BODIPY-Tagged β -Lactams as Selective Quenched Activity-Based Probes to Target Human Neutrophil Elastase

Human Neutrophil Elastase (HNE) plays a vital role in several inflammatory diseases, however its role in the tumour microenvironment and the potential in cancer treatment is still unrevealed. Considering the potential of β-lactams as HNE inhibitors, t he present work describes the development of a synthetic strategy to obtain two different types (Type I and Type II) of quenched activity-based probes (qABPs), using a β-lactam ring as a warhead and BODIPY-FL as a fluorophore. The two types differ in mechanism and relative position between the fluorophore and the quencher moiety. The qABPs synthesized presented IC50 values against HNE lower than 0.5 µM, and high selectivity compared with homologous serine hydrolases. Type II qABPs showed a more efficient turn-on mechanism, and selectively targeted HNE in different cell lysates. The qABP 22 .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint was internalized in U937 cells and in human neutrophils and successfully targeted HNE in both.

Serine hydrolases encompass a large superfamily of enzymes that play key functions in biological processes in mammals, bacteria and viruses. Despite their importance, the role of many serine hydrolases in the development and progress of several diseases remains poorly characterized.(1-3) Human Neutrophil Elastase (HNE) is a serine hydrolase of the chymotrypsin family expressed in polymorphonuclear neutrophils and has been associated with lung related diseases, such as cystic fibrosis, chronic obstructive pulmonary disease and acute respiratory distress syndrome.(4, 5) It has also been recently reported that HNE is present in the tumor microenvironment , promoting tumor growth and metastasis. (6-8) However, the impact of HNE released to the tumor microenvironment on cancer progression remains largely unexplored due to the lack of selective chemical tools to study enzyme localization and dynamics in cells and tissues. Activity-based probes (ABPs) are small molecule tools used in chemical proteomics and cell imaging , incorporating an electrophilic group that covalently react s with catalytic amino acid residues and a reporter tag for detection and enrichment of probe -labelled enzymes. (1, 9) The selectivity displayed by ABPs is strongly dependent on the structural motifs required for optimal molecular recognition by the target enzyme and the intrinsic reactivity of the warhead.(9) ABPs containing a fluorophore tag enable both sensitive gel-based visualization and fluorescence imaging of enzymatic activity , thus being extensively used as reagents for the detection and study enzymes in cells . However, achieving sufficient signal-to-background signal requires extensive washing steps in vitro or clearing times in vivo, in order to reduce the background generated by the intrinsic fluorescence of the probe, which prevents successful application in real-time imaging. A possible solution for this problem is offered by quenched ABPs (qABPs), in which a fluorescence quencher is incorporated as a leaving group. A requirement for this approach is the presence of a warhead with a single leaving group that can be connected to a fluorescence quencher. Synthetically, qABPs must be designed so that, upon engagement with the target enzyme and subsequent catalysis, only the quencher is released, leaving all other essential probe elements intact. A key advantage of qABPs over traditional ABPs is their fluorescence activation exclusively upon target enzyme binding, eliminating background signal from unreacted probe in solution and thereby overcoming major imaging

(Figure 1). (10-13) Although qABPs for cysteine proteases are well .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint established, the development of probes targeting serine hydrolases remains comparatively limited. Figure 1. Activity-Based probes vs quenched Activity-Based Probes in biological applications. The field of qABPs remains relatively underexplored, with only a limited number of reported applications. (14 –23) Specifically for HNE, very few qABPs have been developed to date. The first near‑infrared fluorogenic off-on probe reported for HNE, incorporating a hemicyanine fluorophore, was shown to restore fluorescence upon reaction with HNE (Figure 2A). (24) However, because the fluorophore is released into solution, this strategy is not well suited to gel ‑based assays or pull ‑down experiments. Alternatively, ABPs can incorporate a linker between the reactive group and the tag, which functions not only as a spacer but also as a key determinant of selectivity. A common approach in cysteine ‑targeted ABPP involves introducing a short amino‑acid sequence to direct a broadly reactive chemotype toward a specific protein. Using this principle, Rios et al. (25) developed “triple qABPs” containing three fluorescein moieties paired with three DABSYL quenchers via a peptide sequence , which were selectively cleaved by HNE between isoleucine and norleucine (Figure 2B). While multiple fluorophore/quencher pairs is a well ‑established strategy to reduce background and amplify signal, it substantially increases the overall molecular weight. More recently, cell-permeable qABPs based on the broadly reactive phosphinate ester warhead enabled finetuning of selectivity across different protein targets by varying the amino‑acid sequence between the fluorophore and the linker (Figures 2C ,D), underscoring the critical role of linker des ign in qABP performance. (26, 27) Nevertheless, incorporating peptide-based linkers increases both structural complexity and synthetic demands. . • Fluorescence signal only after reaction with the target enzyme. • Doesn’t need washing steps. Quenched ABP Warhead Linker Fluorophore Quencher • High background fluorescence, even without reaction. • Needs extra washing steps. Traditional ABP Warhead Linker Fluorophore .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint Figure 2.Examples of qABPs that target HNE. A. qABP described by Liu et al. The substitution in the amino xanthene moiety reduces drastically the fluorescence, after hydrolysis by HNE the amino group is release and the fluorescence turned -on. B. Peptide-based “triple qABP” design by Rios et al. the probe presents three fluorophore/quencher pairs connected by a peptide sequence that will be cleaved specific by HNE. C/D. qABPs described by Kahler et al. takes advantage of the electrophilic nature of phosphonate esters as a reactive moiety, the selectivity of the probes is determined by the sequence in the linker. .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint Monobactam moieties have been employed in the inhibition of serine hydrolases, in particular, HNE or Dipeptidyl Peptidases 8 and 9, (28) however, this scaffold has never been reported as a warhead in qABP, which opens an interesting opportunity. . In this study, we developed a synthetic strategy to generate a series of novel quenched activity- based probes (qABPs) featuring a monobactam ring as the warhead and a turn -on mechanism. This design functions as a mechanism -based inhibitor, effectively preventing reaction reversibility and enhancing probe stability. the two different types of qABPs are reported, differing in their activation mechanisms and the form in which the quencher is released. Specifically, in type I, the quencher is released as an acid or analogous moiety, while in type II, the quencher is released as a phenol. In both cases an electrophile is generated in the probe structure that remains attached to the targets enzyme, which can be attacked by a second nucleophile, resulting in an irreversible inhibition mechanism as presented in Figure 3. These probes demonstrated strong inhibition and high selectivity toward HNE, enabling its detection in complex proteomes. Figure 3. General qABP activation mechanism.

and Discussion Design and chemistry. Two different types of qABPs were designed, both with a bodipy as a fluorophore and a dinitrobenzene moiety or cAB40 as quenchers. In the case of type I probes (Figure 4), the synthetic strategy started with azetidinone 1, and through a Barbier-type reaction with propargyl bromide and zinc the alkyne intermediate 2 was obtained. The stereochemistry of the chiral center was maintained and confirmed by 1H NMR. The nitrogen of the ring was hydroxymethylated and the quencher was subsequently coupled under optimized conditions specific to each quencher moiety , resulting in compounds 7i-iii, which were obtained with good yields (>85%). The hydroxyl .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint group was deprotected using tetrabutylammonium fluoride and acetic acid , followed by the click reaction with the previously synthesized bodipy azide 9, resulting in the desired qABPs 10-12 (Figure 5). Figure 4 - Synthetic strategy used to obtain type I qABPs . Compound 1 reacts with propargyl bromide through a Barbie-type reaction to achieve the enantiopure lactam 2. Followed by a hydromethylation of the lactam ring to obtain compound 3. The quencher moiety previously synthesi zed (4-6) was coupled with 3 generating intermediates 7i-iii that was further deprotected (8i-iii) and finally a click reaction was performed to achieve the final qABPs (10-12). .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint Figure 5. Synthesized type I qABPs. All type I probes exhibit as a fluorophore BODIPY-FL, as a quencher 9 and 10 contain a dinitrobenzene moiety, with different linker length and functional group between the linker and the quencher. qABP 11 display as a quencher an antroquinone moiety. Type II qABPs (Figure 6)were designed to increase the reactivity of the warhead, by increasing the pKa of the ring opening, and placing the leaving group that contains the quencher directly attached to the four-membered ring. Like in type I, the synthesis started with azetidinone 1, followed by the substitution of the acetal group by a phenol moiety, previously synthesized. The reaction with carbamate 17 led to alkynes 18i-iii, which were deprotected, and the final probes 20-22 (Figure 7) were obtained via a click reaction with the appropriate BODIPY azide. .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint Figure 6. Synthetic strategy used to obtain type II qABPs . Compound 1 reacts with a phenol intermediate previously synthesized (13-15) that contains the quencher to obtain compounds 16i-iii. Followed by a urea formation on the lactam ring to obtain compounds 18i-iii. Compounds were deprotected (19i-iii) and finally a click reaction was performed to achieve the final qABPs (20-22). Figure 7. Synthesized type II qABPs. Type II probes exhibit as a fluorophore BODIPY-FL, and as a quencher a dinitrobenzene moiety, with different linker length and functional group between the linker and the quencher. Photophysical properties. The photochemical properties were evaluated, and the stability of the probes was analyzed in different pHs. In PBS (pH=7.4) all qABPs were .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint stable up to 48h. To simulate the attack of the catalytic serine residue, increasing concentrations of hydroxide were used as a biomimetic medium and the stability was evaluated through UV spectrophotometry. In the case of type II (Figure 9) qABPs and type I 12 (Figure 8), a shift was observed in the UV-spectra at pH=10 that suggests ring opening followed by release of the quencher moiety. In the case of 10 and 11, this shift was only observed at pH=13. The reaction was monitored by fluorescence scan. For type I qABPs, the fluorescence increase persisted for up to 12 hours, whereas for type II qABPs the reaction proceeded more rapidly, reaching a comparable increase within 2 hours, the difference between the probes tested indicates that in type II the releasing of the quencher is faster and more efficient which constitute a key feature for the desire application. Figure 8. A) UV-Vis spectrum of 10 in PBS at t=0 min. (black) and t=48h (red); B) UV-Vis spectrum of 10 in NaOH 0.1M at t= 0 min (black) and t= 2h (red). Figure 9. A) UV-Vis spectrum of 20 in PBS at t= 0 min. (black) and t= 48h (red); B) UV-Vis spectrum of 20 in pH=10 at t= 0 min (black) and t= 2h (red) To evaluate enzyme activation, porcine pancreatic elastase (PPE) was used, given its high homology with HNE. The compounds were incubated with PPE, and fluorescence was measured over time. With type II qABPs, fluorescence increased up to sixfold within 3 hours (Figure 9); however, with type I qABPs, only a fourfold increase was observed .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint after 19 hours. These results showed that, in the case of type II, the reaction to release the extinguishing fraction was significantly faster compared to type I. (Figure 10) Figure 10. Fluorescence spectrum of 21 in PBS (black) and fluorescence spectrum 3 hours after the addition of PPE (red) The fluorescence quantum yields (ΦF) were calculated for all the compounds (Table 1) using an indirect method. A solution of fluorescein in 0.1M NaOH ( ΦF=89%) was used as a reference . While the azide used i n probe synthesis had a fluorescence quantum yield of 100%, most probes synthesized in this work had a value lower than 11%, suggesting efficient quenching of the probe fluorescence. P robes 11 and 21 were the only exceptions, with a value higher than 20%, which could suggest that the amide bond to the dinitrobenzene moiety decreased quencher efficacy. After activation the fluorescence quantum yield increas ed up to 92%, validating our proposed turn -on mechanism and denoting that the com pounds can be used as qABPs to detect the targets with low background fluorescence (Table 1). Table 1. Fluorescence quantum yields before (PBS) and after activation (NaOH) qABP ΦF (PBS) ΦF (After Activation) Type I 10 4% 54% 11 26% 86% 12 9% 92% Type II 20 11% 79% 21 21% 77% 22 6% 80% After the validation with PPE, we additionally validated probe 22 using pure HNE and human neutrophil lysate . These were incubated with the probe and the generation of fluorescence was measured through time. (Figure 11) .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint Figure 11. Left: Incubation of human neutrophils lysate with 22. Right: Human neutrophil elastase with 22 and 22 in assay buffer. Fluorescence was measure d every minute for 2 hours, using as excitation wavelength 485 nm and emission wavelength 535 nm. Assays were performed in triplicate. In both cases an increase in the fluorescence signal was observed (more significant in the case of pure HNE), confirming the reaction of the probe with the target and sequential release of the quencher moiety. The fluorescence of the probe in the buffer assay was also tested and remained very low and stable during the assay. Biological evaluation. Enzymatic assays were performed with some of the probes against HNE and related enzymes. All probes demonstrated strong activity against HNE, with IC50 values below 0.5 μM. Type I qABPs 10 and 12 showed IC50 values of 0.50 and 0.12 μM, respectively. However, due to the slower quencher release and delayed fluorescence increase compared to Type II probes, a limitation for practical applications, biological evaluation was carried out exclusively with type II qABPs . Type II qABPs showed great selectivity for HNE against related serine proteases, with IC50 values higher than 10 μM for the tested panel (Table 2). Table 2. Activity of the probes against different serine proteases. IC50 (μM) 20 21 22 Chymotrypsin >10 >10 >10 Thrombin >10 >10 >10 Kallikrein >10 >10 >10 Urokinase >10 >10 >10 PPE >10 >10 >10 HNE 0.18 0.13 0.19 The selectivity of the probes to target HNE against selected related enzymes evaluated with pure enzymes is a promising feature for the use of these compounds in the detection of HNE. To get a more complete picture of the reactivity of our compounds in more complex systems, SDS-Page experiments were performed with HNE and by spiking HNE into different proteomes. When incubated with pure HNE and analyzed by gel, .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint fluorescence was only observed in the case of type II probes. (Figure 12A). Importantly, when the enzyme was pre-incubated with ONO -6818, a potent and selective HNE inhibitor, (29) the signal was not observed ( Figure 12B), indicating that the probes are competing for the active site of HNE. Figure 12. A) Incubation of the probes with HNE (left); B) Pre-incubation with ONO-6818 (right) After confirming HNE engagement by gel, human embryonic kidney cell (HEK293) and epidermal carcinoma cell (A431) lysates were spiked with HNE and incubated with type II probes. The probes selectively detected HNE in the HEK293 proteome (Figure 13A). Probe 22, was also tested with decreasing concentrations of HNE, with minimal increase in background labeling and remaining selective for HNE down to 150 nM spiking (Figure 13B). Similarly, the A431 cell lysate experiment showed that all probes of type II could selectively detect HNE (Figure 13C), with probe 22 selectively detecting HNE down to a concentration of 75 nM of spiking (Figure 13D). .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint Figure 13. A) Type II probes incubated with HEK293 lysate spiked with HNE (500 nM); B) 22 incubated with HEK293 lysate spiked with decreasing concentrations of HNE; C) Type II probes incubated with A431 lysate spiked with HNE (500 nM); D) 22 incubated with A431 lysate spiked with decreasing concentrations of HNE. Once the potential of the probes to target HNE was validated in a complex proteome, 22 was incubated with human neutrophil and U937 cell lysates (U937 is monocytic cell line that natively expresses HNE (28)). As presented in Figure 14, in both cases probe 22 was able to identify endogenous HNE in a complex proteome without requiring overexpression. .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint Figure 14. A) Probe 22 incubated with a lysate of human neutrophils, B) Probe 22 incubated with U937 cell lysate. Following these promising results, it was important to understand if the synthesized probes could be internalized into living cells. The internalization was evaluated in U937 cells and in human neutrophils and analyzed by flow cytometry and by imaging, respectively. In U937 cells, by flow cytometry, while control cells (untreated with 22) did not present detectable fluorescence, cells treated with 22 exhibited a marked increase in fluorescent signal, suggesting probe internalization and activation (Figure 14). Figure 15. Flow cytometry of living U937 cells untreated (A) and treated with 22 for 2h (B); in both cases the x-axis represents the fluorescence intensity measure , and the cell count is showed in the y -axis. C. Bars graph (Mean+SD) represents the median fluorescence intensity of the control and the cells treated with 22. In the case of human neutrophils, the internalization was evaluated by fluorescence cell imaging with Operetta CLS High -Content Analyse System , ( Revvity). Significant fluorescence signal in the cytoplasm is observed after incubation of the cells with 22, while the negative control showed no detectable fluorescence (Figure 16), further confirming successful internalization. .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint Figure 16. Incubation of 22 with human neutrophils. Images were taken after 2h of 22 addition. Overall, the synthesized probes presented remarkable selectivity and a strong potential to be used as qABPs to detect HNE in complex proteomes and could become an important tool to reveal the roles of HNE in the tumor microenvironment.

The search for tools that allow us to understand the biological importance of disease- related targets is a continuous effort in the Medicinal Chemistry field. In this work we describe a synthetic method to build qABPs that could selectively target HNE in biological media and in complex proteomes without a complex peptidyl linker. The described qABPs presented high quenching efficacy , with FQY lower than 20%, which increases up to 92% after activation. The activation was demonstrated through fluorescence assays using pure HNE and human neutrophils lysate . The designed probes showed remarkable selectivity for HNE even when compared with other similar serine hydrolases. Detection and activation in biological matrices w ere demonstrated using three different cell lines with and without spiking of HNE , including human neutrophils lysates. Overall, our study delivers a suite of chemical tools that can now be used to study the role of HNE in the tumor microenvironment and to validate HNE as an important target in cancer treatment. Experimental Section

and General Methods Synthesis (3S,4R)-3-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-4-(prop-2-yn-1-yl)azetidin-2- one (2) 3-Bromoprop-1-yne (9.2 M solution in toluene) (1.13ml, 10.44 mmol, 3 eq.), zinc powder (2.27g, 34.79 mmol, 10 eq.) and compound 1 (1g, 3.48 mmol, 1 eq.) in THF (35 ml) were added in a reaction flask under inert atmosphere and the reaction was stirred at reflux temperature for 15 min. The reaction was allowed to cold to room temperature and was filtered through celite. The solvent was evaporated, and ethyl acetate was added. Saturated ammonium chloride solution (80 ml) was added to the reaction mixture and extracted with EtOAc (3 × 80 ml). The organic layer was washed with brine, dried .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint over anhydrous Na 2SO4, filtered, and concentrated under reduced pressure. The obtained residue was purified by flash chromatography using silica -gel to yield a white solid (848 mg, 85%). Adapted from the literature. (30) 1H NMR (300 MHz, CDCl3) δ 6.03 (bs, 1H), 4.20 (qd, J = 6.3, 4.5 Hz, 1H), 3.85 (td, J = 6.2, 2.2 Hz, 1H), 2.90 (ddd, J = 4.4, 2.2, 0.9 Hz, 1H), 2.62 – 2.43 (m, 2H), 2.05 (t, J = 2.6 Hz, 1H), 1.23 (d, J = 6.2 Hz, 3H), 0.87 (s, 9H), 0.07 (s, 3H), 0.07 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 168.1, 79.7, 70.9, 65.1, 63.9, 48.8, 25.7, 24.6, 17.9, -4.3, -5.0. [α]D 24 = −54.5 (c 1.3, CHCl3) (3S,4R)-3-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-1-(hydroxymethyl)-4-(prop-2- yn-1-yl)azetidin-2-one (3) Formaldehyde (35% aqueous solution) (600 µl, 7 mmol, 3.75 eq.), compound 2 (500 mg, 1.9 mmol, 1eq.) and potassium carbonate (129 mg, 0.95 mmol, 0.5eq.) were added to a reaction flask, ethanol (20 ml) was added and the reaction was stirred under reflux for 1 h, then stirred at room temperature overnight. The reaction was filtered, and the solvent was evaporated under reduced pressure. DCM (60 ml) was added to the resulting residue, washed with water and brine (2 × 60 ml), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography using silica-gel to yield a white solid (203 mg, 91%). 1H NMR (300 MHz, CDCl3) δ 4.75 (d, J = 11.6 Hz, 1H), 4.64 (d, J = 11.6 Hz, 1H), 4.19 (qd, J = 6.2, 4.4 Hz, 1H), 3.93 (td, J = 5.5, 2.2 Hz, 1H), 2.94 (dd, J = 4.4, 2.2 Hz, 1H), 2.63 (dd, J = 5.6, 2.6 Hz, 2H), 2.08 (t, J = 2.6 Hz, 1H), 1.21 (d, J = 6.3 Hz, 3H), 0.85 (s, 9H), 0.05 (s, 3H), 0.05 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 167.7, 79.9, 71.4, 65.1, 64.2, 63.1, 52.1, 25.8, 23.1, 22.8, 18.0, -4.2, -4.9. LRMS (ESI) m/ z calcd. for C 15H28NO3Si [M+H]+: 298.5 found: 298. [α]D 24 = −69.4 (c 1.0, CHCl3) 4-nitrophenyl (2 -(2-(2-((2,4-dinitrophenyl)amino)ethoxy)ethoxy)ethyl)carbamate (4) Compound S8 (200 mg, 0.64 mmol, 1.0 eq.) and DMAP (156 mg, 1.28 mmol, 2.0 eq.) were dissolved in acetonitrile (1 ml), the solution was added dropwise to a solution of p- nitrophenyl chloroformate (142 mg, 0.7 mmol, 1.1 eq.) in acetonitrile (1 ml) and the resulting solution was stirred at 50 °C for 3 h. The reaction mixture was then diluted in DCM (10 ml) and washed with 0.5 N HCl (60 ml). The aqueous layer was washed with DCM (5 × 10 ml) and all the organic fractions were collected, dried over Na2SO4, filtered, and evaporated. The residue was purified by flash chromatography using silica -gel to yield a yellow oil (227 mg, 74%). 1H NMR (300 MHz, CDCl3) δ 9.08 (d, J = 2.6 Hz, 1H), 8.88 (bs, 1H), 8.29 – 8.16 (m, 3H), 7.28 (d, J = 9.4 Hz, 2H), 6.92 (d, J = 9.4 Hz, 1H), 5.87 (t, J = 5.3 Hz, 1H), 3.85 (t, J = 5.3 Hz, 2H), 3.76-3.70 (m, 3H), 3.74 – 3.57 (m, 5H), 3.51 (t, J = 5.4 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 156.1, 153.4, 148.4, 144.7, 136.2, 130.5, 126.2, 125.2, 124.4, 122.1, 114.1, 70.8, 70.3, 70.0, 68.3, 43.2, 41.3. .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint Sodium 1-amino-4-((4-(carboxymethyl)phenyl)amino)-9,10-dioxo-9,10- dihydroanthracene-2-sulfonate (cAB40) (6) A solution of bromaminic acid sodium salt (500 mg, 1.23 mmol, 1.12 eq .), 2 -(4- aminophenyl)acetic acid 8 (166 mg, 1.1 mmol, 1 eq.), copper (II) sulfate pentahydrate (45 mg, 0.179 mmol, 0.16 eq.) and sodium carbonate (197 mg, 1.58 mmol, 1.44 eq.) in 50 ml of water was stirred under reflux for 24 h. The reaction colour changed from red to purple/dark blue. The reaction mixture was washed with DCM (3 × 50 ml) and the aqueous layer was evaporated under reduced pressure. The resulting dark solid was dissolved in methanol, filtered and the methanol solution was evaporated in vacuo. The obtained residue was purified by reverse-phase column chromatography to yield a dark blue solid (219 mg, 30%).(31) 1H NMR (300 MHz, DMSO-d6) δ 12.14 (s, 1H), 10.16 (bs, 1H), 8.34 – 8.20 (m, 2H), 7.99 (s, 1H), 7.91 – 7.78 (m, 2H), 7.48 (bs, 1H), 7.31 (d, J = 8.3 Hz, 2H), 7.14 (d, J = 8.3 Hz, 2H), 3.27 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 182.0, 181.7, 144.2, 142.9, 141.7, 136.7, 135.9, 134.1, 133.6, 133.0, 132.7, 130.4, 126.0, 125.9, 122.9, 122.6, 110.7, 109.0, 52.6. LRMS (ESI) m/ z calcd. for C 22H15N2O7S- [M]: 451.4 found: 451.1. ((3S,4R)-3-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-2-oxo-4-(prop-2-yn-1- yl)azetidin-1-yl)methyl (2-(2-(2-((2,4- dinitrophenyl)amino)ethoxy)ethoxy)ethyl)carbamate (7i) Compound 4 (200 mg, 0.42 mmol, 1.0 eq.) and DMAP (102 mg, 0.84 mmol, 2.0 eq.) were dissolved in DCM (1 ml), the solution was added dropwise to a solution of compound 3 (137 mg, 0.46 mmol, 1.1 eq.) in DCM (1 ml) and the resulting solution was stirred for 4 h at room temperature. DCM was added (10 ml) and washed with water and brine (2 x 10 ml), dried over Na2SO4, filtered, and evaporated. The residue was purified by flash chromatography using silica- gel to yield a yellow oil (216 mg, 81%). 1H NMR (300 MHz, CDCl3) δ 9.14 (d, J = 2.7 Hz, 1H), 8.81 (s, 1H), 8.28 (dd, J = 9.5, 2.7 Hz, 1H), 6.94 (d, J = 9.5 Hz, 1H), 5.24 (s, 1H), 5.10 (d, J = 11.6 Hz, 1H), 5.03 (d, J = 11.6 Hz, 1H), 4.22 (qd, J = 6.3, 3.8 Hz, 1H), 4.05 – 3.91 (m, 1H), 3.83 (t, J = 5.2 Hz, 2H), 3.75 – 3.51 (m, 8H), 3.48 – 3.27 (m, 2H), 3.04 (dd, J = 3.8, 2.3 Hz, 1H), 2.81 – 2.58 (m, 2H), 2.01 (t, J = 2.6 Hz, 1H), 1.20 (d, J = 6.3 Hz, 3H), 0.84 (s, 9H), 0.05 (s, 3H), 0.05 (s, 3H). 13C NMR (75 MHz, CDCl 3) δ 168.2, 156.1, 148.4, 136.1, 130.5, 130.4, 124.4, 114.2, 79.0, 71.5, 70.7, 70.3, 70.1, 68.5, 64.6, 63.1, 62.6, 52.1, 43.2, 40.9, 25.8, 25.8, 21.8, 17.9, -4.2, -5.1. [α]D 24 = −46.3 (c 1.0, CHCl3) .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint ((3S,4R)-3-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-2-oxo-4-(prop-2-yn-1- yl)azetidin-1-yl)methyl (3,5 -dinitrophenyl)carbamate (7ii) A solution of 3,5 - dinitrobenzoyl azide S10 (300 mg, 1.27 mmol, 3eq.), in dry toluene (3 ml) was heated to reflux for 3 hours. The reaction was cooled to room temperature and a solution of compound 3 (126 mg, 0.43 mmol, 1 eq.) in dry ACN was added. The reaction was stirred at room temperature overnight. The solvent was evaporated, and the residue was purified by flash chromatography using silica-gel to yield a colourless oil (136 mg, 63%). 1H NMR (300 MHz, CDCl3) δ 9.56 (s, 1H), 8.85 (d, J = 2.0 Hz, 2H), 8.71 (t, J = 2.0 Hz, 1H), 5.33 (d, J = 11.7 Hz, 1H), 5.16 (d, J = 11.7 Hz, 1H), 4.35 – 4.18 (m, 1H), 4.12 (dt, J = 4.3, 2.1 Hz, 1H), 3.24 (dd, J = 4.7, 2.3 Hz, 1H), 3.04 (ddd, J = 17.7, 5.1, 2.7 Hz, 1H), 2.75 (dt, J = 17.7, 2.7 Hz, 1H), 2.07 (t, J = 2.7 Hz, 1H), 1.28 (d, J = 6.2 Hz, 3H), 0.77 (s, 9H), 0.02 (s, 3H), -0.02 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 169.7, 153.7, 149.0, 141.2, 118.3, 112.8, 78.1, 72.2, 65.2, 63.5, 62.1, 53.7, 25.7, 20.9, 17.9, -4.1, -5.1. [α]D 24 = −46.6 (c 1.0, CHCl3) 1-amino-4-((4-(2-(((3S,4R)-3-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-2-oxo-4- (prop-2-yn-1-yl)azetidin-1-yl)methoxy)-2-oxoethyl)phenyl)amino)-9,10-dioxo-9,10- dihydroanthracene-2-sulfonate (7iii) To a solution of cAB40 6 (120 mg, 0.27 mmol, 1 eq.), TBTU (193 mg, 0.8 mmol, 2.2 eq.), HOBt (44 mg, 0.33 mmol, 1.2 eq.) and DIPEA (188 µl, 1.1 mmol, 4 eq.) in dry DMF (1 ml) under inert atmosphere, compound 3 was added and the reaction was stirred for 1 h at 0 ºC and at room temperature overnight. The mixture was purified by flash reverse-phase column chromatography to yield a dark blue solid (103 mg, 65%) . The compound was obtained with trace amounts of starting material. The mixture was used in the following reaction steps without further purification. LRMS (ESI) m/z calcd. for C37H40N3O9SSiNa [M+Na]-:730.2 found: 730. ((3S,4R)-3-((R)-1-hydroxyethyl)-2-oxo-4-(prop-2-yn-1-yl)azetidin-1-yl)methyl (2 -(2- (2-((2,4-dinitrophenyl)amino)ethoxy)ethoxy)ethyl)carbamate (8i) To a solution of compound 2.5 (150 mg, 0.24 mmol, 1 eq.) in THF (1 ml), acetic acid (270 µl, 0.23 mmol, 20 eq.) was added dropwise. Then, a solution of TBAF (1M in THF) (9 ml, 9.41 mmol, 40 eq.) was also added dropwise and stirred under inert atmosphere for 24 h. The reaction was diluted in EtOAc (20 ml) and cold saturated sodium hydrogen carbonated solution (20 ml) was added. The mixture was extracted with EtOAc (3 × 10 ml) and the organic layer was washed with brine (2 × 10 ml), dried over Na 2SO4, filtered and evaporated. The residue was purified by flash chromatography using silica -gel to yie ld a yellow oil (87 mg, 71%). 1H NMR (300 MHz, CDCl3) δ 9.15 (d, J = 2.5 Hz, 1H), 8.82 (s, 1H), 8.28 (dd, J = 9.5, 2.5 Hz, 1H), 6.95 (d, J = 9.5 Hz, 1H), 5.37 (s, 1H), 5.14 (d, J = 11.6 Hz, 1H), .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint 5.03 (d, J = 11.6 Hz, 1H), 4.20 (m, 1H), 3.95 (td, J = 5.0, 2.4 Hz, 1H), 3.83 (t, J = 5.2 Hz, 2H), 3.75 – 3.53 (m, 8H), 3.38 (m, 2H), 3.09 (dd, J = 5.1, 2.4 Hz, 1H), 2.71 (dd, J = 5.0, 2.6 Hz, 2H), 2.05 (t, J = 2.6 Hz, 1H), 1.29 (d, J = 6.4 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 168.4, 156.1, 148.4, 136.0, 130.4, 130.4, 124.3, 114.2, 78.8, 71.6, 70.7, 70.2, 70.0, 68.4, 64.3, 63.2, 62.3, 52.5, 43.2, 40.9, 21.8. [α]D 24 = −34.7 (c 1.0, CHCl3) ((3S,4R)-3-((R)-1-hydroxyethyl)-2-oxo-4-(prop-2-yn-1-yl)azetidin-1-yl)methyl (3,5 - dinitrophenyl)carbamate (8ii) To a solution of compound 7ii (100 mg, 0.20 mmol, 1 eq.) in THF (0.7 ml), acetic acid (170 µl, 2.96 mmol, 15 eq.) was added dropwise. Then, a solution of TBAF (1M in THF) (5.9 ml, 5.92 mmol, 30 eq.) was also added dropwise and stirred under inert atmosphere for 48 h. The reaction was diluted in EtOAc (15 ml) and cold saturated sodium hydrogen carbonated solution (15 ml) was added. The mixture was extracted with EtOAc (3 × 8 ml) and the organic layer was washed with brine (2 × 8 ml), dried over Na2SO4, filtered and evaporated. The residue was purified by flash chromatography using silica-gel to yield a yellow oil (51 mg, 65%). 1H NMR (300 MHz, CDCl3) δ 9.14 (s, 1H), 8.67 (d, J = 1.9 Hz, 2H), 8.59 (t, J = 1.9 Hz, 1H), 5.28 (d, J = 11.7 Hz, 1H), 5.18 (d, J = 11.7 Hz, 1H), 4.22 (s, 1H), 4.13 – 3.97 (m, 1H), 3.17 (dd, J = 4.5, 2.4 Hz, 1H), 2.92 – 2.57 (m, 3H), 2.03 (t, J = 2.4 Hz, 2H), 1.25 (d, J = 6.4 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 170.3, 155.2, 146.1, 139.4, 115.2, 113.6, 79.7, 72.5, 71.5, 66.8, 55.2, 54.6, 20.5, 19.3. [α]D 24 = −47.2 (c 1.0, CHCl3) 1-amino-4-((4-(2-(((3S,4R)-3-((R)-1-hydroxyethyl)-2-oxo-4-(prop-2-yn-1-yl)azetidin- 1-yl)methoxy)-2-oxoethyl)phenyl)amino)-9,10-dioxo-9,10-dihydroanthracene-2- sulfonate tetrabutylammonium salt (8iii). To a solution of compound 7iii (22 mg, 0.03 mmol, 1 eq.) in THF (500 µl ml), acetic acid (13 µl, 0.22 mmol, 7 eq.) was added dropwise. Then, a solution of TBAF (1M in THF ) (420 µl, 14 mmol, 14 eq.) was also added dropwise and stirred under inert atmosphere for 72 h. The solvent was evaporated, and the residue purified by flash reverse-phase column chromatography to yield a blue oil (16 mg, 72%). The compound was isolated as a tetrabutylammonium salt. 1H NMR (300 MHz, Acetone -d6) δ 12.25 (s, 1H), 8.40 – 8.28 (m, 3H), 7.86 – 7.74 (m, 2H), 7.39 (d, J = 8.5 Hz, 2H), 7.29 (d, J = 8.5 Hz, 2H), 5.25 (d, J = 11.4 Hz, 1H), 5.17 (d, J = 11.4 Hz, 1H), 4.04-3.96 (m, 1H), 3.86 (td, J = 4.7, 2.5 Hz, 1H), 3.71 (s, 2H), 3.47 – 3.37 (m, 8H), 3.31 (s, 2H), 3.00 (dd, J = 6.6, 2.4 Hz, 1H), 2.75 (dd, J = 5.0, 2.7 Hz, 1H), 2.69 (dd, J = 4.3, 2.7 Hz, 1H), 2.49 (t, J = 2.7 Hz, 1H), 1.87 – 1.72 (m, 8H), 1.47-1.34 (m, 8H), 1.23 (d, J = 6.3 Hz, 3H), 0.95 (t, J = 7.3 Hz, 12H). .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-(5,5-difluoro-7,9-dimethyl-5H- 4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-3-yl)propanamide (9) Compound S5 (120 mg, 0.41 mmol, 1 eq.) and COMU (184 mg, 0.43 mmol, 1.05 eq.) were dissolved in dry DCM (3 ml). DIPEA (214 µl, 1.23 mmol, 3 eq.) was added and the reaction was stirred at room temperature for 10 min. 11 -Azido-3,6,9-trioxaundecan-1-amine (90 µl, 0.45 mmol, 1.1 eq.) was added and the reaction was stirred for 30 min until all the starting

was consumed. The solvent was evaporated, and the residue was purified by flash chromatography using silica-gel to yield an orange oil (129 mg, 64%). 1H NMR (300 MHz, Acetone-d6) δ 7.52 (d, J = 9.1 Hz, 2H), 7.06 (d, J = 4.0 Hz, 1H), 6.37 (d, J = 4.0 Hz, 1H), 6.25 (s, 1H), 3.72 – 3.56 (m, 16H), 3.23 (t, J = 7.7 Hz, 2H), 2.63 (t, J = 7.7 Hz, 2H), 2.51 (s, 3H), 2.28 (s, 3H). 13C NMR (75 MHz, Acetone -d6) δ 172.5, 159.9, 158.5, 144.6, 135.6, 133.9, 129.2, 125.7, 120.7, 117.1, 70.6, 70.4, 70.4, 70.3, 70.0, 66.6, 39.5, 34.8, 14.4, 10.8. ((2R,3S)-2-((1-(15-(5,5-difluoro-7,9-dimethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'- f][1,3,2]diazaborinin-3-yl)-13-oxo-3,6,9-trioxa-12-azapentadecyl)-1H-1,2,3-triazol-4- yl)methyl)-3-((R)-1-hydroxyethyl)-4-oxoazetidin-1-yl)methyl (2 -(2-(2-((2,4- dinitrophenyl)amino)ethoxy)ethoxy)ethyl)carbamate (10) A solution of compound 8i (25 mg, 0.05 mmol, 1 eq.), copper (II) sulfate (0.2 M aqueous solution) (115 μl, 0.02mmol, 0.48 eq.) and sodium ascorbate (0.2 M aqueous solution) (115 μl, 0.02 mmol, 0.48 eq.) in MeOH (1 ml) was stirred for 10 min. Then, Compound 9 (26 mg, 0.05 mmol, 1.1 eq.) was added and the reaction was stirred at room temperature for 2h. The solvent was evaporated, and the residue was purified by flash chromatography using silica -gel to yield an orange oil (39 mg, 81%). 1H NMR (300 MHz, Acetone-d6) δ 8.96 (d, J = 2.7 Hz, 1H), 8.87 (s, 1H), 8.27 (ddd, J = 9.6, 2.7, 0.6 Hz, 1H), 7.91 (s, 1H), 7.49 (s, 1H), 7.28 (d, J = 9.6 Hz, 2H), 7.03 (d, J = 4.0 Hz, 1H), 6.47 (t, J = 5.7 Hz, 1H), 6.38 (d, J = 4.0 Hz, 1H), 6.24 (s, 1H), 5.14 (d, J = 11.5 Hz, 1H), 5.06 (d, J = 11.5 Hz, 1H), 4.56 – 4.50 (m, 2H), 4.09 – 3.90 (m, 2H), 3.87-3.82 (m, 4H), 3.73 (t, J = 5.1 Hz, 2H), 3.69 – 3.59 (m, 4H), 3.59 – 3.47 (m, 12H), 3.41 – 3.34 (m, 3H), 3.32-3.25 (m, 3H), 3.22 (t, J = 7.6 Hz, 2H), 3.04 – 2.95 (m, 2H), 2.60 (t, J = 7.6 Hz , 2H), 2.50 (s, 3H), 2.28 (s, 3H), 1.06 (d, J = 6.3 Hz, 3H). 13C NMR (75 MHz, Acetone -d6) δ 171.9, 168.0, 164.1, 160.2, 159.3, 156.8, 149.9, 149.5, 144.8, 143.3, 136.4, 135.7, 135.2, 134.3, 131.0, 130.7, 129.5, 125.5, 124.3, 120.9, 117.7, 116.1, 71.0, 71.0, 71.0, 70.9, 70.8, 70.4, 70.0, 69.3, 65.9, 64.1, 63.8, 55.9, 50.6, 43. 9, 41. 4, 39.8, 35. 2, 14.8, 11. 2. HRMS (ESI) m/z calcd. for C 44H60BF2N11O14 [M+H]+: 1016.445 found: 1016.451. [α]D 24 = −64.8 (c 0.7, EtOH) .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint ((2R,3S)-2-((1-(15-(5,5-difluoro-7,9-dimethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'- f][1,3,2]diazaborinin-3-yl)-13-oxo-3,6,9-trioxa-12-azapentadecyl)-1H-1,2,3-triazol-4- yl)methyl)-3-((R)-1-hydroxyethyl)-4-oxoazetidin-1-yl)methyl (3,5- dinitrophenyl)carbamate (11) A solution of compound 8ii (20 mg, 0.05 mmol, 1 eq.), copper (II) sulfate (0.2 M aqueous solution) (122 µl, 0.02mmol, 0.48 eq.) and sodium ascorbate (0.2 M aqueous solution) (122 µl, 0.02 mmol, 0.48 eq.) in MeOH (1 ml) was stirred for 10 min. Then, Compound 9 (27 mg, 0.06 mmol, 1.1 eq.) was added and the reaction was stirred at room temperature for 2h. The solvent was evaporated, and the residue was purified by flash chromatography using silica -gel to yield an orange oil (34 mg, 75%). 1H NMR (300 MHz, Acetone-d6) δ 9.95 (s, 1H), 8.83 (d, J = 2.0 Hz, 2H), 8.51 (t, J = 2.0 Hz, 1H), 7.97 (s, 1H), 7.44 (s, 1H), 7.42 (t, J = 5.5 Hz, 1H), 6.99 (d, J = 4.0 Hz, 1H), 6.35 (d, J = 4.0 Hz, 1H), 6.22 (s, 1H), 5.37 (d, J = 11.5 Hz, 1H), 5.31 (d, J = 11.5 Hz, 1H), 4.59 – 4.47 (m, 2H), 4.18 (ddd, J = 7.2, 4.4, 2.4 Hz, 1H), 4.09-3.97 (m, 1H), 3.86 (t, J = 5.1 Hz, 2H), 3.59-3.49 (m, 11H), 3.46 – 3.34 (m, 2H), 3.20 (t, J = 7.6 Hz, 2H), 3.10 (dd, J = 15.1, 7.8 Hz, 1H), 3.05 – 3.00 (m, 1H), 2.66 – 2.57 (m, 3H), 2.46 (s, 3H), 2.26 (s, 3H), 1.07 (d, J = 6.3 Hz, 3H). 13C NMR (75 MHz, Acetone-d6) δ 172.4, 168.3, 160.2, 159.1, 153.9, 149.5, 144.8, 143.3, 142.5, 135.6, 134.7, 129.5, 125.4, 124.4, 120.9, 118.5, 117.6, 112.5, 71.0, 71.0, 70.9, 70.8, 70.3, 70.0, 65.6, 65.2, 64.3, 55.9, 55.4, 50.7, 39.9, 35.1, 14.8, 11.1. HRMS (ESI) m/z calcd. for C 38H49BF2N10O12 [M-H]-: 885.359 found: 885.355. [α]D 24 = −87.6 (c 0.5, EtOH) 1-amino-4-((4-(2-(((2R,3S)-2-((1-(15-(5,5-difluoro-7,9-dimethyl-5H-4l4,5l4- dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-3-yl)-13-oxo-3,6,9-trioxa-12- azapentadecyl)-1H-1,2,3-triazol-4-yl)methyl)-3-((R)-1-hydroxyethyl)-4-oxoazetidin- 1-yl)methoxy)-2-oxoethyl)phenyl)amino)-9,10-dioxo-9,10-dihydroanthracene-2- sulfonate tetrabutylammonium salt (12) A solution of compound 8iii (14 mg, 16 µmol, 1 eq.), copper (II) sulfate (0.2 M aqueous solution) (39 µl, 8 µmol, 0.48 eq.) and sodium ascorbate (0.2 M aqueous solution) (39 µl, 8 µmol, 0.48 eq.) in MeOH (300 µl) was stirred for 10 min. Then, Compound 9 (9 mg, 18 µmol, 1.1 eq.) was added and the reaction was stirred at room temperature for 2h. The solvent was evaporated, and the residue purified by flash reverse-phase column chromatography to yield a blue oil (15 mg, 83%). 1H NMR (300 MHz, Acetone-d6) δ 12.08 (s, 1H), 8.25 – 8.14 (m, 3H), 7.83 (s, 1H), 7.71 – 7.62 (m, 2H), 7.34 (s, 1H), 7.23 (d, J = 8.5 Hz, 3H), 7.14 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 4.1 Hz, 1H), 6.23 (d, J = 3.9 Hz, 1H), 6.07 (s, 1H), 5.13 (d, J = 11.3 Hz, 1H), 5.07 (d, J = 11.3 Hz, 1H), 4.52 (s, 1H), 4.43 – 4.33 (m, 2H), 3.90 – 3.78 (m, 2H), 3.72 – 3.65 (m, 2H), 3.55 (s, 2H), 3.46 – 3.33 (m, 11H), 3.33 – 3.16 (m, 10H), 3.13 – 3.00 (m, 3H), 2.89 – 2.77 (m, .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint 2H), 2.50 – 2.42 (m, 3H), 2.35 (s, 3H), 2.13 (s, 3H), 1.75 – 1.59 (m, 8H), 1.36-1.20 (m, 8H), 0.94 (d, J = 6.3 Hz, 3H), 0.82 (t, J = 7.3 Hz, 12H). 1-(2-(2-(2-((2,4-dinitrophenyl)amino)ethoxy)ethoxy)ethyl)-3-(4- hydroxyphenethyl)urea (13) Compound 4 (350 mg, 0. 73 mmol, 1.0 eq.) and DMAP (179 mg, 1.46 mmol, 2.0 eq.) were dissolved in DCM ( 2 ml), the solution was added dropwise to a solution of tyramine (110 mg, 0.80 mmol, 1.1 eq.) in DCM ( 2 ml) and the resulting solution was stirred for 3 h at room temperature. DCM was added (20 ml) and washed with water and brine (2 x 20 ml), dried over Na 2SO4, filtered, and evaporated. The residue was purified by flash chromatography using silica -gel to yield a yellow oil (220 mg, 63%). 1H NMR (400 MHz, CDCl3) δ 9.10 (d, J = 2.7 Hz, 1H), 8.97 (s, 1H), 8.30 (dd, J = 9.5, 2.7 Hz, 1H), 7.04 (d, J = 8.1 Hz, 2H), 6.92 (d, J = 9.5 Hz, 1H), 6.74 (d, J = 8.1 Hz, 2H), 6.21 (s, 1H), 5.24 – 5.15 (m, 1H), 5.08 (s, 1H), 3.83 (t, J = 5.2 Hz, 3H), 3.73 (q, J = 3.5 Hz, 2H), 3.67 (q, J = 3.4 Hz, 2H), 3.64 – 3.53 (m, 4H), 3.53 – 3.40 (m, 5H), 2.75 (t, J = 7.0 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 158.6, 154.6, 148.4, 130.7, 129.9, 124.5, 115.3, 114.1, 71.2, 70.9, 70.2, 67.8, 43.0, 41.8, 40.3, 35.5. 1-(3,5-dinitrophenyl)-3-(4-hydroxyphenethyl)urea (14) A solution of 3,5 - dinitrobenzoyl azide S10 (850 mg, 3.59 mmol, 2.5 eq.), in dry toluene (8 ml) was heated to reflux for 3 hours. The reaction was cooled to room temperature and a solution of tyramine (200 mg, 1.45 mmol, 1 eq.) in dry ACN (2 ml) was added. The reaction was stirred at room temperature overnight. The solvent was evaporated, and the residue was purified by flash chromatography using silica-gel to yield a colourless oil (233 mg, 46%). 1H NMR (300 MHz, DMSO-d6) δ 9.55 (s, 1H), 9.16 (s, 1H), 8.69 (d, J = 2.1 Hz, 2H), 8.33 (t, J = 2.1 Hz, 1H), 7.08 – 6.99 (m, 2H), 6.76 – 6.63 (m, 2H), 6.52 (t, J = 5.7 Hz, 1H), 3.33-3.27 (m, 2H), 2.66 (t, J = 7.3 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ 157.2, 154.9, 146.7, 138.7, 132.3, 126.5, 117.1, 116.2, 111.3, 40.9, 34.7. 4-(2-((2,4-dinitrophenyl)amino)ethyl)phenol ( 15) A solution of dinitrofluorobenzene (1,36 ml, 10.8 mmol, 1 eq.) in 40 ml of DCM was added dropwise to a stirring solution of tyramine (1.77 g, 12.9 mmol, 1.2 eq.) in 80 ml of DCM at 0 °C. After the addition was complete, the flask was brought to room temperature and the mixture was stirred for 2 hours. Then, the solvent was evaporated , and the compound was crystalised with hot ethanol to give an orange solid (2.77 g, 85% yield). 1H NMR (300 MHz, DMSO-d6) δ 9.24 (s, 1H), 8.84 (d, J = 2.8 Hz, 1H), 8.80 (t, J = 5.9 Hz, 1H), 8.23 (dd, J = 9.7, 2.8 Hz, 1H), 7.25 (d, J = 9.7 Hz, 1H), 7.17 – 6.99 (m, 2H), 6.75 – 6.53 (m, 2H), 3.74 – 3.56 (m, 2H), .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint 2.83 (t, J = 7.3 Hz, 2H).13C NMR (75 MHz, DMSO-d6) δ 156.4, 148.5, 135.2, 130.4, 130.2, 130.0, 128.7, 124.1, 115.9, 115.7, 45.0, 33.6. 1-(4-(((2R,3R)-3-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-4-oxoazetidin-2- yl)oxy)phenethyl)-3-(2-(2-(2-((2,4-dinitrophenyl)amino)ethoxy)ethoxy)ethyl)urea (16i) To a solution of compound 13 (350 mg, 0.73 mmol, 1 eq.) in dry acetone (1.5 ml) a solution of 1M NaOH (880 μl, 0.88 mmol, 1.2 eq.) was added and the reaction was stirred for 15 minutes. A solution of compound 1 (274 mg, 0.95 mmol, 1.3 eq.) in dry acetone (1 ml) was added to the reaction and the reaction was stirred at room temperature for 20 minutes. The solvent was evaporated and water ( 60 ml) was added. The mixture was extracted with EtOAc (3 × 60 ml) and the organic layer was washed with brine (2 × 60 ml), dried over Na 2SO4, filtered and evaporated. The residue was purified by flash chromatography using silica-gel to yield an orange oil (449 mg, 87%). 1H NMR (300 MHz, CDCl3) δ 9.08 (d, J = 2.7 Hz, 1H), 8.94 (s, 1H), 8.29 (dd, J = 9.6, 2.7 Hz, 1H), 7.21 – 7.10 (m, 2H), 6.92 (d, J = 9.6 Hz, 1H), 6.85 – 6.77 (m, 2H), 6.52 (s, 1H), 5.63 (d, J = 1.2 Hz, 1H), 5.21 – 4.92 (m, 2H), 4.24 (qd, J = 6.3, 3.5 Hz, 1H), 3.82 (t, J = 5.2 Hz, 2H), 3.75 – 3.68 (m, 2H), 3.68 – 3.53 (m, 6H), 3.51 – 3.29 (m, 4H), 3.23 (dd, J = 3.5, 1.2 Hz, 1H), 2.79 (t, J = 7.1 Hz, 2H), 1.25 (d, J = 6.3 Hz, 3H), 0.87 (s, 9H), 0.08 (s, 3H), 0.06 (s, 3H). 13C NMR (75 MHz, CDCl 3) δ 167.7, 162.3, 158.5, 154.7, 148.4, 133.8, 130.7, 130.3, 130.2, 124.4, 116.2, 116.1, 114.2, 71.2, 70.8, 70.1, 67.8, 65.9, 65.4, 64.1, 63.7, 43.0, 41.6, 40.2, 35.6, 25.8, 25.7, 22.5, 21.5, -3.6, -4.3, -5.1. [α]D 24 = −20.8 (c 0.9, CDCl3) 1-(4-(((2R,3R)-3-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-4-oxoazetidin-2- yl)oxy)phenethyl)-3-(3,5-dinitrophenyl)urea (16ii) To a solution of compound 14 (200 mg, 0.58 mmol, 1 eq.) in dry acetone (1 ml) a solution of 1M NaOH (696 μl, 0.70 mmol, 1.2 eq.) was added and the reaction was stirred for 15 minutes. A solution of compound 1 (216 mg, 0.75 mmol, 1.3 eq.) in dry acetone (0.5 ml) was added to the reaction and the reaction was stirred at room temperature for 20 minutes. The solvent was evaporated and water (30 ml) was added. The mixture was extracted with EtOAc (3 × 30 ml) and the organic layer was washed with brine (2 × 30 ml), dried over Na 2SO4, filtered and evaporated. The residue was purified by flash chromatography using silica-gel to yield a yellow oil (258 mg, 78%). 1H NMR (300 MHz, CDCl3) δ 8.53 – 8.45 (m, 3H), 8.43 (t, J = 2.0 Hz, 1H), 7.34 (s, 1H), 7.09 (d, J = 8.6 Hz, 2H), 6.77 (d, J = 8.6 Hz, 2H), 5.78 – 5.69 (m, 1H), 5.55 (d, J = 1.2 Hz, 1H), 4.21 – 4.10 (m, 1H), 3.57 – 3.31 (m, 2H), 3.12 (dd, J = 3.6, 1.2 Hz, 1H), 2.80 – 2.69 (m, 2H), 1.27 – 1.14 (m, 3H), 0.78 (s, 9H), -0.00 (s, 3H), - 0.04 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 163.3, 157.9, 155.2, 148.7, 136.1, 131.5, 130.9, .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint 116.6, 115.5, 113.1, 78.2, 67.1, 59.1, 41.6, 33.6, 26.9, 22.7, 14.2, 4.3. [α]D 24 = −41.5 (c 1.0, CDCl3) (3R,4R)-3-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-4-(4-(2-((2,4- dinitrophenyl)amino)ethyl)phenoxy)azetidin-2-one (16iii) To a solution of compound 15 (400 mg, 1.32 mmol, 1 eq.) in dry acetone ( 2 ml) a solution of 1M NaOH (1.58 ml, 1.58 mmol, 1.2 eq.) was added and the reaction was stirred for 15 minutes. A solution of compound 1 (493 mg, 1.71 mmol, 1.3 eq.) in dry acetone ( 1.5 ml) was added to the reaction and the reaction was stirred at room temperature for 20 minutes. The solvent was evaporated and water (60 ml) was added. The mixture was extracted with EtOAc (3 × 60 ml) and the organic layer was washed with brine (2 × 60 ml), dried over Na 2SO4, filtered and evaporated. The residue was purified by flash chromatography using silica- gel to yield an orange solid (644 mg, 92%). 1H NMR (300 MHz, CDCl3) δ 8.53 – 8.45 (m, 3H), 8.43 (t, J = 2.0 Hz, 1H), 7.34 (s, 1H), 7.09 (d, J = 8.6 Hz, 2H), 6.77 (d, J = 8.6 Hz, 2H), 5.78 – 5.69 (m, 1H), 5.55 (d, J = 1.2 Hz, 1H), 4.21 – 4.10 (m, 1H), 3.57 – 3.31 (m, 2H), 3.12 (dd, J = 3.6, 1.2 Hz, 1H), 2.80 – 2.69 (m, 2H), 1.27 – 1.14 (m, 3H), 0.78 (s, 9H), -0.00 (s, 3H), -0.04 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 166.3, 155.6, 148.3, 131.6, 130.5, 130.3, 129.7, 124.5, 117.0, 114.0, 78.9, 66.2, 64.3, 45.1, 33.5, 25.3, 21.7, 18.1, - 4.8. [α]D 24 = −19.1 (c 1.1, CDCl3) 4-nitrophenyl prop -2-yn-1-ylcarbamate (17) To a solution of p-nitrophenyl chloroformate (1.5 g, 7.5 mmol, 1 eq.) in dry THF (40 ml) at -55°C was slowly added propargylamine (500 mg. 9 mmol, 1.2 eq.), the reaction was stirred at -55°C for 45 min. The reaction was filtered through celite and washed with THF (3x20 ml). The solvent was evaporated and the crude dissolved in Hex/EtOAc 2:1 kept at room temperature for 1h, and after stored at -10°C to crystalize compound 17. The compound was filtered to yield a white solid (1.23 g, 73%). 1H NMR (300 MHz, CDCl3) δ 8.25 (d, J = 9.1 Hz, 2H), 7.33 (d, J = 9.2 Hz, 2H), 5.38 (s, 1H), 4.09 (dd, J = 5.6, 2.5 Hz, 2H), 2.33 (t, J = 2.5 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 155.7, 152.9, 145.0, 125.3, 122.1, 78.2, 72.6, 31.2. (2R,3R)-3-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-2-(4-(13-((2,4- dinitrophenyl)amino)-4-oxo-8,11-dioxa-3,5-diazatridecyl)phenoxy)-4-oxo-N-(prop- 2-yn-1-yl)azetidine-1-carboxamide (18i) To a solution of compound 17 (360 mg, 1.63 mmol, 1 eq.) in dry DCM ( 3 ml), was added DMAP (400 mg, 3.27 mmol, 2 eq.) and the mixture was stirred for 30 minutes. A solution of c ompound 16i (1.15 g, 1.63 mmol, 1 eq.) and triethylamine (228 μl, 1.63 mmol, 1 eq.) was slowly added and the reaction was stirred at room temperature overnight. The solven t was evaporated and the crude was .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint purified by flash chromatography using silica-gel to yield a yellow oil (424 mg, 33%). 1H NMR (300 MHz, CDCl3) δ 9.03 (d, J = 2.7 Hz, 1H), 8.87 (s, 1H), 8.22 (ddd, J = 9.5, 2.7, 0.7 Hz, 1H), 7.13 – 7.04 (m, 4H), 6.87 (d, J = 9.5 Hz, 1H), 6.71 (t, J = 5.6 Hz, 1H), 5.91 (d, J = 1.7 Hz, 1H), 5.09 (s, 1H), 5.00 (s, 1H), 4.22 (qd, J = 6.2, 1.7 Hz, 1H), 4.10 – 3.87 (m, 2H), 3.76 (t, J = 5.2 Hz, 2H), 3.70 – 3.47 (m, 8H), 3.47 – 3.31 (m, 4H), 3.26 (dd, J = 3.0, 1.5 Hz, 1H), 2.73 (t, J = 7.0 Hz, 2H), 2.18 (t, J = 2.5 Hz, 1H), 1.18 (d, J = 6.2 Hz, 3H), 0.76 (s, 9H), -0.00 (s, 3H), -0.04 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 162.3, 158.3, 133.9, 130.3, 130.3, 124.5, 122.1, 116.3, 114.2, 78.7, 71.3, 70.9, 70.2, 67.7, 65.9, 64.4, 64.1, 43.0, 41.6, 40.2, 35.7, 29.7, 25.8, 22.5, 19.8, 1.7, -4.3, -12.6. [α]D 24 = −42.3 (c 1.2, CDCl3) (2R,3R)-3-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-2-(4-(2-(3-(3,5- dinitrophenyl)ureido)ethyl)phenoxy)-4-oxo-N-(prop-2-yn-1-yl)azetidine-1- carboxamide (18ii) To a solution of compound 17 (51 mg, 0.23 mmol, 1 eq.) in dry DCM (0.5 ml) DMAP was added (65 mg, 0.50 mmol, 2 eq.) and the mixture was stirred for 30 minutes. A solution of compound 16ii (160 mg, 0,23 mmol, 1 eq.) and triethylamine (32 μl, 0,23 mmol, 1 eq.) was slowly added and the reaction was stirred at room temperature overnight. The solvent was evaporated and the crude was purified by flash chromatography using silica-gel to yield a yellow solid (53 mg, 29%). 1H NMR (300 MHz, CDCl3) δ 8.50 (t, J = 2.0 Hz, 2H), 7.15 – 7.04 (m, 3H), 6.96 (s, 1H), 6.92 – 6.82 (m, 1H), 6.82 – 6.70 (m, 2H), 5.56 (d, J = 1.2 Hz, 1H), 4.15 (dd, J = 6.3, 3.4 Hz, 1H), 3.62 – 3.34 (m, 2H), 3.13 (dd, J = 3.4, 1.2 Hz, 1H), 2.93 – 2.59 (m, 3H), 2.02 – 1.94 (m, 1H), 1.73 (s, 2H), 1.20 (t, J = 7.2 Hz, 3H), 0.78 (s, 9H), -0.00 (s, 3H), -0.04 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 168.3, 152.9, 155.3, 152.7, 146.7, 135.8, 131.6, 129.9, 120.7, 115.5, 113.1, 79.9, 78.7, 71.3, 69.4, 56.0, 41.4, 34.6, 30.5, 0.7, -5.3, -7.3. [α]D 24 = −31.6 (c 0.9, CDCl3) (2R,3R)-3-((R)-1-((tert-butyldimethylsilyl)oxy)ethyl)-2-(4-(2-((2,4- dinitrophenyl)amino)ethyl)phenoxy)-4-oxo-N-(prop-2-yn-1-yl)azetidine-1- carboxamide (18iii) To a solution of compound 17 (500 mg, 0.27 mmol, 1 eq.) in dry DCM (5 ml) DMAP was added (554 mg, 4.54 mmol, 2 eq.) and the mixture was stirred for 30 minutes. A solution of compound 16iii (1.21 g, 2.27 mmol, 1 eq.) and triethylamine (317 μl, 2.27 mmol, 1 eq.) was slowly added and the reaction was stirred at room temperature 48h. The solvent was evaporated and the crude was purified by flash chromatography using silica -gel to yield a yellow solid (635 mg, 45%). 1H NMR (300 .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint MHz, CDCl3) δ 9.10 (d, J = 2.7 Hz, 1H), 8.56 (s, 1H), 8.23 (dd, J = 9.5, 2.7 Hz, 1H), 7.30 – 7.08 (m, 4H), 6.86 (d, J = 9.5 Hz, 1H), 6.74 (t, J = 5.6 Hz, 1H), 6.01 (d, J = 1.5 Hz, 1H), 4.27 (qd, J = 6.3, 3.0 Hz, 1H), 4.07 – 3.93 (m, 2H), 3.68 – 3.54 (m, 2H), 3.34 (dd, J = 3.0, 1.5 Hz, 1H), 3.00 (t, J = 7.0 Hz, 2H), 2.21 (t, J = 2.5 Hz, 1H), 1.23 (d, J = 6.3 Hz, 3H), 0.79 (s, 9H), 0.04 (s, 3H), 0.00 (s, 3H). 13C NMR (75 MHz, Acetone-d6) δ 167.7, 156.9, 150.2, 149.5, 136.6, 134.5, 131.1, 130.9, 130.1, 129.5, 124.6, 119.9, 116.0, 83.2, 81.1, 72.7, 66.4, 65.2, 62.9, 45.7, 40.0, 38.3, 34.9, 26.3, 22.7, -2.3, -6.7. [α]D 24 = −36.1 (c 1.1, CDCl3) (2R,3R)-2-(4-(13-((2,4-dinitrophenyl)amino)-4-oxo-8,11-dioxa-3,5- diazatridecyl)phenoxy)-3-((R)-1-hydroxyethyl)-4-oxo-N-(prop-2-yn-1-yl)azetidine- 1-carboxamide (19i) To a solution of 18i (60 mg, 0.08 mmol, 1 eq.) in ACN (3 ml), was added a solution of HCl 1M (230 μl, 0.228 mmol, 3 eq.), and the reaction was stirred at room temperature for 6h. The solvent was evaporated and the crude was purified by flash chromatography using silica-gel to yield a yellow oil (39 mg, 73%). 1H NMR (300 MHz, CDCl3) δ 9.07 (d, J = 2.7 Hz, 1H), 8.89 (s, 1H), 8.26 (dd, J = 9.5, 2.7 Hz, 1H), 7.19 – 7.05 (m, 4H), 6.91 (d, J = 9.6 Hz, 1H), 6.74 (t, J = 5.6 Hz, 1H), 5.96 (d, J = 1.6 Hz, 1H), 5.14 (s, 1H), 5.03 (s, 1H), 4.28 – 4.17 (m, 1H), 4.05 (dd, J = 5.6, 2.5 Hz, 2H), 3.80 (t, J = 5.2 Hz, 2H), 3.74 – 3.46 (m, 8H), 3.46 – 3.26 (m, 5H), 2.74 (t, J = 6.9 Hz, 2H), 2.25 (t, J = 2.5 Hz, 1H), 1.31 (d, J = 6.4 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 167.0, 158.6, 155.3, 149.4, 148.5, 136.4, 134.8, 130.7, 130.2, 130.1, 124.5, 118.4, 114.3, 82.7, 79.1, 72.1, 71.2, 70.9, 70.3, 68.0, 65.5, 63.9, 54.0, 43.2, 41.7, 40.4, 29.5. [α]D 24 = −40.8 (c 1.3, CDCl3) (2R,3R)-2-(4-(2-(3-(3,5-dinitrophenyl)ureido)ethyl)phenoxy)-3-((R)-1- hydroxyethyl)-4-oxo-N-(prop-2-yn-1-yl)azetidine-1-carboxamide (19ii) To a solution of 18ii (36 mg, 0.06 mmol, 1 eq.) in ACN (3 ml), was added a solution of HCl 1M (83 μl, 0.08 mmol, 1.5 eq.), and the reaction was stirred at room temperature for 3h. The solvent was evaporated and the crude was purified by flash chromatography using silica-gel to yield an orange oil (26 mg, 81%). 1H NMR (300 MHz, CDCl3) δ 9.16 (s, 1H), 9.08 – 8.97 (m, 2H), 7.72 (d, J = 1.2 Hz, 1H), 7.58 – 7.40 (m, 4H), 6.52 (d, J = 1.5 Hz, 1H), 6.09 (s, 1H), 5.73 (t, J = 5.6 Hz, 1H), 4.77 (s, 1H), 4.54 – 4.43 (m, 3H), 4.32 – 4.20 (m, 2H), 4.01 (dd, J = 13.1, 6.3 Hz, 1H), 3.92 – 3.78 (m, 2H), 2.76 (t, J = 1.8 Hz, 1H), 1.81 (d, J = 6.2 Hz, 3H). 13C NMR (75 MHz, CDCl 3) δ 169.7, 154.9, 155.9, 152.3, 147.7, 138.8, 132.1, 129.7, 117.7, 115.5, 111.6, 75.3, 73.2, 72.3, 64.3, 55.8, 41.9, 34.3, 30.1, 20.9. [α]D 24 = −46.3 (c 1.0, CDCl3) .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint (2R,3R)-2-(4-(2-((2,4-dinitrophenyl)amino)ethyl)phenoxy)-3-((R)-1-hydroxyethyl)-4- oxo-N-(prop-2-yn-1-yl)azetidine-1-carboxamide (19iii) To a solution of 18ii (20 mg, 0.03 mmol, 1 eq.) in ACN (1.5 ml), was added a solution of HCl 1M (48 μl, 0.05 mmol, 1.5 eq.), and the reaction was stirred at room temperature for 3h. The solvent was evaporated and the crude was purified by flash chromatography using silica-gel to yield an orange oil (14 mg, 85%). 1H NMR (300 MHz, CDCl3) δ 9.11 (d, J = 2.7 Hz, 1H), 8.56 (s, 1H), 8.25 (ddd, J = 9.6, 2.7, 0.7 Hz, 1H), 7.32 – 7.13 (m, 4H), 6.87 (d, J = 9.6 Hz, 1H), 6.73 (s, 1H), 6.03 (d, J = 1.6 Hz, 1H), 4.35 – 4.23 (m, 1H), 4.13 – 3.98 (m, 2H), 3.64 (td, J = 7.0, 5.2 Hz, 2H), 3.39 (dd, J = 5.1, 1.6 Hz, 1H), 3.03 (t, J = 6.9 Hz, 2H), 2.26 (t, J = 2.6 Hz, 1H), 1.91 (d, J = 4.7 Hz, 1H), 1.34 (d, J = 6.4 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 166.8, 162.4, 156.8, 156.0, 149.4, 148.3, 133.6, 132.4, 130.5, 130.0, 124.4, 118.8, 114.0, 82.2, 72.2, 65.4, 63.8, 45.0, 34.4, 33.4, 29.6, 22.0. HRMS (ESI) m/z calcd. for C45H54BF2N11O12 [M+H]+: 990.409 found: 990.412. [α]D 24 = −35.6 (c 1.1, CDCl3) (2R,3R)-N-((1-(15-(5,5-difluoro-7,9-dimethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'- f][1,3,2]diazaborinin-3-yl)-13-oxo-3,6,9-trioxa-12-azapentadecyl)-1H-1,2,3-triazol-4- yl)methyl)-2-(4-(13-((2,4-dinitrophenyl)amino)-4-oxo-8,11-dioxa-3,5- diazatridecyl)phenoxy)-3-((R)-1-hydroxyethyl)-4-oxoazetidine-1-carboxamide (20) A solution of compound 19i (29 mg, 0.04 mmol, 1 eq.), copper (II) sulfate (0.2 M aqueous solution) (96 μl, 0.02mmol, 0.48 eq.) and sodium ascorbate (0.2 M aqueous solution) (96 μl, 0.02 mmol, 0.48 eq.) in MeOH (1 ml) was stirred for 10 min. Then, was added compound 9 (30 mg, 0.0 6 mmol, 1. 5 eq.) and the reaction was stirred at room temperature for 2h. The solvent was evaporated, and the residue was purified by flash chromatography using silica-gel to yield an orange oil (31 mg, 64%). 1H NMR (300 MHz, CDCl3) δ 9.02 (d, J = 2.7 Hz, 1H), 8.83 (t, J = 4.9 Hz, 1H), 8.19 (dd, J = 9.6, 2.7 Hz, 1H), 7.71 (s, 1H), 7.15 – 6.99 (m, 4H), 6.89 (d, J = 9.6 Hz, 1H), 6.83 (d, J = 4.0 Hz, 1H), 6.58 (t, J = 5.4 Hz, 1H), 6.23 (d, J = 4.0 Hz, 1H), 6.08 (s, 1H), 5.99 (d, J = 1.5 Hz, 1H), 5.37 (t, J = 5.6 Hz, 1H), 5.20 (t, J = 5.8 Hz, 1H), 4.59 – 4.35 (m, 4H), 4.18 (p, J = 6.2 Hz, 1H), 3.86 – 3.72 (m, 4H), 3.72 – 3.25 (m, 27H), 3.19 (t, J = 7.6 Hz, 2H), 2.71 (t, J = 6.7 Hz, 2H), 2.61 (s, 1H), 2.51 (d, J = 8.1 Hz, 5H), 2.21 (s, 3H), 1.25 (d, J = 7.1 Hz, 3H). [α]D 24 = −35.6 (c 1.1, CDCl3). 13C NMR (101 MHz, Acetone -d6) δ 172.4, 167.5, 160.3, 159.2, 156.3, 150.5, 149.6, 145.1, 144.9, 135.5, 130.7, 130.6, 129.6, 125.6, 124.4, 124.1, 121.0, 119.0, 117.7, 116.1, 83.3, 71.5, 71.1, 71.0, 71.0, 71.0, 70.9, 70.5, 70.1, 69.3, 66.2, 64.1, 55.5, 50.7, 43.9, 43.6, 42.3, 40.7, 40.0, 3 6.5, 35.9, 35.2, 25.1, 22.3, 14.9, 11.2. HRMS (ESI) m/z calcd. for C52H68BF2N13O15 [M+H]+: 1164.509 found: 1164.513. [α]D 24 = −27.5 (c 1.1, CDCl3) .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint (2R,3R)-N-((1-(15-(5,5-difluoro-7,9-dimethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'- f][1,3,2]diazaborinin-3-yl)-13-oxo-3,6,9-trioxa-12-azapentadecyl)-1H-1,2,3-triazol-4- yl)methyl)-2-(4-(2-(3-(3,5-dinitrophenyl)ureido)ethyl)phenoxy)-3-((R)-1- hydroxyethyl)-4-oxoazetidine-1-carboxamide (21) A solution of compound 19ii (24 mg, 0.04 mmol, 1 eq.), copper (II) sulfate (0.2 M aqueous solution) ( 105 μl, 0.02mmol, 0.48 eq.) and sodium ascorbate (0.2 M aqueous solution) (105 μl, 0.02 mmol, 0.48 eq.) in MeOH (1 ml) was stirred for 10 min. Then, was added compound 9 (30 mg, 0.06 mmol, 1.5 eq.) and the reaction was stirred at room temperature for 2h. The solvent was evaporated, and the residue was purified by flash chromatography using silica -gel to yield an orange oil ( 41 mg, 91%). 1H NMR (300 MHz, Acetone-d6) δ 9.17 (s, 1H), 8.63 (d, J = 2.1 Hz, 2H), 8.25 (t, J = 2.1 Hz, 1H), 7.79 (s, 1H), 7.30 (s, 1H), 7.26 (d, J = 7.1 Hz, 1H), 7.18 – 7.05 (m, 5H), 6.86 (d, J = 4.1 Hz, 1H), 6.25 – 6.18 (m, 1H), 6.16 (s, 1H), 6.08 (s, 1H), 5.95 (d, J = 1.5 Hz, 1H), 4.49 – 4.31 (m, 4H), 4.15 – 4.00 (m, 1H), 3.73 (dd, J = 5.6, 4.6 Hz, 2H), 3.52 – 3.29 (m, 14H), 3.29 – 3.16 (m, 3H), 3.12 – 2.97 (m, 2H), 2.50 – 2.40 (m, 3H), 2.34 (s, 3H), 2.13 (s, 3H), 1.21 – 1.11 (m, 3H). 13C NMR (101 MHz, Acetone-d6) δ 171.8, 166.6, 155.7, 154.7, 149.7, 148.7, 144.3, 143.5, 134.1, 129.8, 129.0, 128.7, 124.7, 123.3, 120.1, 118.1, 116.9, 116.9, 109.7, 82.4, 77.9, 70.2, 70.0, 69.5, 69.2, 65.5, 64.1, 63.3, 49.8, 41.0, 39.2, 35.0, 34.3, 24.3, 21.4, 16.9, 13.9, 12.9, 10.3. HRMS (ESI) m/z calcd. for C 46H55BF2N12O13 [M+H]+: 1033.415 found: 1033.419 . [α]D 24 = −64.1 (c 1.2, CDCl3) (2R,3R)-N-((1-(15-(5,5-difluoro-7,9-dimethyl-5H-4λ4,5λ4-dipyrrolo[1,2-c:2',1'- f][1,3,2]diazaborinin-3-yl)-13-oxo-3,6,9-trioxa-12-azapentadecyl)-1H-1,2,3-triazol-4- yl)methyl)-2-(4-(2-((2,4-dinitrophenyl)amino)ethyl)phenoxy)-3-((R)-1- hydroxyethyl)-4-oxoazetidine-1-carboxamide (22) A solution of compound 19iii (18 mg, 0.04 mmol, 1 eq.), copper (II) sulfate (0.2 M aqueous solution) ( 90 μl, 0.02mmol, 0.48 eq.) and sodium ascorbate (0.2 M aqueous solution) (90 μl, 0.02 mmol, 0.48 eq.) in MeOH (1 ml) was stirred for 10 min. Then, was added compound 9 (15 mg, 0.03 mmol, 0.9 eq.) and the reaction was stirred at room temperature for 2h. The solvent was evaporated, and the residue was purified by flash chromatography using silica -gel to yield an orange oil (30 mg, 83%). 1H NMR (300 MHz, CDCl3) δ 9.08 (d, J = 2.6 Hz, 1H), 8.56 (d, J = 5.5 Hz, 1H), 8.21 (dd, J = 9.5, 2.7 Hz, 1H), 7.70 (s, 1H), 7.30 – 7.15 (m, 6H), 7.04 (d, J = 6.8 Hz, 2H), 6.91 – 6.81 (m, 2H), 6.36 (t, J = 5.4 Hz, 1H), 6.25 (d, J = 4.0 Hz, 1H), 6.15 – 6.00 (m, 2H), 4.61 (dd, J = 15.3, 6.3 Hz, 1H), 4.52 – 4.41 (m, 3H), 4.31 – 4.18 (m, 1H), 3.81 (t, J = 5.0 Hz, 2H), 3.68 – 3.43 (m, 10H), 3.43 – 3.31 (m, 4H), 3.23 (t, J = 7.6 Hz, 2H), 3.00 (t, J = 7.0 Hz, 2H), 2.61 – 2.48 (m, 5H), 2.23 (s, 3H), 1.30 (d, J = 6.4 Hz, 3H). 13C NMR (75 MHz, Acetone-d6) δ 176.6, 169.2, 158.9, 151.6, 144.6, 137.8, .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint 133.5, 130.3, 130.3, 124.7, 123.8, 120.2, 118.8, 116.7, 115.4, 115.1, 114.7, 109.2, 109.0, 108.6, 81.3, 70.6, 70.5, 70.4, 69.9, 69.5, 65.7, 63.9, 63.5, 63.2, 50.2, 45.1, 39.4, 35.4, 34.7, 34.2, 24.4, 21.7, 17.8, 15.0, 12.1, 10.5. HRMS (ESI) m/z calcd. for C45H54BF2N11O12 [M+H]+: 990.409 found: 990.412. [α]D 24 = −62.1 (c 1.1, CDCl3) UV-Stability The stability of the compounds was evaluated using UV -Vis spectrophotometry in a Thermo Scientific Evolution 201 UV ‑visible spectrophotometer. In the case of type I qABPs, 3 μl of a 15 mM stock solution in DMSO were diluted in 3 ml of PBS and the UV spectrum was measured over time until 48h, and in the case of type II a stock solution of the probes (15 mM) in DMSO was diluted in PBS/DMSO (final percentage of DMSO 20%) to a concentration of 1μM. All the compounds were also tested at pH 9, 10, 11, 12, and 13 until evidence of reaction was observed. Gel-Based Assays HEK-293 (CRL-1573, ATCC) and A431 (CRL-1555, ATCC) cells were grown in appropriate T75 culture flasks with DMEM medium supplemented with 10% FBS and 1% Penicillin/Streptomycin (Gibco). U-937 (CRL-1593.2, ATCC) cells were grown in appropriate T25 culture flasks with RPMI medium supplemented with 10% FBS and 1% Penicillin/Streptomycin. After incubating for 48 h, cells were detached with trypsin (ThermoFisher Scientific), the volume of the cell suspension was made up to 10 ml with DMEM or RPMI, and the samples were centrifuged at 400 rcf. Human neutrophils were isolated from blood according to the procedure described in Kuang et al. (33) to obtain a stock (1.5 ml) of 4.5x10 6 cells/ml. The stock solution was centrifuged at 400 rcf. Pellets were dissolved in PBS and the lysis was performed with an ultrasonic homogenizer. The concentration of protein in w hole cell lysates w as calculated through a protein concentration assay (DCTM protein assay, BioRad), using as a reference Bovine Serum Albumin (BSA) and was normalized to 1 mg/ ml or 0.6 mg/ml of protein. Regarding labeling experiments, 1 µl of the tested probe (50x concentrated stock solution) or DMSO was incubated for 2 hours with 50 µl of lysate at 37 °C. In the case of competitive assays, 1 µl of ONO-6818 (50X concentrated stock solution) was added and incubated for 30 minutes with 50 µl of lysate at 37 °C, followed by 1 µl of probe (50x concentrated stock solution) for an additional 2 hours. The reaction was quenched by adding 4x gel loading buffer (17 µl). Proteins were resolved by SDS -PAGE (15% acrylamide gel, using 40 V for 30 minutes, followed by approximately 1 hour at 100 V). In-gel fluorescence scanning was performed on a n iBright™ FL1500 (TermoFisher Scientific) using as dyes Alexa .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint Fluor 488 (excitation: 490 nm, emission: 525 nm), Cyanine3 (excitation: 554 nm, emission: 566 nm) and Cyanine5 (excitation: 647 nm, emission: 665 nm). Fluorescence Studies Fluorescence Scan. Fluorescence measurements were done on a SHIMADZU Spectro fluorophotometer RF-6000 instrument. Fluorescence was used to detect the activation with hydroxide, the fluorescence spectra of the samples used in UV -visible were measured before and after activation, obtained using as excitation wavelength the maximum absorption of the probe. The fluoresc ence quantum yield of the synthesized probes was calculated with an indirect method using Fluorescein as a reference (Φ=89% in 0.1M NaOH), as described in the literature. (32) Enzyme assays to observe the reaction with qABPs were performed in 100 mM HEPES buffer (pH 7.4) at 37 °C. A total of 15 µL purified enzyme (20 µM) was added to 3 ml of buffer containing probes ( 1.5 µM). The samples were excited at the maximum of emission of the probes , and the fluorescence intensity enhancement was monitored. Fluorescence kinetics with HNE and neutrophils Fluorescence with pure HNE and neutrophil lysates (prepared in gel-based assays) was performed in a 96-well plate and read in a Anthos multimode fluorometer Zenyth 3100, using as method Fluorescein Top (0.4s), 5 seconds shaking before each reading, and an excitation filter of 485 nm and emission filter of 535 nm. Each well contained 10 µl of pure HNE (stock solution 4 µM) or 50 µl of neutrophils lysate , 1 µl of the probe (stock solution 50 µM) and HEPES buffer (HNE) or PBS (neutrophils lysate) up to 200 µl. The fluorescence was read in 2 minutes interval for 2 hours. The assays were performed in triplicate. Biological activity HNE inhibition activity was carried out, as described in the literature (34), in 200 µl assay buffer containing 50 µl of 80 nM HNE (Merck, Germany, from stock solution 2 µM in 0.05 M acetate buffer, pH 5.5) in assay buffer, 95 µl assay buffer and 5 µl of each concentration of tested inhibitors. After a period of 30 minutes of incubation at 25 ºC the reaction was initiated by the addition of 50 µl of 4 mM chromogenic substrate (N - MeOSuc-Ala-Ala-Pro-Val-pnitroanilide, Sigma, UK) in assay buffer, and activity was monitored at 410 nm for 60 min, at 25 ºC. Due to solubility issues for more concentrated solutions, 90 µl assay buffer were used instead of 95 µl while 5 µl of DMSO were to all solutions used for progress curve evaluation, activity was monitored at 410nm at 25 ºC for 120 min. .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint Selected compounds were evaluated for their ability to inhibit other human serine proteases, namely chymotrypsin, thrombin, kallikrein, urokinase, and Porcine Pancreatic Elastase (PPE), in 200 μL reaction volumes at 25 °C according to the previous ly published experimental methods (35). Briefly, the analysis of chymotrypsin (Calbiochem) inhibition was performed in reaction mixtures containing 0.05 M Tris -HCl, 0.138 M NaCl, pH 8.0, 30 nM human pancreas chymotrypsin, test compounds, and 100 μM substrate (Suc -Ala-Ala-Pro-Phe-7-amino-4-methylcoumarin) (Bachem). The thrombin (Calbiochem) inhibition was evaluated in reaction mixtures containing 0.01 M sodium phosphate, 0.138 M NaCl, 0.1% PEG 6000, pH 7.0, 1.7 U/ ml human plasma thrombin, test compounds, and 50 μM substrate (Z -Gly-Gly-Arg-AMC.HCl) (Bachem). The analysis of kallikrein (Calbiochem) inhibition was performed in reaction mixtures containing 0.05 M Tris-HCl, 0.138 M NaCl, pH 8.0, 2 nM human plasma kallikrein, test compounds, and 50 μM substrate (H -Pro-Phe-Arg-AMC acetate salt) (Bachem). The analysis of urokinase (Calbiochem) inhibition assay was performed in reaction mixtures containing 0.05 M Tris-HCl, 0.138 M NaCl, pH 8.0, 30 U/ml human urine urokinase, test compounds, and 50 μM substrate (Z -Gly-Gly-Arg-AMC.HCl). The analysis of PPE (Calbiochem) inhibition was performed in reaction mixtures containing 100mM HEPES buffer, pH 7.4, and 20 μL of 50 μM PPE, test compounds, and 200 μM substrate (Z-Gly- (MeO-Suc-Ala-Ala-Pro-Val-AMC (Merck, Germany). For all serine proteases activity was measured at excitation and emission wavelengths of 360 and 460 nm, respectively in a microplate reader (FLUOstar Omega, BMG Labtech, Germany). For all compounds tested , each concentration was tested in triplicate, and the concentration of inhibitor that caused 50% inhibition of the enzymatic reaction (IC 50) was determined by non -linear regression using GraphPad PRISM software as previously published (35). Probe Internalization by Flow Cytometry Probe internalization was tested in U-937 cell line in MEM without red phenol, at 100,000 cells per well. Cells were incubated with qAB 22 at 5 µM per triplicate; cells without qABP, were used as negative control. After 2h, cells were analyzed by flow cytometry (Accuri C6 Plus Cytometer, BD Biosciences, Eysins, Switzerland). Viable cells were gated based on forward scatter -area and side scatter -area characteristics. Histograms were generated by plotting the x -axis represents the fluorescence intensity measure in FL1 channel (with 530 nm filter excited by 488 nm laser), and the cell count is showed in the y-axis. .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint Live Cell Imaging Probe internalization was tested in human neutrophils, isolated from blood as it is described above, in Gel-based assay subsection. Frozen neutrophils were thawed and directly seeded in Corning® 384-well Black and Clear Bottom Microplates with 10.000 cells per well in neutrophils buffer (5 mM HEPES, 0.2%BSA in HBSS). Neutrophils were incubated with qABP 22 at 5 µM per triplicate; cells without qABP, were used as negative control. After 2h, live imaging of cells were acquired by Operetta CLS High -Content Analyse System (Revvity, Waltham, Massachusetts, United States) with brightfield and FITC (460-490 nm/500-550 nm excitation/emission) channels, with 63xwater objective.

This project has received funding from the European Union’s Horizon Europe Programme under grant agreement No 101132028, project IMPULSE , and from Fundação para a Ciência e Tecnologia (FCT) trough projects 2022.07857.PTDC and UID/04138/2025 (doi: 10.54499/UID/04138/2025) , and fellowships SFRH/BD/137459/2018 and COVID/BD/153228/2023. We also acknowledge the support from EU-OPENSCREEN and PT-OPENSCREEN. .CC-BY-NC 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 22, 2026. ; https://doi.org/10.64898/2026.03.19.712884doi: bioRxiv preprint

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