A Cyanine Photooxidation/β-Elimination Sequence Enables Near-infrared Uncaging of Aryl Amine Payloads

Article abstract of DOI:10.1111/php.13090

Uncaging strategies that use near-infrared wavelengths can enable the highly targeted delivery of biomolecules in complex settings. Many methods, including an approach we developed using cyanine photooxidation, are limited to phenol-containing payloads. Given the critical role of amines in diverse biological processes, we sought to use cyanine photooxidation to initiate the release of aryl amines. Heptamethine cyanines substituted with an aryl amine at the C4′ position undergo only inefficient release, likely due electronic factors. We then pursued the hypothesis that the carbonyl products derived from cyanine photooxidation could undergo efficient β-elimination. After examining both symmetrical and unsymmetrical scaffolds, we identify a merocyanine substituted with indolenine and coumarin heterocycles that undergoes efficient photooxidation and aniline uncaging. In total, these studies provide a new scheme—cyanine photooxidation followed by β-elimination—through which to design photocages with efficient uncaging properties.

Full text of DOI:10.1111/php.13090

Article type : Research Article
A Cyanine Photooxidation/-Elimination Sequence Enables Near-
Infrared Uncaging of Aryl Amine Payloads
1a
1
1,2
1**
Tsuyoshi Yamamoto, Donald R. Caldwell, Albert Gandioso, Martin J. Schnermann
1
Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute
Frederick, MD 21702 (USA)
2
Seccio de Química Orgànica ,Departament de Química Inorganica i Organica ,Universitat de
Barcelona, Martí i Franques 1-11, E-08028 Barcelona, Spain
a
Current address: Graduate School of Biomedical Sciences, Nagasaki University
Nagasaki, 852-8521 (JAPAN)
*Corresponding author e-mail: martin.schnermann@nih.gov
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/php.13090
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ABSTRACT
Uncaging strategies that use near-infrared (NIR) wavelengths can enable the highly targeted delivery
of biomolecules in complex settings. Many methods, including an approach we developed using
cyanine photooxidation, are limited to phenol-containing payloads. Given the critical role of amines in
diverse biological processes, we sought to use cyanine photooxidation to initiate the release of aryl
amines. Heptamethine cyanines substituted with an aryl amine at the C4’ position undergo only
inefficient release, likely due electronic factors. We then pursued the hypothesis that the carbonyl
products derived from cyanine photooxidation could undergo efficient -elimination. After examining
both symmetrical and unsymmetrical scaffolds, we identify a merocyanine substituted with indolenine
and coumarin heterocycles that undergoes efficient photooxidation and aniline uncaging. In total,
these studies provide a new scheme – cyanine photooxidation followed by -elimination – through
which to design photocages with efficient uncaging properties.
INTRODUCTION
The photocaged/uncaged paradigm is a useful manifold to modulate biological function with high
spatial and temporal control (1, 2). To transition these experiments into increasingly complex
physiological environments, photocages activated with single photon flux of far-red and near-infrared
wavelengths would be highly enabling. Recent efforts have identified some of the first useful small
molecule-based methods in this range. Given the modest photonic energies of these wavelengths, it is
not surprising that these approaches employ a range of chemical mechanisms for bond cleavage (3).
These include the direct cleavage of certain chromophore-payload bonds, the cleavage of carbon-
metal bonds, the use of photoinduced electron transfer (PET)-mediated processes, and the local
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1
2
generation of singlet oxygen ( O ) (4-12)(13). Despite progress, significant reaction discovery efforts
are needed to uncover methods characterized by broad payload flexibility, activation with
wavelengths over 700 nm, and rapid release.
Our group has sought to use cyanine photooxidation as a triggering event to initiate uncaging
1
(
14-16). Molecules of the general type shown in Figure 1A undergo O
2
-mediated oxidative cleavage
between the C2/C1’ and C2’/C3’ positions and then a hydrolysis/cyclization cascade to liberate
phenol payloads (17). The key element of this approach is the altered hydrolytic stability of the C4’-N
bond following oxidative cleavage. We have applied this approach to create NIR-activated antibody-
drug conjugates that release potent anti-tumor agents with in vivo activity (18, 19). Given the critical
role that nucleic acids and other amine-containing molecules play in biological processes (20, 21),
NIR uncaging strategies that release amine payloads have broad potential impact.
Here we detail efforts to apply cyanine photooxidation as an initiating event for amine
uncaging. We find that the direct attachment of aniline payloads to the C4’ position of the
heptamethine cyanine leads to only inefficient release under physiological conditions (Figure 1B). We
then considered whether the oxidative formation of carbonyl products could be harnessed to initiate a
-elimination event. We prepared derivatives, including a symmetrical heptamethine cyanine and
unsymmetrical merocyanines, with suitably positioned carbamate functional groups. Through this
approach, we identify a merocyanine that is highly water soluble and undergoes efficient
photooxidiation with useful uncaging yields.
<
figure 1>
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MATERIALS AND METHODS
Reagents. Unless stated otherwise, reactions were conducted in oven-dried glassware under an
atmosphere of argon using anhydrous solvents (passed through activated alumina columns). All
commercially obtained reagents were used as received. Flash column chromatography was performed
using reꢀersed phase ꢁ1ꢂꢂ ꢃ, 20-40 micron particle size, RediSep® Rf Gold® Reversed-phase C18 or
C18Aq) on a CombiFlash® Rf 200i (Teledyne Isco, Inc., Lincoln, NE).
Apparatus. High-resolution LC/MS analyses were conducted on a Thermo-Fisher ꢄTꢅ-ꢆrbitrap-ꢇꢄ
hybrid mass spectrometer system with an ꢈon ꢉꢊꢇ ꢊꢋꢈ electrospray ion source in negatiꢀe ion modeꢌ
ꢊnalytical ꢄC/ꢉꢍ was performed using a ꢍhimadzu ꢄCꢉꢍ-2ꢂ2ꢂ ꢍingle ꢅuadrupole utilizing a
ꢎineteꢏ 2ꢌꢐ ꢑm C1ꢒ 1ꢂꢂ ꢃ (2.1 x 50 mm) column obtained from Phenomenex, Inc (Torrance, CA).
Runs employed a gradient of 0-90% MeCN/0.1% aqueous formic acid over 4.5 min at a flow rate of
1
13
0
.2 mL/min. H-NMR and C-NMR spectra were recorded on Bruker spectrometers (at 400 or 500
1
MHz or at 100 or 125 MHz) and are reported relative to deuterated solvent signals. Data for H-NMR
spectra are reported as follows: chemical shift ꢁδ ppm), multiplicity, coupling constant ꢁHz), and
1
3
integration. Data for C-NMR spectra are reported in terms of chemical shift. Absorbance curves
were obtained on a Shimadzu UV-2550 spectrophotometer operated by UVProbe 2.32 software.
Samples in 96-well plates (Corning black plate, clear bottom, polystyrene, #3603) were irradiated
using a 690 ± 20 nm LED (L690-66-60, Marubeni America Co.) at a light intensity of 20 mW/cm2 as
measured using a power meter. Fluorescence traces were recorded on a PTI QuantaMaster steady-
state spectrofluorimeter operated by FelixGX 4.2.2 software, with 5 nm excitation and emission slit
widths, and a 0.1 s integration rate. Data analysis and curve fitting were performed using MS Excel
2011 and GraphPad Prism 7.
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Synthesis of Cy7-C4-Cou. In a glove-box, IR-783 (100 mg, 0.13 mmol), 7-amino-4-
trifluoromethyl)chromen-2-one, Coumarin 151 (42.8 mg, 0.19 mmol), Pd (dba) •CHCl (12 mg,
.013 mmol), BrettPhos (14.5 mg, 0.026 mmol), Cs CO (87 mg, 0.26 mmol) and N,N-
(
2
3
3
0
2
3
dimethylformamide (2 mL) were added to an oven dried microwave and the vial was sealed. The
resulting heterogenous mixture was heated to 80 ºC and vigorously stirred for 1 h under inert
atmosphere, after which time LC/MS analysis of the reaction showed completed consumption of IR-
7
83. The reaction was cooled to room temperature and filtered through celite. The flow-through
was diluted with water (3 mL) subjected to reverse-phase column chromatography (C18, 20–60%
MeCN/H
2
O). The solvent was removed in vacuo to afford Cy7-C4-Cou (71 mg, 57% yield) as a green
, 500 MHz) δ 9.26 (s, 1H), 7.90 (d, J = 14.1 Hz, 2H), 7.57 (d, J = 8.1 Hz, 1H),
1
solid. H NMR (DMSO-d
6
7
1
1
1
1
2
.48 (d, J = 7.3 Hz, 2H), 7.39–7.33 (m, 4H), 7.17 (t, J = 7.7 Hz, 2H), 6.94 (br, 1H), 6.77 (br, 1H), 6.60 (s,
H), 6.25 (d, J = 14.1 Hz, 2H), 4.13 (br, 4H), 2.71 (br, 4H),1.93 (br, 4H), 1.80–1.67 (m, 8H), 1.32 (s,
13
2H); C NMR (DMSO-d , 125 MHz)  171.63, 159.40, 156.82, 153.04, 152.97, 142.63, 142.35,
6
41.33, 140.20 (q, J = 30.9 Hz), 128.90, 127.05, 126.67, 124.95, 123.35, 122.77, 121.16, 112.57,
11.62, 110.47, 104.14, 100.82, 100.39, 72.96, 63.54, 51.23, 48.93, 43.97, 27.74, 26.48, 24.51, 23.00,
+
1.38; HRMS (ESI) calculated for C48
H
53
F
N
3 3
O S
8 2
(M ) 920.3221, observed 920.3200.
Synthesis of 4. At 0 ºC, phosphorous oxychloride (1.12 g, 7.32 mmol) was added dropwise to
anhydrous DMF (1.37 mL, 17.7 mmol). After stirring for 30 min, 4-hydroxy-cyclohexanone, 1, (186
mg, 1.62 mmol) in anhydrous DMF (1 mL, 14.5 mmol) was added dropwise to the reaction at 0 ºC
and the mixture was heated at 50 ºC for 1 h in an oil bath. A mixture of aniline/ethanol (50% v/v, 4
mL) was then added dropwise to the mixture at room temperature. The resulting mixture was
poured into ice-cold conc. HCl (10:1 v/v, 22 mL). The mixture was allowed to stand for 5 h at –4 ºC
and then the formed solid was filtrated off, washed with cold water, ethanol and diethyl ether, and
then dried in vacuo to afford chloride 2 as a purple solid (332 mg). The solid was used without
further purification. To a 20-mL vial was added indoleine 3 (500 mg, 1.69 mmol),(22) chloride 2 (192
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mg, 0.57 mmol), sodium acetate (232 mg, 2.80 mmol), ethanol (2 mL) in succession. Acetic
anhydride (286 mg, 2.80 mmol) was added and the reaction was stirred at room temperature for 2
h. The reaction was precipitated into ether (50 mL), the slurry was centrifuged at 3,500 rpm for 5
min and the supernatant was discarded. This procedure was repeated twice. The crude solid was
placed under vacuum for 1 h, and the solid was dissolved in H O (3 mL). The mixture was subjected
2
to reverse-phase chromatography (C18 column, 0 to 100% MeCN/H
2
O). The solvent was removed in
1
vacuo to afford 4 (110 mg, 25% yield) as a green solid. H NMR (Methanol-d
4
, 500 MHz)  8.51 (d, J =
1
4.3 Hz, 2H), 7.54 (d, J = 7.4 Hz, 2H), 7.47–7.40 (m, 4H), 7.30 (t, J = 7.43 Hz, 2H), 4.30 (m, 1H), 4.25 (t,
J = 7.51 Hz, 4H), 2.98 (dd, J = 15.7, 4.0 Hz, 2H), 2.92 (t, J = 6.9 Hz, 4H), 2.78 (dd, J = 15.7, 6.9 Hz, 2H),
1
3
2
1
2
.0 (m, 8H), 1.76 (s, 12H); C NMR (Methanol-d , 125 MHz) 173.00, 148.92, 145.05, 142.21,
4
41.24, 128.53, 125.13, 123.75, 122.04, 111.06, 101.08, 63.53, 50.33, 49.29, 48.10, 43.66, 33.66,
+
6.91, 25.89, 22.22. HRMS (ESI) calculated for C38
H48ClN
O S
2 7 2
(M ) 743.2586, observed 743.2570.
Synthesis of Cy7--Cou. To
a
25-mL pear-shaped flask was added 7-amino-4-
(trifluoromethyl)chromen-2-one (100 mg, 0.44 mmol) and phosgene solution (3 mL, 4.9 mmol;
1
5%wt in toluene) at room temperature. The mixture was stirred for 20 min at room temperature
and then heated to 80 ºC for 13 h. The reaction was cooled and the solvent and excess phosgene
was removed in vacuo to afford 6, which was used without further purification. To the resulting solid
was added a mixture of 4 (20 mg, 26 µmol), dichloromethane/N-methyl-2-pyrrolidone (50%v/v) and
N,N-diisopropylethylamine (45 µL, 0.26 mmol) at 0 ºC. The reaction was stirred for 1 h in an ice bath
and then precipitated in ether (50 mL). The slurry was centrifuged at 3,500 rpm for 5 min and the
supernatant was discarded. This procedure was repeated twice. The crude solid was placed under
vacuum for 1 h, and the solid was dissolved in H O/MeCN (3 mL, 50% v/v). The mixture was
2
subjected to reverse-phase chromatography (C18 column, 0 to 100% MeCN/H O). The solvent was
2
1
removed in vacuo to afford Cy7--Cou (20 mg, 76% yield) as a green solid. H NMR (Methanol-d
4
,
5
00 MHz) d, J =14.2 Hz, 2H), 7.74 (s, 1H), 7.62 (d, J = 8.6 Hz, 1H), d, J = 7.6 Hz, 2H),
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7
7
.46–7.39 (m, 5H), 7.31 (t, J = 7.1 Hz, 2H), 6.72 (s, 1H), 6.38 (d, J = 14.3, 2H), 5.49 (br, 1H), 4.26 (t, J =
.3 Hz, 4H), 3.24–3.02 (m, 4H), 2.89 (t, J = 7.3 Hz, 4H), 2.08–1.90 (m, 8H), 1.76 (d, J = 7.19 Hz, 12H);
13
C NMR (Methanol-d , 125 MHz)  173.34, 159.41, 155.42, 153.27, 148.39, 144.97, 144.06, 142.17,
4
1
1
41.29, 140.76 (q, J = 32.6 Hz), 128.56, 125.33, 125.26, 122.23, 122.05, 115.01, 113.01, 112.96,
11.22, 107.92, 105.36, 101.10, 67.94, 50.37, 49.39, 43.75, 30.78, 26.93, 26.90, 25.96, 22.18. HRMS
+
(ESI) calculated for C49H52ClF
N O
3 3 10
S
2
(M ) 998.2729, observed 998.2711.
Synthesis of 9. To a round-bottom flask was added dichloromethane (30 mL) and pyridine (218 µL,
.7 mmol). After the mixture was cooled to –78 ºC, glycidol (140 µL, 2.7 mmol) and
2
trifluoromethanesulfonic anhydride (454 µL, 2.7 mmol) were added dropwise in succession to the
mixture. The reaction was stirred at –78 ºC for 40 min and was warmed to room temperature. To the
mixture was added hexane (30 mL) and the resulting suspension was subjected to short silica gel
column chromatography (10 g of silica gel with 30 mL of dichloromethane). The solvent was
removed in vacuo and the resulting clear oil was used without further purification. To this oil was
added dichloromethane (10 mL) under inert atmosphere. After the mixture was then cooled to –78 ºC,
2,3,3-trimethylindolenine, 7, (216 µL, 1.35 mmol) and N,N-diisopropylethylamine (235 µL, 1.35
mmol) were added to the solution in succession. The reaction was stirred at –78 ºC for 20 min and
then warmed up to room temperature over 1 h. The reaction was directly subjected to silica gel
column chromatography (0–30% MeOH/DCM). The solvent was removed to afford 9 as a clear oil
1
(
215 mg, 75% yield). H NMR (Chloroform-d, 4ꢂꢂ ꢉHz) δ 7ꢌꢒ2–7.77 (m, 1H), 7.56–7.48 (m, 3H),
5.06 (d, J = 15.1 Hz, 1H) 4.42 (dd, J =15.1, 6.6 Hz, 1H), 3.49 (m, 1H), 2.87 (t, J = 4.3 Hz, 1H), 2.63
1
3
(
dd, J = 4.4, 2.5 Hz, 1H), 1.53 (d, J = 3.6 Hz, 6H); C NMR (Chloroform-d, 100 MHz)  198.89,
141.26, 141.16, 130.14, 129.55, 123.15, 115.59, 54.88, 49.91, 48.72, 45.72, 22.79, 22.68, 14.65;
+
+
HRMS (ESI) calculated for C14H18NO (M ) 216.1383, observed 216.1378.
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Synthesis of 10. To a round-bottom flask was added 9 (670 mg, 3.1 mmol) and dichloromethane (30
mL). To the mixture was added trifluoromethanesulfonic acid (603 µL, 6.8 mmol). The reaction was
stirred for 20 h at room temperature. The resulting solution was washed with saturated sodium
bicarbonate aqueous solution (40 mL) and brine (40 mL). The organic layer was dried over Na
2
SO ,
4
added formic acid (500 µL) and the solvent was removed in vacuo. The resulting oil was subjected to
reverse-phase column chromatography (C18, 0–100% MeCN/H
2
O). The solvent was removed in vacuo
, 5ꢂꢂ ꢉHz) δ 7ꢌꢒꢂ–7.76 (m, 2H),
1
to afford 10 as a clear oil (476 mg, 71% yield). H NMR (Methanol-d
4
13
7
.67–7.63 (m, 2H), 4.59 (m, 1H), 4.43–4.34 (m, 2H), 2.23–2.12 (m, 2H), 1.63 (d, J = 21.8 Hz, 6H); C
NMR (Methanol-d , 126 MHz)  195.51, 141.93, 141.79, 129.86, 128.96, 122.96, 114.14, 60.44,
4
+
+
5
3.67, 51.78, 23.26, 21.86, 21.41, 19.38; HRMS (ESI) calculated for C14
H18NO (M ) 216.1383,
observed 216.1380.
Synthesis of 13. In a round-bottom flask was added N-[5-(phenylamino)-2,4-pentadienylidene]aniline
monohydrochloride, 11, (0.50 g, 1.75 mmol), 1,3-dimethyl barbituric acid, 12, (0.27 g, 1.75 mmol),
sodium acetate (0.14 g, 1.75 mmol) and acetic anhydride (2.5 mL). The flask was evacuated and
flushed with argon. The reaction was stirred at 160 °C for 5 min as the solution color transitioned
2
from dark red to dark brown. The reaction mixture was cooled and concentrated with 2 g of SiO and
then purified by normal phase chromatography (80 g silica column, 0-80% EtOAc/Hexane) to afford
1
1
3 (0.23 g, 37% yield) as a dark brown solid. H NMR (Chloroform-d
1
, 5ꢂꢂ ꢉHz) δ ꢒꢌ1ꢐ (d, J = 13.9
Hz, 1H), 8.04 (d, J = 12.5 Hz, 1H), 7.70 (dd, J = 14.4, 12.4 Hz, 1H), 7.59 – 7.52 (m, 3H), 7.23 – 7.18
1
3
(
(
m, 3H), 5.40 (dd, J = 13.7, 11.4 Hz, 1H), 3.35 (s, 3H), 3.30 (s, 3H), 1.97 (s, 3H); C NMR
Chloroform-d, 125 ꢉHz) δ 1ꢐ9ꢌ24, 1ꢐ2ꢌꢐꢂ, 1ꢐ1ꢌ95, 157ꢌ1ꢒ, 154ꢌ95, 151ꢌ59, 14ꢂꢌ7ꢐ, 13ꢒꢌ23, 13ꢂꢌ59,
20 3 4
129.75, 128.29, 126.76, 113.75, 111.60, 28.57, 27.87, 23.37; HRMS (ESI) calculated for C19H N O
+
(
MH ) 354.1448, observed 354.1444.
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Synthesis of BI--Cou. To a microwave vial was added 10 (60 mg, 0.27 mmol), 13 (75.7 mg, 0.21
mmol), sodium acetate (46 mg, 0.56 mmol) and MeOH/DCM (2 mL, 50% v/v) in succession. The vial
was sealed and heated with a microwave reactor to 75 °C for 1 h as the solution color transitioned
from dark red to blue. The reaction mixture was cooled and concentrated with 0.5 g of silica gel and
then purified by normal phase chromatography (12 g silica column, 0–100% ethyl acetate/hexane) to
afford the intermediate merocyanine as a dark blue solid, which was immediately used for the next.
The intermediate merocyanine in dichloromethane (3 mL) was added to 6 (0.22 mmol, generated as
above) at 0 °C. After addition of N,N-diisopropylethyamine (38 µL, 0.22 mmol) at 0 ºC, the reaction
was stirred for 30 min in an ice bath. Then the reaction was warmed to room temperature and stirred
for 3 h. The reaction mixture was concentrated and purified by normal–phase chromatography (12 g
1
of silica gel, 0–100% EtOAc/hexane) to afford BI--Cou (28 mg, 19% yield) as a dark blue solid. H
NMR (Chloroform-d, 500 MHz,) δ ꢒꢌꢂ5 ꢁd, J = 12.7 Hz, 1H), 7.85 (t, J = 13.7 Hz, 1H), 7.67 (s, 1H),
7
8
.62 (d, J = 8.7 Hz, 1H), 7.49–7.45 (m, 2H), 7.32–7.25 (m, 4H), 7.06 (t, J = 7.2 Hz, 1H), 6.80 (d, J =
.7 Hz, 1H), 6.66 (s, 1H), 6.29 (t, J = 13.0 Hz, 1H), 5.53 (br, 1H), 3.99 (d, J = 13.4 Hz, 1H), 3.88 (d,
1
3
J = 13.0 Hz, 1H), 3.33 (d, J = 17.4 Hz, 6H), 2.78 (s, 2H), 1.66 (s, 6H); C NMR (Chloroform-d, 125
ꢉHz) δ 1ꢐ3ꢌ27, 1ꢐ2ꢌ44, 1ꢐꢂꢌ3ꢐ, 159ꢌ15, 15ꢐꢌꢒ1, 155.42, 151.99, 151.94, 144.53, 142.68, 142.20,
1
1
2
3
41.28 (q, J = 33.1 Hz, CF ), 139.52, 128.25, 126.12, 124.38, 122.68, 122.56, 122.00, 120.37, 120.08,
15.21, 113.66, 109.04, 107.49, 107.36, 106.31, 102.31, 65.96, 47.30, 44.90, 28.75, 28.58, 28.42,
+
7.74; HRMS (ESI) calculated for C36
H
32
F
3
N
4
O
7
(MH ) 689.2218, observed 689.2216.
Synthesis of 15. To a round-bottom flask was added N,N-dimethylformamide (2 mL, 25.8 mmol).
After cooled to 0 ºC, phosphoryl chloride (298 µL, 3.20 mmol) was added dropwise over 5 min. The
reaction was then warmed to room temperature and stirred for 30 min. The reaction was cooled to 0
ºC again and added 2-[6-(diethylamino)-2,3-dihydro-1H-xanthen-9-yl]-benzoic acid 14 (400 mg, 1.07
mmol)(23) in N,N-dimethylformamide (2 mL) dropwise under 0 ºC. The reaction was stirred for 10
min at 0 ºC and for 1 h at room temperature. The reaction was cooled to 0 ºC and quenched with H
2
O
(2 mL) and stirred at room temperature for 3 d. The resulting solution was subjected to reverse-phase
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chromatography (C18, 5–100% MeCN/H
2
O). The solvent was removed in vacuo to afford aldehyde 15
, 400 MHz) δ 1ꢂꢌ29 (s, 1H), 8.05 (dd, J = 1.3, 7.9 Hz, 1H),
1
(
356 mg, 82% yield). H NMR (DMSO-d
6
7.70 (dt, J = 1.4, 7.5 Hz, 1H), 7.58 (dt, J = 1.4 and 7.6 Hz, 1H) , 7.24 (dd, J = 7.6, 1.1 Hz, 1H), 6.57
(d, J = 2.4 Hz, 1H), 6.39 (dd, J =9.0, 2.4 Hz, 1H), 6.32 (d, J = 9.0 Hz, 1H), 3.36 (q, J = 7.0 Hz, 4H),
1
3
2
.27 (t, J = 6.0 Hz, 2H), 2.17–2.02 (m, 2H), 1.58–1.44 (m, 2H), 1.09 (t, J = 7.1 Hz, 6H); C NMR
(DMSO-d
6
, 100 MHz) δ 186.07, 167.65, 161.66, 153.46, 149.59, 140.87, 136.37, 132.81, 131.70,
1
1
30.81, 130.40, 128.95, 126.79, 118.61, 111.21, 110.93, 108.25, 97.11, 44.31, 27.39, 21.65, 20.50,
+
2.92, 12.73. HRMS (ESI) calculated for C25
H26NO
4
(MH ) 404.1856, observed 404.1850.
Synthesis of CI--Cou. To a 20-mL vial was added 15 (292 mg, 0.68 mmol), 10 (220 mg, 1.02 mmol),
sodium acetate (342 mg, 4.07 mmol), ethanol (5 mL) and acetic anhydride (451 µL, 4.07 mmol) in
succession. The reaction was heated at 100 ºC for 2 h. After cooled to room temperature, ethyl
acetate (50 mL) was added to the reaction and the organic layer was washed with saturated sodium
bicarbonate solution (30 mL  2) and brine (30 mL  2). The organic layer was dried over Na
concentrated in vacuo. To the resulting solid was added sodium iodide (4g) in H O/MeOH/DCM (4
mL/1 mL/3 mL) and the solution was stirred for 8 h. To the mixture was added dichloromethane (30
mL) and the solution was washed with H O (30 mL). The resulting organic layer was dried over
Na SO and concentrated in vacuo. The crude mixture was purified by silica gel chromatography (40
2
SO and
4
2
2
2
4
g of silica gel, 0–100% MeOH/dichloromethane) to afford the intermediate secondary alcohol as a
1
green solid (170 mg, 34% yield). H NMR (Methanol-d
4
, 400 MHz) δ ꢒꢌꢐ3 ꢁs, 1H), ꢒꢌꢂ1–7.97 (m, 1H),
7.57–7.51 (m, 3H), 7.43 (t, J = 7.7 Hz, 1H), 7.29 (t, J = 7.3 Hz, 1H), 7.24 (d, J = 7.9 Hz, 1H), 7.15–7.11
(
(
(
m, 1H), 6.94 (dd, J = 9.1, 3.7 Hz, 1H), 6.80–6.76 (m, 1H), 6.63 (br, 1H), 4.49–4.42 (m, 1H), 4.04–3.93
m, 2H), 3.57 (q, J = 7.1 Hz, 4H), 3.12 (d, J = 3.3 Hz, 2H), 2.99–2.89 (m, 2H), 2.55–2.32 (m, 2H), 1.88
13
dd, J = 14.8, 2.4 Hz, 6H), 1.83 (br, 2H), 1.28 (t, J = 7.3 Hz, 6H); C NMR (Methanol-d
4
, 1ꢂꢂ ꢉHz) δ
73.07, 170.63, 164.09, 155.89, 154.29, 151.67, 142.54, 142.44, 140.94, 139.72, 133.74, 129.26,
29.02, 128.85, 128.32, 128.16, 124.64, 121.96, 120.93, 114.39, 113.66, 111.69, 109.54, 108.86,
1
1
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9
4.89, 61.13, 49.09, 44.68, 32.53, 27.90, 27.38, 26.93, 26.36, 21.30, 11.27; HRMS (ESI) calculated for
+
+
C
39
H
N O
41 2 4
(M ) 601.3061, observed 601.3034. To 6 (0.17 mmol, generated as above) was added
the intermediate above (20 mg, 0.033 mmol), dichloromethane and N,N-diisopropylethylamine (45
µL, 0.26 mmol) in succession at 0 ºC. After the reaction was stirred for 1 h in an ice bath, the bath
was removed and for 12 h. To the reaction was added dichloromethane (50 mL) and washed with
saturated aqueous NaHCO
over Na SO and the solvent was removed in vacuo. The mixture was subjected to quick silica gel
chromatography (24 g of silica gel, 30–100% hexane/ethyl acetate, and then 0–100% MeOH/DCM).
The solvent was removed in vacuo to afford a green solid. The solid was dissolved in H O/MeOH
50% v/v, 8 mL) and then sodium iodide (3 g) was added. The colored mixture was stirred for 16 h
under a light–shielded condition. To the solution was added dichloromethane (50 mL) and washed
with H O (50 mL). The organic layer was dried over Na SO and the solvent was removed in vacuo.
3
solution (50 mL  2) and brine (50 mL  2). The organic layer was dried
2
4
2
(
2
2
4
Purification by silica gel chromatography was performed (24 g of silica gel, 30–100% hexane/ethyl
acetate, and then 0–100% MeOH/DCM). The solvent was removed in vacuo to obtain CI--Cou (20
1
mg, 62% yield) as a green solid. H NMR (Methanol-d
4
, 500 MHz) s, 1H), 8.05 (d, J = 7.8 Hz,
1
1
=
3
H), 7.71 (br, 1H), 7.62–7.54 (m, 4H), 7.42 (t, J = 7.6 Hz, 1H), 7.38–7.23 (m, 3H), 7.15 (d, J = 7.1 Hz,
H), 6.91 (d, J = 9.1 Hz, 1H), 6.81 (d, J = 8.8 Hz, 1H), 6.70 (s, 1H), 6.63 (s, 1H), 5.58 (br, 1H), 4.26 (dd, J
14.7 and 70.4 Hz, 2H), 3.58 (q, J = 7.0 Hz, 4H), 3.45 (d, J = 16.0 Hz, 1H), 3.32 (quint, J = 1.7 Hz, 4H),
.24 (d, J = 16.0 Hz, 1H), 3.30–2.84 (m, 2H), 2.46–2.33 (m, 2H), 1.91 (d, J = 14.8 Hz, 6H), 1.77–1.65
13
(
br, 2H), 1.28 (t, J = 7.1 Hz, 6H); C NMR (Methanol-d
4
, 125 MHz)  169.72, 164.24, 159.28, 156.07,
1
1
1
4
8
55.36, 155.04, 152.77, 152.01, 143.72, 142.25, 141.88, 140.83, 140.62, 140.51, 134.28, 129.99,
29.84, 128.80, 128.60, 128.38, 128.33, 125.33, 124.59, 123.12, 122.04, 121.01, 120.39, 114.97,
14.81, 113.76, 113.21, 113.15, 112.17, 109.35, 108.09, 107.25, 105.49, 94.85, 65.34, 49.10, 45.85,
+
+
4.76, 29.54, 28.06, 27.38, 26.93, 26.04, 21.20, 11.27. HRMS (ESI) calculated for C50
H F
45 3
N O
3 7
(M )
56.3204, observed 856.3160.
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Procedure for Determining Photooxidation Efficiency and Uncaging Kinetics. Stock solutions of the
coumarin 151 conjugates Cy7-C4-Cou, Cy7--Cou, BI--Cou, and CI--Cou were diluted into 5%
Dꢉꢍꢆ/pH 7ꢌ4 ꢋBꢍ ꢁ5ꢂ mꢉ) to afford 2 ꢑꢉ solutionsꢌ ꢍamples were dispensed into 96-well plates
(Corning black plate, clear bottom, polystyrene, #3603) and sealed with ThermalSeal RT film (Sigma,
Z707465). Samples in the 96-well plate were irradiated using a 780 LED ± 20 nm (L780-66-60,
Marubeni America Co.) for Cy7-C4-Cou and Cy7--Cou, 690 nm LED (L690-66-60, Marubeni
America Co.) for BI--Cou and 740 nm LED (L740-66-60, Marubeni America Co.) for CI--Cou at a
2
light intensity of 20 mW/cm as measured using a power meter. Single point absorption measurements
at the max and Coumarin 151 fluorescence (ex 372 nm, em 490 nm) were recorded at regular intervals
(
1-30 minutes for 12-150 minutes depending on photobleaching rate) until ablation of the absorbance
signal. Experiments were run in quadruplicate and plotted with error bars derived from the standard
deviation (<5% in all cases). Half-lives were obtained by fitting the curves with one phase decay
parameters in all cases except for CI--Cou, for which two phase decay parameters were used.
Percent yield of Coumarin 151 release was obtained from the input of the experimental fluorescence
values into the equation derived from the standard calibration curve, with 2 M defined as 100% yield
(Figure S1).
Procedure for LC/MS Relative Ion Analysis of Cy7--Cou Photolysis. A 10 mM stock of Cy7--Cou
in DMSO was diluted into water to yield a 10 uM solution. Phenylalanine (40 uM) was used as an
2
internal standard. The photolysis was run for 1 h at 23 ºC with 20 mW/cm 780  20 nm light in a 2
mL vial without a cap. After 1 h, the reaction was capped and keep in the dark. Aliquots were taken at
0
(prior to irradiation), 1 h, 2 h, 6 h, and 14 h and immediately analyzed by a direct loop injection
method with a Shimadzu LCMS-2020 Single Quadrupole instrument (normal resolution) The relative
ion counts in Figure 3C were calculated by integrating the extracted ion chromatogram (EIC) of the
2
m/z of Cy7--Cou, Int-1 + Int-1-H O, Int-2, and C151 and dividing by the ion count of phenylalanine
internal standard.
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RESULTS
Based on our prior studies (Figure 1A), a straightforward approach to this problem entails the
direct attachment of the aniline-containing substrate onto the C4’ position of the heptamethine
scaffold. The preparation of the corresponding aniline-modified cyanines proceeds readily through a
Pd(0)-mediated arylation reaction to provide Cy7-C4-Cou directly from the corresponding C4’-chloro
IR-783 (Figure 2A). The optical properties of Cy7-C4-Cou are consistent with previously prepared
C4’ N-arylated cyanines (Table 1) (24).
<
<
figure 2>
figure 3>
2
Photooxidation of Cy7-C4-Cou using a 780 nm LED (20 mW/cm ) in pH 7.4 PBS w/5%
DMSO proceeds efficiently, with similar kinetics to our prior efforts (t1/2 = 5.7 min, Figure 3B).
However, irradiation leads to only barely detectable release of the fluorescent coumarin 151 at rt over
1
h (<1 %, Figure S2). When Cy7-C4-Cou is subjected to complete photooxidation at rt and then
incubated at 37 °C, modest release occurs over a 24 h time frame (8% vs <1% without irradiation,
Figure 3B, see Figure S3 for background release curves)ꢌ These results suggest that C4’ aniline-
substituted cyanines will only be useful for uncaging applications requiring slow release of the
payload.
We then set out to identify an approach characterized by rapid kinetics. Our prior efforts were
based on the premise that cyanine photooxidation alters the electronic structure of the cyanine
scaffold, thereby enhancing the rate of C4’-N hydrolysis. Another feature of photooxidation is the
formation of a carbonyl product. Release strategies using a carbonyl formation/-elimination
manifold have been reported in several contexts, leading us to consider if similar logic could be
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applied here. (25-28) As an initial entry, we generated Cy7--Cou through carbamate formation from
the corresponding alcohol 4 (Figure 2), which is accessed in two steps from compound 2 (29).
Photooxidation of Cy7--Cou proceeds efficiently (t1/2 = 4.9 min, Figure 4). The release of coumarin
1
51 occurs nearly concurrently with photooxidation at early time points, however, the yield of release
only reached 14% after 20 min, and no significant additional release was observed thereafter (Figure
B). To examine the product mixture being formed in this process, detailed mass spectrometry studies
4
were carried out (Figure 4C). The expected intermediates Int-1 and Int-2 that deriꢀe from C2/C1’ and
C2’/C3’ photooꢏidation, respectiꢀely, were obserꢀed immediately following irradiationꢌ ꢈn following
the mixture over time, we observed that Int-1 and its hydrate persists for several hours. This suggests
that these species are a major and persistent component of the oxidative products derived from Cy7--
Cou. By contrast, Int-2, disappears with apparent concurrent formation of the coumarin 151 payload.
Thus, it appears the -elimination reaction from Int-2 proceeds efficiently, but the corresponding -
elimination from Int-1 is inefficient. In separate work, we haꢀe determined that cleaꢀage at C2/C1’
occurs preferentially to C2’/C3’ cleaꢀage in roughly a 4:1 ratio,(17) which may explain the modest
yield of release.
<
figure 4>
These observations suggested that it might be advantageous to pursue strategies where
cyanine photooxidation occurs with high regioselectivity. To control the site of photooxidation, we
considered using unsymmetrical merocyanines containing an electron-donating indolenine and an
electron-deficient heterocycle partner. This approach was prompted in part by prior studies by Hahn
that found that certain merocyanines undergo regioselective cleavage at the position adjacent to the
more electron-rich heterocycle. (30) We designed molecules that installed a carbamate within a
pendent tetrahydropyridine ring system on the electron-rich indolenine heterocycle. We anticipated
that (Figure 1C) photooxidation, perhaps occurring selectively adjacent to the electron-rich indolenine
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heterocycle, would produce a carbonyl intermediate poised for uncaging. For the electron deficient
unit, we decided to test merocyanines containing barbituric acid and substituted-coumarin
heterocycles. Merocyanines containing various barbituric acid derivatives have been used by Hahn
and others as solvent-sensitive fluorophores.(31) The more recently discovered mixed courmain-
indoleine merocyanines have been used in several in vivo biological contexts and were also chosen for
their improved water solubility.(23, 32)
The synthesis of our target molecules is detailed in Figure 5. The indolenine subunit was
prepared through the alkylation of indolenine 7 with glycidol triflate 8 to access epoxide 9.(33)
Epoxide 9 undergoes cyclization with triflic acid at r.t. to provide alcohol 10.(34) The regiochemistry
of the ring opening reaction to provide 10 was confirmed by COSY analysis (Figure S4). Compound
1
0 could be converted to the merocyanine BI--Cou by addition to polymethine 13, accessed from
commercial 11 and barbituic acid 12, and carbamate formation with isocyanate 6. Similarly, the
coumarin indocyanine hybrid structure CI--Cou could be prepared through formylation of known
1
4(23) to provide 15, which was converted to the merocyanine through the addition of indoleine 10,
followed by carbamate formation with 6.
<
figure 5>
With access to BI--Cou and CI--Cou, we investigated their optical and uncaging properties.
The absorbance/emission maxima of these two molecules, 664/693 nm and 713/729 nm (Table 1),
respectively, is in line with prior molecules in these class. Photooxidation of CI--Cou proceeds
readily with a t1/2 of 3.4 min, with BI--Cou requiring longer irradiation times (t1/2 = 20.2 min). These
highly disparate photobleaching kinetics may reflect differences in aggregation state, with CI--Cou
being mostly monomeric and BI--Cou being partially aggregated in the pH 7.4 PBS buffer used here
(Figure S5A,B). Uncaging kinetics and yield were reasonable in both cases, with CI--Cou releasing
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33% of coumarin 151 after 1 h and BI--Cou releasing 22% after 1 h (Figure 6 and Table 1). Some
additional release (4-5%) is observed over the next two hours in both cases. Of note, similar yields are
obtained with incubation at high temperature (e.g. 37 °C). The analysis of these reactions by mass
spectrometry proved ambiguous, and, in particular, it was difficult to assess the site(s) of oxidative
cleavage. We speculate that incomplete release in this case may reflect either fluorophore oxidation at
1
some position away from desired site(s) or O
2
-mediated oxidation of the uncaged amino coumarin
product.(35) In total, these studies have identified scaffolds, particularly the water-soluble CI--Cou,
that provide a versatile starting point for the development of small molecule photocages capable of
uncaging a range of amine containing payloads.
<
figure 6>
<Table 1>
Conclusion
In conclusion, we report efforts to use cyanine photooxidation as an initiating event for the
release of aniline payloads. Our prior studies had shown that certain secondary amines could be
released following photooxidative cleavage of heptamethine cyanines with useful yields and
reasonable kinetic parameters. These studies illustrate that the release of amine payloads is highly
subject to the electronic properties of the amine. Specifically, placing an aryl amine payload at the
C4’ position does not appear to interfere with cyanine photooxidation, but leads to only inefficient
release (perhaps due to reduced basicity). To develop a more general approach, we have implemented
a photooxidation/-elimination reaction sequence. Embedding a carbamate functional group within
the central carbocyclic ring of symmetric heptamethine cyanines improves the kinetics of the
uncaging process but the release yield was only modest. Mass spectrometry studies suggest this is
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likely due to the selective release from only the minor photooꢏidation product ꢁC2’/C3’ cleaꢀage) and
inefficient release from the major product ꢁC2/C1’ cleaꢀage)ꢌ By contrast merocyanines, and
especially the coumarin containing compound, CI--Cou, undergoes efficient photooxidation and
useful yields of uncaging. It is likely that the improved efficiency of release results from
regioselective photooxidation cleavage reaction(s) to provide carbonyl intermediates that then
undergo -elimination. Additional structural optimization to improve the overall yield of release
would be enabling for future efforts. In total, these studies suggest that the photooxidation/-
elimination framework provides a promising approach through which to develop NIR-activated
photocages.
ACKNOWLEDGEMENTS: We thank Dr. Joseph Barchi, NCI Center for Cancer Research
(NCI-CCR), for NMR assistance and Dr. James Kelley, NCI-CCR, for mass spectrometric analysis. This
work was supported by the Intramural Research Program of the National Institutes of Health (NIH),
NCI-CCR.
SUPPORTING INFORMATION
Additional
supporting
information
may
be
found
online
in
the
Supporting Information section at the end of the article:
Figure S1. Coumarin 151 standard release curve.
Figure S2. Photooxidation and release of Cy7-C4-Cou.
Figure S3. Background Release Curves.
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Figure S4A. Absorbance and normalized emission curves of 2 ꢑꢉ Cy7-C4-Cou (A), Cy7--Cou
B), BI--Cou (C), CI--Cou (D) in ethanol.
(
Figure S4B. Absorbance and normalized emission curves of 2 ꢑꢉ Cy7-C4-Cou (A), Cy7--Cou (B),
BI--Cou (C), CI--Cou (D) in 5%DMSO/PBS (pH 7.
Figure S5. NMR Analysis of 10.
1
13
H and C NMR Spectra
FIGURE CAPTIONS
Figure 1. (A) Key Background and (B) These Studies.
Figure 2. Synthesis of Cy7-C4-Cou and Cy7--Cou.
Figure 3. (A) General scheme for uncaging of Cy7-C4-Cou. (B) Cyanine relative absorbance (780 nm)
(
left) and fluorescence (ex. 372 nm, em. 490 nm, shown as coumarin 151 concentration [M], right)
traces of a solution of Cy7-C4-Cou (2 M, pH 7.4 PBS w/ 5% DMSO) that was irradiated (red boxes,
80 nm ± 20 nm) or not and (black boxes).
7
Figure 4: (A) General scheme for uncaging from Cy7--Cou. (B) Cyanine absorbance (780 nm) (left)
and coumarin fluorescence readings (ex. 372 nm, em. 490 nm, shown as concentration [M], right) of
a solution of Cy7--Cou (2 M, pH 7.4 PBS w/ 5% DMSO) that was irradiated (red boxes, 780 nm ±
20 nm) or not and (black boxes). (C) Mass spectrometry analysis following irradiation of solution
Cy7--Cou (2 M in H O).
2
Figure 5. Synthesis of BI--Cou and CI--Cou.
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Figure 6: Uncaging of BI--Cou (blue) and CI--Cou (red). Absorbance (at the cyanine max) and
fluorescence traces (ex. 372 nm, em. 490 nm, shown as concentration [M] of coumarin 151) for
solutions of BI--Cou (blue) and CI--Cou (red) in pH 7.4 PBS w/ 5% DMSO following irradiation
(solid boxes, 660 nm ± 20 nm for BI--Cou and 690 nm ± 20 nm for CI--Cou) or not (empty boxes).
Table 1. Key parameters.
abs
Cmp.
λ
abs
λ
em
ε
Cyanine
Uncaging
a
a
-1
a
b
b
(nm)
(nm)
(M cm-1)
t
1/2 (min)
yield (%)
5
c
Cy-C4-Cou
779
803
2.38 x 10
5.7
0.6 ± 0.04
d
8.0 ± 0.1
5
c
Cy-β-Cou
BI-β-Cou
781
664
713
805
693
729
2.99 x 10
4.9
16 ± 0.2
22 ± 1.0
33 ± 0.4
5
c
c
1.61 x 10
20.2
3.4
5
CI-β-Cou
1.21 x 10
a
Measured in EtOH.
b
c
As determined in Figures 3, 4, and 6.
Yield after 1 h at room temperature.
d
o
Yield after 24 h at 37 C.
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