Modified Atomic Orbital Overlap: Molecular Level Proof of the Nucleophilic Cleavage Propensity of Dinitrophenol-Based Probes

Article abstract of DOI:10.1021/acs.joc.7b00317

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.

Full text of DOI:10.1021/acs.joc.7b00317

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Article
Modified Atomic Orbital Overlap: Molecular Level Proof of the
Nucleophilic Cleavage Propensity of Dinitrophenol-based Probes
Mangili Venkateswarulu, Sunil Kumar, and Subrata Ghosh
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 06 Apr 2017
Downloaded from http://pubs.acs.org on April 6, 2017
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Modified Atomic Orbital Overlap: Molecular Level Proof of the
Nucleophilic Cleavage Propensity of Dinitrophenolꢀbased Probes
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Mangili Venkateswarulu, Sunil Kumar, and Subrata Ghosh*
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School of Basic Sciences, Indian Institute of Technology Mandi, Mandiꢀ175001, Himachal
Pradesh, 175005, India.
ABSTRACT: Out of six possible positional isomers of dinitrophenol, only 2,4ꢀDNP has
been used extensively by many researchers for developing reactive molecular probes. But the
question remains unanswered: why has 2,4ꢀisomer only emerged as labile protecting group?
To answer this question, six molecular probes using available DNP isomers were developed
and investigated to evaluate the effect of 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
as compared to pꢀNO2 group toward the reactivity of 2,4ꢀDNP based probes. Crystal structure
analysis revealed that the 2p orbital of N atom and the 2p orbital of adjacent ring C atom to
which the o-NO2 is attached are inclined at >30° to each other leading to substantial reduction
in π overlapping (as these two p-orbitals loose coplanar state) resulting in very weak ꢀM
effect of o-NO2 group, whereas the 2p orbitals of N atom of p-NO2 group and adjacent ring C
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atom are almost coplanar (11° inclined to each other) leading to strong π overlap and hence,
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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
group in organic chemistry.1ꢀ2 Since the inception of new methodology for the facile removal
of 2,4ꢀ dinitrophenol (2,4ꢀDNP) protections, this particular protecting group has been widely
explored in polymer and peptide chemistry (Scheme 1a).3ꢀ4 In recent times, 2,4ꢀDNP is being
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.5ꢀ
20
Whether it is deprotection or cleavage of molecular probes into various molecular
fragments exerting optical signal, the removal of 2,4ꢀDNP functionality is done by the attack
of a nucleophile. Some representative cases have been shown in Scheme 1. Surprisingly, out
of six possible positional isomers of dinitrophenol, only 2,4ꢀDNP has been used to establish
all such chemistry.5ꢀ20 In some occasions, 3,5ꢀDNP has been used as protecting group, but it
requires harsh oxidative reaction conditions for its removal.21
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This demonstrates that 2,4ꢀDNP imparts high reactivity toward nucleophilic attack and hence,
no harsh reaction condition is required for its removal. Although, the role of 2,4ꢀDNP as
protecting group has been discussed since long,5ꢀ20 but it has never been investigated with a
mechanistic approach why only 2,4ꢀDNP develops highly reactive molecular probes whereas,
the molecular probes based on other positional isomers are very less reactive? Also, the other
isomeric members of dinitrophenol family remained unexplored in protecting group
chemistry. It may be the case that only 2,4ꢀDNP is an active member in protecting group
chemistry, but still there exists no report to support this wellꢀknown fact. Moreover, if the
electron withdrawing mesomeric effect (ꢀM) of ortho-/para-nitro group (ꢀNO2) is the key in
inducing reactivity, one can expect that 2,3ꢀ; 2,5ꢀ; 3,4ꢀ and 2,6ꢀDNP may also be used to
develop reactive probes as these probes contain orthoꢀ or paraꢀNO2 groups. Thus, this lack of
correlation between DNP isomers as protecting groups and their leaving propensities arouse
interesting query among us, what makes the 2,4ꢀDNP unit as extensively studied leaving
group out of other isomeric DNP family members?
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Scheme 1. (a) Thiolysis of 2,4ꢀdinitrophenyltyrosines by 2ꢀ
mercaptoethanol.3,4 (b) Structure of fluorescent H2S probes based on
dinitrophenyl ether.5ꢀ20
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Based on the first report of effective removal of the 2,4ꢀDNP using mercaptoethanol
nucleophile,3a we sought to understand the above problem at very fundamental level with
experimental evidences. This manuscript describes at the molecular level, the chemistry of
effective atomic orbital overlap behind such differences in the reactivity of DNP based
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.22ꢀ23 To
demonstrate these molecular events, we developed sulfonate ester based six molecular probes
(Figure 1) by reacting dansyl chloride with all possible positional isomers of DNP (Scheme
S1).
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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 reactive nucleophile.24ꢀ26 We focused to monitor the
reaction progress using advanced optical tools such as UVꢀvis absorption and fluorescence
spectroscopy. It was observed that each specific probe followed specific reaction pathway
giving specific optical responses. To gain more insights 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
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structure analysis that the steric factor and the molecular substituent orientation govern the
extent of atomic orbital overlap which eventually control the overall electronic effect and
molecular reactivity. We also employed Single Crystal Xꢀray Diffraction (SCꢀXRD) and
Density Functional Theory (DFT) studies to support our conclusion.
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RESULTS AND DISCUSSION:
As mentioned above, to complete our investigation, we synthesized six dansyl platforms P4ꢀ
P9 where each platform bears unique DNP positional isomer on sulphonyl group (Figure 1).
Each scaffold has dual elelctrophilic sites viz, sulfonate ester and the nitro group. The
nucleophilic attack at sulphonate ester would make the probes as chemodesimeters. It was
clear that the electrophilicity at S atom of sulfonate center was due to the overall electron
withdrawing (EW) effect of 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 entirely 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.
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Figure 2. UVꢀvis (a) and emission spectra (b) of all probes (P4, P5, P6, P7, P8 and
P9) (10 µM) upon addition of NaHS (80 µM) in THF: H2O (1:1, v/v) buffered with
HEPES (1 mM, pH = 7.2).
Initially, the UVꢀvis absorption studies of probe P4ꢀ9 were performed in HEPES buffer
(1mM, pH=7.2) containing 50% THF. Owing to similar chemical structure, all these probes
were found to be stable in buffer solution and displayed similar absorption behaviour. The
fluorescence spectroscopy revealed the nonꢀfluorescent nature of these molecular probes
(Figure S1a and S1b, Table S2). The electronꢀwithdrawing (EW) group induced electron
transfer process inhibited the fluorescence emission, which is also known as PET
(photoinduced electron transfer) or dꢀPET (donorꢀexcitedꢀPET).28,29 After initial
photophysical studies, we studied the optical response of P4ꢀ9 in the presence of H2S
(generated in situ by adding NaHS to water and HS‾ is the active nucleophilic form of H2S)
(Figure 2, S2, S3). Upon addition of H2S, significant changes in UVꢀvis absorption behavior
were observed for P5 only (Figure S2). We observed that the fluorescence spectra of P7
remained unaffected (Figure S3d). Only small enhancement in emission intensity at 495 nm
was observed for P4/P6/P8 (Figure 2, S3a, S3c and S3e). In the case of P9, insignificant
increase in florescence intensity along with ~70 nm red shift (Figure S3f) was observed
(Table S2). The probe P5 exhibited significant changes in absorption and emission spectra
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upon H2S addition (Figure 2, S2b, S3b, S4ꢀS6). In the absorption profile, two new peaks at
~325 and ~475 nm were observed with the disappearance of 348 nm peak (Figure S4).
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Scheme 2. HS¯ꢀMediated chemical changes in the designed DNPꢀprobes.
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To understand the intrigued photophysical changes, products formed after the reaction of
each probe with HS¯ (P4ʹꢀP9ʹ) were successfully isolated from the reaction mixture (Scheme
2) and thoroughly characterized (Figure S7ꢀS24, S56ꢀS73). 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 sulphonyl group leading to cleavage of the probe into
fluorescent entity (P5') and 2,4ꢀDNP unit (Scheme 2). Adding to our surprise, 2,6ꢀDNP
probe (P7) remained unreactive in the presence of HS¯. Whereas, 2,4ꢀDNP based probe P5
underwent fast nucleophilic substitution of 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 P4ʹ/P6ʹ/P8ʹ/P9ʹ (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, 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 oꢀ/pꢀNO2 groups would enhance the
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electron deficiency at Sꢀcentre of sulfonate group through making the Oꢀatom of PhꢀOꢀS
bond of P5 highly electron deficient. Indeed, the sulfonate group of P5 showed high
reactivity towards nucleophilic attack by HS¯. But, if this is true, then the probes P4,P6,P8
which contain atleast one ꢀNO2 group at oꢀ/pꢀ position should have shown similar response
with HS¯ as that of P5. But these probes underwent ꢀNO2 group reduction by H2S and in
addition, P7 which contains two ꢀNO2 groups at ortho positions remained fully silent toward
H2S attack. This unusual reactivity of P4, P6, 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.
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It has been well discussed in organic chemistry that, in general, if the ꢀNO2 group is placed
on metaꢀposition 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
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 ꢀNO2 group is placed on metaꢀposition. As the
mꢀNO2 functionality is four atoms away (three Cꢀatoms and one Oꢀatom) from the Sꢀcenter of
sulfonate group in P4/P6/P8/P9, the –I effect of mꢀNO2 group on Sꢀcenter would be
negligible because the ꢀI effect falls off with distance and hence, the Sꢀcenter could not
acquire sufficiently electron deficient environment to attract nucleophilic attack. As a result,
P4/P6/P8/P9 yielded the reduced product 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.
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To investigate more fundamentally and to gain more insights into molecular level, true
geometries of the probes were required. Thus the single crystals were grown to understand
the geometrical constraints in each probe. All probes were having inverted “V” shape
geometry where sulphonyl group was at bent position (Table S1, Figure 3, S74ꢀS79). The
absence of oꢀNO2 group in the P8 and P9 failed to provide steric crowdedness near sulfonyl
center and as a consequence, it could be seen that dansyl and DNP ring systems face each
other in P8 and P9. But, the probes with orthoꢀsubstitution were found to be twisted with
respect to naphthalene plane mainly because of steric repulsion with the oxygen atoms of
sulphonyl group (Figure 3, S25ꢀS29).
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Figure 4. Schematic showing the atomic orbitals of p–NO2 (in plane and out of plane) with
respect to pꢀorbitals of carbon in aromatic ring in probe (a) P5 and (b) P8.
The simple understanding of mesomeric effect and atomic orbital overlap could tell us that,5
in the DNP unit, orthoꢀ and paraꢀNO2 groups are coupled through ꢀM effect to phenolic
oxygen, while metaꢀNO2 group is not a part of this electronic communication. If looked
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carefully, one can also observe that, in none of these positional isomeric scaffolds, H2S
attacked at the oꢀNO2 and pꢀNO2 groups, but the mꢀNO2 group underwent reduction to amine
functionality. The nonꢀconjugated mꢀNO2 group retained its electrophilic nature and the nonꢀ
steric environment aided hydrogen sulfide mediated reductive attack.30,31 As a result, mꢀNO2
groups got converted into amine functionalities in the cases of P4/P6/P8/P9. But, still the
mystery of silence toward nucleophilic cleavage of o/pꢀnitro substituted probes
(P4/P6/P7/P8) was unanswered.
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The critical analyses of the crystal structures established that the o/pꢀNO2 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 N atom of o/pꢀ
NO2 group and the 2p orbital of ring C atom to which the o/pꢀNO2 groups are attached
become inclined at >33° to each other and hence leads to a substantial reduction in πꢀ
overlapping (Fugure S31). In the cases of P4 and P6, electronic repulsion between the
electron clouds of sulfonate oxygen and oxygen atoms of the oꢀNO2 group prevented the nitro
group from lying in the same plane as the aromatic nucleus and the allowed rotation about Cꢀ
N bond moved ꢀ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 πꢀoverlapping which in turn prevented the electronic interaction with
the aromatic nucleus. Thus, the oꢀNO2 groups in P4/P6 failed to exert ꢀM effect and the
electrophilic environment at Sꢀcenter did not attract nucleophilic attack (Figure S31a, S31b).
Similarly in P8, the steric as well as electronic repulsion between mꢀNO2 and pꢀNO2 group
pushed pꢀNO2 group to move out of plane by 37.4° with respect to aromatic ring plane
(Figure 4b, 5c and 5d), mismatch in planes blocked the mesomeric effect of pꢀNO2 group and
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hence, the S center of sulfonate group did not develop required electron deficiency for
nucleophilic attack.
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The crystal structure analysis also revealed that the oꢀNO2 groups of P7 are not in
conjugation with the ring πꢀcloud because of outꢀofꢀplane orientation of both the oꢀNO2
groups (at 38.4° and 44.7° with respect to aromatic plane) resulting in poor πꢀoverlapping
between these 2p orbitals (Figure S31c, S32e, S32f). In short, it could be said that the oꢀNO2
groups failed to enhance the electrophilicity at S atom because of suffered twist/torsion.
However, in P5 scaffold, the conditions were favourable for active ꢀM effect through the pꢀ
NO2 group as it shares the same plane with phenyl ring of DNP (Figure 4a, 5a and 5b) even
though the oꢀNO2 is out of plane of the aromatic ring. The steric effect free environment
around paraꢀposition allowed the 2p orbital of N atom of pꢀNO2 group and the 2p orbital of
ring C atom to which the pꢀNO2 group is attached to become fairly nearly parallel to each
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other (they are just 11° inclined to each other) leading to effective overlap between these
orbitals which in turn leads to strong –M effect of pꢀNO2 group, and hence the developed
mesomeric interaction through effective delocalization via πꢀorbitals created strong
electrophilicity at the S atom (Figure 4a, 5a and 5b). Interestingly, this is prevented in the
case of oꢀNO2 group as the steric influence inhibited significant overlapping of planes of NO2
group and benzene ring, respectively, (as they are 31.8° inclined to each other) and therefore
the oꢀNO2 group contributes less to the eletrophilicity of Sꢀcenter due to poor mesomeric
electronꢀwithdrawal. Therefore, the molecular level understanding through crystal structure
analysis helped us to establish for the first time that the reactivity of 2,4ꢀDNP based probes or
protection sites is mainly due to pꢀNO2 functionality, whereas oꢀNO2 contributes less toward
the reactivity.
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In order to support the above molecular level understanding, the geometries of P4ꢀP9/P4ʹꢀP9ʹ
were optimized using density functional theory (DFT) (Figure S33ꢀS44).32ꢀ35 In all the cases,
the HOMOs are spread over the N, Nꢀdimethylaminonaphthyl ring whereas the electron
density of the LUMO orbitals are predominantly located on the dinitrobenzene ring. HOMO
to LUMO electronic transitions were accompanied by the photoelectron transfer (PET), and
thus these transitions can be classified as donorꢀexcited PET or dꢀPET which make the probes
weak fluorescent in nature.28,29 Moreover, timeꢀdependent Density Functional Theory (TDꢀ
DFT) calculations were also in close proximity to the experimental absorption band domains
(Figure S45ꢀS55). The MO distribution in P5ʹ 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 (P4ʹ/P6ʹ/P8ʹ) (Figure S40ꢀS44) are
similar to starting DNP isomers P4/P6/P8, and hence these remain nonꢀfluorescent. Although
the MOs change positions in P9ʹ, the dꢀPET process makes the product nonꢀfluorescent in
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nature. These results further suggest that the absence of dꢀPET process in P5' is the major
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cause of the origin of fluorescence.
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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 pseudoꢀfirstꢀorder rate constant was
calculated to be k = 0.1199 minꢀ1 (Figure S56).
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CONCLUSIONS
Often the reactivity of molecular probes, determined experimentally, is explained by
theoretical studies largely due to lack of direct evidences at the molecular level particularly
on reactivity versus extent of atomic orbital overlap. To conclude, we have studied the
unusual high reactivity of 2,4ꢀDNP based molecular probes as compared to all other possible
positional isomeric probes based on DNP. The reason behind unexpected low reactivity of
various DNP based probes, which is beyond our common understanding, has been established
from molecular level understanding and visualization of extent of atomic orbital overlap
through crystal structure analyses. We have shown for the first time that the reactivity of 2,4ꢀ
DNP based probes is largely due to p-NO2 group whereas the contribution of o-NO2 group is
substantially less than expected. This is due to weak overlap between nitrogen p-orbital of o-
NO2 group and the p-orbital of adjacent ring carbon (as these two p-orbitals are not parallel to
each other, rather they are inclined at 33° to each other). All 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 predominance of one
molecular factor over the others. For example, in the case of 2,6ꢀDNP based probe P7 , due to
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strong steric reason, the functional groups are oriented such that both the o-NO2 groups are
outꢀofꢀplane with respect to plane containing aromatic ring which resulted in poor overlap
between nitrogen p-orbital and adjacent carbon p-orbital. Hence, the –M effect of o-NO2
groups are too weak to induce required electron deficiency at Sꢀcenter of P7 for nucleophilic
attack. Similarly, o-NO2 group in P4/P6 and p-NO2 group in P8 have negligible impact on
reactivity due to poor atomic orbitals overlap as the nitrogen p-orbitals are not in parallel
orientation with ring carbon p-orbitals. That o-NO2 group contributes less toward reactivity
of P5 was also supported by the fact that 2,3ꢀ; 2,5ꢀ and 2,6ꢀDNP 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 standard solution to record the UVꢀvis and fluorescence spectra. FTꢀIR spectra in KBr
1
were recorded on a Perkin Elmer Spectrum 2 spectrophotometer. H/13C NMR spectra in
CDCl3 were recorded on JeolꢀECXꢀ500 MHz spectrometer using tetramethyl silane as an
internal standerd. Absorption spectra were recorded with SHIMADZU UVꢀ2450
spectrophotometer. The fluorescence spectra were recorded with Cary Eclipse
spectrophotometer with slit widths of 5 nm for excitation and 5 nm for emission respectively.
HRMSꢀESI spectra were recorded using a quadrupoleꢀtimeꢀofꢀflight mass analyzer on a
Bruker impactꢀHD spectrometer respectively. Single crystal Xꢀray diffraction data were
collected on Agilent SuperNova CCD system.
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Xꢀray Crystal Structure Analysis of P4/P5/P6/P7/P8/P9: Single crystals of
P4/P5/P6/P7/P8/P9 suitable for Xꢀray diffraction were grown in ethanol by its slow
evaporation after one day. Single crystal Xꢀray diffraction data of P4/P5/P6 were measured
using Cu Kα (λ= 1.5406 Å at 298(2) K) source and the probe P4/P5/P6 were measured using
Mo Kα (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
Pꢀ1, 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 structure were
solved by direct methods OLEX2 and refined using fullꢀmatrix leastꢀsquares (F2) on
SHELXLꢀ97.36,37 The positions of all nonꢀhydrogen 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.
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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 B3LYP/6ꢀ31G(d,p)
basis set32,33 with no symmetry constraint using the Gaussian 09 suite of programs.34
Frequency calculation at the same level with the same basis set was performed to ensure that
the geometries correspond to real minima (see in supplementary 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 dry CH2Cl2 under nitrogen at 0 ºC with
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constant stirring. After that, the reaction mixture was left stirring at room temperature and
completion of reaction was judged by the TLC, then the organic solution was washed with
water and the solution was concentrated under reduced pressure to get 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.
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Compound P4: Yield: 83% (65 mg); Melting point: 92ꢀ 96°C; FTꢀIR (KBr, ν in cmꢀ1): 3072,
2951, 2922, 2848, 2664, 2360, 1651, 1613, 1543, 1486, 1458, 1407, 1377, 1338, 1303, 1174,
1
1126, 1061, 961, 941, 817, 719, 565, 501. H NMR (500 MHz, CDCl3): δ 8.70 (d, J = 8.20
Hz, 1H), 8.25 (d, J = 8.90 Hz, 1H), 8.20ꢀ8.18 (m, 1H), 8.05 (d, J = 7.55 Hz, 1H), 7.85ꢀ7.83
(m, 1H), 7.67ꢀ7.64 (m, 2H), 7.55ꢀ7.52 (m, 1H), 7.28ꢀ7.26 (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 calculated for C18H16O7N3S [M+H] 418.0709 and
found 418.0703, [M+K] + 456.0268 and found 456.4405.
Compound P5: Yield: 78% (61 mg); Melting point: 156ꢀ 158°C; FTꢀIR (KBr, ν in cmꢀ1):
2915, 2862, 2749, 1511, 1509, 1453, 1441, 1349, 1303, 1215, 1182, 1146, 967, 856, 826,
1
770, 751, 661, 642 626, 563, 513, 509. H NMR: (500 MHz, CDCl3): δ 8.72ꢀ8.69 (m, 2H),
8.37ꢀ8.33 (m, 2H), 8.16ꢀ8.14 (m, 1H), 7.70ꢀ7.67 (m, 1H), 7.54ꢀ7.51 (m,1H), 7.43 (d, J = 8.95
Hz, 1H), 7.28ꢀ7.26 (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 calculated for C18H15O7N3S [M] 417.0631 and found 417.0607, [M+Na]
440.0528 and found 440.0515, [M+K] + 456.0268 and found 456.0252.
Compound P6: Yield: 81% (63 mg); Melting point: 134ꢀ 136°C; FTꢀIR (KBr, ν in cmꢀ1):
3113, 3094, 2988, 2947, 2840, 2551, 2359, 1938, 1767, 1615, 1547, 1530, 1476, 1404, 1341,
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1309, 1217, 1170, 1150, 1109, 1063, 947, 906, 819, 788, 741, 714, 671, 559, 501. H NMR
(500 MHz, CDCl3): δ 8.70 (d, J = 8.90Hz, 1H), 8.34 (d, J = 8.25Hz, 1H), 8.21ꢀ8.18 (m, 1H),
8.15ꢀ8.13 (m, 1H), 7.99 (d, J = 8.90Hz, 1H), 7.91 (d, J = 2.1Hz, 1H), 7.71ꢀ7.67 (m, 1H), 753ꢀ
7.50 (m, 1H), 7.28 (d, J = 7.55Hz, 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
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(HRMS): m/z calculated for C18H16O7N3S [M+H] + 418.0709 and found 418.0702, [M+K] +
456.0268 and found 456.4410.
Compound P7: Yield: 76 % (59 mg); Melting point: 118ꢀ 126°C; FTꢀIR (KBr, ν in cmꢀ1):
2951, 2853, 2788, 1569, 1534, 1469, 1453, 1413, 1356, 1321, 1214, 1166, 1145, 974, 891,
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833, 796, 761, 691, 653, 629, 568, 529, 512. H NMR (500 MHz, CDCl3): δ 8.72 (d, J =
8.25Hz, 1H), 8.37 (d, J = 8.95Hz, 1H), 8.12ꢀ8.10 (m, 2H), 7.71 (t, J = 8.25Hz, 1H), 7.58 (t, J
= 8.25Hz, 1H), 7.53ꢀ7.50 (m,1H), 7.28ꢀ7.26 (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 calculated for C18H16O7N3S [M+H] + 418.0709 and found 418.0703, [M+Na] +
440.0528 and found 440.0515, [M+K] + 456.0268 and found 456.0259.
Compound P8: Yield: 79% (62 mg); Melting point: 124ꢀ 126°C; FTꢀIR (KBr, ν in cmꢀ1):
2957, 2802, 2359, 2163, 1766, 1572, 1537, 1477, 1437, 1346, 1315, 1204, 1172, 1128, 944,
1
893, 852, 824, 788, 727, 670, 563, 533. H 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), 7.53ꢀ7.50 (m, 1H), 7.426ꢀ7.42 (m, 1H), 7.33ꢀ7.31 (m, 1H), 7.27ꢀ7.26 (m, 1H),
2.91 (s, 6H); 13C 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 calculated for
C18H16O7N3S [M+H] + 418.0709 and found 418.0703.
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Compound P9: Yield:82% (64 mg); Melting point: 118ꢀ 120°C; FTꢀIR (KBr, ν in cmꢀ1):
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3118, 2922, 2850, 2360, 2340, 2193, 1949, 1900, 1743, 1571, 1539, 1453, 1412, 1373, 1337,
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1229, 1203, 1175, 1149, 1066, 972, 946, 897, 819, 796, 722, 626, 585, 487. H NMR (500
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MHz, CDCl3): δ 8.87ꢀ8.86 (m, 1H), 8.68(d, J = 8.25 Hz, 1H), 8.39 (d, J = 8.25 Hz, 1H), 8.12
(d, J = 6.85Hz, 1H), 8.08ꢀ8.07 (m, 2H), 7.76ꢀ7.73 (m, 1H), 7.51ꢀ7.48 (m, 1H), 7.30 (d, J =
7.6Hz, 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 calculated for C18H16O7N3S
[M+H] + 418.0709 and found 418.0703.
General procedure for the synthesis of P4'/P5'/P5"/P6'/P8'/P9': A solution of NaHS (10
equivalent, 0.119 g, 2.14 mmol) in 3 mL H2O was added to a solution of DNPꢀbased probes
(P4/P5/P6/P8/P9) (0.08 g, 0.214 mmol) in 10 mL THF/water (1:1, v/v) under stirring
condition. The reaction mixture was then left stirring at room temperature for 30 min. This
mixture was extracted with DCM, dried over anhydrous Na2SO4, the solvent was removed
under reduced pressure to get the crude product which was purified by column
chromatography on silica gel using suitable eluent.
Compound P4′: (eluent 7: 3 hexane–ethyl acetate mixture); color: yellow solid (0.024 g,
24%). Melting point: 98ꢀ102°C. FTꢀIR (KBr, ν in cmꢀ1): 3349, 3064, 2992, 2948, 2832,
1
2598, 1721, 1631, 1533, 1450, 1373, 1242, 1197, 1056, 1039, 748, 711, 703, 632, 532. H
NMR (500 MHz, CDCl3): δ 8.58 (d, J = 8.9 Hz, 1H), 8.45 (d, J = 8.95Hz, 1H), 8.11 (d, J =
7.55Hz, 1H), 7.68ꢀ7.64 (m, 1H), 7.47ꢀ7.43(m, 1H), 6.90 (t, J = 8.25 Hz, 1H), 6.46ꢀ6.45 (m,
1H), 6.33ꢀ6.32 (m, 1H), 6.18ꢀ6.16 (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 calculated for C18H17O5N3S [M+H] 388.0967, found
388.1046, [M+Na] + 410.0787, found 410.0887.
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Compound P5′: (eluent 9: 1 dichloromethane–methanol mixture); color: light yellow solid
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(0.038 g, 76%). Melting point: 96ꢀ100°C. FTꢀIR (KBr, ν in cmꢀ1): 3712, 3545, 3325, 2972,
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2834, 2275, 1547, 1372, 1335, 1053, 1036, 975, 823, 737, 665, 541. H NMR (500 MHz,
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DMSOꢀd6): δ 8.51 (d, J = 8.95Hz, 1H), 8.17 (d, J = 8.25Hz, 1H), 7.93 (d, J = 7.55Hz, 1H),
7.43ꢀ7.37 (m, 1H), 7.10 (d, J = 7.55Hz, 1H), 2.79 (s, 6H). 13C NMR (DMSOꢀd6): 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): MS
(HRMS): m/z calculated for C12H12NO2S2 [M]ꢀ 266.0309, found 266.0303.
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Compound P5": (eluent 1:1 hexane–ethyl acetate mixture); color: yellow solid (0.021 g,
60%). Melting point: 106ꢀ 110°C. FTꢀIR (KBr, ν in cmꢀ1): 3361, 3262, 3117, 1673, 1539,
1482, 1332, 1253, 1215, 1134, 812, 791, 722, 643, 521, 505. 1H NMR (500 MHz, CDCl3) δ:
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9.07 (d, J = 2.75Hz, 1H), 8.48ꢀ8.45 (m, 1H), 7.36ꢀ7.27 (m, 1H), C NMR (CDCl3): δ 159.0,
+
140.2, 132.6, 131.6, 121.9, 121.2. MS (HRMS): m/z calculated for C6H3O5N2 [MꢀH]
183.0042, found 183.0036.
Compound P6′: (eluent 7: 3 hexane–ethyl acetate mixture); color: yellow solid (0.020 g,
27%). Melting point: 122ꢀ124°C. FTꢀIR (KBr, ν in cmꢀ1): 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 = 8.25Hz, 1H), 8.43 (d, J = 8.95Hz, 1H), 8.17ꢀ8.16
(m, 1H), 7.70ꢀ7.66 (m, 1H), 7.53ꢀ7.49 (m, 2H), 7.33ꢀ7.30 (m, 1H), 7.26ꢀ7.24 (m, 1H), 6.75ꢀ
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 calculated 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); color: yellow solid (0.016 g,
22%). Melting point: 116ꢀ120°C. FTꢀIR (KBr, ν in cmꢀ1): 3329, 3044, 2989, 2952, 2846,
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2534, 1719, 1625, 1536, 1457, 1353, 1253, 1186, 1034, 1026, 737, 715, 714, 652, 522. H
NMR (500 MHz, CDCl3): δ 8.62 (d, J = 8.90Hz, 1H), 8.42 (d, J = 8.25Hz, 1H), 8.07ꢀ8.05 (m,
1H), 7.71ꢀ7.68 (m, 1H), 7.47ꢀ7.43 (m, 2H), 7.26 (d, J = 7.65Hz, 1H), 7.02ꢀ7.00 (m,1H), 6.63
(d, J = 8.95Hz, 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,
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45.4. MS (HRMS): m/z calculated 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); color: yellow solid (0.015 g,
21%). Melting point: 110ꢀ114°C. FTꢀIR (KBr, ν in cmꢀ1): 3342, 3055, 2973, 2930, 2812,
1
2571, 1709, 1658, 1541, 1412, 1354, 1271, 1193, 1039, 1012, 732, 703, 676, 521. H NMR
(500 MHz, CDCl3): δ 8.56 (d, J = 8.25Hz, 1H), 8.44 (d, J = 8.25Hz, 1H), 8.05 (d, J = 7.55Hz,
1H), 7.65ꢀ7.61 (m, 1H), 7.42ꢀ7.39 (m, 1H), 7.22 (d, J = 7.55Hz, 1H), 6.35ꢀ6.31 (m, 2H),
6.08ꢀ6.06 (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 calculated for C18H19O3N3S [M]+ 357.1147, found 357.1509.
ASSOCIATED CONTENT
Supporting Information.
Additional s mentioned in the text. This material is available free of charge via the ACS
Publications website at http://pubs.acs.org. Single crystal Xꢀray diffraction data in CIF, DFT
functional optimized structures and excited state electronic transition characters, and all
compounds characterization data and spectroscopy data (PDF).
AUTHOR INFORMATION
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Corresponding Author
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subrata@iitmandi.ac.in
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ACKNOWLEDGMENT
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This project was funded by the Department of Science and Technology, Government of India
(DST, Grant No. SERB/F/2408/2012ꢀ13). 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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