Selective Chemodosimeter for the Ratiometric Electrochemical Detection of Phosphines

chemosensors

Communication An Organophosphorus(III)-Selective Chemodosimeter for the Ratiometric Electrochemical Detection of Phosphines

Sam A. Spring, Sean Goggins * and Christopher G. Frost Department of Chemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK; [email protected] (S.A.S.); [email protected] (C.G.F.) * Correspondence: [email protected]; Tel.: +44(0)1225-386231; Fax: +44(0)1225-386142

Received: 10 March 2019; Accepted: 3 April 2019; Published: 11 April 2019

Abstract: The high toxicity of phosphine and the use of organophosphines as nerve agent precursors has provoked the requirement for a rapid and reliable detection methodology for their detection. Herein, we demonstrate that a ferrocene-derived molecular probe, armed with an azidobenzene trigger, delivers a ratiometric electrochemical signal selectively in response to organophosphorus(III) compounds and can be accurately measured with an inexpensive, handheld potentiostat. Through an intensive assay optimization process, conditions were found that could determine the presence of a model organophosphine(III) nerve agent precursor within minutes and achieved a limit of detection for triphenylphosphine of just 13 ppm. Due to the portability of the detection system and the excellent stability of the probe in solution, we envisaged that this proof-of-concept of work could easily be taken into the ﬁeld to enable potentially toxic organophosphorus(III) compounds to be detected at the point-of-need.

Keywords: phosphine detection; ratiometric sensing; electrochemical chemodosimeter; point-of-use

1. Introduction Phosphine, the simplest phosphorus(III) compound, is a volatile toxic gas that is utilized in the agricultural industry as a fumigant [1,2]. Alkylphosphines and alkylphosphites also have acute toxicologies similar to phosphine [3–5]. Alkylphosphinites are listed as Schedule 1 compounds under the Chemical Warfare Convention as restricted precursors for the nerve agent VX, and alkylphosphites are listed as Schedule 3. In addition, phosphorus(III) compounds have been widely used in organic synthesis and the pharmaceutical industry, where a diverse number of new phosphorus(III) ligands with unknown toxicologies have been used in cross-coupling reactions, which remain a predominant synthetic tool [6,7]. However, though the toxicology of triphenylphosphine has been studied, with a permitted daily exposure (PDE) of 250 µg reported recently [5], only a few studies have explored the toxicity of less common aryl- and alkylphosphine ligands. In the absence of accurate toxicology, there remains a requirement to detect low-level concentrations of these potentially toxic analytes. The detection of phosphorus(III) compounds can be performed using chemiluminescence [8], gas chromatography–mass spectrometry [9], and flame photometry [10]. However, these methodologies are limited by the requirement for sensitive, lab-based equipment, and the transportation of highly toxic samples to them. Extending the current methods to organophosphorus(III) species has so far been limited to just volatile alkylphosphines until very recently, when nerve-agent mimics were shown to be detected via fluorescence [11]. However, for their adoption into a point-of-care device, the construction of a lightbox is needed [12]. Electrochemistry has emerged as a popular detection method, due to its rapid sample detection rate, and its use of inexpensive equipment, which allows for applications within small,

Chemosensors 2019, 7, 19; doi:10.3390/chemosensors7020019 www.mdpi.com/journal/chemosensors Chemosensors 2019, 7, 19 2 of 8 portable detection systems [13,14]. To combat problems arising from miniaturization, ratiometric electrochemical probes have become more widespread, with the minimization of errors increasing their reliability and reproducibility [15,16]. By obtaining direct conversions from the sample, systematic and sampling errors are reduced. Ferrocene-based probes are speciﬁcally and commonly used, due to their synthetic utility [17–19], aerobic and aqueous stability [20], and tunable oxidation potential [21,22]. Thus, we believe that we could utilize these advantageous properties to synthesize an electrochemical probe purposed for the development of a point-of-care solution for the detection of potentially highly-toxic organophosphorus(III) compounds. Herein, we describe the application of a ferrocene probe to the ratiometric electrochemical detection of organophosphorus(III) compounds, including a model nerve agent precursor, using a commercial, hand-held portable potentiometer.

2. Materials and Methods All reactions were carried out under an atmosphere of nitrogen, in oven-dried glassware, unless otherwise stated. Dichloromethane (DCM), and toluene were dried and degassed by passing through anhydrous alumina columns, using an Innovative Technology Inc. (Carouge, Switzerland) PS-400-7 solvent puriﬁcation system, and were stored under an atmosphere of nitrogen prior to use. 4-Aminobenzyl alcohol was purchased from Alfa Aesar (Heysham, UK). Ferrocenecarboxaldehyde was purchased from Fluorochem (Hadﬁeld, UK). All other chemicals were purchased from Sigma-Aldrich (Gillingham, UK). All chemicals were used as received. Desktop electrochemical analysis was performed by applying a 20 µL sample to a screen-printed electrochemical cell equipped with carbon working and counter electrodes, and a silver (pseudo Ag/AgCl) reference electrode. The potential across the cell was powered by a Metrohm Autolab PGSTAT30 potentiostat controlled by a laptop running General Purpose Electrochemical System (GPES) software in differential pulse mode (modulation = 0.04 s, interval = 0.1 s, initial voltage = −400 mV, end voltage = 600 mV, step potential = 3 mV, modulation amplitude 49.95 mV). Post-scan, a baseline correction (moving average: peak width = 0.03) was performed. Peak integrals were obtained by using the ‘peak search’ function, and conversions were calculated by using the equation:

Conversion (%) = (R product)/(R probe + R product) × 100 (1)

Handheld electrochemical analysis was performed by applying a 20 µL sample to a screen-printed electrochemical cell equipped with carbon working and counter electrodes, and a silver (pseudo Ag/AgCl) reference electrode. The potential across the cell was powered by a PalmSens Emstat3 Blue potentiostat controlled by a tablet, using PS Touch in differential pulse mode (equilibration time = 0 s, initial voltage = −800 mV, end voltage = 200 mV, step potential = 3 mV, pulse potential = 49.95 mV, pulse time = 0.1 s, scan rate = 0.015 V s−1). Currents were obtained using the ‘peak search’ function, and by ﬁnding the maximum current. The currents were calibrated by using Figure S1 to obtain the concentrations, and the conversions were calculated by using the equation:

Conversion (%) = ([product])/([probe] + [product]) × 100 (2)

3. Results

3.1. Concept Inspired by trigger–linker–effector methodology [23,24], benzyl ferrocenylcarbamates have been effectively employed for the ratiometric electrochemical detection of both enzymes, such as β-galactosidase and alkaline phosphatase [25,26], and small molecules, such as ﬂuoride and hydrogen peroxide [27,28]. To achieve our objective of developing a molecular probe for organophosphorus(III) species, we designed benzyl ferrocenylcarbamate 1 with a 4-azido trigger to allow for the chemoselectivity for the target to be attained through a Staudinger reaction. In the presence of the target, we proposed that the formation of iminophosphorane 2 would occur, which, under aqueous Chemosensors 2019, 7 3 of 9

Inspired by trigger–linker–effector methodology [23,24], benzyl ferrocenylcarbamates have been effectively employed for the ratiometric electrochemical detection of both enzymes, such as β- galactosidase and alkaline phosphatase [25,26], and small molecules, such as fluoride and hydrogen peroxide [27,28]. To achieve our objective of developing a molecular probe for organophosphorus(III) species, we designed benzyl ferrocenylcarbamate 1 with a 4-azido trigger to allow for the

Chemosensorschemoselectivity2019, 7, 19for the target to be attained through a Staudinger reaction. In the presence of3 the of 8 target, we proposed that the formation of iminophosphorane 2 would occur, which, under aqueous assay conditions, would be hydrolyzed to give aniline 3; then, a subsequent 1,6-elimination would assayrelease conditions, aminoferrocene would 4 be(Scheme hydrolyzed 1). Indeed, to give it anilinehas since3; then,been abrought subsequent to our 1,6-elimination attention that wouldwhilst releasethis project aminoferrocene was ongoing,4 the(Scheme release1). of Indeed, aminoferrocene it has since 4 from been 4-azidobenzyl brought to our ferrocenylcarbamate attention that whilst 1 thisunder project reductive was ongoing, conditions the releasehad been of aminoferrocenesuccessfully demonstrated4 from 4-azidobenzyl [29]. Due ferrocenylcarbamate to the significantly1 underdifferent reductive electronic conditions environments had been surrounding successfully the demonstrated iron center, aminoferrocene [29]. Due to the 4 signiﬁcantly should have different a lower electronicoxidation environmentspotential (Eox surrounding) compared thewith iron that center, of aminoferrocene1; and, thus it4 shouldshould havebe electrochemically a lower oxidation potentialdistinguishable. (Eox) compared with that of 1; and, thus it should be electrochemically distinguishable.

Scheme 1. Molecular structure of probe 1,, andand itsits proposedproposed mechanismmechanism forfor thethe phosphine-triggeredphosphine-triggered release of aminoferrocene 44.. 3.2. Synthesis, Stability, and Electrochemical Properties of 1 3.2. Synthesis, Stability, and Electrochemical Properties of 1 The synthesis of 1 was successfully achieved with a 77% overall yield via a Curtius rearrangement The synthesis of 1 was successfully achieved with a 77% overall yield via a Curtius of 4-azidobenzyl alcohol, obtained from 4-aminobenzyl alcohol through a Sandmeyer reaction, with rearrangement of 4-azidobenzyl alcohol, obtained from 4-aminobenzyl alcohol through a Sandmeyer ferrocenoyl azide, obtained from ferrocenecarboxylic acid (see Electronic Supporting Information (ESI)). reaction, with ferrocenoyl azide, obtained from ferrocenecarboxylic acid (see Electronic Supporting Once in hand, chemodosimeter 1 remained bench-stable for several months, and more importantly, Information (ESI)). Once in hand, chemodosimeter 1 remained bench-stable for several months, and it remained stable for a month as a solution in acetonitrile at room temperature, allowing for its storage more importantly, it remained stable for a month as a solution in acetonitrile at room temperature, as a ready-to-use solution (Figure S2). A comparison of the electrochemical behavior of probe 1 to allowing for its storage as a ready-to-use solution (Figure S2). A comparison of the electrochemical aminoferrocene 4 via differential pulse voltammetry (DPV) showed that the oxidation potential of behavior of probe 1 to aminoferrocene 4 via differential pulse voltammetry (DPV) showed that the probe 1 was signiﬁcantly higher than the oxidation potential of aminoferrocene 4, by 300 mV (Figure1). oxidation potential of probe 1 was significantly higher than the oxidation potential of aminoferrocene Evidently, the two peaks were fully resolved allowing for conversions to be calculated from the 4, by 300 mV (Figure 1). Evidently, the two peaks were fully resolved allowing for conversions to be integration of the two peaks (Figure S3), using Equation (1) (see Section2 Materials and Methods). calculated from the integration of the two peaks (Figure S3), using Equation 1 (see Section 2 Materials and Methods).

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Chemosensors 2019, 7, 19 4 of 8 Chemosensors 2019, 7 4 of 9

Figure 1. Differential pulse voltammogram obtained for probe 1 (0.5 mM) and aminoferrocene 4 (0.5 mM) in MeCN:Tris buffer (pH 9, 50 mM). FigureFigure 1. 1. DifferentialDifferential pulse pulse voltammogram voltammogram obtained obtained for forprobe probe 1 (0.51 mM)(0.5 mM)and aminoferrocene and aminoferrocene 4 (0.5 4 (0.5mM) mM) in inMeCN:Tris MeCN:Tris buffer buffer (pH (pH 9, 50 9, mM). 50 mM). 3.3. Sensitivity and Selectivity of 1 towards Organophosphorus(III) Compounds 3.3. Sensitivity and Selectivity of 1 towards Organophosphorus(III) Compounds 3.3.To Sensitivity test the and probe’s Selectivity response of 1 towards to phosphorou Organophosphorus(III)s(III) compounds, Compounds triphenylphosphine (PPh3) was

initiallyToTo testselected test the the probe’s asprobe’s a model response response analyte, toto phosphorous(III)asphosphorou it is an easy-to-handles(III) compounds, solid, triphenylphosphine triphenylphosphineand it is the standard (PPh (PPh phosphine3) was3) was initiallyofinitially choice selected inselected the Staudinger as as a a model model reaction analyte,analyte, (Table asas it is 1).an an Ineasy-to-handle easy-to-handleitial conditions solid, solid, were and and inspired it itis isthe the bystandard standard Staudinger phosphine phosphine ligation ofconditions,of choice choice inin with the Staudinger Staudinger10 equivalents reaction reaction of(Table tripheny (Table 1). 1In).itiallphosphine Initial conditions conditions in were a mixed inspired were solvent inspired by Staudinger system by Staudinger ligation of N ,N- ligationdimethylformamideconditions, conditions, with 10 (DMF)/water. with equivalents 10 equivalents Whileof tripheny this of deliver triphenylphosphinelphosphineed a 37% in conversiona mixed in asolvent mixedefficiency system solvent after of60 system minutesN,N- of Nat,dimethylformamide Nroom-dimethylformamide temperature, (DMF)/water.changing (DMF)/water. the While water-miscible this While deliver this co-solvented delivereda 37% conversion to acetonitrile a 37% effici conversion ency(MeCN) after efﬁciency or 60 1,4-dioxane minutes after 60improvedat min room at temperature,the room conversion temperature, changing efficiency changing the water-miscible considerably. the water-miscible co-solventProbe 1 was to co-solvent acetonitrile found to to (MeCN)be acetonitrile insoluble or 1,4-dioxane in (MeCN) alcoholic or 1,4-dioxanesolvents,improved and the improved aminoferrocene conversion the conversioneffi ciency4 was considerably.found efﬁciency to be considerably. unstableProbe 1 wasin both found Probe dimethylsulfoxide to1 wasbe insoluble found to in (DMSO)be alcoholic insoluble and intetrahydrofuransolvents, alcoholic and solvents, aminoferrocene (THF), and thus aminoferrocene preventing4 was found accurate 4towas be unstable found electrochemical to in beboth unstable dimethylsulfoxide analyses, in both so dimethylsulfoxide acetonitrile (DMSO) and was (DMSO)selectedtetrahydrofuran andas the tetrahydrofuran co-solvent (THF), thus moving (THF), preventing forward thus preventing accurate (Table S1).electrochemical accurate Altering electrochemical the analyses, solvent analyses, soratio acetonitrile to soa 3:1 acetonitrile ratiowas of wasacetonitrileselected selected as to asthe water the co-solvent co-solvent greatly moving improved moving forward forwardthe homogene (Table (Table S1).ity S1). Altering of Altering the reactionthe thesolventsolvent mixture, ratio ratio toallowing a to 3:1 a 3:1ratio for ratio moreof of acetonitrilereproducibleacetonitrile to tosampling, water water greatly greatly which improvedimproved in turn improved the homogene homogeneity the reliabilityity of of the the reactionof reaction the method. mixture, mixture, Further allowing allowing increasing forfor more more the reproducibleratioreproducible of acetonitrile sampling, sampling, to water which which was inin turnfoundturn improved to be detrimental the the reliability reliability to the of of thereaction. the method. method. Halving Further Further the increasing equivalents increasing thethe of ratio of acetonitrile to water was found to be detrimental to the reaction. Halving the equivalents of ratiotriphenylphosphine of acetonitrile to improved water was the found accuracy, to be as detrimental we believed to that the this reaction. limited Halving electrode the fouling, equivalents though of triphenylphosphine improved the accuracy, as we believed that this limited electrode fouling, though triphenylphosphinethis alteration lowered improved the conversion the accuracy, down as to we43% believed. The conversion that this limitedcould be electrode increased fouling, to 80% though within this alteration lowered the conversion down to 43%. The conversion could be increased to 80% within this20 minutes, alteration and lowered near-quantitative the conversion conversion down to within 43%. The 60 minutes, conversion by couldwarming be increased the assay tomixture 80% within to 50 20 minutes, and near-quantitative conversion within 60 minutes, by warming the assay mixture to 50 20°C. min,At temperatures and near-quantitative of above 50 conversion°C, solvent loss within and 60 inaccuracies min, by warming in sampling the assayled to unreliable mixture to results. 50 ◦C. °C. At temperatures of above 50 °C, solvent loss and inaccuracies in sampling led to unreliable results. At temperatures of above 50 ◦C, solvent loss and inaccuracies in sampling led to unreliable results. TableTable 1. 1. Optimization Optimization ofof thethe reaction assayassay of of 1 1 (1 (1 mM) mM) with with triphenylphosphine triphenylphosphine (10 (10 mM). mM). Table 1. Optimization of the reaction assay of 1 (1 mM) with triphenylphosphine (10 mM).

Exp. No. Solvent (1 M) Solvent:Water Ratio Conversion 1 Exp.Exp. No. No. Solvent Solvent (1 M) Solvent:Water Solvent:Water Ratio Ratio Conversion Conversion1 1 N,N-dimethylformamide 1 1:1 37% 1 1 N,NN,N-dimethylformamide-dimethylformamide(DMF) (DMF)(DMF) 1:1 1:1 37% 37% 2 1,4-dioxane 1:1 78% 2 2 3 acetonitrile1,4-dioxane1,4-dioxane (MeCN) 1:1 1:1 1:1 78%78% 79% 4 MeCN 3:1 79% 3 3 5 2 acetonitrileacetonitrileMeCN (MeCN) 3:1 1:1 1:1 79%79% 43% 6 3 MeCN 3:1 96% 4 MeCN 3:1 79% 4 MeCN 3:1 79% 1 2 3 Conversion determined by ratiometric electrochemical analysis. Five equivalents of PPh3. 5 equivalents of 52 ◦ MeCN 3:1 43% PPh3 5at2 50 C. MeCN 3:1 43% 63 MeCN 3:1 96% With63 the optimized assay conditionsMeCN in hand, the sensitivity of3:1 probe 1 was further96% examined (Figure2). At superstoichiometric concentrations of triphenylphosphine (2.5 mM and 5 mM), quantitative conversion was achieved within 60 min. No background reactivity was observed in

Chemosensors 2019, 7, 19 5 of 8 the absence of triphenylphosphine. The negligible background rate allowed for the accurate detection, within 60 min, of low concentrations of triphenylphosphine, 50 µM, with a 3% conversion observed. This value is within an order of magnitude of the limit of detection (LOD) for the fluorescence detection of organophosphorus pesticides, using an expensive desktop fluorimeter with highly sophisticated DNA-functionalized nanoparticle detection methodology [30]. This value also corresponds to a LOD of 13 ppm, which is the value of the reported LC50 for rats [2]. At the stoichiometric equivalents of Chemosensorstriphenylphosphine, 2019, 7 a 40% conversion was obtained within 30 min, with no significant further increase5 of 9 in conversion was observed after this period. This is consistent with precedent from the literature, where1 Conversion it has been determined reported that by theratiometric Staudinger electrochemical reaction requires analysis. two 2 Five equivalents equivalents of of triphenylphosphine PPh3. to release3 5 equivalents aniline [ 31of ].PPh3 at 50 °C.

Figure 2. Conversion of probe 1 (1 mM) mM) to to aminoferrocene aminoferrocene 4 after the addition of triphenylphosphine ◦ in MeCN:H2OO (3:1, (3:1, 1M) at 50 °C.C. Error Error bars bars represent represent the standard deviation where n = 3.

WithTo explore the optimized the selectivity assay conditions of probe 1 ,in a hand, selection the ofsensitivity organophosphorus(III) of probe 1 was compoundsfurther examined were (Figurescreened. 2). An At analyte superstoichiometric concentration ofconcentrations 1 mM was chosen, of triphenylphosphine to allow for changes (2.5 in themM rate and of reaction5 mM), quantitativeto be observed conversion (Figure3 was). Expectedly, achieved within electron-rich 60 minutes. arylphosphines No background showed reactivity an increased was observed reaction in therate absence in comparison of triphenylphosphi to that of triphenylphosphinene. The negligible (Figure background S4). However, rate allowed the change for inthe electronics accurate detection,also corresponded within 60 to minutes, a decrease of inlow aminoferrocene concentrations stability, of triphenylphosphine, which resulted in50 a µM, reduced with degree a 3% conversionof reproducibility observed. over This time. value Conversely, is within electron-deficientan order of magnitude arylphosphines of the limit showedof detection a significantly (LOD) for thereduced fluorescence rate of reaction detection in of comparison organophosphorus to triphenylphosphine. pesticides, using In general, an expensive both alkylphosphines desktop fluorimeter and withalkylphosphites highly sophisticated also exhibited DNA-functionalized lower rates of conversion, nanoparticle though detection pleasingly, methodology diethylmethylphosphonite, [30]. This value alsoa precursor corresponds in the to synthesis a LOD of of 13 VX ppm, nerve which gas, delivered is the value a positive of the reported conversion LC50 of 8%,for andrats it[2]. could At the be stoichiometriceasily distinguished equivalents from theof triphenylphosphine, background. Oxygen-sensitive a 40% conversion phosphines was obtained proved towithin be incompatible 30 minutes, with theno significant assay, due further to their increase degradation in conversion in the assay was media. observed Common after this phosphorus(V) period. This is compounds consistent withsuch precedent as phosphate from salts, the literature, potassium where hexafluorophosphate, it has been reported triphenylphosphine that the Staudinger oxide, reaction and requires triethyl phosphonoacetatetwo equivalents of yieldedtriphenylphosp no conversionhine to or release breakdown aniline of [31].1, highlighting its high specificity towards phosphorus(III) species. Other soft nucleophiles such as thiols were also tested and of them, only hydrogen sulfide (H2S) was shown to give any positive conversion with a 10% measurement after 60 min. Neither glutathione nor cysteine, both of which have also been shown to be able to reduce aryl azides [32], afforded any conversion. The robustness of the assay was then challenged by exposing compound 1 to a series of complex samples. Specifically, crude reaction mixtures from various Suzuki cross-coupling reactions were directly injected into the assay (Figure S5). The crude reaction mixture, which contained tetrakis(triphenylphosphine)palladium(0) delivered an 87% conversion rate. This positive result was attributed to the leaching of the organophosphorus(III) ligands, as alternative Pd(0) sources, without phosphine ligands, showed minimal conversion.

Figure 3. Conversion of probe 1 (1 mM) to aminoferrocene 4 60 minutes after the addition of

phosphine in MeCN:H2O (3:1, 1M) at 50 °C. Error bars represent the standard deviation, where n = 3.

(1) PPh3; (2) P(p-tol)3; (3) P(p-MeOPh)3; (4) P(p-CF3Ph)3; (5) P(OiPr)3; (6) MeP(OEt)2; (7) PCy3; (8) P(OPh)3; (9) PCl2Ph; (10) K2HPO4; (11) KPF6; (12) Na2O7P2; (13) triphenylphosphine oxide; (14) triethyl

phosphonoacetate; (15) hydrogen sulfide (H2S); (16) cysteine; (17) glutathione.

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1 Conversion determined by ratiometric electrochemical analysis. 2 Five equivalents of PPh3.

3 5 equivalents of PPh3 at 50 °C.

Figure 2. Conversion of probe 1 (1 mM) to aminoferrocene 4 after the addition of triphenylphosphine

in MeCN:H2O (3:1, 1M) at 50 °C. Error bars represent the standard deviation where n = 3.

With the optimized assay conditions in hand, the sensitivity of probe 1 was further examined (Figure 2). At superstoichiometric concentrations of triphenylphosphine (2.5 mM and 5 mM), quantitative conversion was achieved within 60 minutes. No background reactivity was observed in the absence of triphenylphosphine. The negligible background rate allowed for the accurate detection,Chemosensors within 2019, 7 60 minutes, of low concentrations of triphenylphosphine, 50 µM, with a 63% of 9 conversion observed. This value is within an order of magnitude of the limit of detection (LOD) for the fluorescenceTo explore detectionthe selectivity of organophosphorus of probe 1, a selection pesticides, of organophos using an expensivephorus(III) desktop compounds fluorimeter were withscreened. highly An sophisticated analyte concentration DNA-functionalized of 1 mM was nanoparticle chosen, to detectionallow for methodologychanges in the [30]. rate Thisof reaction value alsoto be corresponds observed (Figure to a LOD 3). Expectedly, of 13 ppm, electron-rich which is the arylphosphines value of the reported showed LC50an increased for rats reaction [2]. At therate stoichiometricin comparison equivalents to that of triphenylphosphineof triphenylphosphine, (Fig aure 40% S4). conversion However, was the obtained change within in electronics 30 minutes, also withcorresponded no significant to a further decrease increase in aminoferrocene in conversion stwasability, observed which after resulted this period. in a reduced This is consistentdegree of withreproducibility precedent fromover the time. literature, Conversely, where electron-dit has beeneficient reported arylphosphines that the Staudinger showed reaction a significantly requires twoChemosensorsreduced equivalents rate2019 of, 7 reactionof, 19 triphenylphosp in comparisonhine toto releasetriphenylphosphine. aniline [31]. In general, both alkylphosphines6 and of 8 alkylphosphites also exhibited lower rates of conversion, though pleasingly, diethylmethylphosphonite, a precursor in the synthesis of VX nerve gas, delivered a positive conversion of 8%, and it could be easily distinguished from the background. Oxygen-sensitive phosphines proved to be incompatible with the assay, due to their degradation in the assay media. Common phosphorus(V) compounds such as phosphate salts, potassium hexafluorophosphate, triphenylphosphine oxide, and triethyl phosphonoacetate yielded no conversion or breakdown of 1, highlighting its high specificity towards phosphorus(III) species. Other soft nucleophiles such as thiols were also tested and of them, only hydrogen sulfide (H2S) was shown to give any positive conversion with a 10% measurement after 60 minutes. Neither glutathione nor cysteine, both of which have also been shown to be able to reduce aryl azides [32], afforded any conversion.

The robustness of the assay was then challenged by exposing compound 1 to a series of complex samples. Specifically, crude reaction mixtures from various Suzuki cross-coupling reactions were directlyFigure injected 3. Conversion into the of probe assay1 (1 (Figure mM) to aminoferroceneS5). The crude4 60 minreaction after themixture, addition which of phosphine contained ◦ tetrakis(triphenylphosin MeCN:H2O (3:1,phine)palladium(0) 1M) at 50 C. Error bars delivered represent an the 87% standard conversion deviation, rate. where This positiven = 3. (1) result PPh3; was i attributedFigure(2) P(p -tol)to3. theConversion3; (3)leaching P(p-MeOPh) of of probethe3 ;organophosphorus(III) (4)1 (1 P( pmM)-CF3 Ph)to 3aminoferrocene; (5) P(O ligands,Pr)3; (6) 4 MeP(OEt)as60 alternativeminutes2; (7)after Pd(0) PCy the3; sources, (8)addition P(OPh) withoutof3 ; (9) PCl Ph; (10) K HPO ; (11) KPF ; (12) Na O P ; (13) triphenylphosphine oxide; (14) triethyl phosphinephosphine 2ligands, in MeCN:H showed2 2O 4(3:1, minimal 1M) at conversion. 650 °C. Error2 bars7 2 represent the standard deviation, where n = 3. phosphonoacetate; (15) hydrogen sulfide (H S); (16) cysteine; (17) glutathione. (1) PPh3; (2) P(p-tol)3; (3) P(p-MeOPh)3; (4)2 P(p-CF3Ph)3; (5) P(OiPr)3; (6) MeP(OEt)2; (7) PCy3; (8) 3.4.3.4. ApplicationsP(OPh)3; (9) PCl towards2Ph; (10) aa Point-of-UsePoint-of-Use K2HPO4; (11) AssayAssay KPF6; with(12)with Na aa HandheldHandheld2O7P2; (13)Potentiostat Potentiostattriphenylphosphine oxide; (14) triethyl phosphonoacetate; (15) hydrogen sulfide (H2S); (16) cysteine; (17) glutathione. ToTo highlighthighlight thethe point-of-usepoint-of-use capabilitycapability ofof probeprobe 11,, thethe modelmodel assayassay waswas testedtested usingusing aa PalmSensPalmSens EmStatEmStat33 Blue handheld potentiostat (Figure S6). The lower sensitivity of the potentiostat required anan increaseincrease inin probeprobe concentrationconcentration toto 1010 mM;mM; however,however, nono otherother modificationsmodifications ofof thethe reaction conditions werewere necessary.necessary. Due to the higher samplesample concentration,concentration, a correspondingcorresponding reductionreduction inin samplesample homogeneityhomogeneity waswas observed,observed, whichwhich resulted inin aa reducedreduced reliabilityreliability ofof thethe conversions,conversions, asas calculatedcalculated throughthrough peakpeak integration.integration. Therefore, inin thisthis instance,instance, thethe currentscurrents werewere measuredmeasured directlydirectly atat specificspecific oxidationoxidation potentials,potentials, and and they they were were used used to to calculate calculate the the conversions conversions via via a calibration a calibration curve curve (Figure (Figure S7). WhileS7). While this doesthis does not takenot take into into account account the diffusionthe diffusion coefficients coefficients for bothfor both probe probe1 and 1 and product product4, we 4, believewe believe that that they they are are similar similar enough enough to to validate validate this this proof-of-principle proof-of-principle experiment. experiment. ToTo testtest theirtheir potentialpotential useuse forfor thethe detectiondetection ofof nervenerve agentagent precursorsprecursors inin thethe field,field, thethe modifiedmodified assayassay waswas testedtested withwith thethe modelmodel precursorprecursor diethylmethylphosphinitediethylmethylphosphinite (Figure(Figure4 ).4).

Figure 4. Conversion of probe 1 (10 mM) to aminoferrocene after the addition of diethylmethylphosphinite ◦ (X mM) in MeCN:H2O (3:1, 1 M) at 50 C. Error bars represent the standard deviation, where n = 3.

Interestingly, the conversion obtained was signiﬁcantly higher (19 ± 3%) than that seen previously with a benchtop potentiostat (8 ± 2%). A similar increase was also observed when a comparative assay was performed while using triphenylphosphine (Figures S8–S10). We believe that the improved reactivity could be attributed to the higher concentration of the probe. More importantly, the tight error bars exhibited demonstrates the excellent reproducibility and reliability of the ratiometric detection Chemosensors 2019, 7, 19 7 of 8 assay. This, coupled with the negligible background rate, highlights the potential for the direct incorporation of the assay into a future point-of-use device.

4. Conclusions In conclusion, we have developed a ferrocene-based probe for the ratiometric electrochemical detection of phosphorus(III) compounds, utilizing the chemoselective Staudinger reaction. The probe showed excellent speciﬁcity for a range of phosphorus(III) compounds, compared against other phosphorus species, creating a highly selective and rapid detection methodology. The negligible background reactivity allowed for the reliable and reproducible detection of a model phosphine, down to 13 ppm. The detection method was successfully applied to the detection of an organophosphorus(III) nerve agent precursor, using a portable handheld potentiostat, demonstrating that the technique could be potentially applied to the rapid detection of nerve agents in the ﬁeld.

Supplementary Materials: The following are available online at http://www.mdpi.com/2227-9040/7/2/19/ s1, which includes the synthetic route for probe 1 (Scheme S1), and all synthetic procedures and NMR spectra for the synthesis of 1 and any intermediates synthesized during the process. Table S1: Assay optimization, Figure S1: Solution stability of probe 1, Figure S2: Kinetic linear transformation of probe 1, Figure S3: Voltammogram overlays, Figure S4: Extended phosphine scope, Figure S5: Complex sample testing, Figure S6: Handheld potentiostat setup, Figure S7: Calibration curve for the handheld potentiostat, Figure S8: Triphenylphosphine assay using the handheld potentiostat, Figures S9&S10: Competitive assays between desktop and handheld potentiostats. Author Contributions: Conceptualization, S.A.S., S.G. and C.G.F.; methodology, S.G.; investigation, S.A.S.; data curation, S.A.S.; writing—original draft preparation, S.A.S.; writing—review and editing, S.G.; supervision, C.G.F. Funding: This research received no external funding. Acknowledgments: The authors would like to thank Professor Frank Marken for loaning the use of the PalmSens Emstat3 Blue potentiostat, and Miss Amelia Langley for her guidance for its use. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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