 2H, H-2′), 3.11–3.02 (m, 2H, H-1′), 2.32 (s, 3H, CH₃ of Ts), 1.87 (s, 3H, CH₃CO), 1.25 (s, 9H, CH₃ of Boc). ¹³C NMR (75 MHz, CD₃OD, δ ppm): 172.3 (CO), 153.9 (CO) 145.7 (C-6), 144.8 (C-2 or C-3), 137.8 (C-5), 136.6 (C_qAr), 129.5, 126.6 (CH-Ar), 82.1 (C-1), 81.9 (C_qBoc), 70.8 (C-4), 58.5 (CH₂), 42.6 (C-2′), 40.2 (C-1′), 26.9 (CH₃ of Boc), 21.1 (CH₃ of Ts), 20.2 (CH₃CO). HRMS (ESI) m/z; found, 517.1425 calcd for C₂₃H₃₀N₂NaO₆S₂ [M + Na]⁺: 517.1437.

4.2.15. tert-Butyl (rac)-2-((2-acetamidoethyl)­thio)-4-(hydroxymethyl)-1-methyl-3-Tosyl-7-azabicyclo[2.2.1]­hepta-2,5-diene-7-carboxylate (17b)

Starting from 17a (200 mg, 0.43 mmol) and N-acetylcysteamine (46 mg, 0.39 mmol) in MeCN following the general procedure, afforded after chromatographic purification with EtOAc compound 17b (70 mg, 0.14 mmol, 36%) as a brown oil. ¹H NMR (300 MHz, CD₃OD, δ ppm): 7.69 (d, 2H, J = 8.2 Hz, Ar–H), 7.30 (d, 2H, J = 8.3 Hz, Ar–H), 6.81 (d, 1H, J = 5.4 Hz, H-5 or H-6), 6.54 (d, 1H, J = 5.8 Hz, H-5 or H-6), 4.46–4.34 (m, 2H, CH₂), 3.06–2.86 (m, 4H, H-1′, H-2′), 2.33 (s, 3H, CH₃ of Ts), 1.91 (s, 3H, CH₃), 1.83 (s, 3H, CH₃CO), 1.27 (CH₃ of Boc). ¹³C NMR (75 MHz, CD₃OD, δ ppm): 171.9, 170.4 (CO), 154.7, 152.2 (C-2, C-3), 145.0 (C_qAr), 144.8, 144.1 (C-5, C-6), 137.9 (C_qAr), 129.4, 127.2 (CH-Ar), 83.2, 82.1 (C-1, C-4), 82.0 (C_qBoc), 59.8 (CH₂), 39.4, 32.5 (C-1′, C-2′), 27.0 (CH₃ of Boc), 21.2 (CH₃ of Ts), 20.2 (CH₃CO), 13.1 (CH₃). HRMS (ESI) m/z; found, 531.1578 calcd for C₂₄H₃₂N₂NaO₆S₂ [M + Na]⁺: 531.1594.

4.2.16. tert-Butyl (rac)-2-((2-acetamidoethyl)­thio)-4-formyl-1-methyl-3-tosyl-7-azabicyclo[2.2.1]­hepta-2,5-diene-7-carboxylate (18b)

Starting from 18a (130 mg, 0.28 mmol) and N-acetylcysteamine (30 mg, 0.25 mmol) in MeCN following the general procedure, afforded after chromatographic purification with EtOAc compound 18b (56 mg, 0.11 mmol, 44%) as a brown oil. ¹H NMR (300 MHz, CD₃OD, δ ppm): 9.92 (s, 1H, CHO), 7.60 (d, 2H, J = 8.6 H, Ar–H), 7.33 (d, 2H, J = 8.2 Hz, Ar–H), 6.97 (d, 1H, J = 5.2 Hz, H-6), 6.62 (dd, 1H, J = 5.6, 1.0 Hz, H-5), 3.07–3.00 (m, 4H, H-1′, H-2′), 2.34 (s, 3H, CH₃ of Ts), 1.89 (s, 3H, CH₃), 1.85 (s, 3H, CH₃CO), 1.28 (s, 9H, CH₃ of Boc). ¹³C NMR (75 MHz, CD₃OD, δ ppm): 190.8 (CHO), 172.0 (CO), 168.2 (CO), 154.3, 150.5 (C-2, C-3), 145.5 (C_qAr), 144.5 (C-5), 140.8 (C-6), 136.9 (C_qAr), 129.7, 127.3 (CH-Ar), 83.9, 83.4 (C-1, C-4), 39.3, 33.2 (C-1′, C-2′), 26.9 (CH₃ of Boc), 21.1 (CH₃ of Ts), 20.2 (CH₃CO), 15.3 (CH₃). HRMS (ESI) m/z; found, 529.1428 calcd for C₂₄H₃₀N₂NaO₆S₂ [M + Na]⁺: 529.1437.

4.2.17. N-(2-(((1R,4S)-3-tosylbicyclo­[2.2.1]­hepta-2,5-dien-2-yl)­thio)­ethyl)­acetamide (19b)

Starting from 19a (400 mg, 1.22 mmol) and N-acetylcysteamine (121 mg, 1.02 mmol) in MeCN following the general procedure, afforded after chromatographic purification with EtOAc compound 19b (140 mg, 0.39 mmol, 38%) as a brown oil. ¹H NMR (300 MHz, CD₃OD, δ ppm): 7.70 (d, 2H, J = 8.4 Hz, Ar–H), 7.39 (d, 2H, J = 8.4 Hz, Ar–H), 6.67–6.62 (m, 1H, H-5 or H-6), 6.58–6.53 (m, 1H, H-5 or H-6), 4.26 (br s, 1H, H-1 or H-4), 3.81 (br s, 1H, H-1 or H-4), 3.39–3.27 (m, 2H, H-1′ or H-2′), 3.17–3.05 (m, 2H, H-1′ or H-2′), 2.44 (s, 3H, CH₃ of Ts), 2.10 (dt, 1H, J = 6.7, 1.4 Hz, CH₂), 2.00 (dt, 1H, J = 6.8, 1.9 Hz, CH₂), 1.96 (s, 3H, CH₃CO). ¹³C NMR (75 MHz, CD₃OD, δ ppm): 172.1, 166.3, 144.4, 142.2, 139.1, 138.0, 137.2, 129.4, 126.7, 68.3, 55.8, 52.8, 40.3, 30.7, 21.1, 20.2. HRMS (ESI) m/z; found, 386.0850 calcd for C₁₈H₂₁NNaO₃S₂ [M + Na]⁺: 386.0855.

4.3. General Procedure for Competition Experiments

To a solution of compound A (1.2 equiv) and B (1.2 equiv) in DMF (20 mL/mmol), phosphate buffer solution (pH 8.0, 50 mM, 25 mL/mmol) and a solution of N-acetylcysteamine (1.0 equiv) in DMF (4 mL/mmol) were added. The mixture was stirred at r.t. for 30 min and then, solvents were evaporated, and the residue was dissolved in EtOAc and washed with water. The aqueous phase was extracted with EtOAc (x3) and the combined organic phases were dried (Na₂SO₄), filtered and concentrated. Purification was performed by silica gel column chromatography (DCM/MeOH 100:1 → 15:1). % Conversion A/B into A'/B' was determined by ¹H NMR of the starting material fraction recovered from the chromatographic purification.

4.3.1. N-(2-((rac)-2-Bromo-3-Tosyl-7-azabicyclo­[2.2.1]­hepta-2,5-dien-7-yl)-2-oxoethyl)-5-(dimethylamino)­naphthalene-1-sulfonamide (29)

To a solution of 4a (100 mg, 0.23 mmol) in DCM (4 mL), TFA (0.5 mL, 7 mmol) was added. The reaction mixture was stirred at r.t. for 30 min and then, the solvent was removed to afford crude unprotected AND. To a cooled (0 °C) solution of this compound (70 mg, 0.21 mmol) in anh. DCM (2 mL), dansylglycine chloride (190 mg, 0.53 mmol) in anh. DCM (5 mL) and Et₃N (75 μL, 0.53 mmol) were added under Ar. The reaction mixture was stirred at r.t for 1.5 h. and then it was diluted with DCM and washed with H₂O. The organic phase was separated, dried, filtered and concentrated. The resulting crude was purified by column chromatography on silica gel (EtOAc/Cy 1:1) to afford 29 (42 mg, 0.068 mmol, 30% three steps, pale yellow solid). ¹H NMR (300 MHz, CDCl₃, 298 K, δ ppm, mixture of rotamers): δ 8.56 (d, 1H, J = 8.7 Hz, Ar–H), 8.25 (d, 1H, J = 8.1 Hz, Ar–H), 8.18 (d, 1H, J = 7.1 Hz, Ar–H), 7.73 (d, 2H, J = 8.1 Hz, Ar–H), 7.60–7.47 (m, 2H, Ar–H), 7.20 (d, 1H, J = 7.2 Hz, Ar–H), 7.00–6.80 (m, 2H, H-5, H-6), 5.68–5.45 (m, 1H, H-1 or H-4), 5.42–5.31 (m, 1H, H-1 or H-4), 3.56 (dd, 1H, J = 15.8, 5.3 Hz, CH₂), 3.35 (dd, 1H, J = 16.0, 3.6 Hz, CH₂), 2.89 (s, 6H, N­(CH₃)₂), 2.43 (s, 3H, CH₃ of Ts). ¹³C NMR (75 MHz, CDCl₃, 298 K, δ ppm, mixture of rotamers): δ 163.5 (CO), 150.2, 146.0.145.7, 144.5 (C_q Ar), 142.6, 140.4 (C-5, C-6), 135.2, 133.9, (C_q Ar), 130.8, 130.4 (CH-Ar), 129.6 (C_q Ar), 129.4, 128.6, 127.8, 123.2, 118.9, 115.5 (CH-Ar), 72.7, 66.7 (C-1, C-4), 45.5 (N­(CH₃)₂), 44.2 (CH₂), 21.8 (CH₃ of Ts). HRESIMS m/z; found, 616.0571 calcd for C₂₇H₂₇ ⁷⁹BrN₃O₅S₂ [M + H]⁺, 616.0570; found, 618.0548 calcd for C₂₇H₂₇ ⁸¹BrN₃O₅S₂ [M + H]⁺, 618.0549.

4.4. Protein Bioconjugation

4.4.1. Synthesis of Bioconjugate A

500 μL protein solution in PBS (pH 7.4) (concentration 74 μM) was reduced for 20 min at 37 °C using 5 equiv of TCEP. After checking completion of the reduction step by LC–MS, 36.5 μL of DMSO were added followed by a solution of compound 29 in DMSO (18.5 μL, 10 mM, 5eq) and the mixture was incubated at rt for 1 h. After accomplishing full conversion (as determined by LC–MS analysis), bioconjugate A was purified by size exclusion chromatography using a 10/300 Superdex Increase 75 GL column (Cytiva, Little Chalfont, UK) and PBS (pH 7.4) as an elution buffer. Calculated mass 16,773 Da, Observed 16,769 Da.

4.4.2. GSH Stability Assay

Four μL of a 10 mM glutathione (GSH) solution (33.1 mg GSH in 1.08 mL PBS, adjusted to pH 7.4) was added at room temperature to 96 μL of bioconjugate A (4 μM in PBS) and mixed thoroughly by pipetting up and down, resulting in a final GSH concentration of 400 μM. The mixture was incubated at 37 °C. Aliquots were taken at 5, 15, 30, 60, 120, and 240 min, and immediately flash-frozen in liquid nitrogen. Samples were analyzed by SDS–PAGE to observe the decrease in fluorescence associated with pyrrole loss (Figure S32, ProQ Emerald 300 channel). The final time point (240 min) was also analyzed by LC–MS (Figure S33), confirming the presence of bioconjugate B (expected: 16,723 Da; found: 16,721 Da) and intermediate bioconjugate C (expected: 17,080 Da; found: 17,076 Da).

4.4.3. Plasma and HSA Stability Studies

The stability of a sample of conjugate A was tested in two separate experiments upon incubation with human plasma (Sigma-Aldrich, Cat. H4522) or human serum albumin (HSA, Sigma-Aldrich Cat. A3782). In short, 10 μL of a 15 μM solution of the conjugate in PBS were added to 40 μL of either human plasma or HSA (50 mg/mL) at room temperature and mixed thoroughly by pipetting up and down. The resulting mixture were incubated at 37 °C. Time points were taken at 0, 1, 2,3 and 4 days, and immediately flash frozen in liquid N₂. Samples were analyzed by SDS–PAGE (Figure S33).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.bioconjchem.5c00371.

• ORTEP of compounds 4a, 6a, 8a, 12a, 15a and 23a; synthesis of starting materials; details for the synthesis and characterization data of 4a-22a; details for competition and fragmentation experiments; bioconjugation and stability studies; ¹H- and ¹³C NMR spectra for new compounds (PDF)

The authors declare no competing financial interest.