Mechanistic investigations reveal that dibromobimane extrudes sulfur from biological sulfhydryl sources other than hydrogen sulfide

Dibromobimane detects sulfide levels as low as 0.6 pM, but reacts in unexpected ways with thiols, as evidenced by mechanistic investigations.


Introduction
5][6] Most biological H 2 S is produced enzymatically from cystathionine-bsynthase (CBS), cystathionine-g-lyase (CSE), and cysteine amino transferase (CAT) working in concert with 3-mercaptosulfurtransferase (3MST), although recent reports have documented H 2 S production for D-cys through a D-amino acid oxidase (DAO) 3MST pathway. 7][10][11] In addition to its different protonation states, sulde can be stored in acid-labile sources, such as iron-sulfur clusters, or in partially-oxidized sulfur pools including hydrodisuldes/per-suldes (RS-SH), hydropolysuldes (RS x -SH), and polysuldes (RS-S x -SR). 12,13These diverse protonation and storage states not only complicate unravelling the multifaceted biological roles of H 2 S, but also complicate H 2 S detection or quantication.
Despite the widespread and accepted emergence of new biological functions of H 2 S, meaningful forward progress has been slowed in many cases by the dearth of appropriate methods of H 2 S detection and quantication.Although the last few years have seen an impressive growth of new reaction-based methods for H 2 S detection, [14][15][16][17][18][19][20][21][22][23][24][25] few of these methods are suitable for quantication of endogenous sulde levels.7][28][29][30] Furthermore, although many of these systems show good selectivity for H 2 S over other reactive sulydryl-containing species, potential side-or competing-reactions oen produce identical products to those generated upon reaction with H 2 S, thus precluding accurate H 2 S quantication in complex samples.This ambiguity, as well as whether such scaffolds

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View Journal | View Issue report on free, acid-labile, 31 or total sulde remains a challenge in further understanding the multifaceted roles of H 2 S.
Direct H 2 S quantication has been maligned by similar challenges.For example, use of the methylene blue method, which was the measurement standard of the eld for many years, requires sample acidication followed by treatment with N,N-dimethyl-p-phenylenediamine and FeCl 3 to generate the methylene blue dye.3][34] Because the human nose is sensitive to aqueous solutions of 1 mM H 2 S, such results do not match well with qualitative observational data. 34dditionally, the reaction conditions required for methylene blue formation, especially treatment with strong acid, can result in liberation of sulde from acid-labile sulfur sources, such as iron-sulfur clusters. 35Furthermore, it has been shown that the methylene blue method is insufficient to differentiate between wild type and heterozygous CSE knock out mice, 36 and has a revised detection limit of 2 mM, which is much less sensitive than the initially indicated detection limit ($10 nM).Taking these limitations into account, many of the measured levels of H 2 S have come under increased scrutiny as new, improved methods for H 2 S measurement are developed.
7][38] In this method, the sample of interest is treated with mBB to trap sulde as sulde dibimane (SdB) (Fig. 1).One key benet of the mBB method is that the analytical selectivity for H 2 S over other thiols can be superimposed at the end of the experiment by chromatographic separation of the different reaction products by HPLC.Additionally, the use of different sample treatment workows allows for the separation and quantication of free, acid-labile, and total sulde thereby allowing for direct investigation of different sulde pools. 370][41][42][43] Despite this prevalence, several limitations exist, including the high mBB loading required to effectively trap all H 2 S and sulydryl nucleophiles, as well as the required trimolecular reaction between H 2 S and two equiv. of mBB.We viewed that use of dibromobimane (dBB), which has two pendant electrophilies on the same uorogenic platform, would serve as a viable strategy to improve the mBB assay.We report here a full study of mBB and dBB sulde quantication, which provides unexpected results regarding the sources from which dBB extracts sulfur in biological samples, and provides a detailed mechanistic analysis of the activity of both mBB and dBB in the presence of other thiol reagents.

Results and discussion
Comparing the mBB and dBB sulde response With the broad use of the mBB method as a sensitive and robust H 2 S quantication method, modications to this system allowing for faster sulde trapping and/or lower trapping agent loadings would provide a signicant benet.Because mBB reacts with any sulydryl-containing nucleophiles, high concentrations of mBB are required to effectively trap H 2 S in the presence of endogenous thiols.Additionally, because reaction with H 2 S initially generates bimane-SH, sufficient concentrations of mBB must be used such that each bimane-SH produced is efficiently converted to SdB prior to HPLC quantication.To overcome such limitations, we viewed dBB as an attractive platform for enhanced sulde quantication.Specically, dBB should react with H 2 S in a 1 : 1 stoichiometry, thus not only improving the reaction kinetics, but also subsequently lowering the overall trapping agent concentration required for effective H 2 S quantication.Recently, we have noted that dibromobimane has been used as a turn-on uorescent sensor for H 2 S; 44,45 however, this use is problematic because the reaction products of dBB with thiols also generate uorescent bimane thioether products, thus precluding uorogenic selectivity for sulde over thiols without prior chromatographic separation of the uorescent components.
For both mBB and dBB, the initial attack of HS À to generate bimane-SH should be fast due to the higher acidity of H 2 S by comparison to thiols.For mBB, the generated bimane-SH must undergo a second bimolecular reaction with mBB to form the SdB product.This reaction is inherently slower than the reaction with sulde due to the decreased nucleophilicity of the bimane sulydryl group by comparison to HS À .For dBB, however, although the initial attack should proceed at the same rate as for mBB, the subsequent attack of the pendant thiol is now transformed into an intramolecular reaction, thus greatly increasing the potential rate of reactivity.To conrm this design hypothesis, we treated 3.3 mM solutions of mBB and dBB with 3.3 mM H 2 S under the conditions used for the mBB method and compared the rates of reaction by uorescence spectroscopy (Fig. S1 †).As expected, the growth of the uorescence signal of the BTE product is faster than that of SdB, thus conrming the importance of the intramolecular reaction manifold for maximizing the rate of sulde trapping.
Having demonstrated that dBB traps H 2 S more quickly than mBB, we next compared the photophysical properties of the SdB and BTE products (Table 1, Fig. S2 †).Treatment of either mBB or dBB with NaSH in CH 3 CN/buffer solutions followed by purication afforded the SdB and BTE products in moderate yield.The absorption maxima (l max ), extinction coefficients (3), emission maxima (l em ), quantum yield (F), and brightness (3 Â F) were measured for both SdB and BTE and are shown in Table 1.As expected, the extinction coefficient for SdB is larger than that of BTE because two bimane uorophores are present in the molecule, thus increasing the absorption cross section.Although the emission maxima of SdB and BTE are similar, the quantum yield of BTE (62%) is signicantly higher than that of SdB (8.3%).This enhancement is likely due to abolishment of internal quenching mechanisms from the two bimane uorophores in SdB.Furthermore, comparing the brightness of SdB and BTE, which normalizes the quantum yield to the relative molar absorptivity of each species, reveals that the BTE product is over four times brighter than SdB.These direct comparisons of the photophysical properties of SdB and BTE suggested that detection limit of BTE should be signicantly lower than that of SdB due to the greater brightness of the BTE product by comparison to SdB.
Based on the photophysical differences between SdB and BTE, we next compared the H 2 S detection limits of mBB and dBB directly.For this comparison, the mBB and dBB reaction products (SdB and BTE, respectively) were compared side-byside under identical conditions, and on the same instrument used in the initial report of the mBB detection limit.Under these identical conditions, BTE has a superior detection limit by comparison to SdB (Fig. 2).Although SdB provides a 2.0 nM detection limit, which is low enough for most practical biological application of sulde detection, BTE provides a 0.6 pM detection limit under identical conditions.This detection limit provides a signicantly larger window for H 2 S detection and quantication and also opens new avenues of H 2 S detection in which low H 2 S levels are present.To the best of our knowledge, the dBB method provides the most sensitive reaction-based method of H 2 S quantication reported to date.

Quantication of exogenous and endogenous H 2 S
To further evaluate the mBB and dBB methods directly, we compared measurements of basal sulde levels in C57BL/6J (wild type) and CSE À/À (CSE KO) mice.Based on previous work, mBB is sufficiently sensitive to differentiate and quantify differential sulde levels in the wild type and homozygous CSE knock out mice.Similarly, the mBB method allows for separation of the free, acid labile, and total sulde pools by either pretreatment with acid or with a reductant. 37For this comparison, both free and total plasma sulde (free + acid labile + bound sulfur) was quantied using the optimized procedures for the mBB assay from identical samples from the same mice.Based on the results, both mBB and dBB clearly differentiate between the C57BL/6J and CSE À/À mice (Fig. 3).In both cases, however, the quantied sulde levels were signicantly different.The mBB method produced sulde levels consistent with previous measurements, however the dBB method provided measured sulde levels that were signicantly higher, suggesting that dBB may extract sulfur from other biological sources to which mBB is unreactive.Additionally, the levels of free sulde measured by dBB are higher than the total sulde levels, which suggests that other volatile sulfur-containing species that react with dBB, but not mBB, are volatilized in the procedure for free sulde measurement, this providing another difference between the mBB and dBB methods.Alternatively, the increased BTE formation could also be due to reaction of dBB with proteins in the plasma, such as albumin, which constitutes the majority of thiols in the plasma, or by extrusion of sulfur from circulating sulfane-sulfur species, such as persuldes or polysuldes.

Comparison of sulfur extrusion by mBB and dBB
Because both mBB and dBB reported identical sulde levels when treated with exogenous sulde sources, we interpreted this result to suggest that dBB was sufficiently reactive to extract sulfur from other sulfur sources, such as thiols.To test this hypothesis, we treated dBB with N-acetyl cysteine (NAC) and monitored the reaction by 1 H NMR spectroscopy.Upon incubation, new 1 H NMR resonances corresponding to the BTE product were observed in the 1 H NMR spectrum and were conrmed by the addition of an authentic sample of BTE (Fig. 4).These results suggest that dBB is sufficiently reactive to extrude sulfur from biological thiols to form BTE, thus articially increasing the measured sulde levels, which is consistent with the increased BTE formation observed from dBB under biological conditions.
To quantify the amount of sulfur extracted from common thiols by dBB, we next investigated and quantied the amount of BTE formed aer treatment with reduced glutathione (GSH) and measured the BTE product by HPLC.Consistent with the 1 H NMR studies, BTE formation was observed by HPLC.To further determine the amount of sulfur extruded from GSH, different concentrations of GSH were added to dBB and the BTE product was quantied by HPLC (Fig. 5).Treatment of mBB with increasing concentrations of GSH ranging from 5 mM to 5 mM only generated low nM concentrations of SdB.By contrast, treatment of dBB with identical GSH concentrations results in generation of micromolar concentrations of BTE.Based on the data, aer a 30 minute incubation, dBB extracts approximately 7.0% of the sulfur from GSH to form BTE. By comparison, under identical conditions the mBB method extruded less than 0.01% sulfur from GSH.These extraction efficiencies not only explain the higher levels of biological sulde detected from dBB but also highlight that mBB does not extract appreciable sulde from endogenous thiol sources.

Mechanistic investigations into dBB sulfur extrusion
Based on these data, we sought to further investigate the mechanism by which sulfur is extruded by dBB.We viewed three possible mechanisms by which dBB could extract sulfur from thiols (Fig. 6).Each mechanism proceeds through an initial nucleophilic attack of the thiol on one of the electrophilic methylbromide groups to generate the thioether.Subsequent intramolecular attack on the second electrophilic methylbromide would generate the cyclic sulfonium intermediate.From this point, we envisioned three potential mechanisms for dealkylation to form the BTE product.If the sulfonium intermediate maintains a sufficiently unhindered a-position, then nucleophilic attack by a second equivalent of the thiol would generate the BTE product and one equivalent of the thioether derived from the incident thiol (Fig. 6c).Alternatively, if the sulfonium has accessible b-hydrogens, the elimination would extrude the BTE product with concomitant formation of a terminal olen and regeneration of one equivalent of the incident thiol (Fig. 6d).The third possible mechanism could include radical fragmentation of the sulfonium intermediate to form the BTE product (Fig. 6e).
To test between these different mechanistic pathways, we chose multiple model thiols to investigate which pathways of sulfur extrusion were operative and monitored the reactions by 1 H NMR spectroscopy.In addition to the biologically-relevant cys, NAC, and GSH we also used other thiols to test specic mechanistic considerations (Fig. 7).All of the thiols, except for thiophenol (PhSH), produced the BTE product, which was identied by 1 H NMR spectroscopy and mass spectrometry. 47ecause tert-butyl thiol generates BTE, we know that nucleophilic attack cannot be the only mechanism of BTE formation because nucleophilic attack on the tertiary carbon is not possible.Similarly, benzyl thiol (BnSH) produced BTE, suggesting that the elimination pathway cannot be the only operative pathway.Consistent with both nucleophilic and elimination pathways leading to BTE formation, treatment of dBB with PhSH, which cannot participate in either of these reaction pathways, failed to produce BTE.If radical fragmentation contributed appreciably to BTE formation, the BTE should have been produced upon treatment with PhSH.To further exclude the radical pathway, we used  cyclopropylmethanethiol-containing 1 as a substrate to monitor BTE formation.If the radical pathway were operative, this substrate would generate a methylcyclopropyl radical, which would quickly react (k > 10 8 s À1 ) to the corresponding openchain product. 48,49Aer treatment of dBB with 1 under identical conditions to those of the other thiol substrates, BTE formation was observed but no cyclopropyl ring opening was observed by 1 H NMR spectroscopy, suggesting that persistent radicals are not formed during the reaction.Similarly, treated dBB with GSH in the presence of DMPO, a radical spin trap, 50 did not produce any spin-trapped product by EPR spectroscopy.Taken together, these results suggest that both the nucleophilic and elimination pathways are operative in the sulfur extrusion of dBB.Consistent with these results, although BTE is stable at neutral pH, it slowly decomposes in acidic conditions, which is consistent with transient protonation of the thioether sulfur followed by nucleophilic attack by thiol (or solvent) at one of the benzylic bimane carbons (Fig. S3 †).
Comparing the overall reactivity and selectivity reveals that dBB is signicantly more sensitive for sulde than is mBB under conditions without other thiols present.If thiols are present, however, dBB is able to extrude sulfur from these thiols with relatively high efficiency (Fig. 8).In such cases in which thiols can be removed from the sample prior to analysis, dBB provides a highly-sensitive method of H 2 S detection and quantication.For biological samples containing other sulydryl containing species, however, mBB is highly efficient for H 2 S quantication.Importantly, mBB very minimally extracts sulfur from thiols, which is not signicant, and can be corrected for by measuring total thiol concentrations in a sample.

Conclusions
Complementing the mBB method, dBB provides a highlysensitive method for sulde quantication with a detection limit of 0.6 pM.In the presence of other sulydryl containing species, however, dBB extracts sulfur from other sources thereby decreasing its delity for H 2 S quantication if other thiols are present.Mechanistic investigations revealed that thiols with aor b-hydrogens react to generate the BTE product.Taken together, these results establish dBB as a highly-sensitive method for H 2 S quantication, but also provide cautions for its use in biological samples in which thiols are present.

Fig. 2
Fig. 2 Comparison of the H 2 S detection limits of the mBB and dBB reaction products SdB and BTE, respectively, using fluorescence HPLC.

Fig. 4 1 H
Fig. 4 1 H NMR spectra of the reaction of dBB (50 mM) with N-acetyl cysteine (NAC, 20 mM) in CD 3 CN.Growth of a new peak (*) at 3.8 ppm corresponds to the BTE product.

Fig. 5
Fig. 5 Quantification of sulfur extrusion from GSH by (a) dBB to form BTE and by (b) mBB to form SdB. BTE and SdB concentrations were quantified by HPLC.GSH concentrations were confirmed by reaction with 4-fluoro-7-sulfobenzofurazan (SBD-F) followed by HPLC quantification.Treatment of dBB with 5 mM GSH resulted in detector saturation (#).

Fig. 6
Fig. 6 Possible reaction routes of dBB reacting with thiols.(a) Addition of one thiol generates the sulfonium thioether.(b) Nucleophilic addition of a thiol generates the dithiol bimane adduct.Possible mechanisms of BTE formation from thiols, including: (c) nucleophilic attack at the aposition of the pendent thiol; (d) elimination from deprotonation of hydrogens in the b-position of the pendant thiols; and (c) radical fragmentation.

Fig. 7
Fig. 7 (a) Reaction of dBB with thiols generates either the bis-thioether or the BTE thioether product.(b) Model thiols used to investigate the mechanism by which BTE is formed.

Fig. 8
Fig. 8 General reaction scheme for (a) mBB and (b) dBB reactivity.Extrusion of sulfur with mBB is inefficient whereas extraction of sulfur with dBB is significantly more efficient.

Table 1
Comparison of the photophysical properties of SdB and BTE a