Photolabile Protecting Groups (PLPG): Synthetic and Biological Applications

Carmin Burrell October 15, 2008

First Examples: Cbz-glycine
O OH

O O

H N

H2, Pd/C or Li°, NH3 or HBr

O H2N OH

O O

H N

O OH

h! (254 nm) Med. P Hg lamp 30 min, H2O 75%
H2N

O OH

Zweifel, G. S.; Nantz, M. H.; Modern Organic Synthesis: An Introduction; University Freeman: New York, 2007, 60. Barltrop, J. A.; Schofield, P.; Tetrahedron Lett., 1962, 16, 697-699.

First Examples: o-Nitrobenzylesters
NO2

acetone h! (>280nm)

NO OH O NO2

O NO2 O2N H O

H Med. P Hg Lamp Pyrex Filter

h!
H O

O2N H O O H

(>280nm)

NO2 H O HO

NO H OH

NO OH O

Leighton, P. A.; Lucy, F. A., J. Chem. Phys., 1934, 2, 756-759. Barltrop, J.A.; Plant, P. J.; Schofield, P.; Chem Comm, 1966, 22, 822-823.

P Hg lamp HO Pyrex Filter R H Ph R' Ph Ph Time(hr) Yield 2.First Examples: o-Nitrobenzylesters NO2 H O R O CCl4 h! (>280nm) R' O R' N O R Med.5 2.. J. Chem Comm... P.0 4. 1966. 822-823. Schofield.5 3.0 17 90 95 87 O Ph C15H31 Ph CH2Ph Barltrop. J. Plant. P. .A. 22.

Mechanism of Deprotection O N O O O O N OH R O O R' O N O H O R O O N R' C R OH O O N O R HO R' O O R' h! R' H R H transfer Barton. A. Tetrahedron Lett. Y.. D.. G... H.. 23. 1962. . 1055-1057. Chow. R. L. Kirby. Cox. W.

Chem. Int. Zinth. S. W. J... W. T.Excitation . F... P.. Gilch. Ed. Schreier. 7901-7904.. Schrader. 2005. 44. Angew.Jablonski Diagram S2 Internal conversion Vibrational relaxation S1 Excitation femto (10-15s) S0 Fluorescence nano (10-9s) pico 10-12s Intersystem crossing T1 Phosphorescence !micro (10-6s) • Excitation promotes a HOMO electron to the LUMO • Rearrangement occurs to cleave the PLPG Laimgruber. Koller. .

V. B. 4765-4769. L. 3301-3307.Applications of PLPGs .. P. B.. Busuyek. . Angew. Ed. G.. Chem. 57.. Tetrahedron. M.. Int. Harran. S.Synthesis of (-)Diazonamide A Me Me HO Me H N O HN O O O Cl O NH NH Me N N Cl (-)-Diazonamide A Snider. 40.. 2001. J. Esser. Li. 2001. Jeong.

NaI. S. 2001..Applications of PLPGs . P. DMF 52% Br CbzHN Br O O Ph OH NO2 OH O Ph OH Li. Harran. Chem. J..Synthesis of (-)Diazonamide A Me Me Me HO Me H N HN N N Cl O O O O Cl O NH NH (-)-Diazonamide A Me HN CbzHN O Me N O CN Na HN Me N O O CN o-NO2C6H4CH2Br K2CO3. Int. Ed. 40.. G. Jeong. Angew. L. 4765-4769. Esser. ..

L.Synthesis of (-)Diazonamide A Me Me Me HO Me H N HN N N Cl O O O O Cl O NH NH (-)-Diazonamide A Me HN CbzHN O Me N O CN CbzHN Me HN O Me N O O O Cl3CCO2H Br O O Ph OH NO2 Ph O O NO2 Br Li. J. Harran. 40. P. . Ed. Esser. Jeong. S. Angew. 4765-4769. 2001.Applications of PLPGs .. Int.. Chem.. G..

L.. toluene/CH2Cl2 0°C!RT Br 83% (2 steps) NO2 Br O O Ph OH NO2 Li. J. Angew.Synthesis of (-)Diazonamide A Me Me Me HO Me H N HN N N Cl O O O O Cl O NH NH (-)-Diazonamide A Me HN CbzHN O Me N O O O N H NHAlMe2 Me HN CbzHN O O Me N HN O NH O O Ph 1. G. 4765-4769.Applications of PLPGs . S. 40.3 eq. Chem.. Int. . 2001.. Jeong.. Ed. P. Harran. Esser.

. Ed. 2001.5 eq NMO 4 Å MS. Int. G.Synthesis of (-)Diazonamide A Me Me Me HO Me H N HN N N Cl O O O O Cl O NH NH (-)-Diazonamide A NH Me HN CbzHN O O Me N HN O Me HN Me N O O HN O NH 5 mol% nPr4NRuO4 1. . 40. P. S. L. Chem. Angew. Esser.. J..Applications of PLPGs . CH2Cl2 78% CbzHN Br O O Ph OH NO2 Br O O Ph O NO2 Li.. 4765-4769. Harran. Jeong.

G. CH2Cl2. P.. 85% Br CbzHN HN Br O OAc O O O Ph O NO2 Ph Li. 40.Applications of PLPGs . 3x10-2M degassed dioxane excess Ac2O. py. J. Harran. Jeong.Synthesis of (-)Diazonamide A Me Me Me HO Me H N HN N N Cl O O O O Cl O NH NH (-)-Diazonamide A NH Me HN CbzHN O O Me N HN O Me Me N O O HN O NH 350nm. . DMAP. S.. Int. 2001. Chem. 4765-4769. Esser. L.. Angew. Ed..

Requirements for PLPGs • A Functionalized Aromatic System • Substituents can allow for use of a specific wavelength radiation • Fast Rate of Cleavage • Reduce the time needed for deprotection • High quantum yields • Stabilize the radicals or ions created after excitation .

73..Y. Org. S.. X. OMe O MeO O R O R' Ph Ph O S O O O OR Wang. J. OR MeO MeO OR NO2 MeO O O Furuta. L. 1193-1200. P. T... Callaway.. H.. Bybee. . W.. 2008.. T. J. Chem. Sugawara. C.C. C. M.. Acad. R.. Org. Kitani. Spencer. 1116-1117. L. T. S. 1995. Dantzker. Wang.. Kakiuchi. J. Wang. Pan.. ChemComm. W. Natl. Tsutsumi. Sci... Morimoto. E.. Hu.Photolabile Protecting Groups (PLPGs) OR MeO O OMe Pirrung. K.. Liang. Y. 6152-6157. 2008. Tsien.. M. Denk. Proc. Dore. M. K. S.... J. Bradley. 60. 2103-2105.. Chem. 1999. K. J. 96.

Requirements for PLPGs OMe N NO2 !"!# $max (nm) 184 n"!* $max (nm) 217 269 251 299 252 330 Lambert.. Lightner. . Prentice-Hall: New Jersey. Organic Structural Spectroscopy.1998. J. 289. F. D. Cooks. Shurvell. G... H.. A. B. R.

Requirements for PLPGs • A Functionalized Aromatic System • Substituents can allow for use of a specific wavelength radiation • Fast Rate of Cleavage • Reduce the time needed for deprotection • High quantum yields .

H. California. . J. 2006. Pan.. Hu.. L. C. X. Dougherty.. Modern Physical Organic Chemistry. 6152-6157. Org.25 Rhodopsin Φ= 0.Requirements for PLPGs Quantum Yield = ! = Number of molecules that undergo cleavage Total number of photons absorbed 0<!<1 OMe OMe Ph Ph O H O Ph Chlorophyll Φ= 0. J..03 3 ! = 0. University Science Books: Sausalito. 971. A.. Chem... V.17 Wang. 73. Wang. Anslyn.66 MeO O H O Ph MeO 2 ! = 0. D. E. 2008. Liang. P. Spencer.

. Chem.. 1964. Soc. J. R. 5277-5281. N2 Φ= 0.Dimethoxybenzoin Carbonates X R4 R3 R2 R1 O benzene h! (>280nm) Med. W.. J. 1971. C. A. M. R. Wilson. J.C. 7222-7228 . P Hg lamp Pyrex Filter R2 R1 O R3 R4 R1 R2 R3 H H H H H H OMe H OMe OAc R4 X Time (hr) Yield H OAc 17 15 H Cl 18 1 H OAc 17 46 MeO O OMe MeCN h! (360nm) Rayonet reactor pyrex filters 7min. 93.. Wilson. J.64 MeO HOAc O OMe 99% Sheenan. Soc. Am. M. Oxford.. Chem. Am. 86.. Sheenan.

J..C. J. Ar MeO HX O OMe OH SH MeO OH OH 88% 95% 90% 94% Pirrung.. M. Org. Chem. Bradley.Dimethoxybenzoin Carbonates X MeO O OMe benzene h! (350nm) Rayonet reactor 1h. . 1116-1117. C.. 1995. 60.

J.. Wirz.Dimethoxybenzoin Carbonates OAc MeO O OMe OAc MeO O OMe MeO O OMe MeO OMe OAc h! MeO O HOAc H OMe O Boudebous. Ko!mrlj. A. B. . "ket. 111.. B.. Chem. Phys. H.. J.. 2007. 2811-2813.

K. Tsutsumi.Thiochromone S. .0X10-2M) 0. Comm. 2008. K.. K.New PLPGs . Morimoto.. T. Sugawara. Chem.S Dioxide O h! (" >280nm) ultra-high P Hg lamp pyrex filter O O OEt O S CD3OD (2. Kakiuchi. 2103-2105.5h S >99% O O OEt O O S O O OR S O O OR Kitani... S. 18.

Thiochromone S. S. K. Morimoto. K..5h EtOH >99% Kitani.. Sugawara.0X10-2M) 0. K. Comm. Kakiuchi.New PLPGs .S Dioxide O h! (" >280nm) ultra-high P Hg lamp pyrex filter O O O OEt S O CD3OD (2.. . 18. 2103-2105. T.. Chem. Tsutsumi. 2008.

Kakiuchi. Chem. K. K.. 2008.. S.New PLPGs . K. Tsutsumi.. 18.S Dioxide O O h! (" >280nm) S O O H O O O O XR CD3OD O S O O O XR CO2 S O O O HXR O O XR S O O O S O O OR S O O OR Kitani. .. T. 2103-2105. Comm. Sugawara. Morimoto.Thiochromone S.

5 >99 (99) Kitani..5 0.5 0. Tsutsumi. K. T. S. Comm..0 >99 >99 >99 Me OH MeO 1.S Dioxide O h! (" >280nm) ultra-high P Hg lamp pyrex filter O O O O -2 R CD3OD (2. Morimoto. K.New PLPGs .0 1..5 1. Sugawara. . 2008. K. Kakiuchi.Thiochromone S.0 >99 (98) 0. 2103-2105.0X10 M) O ROH S O O S O Alcohol Irradiation Time(h) Yield(%) EtOH C8H17OH OH OH Alcohol Irradiation Time(h) Yield(%) 1. Chem.0 H OMe OAc >99 (99) 97 98 H HO HO AcO OAc O OH 0. 18..

. K.0X10 3 R O R H N R' O S O O O S O Amine C4H9NH2 Et2NH NH2 CO2Bn Irradiation Time/h 1. K. S. Morimoto. T.New PLPGs . 18. Comm. Tsutsumi. 2008. 2103-2105. Sugawara.Thiochromone S. Kakiuchi.0 1.0 Yield(%) 97 98 97 Kitani. K...S Dioxide O R' N O h! (" >280nm) ultra-high P Hg lamp pyrex filter -2M) CD OD (2. Chem. .0 1..

New PLPGs - Thiochromone S,S Dioxide
O

h! (" >280nm) ultra-high P Hg lamp pyrex filter
O O O R

O

CD3OD (2.0X10-2M)

RCOOH
S O O

S O

RCOOH C11H23COOH C17H35COOH

Irradiation Time/h 1.0 1.0 1.0
COOH

Yield(%) >99 >99 >99

COOH

1.0

>99

Kitani, S.; Sugawara, K.; Tsutsumi, K.; Morimoto, T.; Kakiuchi, K. Chem. Comm. 2008, 18, 2103-2105.

New PLPGs - Ketones and Aldehydes Generation 1
OMe OMe

!MeO O H O H

!-

!-

O O H H Mulliken Charges

O H

O H

S1 -0.193

S0 -0.143

S1 -0.181

S0 -0.159

S1 -0.168

S0 -0.172

• Ortho - meta effects on the excited state facilitate
heterolytic cleavage
Zimmerman, H. E.; J. Am. Chem. Soc., 1995, 117, 8988-8991 Wang, P.; Hu, H.; Wang,Y.; Org. Lett., 2007, 9, 2831-2833.

Gaussian 03, Revision C.02 CIS/3-21G for excited states HF/3-21G for geometry optimization

Excited State - Ortho - Meta Effect
MO 7 MO 6

MO 5 MO 4

MO 3 MO 2

MO 1

Zimmerman, H. E., J. Am. Chem. Soc.,1995, 117, 8988-8991.

Chem.. P.. E..181 S0 -0. 9.New PLPGs . J.. H. Am.Ketones and Aldehydes Generation 1 OMe OMe !MeO O H O H !- !OMe O O H H Mulliken Charges O H O H MeO S1 -0.159 S1 -0. 2831-2833.143 S1 -0. Soc.meta effects on the excited state facilitate heterolytic cleavage Zimmerman. Org. Revision C.168 S0 -0. H.02 CIS/3-21G for excited states HF/3-21G for geometry optimization . 8988-8991 Wang. 2007. Lett. 1995. 117..Y. Gaussian 03. Wang.193 S0 -0..172 O R1 CH2 O R2 • Ortho . Hu.

.Ketones and Aldehydes Generation 1 OMe MeO O H OMe O h! (>280nm) Med P Hg lamp pyrex sleeve CD3CN/D2O (9:1) 1hr Ph H O Ph 50% h! (>280nm) Med P Hg lamp pyrex sleeve O H O O H Ph CD3CN/D2O (9:1) 1hr Ph h! (>280nm) Med P Hg lamp pyrex sleeve 8% O Ph O O CD3CN/D2O (9:1) H 1hr H Ph Wang. 2007. P. H. Org. Lett.Y. 2831-2833... Hu.. Wang.New PLPGs . 9. .

5 93 86 3.0 91 93 3. P. . Lett. Hu. H. p-TsOH MeO OH OH OMe h! (>280nm) Med.New PLPGs . 2831-2833.5 Wang. 9.0 99 79 0..... 2007. P Hg lamp pyrex filter MeCN/H2O (4:1) CHO MeO O R1 O R2 O O Ph CHO OPh MeO 16 Protection Yield: Deprotection Yield: Time (h): 92 81 4.Ketones and Aldehydes Generation 1 OMe O R1 R2 10 mol%. Org. Wang.Y.

Lett. 9. 2007. Hu.Ketones and Aldehydes Generation 2 O OMe Ph O R1 O R2 OMe Ph OMe Ph O R1 O R2 R1 R2 10 mol%. Org. p-TsOH Ph Ph Ph OH OH h! (>280nm) Med....New PLPGs . P. P Hg lamp pyrex filter MeCN CHO O Ph CHO 8 O OMe MeO Protection Yield: Deprotection Yield: Time (min): 99 90 60 99 89 60 91 86 60 93 84 80 Wang. 1533-1535.. .Y. H. Wang.

Wang. Hu. 2008. C. H.Ketones and Aldehydes Optimized System OMe OMe Ph Ph OH 1 OH OH OH OMe Ph Ph OH OH MeO MeO 2 3 PPG 1 2 3 ! 0.. Spencer. Liang. X. Pan. J. 73. 6152-6157...17 protection yield (%) deprotection yield (%) time (min) 92 99 99 81 90 92 240 60 30 Wang. L. .03 0.11 0.. Chem. Org.New PLPGs . P.. J.

. X. C. 2008. Pan. J. J. 6152-6157. H.Ketones and Aldehydes Optimized System O OMe Ph MeO OH R1 R2 10 mol%. p-TsOH Ph OMe Ph O R1 O R2 O O OPh MeO 16 OH h! (>280nm) Med. Org.New PLPGs .. 73. L.. Wang. Chem. P.. Hu. P Hg lamp MeO pyrex filter MeCN/H2O 10%(v/v) CHO Ph Ph CHO Protection Yield: Deprotection Yield: Time (h): 99 92 30 93 82 44 99 88 25 99 91 25 Wang. Spencer. Liang. ..

X. Org.New PLPGs .. Wang. Spencer. H. J. Chem. J. Pan. .. 73.. Hu.. Liang. 6152-6157. P.Ketones and Aldehydes Optimized System OMe Ph Ph O R2 OMe OMe Ph MeO O R1 O R2 R1 OMe Ph O R2 MeO O R1 h! H2O MeO O Ph Ph h! -H2O MeO OH Ph Ph OH Wang. L. C.. 2008.

2h 23°C. 6152-6157. 2h 23°C. . X. 2h %Recovery 100 100 100 100 100 100 100 94 0 0 100 100 100 91 100 65 47 0 NaBH4 H2. 24h 80°C. 2h 23°C.Ketones and Aldehydes Optimized System Reagent PhLi LiAlH4 Solvent THF THF THF THF MeCN MeCN MeCN MeCN MeCN MeCN MeCN Conditions -78 to 23°C. 24h 80°C.. Pd/C OMe Ph Ph O H O Ph t-BuOK DDQ CAN AcOH TFA HCl (37%) pTsOH MeO Wang. 24h 80°C. 24h 23°C. H. 2h 23°C. Org. 24h 80°C.New PLPGs . L. Pan. 73. 24h 23°C.. J. Wang. Hu.. Liang.. J. Spencer. 24h 80°C. C.. 2h 23°C. 2008. 6h 23°C. 24h 80°C. P. Chem. 24h 80°C. 2h 23°C. 18h 23°C.

Chem. Wang.17 "max 327 297 Wang. Hu. P Hg lamp pyrex filter Ph Yield 99 92 Time 16 30 R1 N(Me)2 OMe R2 H OMe ! 0. J. 73. J.. P. X.Ketones and Aldehydes Amino Acetal R1 h# (>280nm) R2 O H O O H Ph Med... H... Org. 2008. Liang. C. Pan. .New PLPGs . 6152-6157.13 0. Spencer. L.

C. J.. Hu..Orthogonality Using PLPGs OMe Ph Ph O H O Ph N Ph Ph O H O Ph OMe MeO h! MeCN/ MeO H2O (1:1) O Ph Ph O H O Ph H Ph 350nm. Chem. 20min: 98% sunlight. 73.. H. 3h : 98% >89% >96% Wang. Spencer. P.. J. Wang. 6152-6157.. X. 2008. L. Org. Pan. . Liang.

Orthogonality Using PLPGs O O S O O O O O O O O O O O O S O h! (" >280nm) CD3OD.. S. 0.5h 99% S OH O CO2 S O Kitani. Sugawara. 18. Comm. Chem. Morimoto. K. Tsutsumi... K. 2103-2105. K. Kakiuchi.. . T. 2008.

. Chem..Orthogonality Using PLPGs O O mCPBA 85% S O O O OH O S O O O O O OH h! (" >280nm) CD3OD. 18. K. T. 2103-2105. S. . K. Comm. Sugawara. Tsutsumi.. Morimoto. Kakiuchi. 0. 2008.5h 99% HO OH S O O 85% CO2 >99% Kitani. K..

G. C.. Org.. . A. 2002. 67.Orthogonality Using PLPGs OMe O2N MeO O O O n OMe OMe O O n 254 nm 420 nm (%) (%) 0 420 nm 1 2 OMe 3 4 5 O O O 254 nm h!.. Chem. then CH2N2 O2N MeO O n OMe OMe 86 94 94 96 78 92 n 67 83 85 85 87 70 OMe O O O MeO Blanc. Bochet. 5567-5577. J.

• o-Nitrobenzyl is most widely used and has been applied to total syntheses NO2 H O R O R' Summary of Uses of PLPGs in Synthesis • Thiochromone systems could be an alternative although optimization required O S O O O O O R • Acetal protection system for aldehydes and ketones is groundbreaking OMe MeO O R1 • Proof of orthogonality makes PLPG more practical Ph Ph O R2 .

Tsien. W. 1193-1200. T. Proc. Dantzker. M. Bybee. P . J. S. Dore.. Denk. Wang. 1999.Caged Biomolecules • PLPG inactivates the biomolecule until irradiated • Biomolecules that have been caged include: • neurotransmitters. S.. Acad.. 96.. E.Y. J. ATP... M. Natl.. R. Sci. W. T. . Callaway.. L. Glutamate • An effective method to elucidate cellular functions of i biomolecules • Release occurs on a biologically relevant time scale as compared to diffusion (temporal resolution) resolution) • Release can be controlled to a specific area (spacial Furuta.

1997.. L. Avery.C. Proc. W.. Hess. Sci.. 94.Caged Biomolecules . . P. G. Natl. Elegans Li. 5912-5916. USA. Denk. Acad. H..

C. Avery.Caged Biomolecules .. Denk. 5912-5916. 94. P. H. Proc. 1997. L. Sci. . G. USA.. Natl. Acad. W.. Elegans NO2 O COO-O NH3+ COO- h! NO O COOHO O NH3+ COO- Li.. Hess.

. Jones. D. J. S..Light Triggered Molecule-Scale Dosing OCH3 O OCH3 O O O O O O O O OCH3 O O OCH3 O OCH3 OCH3 Acetylsalicylic acid Ibuprofen Ketoprofen McCoy. Gorman. . Am.. C. P. Rooney.. S. R. 2007. C. 9572-9573. P. 129. C. Edwards. Chem.. Soc.

S. Soc... C.. C. .. Rooney. P..Light Triggered Molecule-Scale Dosing 90 Yield of Ibuprofen (%) 60 Off 30 Off 0 0 Off On 10 OCH3 O OCH3 O O Off Off On On Off On On On 20 Time (min) 30 O OCH3 O OCH3 HO 40 h! (365 nm) Hg lamp quartz cuvette Ibuprofen McCoy. Jones. R. 129. Gorman. 9572-9573. Am. J. Edwards. 2007. Chem. D. S. P. C.

Y. 2655-2666.. M. Kimura. 48.5 x 10-4 mg/mL Isotaxel 0. Med. Sohma. 2005. Y. M..Y. Chem... M. Hamada.45 mg/mL Skwarczynski.Taxol® Prodrug O O OH O O O O HO O H O O O N H O OH HO O O H O O O O O O H2N O O O O OH Taxol® 2. Hayashi. Noguchi... T. Kimura. .Y... J. Kiso.

T...Y.45 mg/mL Skwarczynski. Hayashi.Y. M. Kiso.. Hirota. Y. Lett. .. Chem. M.. 2006. Med.. Sohma.Taxol® Prodrug N HCl O O O O NH O O O O O O OH O O O O O OH 430 nm diode laser H2N O O O HO O H O O O O O HO O H O O O Phototaxel < 2. Kimura. S. Noguchi. 16. Bioorg.5 x 10-4 mg/mL Isotaxel 0.. 4492-4496.

Lett.... Kiso. Med. . Y.. Chem. Sohma.. Noguchi. S.Y. 2006. M.Y. Kimura. M. Bioorg. T. 4492-4496. Hirota. Hayashi. 16..Taxol® Prodrug 100 N HCl O O 80 2 O O NH O O O O O O OH 60 (%) O HO O H O O O 40 O 20 0 0 5 10 15 Time (min) 20 25 30 1 O O N H O OH O O O OH N O O 5 OH =1 =2 =5 O HO O H O O O Skwarczynski..

Y. . Hayashi..45 mg/mL Skwarczynski. Chem. Bioorg. S. 2008. 16. Med. Hirota. 5389-5397.. Sohma.Y... M. Noguchi. M. T..Y. Kiso.Taxol® Prodrug O 3 HCl O N NH N H N O O O O OH O N O NH O O O O O O OH O O 365 nm H O O UV lamp O O H2N O O O O HO O H O O O O HO O Phototaxel 2 > 100mg/mL Isotaxel 0... Kimura.

.O O N H O O OH HO O O O O OH Taxol® Prodrug 100 O H O O (%) 80 Taxol® O 60 40 3 HCl O N NH N H N O O 20 O N O NH O O O O O O OH 0 0 10 20 30 Time (min) 40 50 60 O HO O H O O O O = Taxol® = Phototaxel Phototaxel Skwarczynski. T. Med. Kimura... Bioorg.. S.Y.Y. Hirota. Hayashi. Noguchi. 5389-5397.. Sohma. . Chem. Y.. M. 16. Kiso. M. 2008..

UV Damage 100 150 200 250 300 350 Wavelength 400 (nm) X-Rays Ultraviolet radiation UVC UVB UVA Visible light • Preceding PLPGs required 280 . .. N. Toxicol. Pharmacol. 195.. 298-308. 2004. H.365nm radiation for cleavage Matsumura. Y. Appl. Ananthaswamy.

Toxicol. Y. . Suntan. Epidermal Hyperplasia DNA damage Acute UV Cell cycle control Apoptosis DNA Repair p53 induction Maintenance of genomic stability DNA replication and repair Immune Suppression Angiogenesis inhibition Skin aging (Photoaging) Chronic UV DNA damage accumulation Genetic mutations Immune suppression Skin carcinogenesis Matsumura. 195. 2004. Ananthaswamy.. H. Appl.UV Damage Sunburn. 298-308. Pharmacol.. N.

1931 • Theodore Maiman developed the ruby laser -1960 • W. N.1961 microscopy . Spence.Two Photon Excitation • Maria Göppert-Mayer first described non-linear optics . G. 63. L. Garrett observed two photon excited fluorescence in CaF2:Eu2+ crystals.. B. So. P.1980s • Janis Valdemanis and R. Fork described multiphoton • D.R. T. Kean and W.. C.1990 Masters. B. 3-11. Sibbert developed Tisapphire femtosecond laser . Kaiser and C. E. . 2004. Microscopy Research and Technique. P.

M. R. Rev. Drug Deliv. M.. D.... J. 2006. Geisbauer. Michael.. Adv. Madsen. H. 788–808 . D. 58.. Chow.Two Photon Excitation S2 IC S1 Fluorescence S2 IC ISC T1 Phosphorescence Fluorescence S1 ISC T1 Excitation Virtual State Phosphorescence S0 Excitation S0 UV One Photon Excitation IR Two Photon Excitation Oheim.

58.. 770-772. W. D. Drug Deliv.Two Photon Excitation • Utilizes IR radiation instead of UV • Requires concentrated photons and provides increased spacial and temporal control of excitation • Not sensitive to scattering • No out-of-focus absorption • Photobleaching and photodamage is limited to the location of excitation • Increases Depth Penetration Piston. 2006. Adv. Rev.. .

J... Natl. W. Bybee... M... R. Dore. E. 1999. M.. T. Acad. 96. L. T. W. Callaway. Y. J.One Photon UV Two Photon Excitation Two Photon IR Caged Biomolecule Released Biomolecule PLPG Furuta. Dantzker. Proc.. Denk. 1193-1200. Tsien. S. Sci. . S. Wang.

R.Two Photon Excitation Oheim. Chow. Drug Deliv. M. 2006. Michael. . J. Adv. Madsen.. Geisbauer. D. 788–808. Rev. 58. D. H... M....

. Drug Deliv. M.Two Photon Excitation Oheim. Madsen. D.. Geisbauer.. . J.. R. Michael. M. Chow. 788–808. D. H. Rev. 2006. Adv.. 58..

Drug Deliv.. D. 2006.Two Photon Excitation • Utilizes IR radiation instead of UV • Requires concentrated photons and provides increased spacial and temporal control of excitation • Not sensitive to scattering • No out-of-focus absorption • Photobleaching and photodamage is limited to the location of excitation • Increases Depth Penetration Piston. Adv.. 770-772. 58. W. Rev. .

Helmchen..Two Photon Excitation One Photon UV Two Photon IR Denk. F. 932-940. 12. 2005. . Nature Methods.. W.

Rev. Adv.. 770-772. Drug Deliv. . 58.Two Photon Excitation • Utilizes IR radiation instead of UV • Requires concentrated photons and provides increased spacial and temporal control of excitation • Not sensitive to scattering • No out-of-focus absorption • Photobleaching and photodamage is limited to the location of excitation • Increases Depth Penetration Piston. D. W. 2006..

F. .100!m the brain tissue • Used for imaging of slices of • Two Photon Excitation depth in brain tissue: up to 1mm deeper in a tumor • Could lead to excitation Denk. 2005.. 12. Helmchen.. W.Two Photon Excitation • Confocal microscopy depth in brain tissue: 50 . 932-940. Nature Methods.

Org. 6860-6861. 90% H3CO O O cAMP Givens. 60. S. tyrosine. R. B.Coumarin Derivatives O CH2OP(OC2H5)2 CH2Nu h! (360nm) H3CO O O Nu H3CO O O Nu = CH3OH. piperidine.. 106. J. 1984. cysteine.. Am.. 1995. 3953-3956. Chem. T. Ar.. Iwamura. Matuszewski. Furuta. Sugimoto... J. Soc. M. ... "-chymotrypsin NH2 N O P O O O O N O N N NH2 N O P O O O HO N O N N Dioxane/Water (1:1) h! (340nm) Xe lamp 1h. H. M. Chem. Toriagai.

Coumarin Derivatives O MeO MeO O NO2 NH3 CO2 HO O O OAc Cl O O O OAc 1 !-DMNB-glu Cmpd 1 2 3 "(365nm) 0.07 ± 0. Tsien..01 2 !u(740nm (GM)) 3 !u(800nm (GM)) 1. T. .07 ± 0.001 0. J.025 0. Sci.. 1193-1200. L. Bybee. M. Acad.02 Furuta... M.05 1. S. Callaway. Proc.07 0. S..34 ± 0. Wang. 1999. Dore. Natl. E.006 0. Denk. Dantzker. T.Y... J. 96.. R.13 ± 0. W. W.

1653-1656. et. al. Science. M. 1998.. .Cross Sectional Area of Uncaging ("u) • Measures the efficiency of the chromophore to absorb two photons • • Dependent on: • Cross-sectional area of the chromophore 1"u = 10-50 cm4s/photon = 1GM Albota.. 281.

Bybee.Y.. .Coumarin Derivatives O MeO MeO O NO2 NH3 CO2 HO O O OAc Cl O O O OAc 1 !-DMNB-glu Cmpd 1 2 3 "(365nm) 0. Wang. Dore. Natl. T. Callaway. 1193-1200. Denk.025 0. M..07 ± 0. J.13 ± 0. S. S.. Acad.34 ± 0...001 0. W. Dantzker. Proc. J.. L. R..01 2 !u(740nm (GM)) 3 !u(800nm (GM)) 1.07 0. M. W.02 Furuta. Tsien.. Sci.05 1.07 ± 0. 1999. 96. T.006 0. E.

Tsien.065 0. W.01 3.. Callaway.96 ± 0.019 !u(740nm (GM)) 1.99 ± 0..019 0.42 ± 0.24 0. 96.11 0.Y. T. .89 ± 0. Wang. Sci.06 Furuta. J. L.09 0.37 ± 0..1 ± 0.21 !u(800nm (GM)) 0.42 ± 0.. Natl.95 ± 0. S..Coumarin Derivatives OAc Br O O O Br O Br 5 O OAc Br O 4 NH3 CO2 Br O O Br O O O O 6 O O O H N CO2 CO2 O 7 Bhc-glu Cmpd 4 5 6 7 !(365nm) 0. W. 1999. Acad. E..05 0.2 0. Proc. Bybee. T.037 0.. J. R. Dore.. 1193-1200. Denk. Dantzker. M. M. S.

. M. T. J. 1193-1200. . Bybee. Tsien. S. 96. Natl. E. W. M.. Acad.Y. W.Coumarin Derivatives 100 80 60 (%) 40 != 1 "!7 20 0 0 10 20 30 Time (min) 40 50 60 O MeO MeO O NO2 NH3 CO2 Br O O O O O H N CO2 CO2 1 !-DMNB-glu 7 Bhc-glu Furuta.. Sci. J.. L. T. Dantzker. Denk. Wang. 1999.. Dore. Proc.. Callaway.. R.. S.

Geisser. Bendig.. Hagen.. Phys. .. R... 5768-5774.V. J. J. Chem. 111.Coumarin Derivatives .Mechanism of Cleavage OR H C H OR 333nm H3CO CH2 O O H3CO O O SET CH2OH OR H3CO O O H2O H3CO O O HOR Schmidt. D. 2007. A.

Conclusions • Usage is an attractive alternative to classical protecting groups • Technique is still in its infancy and further optimization is required before wide spread usage systems tumors • Has the potential to revolutionize drug delivery • Combined application with fiber optics to target .

Dan. Roozbeh. Wenjing. Atefeh. Petr Klan • Toyin. Ned Jackson • Dr. Arvind.Acknowledgements • Dr. Chryssoula . Babak Borhan • Dr. • All of you for your attention Mercy. Xiaoyong. Sarah. Aman. Camille. Calvin. Sing. Xiaofei.