Indolizine-Cyanine Dyes: Near Infrared Emissive Cyanine Dyes with Increased Stokes Shifts

Article abstract of DOI:10.1021/acs.joc.8b02521

Molecular engineering strategies designed to red-shift cyanine dye absorptions and emissions further into the near-infrared (NIR) spectral region are explored. Through the use of a novel donor group, indolizine, with varying cyanine bridge lengths, dye absorptions and emissions, were shifted deeper into the NIR region than common indoline-cyanines. Stokes shifts resulting from intramolecular steric interactions of up to ?60 nm in many cases were observed and explained computationally. Molecular brightnesses of up to 5800 deep into the NIR region were observed. Structure-property relationships are explored for the six indolizine-cyanine dyes with varying cyanine bridge length and indolizine substituents showing broad absorption and emission tunability. The dyes are characterized by crystallography, and the photophysical properties are probed by varying solvent for absorption and emission studies. Computational data show involvement of the entire indolizine ?-system during light absorption, which suggests these systems can be tunable even further into the NIR region through select derivatizations.

Full text of DOI:10.1021/acs.joc.8b02521

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Article
Indolizine-Cyanine Dyes: Near Infrared Emissive
Cyanine Dyes with Increased Stokes Shifts
Jacqueline Gayton, Shane A. Autry, William Meador, Sean R.
Parkin, Glake Alton Hill, Nathan I Hammer, and Jared H. Delcamp
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02521 • Publication Date (Web): 12 Dec 2018
Downloaded from http://pubs.acs.org on December 13, 2018
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Indolizine-Cyanine Dyes: Near Infrared Emissive Cyanine Dyes with
Increased Stokes Shifts
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a,†
a,†
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b
c
Jacqueline Gayton, Shane A. Autry, William Meador, Sean R. Parkin, Glake Alton Hill Jr.,
a
a,
Nathan I. Hammer, and Jared H. Delcamp *
a
Department of Chemistry and Biochemistry, University of Mississippi, University, MS, 38677
b
Department of Chemistry, University of Kentucky, Lexington, KY, 40506
c
Department of Chemistry, Jackson State University, Jackson, MS, 39217
†
These authors contributed equally.
*
email: delcamp@olemiss.edu
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Abstract: Molecular engineering strategies designed to red-shift cyanine dye absorptions and
emissions further into the near infrared (NIR) spectral region are explored. Through the use of a
novel donor group, indolizine, with varying cyanine bridge lengths, dye absorptions and emissions
were shifted deeper into the NIR region than common indoline-cyanines. Stokes shifts resulting
from intramolecular steric interactions of up to ~60 nm in many cases were observed and explained
computationally. Molecular brightnesses of up to 5,800 deep into the NIR region were observed.
Structure-property relationships are explored for the 6 indolizine-cyanine dyes with varying
cyanine bridge length and indolizine substituents showing broad absorption and emission
tunability. The dyes are characterized by crystallography, and the photophysical properties are
probed by varying solvent for absorption and emission studies. Computational data show
involvement of the entire indolizine π-system during light absorption, which suggests these
systems can be tunable even further into the NIR region through select derivatizations.
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Introduction: Near infrared (NIR) emissive materials are a widely researched class of
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compounds with applications in an array of areas including biological imaging, communications,
and secure displays.2-7 Dye designs based on cyanine structures are ubiquitous because of the
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intense molar absorptivity of these structures in the NIR region. Additionally, the cyanine dye
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absorption wavelength is highly tunable based on methine chain length selection. The tunability,
rapid synthesis, facile bioconjugation, intense molar absorptivities (e), strong NIR emissions, and
high molecular brightness (MB) values make this class of dyes attractive for further exploration as
NIR emissive materials.8-9 In addition to deeper NIR absorption and emission, dyes with large
Stokes shifts and high quantum yields are in high demand.9-12 For many applications, longer
wavelength absorptions allow for access to differential dye properties relative to currently
available dyes, and a larger Stokes shift provides dramatically higher resolution images in
biological applications by reducing background signals.5, 13-14 Importantly, a significant MB is
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needed to give high resolution images with smaller amounts of emissive material. A balance
between long wavelength use, Stokes shifts, and quantum yield must be struck for many functional
materials in the NIR region, since the energy gap rule dictates diminished quantum yields as
wavelengths increase.16-19 Stokes shifts, which arise from molecular reorganizations, also tend to
diminish quantum yields as the Stokes shift increases in magnitude. Introduction of molecular
design elements which can increase Stokes shifts and wavelength of both absorption and emission,
while retaining a reasonable molecular brightness, can addresses a fundamental need in NIR
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emissive dye design. Both the Stokes shift and lower energy absorption/emission challenges can
be addressed by replacing the indoline donor of typical indoline cyanine dyes (such as FDA
approved, commercially available, and widely used indocyanine green ICG) with a fully
conjugated indolizine donor to delocalize the frontier molecular orbitals further (Figure 1). ICG
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serves as a good benchmarking cyanine dye since it is one of the most widely used cyanines over
the last 6 decades with multiple reviews concerning the utility of this compound.21-22 In the specific
case of our study, the single nitrogen heteroatom donor groups of indoline-cyanines can be directly
compared to the single nitrogen heteroatom donor groups of novel indolizine-cyanine dyes since
the donor heteroatom remains constant and a similar number of π-bonds are present in both donor
groups. Additionally, the polycyclic indolizine donor provides a steric presence near the cyanine
bridge, which requires either a deviation from planarity of the π-system or non-ideal bond angles.
Strain such as this in the ground state often affects excited state geometries differently, leading to
increased reorganizations upon photoexcitation and thus enhanced Stokes shifts 23-24
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prior work
small reorganization energy
planar π-system
N
N
HOMO/LUMO localized to cyanine
Na
SO3
O S
3
indocyanine green (ICG)
this work
H
1
0
N
increased reorganization energy
steric strain on π-system
H
7
H
extended HOMO/LUMO heavily
delocalization to indolizine
N
N
ClO4
H
indolizine cyanine
11
N
H
H
7
steric induced dihedral
Figure 1. Comparison of an indoline cyanine dye and an indolizine cyanine dye. Hydrogens are
drawn in for a portion of the indolizine cyanine on the right to show steric interactions. The
indolizines are drawn in a cis conformation with a C7-C10 closest interaction as observed by
crystallography for this specific compound. A second conformation exists for a C7-C11 close
interaction which is also observed via crystallography in an example (see below).
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Recently, our group published a series of squaraine dyes using an indolizine heterocycle as
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a donor in place of an indoline donor. The indolizine donor allows for red shifts farther into the
NIR region due to the proaromatic nature of the indolizine, which generates an aromatic stabilized
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pyridinium ring after electron donation. Additionally, an increased Stokes shift was observed,
due in part to the planarization of aryl substituents on the indolizine ring in the excited state. This
is significant as indoline-squaraine dyes are known for very narrow energy gaps between the
absorption and emission spectrum. Importantly, the molar absorptivities and quantum yields (and
by default the MB, where MB = e ´ F) remained high despite these changes. We reasoned that a
similar strategy could be employed with cyanine bridges in place of the squaraine bridge. The
cyanine-bridged dyes are attractive targets due to the simplistic tunability of the absorption
wavelength by methine-bridge length selection. This type of straightforward absorption spectrum
tuning is attractive, but is challenging to achieve with squaraine bridges. In this work, a series of
dyes utilizing a constant indolizine donor structure with cyanine bridges between the donors of
varying lengths with one, three, and five carbons (C1, C3, and C5) were prepared to probe the
effects of methine bridge length on optical properties (Figure 2). Additionally, the electronic
activity of positions on the indolizine donors are evaluated ranging from electron donating to
electron withdrawing (PhOMeIndz-C5, PhCNIndz-C5, and PhIndzOMe-C5) in order to probe
structure-property relationships on the dye optical properties.
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O
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ClO4
ClO4
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C1
PhOMe-C5
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CN NC
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ClO4
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ClO4
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PhCN-C5
ClO4
ClO4
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C5
O
IndzOMe-C5
O
Figure 2. Target indolizine cyanine dyes.
Results and Discussion: Target dyes C1, C3, and C5 were synthesized from a known
phenyl indolizine (PhIndz), which is available in two steps from commercially available 2-
ethylpyridine (1 when R’ = H) and 2-bromoacetophenone (2 when R = H) via an N-alkylation
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reaction followed by a base catalyzed condensation reaction sequence (Figure 3). PhIndz was
then reacted with methine bridge precursors 4, 5 and 6 in the presence of perchloric acid to give
C1, C3, and C5, respectively, in moderate to high yield (38-85%). PhOMe-C5 and PhCN-C5
follow the same synthetic route as C5 beginning with known indolizine building blocks
PhOMeIndz (3 when R = OMe and R’ = H) and PhCNIndz (3 when R = CN and R’ = H),
respectively. The synthesis of IndzOMe-C5 began with the alkylation of commercially available
2
-ethyl-4-methoxypyridine (1 when R’ = OMe) with 2-bromoacetophenone, followed by
cyclization to give PhIndzOMe. Reaction of PhIndzOMe with iminium salt 6 afforded
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IndzOMe-C5 in low isolated yield, presumably due to the low stability of the indolizine precursor
in the reaction mixture as the starting indolizine was consumed during the reaction. In general, the
starting indolizines were handled with care due to limited stability as they were found to
decompose in solution under air and on silica gel; however, once the indolizine was incorporated
into a cyanine dye the motif became stable to prolonged ambient exposure.
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O
O
R
O
O
4
4
, HClO , NEt3
1
)
Br
N
N
ClO4
room temp., 38%
2
N
R
N
C1
acetone, reflux
O
O
2
) NaHCO (aq)
3
R’
reflux, 37% (R’ = OMe) R’
N
O
1
3
5
, HClO , NEt3
ClO4
4
N
R = H, R' = H (PhIndz)
R = OMe, R' = H (PhOMeIndz)
R = CN, R' = H (PhCNIndz)
R = H, R' = OMe (PhIndzOMe)
room temp., 76%
C3
R
R
O
O
O
6
, HClO , NEt3
4
O
O
O
O
room temp.
ClO
4
4
=
=
5 =
O
O
N
N
O
R'
R'
H
N
H
N
6
R = H, R' = H (C5) 85%
R = OMe, R' = H (PhOMe-C5) 58%
R = CN, R' = H (PhCN-C5) 77%
Cl
R = H, R' = OMe (IndzOMe-C5) 10%
Figure 3. Synthetic route to the target indolizine cyanine dyes. The product conformations are
drawn to match those obtained by crystal structure analysis of C1, C3, and C5 (see below).
With the target dyes in hand, single-crystal X-ray diffraction studies were undertaken to
probe planarity of the dye π-system and to analyze the cyanine bridge bond lengths. We
hypothesized: (1) C1 potentially cannot adopt a conformation with both indolizine heterocycles
co-planar due to steric interactions between the indolizine groups with the short single carbon
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bridge, and (2) both C3 and C5 require some deviation from ideal bond angles or π-system
planarity due to steric interactions between the indolizine and cyanine bridge. To probe these
predictions, crystals suitable for crystallographic studies were grown by slow evaporation of
MeCN from C1, or via vapor diffusion techniques in the cases of C3 and C5. The crystals grew
as thin needles or granules with a metallic like orange luster, as is common for very high molar
absorptivity organic materials. The resolved structures are shown below (Figure 4) with select
properties reported in Table 1.
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Figure 4. Crystal structures of C1, C3, and C5 with co-crystallized solvents and counter ions
omitted. Hydrogens are included in order to emphasize potential steric interactions.
First, the planarity of each derivative was analyzed. As expected, the indolizine groups of
C1 are not co-planar and have π-systems perturbed by a 43° dihedral angle (Table 1). As a result,
C1 adopts a propeller type molecular shape, which significantly affects the molar absorptivity of
this derivative (discussion below). C3 and C5 have lengthened methine chains that can allow for
more co-planar conformations. The indolizine groups can adopt two different cis conformations
or a trans type conformation with the phenyl groups either on the same side of the methine bridge
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or on opposite sides, respectively (see Table S4 for an illustration of the cis conformations). The
trans conformer is the observed conformation of C3 in the crystal structure, with a 175° dihedral
angle (5° from co-planarity). Upon extending the methine chain to a five-carbon segment, the cis
conformer was observed via crystallography, with a deviation of indolizine-indolizine π-faces
from planarity by a 30° dihedral angle. The presence of both the cis and trans isomers of the
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indolizine cyanine dyes as crystals and the H NMR appearing as a single set of peaks suggests
that both isomers are accessible and interconversion between cis and trans isomers is likely
possible as was observed for prior squaraine derivatives.25 Bond length analysis reveals a
reasonably uniform bond length (weak alternation) when the indolizine-cyanine bond is compared
to the bonds of the cyanine bridge. This bond uniformity is indicative of cyanine type structures
and extends from the attachment point of the cyanine bridge to the indolizine across the π-bridge
with lengths varying by less than 0.03 Å for all derivatives. The observed bond lengths (1.37-1.40
Å) are between that of an average single C-C bond (1.54 Å) and a double C-C bond (1.34 Å, Table
1
). This bond length uniformity suggests these systems are more likely to exhibit π-π* optical
transitions than n-π* optical transition and that the non-bonding electron lone pairs, which are
often formally drawn on the nitrogens of the indolizine groups, are delocalized.
Table 1. Select crystal structure geometry measurements.
dye
indz.-indz.
dihedral (°)
C1-C10 (Å)
C10-C11 (Å) C11-C12 (Å)
C1
C3
C5
43
175
30
1.37 (1)
1.38 (7)
1.38 (3)
-
-
-
1.39 (2)
1.40 (2)
1.37 (5)
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Absorption and emission studies were undertaken to analyze the optical properties of the
dyes. First, the molar absorptivity and absorption curve features were analyzed for C1, C3, and
C5 to compare the effect of added methine spacers in several solvents (DCM, DMSO, 1:1
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MeCN:H O, and MeCN) differing in polarity and protic nature (Figure 5, Tables 2, S1 and S2).
2
The absorption maxima were found to shift to longer wavelength with increasing number of
methine groups (e.g. in DMSO lmax values 652, 705, and 819 nm are observed for C1, C3, and
C5, respectively). These maxima are likely from π-π* transitions based on bond length alternations
observed from crystallography, DFT, and the high molar absorptivity values observed (see
discussion below). This is a 23-26 nm red-shift for the C5 derivative relative to the common
indoline cyanine dye, ICG. Additionally, the molar absorptivity increased dramatically as the
cyanine bridge length increased from 1 to 3 or more carbons. The twisted π-system of C1 prevents
-1
-1
a strong low-energy transition, limiting molar absorptivities from 11,000 to 27,000 M cm for C1
depending on solvent selection. C3 and C5 molar absorptivities are in stark contrast to this at up
-1
-1
to 204,000 and 188,000 M cm , respectively. These values are similar to ICG, which has a
-1
-1
maximum measured molar absorptivity of 238,000 M cm . A characteristic cyanine high-energy
2
7
shoulder is observed for each of the dyes and this is attributed to vibronic transitions. Both C3
and C5 exhibit similar molar absorptivity trends with solvents in the following order: DCM >
DMSO > MeCN:H
2
O > MeCN. These trends do not correlate to dielectric constants or dipole of
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Figure 5. The molar absorptivities of C1, C3, and C5 dyes in DCM, DMSO, MeCN, and 1:1
-5
MeCN:H O measured at 1 x 10 M concentrations. See Figure S1 for the full visible range.
2
Individual plots of the absorption and emission of C1, C3, and C5 dyes in the four solvents are in
the SI (Figures S2-S4).
Table 2. The molar absorptivity, absorption maximum, emission maximum, quantum yield, and
Stokes shift for each dye in four solvents.
e
l
max
abs
l
max
emis
Φ
%)
MB
e ´ Φ)
Stokes shift
(nm | eV | cm )
-
1
-
dye
C1
solvent
(M cm
-1
(
(
1
)
(nm) (nm)
MeCN
:1 MeCN:H
DMSO
DCM
MeCN
:1 MeCN:H
DMSO
DCM
MeCN
:1 MeCN:H
DMSO
DCM
MeCN
1:1 MeCN:H
DMSO
27,000
O 11,000
14,000
642
641
652
646
698
698
846
693
725
727
737 1.6
741 <1
848 1.2
840 <1
852 3.5
868 2.2
845 1.3
831
840 2.7
863 1.9
<1
<1
<1
<1
<1
<1
≤300
≤100
≤100
≤200
≤1,300
≤1,300
2,200
≤2,000
1,700
≤1,500
5,800
4,100
1,200
≤1,500
4,400
3,900
56 | 0.15 | 1250
57 | 0.16 | 1270
1
1
1
2
2
2
2
2
a
194 | 0.43 | 3517
47 | 0.13 | 1050
30 | 0.07 | 595
31 | 0.08 | 613
32 | 0.08 | 616
33 | 0.08 | 629
38 | 0.07 | 553
29 | 0.05 | 426
33 | 0.06 | 473
43 | 0.07 | 600
46 | 0.08 | 681
32 | 0.06 | 482
24 | 0.04 | 350
50 | 0.09 | 713
55 | 0.10 | 806
46 | 0.08 | 671
40 | 0.07 | 567
58 | 0.10 | 821
18,000
126,000 695
O 133,000 696
135,000 705
204,000 708
140,000 810
O 151,000 811
166,000 819
188,000 825
C3
C5
94,000
799
O 146,000 799
162,000 816
207,000 813
122,000 799
O 100,000 805
<1
PhOMe-C5
PhCN-C5
DCM
MeCN
1:1 MeCN:H
DMSO
854
851
860 2.3
870 2.0
<1
<1
≤1,200
≤1,000
1,900
84,000
151,000 812
820
DCM
3,000
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MeCN
:1 MeCN:H
DMSO
124,000 819
O 138,000 821
141,000 838
151,000 823
O 238,000 785
211,000 797
181,000 716
216,000 716
849 1.3
859 <1
868 3.6
876 <1
816 9.1
835 17.1
766 2.1
736 9.5
765 16.8
1,600
≤1,400
5,100
<1,500
21,700
36,100
3,800
20,500
2,000
1,700
-
30 | 0.05 | 431
38 | 0.07 | 539
30 | 0.05 | 412
53 | 0.09 | 735
31 | 0.06 | 484
38 | 0.07 | 571
50 | 0.11 | 912
20 | 0.05 | 380
141 | 0.37 | 2954
302 | 0.46 | 3716
305 | 0.48 | 3855
1
1
2
IndzOMe-C5
ICG
DCM
:1 MeCN:H
DMSO
2
PhIn
bis-t-buPhIn
TTD(T)
2
SQ
DCM
DCM
Toluene
Toluene
0
1
2
3
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0
1
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0
1
2
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0
2
SQ
2
12,000
24,000
-
624
TPA-BBTD-TPA
CH1055-PEG
763 1065 7.1
750 1055 0.3
H O
2
Absorption and emission curves for PhOMe-C5, PhCN-C5, and PhIndzOMe-C5 are in the SI
(
Figures S5-S7). Data for ICG were collected under identical conditions as a benchmark. Data for
25
25
24
28
10
PhIn
2
SQ, bis-t-buPhIn
is molar absorptivity. lmax is the absorption curve low energy peak value.
is the emission curve peak value. Φ is the quantum yield. MB is molecular brightness. a
2
SQ, TTD(T)
2
,
TPA-BBTD-TPA, and CH1055-PEG are from
abs
literature reports.
e
emis
l
max
This value is based on the smaller lower energy emission peak. A higher energy emission peak
(715 nm) is also observed which would correspond to a Stokes shift of 63 nm, 0.17 eV.
solvent, which indicates a subtler solvent-dye interaction is occurring to vary molar absorptivities
(
Table S1). C1 follows a different molar absorptivity trend, and this deviation away from the
pattern observed for C3 and C5 may originate from lack of planarity of the π-system of C1.
A host of additional solvents were analyzed for the C1, C3, and C5 dyes to show solvent
effects on absorption wavelength (see Figures 6-8). For each solvent analyzed via absorption
spectroscopy, an emission spectrum in the same solvent was analyzed. For C1, the solvent had a
significant effect on the absorption curve shape, with the shoulder at 600 nm changing intensity
relative to the lower energy peak at 650 nm (Figure 6). THF shows the closest ratio of high-energy
to lower-energy features nearing equal heights, while DCM shows the high-energy feature at about
a 75% of the height of the low-energy feature. All the dyes were found to have a linear absorbance
versus concentration dependence following the Beer-Lambert Law from low concentrations (<1 x
1 -
6
0 M) to high concentrations (>1 x 10 M), which suggests the solvent dependent features in the
-5
C1 absorption spectrum are not due to aggregation. These results show that C1 has significantly
different ground-state solution conformations depending on solvent selection. The emission curve
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shape variation with solvent was even more dramatic. The emission maxima could be tuned over
a ~200 nm range by solvent selection. THF and DMF represent the highest energy emission peaks
near 650 nm, while DMSO, methanol, and ethanol all show peaks at wavelengths >800 nm, with
DMSO being the most red-shifted at 850 nm. This is a 194 nm Stokes shift and represents a very
large reorganization energy (0.43 eV) from the ground state to the excited state. All three of the
solvents with the lowest energy emission spectrum peaks show dual emission behavior with an
emission peak near 700 nm. This type of dichromic behavior has been observed previously with
1
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non-symmetric cyanine dyes. The low-energy emission is the dominant feature in MeOH, while
the high-energy emission is dominant in DMSO. The remaining solvents all show a peak near 700
nm and typically show a shoulder at 750 nm, which mirrors the absorption spectrum, suggesting
the higher energy absorption feature is vibronic in nature. The vast range of emission curve shapes
and peak energies suggests a wide range of geometries are accessible in the excited-state and the
excited-state geometry is exceptionally solvent dependent. The quantum yield for C1 was
measured to be less than 1% in the same four solvents as the molar absorptivity studies (Table 2).
This leads to a low molecular brightness upper limit of ≤300, as is expected from a molecule with
a very large reorganization energy.
For the absorption curve profiles of C3 and C5, solvent effects are not as dramatic as with
C1 (Figures 7 & 8). C3 and C5 show typical cyanine curve shapes with little energetic variation
in the absorption curve maxima based on solvent choice (<20 nm change observed in the 12
solvents examined). The emission curves show a slight increase in solvent effect changes with a
range of about 35 nm observed for C3 and C5. Notably, the most red-shifted and most blue-shifted
solvents change between these two derivatives, suggesting subtle differences in solvent
interactions between these two dyes. Stokes shifts between 30 and 50 nm were observed for these
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30
dyes, which allows for use in some biological imaging applications. Both C3 and C5 show >1%
quantum yields with the highest values (1.6 and 3.5%, respectively) observed in DMSO. A high
MB value of 5,800 is observed for C5. While the MB values for the indolizine-cyanine dyes were
not as high as those reported for the ICG benchmark, it should be noted that these values can be
improved dramatically through enhancing the emission quantum yield by selection of non-
0
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1
5
conjugated substituents, as was recently shown for CH1055-PEG. Compared to other benchmark
materials such as D-A-D dyes based on squaraine (PhIn SQ and bis-t-buPhIn SQ) and
thienothiadiazole (TTD(T) ), C5 is significantly more red shifted by >100 nm in absorption and
2
2
2
emission spectrum maxima. The reorganization energy of C5 is similar to that of the squaraine
derivatives, which have large Stokes shifts for that class of materials (0.07 eV versus 0.05-0.11
eV). Despite being red shifted significantly with a larger Stokes shift, the MB of C5 is higher than
these comparison dyes even with the very high quantum yield of TTD(T) . Compared to one of
2
the most red-shifted imaging materials, TPA-BBTD-TPA (and derivative CH1055-PEG), a
significantly higher MB is observed for C5 with a more red-shifted absorption maximum. Notably,
a significantly more red-shifted emission is present with TPA-BBTD-TPA. Overall, compared to
the benchmark materials analyzed, C5 is a promising dye scaffold for future applications.
The remaining indolizine-cyanine dyes synthesized are analogues of C5 and show
comparable absorption maxima, emission maxima, quantum yields, and MB to the parent
compound in most solvents (Figures S4-S6). These derivatives (PhOMe-C5, PhCN-C5, and
IndzOMe-C5) are dramatically different in substituent electronic nature with PhOMe-C5 and
IndzOMe-C5 having a strong electron donating OMe group and PhCN-C5 having a strong
electron withdrawing group. The lack of change in the absorption and emission spectrum suggests
these derivatives do not have a strong effect on the π-conjugated system (see computational
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discussion below). These positions on the indolizine heterocycle and phenyl group of the
indolizine have little effect on the optical properties of these dyes and are attractive positions for
functionalization to introduce components such as water solubilizing groups or bioconjugation
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1
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8
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0
1
2
3
4
5
6
7
8
9
0
Figure 6. Absorption (left) and emission (right, fit with 0.1 LOESS function) spectra of C1 in
-5
various solvents. Absorption spectra were collected at 1 x 10 M. Emission spectra were collected
with excitation at 633 nm.
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Figure 7. Absorption (left) and emission (right, fit with 0.1 LOESS function) spectra of C3 in
-5
various solvents. Absorption spectra were collected at 1 x 10 M. Emission spectra were
collected with excitation at 633 nm.
0
1
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Figure 8. Absorption (left) and emission (right, fit with 0.1 LOESS function) spectra of C5 in
-5
various solvents. Absorption spectra were collected at 1 x 10 M. Emission spectra were
collected with excitation at 785 nm.
functionality. The C5 derivatives show up to a 41 nm red shift relative to ICG in DMSO, indicating
a deeper NIR light absorption as is desired for many applications (Figures S8 & S9). This red shift
occurs despite a 2 methine shorter bridge for the C5 indolizine cyanine derivatives.
To better understand the role of the indolizine group and the optical properties of these
dyes, calculations were performed using density functional theory (DFT) at the M06-2X/6-
311G(d,p) level with the Gaussian 16 software package.31-32 The SMD solvation model was used
33
during geometry optimizations to better approximate the presence of MeCN. Both the cis and
trans isomers were analyzed since crystallography revealed the presence of cis isomers for C1 and
C5 but the trans isomer of C3. A small energy difference was found between the ground state
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geometries of 1.0-4.4 kcal/mol with the trans isomer slightly lower in energy for C1 and one of
the cis isomers slightly lower in energy for C3 and C5 (see SI). For all derivatives studied the
highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO)
were delocalized across the entire π-system, with significant orbital presence on both the methine
bridge and on the indolizine donors (Figure 9 and S16). It is noteworthy that the HOMO
1
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0
1
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4
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9
0
1
2
3
4
5
6
7
8
9
0
Figure 9. HOMO and LUMO images for C1, C3, and C5 with an isovalue of 0.02.
and LUMO are extended onto the conjugated indolizine heterocycle to a greater extent than is
3
4
observed in the case of indoline cyanines. This suggests the indolizine cyanine dyes may be
electronically tuned more effectively than with indoline cyanines by careful substitution on the
indolizine donor group.
For comparison to experimental trends, time dependent (TD)-DFT calculations were
carried out at the same level of theory as the geometry optimizations (Table 3). TD-DFT shows a
similar trend to the observed experimental data for the cis isomer with a close C7-C11 interaction
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for both lmax and e when compared with the vertical transition energies and oscillator strengths in
MeCN: C1 < C3 < C5 (Table 3, Figure 1). When the different conformers obtained from the crystal
structure analysis are compared computationally, C1 is red shifted relative to C3, and the energy
of the vertical transition is significantly different than the experimental absorption spectrum
maximum. As an example comparison, C5 with the C7-C11 close interaction cis conformer shows
theory and solution experiments within 0.12 eV. When the conformer observed via crystallography
is computationally analyzed the comparison with solution experimentation shows a larger variation
of 0.55 eV. We note that the energy difference between these geometries is small at 2 kcal/mol,
0
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1
and since H NMR shows only a single isomer, rapid interconversion is likely possible. These
transitions were found to be HOMO to LUMO π-π* transitions. To analyze the experimental
emission spectrum data computationally, the excited-state geometry for each derivative was
optimized. Similar to experimental data, C5 was found to have the most red-shifted excited-state
geometry vertical transition (labeled dye* vertical trans. in Table 3) and strongest oscillator
strength (brightest emission). C1 was computationally shown to have the largest Stokes shift,
which is in good agreement with experimental data. Electron density difference mapping of the
three dyes was used to show where electrons are traveling from (red) and to (blue) upon
photoexcitation (Figures 10 & S17). Green areas represent minimal electron movement. The maps
show a delocalization of electron density across the cyanine π-bridge extending onto the full
indolizine π-system in red, and a similar delocalization of areas accepting electron density on
alternating atoms. No significant contribution from the phenyl group is observed on the electron
density difference maps.
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Table 3. TD-DFT values for the ground- and excited-state optimized geometries.
vertical trans. oscillator dye* vertical osc. str. Stokes shift
dye
(
eV, nm)
strength trans. (eV, nm) dye*
(eV, nm)
0.42, 136
0.15, 36
0.17, 64
0.14, 70
a
1
1
1
1
1
1
1
1
1
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2
2
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3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
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C1 cis
2.20, 561
2.33, 533
1.91, 648
1.65, 752
2.08, 596
0.79
1.11
1.49
2.06
1.52
1.78, 697
2.18, 569
1.74, 712
1.51, 822
2.03, 611
0.47
0.97
1.27
1.84
1.75
a
C3 trans
b
C3 cis
b
C5 cis
a,c
C5 cis
0.05, 15
a
b
c
Conformation observed by crystallography. C7-C11 interaction in Figure 1. C7-C10 interaction
in Figure 1.
Figure 10. Electron density difference map. Red is where electrons originate from, blue is where
they travel to upon photoexcitation, and green is neutral.
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1
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The predicted geometry changes from the ground state to the excited state were analyzed
to determine the origin of the Stokes shift in these dyes. The ground state geometry shows a
0
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deviation of bond angles from ideal 120° sp hybridized bong angles to wider values for all isomers
(
122-134° for the first two angles measured, Tables 4 & S3). This is presumably to lower steric
interactions between hydrogen atoms on the indolizine and cyanine bridge (or between the two
indolizines for the case of C1). Despite the bond angle
Table 4. Select computed bond angles, dihedral angles, and atom-atom distances.
dihedral (
°
)
dihedral (
°
)
H-H bond
angle 1 (
°
)
angle 2 (
°
)
angle 3 ( ) angle 4 ( )
° °
dye
indz-cyanine indz-indz distance (Å) GS
GS | ES
GS | ES
GS | ES
GS | ES
GS | ES
15 | 27
GS | ES
35 | 50
15 | 32
22 | 33
| ES
C1
128 | 123
134 | 125
N/A
N/A
N/A
2.7 | 3.6a
1.8, 2.1 | 1.9, 2.1
2.1 | 2.0
C3 128, 123 | 127, 122 131, 126 | 129, 123 120 | 123
C5 122 | 122 127 | 126
2, 10 | 11, 22
7 | 11
122 | 124 123 | 121
a
GS is ground state. ES is excited state. Indicates H-H bond distance is between the two closest
hydrogens of the indolizines. The structure below illustrates the bond angles being analyzed.
2
4
N
3
N
1
H
H
distortion, the nearest hydrogens are still well within a typical hydrogen bond distance (1.5-2.5 Å).
Upon photoexcitation, the bond angles adopt a closer to ideal geometry at 122-130°. These angles
favor increased steric interactions between hydrogen atoms on indolizine and the cyanine bridge
if the π-system were to remain planar. To relieve this increased strain, the indolizine-cyanine
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dihedral angle increases in the excited state by 4°-18° for all the conformers of C3 and C5
derivatives from nearer to planar in the ground state. This results in a less planar π-system, but
does release steric strain, as evidenced by an increase in distance between nearest neighboring
hydrogens of about 0.1 Å (Table S4). Thus, the ground state geometry favors bond angle distortion
with a more ideal dihedral angle across the π-system, while the excited state geometry favors a
distorted dihedral angle across the π-system but gains more ideal bond angles (Figure 11). This
change in geometry explains the increased Stokes shift for these indolizine cyanine derivatives
relative to well-established indoline cyanines.
1
1
1
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1
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9
0
Figure 11. Illustration of geometry changes from the ground state to the excited state.
Conclusions: Six NIR indolizine-cyanine dyes were designed, synthesized and
characterized. UV-Vis-NIR absorption measurements show that increasing the methine bridge
lengths leads to red-shifted absorptions. The indolizine-cyanine dyes show a red-shifted absorption
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5
6
relative to benchmark ICG despite two fewer methines. This shows the critical role the indolizine
groups can play in shifting absorption spectra as a conjugated donor group. The dyes were found
to emit in the NIR region with the sterically congested dye C1 showing the largest solvent effects
and reorganization energies. The 5 methine group bridge of C5 was found to lead to the largest
quantum yields and a high MB value for a dye emitting near 900 nm. Computational studies reveal
that the ground state favors a planarized π-system with distorted bond angles while the excited
state favors the opposite geometry. Importantly, the use of molecularly engineered steric
interactions shows that significant Stokes shifts can be designed into a class of molecules known
for minimal Stokes shifts while keeping a good quantum yield and red shifting the absorption and
emission curves.
0
1
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0
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0
Experimental:
General Information. All commercially obtained reagents and solvents were used as received
without further purification. It should be noted that the dyes were isolated as perchlorate salts, and
perchlorate salts in general have been found explosive is some cases. Care is advised while
handling all perchlorate salts. Thin-layer chromatography (TLC) was conducted with Sorbtech
silica XHL TLC glass backed plates and visualized with UV. Flash column chromatography was
performed using a CombiFlash Rf+ system. RediSep cartridges were charged with silica gel from
1
13
Sobent Tech P60, 40-63 µm (230-400 mesh). H and C NMR spectra were recorded on a Bruker
Avance-300 (300 MHz) spectrometer and a Bruker Avance-500 (500 MHz) spectrometer and are
reported in ppm using solvent as an internal standard (DMSO at 2.50 ppm). Data reported as: s =
singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet, b = broad, ap = apparent, dd
= doublet of doublets; coupling constant(s) in Hz; integration. UV-Vis Spectra were measured
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with a Cary 5000 UV-Vis-NIR spectrometer. HRMS spectra were obtained with a QTOF HRMS
utilizing nanospray ionization. The mass analyzer was set to the 200-2000 Da range. Infrared
spectra were recorded with an Agilent Cary 660 ATR-FTIR. For all derivatives, the excited-state
lifetimes were found to be shorter than the response function of the instrument. The emission of
the C1 and C3 derivatives were measured using a Horiba LabRam spectrometer with a 600
grooves/mm grating, CCD camera detection and an excitation laser of 633 nm. All other dyes were
measured using the same instrument and a 785 nm excitation laser. The quantum efficiency of the
detector was accounted for when measuring emission profiles. The relative quantum yields were
obtained using this equation:
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢍ
h
ꢋꢀꢁꢂꢃꢄꢅ
ꢌꢀꢇꢁꢈꢉꢁꢊꢉ
ꢀꢁꢂꢃꢄꢅ
Fꢀꢁꢂꢃꢄꢅꢆ=ꢆFꢀꢇꢁꢈꢉꢁꢊꢉ
∗
∗
∗
ꢍ
ꢋꢀꢇꢁꢈꢉꢁꢊꢉ ꢌꢀꢁꢂꢃꢄꢅ
h
ꢀꢇꢁꢈꢉꢁꢊꢉ
For the equation above, E is the sum of emission intensities and A is maximum absorbance. h is
3
5
the refractive index of the solvent used and F denotes the quantum yield. The standard used to
obtain the relative quantum yields was Indocyanine Green (ICG) with a quantum yield of 14% in
H
O.36 Diffraction data were collected at 90K on a Bruker D8 Venture dual microsource
2
diffractometer using MoKa X-rays. Data scaling, merging, and absorption correction used well-
established procedures.37-38 Crystal structures were solved and refined using the Shelx programs.39-
4
0
Crystals of C3 included some poorly defined solvent that was accounted for using SQUEEZE.41
(
Z)-1-methyl-3-((1-methyl-2-phenylindolizin-3-yl)methylene)-2-phenyl-3H-indolizin-4-ium
perchlorate (C1): To a round bottom flask equipped with a stir bar, PhIndz (1.00 g, 4.80 mmol)
was added to acetic anhydride (48 mL) followed by perchloric acid (0.48 g, 4.80 mmol). The
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mixture was allowed to stir at room temperature for five minutes. Triethylorthoformate (4) (0.36
g. 2.40 mmol) was added along with triethylamine (0.58 g, 5.76 mmol). The reaction mixture was
allowed to stir at room temperature for 24 hours before being subjected to a silica gel column with
a solvent gradient beginning with 40:60 dichloromethane:hexane and ending with 100%
0
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2
3
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8
9
0
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2
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8
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0
1
dichloromethane. The product was isolated as a red solid (0.48 g, 38%). H NMR (300 MHz,
DMSO-d6) δ 8.50 (s, 2H), 8.08-7.97 (m, 4H), 7.90-7.80 (m, 4H), 7.44-7.37 (m, 8H), 7.3 (t, J =
1
3
-1
6
.8 Hz, 1H), 2.38 (s, 6H). C NMR was not obtained due to low solubility. IR (neat, cm ): 3598,
3
534, 2916, 2665, 2329, 2092, 1777, 1621, 1586, 1464. HRMS m/z calculated for C31 [M-
H
25
N
2
+
°
ClO ] : 425.2018, found 425.2019. Melting Point: 157-162 C.
4
(
Z)-1-methyl-3-((E)-3-(1-methyl-2-phenylindolizin-3-yl)allylidene)-2-phenyl-3H-indolizin-4-ium
perchlorate (C3): To a round bottom flask equipped with a stir bar, PhIndz (0.35 g, 1.69 mmol)
was added to acetic anhydride (17.0 mL) followed by perchloric acid (0.169 g, 1.69 mmol). The
mixture was allowed to stir at room temperature for five minutes. Malonaldehyde bis(dimethyl
acetal) (5) (0.139 g, 0.845 mmol) was added along with triethylamine (0.205 g, 2.03 mmol). The
reaction mixture was allowed to stir at room temperature for 24 hours before being subjected to a
silica gel column with a solvent gradient beginning with a 50:50 dichloromethane:hexane solvent
mixture and ending with 100% dichloromethane. The product was isolated as a gold solid (0.35 g,
1
7
6%). H NMR (300 MHz, DMSO-d6) δ 7.99 (d, J = 8.5 Hz, 2H), 7.76-7.68 (m, 9H), 7.46-7.31
13
(m, 9H), 6.69 (t, J = 13.2 Hz, 1H), 2.19 (s, 6H). C NMR was not obtained due to sparing
-1
solubility. IR (neat, cm ): 3109, 2993, 2817, 2669, 2582, 2330, 2116, 1618, 1555, 1533, 1454.
+
HRMS m/z calculated for C33
H
27
N
2
[M+H] : 451.2174, found 451.2197. Melting Point (dec.):
°
252-253 C.
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(
Z)-1-methyl-3-((2E,4E)-5-(1-methyl-2-phenylindolizin-3-yl)penta-2,4-dien-1-ylidene)-2-phenyl-
3H-indolizin-4-ium perchlorate (C5): To a round bottom flask equipped with a stir bar, PhIndz
1.00 g, 4.80 mmol) was added to acetic anhydride (48.0 mL) followed by perchloric acid (0.48 g,
.80 mmol). The mixture was allowed to stir at room temperature for five minutes.
1
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2
2
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5
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0
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2
3
4
5
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8
9
0
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2
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4
5
6
7
8
9
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8
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2
3
4
5
6
7
8
9
0
(
4
(Phenylamino)pentadienylidene aniline HCl (6) (0.68 g. 2.40 mmol) was added along with
triethylamine (0.58 g, 5.76 mmol). The reaction mixture was allowed to stir at room temperature
for 24 hours before being subjected to a silica gel column with a solvent gradient beginning at
5
0:50 dichloromethane:hexane and ending with 100% dichloromethane. The product was isolated
1
as a gold solid (1.1 g, 82%). H NMR (300 MHz, DMSO-d6) δ 9.18 (d, J = 6.9 Hz, 2H), 8.03 (d,
J = 13.6 Hz, 2H), 7.91 (d, J = 8.6 Hz, 2H), 7.70-7.54 (m, 8H), 7.37-7.25 (m, 8H), 5.91 (t, J = 12.5
13
-1
Hz, 1H), 2.14 (s, 6H). C NMR was not obtained due to sparing solubility. IR (neat, cm ): 3652,
2670, 2564, 2329, 2116, 1925, 1806, 1671, 1617, 1516, 1458. HRMS m/z calculated for C35
H
29
N
2
+
°
[
M] : 477.2331, found 477.2315. Melting Point (dec.): 176-179 C.
(Z)-2-(4-methoxyphenyl)-3-((2E,4E)-5-(2-(4-methoxyphenyl)-1-methylindolizin-3-yl)penta-2,4-
dien-1-ylidene)-1-methyl-3H-indolizin-4-ium perchlorate (PhOMe-C5): To a round bottom flask
equipped with a stir bar, PhOMeIndz (1.08 g, 4.54 mmol) was added to acetic anhydride (45.0
mL) followed by perchloric acid (0.46 g, 4.54 mmol). The mixture was allowed to stir at room
temperature for five minutes. (Phenylamino)pentadienylidene aniline HCl (6) (0.65 g. 2.27 mmol)
was added along with triethylamine (0.55 g, 5.45 mmol). The reaction mixture was allowed to stir
at room temperature for 24 hours before being poured into ether (200 mL). The pure product was
1
insoluble in ether and was isolated by vacuum filtration as a red solid (0.81 g, 58%). H NMR (300
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2
2
2
2
2
2
2
2
3
3
3
3
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3
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3
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4
4
4
4
4
4
4
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5
6
MHz, DMSO-d6) δ 9.16 (d, J = 6.9 Hz, 2H), 7.90-7.85 (m, 2H), 7.70 (t, J = 15.6 Hz, 2H), 7.45-
1
3
7
.29 (m, 8H), 7.13 (d, J = 8.3 Hz, 6H), 6.42 (t, J = 11.5 Hz, 1H), 3.91 (s, 6H), 2.14 (s, 6H). C
-1
NMR was not obtained due to sparing solubility. IR (neat, cm ): 3656, 3581, 3333, 2831, 2656,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
463, 2329, 2116, 1919, 1804, 1754, 1706, 1658, 1607, 1555, 1516, 1465. HRMS m/z calculated
+
°
for C37
H
33
N
2
O [M] : 537.2542, found 537.2516. Melting Point (dec.): 158-161 C.
2
(
Z)-2-(4-cyanophenyl)-3-((2E,4E)-5-(2-(4-cyanophenyl)-1-methylindolizin-3-yl)penta-2,4-dien-
1
-ylidene)-1-methyl-3H-indolizin-4-ium perchlorate (PhCN-C5): To a round bottom flask
equipped with a stir bar, PhCNIndz (1.00 g, 4.31 mmol) was added to acetic anhydride (45.0 mL)
followed by perchloric acid (0.432 g, 4.31 mmol). The mixture was allowed to stir at room
temperature for five minutes. (Phenylamino)pentadienylidene aniline HCl (6) (0.613 g. 2.15
mmol) was added along with triethylamine (0.523 g, 5.17 mmol). The reaction mixture was
allowed to stir at room temperature for 24 hours before being poured into ether (200 mL). The
pure product was insoluble in ether and was isolated by vacuum filtration as a red solid (1.0 g,
1
7
7%). H NMR (300 MHz, DMSO-d6) δ 9.19 (d, J = 6.8 Hz, 2H), 8.05 (d, J = 8.1 Hz, 4H), 7.98
(d, J = 8.6 Hz, 2H), 7.91 (d, J = 14.0 Hz, 2H), 7.76 (t, J = 7.6 Hz, 2H), 7.65 (d, J = 8.1 Hz, 4H),
1
3
7
.44-7.36 (m, 4H), 6.21 (t, J = 13.2 Hz, 1H), 2.18 (s, 6H). C NMR was not obtained due to
-1
sparing solubility. IR (neat, cm ): 3611, 3395, 2354, 2332, 2116, 1619, 1612, 1530, 1527, 1471.
+
HRMS m/z calculated for C37
H
27
N
4
[M] : 527.2236, found 527.2251. Melting Point (dec.): 187-
°
191 C.
(
Z)-1-methyl-3-((E)-3-(1-methyl-2-phenylindolizin-3-yl)allylidene)-2-phenyl-3H-indolizin-4-ium
perchlorate (IndzOMe-C5): Part 1: 7-methoxy-1-methyl-2-phenylindolizine (4, PhIndzOMe): To
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a round bottom flask equipped with a stir bar and a reflux condenser was added 2-ethyl-4-
methoxypyridine (0.39 g, 2.81 mmol) and bromoacetophenone (0.84 g, 4.22 mmol) in acetone
(5.62 mL). The mixture was heated to reflux in an oil bath for 24 hours. The reaction was
monitored by TLC. Upon disappearance of the starting material 2, water (5.62 mL) and sodium
bicarbonate (0.94 g, 11.2 mmol) were added to the reaction mixture. The mixture was heated at
reflux in an oil bath for two hours. The crude product oiled out of solution and was extracted with
dichloromethane to yield a brown oil that was immediately subjected to a rapid plug filtration
through silica gel with 20:80 ethyl acetate:hexanes to yield an off white solid. Due to oxidative
instability, the intermediate was carried forward to the next reaction without any further
purification or characterization. To a round bottom flask equipped with a stir bar under nitrogen,
the intermediate PhIndzOMe (0.25 g, 1.05 mmol) was added to acetic anhydride (10.5 mL)
followed by perchloric acid (0.11 g, 1.05 mmol). The mixture was allowed to stir at room
temperature for five minutes. (Phenylamino)pentadienylidene aniline HCl (6) (0.15 g. 0.52 mmol)
was added along with triethylamine (0.13 g, 1.26 mmol). The reaction mixture was allowed to stir
at room temperature for 3 hours before being subjected to a plug of silica with a solvent gradient
from 100% dichloromethane to 50:50 ethyl acetate:dichloromethane. A final purification was
performed by dissolving the concentrated product in a minimal about of dichloromethane followed
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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3
3
3
3
3
4
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5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
by the addition of diethyl ether:hexanes (50:50) to yield the pure product as a green solid (0.03 g,
1
1
0%). H NMR (300 MHz, DMSO-d6) δ 9.02 (d, J = 7.5 Hz, 2H), 7.73-7.53 (m, 10H), 7.33 (d, J
13
= 7.2 Hz, 4H), 7.15-7.01 (m, 4H), 5.86 (t, J = 14.4 Hz, 1H), 4.01 (s, 6H), 2.10 (s, 6H). C NMR
-1
was not obtained due to sparing solubility. IR (neat, cm ): 3788, 3691, 2917, 2633, 2329, 2116,
+
1
797, 1775, 1630, 1527, 1444. HRMS m/z calculated for C37
H
33
N
2
O
2
[M] : 537.2542, found
°
537.2532. Melting Point (dec.): 162-165 C.
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The authors declare no competing financial interest.
Supporting Information. A table of dielectric constants, dye solubility table, additional
absorption and emission plots, NMR spectra, HOMO-LUMO diagrams, electron density
difference maps, calculated XYZ coordinates, additional crystallography data, and CIF files for
each crystal structure are available in the SI.
Acknowledgements:
The D8 Venture diffractometer at the University of Kentucky was funded by the NSF (MRI CHE-
1
625732). The Horiba LabRam spectrometer at the University of Mississippi was funded by NSF
(MRI CHE-1532079). J.N.G., W.M., G.H. and J.H.D. thank NSF for award OIA-1632825. S.A.
and N.I.H. thank NSF for award OIA-1539035.
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