Reactive species involved in the regioselective photooxidation of heptamethine cyanines

Article abstract of DOI:10.1039/c5sc02396c

Heptamethine cyanines are important near-IR fluorophores used in many fluorescence applications. Despite this utility, these molecules are susceptible to light-promoted reactions (photobleaching) involving photochemically generated reactive oxygen species (ROS). Here, we have sought to define key chemical aspects of this nearly inescapable process. Near-IR photolysis of a model heptamethine cyanine leads to the regioselective oxidative cleavage of the characteristic polyene. We report the first quantitative analysis of the major reaction pathway following either photolysis or exposure to candidate ROS. These studies clearly indicate that only singlet oxygen (¹O₂), and not other feasible ROS, recapitulates the direct photolysis pathway. Computational studies were employed to investigate the regioselectivity of the oxidative cleavage process, and the theoretical ratio is comparable to observed experimental values. These results provide a more complete picture of heptamethine cyanine photooxidation, and provide insight for the design of improved compounds for future applications.

Full text of DOI:10.1039/c5sc02396c

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Reactive species involved in the regioselective
photooxidation of heptamethine cyanines†
Cite this: DOI: 10.1039/c5sc02396c
a
a
b
a
*
*
Roger R. Nani, James A. Kelley, Joseph Ivanic and Martin J. Schnermann
Heptamethine cyanines are important near-IR fluorophores used in many fluorescence applications.
Despite this utility, these molecules are susceptible to light-promoted reactions (photobleaching)
involving photochemically generated reactive oxygen species (ROS). Here, we have sought to define
key chemical aspects of this nearly inescapable process. Near-IR photolysis of a model heptamethine
cyanine leads to the regioselective oxidative cleavage of the characteristic polyene. We report the
first quantitative analysis of the major reaction pathway following either photolysis or exposure to
candidate ROS. These studies clearly indicate that only singlet oxygen (1O2), and not other feasible
ROS, recapitulates the direct photolysis pathway. Computational studies were employed to
investigate the regioselectivity of the oxidative cleavage process, and the theoretical ratio is
comparable to observed experimental values. These results provide a more complete picture of
heptamethine cyanine photooxidation, and provide insight for the design of improved compounds for
future applications.
Received 3rd July 2015
Accepted 6th August 2015
DOI: 10.1039/c5sc02396c
www.rsc.org/chemicalscience
useful are the heptamethine cyanines, which contain a 7-carbon
linker connecting two indolenine heterocycles. The absorption
Introduction
and emission maxima of these molecules are generally around
800 nm, occupying the center of the near-IR window where
tissue penetration is maximized and autouorescence is mini-
mized.7 One example, indocyanine green, is an FDA approved
diagnostic agent used in numerous clinical contexts and new
variants continue to be developed for advanced optical
applications.8–11
The light-induced degradation (photobleaching) of uores-
cent small molecules is a pervasive issue that oen interferes
with modern uorescence techniques. These reactions
generally involve photooxidation pathways in which the
photo-excited molecule interacts with nearly ubiquitous O2
to form reactive oxygen species (ROS).1 Reactions between
the self-generated ROS and the uorophore lead to the loss
of the desired optical signal.2 While the general pathway is
well accepted,3 the precise reactions, and particularly
the identity of the products, involved in these oxidative
events are oen only poorly understood.4,5 A detailed
understanding of these ubiquitous processes would
provide a framework from which to develop improved uo-
rescent scaffolds and to create new uorophore-dependent
applications.6
The photodegradation of cyanines is a frequent issue that
can impede uorescence readout. Not only deleterious, this
chemistry has been used deliberately for productive applica-
tions. We recently demonstrated that the photooxidation of
C40-dialkylamine heptamethine cyanines could be used as the
central element of a biologically compatible near-IR uncaging
approach.12 Other investigators have employed the ROS-
mediated oxidation of cyanines for sensing methods.13,14 Prior
studies examining cyanine photodegradation have shown
that photolysis leads to oxidative cleavage of the polyene
linker to provide carbonyl products (eqn (1)).15–19 These
studies, while providing a key initial description of this
process, have either not addressed or provided conicting
information regarding the observed products and the nature
of the oxidant(s). Other uncertain aspects of cyanine reactivity
include the effect of key ROS and the basis of the high
regioselectivity observed in the oxidative cleavage reaction.
Clear answers to these fundamental questions would enable
efforts that seek to expand the utility of heptamethine
cyanines.
Cyanines are important uorophores that nd extensive use
across a range of modern biological procedures. Particularly
aChemical Biology Laboratory, Center for Cancer Research, National Cancer Institute
at Frederick, Frederick, Maryland 21702, USA. E-mail: martin.schnermann@nih.gov
bAdvanced Biomedical Computing Center, DSITP, Frederick National Laboratory for
Cancer Research, Frederick, Maryland 21702, USA. E-mail: joseph.ivanic@nih.gov
† Electronic supplementary information (ESI) available: Detailed experimental
protocols, HRMS spectra, representative HPLC chromatograms, full details
regarding evaluation of B3LYP and MP2 methods against benchmark and
experiment for the hypothetical model reaction: 3O2 + H2C]CH2 / 2H2C]O,
geometrical conformations considered in comprehensive minima searches of
all Cy7-O2 intermediates, and optimized geometries and energies of
lowest-energy (electronic and free (G298), when determined) species. See DOI:
10.1039/c5sc02396c
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no evidence for the compounds derived from cleavage between
C10/C20 (4 and 5), and C30/C40 (8 and 9), or hydrolysis products
thereof, supporting the notion that this reaction proceeds with
high regioselectivity. We have also shown that 2 and 6 accu-
mulate concurrently with the loss of the signal for 1 and that the
ratio of the two intermediates (ꢁ5 : 1) is essentially constant
throughout the reaction (Fig. S1†). Control experiments have
determined that these photoproducts are stable for the duration
of the photolysis experiment. While we account for a majority
(ꢁ70%) of the material through this method, there is a fraction
of 1 that is not attributed to identiable species. Despite
attempts to purify and characterize other products using HPLC,
we have not been able to account for the balance of the reaction
mixture.
We have carried out further studies on [1 + O2] ions to
determine if these species represent dioxetane intermediates.
As expected, the formation of these ions was observed only upon
near-IR irradiation. MS/MS analysis following direct ow
injection was performed to investigate accessible fragmentation
pathways (Fig. 1B).25 Collision-induced dissociation (CID) of the
[1 + O2] ions generated a product ion spectrum containing high-
resolution masses that correspond to carbonyls 2 ([M + H]+), 3
([M]+), 6 ([M ꢀ H]+, [M + H]+) and 7 ([M]+), among others. These
results provide additional evidence to support the intermediacy
of dioxetanes 10 and 11, as these represent the most reasonable
structures of a [1 + O2] mass that could fragment directly to the
observed carbonyl products.
(1)
To address these questions, we have employed a combina-
tion of experimental and theoretical approaches. Our experi-
mental efforts have centered on mass spectral studies and the
rst quantitative accounting of cyanine oxidation products,
using both direct irradiation and exogenous reactive oxygen
species. Photolysis at the cyanine absorption maximum induces
C–C cleavage at two of the four feasible reaction sites in the
cyanine polyene. We show that only singlet oxygen (1O2),
generated either chemically or through photosensitization, and
not other common ROS, provides the photolysis products in
similar yield. We have also carried out the rst quantum
mechanical computational analysis of the major oxidative
cleavage reaction pathway. Our computational studies have
provided a clear justication for both the origin of the regio-
selectivity (i.e. the relative stability of possible intermediate
dioxetanes) and the relative ratio of the observed products for
the major 1O2-dependent pathway. In total, we have dened key
aspects of this photodegradation process, which is encountered
in nearly any application of these broadly employed molecules.
To investigate the oxidant in this reaction, we have examined
the reactivity of 1 with independently generated ROS. Our
efforts commenced with 1O2, which was generated by two
mechanistically distinct methods. The photosensitizer, tetra-
Results and discussion
Photooxidation: experimental results
We sought to address the regioselectivity of the photooxidative phenylporphyrin zinc (ZnTPP), is well established to cleanly
C–C cleavage, which, in the case of symmetrical heptamethine form 1O2 in high quantum yield (fD ¼ 0.7) upon irradiation with
cyanines, could lead to four regioisomeric product mixtures. 420 nm light.26,27 This method afforded a similar product
Prior reports on several different heptamethine cyanine distribution of oxindole 2 (61–64%) and aldehyde 6 (13–15%) to
substrates provided conicting data with three studies sug- that produced by direct cyanine excitation (entries 2 and 3,
gesting that oxidative cleavage occurs at a single terminal respectively, Table 1). Photolysis using only 420 nm light in the
position in the cyanine polyene,15,20,21 another cleavage occurs at absence of a photosensitizer did induce photooxidation to form
only a central position in the cyanine polyene,22 and two recent 2 and 6, albeit with dramatically slower kinetics (ꢁ15 fold) to
studies, including our own, observing cleavage at two sites.12,23 that observed in the presence of catalytic ZnTPP (Fig. S2†). This
Certainly some of this apparent ambiguity is derived from the result is consistent with a small (ꢁ2% relative to the lmax
)
slightly different substrates and conditions used between the absorption of 1 at 420 nm. To complement the photosensitizer-
different studies. Moreover, these studies oen employed only dependent photooxidation, we examined the impact of 1O2
mass spectral data to assign the photooxidative products, and in generated via the thermal decomposition of endoperoxide 12.28
no instances were reaction yields determined.
We found that exposure of 1 to 200 equivalents of 12 at 34 ꢂC in
We have chosen 1, which represents the simplest heptame- 50% MeCN : H2O produced a high yield of oxindole 2 (71%) and
thine indocyanine, as a model system for our efforts.24 To aldehyde 6 (10%), collectively. Thus, 2 and 6 are formed in
accurately measure yields of reactions run at low micromolar similar yields through direct near-IR photolysis and from
concentrations, we employed triplicate HPLC analysis cali- independently generated 1O2, which was formed either ther-
brated using standard curves generated from independently mally or photochemically. This observation provides signicant
prepared standards of previously known 2 and 6 (see ESI†). support to the notion that 1O2-mediated dioxetane formation is
Exposing a solution of 1 (50% MeCN/H2O, 20 mM) to modest the major pathway involved in cyanine photooxidation.29 Pho-
uence (20 mW cmꢀ2) of 740 nm light for 2 hours produces 2 tosensitized formation of 1O2 is consistent with the small
and 6 in 57% and 12% yields, respectively (Fig. 1A). We have number of prior experimental measurements of heptamethine
also carried out HRMS studies and detected 1 and 7, validating cyanine triplet state emission values (including one measure-
that oxidative cleavage occurred between C2/C10 and C20/C30, as ment on 1), which exceed the requisite 22.6 kcal molꢀ1 needed
1
well as [1 + O2] ions. Careful analysis of the HRMS spectra found for O2 energy transfer.30,31
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Fig. 1 (A) Photoproducts observed by HRMS upon 740 nm light irradiation of 1 and (B) MS/MS [1+O2] adducts.
It has been suggested that cyanine photodegradation may O2cꢀ 17,18,20,32–35 To investigate this possibility, the reactivity of 1
.
involve superoxide (O2cꢀ), generated through electron transfer with these ROS was examined. In the case of OHc (entry 1,
from the triplet state and, potentially, hydrogen peroxide (H2O2) Table 2), 1 was consumed, but formed only trace quantities of 6
or hydroxyl radical (OHc), formed as downstream products of (1–3%) and barely detectable quantities of 2 (<0.5%). We have
Table 1 Yields of 2 and 6 upon reaction of 1 with photolysis or independently generated 1O2
Entry
Conditionsa
Remaining 1b
2b
6b
1
2
3
Dark
740 nm hn
Zn(TPP) 5 mol% 420 nm hn
99.5% (ꢃ1.6)
1.6% (ꢃ0.3)
<0.5%
< 0.5%
<0.5%
56.6% (ꢃ2.8)
11.5% (ꢃ0.4)
62.8% (ꢃ1.7)
14.4% (ꢃ0.7)
4
5.5% (ꢃ0.6)
71.7% (ꢃ0.3)
10.3% (ꢃ0.1)
a
Run at 22 ꢂC in 1 : 1 MeCN : H2O unless noted otherwise. b Determined by HPLC and reported in percent yield. Values are average of experimental
triplicate. Error expressed as standard deviation in parentheses.
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been unable to identify any of the numerous species that appear occurring at only two of four possible sites – was unclear. To
to be formed (see ESI† for representative HPLC traces), sug- address this, we pursued a computational approach. Although
gesting that complex radical-mediated pathways are involved. the distinctive optical properties of heptamethine cyanines have
In contrast, O2cꢀ and H2O2 largely do not react with 1 under been examined computationally in several prior reports,39–43 our
neutral aqueous conditions, even at concentrations that exceed efforts are the rst to examine the reactivity of cyanines.
those attainable by photolysis.36 Critically, control experiments
As a rst step, we computed the total reaction energies
showed that compounds 2 and 6 were unaffected by these or the (electronic) for the four possible cleavage pathways using the
other ROS generating conditions shown in Table 2. These B3LYP and MP2 methods with the 6-31G(d) basis set (see
control experiments ensure that the observed product ratio is Methods section for full details). We point out that we cannot
1
indicative of reactions involving 1, and not of further oxidation explicitly compute MP2 and B3LYP Dg O2 energies since this
of 2 and 6. In total, these results indicate that HOc, H2O2, and electronic state is not a single-reference dominated wave-
O2cꢀ are not likely the major components of direct cyanine function. As such, we have determined 1Dg O2 energies by
photooxidation, with all three providing product mixtures that adjustments (adding experimental electronic energy splitting)
3
ꢀ
are clearly distinct from that obtained upon direct near-IR to readily computed Sg O2 energies (see Methods). A conse-
photolysis. The critical observation is that, while other ROS can quence of this limitation is that we are unable to compute Gibbs
1
generate modest yields of oxindole 2, only O2, formed either free energy changes for these reactions. The p system of 1 is
chemically or photochemically, is capable of generating 6 in completely at (C2v point group) at the B3LYP level of theory but
conversion comparable to that generated by direct photolysis.
very slightly twisted (C2 point group) when using the MP2
Recent studies have imaged oxidative ux by using oxidation method. We have considered both cis and trans isomers (about
of certain cyanines with biological ROS, specicallꢀy hypochlo- the end C–C bond, where relevant) to identify the lowest energy
rite (NaOCl), peroxynitrite (ONOOꢀ), OHc, and O2c .13,14,37 The product aldehydes. Fig. 2 clearly shows that all four reactions
yields of products obtained from such ROS could provide are signicantly exothermic (DEe < ꢀ50 kcal molꢀ1 relative to
1
insights useful in future efforts to develop these methods. reactants 1 + Dg O2), with cleavage at the C2/C10 and C20/C30
Reaction of 1 with NaOCl generated meaningful quantities of sites leading to the most favorable products, having B3LYP-
oxindole 2, and no measureable 6 (entry 6, Table 2). While predicted energies of ꢀ89 kcal molꢀ1 and ꢀ66 kcal molꢀ1
,
exposure of 1 to 150 mM NaONO2 under basic conditions respectively. We also point out that the MP2 and B3LYP results
produced over 30% yield of 2 (entry 7, Table 2), only trace are in excellent agreement. While the two experimentally
amounts of 6 were observed.38 As with entry 1, we were unable to observed cleavage pathways are also the most favored in abso-
identify any other oxidation products formed in entries 6 and 7. lute terms, the observation that all pathways are highly
Taken together with observations of Nagano and coworkers,13 exothermic suggested that this explanation is incomplete.
these results provide additional evidence indicating that the
The experimental results provided additional evidence to
reactivity of heptamethine cyanines is highly dependent on the suggest that O2 addition and subsequent cleavage proceeds via
ROS species.
dioxetane intermediates.44 Accordingly, we investigated whether
the energetics of dioxetane formation is responsible for the
observed regioselectivity. In order to locate the absolute lowest-
energy structures for each of the four possible intermediates, we
performed geometry optimizations starting from a very large
number of initial structures that comprehensively spanned the
Regioselectivity: quantum mechanical computational study
While the studies above claried that the major photooxidation
pathway of heptamethine cyanines involved singlet oxygen, the
basis for the high regioselectivity of this process – with cleavage
Table 2 Reaction of 1 with various ROS and respective yields of 2 and 6
Entry
Conditionsa
Remaining 1b
2b
6b
1 (HOc)
FeCl2 200 mM H2O2 500 mM NaHPO4 pH ¼ 6
H2O2 100 mM PBS pH ¼ 7.4
H2O2 1 mM PBS pH ¼ 7.4
KO2 100 mM
3.4% (ꢃ1.2)
99.5% (ꢃ0.5)
99.0% (ꢃ0.5)
99.2% (ꢃ1.1)
84.1% (ꢃ0.2)
<0.5%
<2.7% (ꢃ0.3)
<0.5%
<0.5%
<0.5%
<0.5%
<0.5%
<0.5%
2 (H2O2)
3 (H2O2)
4 (O2cꢀ)
5 (O2cꢀ)
6 (ClOꢀ)
7 (ONOOꢀ)
<0.5%
KO2 1 mM
10.6% (ꢃ0.1)
19.5% (ꢃ0.7)
32.8% (ꢃ1.0)
<0.6% (ꢃ0.1)
NaOCl 100 mM PBS pH ¼ 7.4
<0.5%
NaONO2 150 mM PBS pH ¼ 9
<0.5%
<0.8% (ꢃ0.1)
a
Run at 22 ꢂC in 1 : 1 MeCN : H2O unless noted otherwise. b Determined by HPLC and reported in percent yield. Values are average of experimental
triplicate. Error expressed as standard deviation in parentheses.
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Table 3 Number of minima located for each possible dioxetane
intermediatea,b,c
10:
13:
11:
14:
Method
(C2/C10)
(C10/C20)
(C20/C30)
(C30/C40)
MP2
B3LYP
12 (6)
4 (4)
6 (6)
18 (9)
4 (4)
21 (12)
4 (4)
5 (5)d
a
6-31G(d) basis set used for minima searches. b See Fig. 1A for carbon
numbering scheme, whereby CX/CY indicates O–O addition locale.
c
d
Numbers in parentheses do not consider N-methyl orientation. At
the B3LYP/CCT level of theory one minimum optimizes to another
leaving only four.
However, one of the intermediate 13 species optimized to
another, leaving only four. Fig. 3B shows the stabilities (elec-
tronic) of the dioxetane intermediates relative to the reactants 1
1
+ Dg 1O2. We nd that the MP2/6-31G(d) and B3LYP/6-31G(d)
results are comparable, predicting similar exothermic stabili-
ties for each intermediate in the order 10 < 11 < 14 < 13,
although MP2 predicts a more stable (by 3.5 kcal molꢀ1) inter-
mediate 10. When advancing to the B3LYP/cc-pVTZ level of
theory, the energetic order of the intermediates is preserved,
however, now only intermediates 10 and 11 are computed to be
Fig. 2 Computed reaction energies (electronic) for the four possible
cleavage pathways of 1 + 1Dg O2 / R ¼ O + [R0 ¼ O]+. Blue, green, red,
and indigo indicate cleavage at C2/C10, C10/C20, C20/C30, and C30/C40
sites, respectively.
potential energy surfaces. For each type of dioxetane interme- exothermic by 2.8 kcal molꢀ1 and 0.7 kcal molꢀ1, respectively.
diate, the only structural restrictions applied were to conserve Therefore, the computational results indicate that 1O2 addition
(i) the initial trans conformation of the two carbon atoms that and subsequent cleavage is energetically favored only via diox-
bind O2 and (ii) the trans backbones of the carbon chains (to etane intermediates 10 and 11. Not only does this prediction
rings) starting from the carbon atoms immediately attached to match which cleavage pathways are observed experimentally
the C(OO)C dioxetane unit. Our reasoning being that O2 addi- (C2/C10 and C20/C30), it also correctly indicates that cleavage at
tion is expected to occur with high stereoselectivity. This (C2/C10) should be the major reaction pathway.
prediction follows from seminal studies by Turro and coworkers
We have also calculated Gibbs free energies (G298) at the MP2/
1
on reactions of related ene-carbamates with O2.45 A full 6-31G(d) and B3LYP/6-31G(d) levels of theory for all discovered
description, with informative illustrations, of the structural minima (B3LYP/6-31G(d) G298 corrections were used to estimate
degrees of freedom that have been considered is provided in the B3LYP/cc-pVTZ G298 energies). For the two methods MP2/6-
ESI.† Geometry searches were performed independently at the 31G(d) and B3LYP/cc-pVTZ it can be the case that the lowest G298
MP2/6-31G(d) and B3LYP/6-31G(d) levels of theory and all structure for an intermediate can vary from the corresponding
discovered minima were conrmed by computations of vibra- lowest electronic energy structure and these differences are
tional frequencies. The numbers of minima located for each shown in Fig. S12 and S13.† Fig. 3C shows computed relative free
class of intermediate and method is summarized in Table 3. We energies (G298) of the dioxetane intermediates where 10 remains
nd that the MP2 method predicts more minima for each the most stable. We nd that when going from the MP2/6-31G(d)
intermediate class than B3LYP (no less than 21 for addition over to B3LYP/6-31G(d) to B3LYP/cc-pVTZ levels of theory that inter-
C30/C40) and includes more dioxetane “twist” conformations mediates 11, 13, and 14 are successively stabilized. Our most
and N-methyl proton orientations (typically staggered). The reliable prediction, B3LYP/cc-pVTZ, indicates that 11 is most
B3LYP/6-31G(d) method identied four minima for each inter- competitive with 10, lying only 1.5 kcal molꢀ1 higher. At room
mediate except the second (addition over C10/C20), for which ve temperature (298 K), this energy difference amounts to a Boltz-
were discovered and in each case only one N-methyl orientation mann ratio of ꢁ13 : 1 that should transfer to the corresponding
was preferred (typically eclipsed).
cleavage products 2 + 3 and 6 + 7. This theoretical prediction is
Fig. 3 illustrates the lowest energy (electronic) B3LYP/6- remarkably similar to the experimentally determined ratio of
31G(d) structures for each of the four possible intermediates. ꢁ5 : 1, especially when taking into account that solvent effects
The lowest energy MP2 structures (provided in ESI†) are have not been considered and energy margins are of the order of
commensurate except for 13 (addition over C10/C20), which a kcal molꢀ1. In fact, the experimentally observed ratio translates
differs by a 180ꢂ rotation about the C2–C10 bond.
to an energy difference (at 298 K) of 0.95 kcal molꢀ1 which is
All 17 dioxetane intermediate minima located using the really not very different from 1.5 kcal molꢀ1 when one
B3LYP/6-31G(d) method were also optimized using the larger acknowledges that quantum chemical accuracy of 1 kcal molꢀ1
cc-pVTZ basis set where lowest energy structures for each of 10, is typically reached only with benchmark methods that are
11, 13 and 14 were commensurate to those shown in Fig. 3A. currently impossible for this sized system.
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Fig. 3 Lowest energy (electronic) B3LYP/6-31G(d) dioxetane intermediate structures (A) and computed stabilities relative to (B) reactants 1 + 1Dg
O2 (electronic) and (C) each other (G298).
mechanistic foundation for our ongoing program harnessing
the cyanine photooxidation reaction as the key step in a near-IR
Conclusions
uncaging method. The systematic modication of the hepta-
methine cyanine scaffold is currently being pursued to access
improved compounds for such applications.
The photooxidation of heptamethine cyanines accompanies
nearly any use of these broadly employed molecules. These
studies, which centered on quantitative accounting of the major
products and detailed computational studies, have provided key
insights into this process. Cyanine photolysis leads to the
regioselective cleavage of the cyanine polyene at two of the four
Methods
possible positions. Only exogenously generated 1O2, and not Experimental
other common ROS, is capable of replicating this reaction
High-resolution LC/MS and MS/MS analyses were conducted on
pathway. We account for ꢁ70% of the molar balance of the
starting cyanine, suggesting that at least this fraction goes
through an energy transfer/1O2 pathway, while leaving open the
possibility that the remainder reacts through other pathways
(perhaps initiated by electron transfer from the triplet state).
Our computational analysis suggests that regioselective
cleavage at only two positions of the polyene, C2/C10 and C20/C30,
can be interpreted as being a consequence of the overall ener-
getics of the dioxetane intermediates. At the highest level exam-
ined, our calculations indicate that O2 addition is exothermic
a Thermo-Fisher LTQ-Orbitrap-XL hybrid mass spectrometer
system with an Ion MAX API electrospray ion source in positive
ion mode. Flow injection analysis followed by collision-induced
dissociation was utilized for MS/MS experiments. A quantitative
HPLC assay to determine yields of 1, 2, and 6 was employed
using an Agilent 1260 Innity instrument. Samples were irra-
diated in HPLC vials using LED light sources obtained from
Marubeni America Corporation or subjected to the reaction
conditions in Tables 1 and 2.
1
only at the observed sites (C2/C10 and C20/C30). By contrast, O2
additions at the remaining two sites, C10/C20 and C30/C40, are less
favorable and may actually be endothermic. Work in progress is
currently exploring the reaction coordinate for singlet oxygen
addition to form the dioxetane intermediates, itself a complex
process in spite of numerous prior investigations.46–48
Quantum chemistry
The calculations in this study were executed using the
GAMESS49 package. We have used the second-order Møller–
Plesset perturbation theory (MP2)50–52 and density functional
theory (DFT)53 methods where, for the latter, the B3LYP54,55
hybrid density functional (containing VWN5 functional) was
utilized with a ne grid of 120 radial and 590 angular Lebedev
points. Open-shell MP2-style calculations for the triplet oxygen
The framework developed here should be of signicant
utility to efforts that seek to improve the photostability of
cyanine uorophores. Moreover, these studies provide
a
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species used the Z-averaged perturbation theory (ZAPT)56–58 a-
vor. The double-zeta 6-31G(d)59 and triple-zeta cc-pVTZ60
Topics in Heterocyclic Chemistry 14, Springer-Verlag,
Berlin, 2008, p. 221.
(spherical functions) basis sets were employed. Geometries 10 R. R. Nani, J. B. Shaum, A. P. Gorka and M. J. Schnermann,
were optimized to tight convergence (maximum Cartesian
Org. Lett., 2015, 17, 302–305.
gradient < 1 ꢄ 10ꢀ5) using analytic gradients and Hessians were 11 A. P. Gorka, R. R. Nani and M. J. Schnermann, Org. Biomol.
computed seminumerically (double differences) using analytic
Chem., 2015, 132, 7584–7598.
gradients for all stationary points located. We note that Fig. S3† 12 A. P. Gorka, R. R. Nani, J. Zhu, S. Mackem and
shows data evaluating the B3LYP and MP2 (including a variety of
open-shell types) methods against benchmark and experiment
M. J. Schnermann, J. Am. Chem. Soc., 2014, 136, 14153–
14159.
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results the ZAPT energy of the 3Sg O2 molecule was used to
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S. H. Wang, Anal. Chem., 2014, 86, 671–677.
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experimental 3Sgꢀ / 1Dg energyꢀdifference (22.639 kcal molꢀ1
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61
to computed energies of the 3Sg O2 system.
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Conflict of interest
The authors declare no competing nancial interest.
Acknowledgements
19 N. J. Patel, E. Manivannan, P. Joshi, T. J. Ohulchanskyy,
R. R. Nani, M. J. Schnermann and R. K. Pandey,
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This project has been funded in whole or in part with federal
funds from the National Cancer Institute, National Institutes of
Health, under Contract No. HHSN261200800001E and by the
Intramural Research Program of the National Institutes of
Health, Center for Cancer Research, and the National Cancer
Institute, National Institutes of Health. The content of this
publication does not necessarily reect the views or policies of
the Department of Health and Human Services, nor does
mention of trade names, commercial products, or organizations
imply endorsement by the U.S. Government.
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