Design, synthesis, and reaction of π-extended coumarin-based new caged compounds with two-photon absorption character in the near-IR region

(1)

HAL Id: hal-01398035

https://hal-univ-rennes1.archives-ouvertes.fr/hal-01398035

Submitted on 24 Nov 2016

HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Design, synthesis, and reaction of π-extended

coumarin-based new caged compounds with two-photon absorption character in the near-IR region

Y. Chitose, Manabu Abe, K. Furukawa, Claudine Katan

To cite this version:

Y. Chitose, Manabu Abe, K. Furukawa, Claudine Katan. Design, synthesis, and reaction of π-extended coumarin-based new caged compounds with two-photon absorption character in the near-IR region.

Chemistry Letters, Chemical Society of Japan, 2016, 45 (10), pp.1186–1188. �10.1246/cl.160586�.

�hal-01398035�

(2)

Des i gn, Synthes i s, and React i on o f π -Extended Coumar i n-based New Caged Compounds w i th Two-photon Absorpt i on Character i n the Near-IR Reg i on

Youhei Chitose,

¹

Manabu Abe,*

¹

Ko Furukawa,

²

and Claudine Katan*

³

1

Department of Chemistry, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526

2

Centre f or Instrumenta l Ana l ys i s, N ii gata Un i vers i ty, 8050 Ikarash i 2-no-cho, N i sh i -ku, N ii gata 950-2181

3

Institut des Sciences Chimiques de Rennes, CNRS, Universite Rennes 1, 35042 Rennes, France

(E-ma il : mabe @ h i rosh i ma-u.ac.jp, c l aud i ne.katan @ un i v-rennes1. f r)

Nove l π -extended coumar i n-based chromophores were de- s i gned w i th two-photon absorpt i on (TPA) character i n the near-IR region. Caged benzoates with a TP-responsive chromophore were synthes i zed, and the i r TP-uncag i ng react i ons were conducted under near-IR light. The 6,7-dimethoxy-substituted derivative had a h i gh TPA cross-sect i on o f 69 GM at 740 nm. The 7-methoxy- substituted derivative showed a high TPA uncaging eﬃciency with a TPA e ﬃ c i ency o f 3.4 GM at 710 nm.

Keywords: Caged compounds | Two-photon absorption | Donor–π–Acceptor

Caged compounds,

¹

the i nact i ve f orms o f b i oact i ve mo l ecu l es formed using a photolabile protecting group (PPG),

²

are now used to e l uc i date the mechan i sm o f b i oact i v i ty o f substances i n var i ous life processes and phenomena. For instance, caged compounds contr i bute to the deve l opment o f new med i c i nes. The PPG i s removed photochem i ca ll y under neutra l cond i t i ons w i thout chem i - cal reagents, thus rapidly releasing the bioactive compound.

Th i s e ﬃ c i ent uncag i ng i s b i o l og i ca ll y use f u l because the t i m i ng, location, and amplitude of photoirradiation can be controlled.

Thus, the photo i nduced re l ease o f b i oact i ve mo l ecu l es has been utilized in various ﬁelds such as neuroscience, cell patterning, or b i omed i ca l sc i ence.

³⁵

Coumarin derivatives have been used to study various functions of biomolecules and for cellular imaging.

⁶

The advan- tages o f coumar i n as a chromophore i nc l ude i ts i nherent f ast response to photolysis, thermal stability, high molar extinction coe ﬃ c i ent, and good ﬂ uorescent property f or mon i tor i ng i ts reactions in vivo.

⁷

However, the recent concern for physiological app li cat i on o f coumar i n-based compounds i s ma i n l y i ts short absorption wavelength. Coumarin itself possesses an absorption wave l ength max i mum o f 274 nm i n methano l .

⁸

Due to th i s short wave l ength, ce ll damages cannot be avo i ded us i ng one-photon absorption (OPA).

⁹

But better penetration to the deeper part of ce ll s

¹⁰

can be a ﬀ orded us i ng chromophores hav i ng s i gn iﬁ cant two- photon absorption (TPA) character in the near-IR region.

^11,12

Chromophores w i th TPA character have attracted much attention because of their application in physiology. Caged compounds w i th TPA character enab l e greater spat i a l contro l o f the re l ease o f b i oact i ve substances.

¹³

For pract i ca l app li cat i ons, a h i gher uncag i ng e ffi c i ency i s necessary to conduct b i o l og i ca l stud i es. The uncag i ng e ffi c i ency f or OP exc i tat i on i s de fi ned as the uncaging quantum yield (Φ

u

) multiplied by the excitation prob- ab ili ty ( ¾ : mo l ar ext i nct i on coe ﬃ c i ent). S i m il ar l y, the e ﬃ c i ency ¤

u

of a TP excitation process is expressed using the TPA cross-section ( ·

2

): ¤

u

= Φ

u

·

2

. The m i n i mum thresho l d va l ue o f TP e ﬃ c i ency for in vivo studies was reported as 3 GM.

¹⁴

In 1999, Furuta et al.

synthesized brominated 7-hydroxycoumarin-4-ylmethyl (Bhc) esters and carbamates w i th TPA character i n the near-IR reg i on, i .e. 650 1350 nm. Its TP e ﬃ c i ency amounts to ca. 1 GM at 740 nm.

¹⁵

In 2012, Zhu et al. reported ¤

u

= 0.25 GM at 800 nm.

¹⁶

In 2013, E lli s-Dav i es et a l . deve l oped a new π -extended caged coumarin molecule that had a TPA maximum at 900 nm.

¹⁷

In th i s study, a coumar i n conta i n i ng a st il bene ske l eton (12 GM at 514 nm)

¹⁸

with a p-nitrophenyl group as the electron- w i thdraw i ng group was des i gned to i ncrease the TPA cross- sections due to its dipolar character (Figure 1).

¹⁹

The cyclic structure should prevent cistrans isomerization in the excited state. A bathochrom i ca ll y sh if ted absorpt i on wave l ength i s a l so expected because of the donorπacceptor conjugated system.

F i rst, the OPA and TPA spectra o f coumar i n chromophores 1a1c were computed at the TD-B3LYP/6-31G//B3LYP/6- 31G l eve l o f theory (F i gure 2; see a l so ESI f or more deta il s).

The predicted absorption maximum of 6,7-dimethoxy-substituted coumar i n 1c i s more red-sh if ted compared to those o f the 7- methoxy-subst i tuted coumar i n 1b and coumar i n der i vat i ve 1a. At this level of theory, a TPA cross-section of ca. 50 GM is computed f or 1a. S i zab l e i ncrease i s observed upon subst i tut i on w i th TPA cross-sections of 170 and 179 GM for 1b and 1c, respectively, in the 740 780-nm spectra l reg i on. The max i ma correspond n i ce l y to twice the OPA maxima (370390 nm). These predictions suggest that TP e l ectron i c exc i tat i on i n the near-IR reg i on i s poss i b l e f or coumar i n der i vat i ves. Thus, the photochem i ca l react i ons o f caged benzoates 2b and 2c w i th π -extended coumar i n der i vat i ves

²⁰

were i nvest i gated exper i menta ll y.

Caged benzoates 2b and 2c were synthesized using a base- cata l yzed Perk i n condensat i on react i on

²¹

(Scheme 1). The UV visible absorption spectrum of 7-methoxy-substituted compound 2b showed the absorpt i on max i mum at 345 nm w i th ¾ = 22726 M

^¹1

cm

^¹1

in DMSO, whereas 6,7-dimethoxy-substituted

O O

HO

Br

O O

Et2N

O O

R¹

R² R³

NO₂

O NHR

OR RO

by Furutaet al. by Ellis-Davies et al.

a: R¹= R² = H b: R¹= OMe, R² = H c: R¹= R²= OMe

1: R³= H; 2: R³= O Ph O

Figure 1. Coumar i n der i vat i ves w i th TPA character.

CL-160586 Received: June 14, 2016 | Accepted: July 1, 2016 | Web Released: July 15, 2016

(3)

compound 2c showed an absorption peak at 369 nm with ¾ = 16902 M

^¹1

cm

^¹1

. As pred i cted by TD-DFT ca l cu l at i ons (F i gure 2), the absorption maximum of 2c exhibited bathochromic and

hypochromic shifts as compared to 2b. The OP photolysis of 2b and 2c was i nvest i gated us i ng a Xe l amp at 360 « 10 nm i n DMSO- d

₆

(Figures 3 and S1). The quantitative uncaging of benzoic acid f rom 2b and 2c was con ﬁ rmed by compar i ng the

¹

H NMR s i gna l s of benzoic acid with those of the photolysate (Figures 3 and S1).

The uncaging quantum yields Φ

u

were determined in DMSO using the photochem i ca l act i nometer f err i oxa l ate coup l ed w i th HPLC analyses. Approximately three-times higher quantum yield for the uncag i ng react i on o f 2b ( Φ

u

= 0.09) than 2c ( Φ

u

= 0.03) was obtained. The OP uncaging eﬃciencies (¾

360

© Φ

u

) of 2b (¾

360

= 18718) and 2c ( ¾

360

= 16102) were 1684 and 483, respect i ve l y.

The lower uncaging eﬃciency of 2c may be attributed to the i ncreased e l ectron i c stab ili ty o f the exc i ted state o f 2b.

²²

The TP-uncag i ng react i ons o f 2b and 2c were a l so i nvest i gated in DMSO using 700-nm, 710-nm, 720-nm, 730-nm, 740-nm, 750- nm, and 760-nm li ghts obta i ned f rom a T i :sapph i re l aser (pu l se width 100 fs, 80 MHz) at an average power of 700 mW. To deter- m i ne the TPA cross-sect i on and e ﬃ c i ency o f 2b and 2c, the rate constants of the TP-uncaging reactions were compared with that of a caged benzoate NPBF-BA (F i gure 4c).

^23,24

The consumpt i on o f 2b and 2c upon TP photolysis was monitored by HPLC (Figure 4).

As shown in Figure 4, the TP-uncaging reaction rates depend on the exc i tat i on wave l ength. The photo l ys i s o f 2b proceeded the fastest at 710 nm (k

710

= 6.5 © 10

^¹6

s

^¹1

), whereas the fastest photo l ys i s rate f or 2c was observed at 740 nm (k

₇₄₀

= 3.9 © 10

^¹6

s

^¹1

). The values of TPA cross-sections at each wavelength were extrapo l ated f rom the rate constant o f TP uncag i ng o f NPBF- BA, 18 GM at 720 nm.

²³

This allowed extrapolation of the TPA cross-sect i on o f 2b to ca. 38 GM at 710 nm, and that o f 2c to ca. 69 GM at 740 nm.

The action spectra of TPA cross-section for 2b and 2c are shown i n F i gure 5. The TP e ﬃ c i ency ¤

u

was determ i ned to be 3.4 GM for 2b at 710 nm, higher than the minimum threshold va l ue o f 3 GM. Compound 2c has a l ower e ﬃ c i ency o f ca. 2.1 GM at 740 nm due to the lower quantum yield of the uncaging reaction, desp i te the h i gher TPA cross-sect i on than 2b. Compared to the prev i ous l y reported NPBF-BA chromophore ( ·

2

= 54 GM and

¤

u

= 5 GM at 740 nm), 6,7-d i methoxy-subst i tuted coumar i n 2c i s super i or i n terms o f TPA cross-sect i on but i n f er i or i n terms o f TP eﬃciency. This is due to its higher dipolar character aﬀorded by the methoxy group subst i tut i on, enhanc i ng the TPA character, but at the expense of a reduced uncaging quantum yield.

In conc l us i on, nove l coumar i n chromophores were des i gned and synthesized, which exhibited eﬃcient reactivity for TP-

(a) 1a

(b) 1b

(c) 1c

Figure 2. Ca l cu l ated OPA (magenta dashed li ne) and TPA (orange so li d li ne) spectra at the TD-B3LYP / 6-31G // B3LYP / 6-31G l eve l o f theory f or (a) 1a, (b) 1b, and (c) 1c.

R

R' OH

NO₂

COOH O

a

O O

NO₂ +

R R'

b

O O

NO₂ R

R'

Br

c 2b,c

Scheme 1. Synthes i s o f 2b and 2c. a) Acet i c anhydr i de, tr i ethy l am i ne, 65 °C to RT, overn i ght, 51 % y i e l d. (b) NBS, benzoy l perox i de, CC l

4

, 90 °C, 15 h, 40 % y i e l d. (c) benzo i c ac i d, K

₂

CO

₃

, KI, DMF, 50 °C, 6 h, 20 % y i e l d.

e) d) c) b) a)

8.0

8.5 7.5 7.0

δ / ppm

hν

Figure 3.

¹

H NMR spectra o f compound 2c i n DMSO-d

₆

(a) be f ore and a f ter (b) 48, (c) 72, and (d) 96 h o f i rrad i at i on at 360 nm; (e)

¹

H NMR spectrum o f benzo i c ac i d i n DMSO-d

₆

.

(4)

uncag i ng react i ons. Compound 2b showed a re l at i ve l y h i gh TP e ﬃ c i ency (3.4 GM). The TPA cross-sect i on (69 GM) o f compound 2c was approx i mate l y tw i ce as l arge as that o f 2b (38 GM). These new TP chromophores have great potential for application in phys i o l og i ca l stud i es.

The NMR and MS measurements were per f ormed at N- BARD, Hiroshima University. This work was supported by a

Grant-in-Aid for Scientiﬁc Research on Innovative Areas “Stimuli- respons i ve Chem i ca l Spec i es f or the Creat i on o f Funct i ona l Molecules (No. 2408)” (JSPS KAKENHI Grant Number JP24109008) f rom the M i n i stry o f Educat i on, Cu l ture, Sports, Sc i ence and Techno l ogy, Japan. C.K. acknow l edges the HPC resources of CINES and of IDRIS under the allocations 2015- [x2015080649] and 2016-[x2016080649] made by GENCI (Grand Equipment National de Calcul Intensif).

Supporting Information is available on http://dx.doi.org/

10.1246/c l .160586.

References and Notes

1 a) J. Engels, E.-J. Schlaeger, J. Med. Chem.

1977,

20, 907. b) J. H.

Kap

l

an, B. Forbush, III, J. F. Ho

ﬀ

man, B

i

ochem

i

stry

1978,

17, 1929.

2 P. K

l

án, T.

Š

o

l

omek, C. G. Bochet, A. B

l

anc, R. G

i

vens, M. Rub

i

na, V. Pop

i

k, A. Kost

i

kov, J. W

i

rz, Chem. Rev.

2013,

113, 119.

3 J. Nakan

i

sh

i

, Y. K

i

kuch

i

, T. Takarada, H. Nakayama, K. Yamaguch

i

, M. Maeda, J. Am. Chem. Soc.

2004,

126, 16314.

4 a) C. Ran, Z. Zhang, J. Hooker, A. Moore, Mo

l

. Imag

i

ng B

i

o

l

.

2012,

14, 156. b) M. Matsuzak

i

, G. C. R. E

lli

s-Dav

i

es, T. Nemoto, Y.

M

i

yash

i

ta, M. I

i

no, H. Kasa

i

, Nat. Neurosc

i

.

2001,

4, 1086.

5 S. Piant, F. Boltze, A. Specht, Opt. Mat. Express

2016,

6, 1679.

6 Y. Zhao, Q. Zheng, K. Dak

i

n, K. Xu, M. L. Mart

i

nez, W.-H. L

i

, J. Am.

Chem. Soc.

2004,

126, 4653.

7 S. Atta, A. Jana, R. Ananthak

i

rshnan, P. S. N. Dhu

l

eep, J. Agr

i

c. Food Chem.

2010,

58, 11844.

8 W. J

i

varamona

i

ku

l

, P. Rashatasakhon, S. Wan

i

chwecharungruang, Photochem. Photob

i

o

l

. Sc

i

.

2010,

9, 1120.

9 C. Br

i

eke, F. Rohrbach, A. Gottscha

l

k, G. Mayer, A. Hecke

l

, Angew.

Chem., Int. Ed.

2012,

51, 8446.

10 M. Paw

li

ck

i

, H. A. Co

lli

ns, R. G. Denn

i

ng, H. L. Anderson, Angew.

Chem., Int. Ed.

2009,

48, 3244.

11 J. Bab

i

n, M. Pe

ll

et

i

er, M. Lepage, J.-F. A

ll

ard, D. Morr

i

s, Y. Zhao, Angew. Chem., Int. Ed.

2009,

48, 3329.

12 M. Noguch

i

, M. Skwarczynsk

i

, H. Prakash, S. H

i

rota, T. K

i

mura, Y.

Hayash

i

, Y. K

i

so, B

i

oorg. Med. Chem.

2008,

16, 5389.

13 G. Bort, T. Ga

ll

avard

i

n, D. Ogden, P. I. Da

l

ko, Angew. Chem., Int. Ed.

2013,

52, 4526.

14 N. I. Kiskin, R. Chillingworth, J. A. McCray, D. Piston, D. Ogden, Eur. B

i

ophys. J.

2002,

30, 588.

15 T. Furuta, S. S.-H. Wang, J. L. Dantzker, T. M. Dore, W. J. Bybee, E. M. Ca

ll

away, W. Denk, R. Y. Ts

i

en, Proc. Nat

l

. Acad. Sc

i

. U.S.A.

1999,

96, 1193.

16 C. Bao, G. Fan, Q. L

i

n, B. L

i

, S. Cheng, Q. Huang, L. Zhu, Org. Lett.

2012,

14, 572.

17 J. P. O

l

son, H.-B. Kwon, K. T. Takasak

i

, C. Q. Ch

i

u, M. J. H

i

g

l

ey, B. L. Sabatini, G. C. R. Ellis-Davies, J. Am. Chem. Soc.

2013,

135, 5954.

18 S. Bo

i

napa

ll

y, B. Huang, M. Abe, C. Katan, J. Noguch

i

, S. Watanabe, H. Kasa

i

, B. Xue, T. Kobayash

i

, J. Org. Chem.

2014,

79, 7822.

19 R. W. Boyd,

Nonlinear Optics, 2nd ed.,2003, p. 515.

do

i

:10.1016

/

B978-012121682-5

/

50013-4.

20 Y. Sakamoto, S. Bo

i

napa

ll

y, C. Katan, M. Abe, Tetrahedron Lett.

2013,

54, 7171.

21 M. A. Musa, L. M. Latinwo, C. Virgile, V. L. D. Badisa, A. J.

Gbadebo, B

i

oorg. Chem.

2015,

58, 96.

22 The mechan

i

sm o

f

uncag

i

ng react

i

on o

f

coumar

i

n der

i

vat

i

ves, wee:

B. Schade, V. Hagen, R. Schm

i

dt, R. Herbr

i

ch, E. Krause, T. Eckardt, J. Bend

i

g, J. Org. Chem.

1999,

64, 9109.

23 N. Komor

i

, S. Jakkampud

i

, R. Moto

i

sh

i

, M. Abe, K. Kamada, K.

Furukawa, C. Katan, W. Sawada, N. Takahash

i

, H. Kasa

i

, B. Xue, T.

Kobayash

i

, Chem. Commun.

2016,

52, 331.

24 S. Jakkampudi, M. Abe, N. Komori, R. Takagi, K. Furukawa, C.

Katan, W. Sawada, N. Takahash

i

, H. Kasa

i

, ACS Omega

2016,

1, 193.

-0.04 -0.03 -0.02 -0.01 0

0 20 40 60 80 ln([2]/[2]0)

Irradiation time / min -0.04

-0.03 -0.02 -0.01 0

0 20 40 60 80 ln([2]/[2]0)

Irradiation time / min 2b TP excitation (a)

(b)

(c)

2c TP excitation

O

NO2

O Ph O NPBF-BA σ2= 18 GM at 720 nm

k760 1.2x10^-6s^-1 k730 1.8x10^-6 s^-1 k720 2.6x10^-6 s^-1 k700 2.8x10^-6 s^-1 k710 3.3x10^-6 s^-1 k750 3.7x10^-6 s^-1 k740 3.9x10^-6s^-1 k760 1.9x10^-6 s^-1

k730 2.2x10^-6s^-1 k720 2.8x10^-6s^-1 k740 3.3x10^-6s^-1 k750 3.6x10^-6 s^-1 k700 5.4x10^-6 s^-1 k710 6.5x10^-6s^-1

Figure 4. T i me pro ﬁl e o f the TP uncag i ng o f (a) 2b and (b) 2c, l n([sub] / [sub]

₀

) vs. i rrad i at i on t i me at wave l engths o f 700, 710, 720, 730, 740, 750, and 760 nm at 700 mW. (c) TP react i on o f NPBF-BA at 720 nm, ·

2

= 18 GM at 720 nm.

0 10 20 30 40 50 60 70 80

700 710 720 730 740 750 760

σσ / GM

Wavelength / nm

2c

2b

Design, synthesis, and reaction of π-extended coumarin-based new caged compounds with two-photon absorption character in the near-IR region

HAL Id: hal-01398035

https://hal-univ-rennes1.archives-ouvertes.fr/hal-01398035

Submitted on 24 Nov 2016

HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Design, synthesis, and reaction of π-extended

coumarin-based new caged compounds with two-photon absorption character in the near-IR region

Y. Chitose, Manabu Abe, K. Furukawa, Claudine Katan

To cite this version:

Y. Chitose, Manabu Abe, K. Furukawa, Claudine Katan. Design, synthesis, and reaction of π-extended coumarin-based new caged compounds with two-photon absorption character in the near-IR region.

Chemistry Letters, Chemical Society of Japan, 2016, 45 (10), pp.1186–1188. �10.1246/cl.160586�.

�hal-01398035�

Des i gn, Synthes i s, and React i on o f π -Extended Coumar i n-based New Caged Compounds w i th Two-photon Absorpt i on Character i n the Near-IR Reg i on

Youhei Chitose,

Manabu Abe,*

Ko Furukawa,

and Claudine Katan*

Department of Chemistry, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526

Centre f or Instrumenta l Ana l ys i s, N ii gata Un i vers i ty, 8050 Ikarash i 2-no-cho, N i sh i -ku, N ii gata 950-2181

Institut des Sciences Chimiques de Rennes, CNRS, Universite Rennes 1, 35042 Rennes, France

(E-ma il : mabe @ h i rosh i ma-u.ac.jp, c l aud i ne.katan @ un i v-rennes1. f r)

Caged compounds,

the i nact i ve f orms o f b i oact i ve mo l ecu l es formed using a photolabile protecting group (PPG),

Th i s e ﬃ c i ent uncag i ng i s b i o l og i ca ll y use f u l because the t i m i ng, location, and amplitude of photoirradiation can be controlled.

Thus, the photo i nduced re l ease o f b i oact i ve mo l ecu l es has been utilized in various ﬁelds such as neuroscience, cell patterning, or b i omed i ca l sc i ence.

Coumarin derivatives have been used to study various functions of biomolecules and for cellular imaging.

The advan- tages o f coumar i n as a chromophore i nc l ude i ts i nherent f ast response to photolysis, thermal stability, high molar extinction coe ﬃ c i ent, and good ﬂ uorescent property f or mon i tor i ng i ts reactions in vivo.

However, the recent concern for physiological app li cat i on o f coumar i n-based compounds i s ma i n l y i ts short absorption wavelength. Coumarin itself possesses an absorption wave l ength max i mum o f 274 nm i n methano l .

Due to th i s short wave l ength, ce ll damages cannot be avo i ded us i ng one-photon absorption (OPA).

But better penetration to the deeper part of ce ll s

can be a ﬀ orded us i ng chromophores hav i ng s i gn iﬁ cant two- photon absorption (TPA) character in the near-IR region.

Chromophores w i th TPA character have attracted much attention because of their application in physiology. Caged compounds w i th TPA character enab l e greater spat i a l contro l o f the re l ease o f b i oact i ve substances.

For pract i ca l app li cat i ons, a h i gher uncag i ng e ffi c i ency i s necessary to conduct b i o l og i ca l stud i es. The uncag i ng e ffi c i ency f or OP exc i tat i on i s de fi ned as the uncaging quantum yield (Φ

) multiplied by the excitation prob- ab ili ty ( ¾ : mo l ar ext i nct i on coe ﬃ c i ent). S i m il ar l y, the e ﬃ c i ency ¤

of a TP excitation process is expressed using the TPA cross-section ( ·

): ¤

= Φ

·

. The m i n i mum thresho l d va l ue o f TP e ﬃ c i ency for in vivo studies was reported as 3 GM.

In 1999, Furuta et al.

synthesized brominated 7-hydroxycoumarin-4-ylmethyl (Bhc) esters and carbamates w i th TPA character i n the near-IR reg i on, i .e. 650 ­ 1350 nm. Its TP e ﬃ c i ency amounts to ca. 1 GM at 740 nm.

In 2012, Zhu et al. reported ¤

= 0.25 GM at 800 nm.

In 2013, E lli s-Dav i es et a l . deve l oped a new π -extended caged coumarin molecule that had a TPA maximum at 900 nm.

In th i s study, a coumar i n conta i n i ng a st il bene ske l eton (12 GM at 514 nm)

with a p-nitrophenyl group as the electron- w i thdraw i ng group was des i gned to i ncrease the TPA cross- sections due to its dipolar character (Figure 1).

The cyclic structure should prevent cis­trans isomerization in the excited state. A bathochrom i ca ll y sh if ted absorpt i on wave l ength i s a l so expected because of the donor­π­acceptor conjugated system.

F i rst, the OPA and TPA spectra o f coumar i n chromophores 1a­1c were computed at the TD-B3LYP/6-31G*//B3LYP/6- 31G * l eve l o f theory (F i gure 2; see a l so ESI f or more deta il s).

were i nvest i gated exper i menta ll y.

Caged benzoates 2b and 2c were synthesized using a base- cata l yzed Perk i n condensat i on react i on

(Scheme 1). The UV ­ visible absorption spectrum of 7-methoxy-substituted compound 2b showed the absorpt i on max i mum at 345 nm w i th ¾ = 22726 M

cm

in DMSO, whereas 6,7-dimethoxy-substituted

Figure 1. Coumar i n der i vat i ves w i th TPA character.

compound 2c showed an absorption peak at 369 nm with ¾ = 16902 M

cm

. As pred i cted by TD-DFT ca l cu l at i ons (F i gure 2), the absorption maximum of 2c exhibited bathochromic and

hypochromic shifts as compared to 2b. The OP photolysis of 2b and 2c was i nvest i gated us i ng a Xe l amp at 360 « 10 nm i n DMSO- d

(Figures 3 and S1). The quantitative uncaging of benzoic acid f rom 2b and 2c was con ﬁ rmed by compar i ng the

H NMR s i gna l s of benzoic acid with those of the photolysate (Figures 3 and S1).

The uncaging quantum yields Φ

were determined in DMSO using the photochem i ca l act i nometer f err i oxa l ate coup l ed w i th HPLC analyses. Approximately three-times higher quantum yield for the uncag i ng react i on o f 2b ( Φ

= 0.09) than 2c ( Φ

= 0.03) was obtained. The OP uncaging eﬃciencies (¾

© Φ

) of 2b (¾

= 18718) and 2c ( ¾

= 16102) were 1684 and 483, respect i ve l y.

The lower uncaging eﬃciency of 2c may be attributed to the i ncreased e l ectron i c stab ili ty o f the exc i ted state o f 2b.

The consumpt i on o f 2b and 2c upon TP photolysis was monitored by HPLC (Figure 4).

As shown in Figure 4, the TP-uncaging reaction rates depend on the exc i tat i on wave l ength. The photo l ys i s o f 2b proceeded the fastest at 710 nm (k

= 6.5 © 10

s

), whereas the fastest photo l ys i s rate f or 2c was observed at 740 nm (k

= 3.9 © 10

s

). The values of TPA cross-sections at each wavelength were extrapo l ated f rom the rate constant o f TP uncag i ng o f NPBF- BA, 18 GM at 720 nm.

This allowed extrapolation of the TPA cross-sect i on o f 2b to ca. 38 GM at 710 nm, and that o f 2c to ca. 69 GM at 740 nm.

The action spectra of TPA cross-section for 2b and 2c are shown i n F i gure 5. The TP e ﬃ c i ency ¤

synthesized brominated 7-hydroxycoumarin-4-ylmethyl (Bhc) esters and carbamates w i th TPA character i n the near-IR reg i on, i .e. 650 1350 nm. Its TP e ﬃ c i ency amounts to ca. 1 GM at 740 nm.

The cyclic structure should prevent cistrans isomerization in the excited state. A bathochrom i ca ll y sh if ted absorpt i on wave l ength i s a l so expected because of the donorπacceptor conjugated system.

F i rst, the OPA and TPA spectra o f coumar i n chromophores 1a1c were computed at the TD-B3LYP/6-31G//B3LYP/6- 31G l eve l o f theory (F i gure 2; see a l so ESI f or more deta il s).

(Scheme 1). The UV visible absorption spectrum of 7-methoxy-substituted compound 2b showed the absorpt i on max i mum at 345 nm w i th ¾ = 22726 M

Figure 2. Ca l cu l ated OPA (magenta dashed li ne) and TPA (orange so li d li ne) spectra at the TD-B3LYP / 6-31G // B3LYP / 6-31G l eve l o f theory f or (a) 1a, (b) 1b, and (c) 1c.