Silicon Substitution in Oxazine Dyes Yields Near-Infrared Azasiline Fluorophores That Absorb and Emit beyond 700 nm

Article abstract of DOI:10.1021/acs.orglett.8b01786

Exchanging the bridging oxygen atom in rhodamine dyes with a dimethylsilyl group red-shifts their excitation and emission spectra, transforming orange fluorescent rhodamines into far-red Si-rhodamines. To study the effect of this substitution in other dye

Full text of DOI:10.1021/acs.orglett.8b01786

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Silicon Substitution in Oxazine Dyes Yields Near-Infrared Azasiline
Fluorophores That Absorb and Emit beyond 700 nm
Adam Choi and Stephen C. Miller*
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts
01605, United States
S
* Supporting Information
ABSTRACT: Exchanging the bridging oxygen atom in rhod-
amine dyes with a dimethylsilyl group red-shifts their excitation
and emission spectra, transforming orange fluorescent rhod-
amines into far-red Si-rhodamines. To study the effect of this
substitution in other dye scaffolds, synthetic approaches to
incorporate silicon into the bridging position of oxazine dyes
were developed. The fluorescence of the compact azasiline dyes
ASiFluor710 and ASiFluor730 is red-shifted by 57−83 nm from
that of Oxazine 1.
luorescent probes are among the simplest and most
convenient chemical tools used to investigate biological
F
processes. In particular, near-infrared (NIR) fluorophores with
excitation and emission between 650 and 900 nm are
becoming increasingly popular. Unlike visible-light fluoro-
phores, NIR light can effectively penetrate live tissues,
lessening phototoxicity and improving contrast.1 Although
the benefits of employing NIR fluorophores are well
recognized, NIR scaffolds have been dominated by just a few
structural classes, particularly cyanines.2 Expanding the
repertoire of scaffolds for constructing NIR fluorophores and
chromophores can improve our ability to fine-tune spectral
properties and develop novel chemical sensors.
Substitution of the bridging oxygen in xanthene fluorophores
with silicon or other group 14 elements has been shown to
dramatically red-shift their excitation and emission wavelengths
and, thus, has received much attention in the past decade
(Figure 1).3,4 Among the group 14 elements, silicon produces
the most red-shifted fluorophores.5 For example, the excitation
and emission wavelengths of tetramethylrhodamine (548ex/
572em) are red-shifted by ∼90 nm in Si-tetramethylrhodamine
(643ex/662em).6 Moreover, Si-rhodamines (Figure 1) retain the
high photostability of rhodamines with quantum yields
comparable to those of popular near-IR cyanine fluoro-
phores.7,8 Hence, numerous Si-rhodamines have been reported
with advantageous properties for live cell and in vivo imaging
techniques.9−12
Clearly, Si incorporation has expanded the applications and
potential of rhodamine fluorophores, and the incorporation of
other elements at this position is a burgeoning area of
research.13−16 Less well studied is the effect of these
modifications on other dye scaffolds. Oxazine dyes already
have typical excitation and emission wavelengths around 640−
660 nm.17 We hypothesized that silicon substitution in
oxazines would yield analogously red-shifted “Si-oxazine”
(azasiline) dyes (Figure 1).
Figure 1. Scope of this work.
Here, we report the synthesis and photophysical character-
istics of two azasiline fluorophores with excitation and
emission wavelengths over 700 nm.
On the basis of the structural similarities between oxazines
and the envisioned azasilines, we first designed a synthetic
scheme modeled after known synthetic routes to oxazine
dyes17,18 (Scheme 1). Silane 1 was obtained by treating 3-
bromo-N,N-dimethylaniline with n-BuLi and dichlorodime-
thylsilane.19 Analogously to oxazine dye synthesis, a diazoni-
trobenzene moiety was added to yield 2. However, acid-
catalyzed cyclization conditions that readily form oxazine dyes
failed for the corresponding azasilines. The lack of reaction
could reflect the lowered nucleophilicity of the aryl silane
compared to the aryl ether and/or the longer C−Si bond
length.
Received: June 7, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b01786
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Organic Letters
Letter
Scheme 1. Modeling Azasiline Synthesis after Oxazines
Scheme 3. Synthesis of a Difluoroazasiline (ASiFluor710)
We next adopted a synthetic route predicated on the
synthesis of azasilines as blue fluorescent emitters for
OLEDs.20,21 In this revised scheme, diphenylamine was
tetrabrominated with NBS to yield 3 (Scheme 2). The
To our delight, 13 readily oxidized and precipitated out of
solution during I2 treatment. Simple filtering and washing of
the precipitant with hexanes and methanol afforded pure
product. In ethanol, the dye exhibited absorption and
fluorescence at wavelengths >700 nm (Figure S1), with an
extinction coefficient of 60000 M−1 cm−1 and quantum yield of
0.11 (Figure S2). Unfortunately, the dye slowly degraded in
PBS (Figure S3). LC/MS analysis of ASiFluor710 incubated in
PBS for 48 h revealed a new peak with absorbance maxima of
∼640 nm and a mass of 306, compared to 370 for the parent
dye (Figure S3b). 1H and 19F NMR indicated the loss of both
fluorines and the dimethylsilyl group. The data are consistent
with the unexpected rearrangement to oxazine dye 13′,
highlighting the need to further explore the unique chemistry
of silicon, especially in the context of multifunctional
heterocycles.22
Although the incorporation of the π-donor fluorine allowed
the synthesis of an azasiline dye, its reactivity in SNAr reactions
likely leads to fluorine substitution and subsequent fluoride-
mediated reaction with the silyl group. Methyl groups are more
benign electron donors that may help stabilize the oxidized
azasiline dye without this liability. To access the dimethyl
azasiline derivative, we first followed the same general synthetic
route used to synthesize 13. However, that scheme failed at the
PMB protection step, presumably due to the increased steric
hindrance posed by the methyl groups. We therefore revisited
our original synthetic scheme and postulated that cyclization
could be achieved via Buchwald−Hartwig coupling (Scheme
4). First, Buchwald−Hartwig coupling of azetidine with 3,5-
dibromotoluene yielded 14. The symmetric silane 15 was then
formed by lithium−halogen exchange and treatment with
dichlorodimethylsilane. Subsequent dibromination with NBS
produced silane 16, which underwent Buchwald−Hartwig
coupling with 4-methoxybenzylamine to effect ring closure,
Scheme 2. Attempted Synthesis of an Azetidenyl Azasiline
nitrogen was then protected with a PMB group using NaH to
yield 4. Lithium−halogen exchange with n-BuLi followed by
dichlorodimethylsilane treatment afforded the cyclized product
5.
Azetidines, which have been shown to increase the quantum
yields of rhodamines,6 were then installed using Buchwald−
Hartwig coupling to yield the protected leuco-dye 6. However,
attempted deprotection of 6 with TFA to yield azasiline 7
resulted in decomposition after solvent removal. In an
alternative method for deprotection, iodine treatment of 6 in
methanol yielded the crude putative azasiline fluorophore as a
green compound, and HPLC purification encouragingly
revealed a peak with an absorption maxima of 730 nm.
However, upon solvent removal, decomposition again occurred
to yield a possibly polymeric product that was insoluble in
acetone, chloroform, methanol, water, and DMSO.
Stymied by the apparent instability of 7, we next tested
whether the stability of the azasiline dye could be modulated
by altering the sterics and/or electronics of the azasiline with
different substituents. Two different derivatives were designed:
one that includes inductively withdrawing but π-donating
fluorine groups10 (13) and one that contains bulkier and
electron-donating methyl groups (18).
Scheme 4. Synthesis of a Dimethylazasiline (ASiFluor730)
To access the difluoro azasiline 13, bis(2-fluorophenyl)-
amine 8 was synthesized through Buchwald−Hartwig coupling
of 2-fluoroaniline with 1-bromo-2-fluorobenzene (Scheme 3).
As with diphenylamine in Scheme 2, 8 was tetrabrominated,
protected with PMB and cyclized to yield the core brominated
azasiline. Transformation to the azetidine leuco-dye 12 was
then performed by Buchwald−Hartwig amination. Depro-
tection and oxidation were achieved in one step using I2 in
methanol to give the final difluorinated azasiline 13
(ASiFluor710).
B
DOI: 10.1021/acs.orglett.8b01786
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yielding the protected leuco dye 17. Iodine treatment of 17
yielded the final product 18 (ASiFluor730).
ASiFluor730 (18) fluoresces at >730 nm in both organic and
aqueous solvents (Table 1 and Figure S1). Gratifyingly, HPLC
analogues of oxazine dyes. Although not as bright as cyanines,
ASiFluor730 is one of the most compact near-IR fluorophores
that absorbs beyond 700 nm (Table S1). The NIR excitation
and emission wavelength, photostability, and compact size
show promise for the future development of azasiline-based
optical probes and suggests that silicon can be incorporated
more broadly into chromophore scaffolds to further expand the
diversity of NIR fluorophores, chromophores, and luminescent
molecules.28−30
Table 1. Photophysical Properties of Azasilines
ASSOCIATED CONTENT
* Supporting Information
■
a
S
λabs
(nm)
λem
(nm)
ε
b
R
solvent
EtOH
PBS
(M−1 cm−1
)
Φ
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b01786.
a
CH3
732
725
725
730
736
701
709
745
739
739
745
747
734
719
121000
53000
70000
95000
109000
46000
0.11
0.01
0.009
0.06
0.13
0.11
0.11
H2O
Experimental procedures, supplemental tables and
figures, and full spectroscopic data for all new
compounds (PDF)
0.05% Tween-20
ethylene glycol
1,4-dioxane
EtOH
b
F
60000
AUTHOR INFORMATION
a
b
■
ε values were rounded to the nearest thousands. Φ (relative
Corresponding Author
*E-mail: stephen.miller@umassmed.edu.
ORCID
quantum yields) were calculated with 1,1′,3,3,3′,3′-hexamethylindo-
tricarbocyanine iodide (HITCI, a) and Cy5.5 (b) as standards.2,15,16
.
and LC/MS analyses of 18 incubated in PBS for 24 h showed
no degradation (Figure S3). The quantum yield and molar
extinction coefficient of 18 were calculated to be 0.11 and
121000 M−1 cm−1 in ethanol (Table 1), which is comparable
to that of oxazine 1 (0.14 and 118000 M−1 cm−1).23,24 In
ethylene glycol and 1,4-dioxane, 18 has quantum yields of 0.13
and 0.11, respectively. However, the quantum yield of 18
dropped to approximately 0.01 in deionized water and PBS,
which is 11-fold lower than in ethanol (Table 1 and Figure S2).
The addition of 0.05% Tween-20 increased the quantum yield
to 0.06, suggesting that the reduced quantum yield in aqueous
solvent is due in part to aggregation.25 (Table 1 and Figure
S2). Future incorporation of polar groups such as sulfonates
should increase aqueous solubility and may, in turn, increase
quantum yields in water.26,27
Lastly, we compared the photostability of the ASiFluors to
Oxazine 1 and Cy5.5. Each fluorophore was subjected to
continuous irradiation at its peak absorption wavelength for 1
h. All fluorophores were photostable in ethanol. However, in
PBS, Cy5.5 lost about 60% of its initial fluorescence, whereas
Oxazine 1 and ASiFluor730 were unaffected (Figure 2).
In conclusion, we have developed synthetic approaches to a
new class of near IR-fluorophores based on azasilines, the Si
Stephen C. Miller: 0000-0001-7154-7757
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank members of the Kelch and Ryder laboratories at
UMass Medical School for the use of equipment. This work
was supported in part by a grant from the US National
Institutes of Health (DA039961).
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DOI: 10.1021/acs.orglett.8b01786
Org. Lett. XXXX, XXX, XXX−XXX