Modified Atomic Orbital Overlap: Molecular Level Proof of the

Apr 6, 2017 - Out of six possible positional isomers of dinitrophenol, only 2,4-DNP has been used extensively by many researchers for developing react...
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Modified Atomic Orbital Overlap: Molecular Level Proof of the Nucleophilic Cleavage Propensity of Dinitrophenol-Based Probes Mangili Venkateswarulu, Sunil Kumar, and Subrata Ghosh* School of Basic Sciences, Indian Institute of Technology Mandi, Mandi, Himachal Pradesh 175005, India S Supporting Information *

ABSTRACT: Out of six possible positional isomers of dinitrophenol, only 2,4-DNP has been used extensively by many researchers for developing reactive molecular probes. But the question remains unanswered: why has only the 2,4-isomer emerged as a labile protecting group? To answer this question, six molecular probes using available DNP isomers were developed and investigated to evaluate the effect of the extent of atomic orbital overlap on their reactivity. We have proved for the first time at the molecular level that the o-NO2 group contributes less compared to the p-NO2 group toward the reactivity of 2,4-DNP-based probes. Crystal structure analysis revealed that the 2p orbital of N atom and the 2p orbital of the adjacent ring C atom to which the o-NO2 is attached are inclined at >30° to each other, leading to substantial reduction in π overlap (as these two p-orbitals loose coplanar state) resulting in a very weak −M effect of the o-NO2 group, whereas the 2p orbitals of the N atom of the p-NO2 group and the adjacent ring C atom are almost coplanar (11° inclined to each other), leading to strong π overlap. Hence, the p-NO2 group contributes largely toward the molecular reactivity through its −M effect.

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INTRODUCTION The scissile nature and synthetic feasibility are the fundamental requirements of protecting groups in organic chemistry.1,2 Since the inception of new methodology for the facile removal of 2,4- dinitrophenol (2,4-DNP) protection, this particular protecting group has been widely explored in polymer and peptide chemistry (Scheme 1a).3,4 In recent times, 2,4-DNP has been widely used in designing and developing various emissive and reactive molecular probes to detect and quantify a large number of analytes including hydrogen sulfide, biothiols, etc. as well as to image those analytes inside biological systems to understand different bioevents.5−20 Whether it is deprotection or cleavage of molecular probes into various molecular fragments exerting an optical signal, the removal of 2,4-DNP functionality is done by the attack of a nucleophile. Some representative cases are shown in Scheme 1. Surprisingly, out of six possible positional isomers of dinitrophenol, only 2,4-DNP has been used to establish all such chemistry.5−20 On some occasions, 3,5-DNP has been used as a protecting group, but it requires harsh oxidative reaction conditions for its removal.21 This demonstrates that 2,4-DNP imparts high reactivity toward nucleophilic attack, and hence, harsh reaction conditions are not required for its removal. Although the role of 2,4-DNP as a protecting group has been discussed for a long time,5−20 it has never been investigated with a mechanistic approach as to why only 2,4-DNP develops highly reactive molecular probes whereas the molecular probes based on other positional isomers are very less reactive? In addition, the other isomeric members of the dinitrophenol family remained © 2017 American Chemical Society

unexplored in protecting group chemistry. It may be the case that only 2,4-DNP is an active member in protecting group chemistry, but still there exists no report to support this wellknown fact. Moreover, if the electron-withdrawing mesomeric effect (−M) of the o-/p-nitro group (−NO2) is the key in inducing reactivity, one can expect that 2,3-, 2,5-, 3,4-, and 2,6DNP may also be used to develop reactive probes as these probes contain o- or p-NO2 groups. Thus, this lack of correlation between DNP isomers as protecting groups and their leaving propensities arouse interesting queries among us, including what makes the 2,4-DNP unit the most extensively studied leaving group out of other isomeric DNP family members? Based on the first report of effective removal of 2,4-DNP using mercaptoethanol nucleophile,3 we sought to understand the above problem at a very fundamental level with experimental evidence. This manuscript describes, at the molecular level, the chemistry of effective atomic orbital overlap behind such differences in the reactivity of DNPbased probes. We have been successful, through visualization at the molecular level, in explaining how molecular conformation and sterically modified atomic orbital overlap tune the electronic parameters and eventually control the molecular properties and reactivity.22,23 To demonstrate these molecular events, we developed six sulfonate ester based molecular probes Received: February 15, 2017 Published: April 6, 2017 4713

DOI: 10.1021/acs.joc.7b00317 J. Org. Chem. 2017, 82, 4713−4720

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Scheme 1. (a) Thiolysis of 2,4-Dinitrophenyltyrosines by 2-Mercaptoethanol.3,4 (b) Structure of Fluorescent H2S Probes Based on Dinitrophenyl Ether5−20

We also employed single-crystal X-ray diffraction (SC-XRD) and density functional theory (DFT) studies to support our conclusion.

(Figure 1) by reacting dansyl chloride with all possible positional isomers of DNP (Scheme S1).

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RESULTS AND DISCUSSION As mentioned above, to complete our investigation, we synthesized six dansyl platforms P4−P9 where each platform bears a unique DNP positional isomer on the sulfonyl group (Figure 1). Each scaffold has dual elelctrophilic sites, viz., sulfonate ester and the nitro group. The nucleophilic attack at the sulfonate ester would make the probes chemodesimeters. It was clear that the electrophilicity at the S atom of the sulfonate center was due to the overall electron-withdrawing (EW) effect of the nitroaromatic ring, which is largely governed by the −M effect of o- and/or p-NO2 groups or any EW substituent.27 However, isomer specific preferential nucleophilic attack of H2S was observed in our study, which increased our curiosity to understand how the positions of −NO2 groups drive the reactivity of these probes toward the nucleophilic attack. The reaction progress and the reactivity of each probe were monitored by studying the optical responses (both absorption and fluorescence) of the probes in the absence and presence of H2S, and the optical responses were then correlated with the chemical structures of the probes. Initially, the UV−vis absorption studies of P4−P9 were performed in HEPES buffer (1 mM, pH = 7.2) containing 50% THF. Owing to their similar chemical structure, all these probes were found to be stable in buffer solution and displayed similar absorption behavior. The fluorescence spectroscopy revealed the nonfluorescent nature of these molecular probes (Figures

Figure 1. Molecular structures of the probes P4−P9.

To investigate the molecular reactivity of these positional isomeric probes as well as the reaction dynamics, we chose H2S as the reactive nucleophile.24−26 We focused on monitoring the reaction progress using advanced optical tools such as UV−vis absorption and fluorescence spectroscopy. It was observed that each specific probe followed a specific reaction pathway to give specific optical responses. To gain more insight into nucleophilic cleavage process, the reaction products were isolated and fully characterized to know their chemical structures. During the course of this investigation, we have been able to establish through molecular structure analysis that the steric factor and the molecular substituent orientation govern the extent of atomic orbital overlap which eventually controls the overall electronic effect and molecular reactivity.

Figure 2. UV−vis (a) and emission spectra (b) of all probes (P4−P9) (10 μM) upon addition of NaHS (80 μM) in THF/H2O (1:1, v/v)-buffered with HEPES (1 mM, pH = 7.2). 4714

DOI: 10.1021/acs.joc.7b00317 J. Org. Chem. 2017, 82, 4713−4720

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The Journal of Organic Chemistry Scheme 2. HS−-Mediated Chemical Changes in the Designed DNP Probes

Figure 3. ORTEP diagram of isomeric probes P4 (a), P5 (b), P6 (c), P7 (d), P8 (e), and P9 (f) showing an atomic crystallographic numbering scheme. Thermal ellipsoids are scaled to the 50% probability level.

S1a and S1b, Table S2). The electron-withdrawing (EW) group induced electron-transfer process inhibited the fluorescence emission, which is also known as PET (photoinduced electron transfer) or d-PET (donor-excited-PET).28,29 After initial photophysical studies, we studied the optical response of P4−P9 in the presence of H2S (generated in situ by adding NaHS to water and HS− is the active nucleophilic form of H2S) (Figure 2 and Figures S2 and S3). Upon addition of H2S, significant changes in UV−vis absorption behavior were observed for P5 only (Figure S2). We observed that the fluorescence spectra of P7 remained unaffected (Figure S3d). Only a small enhancement in emission intensity at 495 nm was observed for P4/P6/P8 (Figure 2 and Figure S3a,c,e). In the case of P9, insignificant increase in florescence intensity along with a ∼70 nm red shift (Figure S3f) was observed (Table S2). The probe P5 exhibited significant changes in absorption and emission spectra upon H2S addition (Figure 2 and Figures S2b, S3b, and S4−S6). In the absorption profile, two new peaks at ∼325 and ∼475 nm were observed with the disappearance of the 348 nm peak (Figure S4). To understand the intriguing photophysical changes, products formed after the reaction of each probe with HS−

(P4′−P9′) were successfully isolated from the reaction mixtures (Scheme 2) and thoroughly characterized (Figures S7−S24 and S56−S73). It was observed that HS− either reduced a particular −NO2 group to yield an amine derivative (P4′, P6′, P8′, P9′) or performed nucleophilic substitution at a sulfonyl group leading to cleavage of the probe into the fluorescent entity (P5′) and 2,4-DNP unit (Scheme 2). Adding to our surprise, the 2,6-DNP probe (P7) remained unreactive in the presence of HS−, whereas a 2,4-DNP-based probe P5 underwent fast nucleophilic substitution of the DNP unit with HS− and the formed product exhibited promising optical changes (Scheme 2). Further, the optical responses of P4/P6/ P8/P9 established the weak fluorescence enhancement due to the formation of amine derivatives P4′/P6′/P8′/P9′ (Scheme 2). The strong fluorescence enhancement in P5 solution was due to the formation of green emissive dansyl fluorophore (P5′) formed after the thiolytic nucleophilic cleavage of P5 (Scheme 2 and Scheme S2). From our common understanding on the effect of electronic parameters of functional groups in increasing electron deficiency of a reaction site, one could expect that the −M effect of o-/p-NO2 groups would enhance the electron deficiency at the S-center of the sulfonate group by 4715

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Table 1. Angle of Deviation from Co-planarity of p-Orbitals of C (NO2-Attached Aromatic Ring) and N (−NO2), Mesomeric Interaction, and Nucleophilic Cleavage

θ (deg) probe

ortho

P4 P5 P6 P7 P8 P9

84.6 31.8 34.9 38.4, 44.7

−M effect

2p orbital overlap para 11

ortho weak weak weak weak

37.4

para strong

weak

ortho weak weak weak weak

para strong

weak

nucleophilic cleavage no yes no no no no

consequence, it could be seen that dansyl and DNP ring systems face each other in P8 and P9. However, the probes with ortho-substitution were found to be twisted with respect to the naphthalene plane mainly because of steric repulsion with the oxygen atoms of the sulfonyl group (Figure 3 and Figures S25−S29). The simple understanding of mesomeric effect and atomic orbital overlap could tell us that,5 in the DNP unit, o- and pNO2 groups are coupled through the −M effect to phenolic oxygen, while the m-NO2 group is not a part of this electronic communication. If examined carefully, one can also observe that in none of these positional isomeric scaffolds did H2S attack at the o-NO2 and p-NO2 groups, but the m-NO2 group underwent reduction to amine functionality. The nonconjugated m-NO2 group retained its electrophilic nature, and the nonsteric environment aided hydrogen sulfide mediated reductive attack.30,31 As a result, m-NO2 groups were converted into amine functionalities in the cases of P4/P6/P8/P9. Still, the mystery of silence toward nucleophilic cleavage of o/p-nitrosubstituted probes (P4/P6/P7/P8) was unanswered. The critical analyses of the crystal structures established that the o/p-NO2 groups of P4/P6/P7/P8 lost coplanarity with respect to the aromatic ring to which they are attached (Figure S30), largely due to steric interaction as well as electronic repulsion between the oxygen atoms of nitro group and nearby groups. As a result, the 2p orbital of the N atom of o/p-NO2 group and the 2p orbital of the ring C atom to which the o/pNO2 groups are attached become inclined at >33° to each other and hence lead to a substantial reduction in π-overlap (Figure S31). In the cases of P4 and P6, electronic repulsion between the electron clouds of sulfonate oxygen and oxygen atoms of the o-NO2 group prevented the nitro group from lying in the same plane as the aromatic nucleus and the allowed rotation about the C−N bond to move −NO2 groups out of the plane by 84.6° and 34.9°, respectively (Figure S32). Such mismatch in planes of 2p orbitals of carbon (of benzene) and N (of −NO2) induces substantial reduction in π-overlap, which in turn prevented the electronic interaction with the aromatic nucleus (Table 1). Thus, the o-NO2 groups in P4/P6 failed to exert −M effect, and the electrophilic environment at the Scenter did not attract nucleophilic attack (Figure S31a,b). Similarly, in P8, the steric as well as electronic repulsion between the m-NO2 and p-NO2 group pushed the p-NO2 group

making the O atom of the Ph−O−S bond of P5 highly electron deficient. Indeed, the sulfonate group of P5 showed high reactivity toward nucleophilic attack by HS−. But, if this is true, then the probes P4, P6, and P8, which contain at least one −NO2 group at the ortho/para position, should have shown a response with HS− aimilar to that of P5. But these probes underwent −NO2 group reduction by H2S, and in addition, P7, which contains two −NO2 groups at the ortho positions, remained fully silent toward H2S attack. This unusual reactivity of P4, P6, and P8 with HS− was beyond our common understanding and indicated that the substituent position governs the molecular conformation and responsible for molecular reactivity. This made us curious to investigate the actual reason for this unusual reactivity through molecular level understanding. It has been well discussed in organic chemistry that, in general, if the −NO2 group is placed on the meta position with respect to an electron-deficient substituent in an aromatic compound, the −NO2 group undergoes reduction in the presence of H2S leading to the formation of the corresponding amine derivative.30,31 Hence, the conversion of P4, P6, and P8 to the respective amines P4′, P6′, and P8′ in the presence of H2S seemed quite logical. Moreover, the only active electronic effect is the − I effect when the −NO2 group is placed on the meta position. As the m-NO2 functionality is four atoms away (three C atoms and one O atom) from the S-center of sulfonate group in P4/P6/P8/P9, the −I effect of the m-NO2 group on the S-center would be negligible because the −I effect falls off with distance and, hence, the S-center could not acquire a sufficiently electron-deficient environment to attract nucleophilic attack. As a result, P4/P6/P8/P9 yielded the reduced products upon completion of the reaction, but the formation of different products from P5 and P8 in the presence of HS− made us curious to investigate the substitution effect in each probe from the scratch. To investigate more fundamentally and to gain more insight into the molecular level, true geometries of the probes were required. Thus, single crystals were grown to understand the geometrical constraints in each probe. All probes were having an inverted “V” shape geometry where the sulfonyl group was at the bent position (Table S1, Figure 3, and Figures S74− S79). The absence of o-NO2 group in the P8 and P9 failed to provide steric crowdedness near the sulfonyl center, and as a 4716

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Figure 4. Schematic showing the atomic orbitals of p-NO2 (in plane and out of plane) with respect to p-orbitals of carbon in the aromatic ring in probe (a) P5 and (b) P8.

and hence, the developed mesomeric interaction through effective delocalization via π-orbitals created strong electrophilicity at the S atom (Figures 4a and 5a,b). Interestingly, this is prevented in the case of o-NO2 group as the steric influence inhibited significant overlapping of planes of the NO2 group and benzene ring, respectively, (as they are 31.8° inclined to each other), and therefore, the o-NO2 group contributes less to the eletrophilicity of the S-center due to poor mesomeric electron withdrawal. Therefore, the molecular level understanding through crystal structure analysis helped us to establish for the first time that the reactivity of 2,4-DNPbased probes or protection sites is mainly due to p-NO2 functionality, whereas o-NO2 contributes less toward the reactivity. In order to support the above molecular level understanding, the geometries of P4−P9/P4′−P9′ were optimized using DFT (Figures S33−S44).32−35 In all cases, the HOMOs are spread over the N,N-(dimethylamino)naphthyl ring, whereas the electron density of the LUMO orbitals is predominantly located on the dinitrobenzene ring. HOMO to LUMO electronic transitions were accompanied by PET, and thus, these transitions can be classified as donor-excited PET or dPET, which make the probes weak fluorescent in nature.28,29 Moreover, time-dependent density functional theory (TDDFT) calculations were also in close proximity to the experimental absorption band domains (Figures S45−S55). The MO distribution in P5′ suggested the side-by-side arrangement of HOMO and LUMO which was the key behind the strong fluorescence enhancement of P5′. The molecular orbital distributions in amine products (P4′/P6′/P8′) (Figures S40−S44) are similar to those of starting DNP isomers P4/P6/ P8, and hence, these remain nonfluorescent. Although the MOs change positions in P9′, the d-PET process makes the product nonfluorescent in nature. These results further suggest that the absence of a d-PET process in P5′ is the major cause of the origin of fluorescence. The nucleophilic cleavage rate of P5 (10 μM) was studied in the presence of HS− (66 μM). The reaction progress was monitored by measuring the change in fluorescence intensity (λex = 348 nm and λem = 505 nm) at different time intervals after the addition of HS− (Figure S56). The reaction was complete within 30 min, and the pseudo-first-order rate constant was calculated to be k = 0.1199 min−1 (Figure S56).

out of plane by 37.4° with respect to the aromatic ring plane (Figure 4b and 5c,d), mismatch in planes blocked the mesomeric effect of p-NO2 group, and hence, the S center of sulfonate group did not develop the required electron deficiency for nucleophilic attack. The crystal structure analysis also revealed that the o-NO2 groups of P7 are not in conjugation with the ring π-cloud because of out-of-plane orientation of both of the o-NO2 groups (at 38.4° and 44.7° with respect to aromatic plane), resulting in poor π-overlap between these 2p orbitals (Figures S31c and S32e,f). In short, it could be said that the o-NO2 groups failed to enhance the electrophilicity at the S atom because of suffered twist/torsion. However, in the P5 scaffold, the conditions were favorable for an active −M effect through the p-NO2 group as it shares the same plane with the phenyl ring of DNP (Figure 4a and 5a,b) even though the o-NO2 is out

Figure 5. Interplanar angle between DNP system and (a) o-NO2 group of P5, (b) p-NO2 group of P5, (c) m-NO2 group of P8, and (d) p-NO2 group of P8, respectively.

of the plane of the aromatic ring. The steric effect free environment around the para-position allowed the 2p orbital of the N atom of the p-NO2 group and the 2p orbital of the ring C atom to which the p-NO2 group is attached to become fairly nearly parallel to each other (they are just 11° inclined to each other), leading to effective overlap between these orbitals, which in turn leads to a strong −M effect of the p-NO2 group, 4717

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X-ray diffraction data of P4/P5/P6 were measured using a CuKα (λ= 1.5406 Å at 298(2) K) source, and the probe P4/P5/P6 was measured using a MoKα (λ= 0.71073 Å at 298(2) K) source which revealed that P4 crystallizes in orthorhombic P21/n, P5 in monoclinic P212121, P6 in orthorhombic P21/c, P7 in orthorhombic P-1, P8 in orthorhombic P21/c, and P9 in orthorhombic P212121 space groups. Data were collected by standard “CrysalisPro” Software (online version), and reduction was undertaken with CrysalisPro Software (offline version). All calculations and molecular structures were solved by direct methods OLEX2 and refined using full-matrix least-squares (F2) on SHELXL-97.36,37 The positions of all non-hydrogen atoms were located and refined anisotropically. After that, hydrogen atoms were obtained from the residual density map and refined with isotropic thermal parameters. All parameters of crystal structures are given in Table S1. DFT Calculations. In order to understand the geometry and sensing mechanism, reactants (P4/P5/P6/P7/P8/P9) and their derivatives (P4′/P5′/P6′/P8′/P9′) were optimized at DFT with a B3LYP/6-31G(d,p) basis set32,33 with no symmetry constraint using the Gaussian 09 suite of programs.34 Frequency calculations at the same level with the same basis set were performed to ensure that the geometries correspond to real minima (see the Supporting Information). Chemcraft software along with Gauss view were used for visualization.35 General Procedure for the Synthesis of DNP-Based Probes. Triethylamine (0.055 g, 0.546 mmol) was added dropwise to a mixture of dansyl chloride (0.050 g, 0.185 mmol) and dinitrophenol (0.041 g, 0.223 mmol) in 10 mL of dry CH2Cl2 under nitrogen at 0 °C with constant stirring. After that, the reaction mixture was allowed to stir at room temperature, completion of the reaction was judged by TLC, the organic solution was washed with water, and the solution was concentrated under reduced pressure to obtain the crude product, which was purified by column chromatography on silica gel (eluent: 8:2 hexane−ethyl acetate mixture) to afford pure product as a solid. Compound P4. Yield: 83% (65 mg). Melting point: 92−96 °C. FTIR (KBr, ν in cm−1): 3072, 2951, 2922, 2848, 2664, 2360, 1651, 1613, 1543, 1486, 1458, 1407, 1377, 1338, 1303, 1174, 1126, 1061, 961, 941, 817, 719, 565, 501. 1H NMR (500 MHz, CDCl3): δ 8.70 (d, J = 8.20 Hz, 1H), 8.25 (d, J = 8.90 Hz, 1H), 8.20−8.18 (m, 1H), 8.05 (d, J = 7.55 Hz, 1H), 7.85−7.83 (m, 1H), 7.67−7.64 (m, 2H), 7.55−7.52 (m, 1H), 7.28−7.26 (m, 1H), 2.91 (s, 6H). 13C NMR: δ 152.1, 142.0, 140.7, 133.5, 131.2, 131.1, 129.9, 129.8, 129.7, 129.6, 129.3, 122.9, 122.7, 118.5, 116.0, 45.3. MS (HRMS): m/z calcd for C18H16O7N3S [M + H] + 418.0709, found 418.0703, [M + K]+ 456.0268 and found 456.4405. Compound P5. Yield: 78% (61 mg). Melting point: 156- 158 °C. FT-IR (KBr, ν in cm−1): 2915, 2862, 2749, 1511, 1509, 1453, 1441, 1349, 1303, 1215, 1182, 1146, 967, 856, 826, 770, 751, 661, 642 626, 563, 513, 509. 1H NMR: (500 MHz, CDCl3): δ 8.72−8.69 (m, 2H), 8.37−8.33 (m, 2H), 8.16−8.14 (m, 1H), 7.70−7.67 (m, 1H), 7.54− 7.51 (m,1H), 7.43 (d, J = 8.95 Hz, 1H), 7.28−7.26 (m, 1H), 2.92 (s, 6H). 13C NMR: δ 152.1, 146.1, 145.1, 142.7, 133.5, 131.4, 129.9, 129.8, 129.7, 129.7, 128.3, 125.6, 122.8, 121.5, 118.6, 116.0, 45.4. MS (HRMS): m/z calcd for C18H15O7N3S [M]+417.0631, found 417.0607, [M + Na] + 440.0528, found 440.0515, [M + K] + 456.0268, found 456.0252. Compound P6. Yield: 81% (63 mg). Melting point: 134−136 °C. FT-IR (KBr, ν in cm−1): 3113, 3094, 2988, 2947, 2840, 2551, 2359, 1938, 1767, 1615, 1547, 1530, 1476, 1404, 1341, 1309, 1217, 1170, 1150, 1109, 1063, 947, 906, 819, 788, 741, 714, 671, 559, 501. 1H NMR (500 MHz, CDCl3): δ 8.70 (d, J = 8.90 Hz, 1H), 8.34 (d, J = 8.25 Hz, 1H), 8.21−8.18 (m, 1H), 8.15−8.13 (m, 1H), 7.99 (d, J = 8.90 Hz, 1H), 7.91 (d, J = 2.1 Hz, 1H), 7.71−7.67 (m, 1H), 753−7.50 (m, 1H), 7.28 (d, J = 7.55 Hz, 1H), 2.92 (s,6H). 13C NMR: δ 152.1, 149.4, 146.5, 142.0, 133.5, 131.4, 129.8, 129.7, 129.4, 126.5, 122.8, 122.1, 120.3, 118.4, 116.0, 45.3. MS (HRMS): m/z calcd for C18H16O7N3S [M + H]+ 418.0709, found 418.0702, [M + K]+ 456.0268, found 456.4410. Compound P7. Yield: 76% (59 mg). Melting point: 118−126 °C. FT-IR (KBr, ν in cm−1): 2951, 2853, 2788, 1569, 1534, 1469, 1453,

CONCLUSIONS Often the reactivity of molecular probes, determined experimentally, is explained by theoretical studies largely due to lack of direct evidence at the molecular level particularly on reactivity versus extent of atomic orbital overlap. To conclude, we have studied the unusual high reactivity of 2,4-DNP-based molecular probes as compared to all other possible positional isomeric probes based on DNP. The reason behind the unexpected low reactivity of various DNP based probes, which is beyond our common understanding, has been established from molecular level understanding and visualization of the extent of atomic orbital overlap through crystal structure analyses. We have shown for the first time that the reactivity of 2,4-DNP-based probes is largely due to the p-NO2 group, whereas the contribution of the o-NO2 group is substantially less than expected. This is due to weak overlap between the nitrogen p-orbital of the o-NO2 group and the p-orbital of the adjacent ring carbon (as these two p-orbitals are not parallel to each other; rather they are inclined at 33° to each other). All of the experimental data led us to conclude that a delicate balance between steric factor, functional group orientation, and electronic parameters govern the reactivity of DNP-based probes/protection site. Hence, the unexpected reactivity of certain positional isomers is mainly due to the predominance of one molecular factor over the others. For example, in the case of 2,6-DNP-based probe P7, due to strong steric reasons, the functional groups are oriented such that both of the o-NO2 groups are out-of-plane with respect to the plane containing the aromatic ring, which resulted in poor overlap between the nitrogen p-orbital and the adjacent carbon p-orbital. Hence, the −M effect of o-NO2 groups is too weak to induce the required electron deficiency at the S-center of P7 for nucleophilic attack. Similarly, the o-NO2 group in P4/P6 and the p-NO2 group in P8 have negligible impact on reactivity due to the poor atomic orbital overlap as the nitrogen p-orbitals are not in parallel orientation with ring carbon p-orbitals. That the o-NO2 group contributes less toward the reactivity of P5 was also supported by the fact that 2,3-, 2,5-, and 2,6-DNP-based probes P4/P6/ P7, all of which contain o-NO2 group(s) in their molecular architecture, are silent toward nucleophilic cleavage in the presence of H2S. Theoretical studies also supported the structural orientation, optical responses, experimental observations, and conclusions.

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EXPERIMENTAL SECTION

General Information. All chemicals were purchased from Merck, S.D. Fine, and Sigma-Aldrich and were used without further purification. THF (AR grade) was used for the spectral studies. H2S as NaHS (10 mM) (HS− is the active nucleophilic form of H2S) in H2O was used as the standard solution to record the UV−vis and fluorescence spectra. FT-IR spectra in KBr were recorded on a PerkinElmer Spectrum 2 spectrophotometer. 1H/13C NMR spectra in CDCl3 were recorded on a Jeol-ECX-500 MHz spectrometer using tetramethylsilane as an internal standerd. Absorption spectra were recorded with a SHIMADZU UV-2450 spectrophotometer. The fluorescence spectra were recorded with a Cary Eclipse spectrophotometer with slit widths of 5 nm for excitation and 5 nm for emission, respectively. HRMS-ESI spectra were recorded using a quadrupoletime-of-flight mass analyzer on a Bruker impact-HD spectrometer, respectively. Single-crystal X-ray diffraction data were collected on a Agilent SuperNova CCD system. X-ray Crystal Structure Analysis of P4/P5/P6/P7/P8/P9. Single crystals of P4/P5/P6/P7/P8/P9 suitable for X-ray diffraction were grown in ethanol by slow evaporation after 1 day. Single-crystal 4718

DOI: 10.1021/acs.joc.7b00317 J. Org. Chem. 2017, 82, 4713−4720

Article

The Journal of Organic Chemistry 1413, 1356, 1321, 1214, 1166, 1145, 974, 891, 833, 796, 761, 691, 653, 629, 568, 529, 512. 1H NMR (500 MHz, CDCl3): δ 8.72 (d, J = 8.25 Hz, 1H), 8.37 (d, J = 8.95 Hz, 1H), 8.12−8.10 (m, 2H), 7.71 (t, J = 8.25 Hz, 1H), 7.58 (t, J = 8.25 Hz, 1H), 7.53−7.50 (m,1H), 7.28−7.26 (m, 2H), 2.92 (s,6H). 13C NMR: δ 151.9, 134.8, 133.6, 131.1, 130.0, 129.9, 129.7, 129.5, 129.2, 127.5, 122.8, 118.8, 115.9, 45.3. MS (HRMS): m/z calcd for C18H16O7N3S [M + H]+ 418.0709, found 418.0703, [M + Na]+ 440.0528, found 440.0515, [M + K]+ 456.0268, found 456.0259. Compound P8. Yield: 79% (62 mg). Melting point: 124−126 °C. FT-IR (KBr, ν in cm−1): 2957, 2802, 2359, 2163, 1766, 1572, 1537, 1477, 1437, 1346, 1315, 1204, 1172, 1128, 944, 893, 852, 824, 788, 727, 670, 563, 533. 1H NMR (500 MHz, CDCl3): δ 8.67 (d, J = 8.95 Hz, 1H), 8.34 (d, J = 8.20 Hz, 1H), 8.13 (d, J = 7.55 Hz, 1H), 7.83 (d, J = 8.25 Hz, 1H), 7.71 (t, J = 8.2 Hz, 1H), 7.53−7.50 (m, 1H), 7.426− 7.42 (m, 1H), 7.33−7.31 (m, 1H), 7.27−7.26 (m, 1H), 2.91 (s, 6H). 13 C NMR: δ 152.4, 152.2, 143.6, 140.3, 133.4, 131.7, 129.8, 129.8, 129.7, 129.3, 126.6, 126.6, 123.0, 119.0, 118.3, 116.0, 45.3. MS (HRMS): m/z calcd for C18H16O7N3S [M + H]+ 418.0709, found 418.0703. Compound P9. Yield: 82% (64 mg). Melting point: 118−120 °C. FT-IR (KBr, ν in cm−1): 3118, 2922, 2850, 2360, 2340, 2193, 1949, 1900, 1743, 1571, 1539, 1453, 1412, 1373, 1337, 1229, 1203, 1175, 1149, 1066, 972, 946, 897, 819, 796, 722, 626, 585, 487. 1H NMR (500 MHz, CDCl3): δ 8.87−8.86 (m, 1H), 8.68(d, J = 8.25 Hz, 1H), 8.39 (d, J = 8.25 Hz, 1H), 8.12 (d, J = 6.85 Hz, 1H), 8.08−8.07 (m, 2H), 7.76−7.73 (m, 1H), 7.51−7.48 (m, 1H), 7.30 (d, J = 7.6 Hz, 1H), 2.92 (s,6H); 13C NMR: δ 152.4, 150.2, 148.6, 133.4, 131.7, 129.9, 129.8, 129.7, 129.2, 123.1, 122.9, 118.3, 117.0, 116.1, 45.3. MS (HRMS): m/z calcd for C18H16O7N3S [M + H]+ 418.0709, found 418.0703. General Procedure for the Synthesis of P4′/P5′/P5″/P6′/P8′/ P9′. A solution of NaHS (10 equiv, 0.119 g, 2.14 mmol) in 3 mL of H2O was added to a solution of DNP-based probes (P4/P5/P6/P8/ P9) (0.08 g, 0.214 mmol) in 10 mL of THF/water (1:1, v/v) under stirring. The reaction mixture was then allowed to stir at room temperature for 30 min. This mixture was extracted with DCM and dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure to obtain the crude product, which was purified by column chromatography on silica gel using a suitable eluent. Compound P4′. Eluent 7:3 hexane−ethyl acetate mixture. Yellow solid (0.024 g, 24%). Melting point: 98−102 °C. FT-IR (KBr, ν in cm−1): 3349, 3064, 2992, 2948, 2832, 2598, 1721, 1631, 1533, 1450, 1373, 1242, 1197, 1056, 1039, 748, 711, 703, 632, 532. 1H NMR (500 MHz, CDCl3): δ 8.58 (d, J = 8.9 Hz, 1H), 8.45 (d, J = 8.95 Hz, 1H), 8.11 (d, J = 7.55 Hz, 1H), 7.68−7.64 (m, 1H), 7.47−7.43(m, 1H), 6.90 (t, J = 8.25 Hz, 1H), 6.46−6.45 (m, 1H), 6.33−6.32 (m, 1H), 6.18−6.16 (m, 1H), 2.90 (s, 6H). 13C NMR (CDCl3): δ 131.8, 131.7, 131.1, 130.0, 129.7, 128.9, 123.0, 119.6, 115.5, 115.1, 113.6, 112.7, 111.5, 108.6, 108.2, 106.7, 45.44. MS (HRMS): m/z calcd for C18H17O5N3S [M + H]+ 388.0967, found 388.1046, [M + Na]+ 410.0787, found 410.0887. Compound P5′. Eluent 9:1 dichloromethane−methanol mixture. Color: light yellow solid (0.038 g, 76%). Melting point: 96−100 °C. FT-IR (KBr, ν in cm-1): 3712, 3545, 3325, 2972, 2834, 2275, 1547, 1372, 1335, 1053, 1036, 975, 823, 737, 665, 541. 1H NMR (500 MHz, DMSO-d6): δ 8.51 (d, J = 8.95 Hz, 1H), 8.17 (d, J = 8.25 Hz, 1H), 7.93 (d, J = 7.55 Hz, 1H), 7.43−7.37 (m, 1H), 7.10 (d, J = 7.55 Hz, 1H), 2.79 (s, 6H). 13C NMR (DMSO-d6): δ 150.4, 144.3, 130.4, 128.8, 125.4, 125.2, 124.3, 123.6, 122.5, 113.8, 45.1. MS (HRMS): m/z calcd for C12H12NO2S2 [M]− 266.0309, found 266.0303. Compound P5″. Eluent 1:1 hexane−ethyl acetate mixture. Yellow solid (0.021 g, 60%). Melting point: 106−110 °C. FT-IR (KBr, ν in cm−1): 3361, 3262, 3117, 1673, 1539, 1482, 1332, 1253, 1215, 1134, 812, 791, 722, 643, 521, 505. 1H NMR (500 MHz, CDCl3) δ: 9.07 (d, J = 2.75 Hz, 1H), 8.48−8.45 (m, 1H), 7.36−7.27 (m, 1H). 13C NMR (CDCl3): δ 159.0, 140.2, 132.6, 131.6, 121.9, 121.2. MS (HRMS): m/z calcd for C6H3O5N2 [M − H]+ 183.0042, found 183.0036. Compound P6′. Eluent 7:3 hexane−ethyl acetate mixture. Yellow solid (0.020 g, 27%). Melting point: 122−124 °C. FT-IR (KBr, ν in

cm-1): 3341, 3029, 2979, 2936, 2872, 2586, 2373, 1769, 1656, 1537, 1436, 1361, 1213, 1176, 1033, 1079, 752, 701, 692, 642, 512. 1H NMR (500 MHz, CDCl3): δ 8.66 (d, J = 8.25 Hz, 1H), 8.43 (d, J = 8.95 Hz, 1H), 8.17−8.16 (m, 1H), 7.70−7.66 (m, 1H), 7.53−7.49 (m, 2H), 7.33−7.30 (m, 1H), 7.26−7.24 (m, 1H), 6.75−6.74 (m, 1H), 4.19 (s, 2H), 2.91 (s, 6H). 13C NMR (CDCl3): δ 152.2, 146.6, 140.6, 140.5, 132.8, 131.4, 130.5, 129.8, 129.8, 129.6, 123.0, 122.9, 118.5, 115.8, 112.7, 111.2, 45.3. MS (HRMS): m/z calcd for C18H17O5N3S [M + H]+ 388.0967, found 388.0978, [M + Na]+ 410.0787, found 410.0707. Compound P8′. Eluent 7:3 hexane−ethyl acetate mixture. Yellow solid (0.016 g, 22%). Melting point: 116−120 °C. FT-IR (KBr, ν in cm-1): 3329, 3044, 2989, 2952, 2846, 2534, 1719, 1625, 1536, 1457, 1353, 1253, 1186, 1034, 1026, 737, 715, 714, 652, 522. 1H NMR (500 MHz, CDCl3): δ 8.62 (d, J = 8.90 Hz, 1H), 8.42 (d, J = 8.25 Hz, 1H), 8.07−8.05 (m, 1H), 7.71−7.68 (m, 1H), 7.47−7.43 (m, 2H), 7.26 (d, J = 7.65 Hz, 1H), 7.02−7.00 (m,1H), 6.63 (d, J = 8.95 Hz, 1H), 6.04 (s, 2H), 2.91 (s, 6H). 13C NMR (CDCl3): δ 152.0, 143.3, 139.1, 132.4, 131.5, 130.8, 130.5, 130.1, 129.9, 129.7, 129.3, 122.9, 119.4, 119.3, 119.8, 115.7, 45.4. MS (HRMS): m/z calcd for C18H17O5N3S [M + H]+ 388.0967, found 388.0969, [M + Na]+ 410.0787, found 410.0780. Compound P9′. Eluent 1:1 hexane−ethyl acetate mixture). Yellow solid (0.015 g, 21%). Melting point: 110−114 °C. FT-IR (KBr, ν in cm-1): 3342, 3055, 2973, 2930, 2812, 2571, 1709, 1658, 1541, 1412, 1354, 1271, 1193, 1039, 1012, 732, 703, 676, 521. 1H NMR (500 MHz, CDCl3): δ 8.56 (d, J = 8.25 Hz, 1H), 8.44 (d, J = 8.25 Hz, 1H), 8.05 (d, J = 7.55 Hz, 1H), 7.65−7.61 (m, 1H), 7.42−7.39 (m, 1H), 7.22 (d, J = 7.55 Hz, 1H), 6.35−6.31 (m, 2H), 6.08−6.06 (m, 1H), 3.2 (s, 4H), 2.8 (s, 6H). 13C NMR (CDCl3): δ 151.7, 142.8, 135.5, 133.3, 131.6, 131.1, 131.0, 130.0, 129.5, 128.8, 122.9, 119.5, 116.3, 115.4, 112.7, 110.0, 45.3. MS (HRMS): m/z calcd for C18H19O3N3S [M]+ 357.1147, found 357.1509.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.7b00317. X-ray data for P4−P9 (CIF) Single-crystal X-ray diffraction data, DFT functional optimized structures and excited-state electronic transition characters, and characterization data and spectroscopic data for all compounds (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Subrata Ghosh: 0000-0002-8030-4519 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was funded by the Department of Science and Technology, Government of India (DST, Grant No. SERB/F/ 2408/2012-13). M.V. thanks to Council of Scientific and Industrial Research (CSIR), Government of India for research fellowship. Sunil Kumar acknowledges the University Grant Commission (UGC) for the doctoral fellowship. Thanks to the Director of IIT Mandi for research facilities. Thanks to Advanced Materials Research Center (AMRC), IIT Mandi, for technical support and analytical facilities.

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DOI: 10.1021/acs.joc.7b00317 J. Org. Chem. 2017, 82, 4713−4720

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