Selective molecular imaging of viable cancer cells with pH ... · Barrett3, Mako Kamiya1, Tetsuo Nagano 1, Toshiaki Watanabe 4, Akira Hasegawa 4, Peter L. Choyke3 and Hisataka Kobayashi3*

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Selective molecular imaging of viable cancer cells with pH-activatable fluorescence

probes

Yasuteru Urano1,2*, Daisuke Asanuma1, Yukihiro Hama3, Yoshinori Koyama3, Tristan

Barrett3, Mako Kamiya1, Tetsuo Nagano1, Toshiaki Watanabe4, Akira Hasegawa4, Peter L.

Choyke3 and Hisataka Kobayashi3*

1. Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo,

Bunkyo, Tokyo 113-0033, JAPAN

2. PRESTO, Japan Science and Technology Agency, 3-5 Sanbancho, Chiyoda, Tokyo

102-0075, JAPAN

3. Molecular Imaging Program, Center for Cancer Research, National Cancer Institute,

NIH, Bldg. 10, Room 1B40, MSC 1088, 10 Center Dr., Bethesda, MD 20892-1088,

USA

4. Molecular Diagnostic Technology Group, Advanced Core Technology Department,

Research and Development Division, Olympus Corporation, 2-3 Kuboyama-cho,

Hachioji, Tokyo 192-8512, JAPAN

* To whom correspondence should be addressed. E-mail: [email protected] (Y.U.),

[email protected] (H.K.)

Nature Medicine: doi:10.1038/nm.1854

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Supplementary Information

Supplementary Table 1: Photo-physical Properties of acidic pH-activatable BODIPYs

aMeasured at pH 1.47 for 3a and at pH 2.00 for 3b, 3c and 3d bMeasured at pH 9.00 for 3a, 3b, 3c and 3d cCalculated by using fluorescein (Φfl = 0.85) as reference dDetermined with Henderson-Hasselbach equation using fluorescence quantum yield Supplementary Table 2: Comparison of tumor-to-background(heart) ratio between the “always on”

and the “activatable” DiEtNBODIPY probe

λabs, max λem, max Stokes' shift ε[nm] [nm] [nm] [M-1cm-1]

N -protonateda 520 533 13 72000 0.55N -nonprotonatedb 519 536 17 71000 < 0.01




PhBDP (3e) - 520 534 14 73000 0.56 -

H2NBDP (3a)

DiMeNBDP (3b)

DiEtNBDP (3d) 6.0

4.3

3.8

EtMeNBDP (3c) 5.2

Compound Form pK adΦfl

c

"Always on" "Activatable" BG signal

Mouse set

average signalintensity oftumor

pixel size oftumor

average signalintensity ofheart Heart-BG Tumor-BG Lung DeBDP pixel size lung Heart DeBDP Heart-BG Lung-BG

M2M5 122.38 242 12.41 10.88 120.85 43.93 258 1.46 <0 42.4 1.5373.54 260 13.7 12.43 72.27 54.38 249 1.39 0.12 53.11 1.27

M3M4 139.79 260 36.18 11.23 114.84 77.63 252 11.31 <0 52.68 24.95229.61 257 34.31 2.72 198.02 103.67 258 14.03 <0 72.08 31.59

M1M3 254.25 259 67.27 51.54 238.52 84.24 248 18.42 2.69 68.51 15.73178.85 255 55.32 40.48 164.01 59.58 251 14.73 <0 44.74 14.84

M2M3 190.38 254 39.16 13.29 164.51 95.88 260 20.65 <0 70.01 25.87128.81 260 40.58 20.81 109.04 121.19 256 14.35 <0 101.42 19.77

M2M3 255.59 255 54.28 35.16 236.47 145.04 258 20.31 1.19 125.92 19.12341.91 250 44.4 26.39 323.9 61.64 246 13.97 <0 43.63 18.01

M2M3 181.43 255 40.24 18.07 159.26 127.66 255 21.15 <0 105.49 22.17172.08 254 37.23 17.14 151.99 139.72 259 21.54 1.45 119.63 20.09

M1M2 253.16 246 30.17 12.21 235.2 103.54 257 9.82 <0 85.58 17.96173.7 256 26.12 8.74 156.32 83.83 241 13.38 <0 66.45 17.38

Average 254.5 20.1 174.7 253.4 0.4 75.1SD 5.3 13.7 66.2 5.7 1.1 28.3

AverageTumor/Heartintensity 8.698993205

AverageTumor/Heartintensity 192.9633028

"Activatable"/"Always on" 22.18225698


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Supplementary Table 3: Summary of the sensitivity/specificity results for both “always on” and

the “activatable” DiEtNBODIPY probes.

Sensitivity and Specificity 2-color tumor model (HER2+ or RFP+/HER2-) injected always ON vs activatable

11 sets 22 mice 44 lungs 941 tumors (0.5-2mm )

Green Yellow (Both R&G) G+Y No color Red N+R G+N Y+R ALL always ON-BODIPY activatable (DiEt)-BODIPY

153 48 201 140 3 1430 267 267 0 329 329

153 315 468 140 332 472 Sensitivity 100.0% Sensitivity 100.0% Specificity 84.8% Specificity 99.1% Accuracy 89.7% Accuracy 99.4%


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Supplementary Movie 1: Reversible and real-time activation of DiEtNBODIPY-conjugated

trastuzumab by altering the pH.

At first, each test tube contained the same neutral pH (7.4) solution of the probe, and both were excited

with UV lamp. To the right, diluted HCl solution was added to show bright green fluorescence. Further,

the addition of diluted NaOH solution to neutralize the pH caused reversible quenching of the

fluorescence.

Supplementary Movies 2-4: The real-time fluorescence endoscopy of peritoneal disseminated

ovarian cancer with activatable DiEtNBODIPY-tagged galactosyl serum albumin (GSA) probe in

live mice.

Tiny disseminated implants are clearly visualized in a live mouse. White light images are on the left side

and fluorescence images on the right side. Excitation and emission wavelength were 450-480 nm and

516-556 nm, respectively.

Supplementary Movie 5: Reversible and real-time activation of the pH-activatable rhodamine

probe by altering the pH.

At first, each test tube contained the same neutral pH (7.4) solution of the probe, and both were excited

with UV lamp. To the right, diluted HCl solution was added to show bright orange-pink fluorescence.

Further, the addition of diluted NaOH solution to neutralize the pH caused reversible quenching of the

fluorescence.


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Supplementary figure 1: Activatable GSA probe can internalize into SHIN3 cells quicker than the

activatable Herceptin probe.

Serial fluorescence microscopy images of SHIN3 ovarian cancer cells. Fluorescent microscopy as well

as differential interference contrast (DIC) imaging was performed after 30 minutes, 1 and 3 hours

incubation with 200 nmol/L GSA-BDP (8PhBODIPY-tagged GSA; “always-on”) or GSA-detBDP

(DiEtNBODIPY-tagged GSA; “activatable”). The fluorescent dots produced by GSA-detBDP were

initially very small and minimally fluorescent (30 min), but became marked and bright at 1h or later

after incubation. Unlike the temporal changes observed in GSA-detBDP, GSA-BDP showed a minimal

change in the size and the fluorescence intensity of intracellular dots.


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Supplementary figure 2: Activatable GSA probe can clearly visualize tiny SHIN3 implants without

fluid background within 3 hours after injection.

In vivo targeted spectral imaging of living and dying cancer cells. Spectral fluorescence imaging of the

peritoneal cavities as well as the closeup peritoneal membranes of tumor-bearing mice was performed 3

hours after intraperitoneal injection of 700 pmol GSA-BDP (8PhBODIPY-tagged GSA; “always-on”)

and GSA-detBDP (DiEtNBODIPY-tagged GSA; “activatable”). (A) Spectral unmixed autofluorescence

(upper), GSA-BDP/GSA-detBDP fluorescence (middle) and composite (lower) images. Aggregated

tumor foci (yellow arrows) as well as the small cancer implants (yellow arrowheads) were clearly

detected by GSA-detBDP. However, GSA-BDP failed to depict the cancer foci (blue arrow) because of

the signal intensities from the background (blue arrowheads).


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Supplementary figure 3: Activatable GSA probe can clearly visualize tiny SHIN3 implants without

fluid background on the peritoneal membrane.

White light and spectral composite images of closeup peritoneal membranes instilled with GSA-BDP

(8PhBODIPY-tagged GSA; “always-on”) or GSA-detBDP (DiEtNBODIPY-tagged GSA; “activatable”).

White light images were not conclusive in either fluorophore, but the composite image of GSA-detBDP

clearly visualized submillimeter cancer foci with minimal background signals. GSA-BDP also

accumulated into the cancer foci, but due to the high background signals, clear delineation of cancer foci

was difficult.


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Supplementary figure 4: Activatable GSA probe can achieve real-time monitoring damages of the

cancer cell .

Side-by-side dynamic comparison study of tumor-implanted peritoneal membranes after sacrifice.

Immediately (0 min) after sacrifice, small cancer foci (arrowheads) were clearly visualized by both

GSA-BDP and GSA-detBDP (upper left). A region of interest (ROI) was drawn inside the bowel (lower

left), and the serial fluorescence intensity of each ROI was measured every 5 minutes (lower right).

GSA-BDP showed minimal fluorescence intensity changes even 60 minutes after sacrifice, but

GSA-detBDP showed marked decrease in signal intensity at 60 minutes after sacrifice. The signal

intensity of GSA-detBDP, which was 1.20 in arbitrary unit (a.u.) immediately after sacrifice, consistently

decreased to 0.37 (a.u.) at 60 minutes after sacrifice (70% reduction). Thus, the small cancer foci

(arrowheads), which were clearly visualized immediately after sacrifice (upper left), became almost

undetectable 60 minutes after sacrifice (upper right).


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Synthesis

Methyl 2,4-dimethyl-3-pyrrolepropionate (1)

Methyl 5-(benzyloxycarbonyl)-2,4-dimethyl-3-pyrrolepropionate (1.55 g, 4.91 mmol) was dissolved in

150 mL of acetone containing 10% palladium-carbon. The resulting solution was stirred under H2 at

ambient temperature for 12 hr. The reaction solution was then filtered and evaporated. The residue was

immediately dissolved in 10 mL of TFA and stirred under an Ar atmosphere at ambient temperature for

10 min. 30 mL of dichloromethane was then added and the resulting solution was washed with H2O and

1 M NaHCO3 aq., dried over anhydrous sodium sulfate, filtered, and evaporated, affording 1 (0.835 g,

94%) as a slightly brown oil. 1H NMR (300 MHz, CDCl3) δ 2.02 (s, 3H, NHCHCCH3), 2.16 (s, 3H,

NHCCH3), 2.42-2.48 (m, 2H, COCH2), 2.69-2.74 (m, 2H, COCH2CH2), 3.66 (s, 3H, OCH3), 6.36 (s, 1H,

NHCH), 7.64 (br s, 1H, NH). 13C NMR (75 MHz, CDCl3) δ 10.22, 11.09, 19.85, 35.26, 51.37, 113.0,

116.5, 117.7, 124.1, 173.9. MS (ESI+) m/z 182 [M+H]+.

1,3,5,7-Tetramethyl-2,6-bis-(2-methoxycarbonylethyl)-8-(4-aminophenyl)-4,4-difluoro-4-bora-3a,4

a- diaza-s-indacene (2a)

1 (0.542 g, 2.99 mmol) and 4-aminobenzaldehyde (0.153 g, 1.49 mmol) were dissolved in 300 mL of


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dichloromethane containing a catalytic amount of TFA. The resulting mixture was stirred overnight at

ambient temperature under an Ar atmosphere. Tetrachloro-1,4-benzoquinone (p-chloranil) (0.361 g, 1.47

mmol) was added, and stirring was continued for 10 min. The reaction mixture was washed with H2O,

dried over anhydrous sodium sulfate, filtered, and evaporated. Repeated column chromatography over

aluminum oxide using dichloromethane/methanol (9:1) containing 1% triethylamine as the eluent

yielded a red-green compound. The compound thus obtained was dissolved in 100 mL of toluene

containing N,N-diisopropylethylamine (DIEA) (3 mL), and the resulting solution was stirred at ambient

temperature. BF3•OEt2 (3 mL) was then slowly added, and stirring was continued for 10 min. The

reaction mixture was washed with H2O, dried over anhydrous sodium sulfate, filtered, and evaporated.

The crude compound was purified by column chromatography over silica gel using

dichloromethane/methanol (95:5) as the eluent, affording 2a (40.1 mg, 5.2%) as a red compound. 1H

NMR (300 MHz, CDCl3) δ 1.41 (s, 6H, NCCCH3), 2.33-2.28 (m, 4H, COCH2), 2.53 (s, 6H, NCCH3),

2.61-2.67 (m, 4H, COCH2CH2), 3.65 (s, 6H, OCH3), 3.93 (br s, 2H, NH2), 6.76-6.78 (m, 2H,

NH2CCHCH), 6.96-6.99 (m, 2H, NH2CCH). 13C NMR (75 MHz, CDCl3) δ 11.99, 12.42, 19.23, 34.15,

51.54, 115.3, 124.7, 128.7, 128.9, 131.4, 139.5, 141.8, 147.1, 153.4, 173.0. HRMS (ESI+) Calcd for

[M+Na]+ m/z 534.23516, Found 534.23846 (Δ 3.30 mmu).


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1,3,5,7-Tetramethyl-2,6-bis-(2-carboxyethyl)-8-(4-aminophenyl)-4,4-difluoro-4-bora-3a,4a-diaza-

s-indacene (3a)

2a (40.1 mg, 78.4 μmol) was dissolved in 1 mL of dichloromethane. To the resulting solution were

added 20 mL of methanol and 5 mL of 1N NaOH aq., successively. The reaction solution was stirred

overnight at ambient temperature. 30 mL of H2O was added, and the reaction solution was washed with

dichloromethane three times. The aqueous phase was then acidified with 1 N HCl aq. (~1 mL) until the

solution emitted green fluorescence on UV excitation at 365 nm, followed by extraction with

dichloromethane five times. The dichloromethane extract was dried over anhydrous sodium sulfate,

filtered, and evaporated. The crude compound was then purified twice by semi-preparative HPLC under

the following conditions: A/B = 50/50 (0 min) to 0/100 (20 min), then A/B = 70/30 (0 min) to 0/100 (30

min) (solvent A: H2O, 0.1% TFA; solvent B: acetonitrile/H2O = 80/20, 0.1% TFA). The aqueous

fractions containing the desired product were extracted with dichloromethane three times. The

dichloromethane extract was dried over anhydrous sodium sulfate, filtered, and evaporated, affording 3a

(32.0 mg, 84%) as an orange powder. 1H NMR (300 MHz, CD3OD) δ 1.37 (s, 6H, NCCCH3), 2.24 (t, 4H,

J = 7.4, 8.0 Hz, COCH2), 2.38 (s, 6H, NCCH3), 2.54 (t, 4H, J = 7.4, 8.0 Hz, COCH2CH2), 6.73-6.75 (m,

2H, NCCHCH), 6.83-6.86 (m, 2H, NCCH). 13C NMR (75 MHz, CD3OD) δ 12.46, 12.65, 20.34, 35.28,

116.6, 125.2, 130.1, 130.4, 132.7, 140.8, 144.0, 150.2, 154.6, 176.5. HRMS (ESI+) Calcd for [M+Na]+


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m/z 506.20386, Found 506.20729 (Δ 3.43 mmu).

1,3,5,7-Tetramethyl-2,6-bis-(2-methoxycarbonylethyl)-8-[4-(N,N-dimethylamino)phenyl]-4,4-difluo

ro- 4-bora-3a,4a-diaza-s-indacene (2b)

1 (0.574 g, 3.17 mmol) and 4-(N,N-dimethylamino)benzaldehyde (0.236 g, 1.58 mmol) were dissolved

in 300 mL of dichloromethane containing a catalytic amount of TFA. The resulting mixture was stirred

for a day at ambient temperature under an Ar atmosphere. p-Chloranil (0.384 g, 1.56 mmol) was added,

and stirring was continued for 10 min. The reaction mixture was washed with H2O, dried over anhydrous

sodium sulfate, filtered, and evaporated. Repeated column chromatography over aluminum oxide using

dichloromethane/methanol (9:1) containing 1% triethylamine as the eluent yielded a green compound.

The compound thus obtained was dissolved in 100 mL of toluene containing DIEA (5 mL), and the

resulting solution was stirred at ambient temperature. BF3•OEt2 (5 mL) was then slowly added, and

stirring was continued for 10 min. The reaction mixture was washed with H2O, dried over anhydrous

sodium sulfate, filtered, and evaporated. The crude compound was purified by repeated column

chromatography over silica gel using dichloromethane/methanol (97:3, 99:1, then 100:0) as the eluent,

affording 2b (228 mg, 27%) as a brown compound. 1H NMR (300 MHz, CDCl3) δ 1.40 (s, 6H,

NCCCH3), 2.33-2.38 (m, 4H, COCH2), 2.53 (s, 6H, NCCH3), 2.61-2.67 (m, 4H, COCH2CH2), 3.02 (s,


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6H, NCH3), 3.65 (s, 6H, OCH3), 6.75-6.80 (m, 2H, NCCHCH), 7.01-7.06 (m, 2H, NCCH). 13C NMR

(75 MHz, CDCl3) δ 12.11, 12.51, 19.35, 34.26, 40.32, 51.62, 112.4, 122.5, 128.7, 128.8, 131.6, 139.6,

142.4, 150.7, 153.3, 173.1. HRMS (ESI+) Calcd for [M+Na]+ m/z 562.26646, Found 562.26315 (Δ -3.32

mmu).

1,3,5,7-Tetramethyl-2,6-bis-(2-carboxyethyl)-8-[4-(N,N-dimethylamino)phenyl]-4,4-difluoro-4-

bora-3a,4a-diaza-s-indacene (3b)

2b (50.8 mg, 94.2 μmol) was dissolved in 1 mL of dichloromethane. To the resulting solution were

added 20 mL of methanol and 5 mL of 1N NaOH aq., successively. The reaction solution was stirred for

2 h at ambient temperature. 30 mL of H2O was added, and the reaction solution was washed with



dichloromethane three times. The dichloromethane extract was dried over anhydrous sodium sulfate,

filtered, and evaporated. The crude compound was then purified by semi-preparative HPLC under the

following conditions: A/B = 60/40 (0 min) to 0/100 (30 min) (solvent A: H2O, 0.1% TFA; solvent B:

acetonitrile/H2O = 80/20, 0.1% TFA). The aqueous fractions containing the desired product were

extracted with dichloromethane three times. The dichloromethane extract was dried over anhydrous


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sodium sulfate, filtered, and evaporated, affording 3b (40.3 mg, 84%) as a reddish powder. 1H NMR

(300 MHz, CD3OD + DMF-d7) δ 1.34 (s, 6H, NCCCH3), 2.21-2.26 (m, 4H, COCH2), 2.39 (s, 6H,

NCCH3), 2.51-2.56 (m, 4H, COCH2CH2), 2.91 (s, 6H, NCH3), 6.77-6.80 (m, 2H, NCCHCH), 6.94-6.97

(m, 2H, NCCH); 13C NMR (75 MHz, CD3OD + DMF-d7) δ 12.56, 12.84, 20.34, 35.27, 40.54, 113.6,

123.5, 130.1, 130.6, 132.7, 140.8, 143.9, 152.5, 154.6, 175.9; HRMS (ESI-) Calcd for [M-H]- m/z

510.23757, Found 510.23776 (Δ 0.19 mmu).

1,3,5,7-Tetramethyl-2-(2-carboxyethyl)-6-(2-succinimidyloxycarbonylethyl)-8-[4-(N,N-dimethylami

no)- phenyl]-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (4b)

3b (11.1 mg, 21.7 μmol) was dissolved in 2 mL of N,N-dimethylformamide (DMF) and the resulting

solution was cooled down to 0 °C. To the reaction solution were added 100 mM N-hydroxysuccinimide

(NHS) in DMF and 100 mM water-soluble carbodiimide (WSCD) in DMF (each 32.6 μmol). The

reaction mixture was stirred at 0 °C, then allowed to warm gradually to ambient temperature. After 24 h,

the reaction mixture was concentrated in vacuo. The crude compound was then purified by

semi-preparative HPLC under the following conditions: A/B = 50/50 (0 min) to 0/100 (20 min) (solvent

A: H2O, 0.1% TFA; solvent B: acetonitrile/H2O = 80/20, 0.1% TFA). The aqueous fractions containing

the desired product were extracted with dichloromethane three times. The dichloromethane extract was


15

dried over anhydrous sodium sulfate, filtered, and evaporated, affording 4b (2.6 mg, 20%) as a red

powder. Recovery 41%. HRMS (ESI+) Calcd for [M+Na]+ m/z 631.25154, Found 631.25518 (Δ 3.64

mmu).

1,3,5,7-Tetramethyl-2,6-bis-(2-methoxycaronylethyl)-8-[4-(N,N-diethylamino)phenyl]-4,4-difluoro-

4- bora-3a,4a-diaza-s-indacene (2c)

1 (0.542 g, 2.99 mmol) and 4-(N,N-diethylamino)benzaldehyde (0.265 g, 1.49 mmol) were dissolved in

300 mL of dichloromethane containing a catalytic amount of TFA. The resulting mixture was stirred

overnight at ambient temperature under an Ar atmosphere. p-Chloranil (0.370 g, 1.51 mmol) was added,

and stirring was continued for 10 min. The reaction mixture was washed with H2O, dried over anhydrous

sodium sulfate, filtered, and evaporated. Repeated column chromatography over aluminum oxide using

dichloromethane/methanol (95:5, 98:2, and 100:0) containing 1% triethylamine as the eluent yielded a

greenish amorphous compound. The compound thus obtained was dissolved in 100 mL of toluene

containing DIEA (5 mL), and the resulting solution was stirred at ambient temperature. BF3•OEt2 (5 mL)

was then slowly added, and stirring was continued for 10 min. The reaction mixture was washed with

H2O, dried over anhydrous sodium sulfate, filtered, and evaporated. The crude compound was purified

by repeated column chromatography over silica gel by using dichloromethane/methanol (95:5, 98:2, and


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100:0) as the eluent, affording 2c (136 mg, 16%) as an orange powder. 1H NMR (300 MHz, CDCl3) δ

1.22 (t, 6H, J = 7.0 Hz, NCH2CH3), 1.44 (s, 6H, NCCCH3), 2.36 (t, 4H, J = 7.3, 8.4 Hz, COCH2), 2.53 (s,

6H, NCCH3), 2.65 (t, 4H, J = 7.3, 8.4 Hz, COCH2CH2), 3.41 (q, 4H, J = 7.0 Hz, NCH2), 3.65 (s, 6H,

OCH3), 6.74 (d, 2H, J = 8.6 Hz, NCCHCH), 6.99 (d, 2H, J = 8.6 Hz, NCCH); 13C NMR (75 MHz,

CDCl3) δ 12.11, 12.30, 12.45, 19.34, 34.25, 44.31, 51.56, 112.0, 121.6, 128.6, 129.0, 131.7, 139.6, 142.6,

148.2, 153.1, 173.1; HRMS (ESI+) Calcd for [M+H]+ m/z 568.31582, Found 568.31626 (Δ 0.44 mmu).

1,3,5,7-Tetramethyl-2,6-bis-(2-carboxyethyl)-8-[4-(N,N-diethylamino)phenyl]-4,4-difluoro-4-bora-

3a,4a-diaza-s-indacene (3c)

2c (136 mg, 239 μmol) was dissolved in 3 mL of dichloromethane. To the resulting solution were added

20 mL of methanol and 5 mL of 1 N NaOH aq., successively. The reaction solution was stirred for 4 hr at

ambient temperature. 30 mL of H2O was added, and the reaction solution was washed with



dichloromethane three times. The dichloromethane extract was dried over anhydrous sodium sulfate,

filtered, and evaporated. The crude compound was then purified by PLC using dichloromethane/acetone

(1:1) as the eluent, affording 3c (118 mg, 91%) as an orange powder. 1H NMR (300 MHz, CD3OD) δ


17

1.10 (t, 6H, J = 7.0 Hz, NCH2CH3), 1.39 (t, 6H, NCCCH3), 2.25 (t, 4H, J = 7.5, 7.9 Hz, COCH2), 2.39 (s,

6H, NCCH3), 2.56 (t, 4H, J = 7.5, 7.9 Hz, COCH2CH2), 3.33 (q, 4H, J = 7.0 Hz, NCH2), 6.75 (d, 2H, J =

8.8 Hz, NCCHCH), 6.93 (d, 2H, J = 8.8 Hz, NCCH); 13C NMR (75 MHz, CD3OD) δ 12.47, 12.66, 20.38,

35.31, 45.40, 113.3, 122.8, 130.3 (representing two different carbons), 132.8, 140.8, 144.2, 149.7, 154.4,

176.5; HRMS (ESI-) Calcd for [M-H]- m/z 538.26887, Found 538.26446 (Δ -4.40 mmu).

1,3,5,7-Tetramethyl-2-(2-carboxyethyl)-6-(succinimidyloxycarbonylethyl)-8-[4-(N,N-diethylamino)

- phenyl]-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (4c)

3c (25.7 mg, 47.6 μmol) was dissolved in 2 mL of N,N-dimethylformamide (DMF) and the resulting

solution was cooled to 0 °C. To the reaction solution were added 100 mM NHS in DMF and 100 mM

WSCD in DMF (each 47.6 μmol). The reaction mixture was stirred at 0 °C, then allowed to warm

gradually to ambient temperature. After 14 h, the reaction mixture was concentrated in vacuo. The crude

compound was then purified by PLC using dichloromethane/acetone (1:1) as the eluent, affording 4c

(13.4 mg, 44%) as a red powder. HRMS (ESI+) Calcd for [M+H]+ m/z 637.30090, Found 637.30278 (Δ

1.89 mmu).

1,3,5,7-Tetramethyl-2,6-bis-(2-methoxycarbonylethyl)-8-phenyl-4,4-difluoro-4-bora-3a,4a-diaza-s-


18

indacene (2d)

1 (0.634 g, 3.50 mmol) and benzaldehyde (0.185 g, 1.74 mmol) were dissolved in 300 mL of

dichloromethane containing a catalytic amount of TFA. The resulting mixture was stirred overnight at

ambient temperature under an Ar atmosphere. p-Chloranil (0.428 g, 1.74 mmol) was added, and stirring

was continued for 10 min. The reaction mixture was washed with H2O, dried over anhydrous sodium

sulfate, filtered, and evaporated. Repeated column chromatography over aluminum oxide using

dichloromethane containing 1% triethylamine as the eluent yielded a brown oil. The compound thus

obtained was dissolved in 100 mL of toluene containing DIEA (5 mL), and the resulting solution was

stirred at ambient temperature. BF3•OEt2 (5 mL) was then slowly added, and stirring was continued for

10 min. The reaction mixture was washed with H2O, dried over anhydrous sodium sulfate, filtered, and

evaporated. The crude compound was purified by column chromatography over silica gel using

dichloromethane as the eluent, affording 2d (273 mg, 32%) as a green compound. 1H NMR (300 MHz,

CDCl3) δ 1.29 (s, 6H, NCCCH3), 2.32-2.38 (m, 4H, COCH2), 2.54 (s, 6H, NCCH3), 2.61-2.66 (m, 4H,

COCH2CH2), 3.65 (s, 6H, OCH3), 7.25-7.28 (m, 2H, benzene), 7.46-7.49 (m, 3H, benzene). 13C NMR

(75 MHz, CDCl3) δ 11.77, 12.59, 19.29, 34.18, 51.63, 128.0, 128.9, 129.1, 130.9, 135.4, 139.4, 140.9,

154.0, 173.0. HRMS (ESI+) Calcd for [M+Na]+ m/z 519.22426, Found 519.22433 (Δ 0.07 mmu).


19

1,3,5,7-Tetramethyl-2,6-bis-(2-carboxyethyl)-8-phenyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene

(3d)

2d (40.1 mg, 78.4 μmol) was dissolved in 1 mL of dichloromethane. To the resulting solution were

added 20 mL of methanol and 5 mL of 1 N NaOH aq, successively. The reaction solution was stirred

overnight at ambient temperature. 30 mL of H2O was added, and the reaction solution was washed with

dichloromethane three times. The aqueous phase was then acidified with 1 N HCl aq (~1 mL) until the


dichloromethane five times. The dichloromethane extract was dried over anhydrous sodium sulfate,

filtered, and evaporated. The crude compound was then purified twice by semi-preparative HPLC under

the following conditions: A/B = 50/50 (0 min) to 0/100 (20 min), then A/B = 70/30 (0 min) to 0/100 (30

min) (solvent A: H2O, 0.1% TFA; solvent B: acetonitrile/H2O = 80/20, 0.1% TFA). The aqueous

fractions containing the desired product were extracted with dichloromethane three times. The

dichloromethane extract was dried over anhydrous sodium sulfate, filtered, and evaporated, affording 3d

(32.0 mg, 84%) as an orange powder. 1H NMR (300 MHz, CD3OD) δ 1.19 (s, 6H, NCCCH3), 2.23 (t, 4H,

J = 8.1 Hz, COCH2), 2.40 (s, 6H, NCCH3), 2.55 (t, 4H, J = 8.1 Hz, COCH2CH2), 7.21-7.46 (m, 5H,

benzene). 13C NMR (75 MHz, CD3OD/NaOD) δ 12.19 (representing two different carbons), 22.01,

39.34, 129.5, 130.2, 130.4, 132.0, 132.2, 136.9, 140.4, 142.1, 155.2, 181.8. HRMS (ESI+) Calcd for


20

[M+Na]+ m/z 491.19296, Found, 491.18910 (Δ -3.87 mmu)

1,3,5,7-Tetramethyl-2-(2-carboxyethyl)-6-(succinimidyloxycarbonylethyl)-8-phenyl-4,4-difluoro-4-

bora- 3a,4a-diaza-s-indacene (4d)

3d (12.4 mg, 26.5 μmol) was dissolved in 1 mL of N,N-dimethylformamide (DMF) and the resulting

solution was cooled to 0 °C. To the reaction solution were added 100 mM NHS in DMF and 100 mM

WSCD in DMF (each 39.7 μmol). The reaction mixture was stirred at 0 °C, then allowed to warm

gradually to ambient temperature. After 24 h, the reaction mixture was concentrated in vacuo. The crude

compound was then purified by semi-preparative HPLC under the following conditions: A/B = 50/50 (0

min) to 0/100 (20 min) (solvent A: H2O, 0.1% TFA; solvent B: acetonitrile/H2O = 80/20, 0.1% TFA).

The aqueous fractions containing the desired product were extracted with dichloromethane three times.

The dichloromethane extract was dried over anhydrous sodium sulfate, filtered, and evaporated,

affording 4d (4.0 mg, 27%) as a red powder. Recovery 24%. HRMS (ESI-) Calcd for [M-H]- m/z

564.21175, Found 564.21392 (Δ 2.18 mmu)


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