- Email: [email protected]
Sensors for Biological Thiols
8.20
Sensors for Biological Thiols
RM Strongin and JO Escobedo, Portland State University, Portland, OR, United States Ó 2017 Elsevier Ltd. All rights reserved.
8.20.1 8.20.1.1 8.20.1.1.1 8.20.1.1.2 8.20.1.1.3 8.20.1.2 8.20.1.2.1 8.20.1.2.2 8.20.2 8.20.2.1 8.20.2.1.1 8.20.2.1.2 8.20.2.1.3 8.20.2.1.4 8.20.2.1.5 8.20.2.2 8.20.2.2.1 8.20.2.2.2 8.20.2.2.3 8.20.2.2.4 8.20.2.2.5 8.20.3 References
8.20.1
Introduction Standard Biological Thiol Detection Methods Electrochemical methods Sensors employing biological materials Mass spectrometry and chromatography Universal Thiol-Reactive Chromophores and Fluorophores Conventional thiol-reactive probes More recent examples of universal thiol-reactive probes Selective Thiol Sensors: Opportunities for Improved Diagnostics Glutathione The significance of monitoring GSH whole blood levels Traditional methods for monitoring GSH whole blood levels Recently reported fluorescent reagents for imaging GSH in cells Designing a reagent for GSH diagnostics Designing a blood spot test using a GSH-selective fluorescent reagent Homocysteine The significance of monitoring Hcy levels A gap in Hcy monitoring in clinical studies Methods for monitoring Hcy and metabolite levels Selective indicators for Hcy and metabolites A simple supramolecular approach for enhancing Hcy over Cys selectivity in aldehyde-functionalized fluorescein dyes Summary and Conclusions
435 436 436 436 436 436 436 437 437 437 437 438 438 439 441 441 442 442 442 443 443 444 444
Introduction
The most abundant thiol amino acids in humans are cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). They have attracted great recent interest in the field of sensor and probe development, as each plays a major role in human health and disease. Chemists in the sensor and supramolecular fields have, relatively recently, embraced the challenges inherent in designing reagents that enable the selective imaging or determination of each specific amino thiol in natural media. Fluorescent chemosensors, dosimeters, and probes have been designed to overcome issues relating to not only optimizing responses, kinetics and compatibility with biological fluids, but also to address the very similar structures and reactivity of Cys, Hcy, and GSH and their varying relative abundance in native media (Fig. 1). The purpose of this chapter is twofold. It highlights (i) relatively under-addressed biomedical issues and opportunities in the thiol sensor field and (ii) sensors relying on non-covalent interactions to enable selectivity among each of the respective major biothiols in natural media. It does not summarize the biological thiol sensor field, which has been reviewed in detail previously.1Rather, it highlights the opportunities that still exist in this field.
Figure 1 Homocysteine (Hcy), cysteine (Cys), and glutathione (GSH) are the major low molecular weight biological thiol compounds. Note that Hcy and Cys differ by only one methylene in their side chains, and that Cys is the internal amino acid of the tripeptide GSH. Their similar structures render it challenging to create synthetic probes and indicators that exhibit selectivity for just one of these analytes. However, the fact that each is associated with distinctive significant physiology has prompted recent efforts in the abiotic sensor field to enable monitoring of each of these respective biothiols.
Comprehensive Supramolecular Chemistry II, Volume 8
http://dx.doi.org/10.1016/B978-0-12-409547-2.12624-1
435
436
Sensors for Biological Thiols
8.20.1.1
Standard Biological Thiol Detection Methods
Thiol detection procedures have generally involved chromatographic separations, immuno- and enzymatic assays, electrochemical, mass spectrometric, and flow injection technology. Each method has well-known strengths and limitations that are important to consider in designing functional sensing or probe reagents to either supplant or complement these methods.
8.20.1.1.1
Electrochemical methods
Electrochemical detection may be complicated by interference from oxidizable impurities. Electrochemical detection of thiols by Capillary Electrophoresis requires precision electrode alignment and isolating the detector from the separation voltage. Amperometric postcolumn detection of Cys and Hcy may suffer from low selectivity and high background current as Cys exhibits irreversible oxidation requiring positive overpotential. Small volumes in the separation capillaries in Capillary Electrophoresis require that the detector be placed in-line to minimize line broadening. Good sensitivity often requires dual electrode configurations. The stability of the detection cell components is another concern, depending on the type of analysis. Mercury and mercury amalgam electrodes have been widely used for thiols but have limitations including toxicity and poor stability. Chemically modified electrodes require complex preparation, can exhibit poor stability, and need well-controlled working conditions.
8.20.1.1.2
Sensors employing biological materials
Immunoassays require specialized detection systems such as fluorescent polarization (FP). The assays can be time consuming and have lower precision than enzymatic assays. Many enzymes are relatively unstable (low shelf life compared to common organic reagents). Enzyme cycling can exhibit advantages over immunoassays such as less sample pretreatment and run time as well as utility with routine clinical lab instrumentation. Radioimmunoassays involve toxic substances. STE (Substrate-Trapping-Enzyme) technology necessitates a batch chromatography step and exhibits relatively low precision. In general, biomaterials are relatively expensive and methods relying upon them often require tedious sample preparation and handling as compared to fully synthetic molecular probes.
8.20.1.1.3
Mass spectrometry and chromatography
Mass spectrometry coupled to HPLC or GC requires relatively tedious procedures that are less suitable for routine diagnostic applications. Gas chromatography–electron capture detection and flame photometry detection require involved sample preparations and/or high operating temperatures. Trap & Release Membrane Introduction Mass Spectrometry (T&R MIMS) requires time-consuming derivatizations and sophisticated instrumentation rendering it less suitable for routine analysis.
8.20.1.2 8.20.1.2.1
Universal Thiol-Reactive Chromophores and Fluorophores Conventional thiol-reactive probes
The aminothiols are essentially colorless and nonfluorescent in the visible spectral region. Thus, thiol-reactive optical probes have mainly been used as fluorescent labels for biomolecules via conjugation to their Cys residues, or in conjunction with separation techniques. The Molecular Probes Handbook has an entire chapter devoted to commercially available thiol-reactive probes.2 The majority are based on alkylation reactions of the nucleophilic sulfhydryl moiety and thus do not enable discrimination among GSH, Cys, or Hcy. Examples of drawbacks and limitations of some of the most well-known thiol probe types are summarized in Table 1. End users and inventors should also be aware that universal thiol-reactive probes are sometimes marketed or reported as GSH-selective. This is based on an assumption that, since GSH is the most abundant (mM levels) thiol in cells, any signaling from an interaction with Hcy or Cys would be negligible. This is likely true in cells for many probes, though there is a lack of published data to support such an assumption. However, in fluids such as human plasma, GSH is less abundant than Cys, and its detection by a universal thiol reagent will encounter interference from Cys and GSH.
Table 1
Drawbacks and limitations of some well-known thiol probes
Monobromobimane Labeling (i) Unstable at room temperature and in H2O; (ii) batch-to-batch varying impurity levels; (iii) fluorescent hydrolysis products produced upon labeling; (iv) gradient and relatively complex elution chromatography needed Iodoacetamide labeling (i) Cross-reactivity with histidine, tyrosine, and methionine; (ii) promotes loss of NH3 upon reaction with Cys residues; (iii) reduces mass spectrometric sensitivity O-phthalaldehyde labeling (i) Highly pH sensitive reaction; (ii) thiol adduct exhibits high photoinstability; (iii) cross-reactivity with many other amino acids Maleimide labeling (i) Hydrolysis peaks are encountered at the beginning and end of chromatographic elution; (ii) cross-linking to amines; (iii) unwanted rearrangements of conjugates Hexaiodoplatinate labeling Broad cross-reactivity with interferences, including thioethers, thiazolidines, and ascorbic acid
Sensors for Biological Thiols
8.20.1.2.2
437
More recent examples of universal thiol-reactive probes
Although probes that react with sulfhydryl-containing molecules are broadly available, there are many ongoing efforts toward alternatives. Several examples are based on conjugate addition of the sulfhydryl to a probe that is functionalized with a conjugate acceptor moiety. For instance, thiol addition to a Tb(III) complex was used in monitoring the enzymatic reduction of glutathione disulfide (GSSG) to GSH.3 In addition, conjugate addition to a squaraine dye resulted in successful monitoring of the elevation of blood thiol levels upon smoking.4 The conjugate reaction also enabled a functionalized coumarin to exhibit excellent kinetics (< 1 min reaction with thiols at RT) and also label protein-containing thiols efficiently at low (ng) levels.5 A probe functioning via conjugate addition, in conjunction with hydrogen bonding, was successfully employed for ratiometric imaging of GSH in cells.6 The nucleophilic reactivity of the sulfhydryl group has also led to probes functioning by other mechanisms. The reaction of aminothiols via an SNAr mechanism was used to create thiol-reactive fluorescein and cresyl violet derivatives. They were used in monitoring cholinesterase and glutathione-S-transferase activity, respectively. Thiol-reactive probes based on disulfide exchange reactions were used for two-photon imaging in cells and tissue7 as well as in a targeted in vivo biodistribution imaging application.8 Another disulfide probe was also shown to have utility in the radiometric detection of mitochondrial thiols.9Probes that fluoresce based on thiol cleavage of an Se-N bond have been used to label protein thiols10 and to monitor cellular redox status via reacting with each of GSH and H2O2.11 Nucleophilic addition to a sulfonate probe enabled thiol monitoring in the near-infra red spectral region.12
8.20.2
Selective Thiol Sensors: Opportunities for Improved Diagnostics
8.20.2.1
Glutathione
GSH is a major natural antioxidant. It is a tripeptide possessing an internal Cys residue. It is the most abundant low molecular weight biological thiol found in cells, present at mM levels (Cys and Hcy levels in cells are in the mM range). It is a major antioxidant, playing several protective roles in human health. For example, GSH scavenges reactive oxygen species as well as free radicals via direct reactions. It exchanges its sulfhydryl hydrogen for a free radical located on other molecules in a natural biological repair processes. It is produced in virtually all organs and is present in all tissues. GSH also plays a role in significant physiological processes such as DNA synthesis, amino acid transport, and xenobiotic detoxification. It also functions as a key enzyme substrate and cofactor, and regulates nitric oxide production.13,14 The ratio of reduced (GSH, thiol form) to oxidized (GSSG, disulfide form) is indicative of cellular redox status, and is a well-known measure of oxidative stress. In healthy cells and tissues the vast majority (> 90%) exists in the reduced (GSH) form.15
8.20.2.1.1
The significance of monitoring GSH whole blood levels
GSH is associated with a relatively large number of diseases. Importantly, it is generally agreed that GSH deficiency is a risk factor for chronic disease (Fig. 2). Chronic diseases are characterized by long duration and relatively slow progression. They include heart
Figure 2 Blood glutathione content in chronic diseases. Adapted from Lang, 2000. Values are expressed as mean standard error of the mean with units of mmol/1010 red blood cells and converted to mM which is presented on the secondary axis. GSH levels are decreased by 20% to 50% for chronically ill patients, which corresponds to a 300 mM decrease from approximately 1000 mM in healthy control subjects to an average of 700 mM in chronic patients.
438
Sensors for Biological Thiols
disease, stroke, cancer, chronic respiratory diseases, and diabetes. Collectively, they are by far the leading cause of mortality in the world, accounting for > 60% of all deaths. GSH is used as a biomarker to follow disease progression and treatment; however, healthy and disease levels that have been reported by various research groups have been in poor inter- and intra-laboratory agreement. For example, according to one author “it is evident that the measured concentrations . are highly divergent among different research groups, so these findings should be reconsidered once (there is) a general agreement about the physiological levels.”16 This issue of inconsistency has been attributed to the variability in analytical methods, the use of different matrices (plasma vs. blood), sample oxidation, different sample preparations, and a lack of general standardized techniques that persists to date.17 Despite the challenges of GSH monitoring, there is great current interest in its use as a risk factor for monitoring major disease. The range of GSH level depletion associated with specific chronic diseases is shown in Fig. 2.18 It varies from 20% to 50%, with an average of > 30%. This corresponds to a drop from approximately 1 mM (healthy levels) to an average of 0.7 mM. In a striking example of the potential diagnostic value of whole blood GSH levels, French researchers found that whole blood GSH testing led to earlier detection of asymptomatic patients having structural cardiac abnormalities, as compared to more traditional markers. Specifically, depletion in systemic blood GSH occurred prior to any detectable elevation in soluble TNF receptor-1 (sTNFR1), a well-known biomarker of symptomatic heart failure severity that, like GSH, is associated with oxidative stress. The researchers in this study reported GSH decreases as high as 58% in patients as compared to healthy controls.19 Other examples of GSH monitoring, all involving c.20–50% decreases, include (i) birth defects and complications during pregnancy, (ii) monitoring the effect of postoperative therapy, (iii) end-stage renal disease (ESRD) therapy, (iv) mitochondrial diseases, and (v) autism. For example, in the case of pregnancy and birth defects the measurement of whole blood GSH levels, along with Hcy levels (vide infra), was used to monitor preeclampsia and normotensive pregnancies.20 Antepartum levels of GSH as well as the free-to-oxidized ratios of Hcy were lower in pre-eclampsia and normotensive pregnancy when compared with corresponding postpartum values. Whole blood GSH measurements have also been used to monitor the responses of patients to glutamine (GLN) therapy following open heart surgery. This study was designed to potentially explain the shorter hospital stays as a function of normalized GSH levels after GLN supplementation.21 The restoration of depleted GSH levels has also been targeted as a therapeutic strategy in ESRD patients treated with hemodialysis.22 Levels of whole blood GSH in peritoneal dialysis patients have been used to monitor therapeutic outcomes.23 GSH monitoring is also a promising means to diagnose mitochondrial disorders, and, in clinical trials, to monitor autism by indexing the imbalance of oxidative and anti-oxidative stress systems in autistic patients.24 A review article on the significance of GSH monitoring in hemodialysis patients again underscores the fact that, despite the imprecision of current GSH assays, the need for GSH monitoring is too significant to overlook. “Despite some discrepant findings due to differences in . type of blood specimens tested (whole blood, plasma, erythrocytes or white blood cells) and, last but not least, measurement precision for glutathione system components, there is a general consensus that . the generally observed marked deficit of GSH appears . relevant to disease . Therapeutic approaches aimed at an upregulation of intracellular thiol concentration, which controls key molecular mechanisms of cell life, have already led to the demonstration of promising beneficial effects in terms of cardiovascular complications and anaemia correction.”22
8.20.2.1.2
Traditional methods for monitoring GSH whole blood levels
Many current laboratory methods use chromatography,22 relatively fragile materials, and/or a relatively high degree of sample processing.25,26 As noted above, sample handling, degradation, and related issues have led to significant concerns over measurement reproducibility.27 A current gold standard for GSH monitoring involves the glutathione-S-transferase catalyzed reaction of monochlorobimane and GSH. This is a highly selective enzyme-promoted reaction for GSH with specificity over all other biological thiols. The bimane fluorogens are useful in that they are nonfluorescent until reacted with thiols. However, monochlorobimane requires skilled handling and specialized storage as it is unstable at RT (storage is required below 25 C), unstable in water, and can only be exposed to ambient light for very short periods of time. Moreover, its hydrolysis products are also fluorescent and can cause artificially elevated readings. GSH kits that are commercially available generally use multistep procedures and fragile materials that require storage below 20 C, problematic for emerging nations. Most kits are also not selective for GSH over other thiols, and additionally note that Cys and DTT (dithiothreitol, a common thiol reducing agent) are interferents. For example, in the ThiolTrackerTM violet kit marketed for imaging GSH, the technical bulletin states that it reacts with the other thiols as well as GSH. Should simple, rapid, and inexpensive indicator-based methods for whole blood GSH enable limited or no sample handling, and prove straightforward enough for broad acceptance and standardization, there would be “fewer manipulations, resulting in fewer sources of error, shorter times to results, and reduced health hazards due to the handling of samples . such capabilities will be valuable in emergency, bedside, and point-of-care testing.”28,29
8.20.2.1.3
Recently reported fluorescent reagents for imaging GSH in cells
The majority of recently reported GSH-reactive probes have been designed and tested for cell imaging, rather than diagnostic applications. Many are useful examples of universal thiol-reactive reagents (vide supra)30–32,6 including a unique, new reversible conjugate addition probe enabling GSH quantitation in live cells.33 There are also examples of promising probes for GSH imaging
Sensors for Biological Thiols
439
Scheme 1 Examples of conjugate addition reactions of Cys, Hcy, and GSH to acrylates, followed by addition–elimination and loss (in the above cases) of MeOH to afford heterocyclic products. The conjugate addition step can readily occur in the case of each biothiol; however, cyclization is favored in the case of Cys, which occurs via a seven-membered ring. Scheme 2 shows an example of how this mechanism led to a Cys-selective sensor.
that exhibit GSH selectivity. Examples include a bis-spiropyran dipolar molecule receptor34 and a cyanine-derived near-infra red active agent.35
8.20.2.1.4
Designing a reagent for GSH diagnostics
To address the lack of GSH-selective probes for diagnostics, as a starting point, our group examined two of the probes that we had in hand for selective Cys or Hcy detection. These were coinvented by our colleague, Professor Xiaofeng Yang, a visiting Professor who spent a year in our research group from the College of Chemistry and Materials Science, Northwest University. These sensors functioned by a tandem conjugate addition/cyclization sequence (Scheme 1). One was a (hydroxymethoxyphenyl)benzothiazole-based probe that operated by a combined photo-induced electron transfer (PET) and excited-state intramolecular proton transfer mechanism. It enabled simultaneous, selective detection of both Cys and Hcy.36 The other was a seminaphthofluorescein (SNF)-based probe for long wavelength, highly selective detection of Cys. It coupled a conjugate addition/cyclization mechanism to a xanthene dye spirolactone-opening reaction (Scheme 2).37
Scheme 2 Cys addition to the SNF diacrylate leads to the release of the SNF fluorophore selectively over Hcy and GSH which require less favorable 8- and 12-membered heterocyclic ring formation.
440
Sensors for Biological Thiols
Figure 3 GSH binds to CTAB micelles via its carboxylate anions. This creates a favorable orientation for conjugate addition between its sulfhydryl and the resorufin acrylate fluorophore.
Neither of these probes afforded selective GSH signaling due to the requirement of a 12-membered ring intermediate in the mechanism (Scheme 1). To achieve selective detection of GSH over Cys and Hcy, one would need to employ chemistry that promoted large ring formation. Interestingly, it had been reported earlier that cetyltrimethylammonium bromide (CTAB) micelles can catalyze the intramolecular ring closure of larger rings, reversing the expected kinetics based on ring size.38 It is also known that GSH binds to the surface of cationic CTAB micelles via its two carboxylate anions, thereby orienting the internal sulfhydryl toward the interior of the micelle, potentially bringing it in proximity to a micelle-bound reagent.39 However, in the case of the SNF dye, the use of surfactants did not contribute to GSH-induced signaling but instead enhanced the kinetics of Cys detection. We reasoned that a relatively smaller, more planar dye structure compared to SNF probe might be better accommodated in the micelle interior while minimizing perturbations to micelle structure and supramolecular binding properties. We thus reacted resorufin with acryloyl chloride and attained a simple monoacrylate probe (Figs. 3 and 4) for selective detection of GSH in CTAB medium. The resorufin acrylate reacted instantly with GSH in the presence of CTAB, and with excellent selectivity compared to Cys and GSH (Fig. 5). Conversely, in solutions without added CTAB, this same probe functioned as a selective Cys sensor.
Figure 4 Resorufin acrylate, in the presence of CTAB-derived micelles, affords an instant, selective color and fluorescence change due to the presence of GSH via the mechanism shown in Scheme 1. Without added CTAB, it is Cys-selective.
Sensors for Biological Thiols
Figure 5
441
Schematic of a simple reduction/deproteinization step used DBS whole blood determination of GSH.
In human blood plasma, standard addition using the resorufin acrylate combined with CTAB showed that the GSH content was 3.24 0.14 mM, well within the reported GSH concentration range for human plasma samples from healthy individuals (1–6 mM).40 Recoveries of the known spiked amounts of GSH were between 99.2% and 102.3%, with good precision.41
8.20.2.1.5
Designing a blood spot test using a GSH-selective fluorescent reagent
Dried blood spot assays for GSH have previously been hampered by the optical properties of blood hemoglobin (Hb). Samples have been diluted 5000-fold to address this issue.42 Another approach has been the use of nonoptimal excitation wavelength for the dyes. Both of these strategies negatively influence signal-to-noise ratios and limits of detection. In a specific example of how hemoglobin interference affects measurements, it was observed that the fluorescence signal of monochlorobimane (MCB) in a 100 mM GSH standard solution decreased upon addition of 0.1% red blood cells, even though RBCs contain mM levels of GSH. MCB excitation at a nonoptimal wavelength minimized this issue, resulting in an increase in signal in the presence of 0.1% RBCs as compared to the MCB/GSH standard; however, nonoptimal excitation also decreased the overall signal.25 The dilution and suboptimal wavelength approaches do not completely remove the interference. Moreover, natural variability in hemoglobin concentration (120–180 mg/L) can still introduce errors, especially if variability in Hb concentration contributes to a total absorbance greater than 0.1 at the excitation wavelength, which will lead to nonlinear fluorescence responses. A small volume of blood dries rapidly on paper. The cells burst and leak GSH stores. As noted above, approximately three decades ago extreme sample dilution (e.g., 5000-fold) was used to minimize hemoglobin (Hb) interference in the determination of GSH in dried blood spots (DBS) on filter paper. Hb signal can also be minimized via a commercial product, HemogloBindÔ. It can isolate and remove up to 90% of blood Hb. However, it is relatively expensive and the residual Hb can still exhibit spectral overlap. To address these issues, we have developed a relatively simple method for DBS detection and quantitation of whole blood GSH. In addition to other proteins, the method removes Hb. Deproteinization is straightforward, accomplished via filtration through Sephadex. This is being compared to the use of acids and organic solvents for deproteinization. There is also no centrifugation step required. Due to the simplicity and user-friendliness of this technique it has potential for minimally invasive GSH DBS monitoring. It allows for analysis of samples mailed to centralized labs, as 24 h after spotting and drying was determined to be optimal for analysis, in the first generation method. Fig. 5 shows a schematic of the procedure. Briefly, a 30 mL blood sample is spotted on filter paper and dried for 24 h. The sample is extracted from the paper with buffer, treated with a commercial thiol reducing gel and passed through a PD MiniTrapÔ G-25 SephadexÔ column. When six 0.3 mL fractions were collected, the first three contained all of the Hb and other proteins, and the fourth and fifth fractions contained all of the sample-derived GSH. The concentration of GSH in a DBS of bovine blood was determined by standard addition. The value was 1.35 0.16 mM, which falls within the expected range of reported intracellular GSH levels. Recoveries of spiked GSH were between 94.0% and 108.6%, with suitable precision.43
8.20.2.2
Homocysteine
Hcy is a one-carbon homolog of Cys (Fig. 1). The majority (c.80%) of Hcy present in humans are protein bound. However, unlike Cys, Hcy is typically incorporated into proteins posttranslationally, via disulfide bonds to Cys residues or via amide bonds to Lys residues. Moreover, it is also a risk factor, at elevated levels, for several major pathologies including cardiovascular disease (CVD), birth defects, osteoporosis, Alzheimer’s, and renal failure among others. Despite decades of study, Hcy research remains controversial. There is no general agreement on Hcy’s association with disease.44,45 Hcy is a product of methionine metabolism. It is cytotoxic to cells if it over-accumulates. It is remethylated to methionine or converted to Cys by a transsulfuration pathway. Hcy is also metabolized to the thioester homocysteine thiolactone (HTL) in an
442
Sensors for Biological Thiols
Scheme 3 The reaction of HTL with the 3-amine moiety of protein lysine residues results in a post translational modification (PTM) known as N-Hcyprotein that alters protein function. This PTM leads to protein aggregates, free radicals, and carbonyls as well as degradation to N3 -Hcy-Lys isopeptide.
error-editing reaction in protein biosynthesis, when Hcy is selected in place of Met by methionyl-tRNA synthetase. HTL, known to be cytotoxic in experimental animals and cell cultures, reacts with protein lysine residues generating N-Hcy-protein (Scheme 3), which alters protein structure and function.
8.20.2.2.1
The significance of monitoring Hcy levels
When its metabolism is impaired, elevated intracellular Hcy and its metabolites are exported to the bloodstream. Higher extracellular Hcy and metabolite levels in plasma and urine are thus a function of lower activities of methionine synthase and cystathionine b-synthase, and (cofactor) folate, vitamin B12, or B6 deficiency. Hyperhomocysteinemia (HHcy) is typically defined as a condition in which total plasma homocysteine (tHcy), the sum of reduced (free sulfhydryl) and oxidized (disulfide) forms of Hcy, exceeds c.12 mM. Elevated disease risk is generally assumed when levels of plasma tHcy are above the HHcy threshold. Hcy is a substrate for toxic metabolites (Scheme 3). Homocysteine thiolactone (HTL) is a cyclic thioester that is more reactive and potentially more toxic than Hcy to human endothelial cells46 and to mice.47 HTL is excreted at sub-mM levels in human urine48 at disease levels. This latter value is in the range of circulating Hcy levels (circulating HTL levels are in the nM range). There is strong evidence that N-Hcy-protein is associated with coronary artery disease (CAD), induces cell death, causes autoimmune responses, and interferes with blood clotting.49 N3-Hcy-Lys is also a potentially useful independent clinical predictor of heart attack within 12 h of symptom presentation.50 Our group has also reported that N-Hcy-protein formation promotes free radical damage to proteins and can lead to protein carbonyl formation.51 Extensive large-scale clinical trials, spanning decades and tens of thousands of patients, have, however, not established a definitive relationship between Hcy lowering using vitamin therapy and reduced CVD risk. The dichotomy between the observational evidence that Hcy is linked to CVD (and to risk for stroke, venous thromboembolism, Alzheimer’s disease, neural tube defects and complications during pregnancy, inflammatory bowel disease and osteoporosis as well as several other major disorders) and the unclear outcomes of clinical trials based on vitamin therapy for Hcy lowering has rendered Hcy’s role in disease a controversial topic.44,45
8.20.2.2.2
A gap in Hcy monitoring in clinical studies
The term “total homocysteine (tHcy)” (vide supra) is actually a misnomer. In current clinical protocols used to measure tHcy levels, at least half of the Hcy in subjects may not be monitored. For example, Hcy-thiolactone (HTL) is found in all human cells, is more toxic than Hcy, and reacts readily with protein Lys residues affording N-Hcy-protein, and proteolytic degradation of N-Hcy-protein affords the isopeptide N3-Hcy-Lys. Levels of N-Hcy-protein and N3-Hcy-Lys are in the mM range in circulation, in range of those of “tHcy.” However, since these HTL-derived residues are amide-, rather than disulfide-bound, they are missed via the current clinical screens for Hcy. There thus appears a need for improved clinical monitoring of Hcy in conjunction with currently overlooked metabolites, which are present at levels in the range of tHcy, particularly in light of the fact that recent studies have not led to consensus on Hcy’s role in major human disease.
8.20.2.2.3
Methods for monitoring Hcy and metabolite levels
Hcy can be monitored via HPLC. It is relatively low throughput, requiring laborious sample pretreatment. HPLC testing can only be run sequentially, with each run taking approximately 10–30 min. The Axis-Shield immunoassay method requires instruments that can handle multiple reagents per test, as well as relatively specialized FP detection. Time required is approximately 30–45 min per test. The precision of the immunoassay is often lower than that of HPLC or enzymatic assays. Diazyme has created a commercial enzyme cycling test for Hcy that is relatively faster, less expensive, and less labor intensive than HPLC and immunoassays. Of the commercial tests, HPLC can monitor Hcy levels along with other structurally related analytes, such as Cys. HPLC methods have also been reported for determining levels of HTL52 and N-Hcy-protein53,54 as well as N3-Hcy-Lys.55
Sensors for Biological Thiols
8.20.2.2.4
443
Selective indicators for Hcy and metabolites
Reagents that afford a selective optical response corresponding to Hcy levels and/or its metabolites could facilitate basic research, clinical studies, and diagnostics in this field. It has been shown that such approaches can be relatively rapid and may obviate the need for sample preparation for Hcy determination56,57 and can be applied to N-Hcy-protein determination,51 thereby enabling higher throughput and less labor than currently possible, and without biological reagents. Overall, there have been fewer reports of optical sensing agents that are selective for Hcy as compared to Cys and GSH. Circulating Cys levels are typically 15–20 times higher than those of Hcy. Therefore, the selective detection of Hcy by abiotic chemosensors or dosimeters has been challenging. To the best of our knowledge there is only one report of an optical indicator method for the detection of an Hcy (HTL) by-product, N-Hcy-protein.51 Apart from redox-based Hcy-based selective indicators,56–58 the reaction of aldehyde fluorophores with Hcy and Cys to form thiazinanes and thiazolidine heterocycles, respectively,59 has been incorporated into the design of Hcy probes. Recent Hcy reagents include, for example, an iridium complex60 and the pyrene61 fluorophore. There are still challenges in the field including enhanced sensitivity at physiological thiol levels, selectivity, fluorescence via short wavelength excitation, and/or non-aqueous solvents.
8.20.2.2.5
A simple supramolecular approach for enhancing Hcy over Cys selectivity in aldehyde-functionalized fluorescein dyes
In 2004 our group published the selective detection of Cys and Hcy by fluorescein dialdehyde (FDA) (Scheme 4) over GSH and all other thiols and amino acids tested at the time. Upon reaction with either Hcy or Cys, fluorescent quenching as well as a color change was observed, due to stable 6- or 5-membered ring heterocycle formation. The reaction and the optical changes it produced were investigated at the time at high solution pH values. Four years later, Kim and coworkers reported that the lone pairs on thiazinane and thiazolidine heterocycles formed via reaction of a coumarin aldehyde could result in photo-induced electron transfer (PET) from the heterocycle amine lone pairs to the fluorophore.62 A similar PET process was likely also the cause of the quenching of FDA upon its reaction with Hcy and Cys. In addition, in the case of fluorescein, emission is enhanced upon deprotonation of the fluorophore eOH group. In order to attain selectivity between Hcy and Cys one would have to preferentially protonate either the Hcy- or Cys-derived heterocycle not only to shut off the PET quenching process but also to favor negative charge buildup on the fluorophore oxygen via proximal ammonium cation formation (Scheme 4).
Scheme 4 The reaction of fluorescein dialdehyde (FDA) with Cys and Hcy. FDA as well as the mono- and trialdehyde homologs can be tuned for selective Hcy signal enhancement by simply tuning the solution pH to 6.0 which results in selective protonation of the thiazinane over the thiazolidine. Selective ammonium ion creation leads to two synergistic effects: (i) PET quenching that was derived from the amine lone pairs is turned off and (ii) enhancement of negative charge on the proximal fluorophore oxygen, whose ionization state modulates fluorescence emission.
444
Sensors for Biological Thiols
Fortunately, due to their different ring sizes and the associated hybridization effects, thiazinane and thiazolidine ammonium cations have different pKas. At pH 6 the thiazolidine amine and ammonium forms are each significant at equilibrium, resulting in essentially no signal (enhancement and quenching canceled) derived from the presence of Cys. Conversely, the Hcy-derived thiazinane existed mainly in the ammonium form at pH 6 affords fluorescent enhancement selectively for Hcy. Further tuning of fluorophore structures, by accounting for neighboring group effects on charge states as well as quenching/enhancement mechanisms, will enable optimized Hcy signaling as well as related selective Hcy aminothiol metabolite detection.
8.20.3
Summary and Conclusions
There were two main goals for this chapter. The first was to demonstrate how the selectivity of covalent-based universal thiol sensing could be rendered selective for each of the major aminothiols by judicious incorporation of simple supramolecular strategies. In the case of GSH, a highly rapid and selective sensor was rendered GSH-selective by a catalytic micelle strategy. In the case of Hcy, pH-based tuning of the ionization states of the Cys and Hcy heterocyclic reaction products led to favorable electrostatic interactions between the Hcy product and the fluorophore, in addition to selectively turning off PET quenching. The second goal was to shed light on opportunities and needs in areas that have received relatively less attention from the thiol sensing community. In the case of GSH, there is a general need for selective diagnostic reagents for many specific applications. In the Hcy detection field, there is still a need for optimized probes and sensors. Moreover, targeting Hcy downstream products with optical reagents remains a relatively untapped area for future investigation.
References 1. (a) Peng, H.; Chen, W.; Cheng, Y.; Hakuna, L.; Strongin, R.; Wang, B. Sensors 2012, 12 (11), 15907–15946; (b) Jung, H. S.; Chen, X.; Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2013, 42 (14), 6019–6031; (c) Niu, L.-Y.; Chen, Y.-Z.; Zheng, H.-R.; Wu, L.-Z.; Tung, C.-H.; Yang, Q.-Z. Chem. Soc. Rev. 2015, 44 (17), 6143–6160; (d) Wang, K.; Peng, H.; Wang, B. J. Cell. Biochem. 2014, 115 (6), 1007–1022. 2. Johnson, I.; Spence, M. The Molecular Probes Handbook; Life Technologies Corporation: Carlsbad, 2010. 3. McMahon, B. K.; Gunnlaugsson, T. J. Am. Chem. Soc. 2012, 134 (26), 10725–10728. 4. Sreejith, S.; Divya, K. P.; Ajayaghosh, A. Angew. Chem. 2008, 120 (41), 8001–8005. 5. Yi, L.; Li, H.; Sun, L.; Liu, L.; Zhang, C.; Xi, Z. Angew. Chem. Int. Ed. 2009, 48 (22), 4034–4037. 6. Kim, G.-J.; Lee, K.; Kwon, H.; Kim, H.-J. Org. Lett. 2011, 13 (11), 2799–2801. 7. Lee, J. H.; Lim, C. S.; Tian, Y. S.; Han, J. H.; Cho, B. R. J. Am. Chem. Soc. 2010, 132 (4), 1216–1217. 8. Lee, M. H.; Han, J. H.; Kwon, P.-S.; Bhuniya, S.; Kim, J. Y.; Sessler, J. L.; Kang, C.; Kim, J. S. J. Am. Chem. Soc. 2012, 134 (2), 1316–1322. 9. Lim, C. S.; Masanta, G.; Kim, H. J.; Han, J. H.; Kim, H. M.; Cho, B. R. J. Am. Chem. Soc. 2011, 133 (29), 11132–11135. 10. Tang, B.; Xing, Y.; Li, P.; Zhang, N.; Yu, F.; Yang, G. J. Am. Chem. Soc. 2007, 129 (38), 11666–11667. 11. Xu, K.; Qiang, M.; Gao, W.; Su, R.; Li, N.; Gao, Y.; Xie, Y.; Kong, F.; Tang, B. Chem. Sci. 2013, 4 (3), 1079–1086. 12. Maity, D.; Govindaraju, T. Org. Biomol. Chem. 2013, 11 (13), 2098–2104. 13. Meister, A.; Anderson, M. E. Annu. Rev. Biochem. 1983, 52 (1), 711–760. 14. Perricone, C.; De Carolis, C.; Perricone, R. Autoimmun. Rev. 2009, 8 (8), 697–701. 15. Franco, R.; Schoneveld, O.; Pappa, A.; Panayiotidis, M. Arch. Physiol. Biochem. 2007, 113 (4-5), 234–258. 16. Iwasaki, Y.; Saito, Y.; Nakano, Y.; Mochizuki, K.; Sakata, O.; Ito, R.; Saito, K.; Nakazawa, H. J. Chromatogr. B 2009, 877 (28), 3309–3317. 17. Fahrenholz, T.; Wolle, M. M.; Kingston, H. S.; Faber, S.; Kern, J. C.; Pamuku, M.; Miller, L.; Chatragadda, H.; Kogelnik, A. Anal. Chem. 2015, 87 (2), 1232–1240. 18. Lang, C. A.; Mills, B. J.; Mastropaolo, W.; Liu, M. C. J. Lab. Clin. Med. 2000, 135 (5), 402–405. 19. Damy, T.; Kirsch, M.; Khouzami, L.; Caramelle, P.; Le Corvoisier, P.; Roudot-Thoraval, F.; Dubois-Randé, J.-L.; Hittinger, L.; Pavoine, C.; Pecker, F. PLoS ONE 2009, 4 (3), e4871. 20. Raijmakers, M. T.; Roes, E. M.; Poston, L.; Steegers, E. A.; Peters, W. H. Eur. J. Obstet. Gynecol. Reprod. Biol 2008, 138 (1), 39–44. 21. Engel, J.; Mühling, J.; Kwapisz, M.; Heidt, M. Acta Anaesthesiol. Scand. 2009, 53 (10), 1317–1323. 22. Santangelo, F.; Witko-Sarsat, V.; Drüeke, T.; Descamps-Latscha, B. Nephrol. Dial. Transplant. 2004, 19 (8), 1951–1955. 23. Moberly, J. B.; Logan, J.; Borum, P. R.; Story, K. O.; Webb, L. E.; Jassal, S. V.; Mupas, L.; Rodela, H.; Alghamdi, G. A.; Moran, J. E. J. Am. Soc. Nephrol. 1998, 9 (6), 1093–1099. 24. Atkuri, K. R.; Cowan, T. M.; Kwan, T.; Ng, A.; Herzenberg, L. A.; Herzenberg, L. A.; Enns, G. M. Proc. Natl. Acad. Sci. 2009, 106 (10), 3941–3945. 25. Raththagala, M.; Root, P. D.; Spence, D. M. Anal. Chem. 2006, 78 (24), 8556–8560. 26. Rahman, I.; Kode, A.; Biswas, S. K. Nat. Protoc. 2006, 1 (6), 3159–3165. 27. Rossi, R.; Milzani, A.; Dalle-Donne, I.; Giustarini, D.; Lusini, L.; Colombo, R.; Di Simplicio, P. Clin. Chem. 2002, 48 (5), 742–753. 28. Abugo, O. O.; Herman, P.; Lakowicz, J. R. J. Biomed. Opt. 2001, 6 (3), 359–365. 29. Abugo, O. O.; Nair, R.; Lakowicz, J. R. Anal. Biochem. 2000, 279 (2), 142–150. 30. Xu, J.-P.; Song, Z.-G.; Fang, Y.; Mei, J.; Jia, L.; Qin, A. J.; Sun, J. Z.; Ji, J.; Tang, B. Z. Analyst 2010, 135 (11), 3002–3007. 31. Lu, C.; Li, Q.; Chen, S.; Zhao, L.; Zheng, Z. Talanta 2011, 85 (1), 476–481. 32. Ahn, Y.-H.; Lee, J.-S.; Chang, Y.-T. J. Am. Chem. Soc. 2007, 129 (15), 4510–4511. 33. Jiang, X.; Yu, Y.; Chen, J.; Zhao, M.; Chen, H.; Song, X.; Matzuk, A. J.; Carroll, S. L.; Tan, X.; Sizovs, A. ACS Chem. Biol. 2015, 10 (3), 864–874. 34. Shao, N.; Jin, J.; Wang, H.; Zheng, J.; Yang, R.; Chan, W.; Abliz, Z. J. Am. Chem. Soc. 2009, 132 (2), 725–736. 35. Yin, J.; Kwon, Y.; Kim, D.; Lee, D.; Kim, G.; Hu, Y.; Ryu, J.-H.; Yoon, J. J. Am. Chem. Soc. 2014, 136 (14), 5351–5358. 36. Yang, X.; Guo, Y.; Strongin, R. M. Angew. Chem. Int. Ed. 2011, 50 (45), 10690–10693. 37. Yang, X.; Guo, Y.; Strongin, R. M. Org. Biomol. Chem. 2012, 10 (14), 2739–2741. 38. Wei, L.; Lucas, A.; Yue, J.; Lennox, R. B. Langmuir 1991, 7 (7), 1336–1339. 39. Huang, X.; Dong, Z.; Liu, J.; Mao, S.; Xu, J.; Luo, G.; Shen, J. Langmuir 2007, 23 (3), 1518–1522. 40. Wu, G.; Fang, Y.-Z.; Yang, S.; Lupton, J. R.; Turner, N. D. J. Nutr. 2004, 134 (3), 489–492.
Sensors for Biological Thiols
41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62.
445
Guo, Y.; Yang, X.; Hakuna, L.; Barve, A.; Escobedo, J. O.; Lowry, M.; Strongin, R. M. Sensors 2012, 12 (5), 5940–5950. Orfanos, A. P.; Naylor, E. W.; Guthrie, R. Anal. Biochem. 1980, 104 (1), 70–74. Hakuna, L.; Doughan, B.; Escobedo, J. O.; Strongin, R. M. Analyst 2015, 140 (10), 3339–3342. (a) Smulders, Y. M.; Blom, H. J. J. Inherit. Metab. Dis. 2011, 34 (1), 93–99; (b) Debreceni, B.; Debreceni, L. Cardiovasc. Ther. 2014, 32 (3), 130–138. Joseph, J.; Loscalzo, J. Nutrients 2013, 5 (8), 3235–3256. Kerkeni, M.; Tnani, M.; Chuniaud, L.; Miled, A.; Maaroufi, K.; Trivin, F. Mol. Cell. Biochem. 2006, 291 (1-2), 119–126. Borowczyk, K.; Shih, D. M.; Jakubowski, H. J. Alzheimers Dis. 2012, 30 (2), 225–231. Chwatko, G.; Jakubowski, H. Clin. Chem. 2005, 51 (2), 408–415. Jakubowski, H. J. Nutr. 2006, 136 (6), 1741S–1749S. Zaa˛ bczyk, M.; G1owacki, R.; Machnik, A.; Heród, P.; Kazek, G.; Jakubowski, H.; Undas, A. Clin. Chem. Lab. Med. 2011, 49 (4), 729–735. Sibrian-Vazquez, M.; Escobedo, J. O.; Lim, S.; Samoei, G. K.; Strongin, R. M. Proc. Natl. Acad. Sci. 2010, 107 (2), 551–554. Chwatko, G.; Jakubowski, H. Anal. Biochem. 2005, 337 (2), 271–277. Jakubowski, H. Anal. Biochem. 2008, 380 (2), 257–261. G1owacki, R.; Bald, E.; Jakubowski, H. Amino Acids 2011, 41 (1), 187–194. Mazur, P.; Kozynacka, A.; G1owacki, R.; Pfitzner, R.; Fijorek, K.; Sadowski, J.; Undas, A. Eur. J. Vasc. Endovasc. Surg. 2012, 43 (5), 588–593. Hakuna, L.; Escobedo, J. O.; Lowry, M.; Barve, A.; McCallum, N.; Strongin, R. M. Chem. Commun. 2014, 50 (23), 3071–3073. Escobedo, J. O.; Wang, W.; Strongin, R. M. Nat. Protoc. 2006, 1 (6), 2759–2762. Peng, H.; Wang, K.; Dai, C.; Williamson, S.; Wang, B. Chem. Commun. 2014, 50 (89), 13668–13671. Rusin, O.; Luce, N., St.; Agbaria, R. A.; Escobedo, J. O.; Jiang, S.; Warner, I. M.; Dawan, F. B.; Lian, K.; Strongin, R. M. J. Am. Chem. Soc. 2004, 126 (2), 438–439. Chen, H.; Zhao, Q.; Wu, Y.; Li, F.; Yang, H.; Yi, T.; Huang, C. Inorg. Chem. 2007, 46 (26), 11075–11081. Lee, H. Y.; Choi, Y. P.; Kim, S.; Yoon, T.; Guo, Z.; Lee, S.; Swamy, K.; Kim, G.; Lee, J. Y.; Shin, I. Chem. Commun. 2014, 50 (53), 6967–6969. Kim, T.-K.; Lee, D.-N.; Kim, H.-J. Tetrahedron Lett. 2008, 49 (33), 4879–4881.
Sensors for Biological Thiols
RM Strongin and JO Escobedo, Portland State University, Portland, OR, United States Ó 2017 Elsevier Ltd. All rights reserved.
8.20.1 8.20.1.1 8.20.1.1.1 8.20.1.1.2 8.20.1.1.3 8.20.1.2 8.20.1.2.1 8.20.1.2.2 8.20.2 8.20.2.1 8.20.2.1.1 8.20.2.1.2 8.20.2.1.3 8.20.2.1.4 8.20.2.1.5 8.20.2.2 8.20.2.2.1 8.20.2.2.2 8.20.2.2.3 8.20.2.2.4 8.20.2.2.5 8.20.3 References
8.20.1
Introduction Standard Biological Thiol Detection Methods Electrochemical methods Sensors employing biological materials Mass spectrometry and chromatography Universal Thiol-Reactive Chromophores and Fluorophores Conventional thiol-reactive probes More recent examples of universal thiol-reactive probes Selective Thiol Sensors: Opportunities for Improved Diagnostics Glutathione The significance of monitoring GSH whole blood levels Traditional methods for monitoring GSH whole blood levels Recently reported fluorescent reagents for imaging GSH in cells Designing a reagent for GSH diagnostics Designing a blood spot test using a GSH-selective fluorescent reagent Homocysteine The significance of monitoring Hcy levels A gap in Hcy monitoring in clinical studies Methods for monitoring Hcy and metabolite levels Selective indicators for Hcy and metabolites A simple supramolecular approach for enhancing Hcy over Cys selectivity in aldehyde-functionalized fluorescein dyes Summary and Conclusions
435 436 436 436 436 436 436 437 437 437 437 438 438 439 441 441 442 442 442 443 443 444 444
Introduction
The most abundant thiol amino acids in humans are cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). They have attracted great recent interest in the field of sensor and probe development, as each plays a major role in human health and disease. Chemists in the sensor and supramolecular fields have, relatively recently, embraced the challenges inherent in designing reagents that enable the selective imaging or determination of each specific amino thiol in natural media. Fluorescent chemosensors, dosimeters, and probes have been designed to overcome issues relating to not only optimizing responses, kinetics and compatibility with biological fluids, but also to address the very similar structures and reactivity of Cys, Hcy, and GSH and their varying relative abundance in native media (Fig. 1). The purpose of this chapter is twofold. It highlights (i) relatively under-addressed biomedical issues and opportunities in the thiol sensor field and (ii) sensors relying on non-covalent interactions to enable selectivity among each of the respective major biothiols in natural media. It does not summarize the biological thiol sensor field, which has been reviewed in detail previously.1Rather, it highlights the opportunities that still exist in this field.
Figure 1 Homocysteine (Hcy), cysteine (Cys), and glutathione (GSH) are the major low molecular weight biological thiol compounds. Note that Hcy and Cys differ by only one methylene in their side chains, and that Cys is the internal amino acid of the tripeptide GSH. Their similar structures render it challenging to create synthetic probes and indicators that exhibit selectivity for just one of these analytes. However, the fact that each is associated with distinctive significant physiology has prompted recent efforts in the abiotic sensor field to enable monitoring of each of these respective biothiols.
Comprehensive Supramolecular Chemistry II, Volume 8
http://dx.doi.org/10.1016/B978-0-12-409547-2.12624-1
435
436
Sensors for Biological Thiols
8.20.1.1
Standard Biological Thiol Detection Methods
Thiol detection procedures have generally involved chromatographic separations, immuno- and enzymatic assays, electrochemical, mass spectrometric, and flow injection technology. Each method has well-known strengths and limitations that are important to consider in designing functional sensing or probe reagents to either supplant or complement these methods.
8.20.1.1.1
Electrochemical methods
Electrochemical detection may be complicated by interference from oxidizable impurities. Electrochemical detection of thiols by Capillary Electrophoresis requires precision electrode alignment and isolating the detector from the separation voltage. Amperometric postcolumn detection of Cys and Hcy may suffer from low selectivity and high background current as Cys exhibits irreversible oxidation requiring positive overpotential. Small volumes in the separation capillaries in Capillary Electrophoresis require that the detector be placed in-line to minimize line broadening. Good sensitivity often requires dual electrode configurations. The stability of the detection cell components is another concern, depending on the type of analysis. Mercury and mercury amalgam electrodes have been widely used for thiols but have limitations including toxicity and poor stability. Chemically modified electrodes require complex preparation, can exhibit poor stability, and need well-controlled working conditions.
8.20.1.1.2
Sensors employing biological materials
Immunoassays require specialized detection systems such as fluorescent polarization (FP). The assays can be time consuming and have lower precision than enzymatic assays. Many enzymes are relatively unstable (low shelf life compared to common organic reagents). Enzyme cycling can exhibit advantages over immunoassays such as less sample pretreatment and run time as well as utility with routine clinical lab instrumentation. Radioimmunoassays involve toxic substances. STE (Substrate-Trapping-Enzyme) technology necessitates a batch chromatography step and exhibits relatively low precision. In general, biomaterials are relatively expensive and methods relying upon them often require tedious sample preparation and handling as compared to fully synthetic molecular probes.
8.20.1.1.3
Mass spectrometry and chromatography
Mass spectrometry coupled to HPLC or GC requires relatively tedious procedures that are less suitable for routine diagnostic applications. Gas chromatography–electron capture detection and flame photometry detection require involved sample preparations and/or high operating temperatures. Trap & Release Membrane Introduction Mass Spectrometry (T&R MIMS) requires time-consuming derivatizations and sophisticated instrumentation rendering it less suitable for routine analysis.
8.20.1.2 8.20.1.2.1
Universal Thiol-Reactive Chromophores and Fluorophores Conventional thiol-reactive probes
The aminothiols are essentially colorless and nonfluorescent in the visible spectral region. Thus, thiol-reactive optical probes have mainly been used as fluorescent labels for biomolecules via conjugation to their Cys residues, or in conjunction with separation techniques. The Molecular Probes Handbook has an entire chapter devoted to commercially available thiol-reactive probes.2 The majority are based on alkylation reactions of the nucleophilic sulfhydryl moiety and thus do not enable discrimination among GSH, Cys, or Hcy. Examples of drawbacks and limitations of some of the most well-known thiol probe types are summarized in Table 1. End users and inventors should also be aware that universal thiol-reactive probes are sometimes marketed or reported as GSH-selective. This is based on an assumption that, since GSH is the most abundant (mM levels) thiol in cells, any signaling from an interaction with Hcy or Cys would be negligible. This is likely true in cells for many probes, though there is a lack of published data to support such an assumption. However, in fluids such as human plasma, GSH is less abundant than Cys, and its detection by a universal thiol reagent will encounter interference from Cys and GSH.
Table 1
Drawbacks and limitations of some well-known thiol probes
Monobromobimane Labeling (i) Unstable at room temperature and in H2O; (ii) batch-to-batch varying impurity levels; (iii) fluorescent hydrolysis products produced upon labeling; (iv) gradient and relatively complex elution chromatography needed Iodoacetamide labeling (i) Cross-reactivity with histidine, tyrosine, and methionine; (ii) promotes loss of NH3 upon reaction with Cys residues; (iii) reduces mass spectrometric sensitivity O-phthalaldehyde labeling (i) Highly pH sensitive reaction; (ii) thiol adduct exhibits high photoinstability; (iii) cross-reactivity with many other amino acids Maleimide labeling (i) Hydrolysis peaks are encountered at the beginning and end of chromatographic elution; (ii) cross-linking to amines; (iii) unwanted rearrangements of conjugates Hexaiodoplatinate labeling Broad cross-reactivity with interferences, including thioethers, thiazolidines, and ascorbic acid
Sensors for Biological Thiols
8.20.1.2.2
437
More recent examples of universal thiol-reactive probes
Although probes that react with sulfhydryl-containing molecules are broadly available, there are many ongoing efforts toward alternatives. Several examples are based on conjugate addition of the sulfhydryl to a probe that is functionalized with a conjugate acceptor moiety. For instance, thiol addition to a Tb(III) complex was used in monitoring the enzymatic reduction of glutathione disulfide (GSSG) to GSH.3 In addition, conjugate addition to a squaraine dye resulted in successful monitoring of the elevation of blood thiol levels upon smoking.4 The conjugate reaction also enabled a functionalized coumarin to exhibit excellent kinetics (< 1 min reaction with thiols at RT) and also label protein-containing thiols efficiently at low (ng) levels.5 A probe functioning via conjugate addition, in conjunction with hydrogen bonding, was successfully employed for ratiometric imaging of GSH in cells.6 The nucleophilic reactivity of the sulfhydryl group has also led to probes functioning by other mechanisms. The reaction of aminothiols via an SNAr mechanism was used to create thiol-reactive fluorescein and cresyl violet derivatives. They were used in monitoring cholinesterase and glutathione-S-transferase activity, respectively. Thiol-reactive probes based on disulfide exchange reactions were used for two-photon imaging in cells and tissue7 as well as in a targeted in vivo biodistribution imaging application.8 Another disulfide probe was also shown to have utility in the radiometric detection of mitochondrial thiols.9Probes that fluoresce based on thiol cleavage of an Se-N bond have been used to label protein thiols10 and to monitor cellular redox status via reacting with each of GSH and H2O2.11 Nucleophilic addition to a sulfonate probe enabled thiol monitoring in the near-infra red spectral region.12
8.20.2
Selective Thiol Sensors: Opportunities for Improved Diagnostics
8.20.2.1
Glutathione
GSH is a major natural antioxidant. It is a tripeptide possessing an internal Cys residue. It is the most abundant low molecular weight biological thiol found in cells, present at mM levels (Cys and Hcy levels in cells are in the mM range). It is a major antioxidant, playing several protective roles in human health. For example, GSH scavenges reactive oxygen species as well as free radicals via direct reactions. It exchanges its sulfhydryl hydrogen for a free radical located on other molecules in a natural biological repair processes. It is produced in virtually all organs and is present in all tissues. GSH also plays a role in significant physiological processes such as DNA synthesis, amino acid transport, and xenobiotic detoxification. It also functions as a key enzyme substrate and cofactor, and regulates nitric oxide production.13,14 The ratio of reduced (GSH, thiol form) to oxidized (GSSG, disulfide form) is indicative of cellular redox status, and is a well-known measure of oxidative stress. In healthy cells and tissues the vast majority (> 90%) exists in the reduced (GSH) form.15
8.20.2.1.1
The significance of monitoring GSH whole blood levels
GSH is associated with a relatively large number of diseases. Importantly, it is generally agreed that GSH deficiency is a risk factor for chronic disease (Fig. 2). Chronic diseases are characterized by long duration and relatively slow progression. They include heart
Figure 2 Blood glutathione content in chronic diseases. Adapted from Lang, 2000. Values are expressed as mean standard error of the mean with units of mmol/1010 red blood cells and converted to mM which is presented on the secondary axis. GSH levels are decreased by 20% to 50% for chronically ill patients, which corresponds to a 300 mM decrease from approximately 1000 mM in healthy control subjects to an average of 700 mM in chronic patients.
438
Sensors for Biological Thiols
disease, stroke, cancer, chronic respiratory diseases, and diabetes. Collectively, they are by far the leading cause of mortality in the world, accounting for > 60% of all deaths. GSH is used as a biomarker to follow disease progression and treatment; however, healthy and disease levels that have been reported by various research groups have been in poor inter- and intra-laboratory agreement. For example, according to one author “it is evident that the measured concentrations . are highly divergent among different research groups, so these findings should be reconsidered once (there is) a general agreement about the physiological levels.”16 This issue of inconsistency has been attributed to the variability in analytical methods, the use of different matrices (plasma vs. blood), sample oxidation, different sample preparations, and a lack of general standardized techniques that persists to date.17 Despite the challenges of GSH monitoring, there is great current interest in its use as a risk factor for monitoring major disease. The range of GSH level depletion associated with specific chronic diseases is shown in Fig. 2.18 It varies from 20% to 50%, with an average of > 30%. This corresponds to a drop from approximately 1 mM (healthy levels) to an average of 0.7 mM. In a striking example of the potential diagnostic value of whole blood GSH levels, French researchers found that whole blood GSH testing led to earlier detection of asymptomatic patients having structural cardiac abnormalities, as compared to more traditional markers. Specifically, depletion in systemic blood GSH occurred prior to any detectable elevation in soluble TNF receptor-1 (sTNFR1), a well-known biomarker of symptomatic heart failure severity that, like GSH, is associated with oxidative stress. The researchers in this study reported GSH decreases as high as 58% in patients as compared to healthy controls.19 Other examples of GSH monitoring, all involving c.20–50% decreases, include (i) birth defects and complications during pregnancy, (ii) monitoring the effect of postoperative therapy, (iii) end-stage renal disease (ESRD) therapy, (iv) mitochondrial diseases, and (v) autism. For example, in the case of pregnancy and birth defects the measurement of whole blood GSH levels, along with Hcy levels (vide infra), was used to monitor preeclampsia and normotensive pregnancies.20 Antepartum levels of GSH as well as the free-to-oxidized ratios of Hcy were lower in pre-eclampsia and normotensive pregnancy when compared with corresponding postpartum values. Whole blood GSH measurements have also been used to monitor the responses of patients to glutamine (GLN) therapy following open heart surgery. This study was designed to potentially explain the shorter hospital stays as a function of normalized GSH levels after GLN supplementation.21 The restoration of depleted GSH levels has also been targeted as a therapeutic strategy in ESRD patients treated with hemodialysis.22 Levels of whole blood GSH in peritoneal dialysis patients have been used to monitor therapeutic outcomes.23 GSH monitoring is also a promising means to diagnose mitochondrial disorders, and, in clinical trials, to monitor autism by indexing the imbalance of oxidative and anti-oxidative stress systems in autistic patients.24 A review article on the significance of GSH monitoring in hemodialysis patients again underscores the fact that, despite the imprecision of current GSH assays, the need for GSH monitoring is too significant to overlook. “Despite some discrepant findings due to differences in . type of blood specimens tested (whole blood, plasma, erythrocytes or white blood cells) and, last but not least, measurement precision for glutathione system components, there is a general consensus that . the generally observed marked deficit of GSH appears . relevant to disease . Therapeutic approaches aimed at an upregulation of intracellular thiol concentration, which controls key molecular mechanisms of cell life, have already led to the demonstration of promising beneficial effects in terms of cardiovascular complications and anaemia correction.”22
8.20.2.1.2
Traditional methods for monitoring GSH whole blood levels
Many current laboratory methods use chromatography,22 relatively fragile materials, and/or a relatively high degree of sample processing.25,26 As noted above, sample handling, degradation, and related issues have led to significant concerns over measurement reproducibility.27 A current gold standard for GSH monitoring involves the glutathione-S-transferase catalyzed reaction of monochlorobimane and GSH. This is a highly selective enzyme-promoted reaction for GSH with specificity over all other biological thiols. The bimane fluorogens are useful in that they are nonfluorescent until reacted with thiols. However, monochlorobimane requires skilled handling and specialized storage as it is unstable at RT (storage is required below 25 C), unstable in water, and can only be exposed to ambient light for very short periods of time. Moreover, its hydrolysis products are also fluorescent and can cause artificially elevated readings. GSH kits that are commercially available generally use multistep procedures and fragile materials that require storage below 20 C, problematic for emerging nations. Most kits are also not selective for GSH over other thiols, and additionally note that Cys and DTT (dithiothreitol, a common thiol reducing agent) are interferents. For example, in the ThiolTrackerTM violet kit marketed for imaging GSH, the technical bulletin states that it reacts with the other thiols as well as GSH. Should simple, rapid, and inexpensive indicator-based methods for whole blood GSH enable limited or no sample handling, and prove straightforward enough for broad acceptance and standardization, there would be “fewer manipulations, resulting in fewer sources of error, shorter times to results, and reduced health hazards due to the handling of samples . such capabilities will be valuable in emergency, bedside, and point-of-care testing.”28,29
8.20.2.1.3
Recently reported fluorescent reagents for imaging GSH in cells
The majority of recently reported GSH-reactive probes have been designed and tested for cell imaging, rather than diagnostic applications. Many are useful examples of universal thiol-reactive reagents (vide supra)30–32,6 including a unique, new reversible conjugate addition probe enabling GSH quantitation in live cells.33 There are also examples of promising probes for GSH imaging
Sensors for Biological Thiols
439
Scheme 1 Examples of conjugate addition reactions of Cys, Hcy, and GSH to acrylates, followed by addition–elimination and loss (in the above cases) of MeOH to afford heterocyclic products. The conjugate addition step can readily occur in the case of each biothiol; however, cyclization is favored in the case of Cys, which occurs via a seven-membered ring. Scheme 2 shows an example of how this mechanism led to a Cys-selective sensor.
that exhibit GSH selectivity. Examples include a bis-spiropyran dipolar molecule receptor34 and a cyanine-derived near-infra red active agent.35
8.20.2.1.4
Designing a reagent for GSH diagnostics
To address the lack of GSH-selective probes for diagnostics, as a starting point, our group examined two of the probes that we had in hand for selective Cys or Hcy detection. These were coinvented by our colleague, Professor Xiaofeng Yang, a visiting Professor who spent a year in our research group from the College of Chemistry and Materials Science, Northwest University. These sensors functioned by a tandem conjugate addition/cyclization sequence (Scheme 1). One was a (hydroxymethoxyphenyl)benzothiazole-based probe that operated by a combined photo-induced electron transfer (PET) and excited-state intramolecular proton transfer mechanism. It enabled simultaneous, selective detection of both Cys and Hcy.36 The other was a seminaphthofluorescein (SNF)-based probe for long wavelength, highly selective detection of Cys. It coupled a conjugate addition/cyclization mechanism to a xanthene dye spirolactone-opening reaction (Scheme 2).37
Scheme 2 Cys addition to the SNF diacrylate leads to the release of the SNF fluorophore selectively over Hcy and GSH which require less favorable 8- and 12-membered heterocyclic ring formation.
440
Sensors for Biological Thiols
Figure 3 GSH binds to CTAB micelles via its carboxylate anions. This creates a favorable orientation for conjugate addition between its sulfhydryl and the resorufin acrylate fluorophore.
Neither of these probes afforded selective GSH signaling due to the requirement of a 12-membered ring intermediate in the mechanism (Scheme 1). To achieve selective detection of GSH over Cys and Hcy, one would need to employ chemistry that promoted large ring formation. Interestingly, it had been reported earlier that cetyltrimethylammonium bromide (CTAB) micelles can catalyze the intramolecular ring closure of larger rings, reversing the expected kinetics based on ring size.38 It is also known that GSH binds to the surface of cationic CTAB micelles via its two carboxylate anions, thereby orienting the internal sulfhydryl toward the interior of the micelle, potentially bringing it in proximity to a micelle-bound reagent.39 However, in the case of the SNF dye, the use of surfactants did not contribute to GSH-induced signaling but instead enhanced the kinetics of Cys detection. We reasoned that a relatively smaller, more planar dye structure compared to SNF probe might be better accommodated in the micelle interior while minimizing perturbations to micelle structure and supramolecular binding properties. We thus reacted resorufin with acryloyl chloride and attained a simple monoacrylate probe (Figs. 3 and 4) for selective detection of GSH in CTAB medium. The resorufin acrylate reacted instantly with GSH in the presence of CTAB, and with excellent selectivity compared to Cys and GSH (Fig. 5). Conversely, in solutions without added CTAB, this same probe functioned as a selective Cys sensor.
Figure 4 Resorufin acrylate, in the presence of CTAB-derived micelles, affords an instant, selective color and fluorescence change due to the presence of GSH via the mechanism shown in Scheme 1. Without added CTAB, it is Cys-selective.
Sensors for Biological Thiols
Figure 5
441
Schematic of a simple reduction/deproteinization step used DBS whole blood determination of GSH.
In human blood plasma, standard addition using the resorufin acrylate combined with CTAB showed that the GSH content was 3.24 0.14 mM, well within the reported GSH concentration range for human plasma samples from healthy individuals (1–6 mM).40 Recoveries of the known spiked amounts of GSH were between 99.2% and 102.3%, with good precision.41
8.20.2.1.5
Designing a blood spot test using a GSH-selective fluorescent reagent
Dried blood spot assays for GSH have previously been hampered by the optical properties of blood hemoglobin (Hb). Samples have been diluted 5000-fold to address this issue.42 Another approach has been the use of nonoptimal excitation wavelength for the dyes. Both of these strategies negatively influence signal-to-noise ratios and limits of detection. In a specific example of how hemoglobin interference affects measurements, it was observed that the fluorescence signal of monochlorobimane (MCB) in a 100 mM GSH standard solution decreased upon addition of 0.1% red blood cells, even though RBCs contain mM levels of GSH. MCB excitation at a nonoptimal wavelength minimized this issue, resulting in an increase in signal in the presence of 0.1% RBCs as compared to the MCB/GSH standard; however, nonoptimal excitation also decreased the overall signal.25 The dilution and suboptimal wavelength approaches do not completely remove the interference. Moreover, natural variability in hemoglobin concentration (120–180 mg/L) can still introduce errors, especially if variability in Hb concentration contributes to a total absorbance greater than 0.1 at the excitation wavelength, which will lead to nonlinear fluorescence responses. A small volume of blood dries rapidly on paper. The cells burst and leak GSH stores. As noted above, approximately three decades ago extreme sample dilution (e.g., 5000-fold) was used to minimize hemoglobin (Hb) interference in the determination of GSH in dried blood spots (DBS) on filter paper. Hb signal can also be minimized via a commercial product, HemogloBindÔ. It can isolate and remove up to 90% of blood Hb. However, it is relatively expensive and the residual Hb can still exhibit spectral overlap. To address these issues, we have developed a relatively simple method for DBS detection and quantitation of whole blood GSH. In addition to other proteins, the method removes Hb. Deproteinization is straightforward, accomplished via filtration through Sephadex. This is being compared to the use of acids and organic solvents for deproteinization. There is also no centrifugation step required. Due to the simplicity and user-friendliness of this technique it has potential for minimally invasive GSH DBS monitoring. It allows for analysis of samples mailed to centralized labs, as 24 h after spotting and drying was determined to be optimal for analysis, in the first generation method. Fig. 5 shows a schematic of the procedure. Briefly, a 30 mL blood sample is spotted on filter paper and dried for 24 h. The sample is extracted from the paper with buffer, treated with a commercial thiol reducing gel and passed through a PD MiniTrapÔ G-25 SephadexÔ column. When six 0.3 mL fractions were collected, the first three contained all of the Hb and other proteins, and the fourth and fifth fractions contained all of the sample-derived GSH. The concentration of GSH in a DBS of bovine blood was determined by standard addition. The value was 1.35 0.16 mM, which falls within the expected range of reported intracellular GSH levels. Recoveries of spiked GSH were between 94.0% and 108.6%, with suitable precision.43
8.20.2.2
Homocysteine
Hcy is a one-carbon homolog of Cys (Fig. 1). The majority (c.80%) of Hcy present in humans are protein bound. However, unlike Cys, Hcy is typically incorporated into proteins posttranslationally, via disulfide bonds to Cys residues or via amide bonds to Lys residues. Moreover, it is also a risk factor, at elevated levels, for several major pathologies including cardiovascular disease (CVD), birth defects, osteoporosis, Alzheimer’s, and renal failure among others. Despite decades of study, Hcy research remains controversial. There is no general agreement on Hcy’s association with disease.44,45 Hcy is a product of methionine metabolism. It is cytotoxic to cells if it over-accumulates. It is remethylated to methionine or converted to Cys by a transsulfuration pathway. Hcy is also metabolized to the thioester homocysteine thiolactone (HTL) in an
442
Sensors for Biological Thiols
Scheme 3 The reaction of HTL with the 3-amine moiety of protein lysine residues results in a post translational modification (PTM) known as N-Hcyprotein that alters protein function. This PTM leads to protein aggregates, free radicals, and carbonyls as well as degradation to N3 -Hcy-Lys isopeptide.
error-editing reaction in protein biosynthesis, when Hcy is selected in place of Met by methionyl-tRNA synthetase. HTL, known to be cytotoxic in experimental animals and cell cultures, reacts with protein lysine residues generating N-Hcy-protein (Scheme 3), which alters protein structure and function.
8.20.2.2.1
The significance of monitoring Hcy levels
When its metabolism is impaired, elevated intracellular Hcy and its metabolites are exported to the bloodstream. Higher extracellular Hcy and metabolite levels in plasma and urine are thus a function of lower activities of methionine synthase and cystathionine b-synthase, and (cofactor) folate, vitamin B12, or B6 deficiency. Hyperhomocysteinemia (HHcy) is typically defined as a condition in which total plasma homocysteine (tHcy), the sum of reduced (free sulfhydryl) and oxidized (disulfide) forms of Hcy, exceeds c.12 mM. Elevated disease risk is generally assumed when levels of plasma tHcy are above the HHcy threshold. Hcy is a substrate for toxic metabolites (Scheme 3). Homocysteine thiolactone (HTL) is a cyclic thioester that is more reactive and potentially more toxic than Hcy to human endothelial cells46 and to mice.47 HTL is excreted at sub-mM levels in human urine48 at disease levels. This latter value is in the range of circulating Hcy levels (circulating HTL levels are in the nM range). There is strong evidence that N-Hcy-protein is associated with coronary artery disease (CAD), induces cell death, causes autoimmune responses, and interferes with blood clotting.49 N3-Hcy-Lys is also a potentially useful independent clinical predictor of heart attack within 12 h of symptom presentation.50 Our group has also reported that N-Hcy-protein formation promotes free radical damage to proteins and can lead to protein carbonyl formation.51 Extensive large-scale clinical trials, spanning decades and tens of thousands of patients, have, however, not established a definitive relationship between Hcy lowering using vitamin therapy and reduced CVD risk. The dichotomy between the observational evidence that Hcy is linked to CVD (and to risk for stroke, venous thromboembolism, Alzheimer’s disease, neural tube defects and complications during pregnancy, inflammatory bowel disease and osteoporosis as well as several other major disorders) and the unclear outcomes of clinical trials based on vitamin therapy for Hcy lowering has rendered Hcy’s role in disease a controversial topic.44,45
8.20.2.2.2
A gap in Hcy monitoring in clinical studies
The term “total homocysteine (tHcy)” (vide supra) is actually a misnomer. In current clinical protocols used to measure tHcy levels, at least half of the Hcy in subjects may not be monitored. For example, Hcy-thiolactone (HTL) is found in all human cells, is more toxic than Hcy, and reacts readily with protein Lys residues affording N-Hcy-protein, and proteolytic degradation of N-Hcy-protein affords the isopeptide N3-Hcy-Lys. Levels of N-Hcy-protein and N3-Hcy-Lys are in the mM range in circulation, in range of those of “tHcy.” However, since these HTL-derived residues are amide-, rather than disulfide-bound, they are missed via the current clinical screens for Hcy. There thus appears a need for improved clinical monitoring of Hcy in conjunction with currently overlooked metabolites, which are present at levels in the range of tHcy, particularly in light of the fact that recent studies have not led to consensus on Hcy’s role in major human disease.
8.20.2.2.3
Methods for monitoring Hcy and metabolite levels
Hcy can be monitored via HPLC. It is relatively low throughput, requiring laborious sample pretreatment. HPLC testing can only be run sequentially, with each run taking approximately 10–30 min. The Axis-Shield immunoassay method requires instruments that can handle multiple reagents per test, as well as relatively specialized FP detection. Time required is approximately 30–45 min per test. The precision of the immunoassay is often lower than that of HPLC or enzymatic assays. Diazyme has created a commercial enzyme cycling test for Hcy that is relatively faster, less expensive, and less labor intensive than HPLC and immunoassays. Of the commercial tests, HPLC can monitor Hcy levels along with other structurally related analytes, such as Cys. HPLC methods have also been reported for determining levels of HTL52 and N-Hcy-protein53,54 as well as N3-Hcy-Lys.55
Sensors for Biological Thiols
8.20.2.2.4
443
Selective indicators for Hcy and metabolites
Reagents that afford a selective optical response corresponding to Hcy levels and/or its metabolites could facilitate basic research, clinical studies, and diagnostics in this field. It has been shown that such approaches can be relatively rapid and may obviate the need for sample preparation for Hcy determination56,57 and can be applied to N-Hcy-protein determination,51 thereby enabling higher throughput and less labor than currently possible, and without biological reagents. Overall, there have been fewer reports of optical sensing agents that are selective for Hcy as compared to Cys and GSH. Circulating Cys levels are typically 15–20 times higher than those of Hcy. Therefore, the selective detection of Hcy by abiotic chemosensors or dosimeters has been challenging. To the best of our knowledge there is only one report of an optical indicator method for the detection of an Hcy (HTL) by-product, N-Hcy-protein.51 Apart from redox-based Hcy-based selective indicators,56–58 the reaction of aldehyde fluorophores with Hcy and Cys to form thiazinanes and thiazolidine heterocycles, respectively,59 has been incorporated into the design of Hcy probes. Recent Hcy reagents include, for example, an iridium complex60 and the pyrene61 fluorophore. There are still challenges in the field including enhanced sensitivity at physiological thiol levels, selectivity, fluorescence via short wavelength excitation, and/or non-aqueous solvents.
8.20.2.2.5
A simple supramolecular approach for enhancing Hcy over Cys selectivity in aldehyde-functionalized fluorescein dyes
In 2004 our group published the selective detection of Cys and Hcy by fluorescein dialdehyde (FDA) (Scheme 4) over GSH and all other thiols and amino acids tested at the time. Upon reaction with either Hcy or Cys, fluorescent quenching as well as a color change was observed, due to stable 6- or 5-membered ring heterocycle formation. The reaction and the optical changes it produced were investigated at the time at high solution pH values. Four years later, Kim and coworkers reported that the lone pairs on thiazinane and thiazolidine heterocycles formed via reaction of a coumarin aldehyde could result in photo-induced electron transfer (PET) from the heterocycle amine lone pairs to the fluorophore.62 A similar PET process was likely also the cause of the quenching of FDA upon its reaction with Hcy and Cys. In addition, in the case of fluorescein, emission is enhanced upon deprotonation of the fluorophore eOH group. In order to attain selectivity between Hcy and Cys one would have to preferentially protonate either the Hcy- or Cys-derived heterocycle not only to shut off the PET quenching process but also to favor negative charge buildup on the fluorophore oxygen via proximal ammonium cation formation (Scheme 4).
Scheme 4 The reaction of fluorescein dialdehyde (FDA) with Cys and Hcy. FDA as well as the mono- and trialdehyde homologs can be tuned for selective Hcy signal enhancement by simply tuning the solution pH to 6.0 which results in selective protonation of the thiazinane over the thiazolidine. Selective ammonium ion creation leads to two synergistic effects: (i) PET quenching that was derived from the amine lone pairs is turned off and (ii) enhancement of negative charge on the proximal fluorophore oxygen, whose ionization state modulates fluorescence emission.
444
Sensors for Biological Thiols
Fortunately, due to their different ring sizes and the associated hybridization effects, thiazinane and thiazolidine ammonium cations have different pKas. At pH 6 the thiazolidine amine and ammonium forms are each significant at equilibrium, resulting in essentially no signal (enhancement and quenching canceled) derived from the presence of Cys. Conversely, the Hcy-derived thiazinane existed mainly in the ammonium form at pH 6 affords fluorescent enhancement selectively for Hcy. Further tuning of fluorophore structures, by accounting for neighboring group effects on charge states as well as quenching/enhancement mechanisms, will enable optimized Hcy signaling as well as related selective Hcy aminothiol metabolite detection.
8.20.3
Summary and Conclusions
There were two main goals for this chapter. The first was to demonstrate how the selectivity of covalent-based universal thiol sensing could be rendered selective for each of the major aminothiols by judicious incorporation of simple supramolecular strategies. In the case of GSH, a highly rapid and selective sensor was rendered GSH-selective by a catalytic micelle strategy. In the case of Hcy, pH-based tuning of the ionization states of the Cys and Hcy heterocyclic reaction products led to favorable electrostatic interactions between the Hcy product and the fluorophore, in addition to selectively turning off PET quenching. The second goal was to shed light on opportunities and needs in areas that have received relatively less attention from the thiol sensing community. In the case of GSH, there is a general need for selective diagnostic reagents for many specific applications. In the Hcy detection field, there is still a need for optimized probes and sensors. Moreover, targeting Hcy downstream products with optical reagents remains a relatively untapped area for future investigation.
References 1. (a) Peng, H.; Chen, W.; Cheng, Y.; Hakuna, L.; Strongin, R.; Wang, B. Sensors 2012, 12 (11), 15907–15946; (b) Jung, H. S.; Chen, X.; Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2013, 42 (14), 6019–6031; (c) Niu, L.-Y.; Chen, Y.-Z.; Zheng, H.-R.; Wu, L.-Z.; Tung, C.-H.; Yang, Q.-Z. Chem. Soc. Rev. 2015, 44 (17), 6143–6160; (d) Wang, K.; Peng, H.; Wang, B. J. Cell. Biochem. 2014, 115 (6), 1007–1022. 2. Johnson, I.; Spence, M. The Molecular Probes Handbook; Life Technologies Corporation: Carlsbad, 2010. 3. McMahon, B. K.; Gunnlaugsson, T. J. Am. Chem. Soc. 2012, 134 (26), 10725–10728. 4. Sreejith, S.; Divya, K. P.; Ajayaghosh, A. Angew. Chem. 2008, 120 (41), 8001–8005. 5. Yi, L.; Li, H.; Sun, L.; Liu, L.; Zhang, C.; Xi, Z. Angew. Chem. Int. Ed. 2009, 48 (22), 4034–4037. 6. Kim, G.-J.; Lee, K.; Kwon, H.; Kim, H.-J. Org. Lett. 2011, 13 (11), 2799–2801. 7. Lee, J. H.; Lim, C. S.; Tian, Y. S.; Han, J. H.; Cho, B. R. J. Am. Chem. Soc. 2010, 132 (4), 1216–1217. 8. Lee, M. H.; Han, J. H.; Kwon, P.-S.; Bhuniya, S.; Kim, J. Y.; Sessler, J. L.; Kang, C.; Kim, J. S. J. Am. Chem. Soc. 2012, 134 (2), 1316–1322. 9. Lim, C. S.; Masanta, G.; Kim, H. J.; Han, J. H.; Kim, H. M.; Cho, B. R. J. Am. Chem. Soc. 2011, 133 (29), 11132–11135. 10. Tang, B.; Xing, Y.; Li, P.; Zhang, N.; Yu, F.; Yang, G. J. Am. Chem. Soc. 2007, 129 (38), 11666–11667. 11. Xu, K.; Qiang, M.; Gao, W.; Su, R.; Li, N.; Gao, Y.; Xie, Y.; Kong, F.; Tang, B. Chem. Sci. 2013, 4 (3), 1079–1086. 12. Maity, D.; Govindaraju, T. Org. Biomol. Chem. 2013, 11 (13), 2098–2104. 13. Meister, A.; Anderson, M. E. Annu. Rev. Biochem. 1983, 52 (1), 711–760. 14. Perricone, C.; De Carolis, C.; Perricone, R. Autoimmun. Rev. 2009, 8 (8), 697–701. 15. Franco, R.; Schoneveld, O.; Pappa, A.; Panayiotidis, M. Arch. Physiol. Biochem. 2007, 113 (4-5), 234–258. 16. Iwasaki, Y.; Saito, Y.; Nakano, Y.; Mochizuki, K.; Sakata, O.; Ito, R.; Saito, K.; Nakazawa, H. J. Chromatogr. B 2009, 877 (28), 3309–3317. 17. Fahrenholz, T.; Wolle, M. M.; Kingston, H. S.; Faber, S.; Kern, J. C.; Pamuku, M.; Miller, L.; Chatragadda, H.; Kogelnik, A. Anal. Chem. 2015, 87 (2), 1232–1240. 18. Lang, C. A.; Mills, B. J.; Mastropaolo, W.; Liu, M. C. J. Lab. Clin. Med. 2000, 135 (5), 402–405. 19. Damy, T.; Kirsch, M.; Khouzami, L.; Caramelle, P.; Le Corvoisier, P.; Roudot-Thoraval, F.; Dubois-Randé, J.-L.; Hittinger, L.; Pavoine, C.; Pecker, F. PLoS ONE 2009, 4 (3), e4871. 20. Raijmakers, M. T.; Roes, E. M.; Poston, L.; Steegers, E. A.; Peters, W. H. Eur. J. Obstet. Gynecol. Reprod. Biol 2008, 138 (1), 39–44. 21. Engel, J.; Mühling, J.; Kwapisz, M.; Heidt, M. Acta Anaesthesiol. Scand. 2009, 53 (10), 1317–1323. 22. Santangelo, F.; Witko-Sarsat, V.; Drüeke, T.; Descamps-Latscha, B. Nephrol. Dial. Transplant. 2004, 19 (8), 1951–1955. 23. Moberly, J. B.; Logan, J.; Borum, P. R.; Story, K. O.; Webb, L. E.; Jassal, S. V.; Mupas, L.; Rodela, H.; Alghamdi, G. A.; Moran, J. E. J. Am. Soc. Nephrol. 1998, 9 (6), 1093–1099. 24. Atkuri, K. R.; Cowan, T. M.; Kwan, T.; Ng, A.; Herzenberg, L. A.; Herzenberg, L. A.; Enns, G. M. Proc. Natl. Acad. Sci. 2009, 106 (10), 3941–3945. 25. Raththagala, M.; Root, P. D.; Spence, D. M. Anal. Chem. 2006, 78 (24), 8556–8560. 26. Rahman, I.; Kode, A.; Biswas, S. K. Nat. Protoc. 2006, 1 (6), 3159–3165. 27. Rossi, R.; Milzani, A.; Dalle-Donne, I.; Giustarini, D.; Lusini, L.; Colombo, R.; Di Simplicio, P. Clin. Chem. 2002, 48 (5), 742–753. 28. Abugo, O. O.; Herman, P.; Lakowicz, J. R. J. Biomed. Opt. 2001, 6 (3), 359–365. 29. Abugo, O. O.; Nair, R.; Lakowicz, J. R. Anal. Biochem. 2000, 279 (2), 142–150. 30. Xu, J.-P.; Song, Z.-G.; Fang, Y.; Mei, J.; Jia, L.; Qin, A. J.; Sun, J. Z.; Ji, J.; Tang, B. Z. Analyst 2010, 135 (11), 3002–3007. 31. Lu, C.; Li, Q.; Chen, S.; Zhao, L.; Zheng, Z. Talanta 2011, 85 (1), 476–481. 32. Ahn, Y.-H.; Lee, J.-S.; Chang, Y.-T. J. Am. Chem. Soc. 2007, 129 (15), 4510–4511. 33. Jiang, X.; Yu, Y.; Chen, J.; Zhao, M.; Chen, H.; Song, X.; Matzuk, A. J.; Carroll, S. L.; Tan, X.; Sizovs, A. ACS Chem. Biol. 2015, 10 (3), 864–874. 34. Shao, N.; Jin, J.; Wang, H.; Zheng, J.; Yang, R.; Chan, W.; Abliz, Z. J. Am. Chem. Soc. 2009, 132 (2), 725–736. 35. Yin, J.; Kwon, Y.; Kim, D.; Lee, D.; Kim, G.; Hu, Y.; Ryu, J.-H.; Yoon, J. J. Am. Chem. Soc. 2014, 136 (14), 5351–5358. 36. Yang, X.; Guo, Y.; Strongin, R. M. Angew. Chem. Int. Ed. 2011, 50 (45), 10690–10693. 37. Yang, X.; Guo, Y.; Strongin, R. M. Org. Biomol. Chem. 2012, 10 (14), 2739–2741. 38. Wei, L.; Lucas, A.; Yue, J.; Lennox, R. B. Langmuir 1991, 7 (7), 1336–1339. 39. Huang, X.; Dong, Z.; Liu, J.; Mao, S.; Xu, J.; Luo, G.; Shen, J. Langmuir 2007, 23 (3), 1518–1522. 40. Wu, G.; Fang, Y.-Z.; Yang, S.; Lupton, J. R.; Turner, N. D. J. Nutr. 2004, 134 (3), 489–492.
Sensors for Biological Thiols
41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62.
445
Guo, Y.; Yang, X.; Hakuna, L.; Barve, A.; Escobedo, J. O.; Lowry, M.; Strongin, R. M. Sensors 2012, 12 (5), 5940–5950. Orfanos, A. P.; Naylor, E. W.; Guthrie, R. Anal. Biochem. 1980, 104 (1), 70–74. Hakuna, L.; Doughan, B.; Escobedo, J. O.; Strongin, R. M. Analyst 2015, 140 (10), 3339–3342. (a) Smulders, Y. M.; Blom, H. J. J. Inherit. Metab. Dis. 2011, 34 (1), 93–99; (b) Debreceni, B.; Debreceni, L. Cardiovasc. Ther. 2014, 32 (3), 130–138. Joseph, J.; Loscalzo, J. Nutrients 2013, 5 (8), 3235–3256. Kerkeni, M.; Tnani, M.; Chuniaud, L.; Miled, A.; Maaroufi, K.; Trivin, F. Mol. Cell. Biochem. 2006, 291 (1-2), 119–126. Borowczyk, K.; Shih, D. M.; Jakubowski, H. J. Alzheimers Dis. 2012, 30 (2), 225–231. Chwatko, G.; Jakubowski, H. Clin. Chem. 2005, 51 (2), 408–415. Jakubowski, H. J. Nutr. 2006, 136 (6), 1741S–1749S. Zaa˛ bczyk, M.; G1owacki, R.; Machnik, A.; Heród, P.; Kazek, G.; Jakubowski, H.; Undas, A. Clin. Chem. Lab. Med. 2011, 49 (4), 729–735. Sibrian-Vazquez, M.; Escobedo, J. O.; Lim, S.; Samoei, G. K.; Strongin, R. M. Proc. Natl. Acad. Sci. 2010, 107 (2), 551–554. Chwatko, G.; Jakubowski, H. Anal. Biochem. 2005, 337 (2), 271–277. Jakubowski, H. Anal. Biochem. 2008, 380 (2), 257–261. G1owacki, R.; Bald, E.; Jakubowski, H. Amino Acids 2011, 41 (1), 187–194. Mazur, P.; Kozynacka, A.; G1owacki, R.; Pfitzner, R.; Fijorek, K.; Sadowski, J.; Undas, A. Eur. J. Vasc. Endovasc. Surg. 2012, 43 (5), 588–593. Hakuna, L.; Escobedo, J. O.; Lowry, M.; Barve, A.; McCallum, N.; Strongin, R. M. Chem. Commun. 2014, 50 (23), 3071–3073. Escobedo, J. O.; Wang, W.; Strongin, R. M. Nat. Protoc. 2006, 1 (6), 2759–2762. Peng, H.; Wang, K.; Dai, C.; Williamson, S.; Wang, B. Chem. Commun. 2014, 50 (89), 13668–13671. Rusin, O.; Luce, N., St.; Agbaria, R. A.; Escobedo, J. O.; Jiang, S.; Warner, I. M.; Dawan, F. B.; Lian, K.; Strongin, R. M. J. Am. Chem. Soc. 2004, 126 (2), 438–439. Chen, H.; Zhao, Q.; Wu, Y.; Li, F.; Yang, H.; Yi, T.; Huang, C. Inorg. Chem. 2007, 46 (26), 11075–11081. Lee, H. Y.; Choi, Y. P.; Kim, S.; Yoon, T.; Guo, Z.; Lee, S.; Swamy, K.; Kim, G.; Lee, J. Y.; Shin, I. Chem. Commun. 2014, 50 (53), 6967–6969. Kim, T.-K.; Lee, D.-N.; Kim, H.-J. Tetrahedron Lett. 2008, 49 (33), 4879–4881.