Acceptor Influence on Thiolate Sensing by Hemicyanine Dyes - The

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Acceptor Influence on Thiolate Sensing by Hemicyanine Dyes Andrew J. Chung, Prashant S Deore, M. Sameer Al-Abdul-Wahid, Mohamed Aboelnga, Stacey D Wetmore, and Richard A. Manderville J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b00066 • Publication Date (Web): 21 Jan 2019 Downloaded from http://pubs.acs.org on January 21, 2019

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The Journal of Organic Chemistry

Acceptor Influence on Thiolate Sensing by Hemicyanine Dyes Andrew J. Chung,† Prashant S. Deore,† Sameer Al-Abdul-Wahid,† Mohamed Aboelnga,‡ Stacey D. Wetmore,‡,* and Richard A. Manderville†,* †

Departments of Chemistry and Toxicology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada

‡

Department of Chemistry & Biochemistry, University of Lethbridge, Lethbridge, Alberta, T1K 3M4, Canada

ABSTRACT: Promoting selective interactions between a nucleophile and electrophilic dye in complex environments is a central goal in nucleophilic chemosensor development. Commonly employed dyes are hemicyanines containing either the N-methylbenzothiazolium (Btz) or the N-methyl-3,3-dimethylindolium (Ind) acceptors. The dyes are related to ,unsaturated carbonyls and contain two sites of reactivity (C2 vs. C4) with the C2-site directly attached to the quaternary nitrogen possessing greater electrophilicity. We demonstrate the regioselectivity between reactions of sodium thiomethoxide (NaSMe) with two electrophilic hemicyanine dyes bearing Btz (1) or Ind (2) in dipolar aprotic solvent-water mixtures. Adduct complexation was followed by NMR spectroscopy and structures were optimized in the gas phase to estimate relative adduct stability. The key results include finding a preference for thiolate attachment at the C 4-site to generate an enamine adduct with no evidence for attachment at the more electrophilic C2-position. Equilibration between NaSMe and water also affords NaOH that displays a thermodynamic preference for C2-attachment. Dye 1 containing the Btz moiety exhibits greater selectivity for thiolate addition, with dye 2 being more reactive toward adventitious water to generate OH-adducts. Our data affords diagnostic 1H/13C NMR adduct signals, regioselectivity for various dye/nucleophile combinations and suggests use of the Btz acceptor for direct thiolate detection.

INTRODUCTION. Optical probes are highly sought after for real-time quantification of nucleophilic species in complex matrices.1,2 A common strategy for sensing nucleophiles, such as thiols/thiolates,3-6 toxic cyanide7-10 and hydroxide11 is to construct an electrophilic dye that can react specifically with the nucleophile to generate an optical readout. Hemicyanines are a family of dyes that suit this purpose and feature a donor--acceptor (D--A) motif.12 The acceptor is a positively charged nitrogen heterocycle that is connected via a conjugated system to the aromatic donor bearing a terminal hydroxyl, alkoxy, or amino group. The two acceptors that appear to be used interchangeably for nucleophile sensing are the N-methylbenzothiazolium (Btz) or the N-methyl-3,3-dimethylindolium (Ind) moiety (Figure 1A). When connected to a suitable donor the resulting hemicyanine displays strong visible absorbance with quenched fluorescence in polar protic solvents due to an excited-state intramolecular charge transfer (ICT) process. Nucleophilic attachment provides a basis for detection by blocking ICT to turn-on emission and cause a substantial blue-shift in absorbance that can lead to loss of dye color.12 Despite efforts in creating hemicyanines and hybrids thereof that appear to exhibit impressive nucleophile sensing characteristics, even in cellular matrices,3-6 the adduct structures are seldom rigorously characterized.12 This lack of product identification generates ambiguity

Figure 1. (A) Schematic of hemicyanine dyes with typical donor (D) and acceptor (A) motifs. (B) Nucleophilic (Nu) reaction pathways for a hemicyanine with two sites (C2 and C4) for covalent modification. (C) Structures of hemicyanines (1, 2) used in this study.

regarding sensor selectivity, which may be generating false-positive readouts due to reactions with non-target nucleophiles. It also hinders our understanding of hemicyanine reactivity, which have potential to react with various nucleophiles (Nu) to give rise to a diverse number of products (Figure 1B). Nu attachment can occur at the C2or C4-sites and how the nature of the nucleophile and dye structure dictates site-preference is uncertain. For the C4-adduct, it is also uncertain which tautomer (enamine vs. iminium ion4,13) is favored, and it is conceivable for the iminium ion to react with a second equivalent of nucleo-

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phile to produce a C2,C4-diadduct.12,14 Mass spectral determinations also strongly favor cationic dye recovery from the neutral adduct.3 As a further complication, nucleophilic reactions with hemicyanines are typically carried out in water–dipolar aprotic solvent mixtures with DMF, CH3CN or DMSO added to increase nucleophilic reactivity and promote covalent attachment to the dye.8,15,16 However, under these conditions, equilibration between H2O and the added nucleophile can produce hydroxide, which may react with the dye to afford OHadducts that also lack ICT.11 Consequently, model studies aimed at providing a deeper understanding of hemicyanine dye reactivity toward nucleophilic species may assist in the design of more robust chemosensors. Herein, we describe the nucleophilic reactions of two representative hemicyanines containing the Btz (1) and Ind (2) acceptor moieties attached to a donor bearing an exocyclic amine (Figure 1C) in water-dipolar aprotic solvent mixtures. The dyes appeared to exhibit almost identical sensing characteristics, as monitored by optical (UVvis/fluorescence) spectroscopy, with certain thiolates and hydroxide able to block the dye ICT, which causes loss of the visible dye absorbance and produces new absorbance bands in the UV. However, the nucleophile addition products displayed diverse optical properties that suggested acceptor influence on the nature of the products produced. This acceptor influence was further explored using two-dimensional NMR spectroscopy to conduct product analysis for the dye reactions with the target thiolate, sodium thiomethoxide (NaSMe). Our findings provide unambiguous structural evidence for the C4- and C2-adducts - affording diagnostic NMR fingerprints for assignment of the two adduct types. We have also utilized density function theory (DFT) calculations to provide an estimate of the relative adduct stabilities for reactions of MeS− and HO− with 1 and 2. Our results provide insight into the regioselectivity displayed by the dye/Nu combinations and highlight the Btz moiety as a better acceptor choice than Ind for thiolate detection strategies using electrophilic hemicyanine dye derivatives. RESULTS AND DISCUSSION. Optical Analysis of Dye-Nu Interactions. The reactivity of 1 and 2 toward neutral and anionic nucleophiles were initially evaluated by UV-vis spectroscopy in aqueous acetonitrile solution (CH3CN-H2O, 9:1, v:v) at ambient temperature (Figure 2); conditions previously employed to monitor selective reactivity of a hybrid coumarinhemicyanine dye toward cyanide.8 The hemicyanine dye 1 exhibits a strong ICT band centered at ~ 525 nm ( = 3.7 x 104 M−1cm−1).17 Loss of the ICT band is indicative of nucleophile attachment,8 and of the nucleophiles tested (Figure 2A), only the anionic NaSMe and NaOH reacted with the dye to cause loss of the 525 nm band and generate new absorption bands in the UV (Figure 2B). Reaction of 1 with NaSMe afforded two broad UV bands centered at 390 nm and 300 nm, while the reaction with NaOH displayed a sharper UV band peaking at 360 nm.

Figure 2. (A) Absorbance ratio at 525 nm of 1 (10 M) upon addition of various nucleophiles (100 equiv.). (B) UV-vis spectra of 1 (10 M) in the absence and presence of 100 equiv. NaOH and NaSMe. Spectra were acquired in CH3CN-H2O (9:1, v:v) after mixing at room temperature for 5 min.

Under analogous conditions the hemicyanine 2 (max = 540 nm,  = 5.4 x 104 M−1cm−1) also displayed selective responses toward NaSMe and NaOH, (Figure S1A, Supporting Information (SI)). However, in this instance the solutions containing 2 with excess NaOH and NaSMe produced UV bands peaking at ~ 300 nm with similar intensities. Interestingly, the reaction of 2 with NaOH afforded a solution with  = 290 nm, for a blue-shift of 70 nm compared to reaction of NaOH with 1, suggesting different product structures. The fluorescent properties of the dyes in the absence and presence of excess NaSMe and NaOH is depicted in Figure 3. Dye 1 (ex = 525 nm, em = 595 nm, red traces, Figure 3A) exhibited greater emission intensity than solutions containing the added nucleophiles. In the presence of excess NaOH the product displayed blue emission at 457 nm following excitation at 360 nm (green traces, Figure 3A), while excitation at 311 nm for the solution containing NaSMe produced dual emission with peaks at 385 and 437 nm (blue traces, Figure 3A). In contrast, dye 2 (ex = 540 nm, em = 590 nm, red traces, Figure 3B) was less emissive than its nucleophilic addition products. The NaOH adduct displayed relatively bright emission at 379 nm following excitation at ~ 300 nm (green traces, Figure 3B) that is blue-shifted by 78 nm compared to the corresponding adduct of 1. The NaSMe adduct displayed weaker emission intensity at 384 nm (blue traces, Figure 3B). Thus, while the dyes displayed almost identical sensing abilities (i.e. loss of the ICT band), the optical properties of the products indicated diverse product formation that was dictated by the dye acceptor moiety.


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Figure 3. Excitation and emission spectra of hemicyanine dyes ((10 M) (A) 1, (B) 2, in the absence and presence of 100 equiv. NaOH or NaSMe. Spectra were obtained in CH3CNH2O (9:1, v/v) and acquired after mixing at room temperature for 5 min.

NaSMe Complexation Monitored by NMR. To further determine the acceptor influence on product formation mediated by dyes 1 and 2 in the presence of NaSMe as the target thiolate nucleophile, a 600-MHz NMR study was carried out in DMSO-d6-D2O solvent mixtures ( relative to CD2HSOCD3 (2.50) in ppm, J in hertz; see Table 1). Depending on the amount of D2O added, these conditions also afforded hydroxide production and the products that arise as a consequence are also described. Addition of 5 equiv. NaSMe in D2O to a DMSO-d6 solution of 1 (final solution DMSO-d6-D2O, 9:1, v/v) resulted in the disappearance of the 1H dye signals for the trans doublebond (Ha, 8.06, Hb, 7.61, J = 15.2, Figure S2A) and the simultaneous appearance of a new set of doublets upfield at 4.25 and 4.72 (J = 9.6, Figure S2B). Unambiguous assignment of the structure as the enamine C4-SMe adduct was obtained through 1H-13C correlations (Figure 4). The sp3 Ha proton that is directly attached to C4 (51.1 ppm, Figure 4A) revealed through-bond correlations with C2 (143.9), C5 (129.0), C3 (89.3) and SMe (C6, 13.9) (Figure 4C), while the SMe signal (3H, 1.90, s) also displayed a through-bond correlation with C4 (Figure 4B), confirming MeS− attachment to the C4-site of the dye. Also consistent with the enamine tautomer for the C4-SMe adduct, the 1H/13C signals of the sp3 N1-Me resonated at 3.13/30.0, while for dye 1 the quaternary N1-Me was observed at 4.22/35.3.

1

13

Figure 4. NMR characterization of the C4-SMe adduct of 1 in DMSO-d6-D2O (9:1, v/v): (A) H- C HSQC spectrum highlighting 13 1 13 1 13 C resonances (C4, C3) for Ha and Hb; (B) H- C HMBC spectrum revealing through-bond correlation between SMe-C4; (C) H- C 1 13 HMBC spectrum revealing through-bond correlations between Ha and Hb with C2, C5, C3 and C6; (D) H- C HSQC spectrum 13 highlighting the C resonance (C6) of SMe.



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Scheme 1. Proposed pathways for reaction of 1 with NaSMe in DMSO-D2O mixtures.

The reaction of 5 equiv. of NaSMe with 1 was also carried out in 1:1 (v/v) DMSO-d6-D2O (Figure S2C). The major product displayed a pair of doublets for a trans double-bond at 7.20 and 5.88 (J = 15.7), that was initially ascribed to a C2-adduct. However, the 13C NMR spectrum contained a signal at 169.3 ppm for C2 (Figure S3, SI) that exhibited through-bond correlations (Figure 5) with Hb (5.88) and N1Me (3.06). No 1H-13C correlations were observed between the NMR signals for the dye and the added NaSMe. These observations suggested initial formation of the C2-OH solvolysis adduct that underwent ringopening to afford the amide with a C2-carbonyl (Scheme 1). In this instance it was possible to analyze the hydrolysis product using negative ionization electrospray mass spectrometry (ESI−), which afforded the expected amide parent ion at [M – H]− = 341 (Figure S3, SI), for a mass of 342 that is 17 mass units (OH) heavier than 1 (m/z 325). For dye 2 addition of 5 equiv. NaSMe produced the same product in 1:1 and 9:1 DMSO-d6-D2O mixtures (Figure S4, SI). In 1:1 DMSO-d6-D2O (Figure S4C) the adduct displayed a pair of doublets for the trans double-bond at 5.98 and 6.59 (J = 16.1), suggesting formation of a C2adduct. Its C2 resonance occurred at 101.2 ppm, which is similar in chemical shift to the sp3-spirocarbon of the closed-form of indolino-benzospiropyran dyes that contain an attached phenolic oxygen atom and resonate at ~ 104 ppm in CDCl3.18 The 1H-13C HMBC spectrum of the reaction mixture revealed through-bond correlations between C2 (101.2) with Hb (6.59), Ha (5.98), N1-Me (2.55) and the two C-methyl groups of the Ind moiety (Figure S5, SI). No correlation was observed between C2 and the Me protons of the added NaSMe anion, suggesting that the dye had undergone solvolysis to produce the C2-OH adduct. In this case it is not feasible for the C2-OH adduct of 2 to undergo ring-opening to afford an amide due to the lack of a sufficient leaving group. The ionization state of the C2-OH adduct was also uncertain due to the lack of an observable OH peak. This outcome suggested that 2 is more prone to solvolysis than 1 given that it afforded the C2-OH adduct in 9:1 DMSO-d6-D2O (Figure S4, SI); conditions in which 1 produced the C4-SMe adduct as the sole product (Figure S2B). To limit solvolysis in reaction of 2 with NaSMe, the reaction was carried in DMSO-d6 with no D2O added to the

Figure 5. NMR characterization of the ring-opened amide C2=O hydrolysis product of 1 in DMSO-d6-D2O (1:1, v/v): sec1 13 tions of the H- C HMBC spectrum highlighting throughbond correlations between Hb with C4 and C2 and between the N1Me with C5 and C2.

reaction mixture. Under these conditions, two major sets of doublets in a 2:1 ratio were observed in the 4.5-5.5 ppm region (Figure S6B). The major adduct (labeled Ha, Hb in Figure S6B) displayed doublets at 4.58 and 4.99 (J = 11.9), while the minor adduct (Ha′, Hb′) revealed doublets at 4.77 and 5.33 (J = 11.9), suggesting formation of two enamine C4-adducts. Interestingly, 1H-13C correlations revealed that the sp3 Ha, Ha′ protons of the two C4-adducts resonate downfield of the sp2-enamine Hb, Hb′ protons (Figure 6A). This observation contrasted with the corresponding chemical shifts for the C4-SMe adduct produced by 1 (Figure 4A). For the major adduct, Ha (4.99) that is directly attached to C4 (46.8) exhibited through-bond correlations with C2 (153.2), C5 (130.0), C3 (96.1) and SMe (C6, 14.5) (Figure 6B). The more upfield doublet Hb (4.58) that is directly attached to the enamine C3 (96.1) (Figure 6A) revealed through-bond correlations with C2 (153.2), C5 (130.0), C7 (42.0) and the N1Me (29.1) (Figure 6B). These correlations confirmed that the major enamine product was the C4-SMe adduct. For the minor enamine product, Ha′ (5.33) is directly attached to the sp3-C4′ that resonates at 66.1 ppm, which is considerably shifted downfield compared to the sp3-C4 (46.8) of the C4-SMe adduct (Figure 6A). As chemical shift references, it is diagnostic that the sp3-methylene carbon of benzyl alcohol resonates ~ 65 ppm,19 while the methylene carbon of benzyl methyl sulfides resonate upfield at ~ 40 ppm.20 The Ha′ proton displayed throughbond correlations with C2′ (157.3), C5′, C6′ and C3′ (85.6), but failed to exhibit a correlation with the SMe group. The more upfield enamine Hb′ proton (4.77) is directly attached to C3′ (85.6, Figure 6A) and exhibits correlations with C2′ (157.3) and C7′ (42.0). These correlations coupled with the downfield shift of C4′ (66.1) and the lack of correlation to SMe suggested formation of the C4-OH adduct


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Table 1. Diagnostic NMR Features of Hemicyanines (1, 2), Thiomethoxide and Hydroxide Adducts. a

dye/adduct b

1, R = S

N1CH3

C2

Hb/C3

Ha/C4

SMe f

4.22, s/35.3

171.8

7.61, d, J=15.2/105.7

8.06, d, J=15.2/149.6

na

b C(CH3)2 c

3.96, s/32.8

178.9

7.24, d, J=15.7/104.5

8.30, d, J=15.7/153.7

na

3.13, s/30.0

143.9

4.72, d, J=9.6/89.3

4.25, d, J=9.6/51.1

1.90, s/13.9

b

2.99, s/29.1

153.2

4.58, d, J=11.9/96.1

4.99, d, J=11.9/46.8

1.90, s/14.5

2, C4-OH

3.08, s/29.1

157.3

4.77, d, J=11.9/85.6

5.33, d, J=11.9/66.1

na

d,e

3.06, s/36.3

169.3

5.88, d, J=15.7/115.4

7.20, d, J=15.7/141.5

na

2.55, s/29.6

101.2

5.98, d, J=16.1/124.5

6.59, d, J=16.1/132.3

na

2, R =

1, C4-SMe

2, C4-SMe

b

1, C2=O

e

2, C2-OH a

1

13

Chemical shifts are given in ppm measured at 600 ( H)/150 ( C) MHz; coupling constants are in hertz, obtained at ambient e f b c d temperature. DMSO-d6. DMSO-d6-D2O (9:1, v/v). Ring-opened amide product. DMSO-d6-D2O (1:1, v/v). Not applicable.

Scheme 2. Proposed pathways for reaction of 2 with NaSMe in DMSO and DMSO-D2O mixtures.

Figure 6. NMR characterization of the C4-SMe and C4-OH 1 13 adducts of 2 in DMSO-d6: (A) H- C HSQC spectrum high13 lighting C resonances (C4, C3) for Ha, Hb of C4-SMe and (C4′, 1 13 C3′) for Ha′, Hb′ of C4-OH; (B) H- C HMBC spectrum revealing through-bond correlations between Ha, Ha′ and Hb, Hb′ with nearby carbons.

that can be ascribed to solvolytic processes involving adventitious water in DMSO-d6. Thus, as outlined in Scheme 2, the reaction of 2 with NaSMe in DMSO also favors formation of the C4-SMe adduct as the target product, but can produce both the C4-OH and C2-OH solvolysis adducts.

Diagnostic 1H/13C NMR chemical shifts and coupling constants for dyes 1 and 2 along with the observed NaSMe and NaOH adducts are summarized in Table 1. Our NMR analysis of the products demonstrated a preference for NaSMe attachment at the C4-site of both dyes to afford the enamine tautomer. No evidence for an iminium ion tautomer4,13 and C2,C4-diadducts14 was obtained. Also, no evidence was found for a C2-SMe adduct with either dye, despite examples in the literature for C2-thiolate formation.3 The key to these assignments was 1H—13C correlations to confirm SMe attachment to the dye. Dye 2 was found to be more susceptible than 1 to solvolysis and NMR evidence was observed for formation of both C4-OH and C2-OH adducts (Scheme 2), with the C2-OH adduct being favored with increased D2O content. For 1, solvolytic pathways produced the ring-opened amide product (1, C2=O, Scheme 1), which was believed to stem from initial formation of the C2-OH adduct, although NMR peaks for the putative C2-OH adduct were not detected. For these hydrolysis products 13C chemical shifts were much more



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diagnostic of adduct structure than the corresponding 1H signals. Conjugation of the amide with the donor moiety in the ring-opened product also provided a rationale for the red-shifted emission (457 nm) and absorption maxima (360 nm) of 1 in the presence of excess NaOH (Figures 2 and 3). In comparison, reaction of 2 with excess NaOH, which preferentially produces the C2-OH adduct in DMSO-D2O (9:1, v/v), and presumably in CH3CN-H2O (9:1) afforded blue-shifted maxima (300 nm) due to the lack of conjugation between the donor and acceptor. Reaction Pathways: Kinetic vs. Thermodynamic Control. Characterizing products derived from nucleophilic attachment to electrophiles containing two or more sites of reactivity has constituted a vast area of research, due, in part, to the interesting structure-reactivity relationships that have emerged from such studies. Classic examples include ,-unsaturated carbonyl compounds and picryl ether sytems, such as 2,4,6-trinitroanisole (TNA). For unsaturated carbonyls, the carbonyl carbon is the most electrophilic site, while the coefficient of the Lowest Unoccupied Molecular Orbital (LUMO) is larger at the -carbon.21 According to the Hard and Soft, Acids and Bases (HSAB) theory of Pearson, soft nucleophiles that have large atomic radii with valence electrons that are highly polarizable tend to preferentially react at the carbon of ,-unsaturated carbonyls.22 In contrast, harder nucleophiles that possess smaller atomic radii, more localized charge and greater electronegativity tend to react at the more electrophilic carbonyl carbon.21 The product from attachment at the -carbon is usually thermodynamically favored because it still contains the strong carbonyl bond.21 TNA also displays diverse reactivity toward various nucleophiles. For example, alkoxides react with TNA to produce an initial -adduct at the unsubstituted C3position that gives way over time to the thermodynamically more stable C1-adduct.23 In contrast, ambident phenoxide reacts as an oxygen-centered nucleophile preferentially at the C1-site bearing the methoxy substituent to afford the C1-adduct as the product of both kinetic and thermodynamic control.23 These regioselectivity patterns have been classified as K3T1 for alkoxides/TNA and K1T1 for phenoxide/TNA.24 The NMR analysis of the present hemicyanine dye/Nu combinations also demonstrate diverse reactivity, which will be classified according to the terminology outlined for picryl ether/Nu systems. To assist in this analysis, DFT calculations were carried out to predict structures and relative energies for the various dye/Nu adduct combinations (Figure 7). For reaction of NaSMe with dye 1, the DFT calculations predict that the C4-SMe adduct is more stable than the C2-SMe adduct by 27.2 kJ/mol (Figure 7A). The C4SMe adduct was the only species detected by NMR for reaction of 1 with NaSMe in 9:1 DMSO-D2O (Figure 4b). In contrast, the calculations suggest that for 1/OH, the C2OH adduct is more stable than the C4-OH adduct by 7.8

Figure 7. B3LYP-D3(BJ)/6-31+G(d,p) structures and relative energies (kJ/mol) for the various dye/Nu combinations (A) 1/SMe, (B) 1/OH, (C) 2/SMe, and (D) 2/OH.

kJ/mol. Furthermore, the calculations indicate that the ring-opened amide is more stable than the C2-OH adduct by 7.7 kJ/mol, suggesting that the amide is more stable than the C4-OH adduct by 15.5 kJ/mol (Figure 7B). The ring-opened amide was the only hydrolysis product observed by NMR. Thus, the DFT predicted relative thermodynamic stability of the products generated from dye 1 directly correlate with our NMR data. These findings also appear consistent with the HSAB theory of reactivity. The thiolate would be expected to favor attachment at the softer C4-site, while the harder more basic NaOH would favor attachment to the more electrophilic C2-site. Thus, our NMR data combined with the DFT calculations imply that 1/SMe displays K4T4 behavior, in which the C4adduct is the product of both kinetic and thermodynamic control. In contrast, 1/OH displays K2T2 behavior, in which OH attachment to the electrophilic C2-site is kinetically favored and the resulting adduct undergoes ringopening to afford the thermodynamically stable amide. The only alternative behavior for 1/OH would be K4T2, in which the C4-OH adduct is kinetically-preferred, but forms prior to the first NMR observation and decomposes too rapidly to detect. This scenario seems unlikely because the corresponding C4-OH adduct of dye 2 was ob-


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served by NMR and was relatively stable, permitting analysis by 2D NMR (Figure 6). Replacement of the Btz with the Ind acceptor had a dramatic impact on nucleophilic reactivity. First, 2 was much more susceptible to hydrolysis than 1, favoring the C2-OH adduct in 9:1 DMSO-D2O as the only detectable product upon addition of 5 equiv. of NaSMe (Figure S4, SI). Under analogous conditions, dye 1 only produced the anticipated thiolate adduct (C4-SMe). One would expect both dyes to favor thiolate attachment because the equilibrium between water and NaSMe strongly favors the thiolate (pKa of alkyl thiols ~ 10 in water25 versus 15.7, pKa = 5.3). In DMSO, the equilibrium favors the thiolate even more (pKa of alkyl thiols ~ 1725 versus 32,26pKa = 15); however, now nucleophilicity mirrors basicity and hydroxide is significantly more reactive than NaSMe. On the basis of 13C NMR chemical shifts (Table 1), C2 of dye 1 (171.8 ppm) is less electrophilic than C2 of 2 (178.9 pm) due to the presence of the electron-donating sulfur atom within the Btz acceptor. The greater C2-electrophilicity of dye 2 coupled with the superior stability of the C2-OH adduct compared to the C4-SMe adduct, must provide the driving force for formation of the solvolysis product. In an effort to eliminate hydrolysis of 2, its reaction with NaSMe was carried out in DMSO containing no added D2O. Even under these conditions a hydrolysis product was detected, but it was C4-OH instead of the anticipated C2-OH adduct. The DFT calculations predict a thermodynamic preference for C2-OH by 10.1 kJ/mol (Figure 7D). Thus, 2/OH is classified as K4T2, in which the C4-OH adduct is kinetically-preferred, but the ultimate product of thermodynamic control is the C2-OH species. A kinetic factor favoring C4-adduct formation for 2 is steric hindrance to approach of the nucleophile at the C2-site that contains the adjacent sp3-carbon with two methyl groups. In reaction of 2 with NaSMe in DMSO, the C4-SMe adduct was detected as the major product. No evidence for the C2-SMe adduct was found, even though it is predicted by DFT calculations to be the thermodynamically more stable species (10.3 kJ/mol, Figure 7C). Formation of the C4-SMe adduct is expected to be kinetically favored due to steric hindrance at C2. However, a thermodynamic factor favoring the C2-SMe adduct is that the trans double-bond remains conjugated with the aromatic donor moiety. Thus, 2/SMe is tentatively ascribed to K4T2 behavior. In this case, the lack of observation of the C2-SMe adduct may be due to the strong tendency of 2 to undergo hydrolysis. Under this scenario, isomerization of the C4SMe adduct into the thermodynamically favored C2-SMe adduct would be intercepted by formation of the more stable hydrolysis products. CONCLUSIONS The current study has allowed us to conclude the following: (1) hemicyanine dyes 1 and 2 can react with the thiolate NaSMe to generate an enamine C4-SMe adduct. No evidence was found for formation of a C2-SMe adduct for either dye. (2) Dye 2 containing the N-methyl-3,3-

dimethylindolium (Ind) acceptor moiety is more electrophilic than dye 1 with the N-methylbenzothiazolium (Btz) acceptor. Consequently, it is less selective for thiolate attachment and has a stronger tendency to undergo solvolysis with H2O to generate the C4-OH adduct as the product of kinetic control and the C2-OH adduct as the ultimate product of thermodynamic control. (3) Dye 1 also undergoes solvolysis with H2O, but in this case the only solvolysis product detected was a ring-opened amide (C2=O), presumably stemming from initial HO attachment at the C2-site of 1. (4) Dye 1 has proven to be a better choice for direct thiolate detection because in 90:10 (v/v) DMSO-D2O the only product detected by NMR was the C4-SMe adduct. DFT calculations also predict that the C4-SMe adduct of 1 is more stable than the putative C2SMe adduct, suggesting that it is both kinetically and thermodynamically favored. In contrast, under analogous conditions the only adduct detected for reaction of dye 2 with NaSMe was the thermodynamically favored solvolysis C2-OH adduct with no evidence for peaks representing the C4-SMe adduct. The better selectivity displayed by dye 1 suggests that Btz is a better acceptor choice in construction of hemicyanine dyes for thiolate detection strategies because the Ind acceptor is much more prone to solvolysis. EXPERIMENTAL SECTION Materials and Methods. 2-Methylbenzothiazole, 2,3,3trimethylindolenine, N-methyl-N-(2-hydroxyethyl)-4aminobenzaldehyde and methyl iodide were obtained from commercial sources and used as received. Treatment of 2-methylbenzothiazole or 2,3,3trimethylindolenine with methyl iodide (1.5 equiv.) in acetonitrile at reflux was used to prepare N-methyl-2methylbenzothiazolium iodide and N-methyl-2,3,3trimethylindolenium iodide, as described previously.27 All UV-vis and fluorescence emission/excitation spectra were obtained at room temperature in CH3CN-H2O (9:1, v/v). Initial spectra of the dyes (10 M) were acquired and then 100 equiv. of nucleophile (Nu) was added to the cuvette from a stock water solution. Spectra of the dye/Nu combination were then acquired after mixing at room temperature for 5 min. Mass spectra were acquired on either a quadrupole ion trap or on a Q-TOF high-resolution (HRMS) instrument, both using an electrospray ionization source. Hemicyanine dye synthesis. Dyes 1 and 2 were synthesized by the Knoevenagel condensation reaction of Nmethyl-N-(2-hydroxyethyl)-4-aminobenzaldehyde (268 mg, 1.49 mmol) with either N-methyl-2methylbenzothiazolium iodide (334 mg, 1.15 mmol) for the synthesis of 1, or N-methyl-2,3,3-trimethylindolenium iodide (346 mg, 1.15 mmol) for the synthesis of 2, catalyzed by piperidine in ethanol at 80 °C, as outlined previously for the synthesis of 1.17 Following precipitation with diethyl ether, both compounds were isolated as purple powders in 353 mg (68%) and 201 mg (38%) yields, respectively.



E-2-(4-((2-hydroxyethyl)(methyl)amino)styryl)-3methylbenzo[d]thiazol-3-ium iodide (1): mp. 223-225 °C; 1 H-NMR (DMSO-d6, 600 MHz)  3.12 (3H, s), 3.60 (4H, m), 4.23 (3H, s), 4.82 (1H, t, J=5.2 Hz), 6.88 (2H, d, J=8.9 Hz), 7.61 (1H, d, J=15.3 Hz), 7.68 (1H, m), 7.79 (1H, m), 7.90 (2H, d, J=8.9 Hz), 8.06 (1H, d, J=15.3 Hz), 8.09 (1H, d, J=8.4 Hz), 8.30 (1H, d, J=7.6 Hz); 13C{1H}-NMR (DMSO-d6, 150 MHz)  35.0, 38.4, 53.4, 57.8, 105.5, 111.5, 115.4, 120.8, 123.3, 126.2, 126.9, 128.3, 132.4, 141.4, 149.6, 152.6, 170.8; HRMS (ESI/Q-TOF) m/z: [M]+ Calcd for C19H21N2OS+ 325.1369; Found: 325.1357. E-2-(4-((2-hydroxyethyl)(methyl)amino)styryl)-1,3,3trimethyl-3H-indol-1-ium iodide (2): mp. 216-218 °C; 1HNMR (DMSO-d6, 600 MHz)  1.75 (6H, s), 3.17 (3H, s), 3.64 (4H, m), 3.97 (3H, s), 4.85 (1H, t, J=5.3 Hz), 6.92 (2H, d, J=9.0 Hz), 7.24 (1H, d, J=15.7 Hz), 7.48 (1H, m), 7.55 (1H, m), 7.70 (1H, d, J=8.0 Hz), 7.78 (1H, d, J=7.4 Hz), 8.06 (2H, d, J=8.4 Hz), 8.30 (1H, d, J=15.7 Hz); 13C{1H}-NMR (DMSOd6, 150 MHz) 26.7, 33.5, 51.3, 54.5, 58.9, 105.4, 112.8, 114.0, 122.7, 123.1, 127.9, 129.2, 142.6, 143.0, 154.4, 154.6, 179.9; HRMS (ESI/Q-TOF) m/z: [M]+ = Calcd for C22H27N2O+: 335.2118; Found: 335.2114. NMR Experiments. NMR measurements were performed on a spectrometer with a 1H operating frequency of 600 MHz equipped with a cryogenically-cooled (TCI) 5 mm probe. The sample temperature was regulated at 298.0 ± 1 K. One-dimensional 1H experiments were typically performed with 16 scans and a relaxation delay of 10 seconds between scans, for a total acquisition time of 3.3 minutes, while one-dimensional 13C-proton-decoupled (13C{1H}) experiments were typically performed with 256 scans and a relaxation delay of 5 seconds between scans, for a total acquisition time of 26 minutes. 1H-13C HSQC experiments were typically collected with 384 increments spanning 165 ppm in the indirect dimension, a relaxation delay of 1.5 seconds between scans, and 2 scans per increment, for a total acquisition time of ~ 20 minutes. 1H13 C HMBC experiments were typically collected with 256 increments spanning 220 ppm in the indirect dimension, a relaxation delay of 1.5 seconds between scans, and 4 scans per increment, for a total acquisition time of 30 minutes. The 1H-13C HMBC experiments were optimized for a long-range coupling constant of 8 Hz, while a onebond filter was used to attenuate one-bond J-couplings between 130-180 Hz. For a typical NMR experiment, a solution of the dye in DMSO-d6 was added to a 5 mm diameter NMR tube (~ 15 mM in 600 L DMSO-d6). An initial 1H NMR spectrum of the dye was recorded with the DMSO-d5H peak in the solvent serving as the chemical shift reference (2.50 ppm relative to TMS). Injection of the relevant quantity of NaSMe prepared in D2O (50 L for 5 equiv.) initiated the reaction. The contents in the NMR tube were thoroughly mixed and then various NMR spectra were recorded as rapidly as possible. Computational Details. Several possible conformations of 2 were explored at the B3LYP-D3(BJ)/631+G(d,p) level of theory in the gas phase. Seven unique conformations were identified, which mainly differed in the orientation of the terminal hydroxyethyl moiety at-

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tached to the exocyclic amine. The most stable conformation for 2 was then used to generate the starting structure for 1, as well as the corresponding adducts in order to maintain a consistent orientation of the hydroxyethyl group. All models were optimized using B3LYP-D3(BJ/631+G(d,p) and frequency calculations were performed at the same level of theory to ensure that all optimized structures correspond to local minima. Relative energies were determined using B3LYP-D3(BJ)/6-311++G(2df,p). All calculations were performed using Gaussian 09.28

ASSOCIATED CONTENT Supporting Information. Figures S1-S6 described in the text, NMR spectra of synthetic dyes and Cartesian coordinates for the DFT structures. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author ‡

S.D.W. e-mail: [email protected]

†

R.A.M. e-mail: [email protected]

ACKNOWLEDGMENT Financial support for this research was provided by the Natural Sciences and Engineering Research Council (NSERC) of Canada, the Canada Foundation for Innovation, Ontario Innovation Trust Fund and the Board of Governors Research Chair Program at the University of Lethbridge. This research has been facilitated by use of computing resources provided by WestGrid and Compute/Calcul Canada.

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SYNOPSIS TOC


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Acceptor Influence on Thiolate Sensing by Hemicyanine Dyes - The

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