A unified framework for the incorporation of bioorthogonal compound

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A unified framework for the incorporation of bioorthogonal compound exposure probes within biological compartments Benjamin Spangler, Shengtian Yang, Christopher M. Baxter Rath, Folkert Reck, and Brian Y. Feng ACS Chem. Biol., Just Accepted Manuscript • Publication Date (Web): 25 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019

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ACS Chemical Biology

A unified framework for the incorporation of bioorthogonal compound exposure probes within biological compartments Benjamin Spangler1*, Shengtian Yang1, Christopher M. Baxter Rath1, Folkert Reck1, and Brian Y. Feng1 Abstract: Compartmentalization is a crucial facet of many biological systems and key aspects of cellular processes rely on spatial segregation within the cell. While many drug targets reside in specific intracellular compartments, the tools available for assessing compound exposure are generally limited to whole-cell measurements. To address this gap we recently developed a bioorthogonal chemistry-based method to assess compartment-specific compound exposure and demonstrated its use in Gram-negative bacteria. To expand the applicability of this approach we report here novel bioorthogonal probe modalities which enable diverse probe incorporation strategies. The probes we developed utilize a cleavable thiocarbamate linker to connect localizing elements such as metabolic substrates to a cyclooctyne moiety which enables the detection of azide-containing molecules. Adducts between the probe and azide-bearing compounds can be recovered and affinity purified after exposure experiments, thus facilitating the mass-spectrometry based analysis used to assess compound exposure. The bioorthogonal system reported here thus provides a valuable new tool for interrogating compartment-specific compound exposure in a variety of biological contexts while retaining a simple and unified sample preparation and analysis workflow.

Novartis Institutes for BioMedical Research; Emerville, CA, 94608; USA. *Corresponding author: [email protected] ACS Paragon Plus Environment

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A major hurdle to the translation of potent biochemical hits into compounds with good cellular activity is achieving sufficient on-target exposure for intracellular targets. The commonly used assays for measuring compound permeability (PAMPA, MDCK, Caco-2) and mass spectrometry (MS) based methods for measuring cellular compound exposure1,2 generally do not provide measures for the free levels of compound exposure which are thought to drive target occupancy within cells.3–5 The application of methods which approximate free intracellular concentrations can help reconcile the disconnect between bulk cellular accumulation assays and observed efficacies;6,7 however, these approaches can still fail to resolve discrepancies when targets reside within intracellular compartments. More demanding approaches such as SIMS8,9 and single-cell sampling methods10,11 can provide better resolution of compound exposure in cells, however significant limitations on sensitivity and throughput hamper the systematic application of these technologies to study compound exposure. New tools capable of measuring on-target compound exposure independent of activity within multi-compartmental cellular contexts would thus provide significant benefit to drug discovery efforts.3 To address these challenges we recently reported the development of a novel activity-independent technology for the compartment-specific determination of compound exposure profiles.12 This technology utilizes compartmentally-localized bioorthogonal cyclooctynes as probes to measure the local exposure of azide-bearing compounds via relative quantification of in situ click-product formation. We have termed this approach Bioorthogonal in situ Subcellular Compound Exposure Profiling (BiSCEP) and demonstrated its application in Gram-negative bacteria with biotin-based bicyclo[6.1.0]nonyne (BCN) probes that were localized via compartment-specific streptavidin expression in the cytoplasmic and periplasmic compartments of E. coli. This approach provided a sensitive and accurate means of measuring compartment-specific compound exposure in E. coli; however, the need to genetically engineer localized streptavidin-expressing strains limits the

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application of this technology. To expand the BiSCEP platform we thus sought to develop a new bioorthogonal moiety which could enable additional labelling strategies. Many alternative probe incorporation strategies such as metabolic incorporation and site-specific protein labelling would result in the probe being covalently incorporated into endogenous enzymes or cellular structures. To utilize these approaches for BiSCEP assays we thus required a reliable means of releasing the probes prior to MS-based exposure analyses. To address these requirements we report here the development of a cleavable thiocarbamate linker which can be paired with cyclooctyne probe moieties. We show that this linker has no impact on the reactivity of the cyclooctyne probes while providing a biologically stable linkage that can be efficiently cleaved to release analytes of interest. This linker enables metabolic incorporation of cyclooctyne probes which can then be recovered and affinity purified after exposure experiments, facilitating the MSbased analyses used to assess compound exposure. The new bioorthogonal systems reported here thus provide valuable new tools for interrogating compound exposure which could be readily applied to diverse probe incorporation strategies to enable compartment-specific compound exposure analysis in a variety of biological contexts.

Results and Discussion: In order to apply BCN probes as tools for measuring compartmental compound exposure they must first be localized to the compartments of interest. These probes thus consist of three elements: the BCN itself, a localization moiety (e.g. metabolic substrate, biotin), and a linker to join the two (Figure 1a). To expand the types of localization strategies that could be applied for these approaches we required a novel linker which would enable release of the BCN after covalent incorporation into cellular components. We reasoned such a linker would need to be stable in intracellular environments but readily cleavable within cellular lysates after exposure experiments were completed. The linker and probe moieties further needed to be as small as possible to avoid



interfering with the use of metabolic incorporation strategies using endogenous enzymes. Finally, we reasoned the ability to affinity purify and concentrate our analytes of interest from the complicated matrix of cellular lysates would simplify analysis and improve the sensitivity of the assay. To satisfy these criteria we selected a thiocarbamate moiety as a minimal linker which should be stable in many intracellular compartments yet readily cleavable via basic hydrolysis.13 The resulting thiol could then be captured using reactive resins for affinity purification,14,15 thus enabling a “Click-Cleave-Capture” (C3) approach for utilizing BCN probes and recovering analytes for exposure analyses (Figure 1a).

Figure 1: (a) Schematic of using a “click-cleave-capture” (C3) approach to enable metabolic incorporation of recoverable BCN probes for compartmental exposure assays. (b) Synthesis of BCNthiocarbamate conjugates from commercially available BCN alcohol 1.


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Synthesis of BCN thiocarbamates. To afford a thiocarbamate linkage of BCN to substrates for covalent probe incorporation we first sought to convert the alcohol of 1 — a commonly used attachment point for further transformations — into a thiol. Under appropriate conditions, Mitsunobu reaction of alcohol 1 with thioacetic acid gave the thioester 2 in good purity and yield (67%) (Figure 1b). We found it was important to add only one equivalent of thioacetic acid dropwise to the betaine of 1, as the alkyne moiety of BCN analogs can be labile in the presence of thiols.16 Subsequent deacetylation of 2 to afford the desired thiol intermediate (3) proved challenging. We found standard acidic deprotection of the acetyl group failed to produce the desired product. As the same conditions readily afforded the desired thiol when the BCN had previously undergone a click reaction with benzyl azide, we determined that these conditions were incompatible with the strained alkyne of the BCN (Supplementary Scheme 1). While this instability significantly limits the synthetic strategies that can be applied in the presence of BCN moieties, it is poorly represented in the literature with only one previously noted observation which provided no structural evidence for the undesired product formed upon acid mediated degradation of the alkyne17. We found BCNconjugates such as 4 to be so labile to acidic conditions that HPLC fractions containing 0.1% trifluoracetic acid (TFA) buffer could not be concentrated (under reduced pressure at 40 °C) without inducing degradation of the alkyne bond. NMR analysis of the resulting material showed that the initial putative hydration product rearranges to the corresponding cyclooctanone 5 (Scheme 1, Supplementary Figure 1). Compound 5 is not reactive towards azide probes in the subsequent click-reactions and thus care should be exercised when exposing BCN analogs to acidic conditions.



Scheme 1: Acid mediated degradation pathway for BCN conjugates such as 4 in to inactive vinyl alcohol and ketone species (5). Attempts to cleave the thioester under reducing conditions with LAH also failed to provide detectable product formation, presumably due to the free thiol being unstable under these conditions and prone to polymerization via thiol-yne addition and/or disulfide formation over time. We thus chose to pursue a one-pot conversion of acetate 2 to p-nitrophenylcarbonate 6 to mitigate these potential liabilities. Initial efforts utilizing potassium tert-butoxide (KOtBu) at 0 °C gave the desired product but in poor yields (15%). Ultimately, the use of sodium methoxide (NaOMe) at 0 °C in place of KOtBu gave the best yields (39%) and provided synthetically useful quantities of the key intermediate (6) (Figure 1b). Careful monitoring of the hydrolysis step and rapid quenching of the produced thiolate anion was found to be important in achieving improved yields of the desired product (6). Conversion of 6 to the desired thiocarbamates was then readily achieved by introducing amines of interest in the presence of triethylamine (TEA) (36 – 62%) (Figure 1b).

In vitro characterization of BCN-thiocarbamate conjugates. In order to study the reactivity and stability of BCN-thiocarbamates we synthesized a BCN conjugate of the Dansyl fluorophore with a thiocarbamate linker (7) and compared its reactivity to that of the corresponding carbamate analog (8) using fluorescence-based reaction monitoring (Figure 2a, Supplementary Scheme 2). When exposed to various concentrations of an azidebearing fluorescent quencher (Dabsyl-azide) in E. coli lysate both compounds showed time and concentration dependent decreases in fluorescence relative to a non-cyclooctyne control (9,


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Supplementary Scheme 2), consistent with the reactions proceeding to give the quenched clickproducts (Figure 2b). There was no significant difference in the reactivity of 7 and 8 with Dabsylazide, indicating that the reactivity of the cyclooctyne with azides is not significantly impacted by variations in the linker (Figure 2d). Furthermore, once quenched there was no evidence of increasing fluorescence in samples with 7 or 8 over the course of the experiment, indicating that the thiocarbamate conjugates are stable in E. coli lysate for greater than 18 h at 37 °C.

Figure 2: (a) Scheme for the use of Dansyl-BCN conjugates as fluorescent reporters for monitoring click reactions with Dabsyl-azide and the stability of the resulting products. (b) Change in the fluorescence intensity of 10 μM solutions of 7 or 8 in E. coli lysate over the course of 18 h at 37 °C when exposed to 50 μM Dabsyl-azide. Data were normalized to the fluorescence of an unreactive Dansyl control conjugate (9) to account for reaction independent quenching. (c) The quenched



samples were exposed to 0.1 N NaOH and monitored for changes in fluorescence compared to 9 over time. (d) The rates of click reaction with 7 and 8 as a function of the concentration of Dabsylazide as determined by the initial rate method, expressed as the percent change in fluorescence per minute. (e) The rates of cleavage for the quenched species of 7 and 8 as a function of NaOH concentration as determined by the initial rate method, expressed as the percent change in fluorescence per minute. All experiments were conducted in quadruplicate and error bars represent SEM (n = 4).

In contrast, when the quenched species were subsequently exposed to sodium hydroxide (NaOH) the thiocarbamate conjugate (7) was observed to cleave rapidly to release the fluorescent amine while the carbamate linkage (8) remained intact (Figure 2c). The rate of thiocarbamate cleavage was dependent on the concentration of NaOH used (Figure 2e) and 0.1 N NaOH exposure gave complete hydrolysis of the thiocarbamate bond within 5 minutes at room temperature. These data thus demonstrate that thiocarbamates can be used as cleavable-linkers for BCN-conjugates without adversely impacting the reactivity of the BCN-probe or the stability of the conjugates in relevant biological matrices.

Optimizing thiol capture for MS analysis of azide exposure. Having confirmed that thiocarbamates could be used as cleavable linkers for BCN-conjugates we next sought to optimize protocols for the detection of the thiol analytes released after thiocarbamate hydrolysis. To enable comparisons to our previous methods we synthesized a Biotin-BCN thiocarbamate conjugate (10) and benzyl azide click-product (11) which could be utilized for in vitro and bacterial assays to compare the detection of azides by our previous methods to the click-cleave-capture (C3) strategy used here (Figure 3a, Supplementary Scheme 3). We first explored the detection limits of thiols released upon thiocarbamate cleavage after different affinity purification strategies. Without any affinity purification steps the thiol released upon cleavage of 11 was detectable at low nM concentrations (Figure 3b). However, there was a high degree of variability in the data, perhaps due the potential of the samples to oxidize to disulfide


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side-products. Furthermore, the unreacted BCN-thiol released after cleavage of 10 was not observable at all under these conditions (Supplementary Figure 2). Maleimide-based thiol capture beads (MA-beads, Supplementary Figure 2a) similar to those previously reported for the enrichment of thiol bearing metabolites14 enabled detection of the unreacted BCN; however, signal intensity for the product of thiol capture from 11 was lower than that observed without the beads (Figure 3b, Supplementary Figure 2). Analysis of the bead flow-through showed that some thiol remained uncaptured despite substantial optimization of thiol capture conditions with these beads (SupplementarySupplementary Figures 2 and 3). Further analysis suggested incomplete release of analytes from the beads was also limiting signal. Extensive attempts to optimize the trypsinization and elution protocols used for these beads failed to significantly improve the detection limits of thiols with these beads (Supplementary Figure 3). We thus turned towards disulfide-based thiopropyl sepharose bead capture (Supplementary Figure 2). In contrast to the MA-beads, this route proved to be a robust (low variability) and sensitive means of recovering thiol analytes from lysates with a linear range of detection from 1 nM to 10 μM in E. coli lysate after cleavage of 11 (Figure 3b). These beads were thus used for the affinity purification of thiol analytes in subsequent experiments. C3 methodology provides a robust means of azide detection in vitro and in cellular assays. With a reliable means of analyzing the thiol-based analytes released after thiocarbamate cleavage in hand, we next compared the detection efficiency of this method with our previous approach12 in a cell-free assay. BCN-thiocarbamate 10 was exposed to dose responses of either azidothymidine (AZT) or propidium monoazide (PMA) in E. coli lysate for 2 h then either processed for direct MS analysis or cleaved with 0.1 N NaOH and subjected to affinity purification of the resulting thiolbearing analytes. As expected, the more reactive aromatic azide (PMA) gave significantly more signal for the click product with BCN in vitro than the aliphatic azide (AZT);18 however, both



compounds had limits of detection in the single digit nM range consistent with our previous observations using biotin-BCN conjugates (Figure 3c).12 Importantly, we found that signal for the formation of click-products from either azide was essentially identical between the two methods of analysis (Figure 3c). These findings thus indicate that the additional processing steps required to release and capture thiol analytes from thiocarbamate conjugates do not negatively impact the detection of azide bearing molecules in vitro. Should additional sensitivity be required for particular applications we found that the affinity purification enabled by the beads could be used to enhance the detection of limited quantities of the thiol analytes (Figure 3d).

Figure 3: (a) Scheme for the use of Biotin-BCN thiocarbamate conjugates to study MS based detection of azide-bearing compounds. (b) Signal for thiols and thiol capture products released from hydrolysis of 11 as a function of the concentration of 11. (c) Signal for the click products of AZT and PMA with 20 μM 10 after a 3 h incubation at 37 °C as a function of azide concentration. The samples were split in half and either processed for MS analysis as intact conjugates by our previous methods (Std) or cleaved and captured for affinity purification (C3). Integrated ion intensities were normalized to signal from the internal control for each sample (200 nM 11). (d)


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Signal for thiol released from hydrolysis of 40 nM of 11 as a function of the volume of sample affinity purified on thiopropyl sepharose beads eluting with 25 mM DTT in 100 mM Tris-HCl, pH = 8. (e) The observed exposure of PMA and 7N3HC relative to AZT (10 μM, 2 h, 37 °C) in the cytoplasm and periplasm of WT and ΔtolC E. coli strains as determined by C3 analysis of bacteria loaded with probe 10. Signal for each click-product was normalized to in vitro control reactions and AZT signal in the same compartment and strain to provide ‘Relative Exposure’ values as previously described11. All data depict mean values from experiments conducted in triplicate with error bars representing SD (n=3). The structures of the assayed compounds are shown to the right of the figure. See Supplementary table 1 for information on the analytes monitored for each experiment.

To test whether the thiocarbamate linker had an impact on signal for azide exposure in cellular assays we compared the observed compartmental exposure of a small set of representative compounds when assayed by either methodology in streptavidin expressing E. coli. We observed the same trends for azide exposure in each compartment of the bacteria regardless of the sample processing used (Supplementary Figure 4). Compared to AZT (our positive control for exposure), both PMA and the coumarin azide (7N3HC) had limited exposure in WT E. coli which increased in the absence of tolC mediated efflux (E. coli-ΔtolC), consistent with our previous observations12 and literature precedent for these compounds (Figure. 3e).19,20 PMA also displayed poor distribution into the cytoplasm, indicative of the additional barrier the inner membrane presents to this positively charged compound. These findings correspond well with our previous observations using Biotin-BCN carbamate conjugates12 thus confirming the thiocarbamate cleavage and thiol capture protocols are compatible with experiments of compartmental compound exposure in E. coli.

Thiocarbamate conjugates can be metabolically incorporated in E. coli. We next sought to leverage the cleavable feature of the thiocarbamate linker to enable metabolic incorporation of BCN probes in bacteria for use in BiSCEP assays. Recent reports had indicated that BCN-D-alanine derivatives (4) could be metabolically incorporated into bacterial peptidoglycan (PG) via permissive transpeptidases.21 We reasoned that the single atom substitution required to



produce a BCN-D-alanine thiocarbamate analog would be tolerated by the same enzymes thus allowing us to metabolically label the periplasmic space of E. coli with BCN probes. The requisite conjugate (12) was thus synthesized in two steps from 6 (Figure 4a).

Figure 4: (a) Synthesis of BCN-D-alanine thiocarbamate analog 12 and structure of fluorescent azide 5R110-N3. (b)Representative fluorescence microscopy images of WT E. coli after labelling with 200 μM 12 and click-reaction with 20 μM 5R110-N3 (green). Bacterial membranes were visualized with FM4-64FX dye (Red) and DNA was visualized with Hoechst 34580 nuclear stain (blue). Scale bars represent 2 μm. (c) Flow cytometry histograms for the fluorescence intensity of WT E. coli treated with the indicated concentrations of probe 12 prior to 5R110-N3 exposure (n =


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100,000 bacteria/condition). (d) Average signal for bacterial fluorescence by flow cytometry as a function of the concentration of 12 used for probe incorporation. Signal is represented as fold change from DMSO treated bacteria in experiments conducted in triplicate. Error bars represent SD (n = 3).

To confirm probe 12 was incorporated into the PG of E. coli we exposed probe treated bacteria to a fluorescent azide (5R110-N3) known to achieve exposure to both the cytoplasmic and periplasmic compartment of the bacteria.12 After washing away unreacted azide, we stained the bacteria for membrane and nuclear components to differentiate the periplasmic and cytoplasmic compartments and used fluorescence microscopy to image the bacteria for fluorescence localization. We found signal for the incorporated probe co-localized well with membrane staining consistent with the periplasmic localization expected upon PG incorporation21 (Figure 4b). Interestingly, we noticed small foci of fluorescence signal in some of the bacteria which had been treated with 12. These foci were observed in both the 5R110-N3 and the membrane channels suggesting the modification of PG may have an impact on bacterial membrane structure at higher concentrations. To explore the dose-dependence of probe incorporation we used the same methods paired with flow cytometry analysis of the labelled bacteria (Figure 4c). As bacteria were exposed to increasing concentrations of 12 dose-dependent increases in fluorescence were observed up to 0.5 mM concentrations in WT E. coli. When the same experiment was performed with E. coli-ΔtolC an overall increase in fluorescence signal was observed (Figure 4d). While we observed no significant impact of probe treatment on bacterial growth in WT E. coli up to 0.5 M concentrations, E. coli-ΔtolC did show a growth defect when treated with concentrations of 12 over 60 μM, suggesting that probe incorporation can have a detrimental effect on growth at higher concentrations (Supplementary Figure 5). Importantly, signal for probe incorporation was significantly diminished when an L-alanine derivative (13) was applied in place of 12 consistent with the known specificity of bacterial transpeptidases for D-amino acids (Supplementary Figure 6).22,23 Taken



together these findings confirm the thiocarbamate isostere (12) of the previously reported BCN-Dalanine carbamate analog (4)21 can be applied to metabolically label bacterial PG; however, care should be taken to ensure the incorporation conditions used do not significantly impact bacterial viability.

Metabolic incorporation of BCN probes enables multi-compartmental analysis of compound exposure. In order to study the impact of probe incorporation on the bacterial permeability barriers and compound exposure we combined the use of metabolic probe 12 with our previously reported Biotin-BCN carbamate probe 14 as a complementary means of assaying compartmental compound exposure. As the two methodologies have orthogonal sample processing steps and produce different analytes we reasoned we could simultaneously label the periplasm and cytoplasm of E. coli with BCN probes by exposing cytoplasmic streptavidin-expressing E. coli strains to 12 and 14 in tandem (Figure 5a). After washing away unincorporated probes and conducting azide exposure experiments the bacteria could be lysed and the thiocarbamate based 12 (and click-products thereof) could be cleaved and captured while the carbamate based 14 remained in the bead flow through. Mass-spectrometry analysis of the flow through and eluents after thiol-bead affinity purification would then enable differential analysis of the exposure of azides to each probe. We first applied this strategy to study the incorporation of 12 in the periplasm. As bacteria were exposed to increasing concentrations of 12, corresponding increases in the amount of AZT clickproducts formed in the periplasm were observed from the thiol-capture samples (Figure 5b). Signal in both strains saturated above 100 μM concentrations and more probe appeared to be incorporated in the E. coli-ΔtolC mutant overall, consistent with the fluorescence-based studies. In contrast, analysis of the biotin-conjugates in the flow through from the same samples showed no significant changes in click-products originating from AZT exposure to 14 in the cytoplasm when


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bacteria were treated with concentrations of 12 at or below 50 μM (Figure 5c). While concentrations of 12 greater than 50 μM had no impact on observed azide exposure in the E. coliΔtolC mutant they did cause a modest but significant increase in the signal observed for cytoplasmic AZT exposure in WT E. coli. This increase seems to be driven at least in part by increased incorporation of 14 at higher concentrations of 12, likely due to both probes being efflux substrates, with competition for efflux transporters increasing effective intracellular probe concentrations (Supplementary Figure 7a). The same trends were also observed in bacteria treated with PMA (Supplementary Figure 7b). Based on these findings and the previous observations of growth inhibition in bacteria treated with higher concentrations of 12 we selected 50 μM concentrations of 12 for future experiments as the compound showed no significant impact on bacterial viability or permeability under these conditions while still providing sufficient probe incorporation for subsequent exposure analyses.



Figure 5: (a) Scheme for dual probe incorporation of 12 and 14 for use in multiplexed assays of compartmental compound exposure. This graphic was adapted from our previous work.11 (b) Signal for AZT click-products derived from 12 (periplasmic) in bacteria after azide exposure assays (10 μM, 2 h, 37 °C) and C3 processing as a function of the concentration of 12 used for probe loading. Data are shown as the fold change in signal over background from control samples treated with vehicle (DMSO) in place of 12. (c) Signal for AZT click-products derived from 14 (cytoplasmic) in bacteria as a function of the concentration of 12 used for probe loading in the periplasmic compartment. Data are displayed as in (b). (d) Comparison of signal for AZT click-products derived


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from 12 when bacteria from the indicated strains were assayed with 12 alone or in combination with 14 for dual probe incorporation. Data are displayed as fold change from signal in the single probe samples to display the impact of probe multiplexing on signal generation. (e) Relative exposure of AZT and PMA control compounds in WT and E. coli-ΔtolC mutants as determined by multiplexed exposure analysis with 12 (periplasmic) and 14 (cytoplasmic). Data are represented as relative exposure compared to AZT in each compartment and strain as described in the methods. Data in all figures represent the mean values from experiments conducted in triplicate with error bars depicting SD (n = 3). See Supplementary table 1 for information on the analytes monitored for each experiment.

To further explore the impact the probes have on observed compound exposure we analyzed the production of click-products resulting from AZT exposure to 12 in the presence or absence of 14 localized to the cytoplasmic or periplasmic compartments via streptavidin expression. We found that the amount of AZT-click product formed with 12 was unchanged regardless of the presence of 14, even within the same compartment (Figure 5d). In our previous work, we reasoned the cyclooctyne probes would not significantly impact compound exposure as the rate of the click reaction in the compartments (t1/2 of hours) was much slower than the kinetics of compound permeation in the bacteria.12 These findings support this hypothesis; were the probes acting as irreversible sinks to sequester azide-bearing compounds then the presence of 14 would be expected to reduce the exposure of AZT to 12 (and vice versa), resulting in a decrease in the formation of click-products which we did not observe in our experiments. We were thus encouraged to find that — at the probe concentrations used in these assays — the presence of BCN probes does not significantly impact the observed exposure for azide-bearing compounds. Given that neither probe appeared to alter the observed exposure of azide-bearing compounds to the other we determined that these two approaches could be applied orthogonally to simultaneously measure the exposure of azide bearing compounds to the periplasmic and cytoplasmic compartments of E. coli. We thus treated cytoplasmic streptavidin expressing strains in WT and ΔtolC backgrounds with both 12 and 14 for simultaneous probe incorporation. After washing away the unincorporated probes the bacteria were then exposed to azide bearing



compounds and compartmental compound exposure was assessed after C3 processing. Analysis of the exposure of the control compounds AZT and PMA demonstrated that this method gave comparable results to those obtained with independent measures of compartmental exposure with biotin-based probes (Figure 5e, Supplementary Figure 4). The orthogonality of these two assays thus provides the opportunity to simultaneously interrogate compound exposure in multiple compartments.

Concluding remarks. Overall, these findings demonstrate that thiocarbamate conjugates can be utilized for the covalent incorporation of BCN probes which can then be recovered from lysates via thiocarbamate cleavage and affinity purification of the resulting thiols. This approach could be envisioned to be applicable to label various compartments throughout diverse biological systems by utilizing permissive endogenous enzyme and pathways such as those involved in pantothenic acid uptake and ACP labelling24–26 or vitamin B-12 transport27. These approaches can have significant value; the D-ala probe described here (12) provides a new means of interrogating compound exposure in the periplasm of Gram-negative bacteria which does not require genetic engineering and thus should be readily applicable, particularly in the study of the impact genetic changes (e.g. permeability mutants) can have on compound exposure. Moreover, this approach is not limited to metabolic routes of probe incorporation; techniques that allow for unnatural amino acid incorporation in target proteins provide the tantalizing opportunity to siteselectively incorporate BCN probes into proteins of interest.28 Given that BCN carbamate analogs have already been successfully utilized for these approaches,29,30 it is reasonable to expect that thiocarbamate analogs may be similarly tolerated.


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For probe incorporation strategies to be of utility they must be able to specifically localize significant amounts of the BCN probes to the compartments of interest such that they retain their ability to react with azide-bearing compounds. As previously noted, BCN moieties have incompatibilities with particularly acidic or thiol rich conditions; thus, compartments with such environments may be unsuitable for this approach. Experiments with known control compounds such as AZT and PMA should thus always be conducted to assess the viability of a given method of probe incorporation. Notably, while the cytoplasm of E. coli is a reducing environment31, the increased prevalence of free thiols in this compartment compared to the periplasm has not been observed to significantly quench the probes localized there or impede their use in our studies. Thus, the chemical liabilities of these compounds under synthetic conditions may be less relevant under biologically relevant conditions. When using thiocarbamate linked-probes, the chemical matter being studied should also be considered in the context of the hydrolysis conditions used to cleave the linker. Base labile functional groups such as esters are likely to be partially or completely hydrolyzed under the 0.1 N NaOH conditions used for linker cleavage. While this is not necessarily prohibitive to exposure analyses — provided predictable products are produced and can be monitored and controlled for with in vitro experiments — it will add complexity to the analysis. The requirement for azide labelled compounds also imparts certain limitations on the applications of these technologies. While the azide represents a minimal mark in terms of changes to the physicochemical properties of the parent compound, its potential to impact the exposure of the compound cannot be ruled out. Thus, we have found the most useful analyses to be driven by relative comparisons of how changes within a related series of compounds (with the azide at a fixed position) impact compartmental exposure compared to a parent or lead compound.12 This approach enables activity-independent optimization of compartmental exposure profiles which we have found to be valuable.



Regardless of the route of probe incorporation or the ultimate localization of the probes in cells the thiocarbamate linkers described here provide for a unified sample processing protocol. Once these linkers are cleaved, the resulting thiol analytes can be affinity purified and analyzed by MS for the presence of click-products arising from compound exposure. The same analytes are generated in this approach even if divergent probes are used to enable different localizations; thus, the analytical processing is retained between probes, minimizing the need to develop new workflows for each probe generated. The ability to affinity-purify the generated analytes also provides a built-in option for signal amplification in contexts where assay sensitivity is lacking for a given application. While we found additional sensitivity was not required for the exposure analyses done here, the ability to affinity purify analytes further provides a convenient means of separating analytes generated from metabolic probes from those generated from Biotin-BCN probes when these two technologies have been paired in multiplexed experiments. This approach may be particularly useful for ratio-metric analyses of compound exposure between compartments (e.g. for the study of endosomal escape in drug delivery strategies), while minimizing the number of assays required to assess exposure. In conclusion, these studies provide the framework for expanding BiSCEP assays across biological compartments and a means of simultaneously monitoring compound exposure profiles in the cytoplasmic and periplasmic compartments of Gram-negative bacteria via multiplexed applications of metabolic and genetic probe incorporation strategies. We anticipate future applications will enable the generation of more nuanced models of compound exposure determinants. These tools may also find utility in the development of drug-delivery strategies where quantification of delivery efficiency would be useful in guiding optimization of these technologies. In addition to adding to the BiSCEP platform and facilitating its application across bacteria with the D-ala probe described here, the thiocarbamate linker we present here should be a generally useful addition to the toolkit of bioorthogonal chemistries. We hope this work enables further applications of these powerful tools.


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Supporting information: Supplementary figures, schemes, and tables, full experimental methods, and synthetic protocols and compound characterizations can be found in the supporting information. This material is available free of charge via the Internet.

Acknowledgments: The authors would like to thank C. Lee for helpful input in the preparation of this manuscript.

Funding: This work was funded the Novartis Institutes for BioMedical Research. B.S. was supported by the Novartis Post-doctoral program.

Author Contributions: Conceptualization, B.S., C.R., and B.Y.F.; Methodology, B.S.; Investigation, B.S. and S.Y.; Resources, B.S.; Writing – Original Draft, B.S.; Writing – Review & Editing, B.S., B.Y.F., and F.R.; Visualization, B.S.; Supervision, B.Y.F. and F.R.

Declaration of Interests: The authors are all current or former employees of Novartis.

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A unified framework for the incorporation of bioorthogonal compound

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