photolabile protecting groups (plpg): synthetic …...requirements for plpgs •a functionalized...
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Photolabile Protecting Groups (PLPG): Synthetic and Biological Applications
Carmin BurrellOctober 15, 2008
First Examples: Cbz-glycine
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.
HN
O
OHO
O H2NOH
OH2, Pd/C
orLi°, NH3
orHBr
HN
O
OHO
O H2N OH
Ohν (254 nm)
Med. P Hg lamp
30 min, H2O75%
First Examples: o-Nitrobenzylesters
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.
NO2
H
O
acetonehν (>280nm)
Med. P Hg LampPyrex Filter
NO
OH
O
NO2
H
O
O2N
H
O
hν
(>280nm)
NO2
H
O
O2N
H
O
NO2
H
O
NO
H
OHHO
NO
OH
O
First Examples: o-Nitrobenzylesters
Barltrop, J.A.; Plant, P. J.; Schofield, P.; Chem Comm, 1966, 22, 822-823.
R
OHNO2
R'
O
CCl4hν (>280nm)
Med. P Hg lampPyrex Filter
HO
O
R'
N O
O
R
R R' Time(hr) Yield
H Ph 2.5 17
Ph Ph 2.5 90
Ph C15H31 3.0 95
Ph CH2Ph 4.0 87
Mechanism of Deprotection
Barton, D. H. R.; Chow, Y. L.; Cox, A.; Kirby, G. W.; Tetrahedron Lett., 1962, 23, 1055-1057.
NO
OO
R
N O
O
H
R
NOH
O
CR
OHNO
hνR'
OH
O R'
O
O R'
O
OR'
O
H
transfer HO R'
ON O
R
OR
Excitation - Jablonski Diagram
• Excitation promotes a HOMO electron to the LUMO
• Rearrangement occurs to cleave the PLPG
Laimgruber, S.; Schreier, W. J.; Schrader, T.; Koller, F.; Zinth, W.; Gilch, P.; Angew. Chem. Int. Ed. 2005, 44, 7901-7904.
Excitationfemto(10-15s)
InternalconversionVibrational relaxation
Intersystem crossing
Fluorescencenano (10-9s)
Phosphorescence≥micro (10-6s)
pico 10-12sS2
S1
S0
T1
Applications of PLPGs - Synthesis of (-)-Diazonamide A
HN
O
Me Me
HN
OHO
Me MeN
O
O NH NH
O
N Cl
Cl
(-)-Diazonamide A
Snider, B. B.; Busuyek, M. V.; Tetrahedron, 2001, 57, 3301-3307.Li, J.; Jeong, S.; Esser, L.; Harran, P. G.; Angew. Chem. Int. Ed. 2001, 40, 4765-4769.
Li, J.; Jeong, S.; Esser, L.; Harran, P. G.; Angew. Chem. Int. Ed. 2001, 40, 4765-4769.
HN
O
Me Me
HN
OHO
Me MeN
O
O NH NH
O
N Cl
Cl
(-)-Diazonamide A
Applications of PLPGs - Synthesis of (-)-Diazonamide A
HN
OCbzHN
MeMe
O
N
OHOPh
CN
OHBr
o-NO2C6H4CH2Br
K2CO3, NaI, DMF52%
HN
OCbzHN
MeNa
O
N
OHOPh
CN
OBr
NO2
Applications of PLPGs - Synthesis of (-)-Diazonamide A HN
O
Me Me
HN
OHO
Me MeN
O
O NH NH
O
N Cl
Cl
(-)-Diazonamide A
Li, J.; Jeong, S.; Esser, L.; Harran, P. G.; Angew. Chem. Int. Ed. 2001, 40, 4765-4769.
HN
OCbzHN
MeMe
O
N
OHOPh
CN
OBr
NO2
Cl3CCO2H
HN
OCbzHN
MeMe
O
N
OPh
OBr
NO2
O
O
Applications of PLPGs - Synthesis of (-)-Diazonamide A
Li, J.; Jeong, S.; Esser, L.; Harran, P. G.; Angew. Chem. Int. Ed. 2001, 40, 4765-4769.
HN
O
Me Me
HN
OHO
Me MeN
O
O NH NH
O
N Cl
Cl
(-)-Diazonamide A
HN
OCbzHN
MeMe
O
N
OPh
OBr
NO2
O
O
NH
NHAlMe2
1.3 eq, toluene/CH2Cl20°C→RT
83% (2 steps)
HN
OCbzHN
MeMe
O
N
OHOPh
OBr
NO2
HN
O
NH
Applications of PLPGs - Synthesis of (-)-Diazonamide A
Li, J.; Jeong, S.; Esser, L.; Harran, P. G.; Angew. Chem. Int. Ed. 2001, 40, 4765-4769.
HN
O
Me Me
HN
OHO
Me MeN
O
O NH NH
O
N Cl
Cl
(-)-Diazonamide A
HN
OCbzHN
MeMe
O
N
OHOPh
OBr
NO2
HN
O
NH
5 mol% nPr4NRuO41.5 eq NMO
4 Å MS, CH2Cl278%
HN
OCbzHN
MeMe
O
N
OOPh
OBr
NO2
HN
O
NH
Li, J.; Jeong, S.; Esser, L.; Harran, P. G.; Angew. Chem. Int. Ed. 2001, 40, 4765-4769.
HN
O
Me Me
HN
OHO
Me MeN
O
O NH NH
O
N Cl
Cl
(-)-Diazonamide A
Applications of PLPGs - Synthesis of (-)-Diazonamide A
HN
OCbzHN
MeMe
O
N
OOPh
OBr
NO2
HN
O
NH
350nm, 3x10-2Mdegassed dioxane
excess Ac2O, py,DMAP, CH2Cl2, 85%
HN
OCbzHN
MeMe
O
N
OPh
OBr
HN
O
NH
OAc
Requirements for PLPGs
• A Functionalized Aromatic System
• Substituents can allow for use of a specific wavelength radiation
• Stabilize the radicals or ions created after excitation
• Fast Rate of Cleavage
• Reduce the time needed for deprotection
• High quantum yields
Photolabile Protecting Groups (PLPGs)
Wang, P.; Wang, Y.; Hu, H.; Spencer, C.; Liang, X.; Pan, L. J. Org. Chem. 2008, 73, 6152-6157.
Kitani. S.; Sugawara, K.; Tsutsumi, K.; Morimoto, T.; Kakiuchi, K.; ChemComm.
2008, 2103-2105.
Furuta, T.; Wang, S. S.; Dantzker, J. L.; Dore, T. M.; Bybee, W. J.; Callaway, E. M.; Denk, W.; Tsien, R. Y.; Proc. Natl.
Acad. Sci. 1999, 96, 1193-1200.
MeO
OMe
O O
PhPh S
O
OO O
R R'
OR
O
O
OR
OMe
MeO
NO2 O
MeO
MeO MeO
OR
O
OR
Pirrung, M.C.; Bradley, J. C., J. Org. Chem., 1995, 60,
1116-1117.
Requirements for PLPGs
OMe N NO2
π→π∗ λmax (nm) 184 217 251 252n→π* λmax (nm) 269 299 330
Lambert, J. B.; Shurvell, H. F.; Lightner, D. A.; Cooks, R. G.; Organic Structural Spectroscopy, Prentice-Hall: New Jersey,1998, 289.
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
Wang, P.; Wang, J.; Hu, H.; Spencer, C.; Liang, X.; Pan, L. J. Org. Chem. 2008, 73, 6152-6157.Anslyn, E. V.; Dougherty, D. A.; Modern Physical Organic Chemistry; University Science Books: Sausalito,
California, 2006, 971.
OMe
O OH Ph
MeO
OMe
O O
PhPh
H Ph
MeO
2Φ = 0.03
3Φ = 0.17
Chlorophyll Φ= 0.25
Rhodopsin Φ= 0.66
Requirements for PLPGsNumber of molecules that undergo cleavage
Total number of photons absorbedQuantum Yield = Φ =
0 < Φ < 1
Sheenan, J.C.; Wilson, R. M., J. Am. Chem. Soc., 1964, 86, 5277-5281.Sheenan, J. C.; Wilson, R. M.; Oxford, A. W, J. Am. Chem. Soc., 1971, 93, 7222-7228
Dimethoxybenzoin Carbonates
Φ= 0.64
MeO
OMe
O
OAc MeCNhν (360nm)
Rayonet reactorpyrex filters
7min, N2
O
MeO
OMe
HOAc
99%
O
X
R2
R1
R3
R4
benzenehν (>280nm)
Med. P Hg lampPyrex Filter
OR2
R1 R3
R4
R1 R2 R3 R4 X Time (hr) Yield H H H H OAc 17 15 H H H H Cl 18 1 OMe H OMe H OAc 17 46
Pirrung, M.C.; Bradley, J. C., J. Org. Chem., 1995, 60, 1116-1117.
Dimethoxybenzoin Carbonates
MeO
OMe
O
X
OH SH
MeO
OHOH
benzenehν (350nm)
Rayonet reactor1h, Ar
O
MeO
OMe
HX
88% 95% 90% 94%
Boudebous, H.; Košmrlj, B.; Šket, B.; Wirz, J., J. Phys. Chem. A., 2007, 111, 2811-2813.
Dimethoxybenzoin Carbonates
MeO
OMe
O
OAcMeO
OMe
O
OAc
O
MeO
OMe
OAc
O
MeO
OMeH
O
MeO
OMe
HOAc
hν
New PLPGs - Thiochromone S,S Dioxide
Kitani, S.; Sugawara, K.; Tsutsumi, K.; Morimoto, T.; Kakiuchi, K. Chem. Comm. 2008, 18, 2103-2105.
S
O
O OR
OS
O
O OR
O
S
O
O OEt
O
hν (λ >280nm)ultra-high P Hg lamp
pyrex filter
CD3OD (2.0X10-2M)0.5h
S
O
O OEt
O>99%
Kitani, S.; Sugawara, K.; Tsutsumi, K.; Morimoto, T.; Kakiuchi, K. Chem. Comm. 2008, 18, 2103-2105.
New PLPGs - Thiochromone S,S Dioxide
S
O
OO O
OEt
O
EtOH>99%
hν (λ >280nm)ultra-high P Hg lamp
pyrex filter
CD3OD (2.0X10-2M)0.5h
Kitani, S.; Sugawara, K.; Tsutsumi, K.; Morimoto, T.; Kakiuchi, K. Chem. Comm. 2008, 18, 2103-2105.
New PLPGs - Thiochromone S,S Dioxide
S
O
OO O
XR
OS
O
OO O
XR
O
S
O
O OO XR
O S
O
O O
CO2 HXR
H
hν (λ >280nm)
CD3OD
S
O
O OR
OS
O
O OR
O
Kitani, S.; Sugawara, K.; Tsutsumi, K.; Morimoto, T.; Kakiuchi, K. Chem. Comm. 2008, 18, 2103-2105.
New PLPGs - Thiochromone S,S Dioxide
S
O
OO O
O
O
CD3OD (2.0X10-2M)R S
O
O O
ROH
Alcohol Irradiation Time(h) Yield(%)EtOH 0.5 >99C8H17OH 0.5 >99
0.5 >99OH
1.0 97OH
OH 1.0 98
OH1.0 >99 (98)
OMeMe
HOH H
1.0 >99 (99)
O OMe
OAcAcOOAc
HO 0.5 >99 (99)
Alcohol Irradiation Time(h) Yield(%)
hν (λ >280nm)ultra-high P Hg lamp
pyrex filter
Kitani, S.; Sugawara, K.; Tsutsumi, K.; Morimoto, T.; Kakiuchi, K. Chem. Comm. 2008, 18, 2103-2105.
New PLPGs - Thiochromone S,S Dioxide
S
O
OO O
N
O
CD3OD (2.0X10-2M)R S
O
O O
R'
R NH
R'
1.0 97
1.0 98
1.0 97
Amine Irradiation Time/h Yield(%)
C4H9NH2
Et2NHNH2
CO2Bn
hν (λ >280nm)ultra-high P Hg lamp
pyrex filter
Kitani, S.; Sugawara, K.; Tsutsumi, K.; Morimoto, T.; Kakiuchi, K. Chem. Comm. 2008, 18, 2103-2105.
New PLPGs - Thiochromone S,S Dioxide
S
O
OO O
R
O
CD3OD (2.0X10-2M)RCOOH
S
O
O O
RCOOH Irradiation Time/h Yield(%)
C11H23COOH 1.0 >99
C17H35COOH 1.0 >99
1.0 >99COOH
COOH 1.0 >99
hν (λ >280nm)ultra-high P Hg lamp
pyrex filter
New PLPGs - Ketones and AldehydesGeneration 1
• Ortho - meta effects on the excited state facilitate heterolytic cleavage
Zimmerman, H. E.; J. Am. Chem. Soc., 1995, 117, 8988-8991Wang, P.; Hu, H.; Wang, Y.; Org. Lett., 2007, 9, 2831-2833.
OMe
O OH H
OMe
O OH H
MeOO OH H
S1 S0
-0.181 -0.159
S1 S0
-0.193 -0.143
S1 S0
-0.168 -0.172
δ- δ- δ-
Mulliken Charges
Gaussian 03, Revision C.02 CIS/3-21G for excited states
HF/3-21G for geometry optimization
Excited State - Ortho - Meta Effect
Zimmerman, H. E., J. Am. Chem. Soc.,1995, 117, 8988-8991.
MO 1
MO 3
MO 5
MO 7
MO 2
MO 4
MO 6
New PLPGs - Ketones and AldehydesGeneration 1
• Ortho - meta effects on the excited state facilitate heterolytic cleavage
Zimmerman, H. E.; J. Am. Chem. Soc., 1995, 117, 8988-8991Wang, P.; Hu, H.; Wang, Y.; Org. Lett., 2007, 9, 2831-2833.
OMe
O OH H
OMe
O OH H
MeOO OH H
S1 S0
-0.181 -0.159
S1 S0
-0.193 -0.143
S1 S0
-0.168 -0.172
δ- δ- δ-
Mulliken Charges
Gaussian 03, Revision C.02 CIS/3-21G for excited states
HF/3-21G for geometry optimization
OMe
OCH2O
R1 R2
MeO
Wang, P.; Hu, H.; Wang, Y.; Org. Lett., 2007, 9, 2831-2833.
New PLPGs - Ketones and AldehydesGeneration 1
O OH
OMe
O OH
OMe
O OH
MeO
Ph
Ph
Ph
hν (>280nm)Med P Hg lamp
pyrex sleeve
CD3CN/D2O (9:1)1hr
H Ph
O
H Ph
O
H Ph
O
50%
8%
hν (>280nm)Med P Hg lamp
pyrex sleeve
CD3CN/D2O (9:1)1hr
hν (>280nm)Med P Hg lamp
pyrex sleeve
CD3CN/D2O (9:1)1hr
Wang, P.; Hu, H.; Wang, Y.; Org. Lett., 2007, 9, 2831-2833.
New PLPGs - Ketones and AldehydesGeneration 1
R1
O
R2
hν (>280nm)Med. P Hg lamp
pyrex filterMeCN/H2O (4:1)
OMe
OH OHMeO
10 mol%, p-TsOH
OMe
O OR1 R2
MeO
Ph CHO
CHO
OPh MeO
O
O
16
Protection Yield: 92 99 93 91
Deprotection Yield: 81 79 86 93
Time (h): 4.0 0.5 3.0 3.5
Wang, P.; Hu, H.; Wang, Y.; Org. Lett., 2007, 9, 1533-1535.
New PLPGs - Ketones and AldehydesGeneration 2
O
MeO
OCHO
OMe
8
Protection Yield: 99 99 91 93
Deprotection Yield: 90 89 86 84
Time (min): 60 60 60 80
OMe
OH OH
PhPh
OMe
O OR1 R2
PhPh
Ph CHO
R1
O
R2
hν (>280nm)Med. P Hg lamp
pyrex filterMeCN
10 mol%, p-TsOHOMe
O OR1 R2
PhPh
Wang, P.; Wang, J.; Hu, H.; Spencer, C.; Liang, X.; Pan, L. J. Org. Chem. 2008, 73, 6152-6157.
New PLPGs - Ketones and AldehydesOptimized System
OMe
OH OH
PhPh
MeOOH OH
OMe
1 2
OMe
MeOOH OH
PhPh
3
PPG Φ protection yield (%) deprotection yield (%) time (min)
1 0.03 92 81 2402 0.11 99 90 603 0.17 99 92 30
New PLPGs - Ketones and AldehydesOptimized System
Wang, P.; Wang, J.; Hu, H.; Spencer, C.; Liang, X.; Pan, L. J. Org. Chem. 2008, 73, 6152-6157.
OMe
OH OHMeO
OMe
O OR1 R2
MeO
Ph CHO
CHO
OPh MeO
O
O
16
Protection Yield: 99 93 99 99
Deprotection Yield: 92 82 88 91
Time (h): 30 44 25 25
PhPh PhPh
R1
O
R2
hν (>280nm)Med. P Hg lamp
pyrex filterMeCN/H2O 10%(v/v)
10 mol%, p-TsOH
Wang, P.; Wang, J.; Hu, H.; Spencer, C.; Liang, X.; Pan, L. J. Org. Chem. 2008, 73, 6152-6157.
New PLPGs - Ketones and AldehydesOptimized System
OMe
MeOO OR1 R2
PhPh
hν
OMe
MeOO OR1 R2
PhPh
R1 R2O
OMe
MeOO
Ph
Ph
H2O
hν-H2O
OMe
MeOOH OH
PhPh
Wang, P.; Wang, J.; Hu, H.; Spencer, C.; Liang, X.; Pan, L. J. Org. Chem. 2008, 73, 6152-6157.
OMe
O O
PhPh
H Ph
MeO
New PLPGs - Ketones and AldehydesOptimized System
Reagent Solvent Conditions %RecoveryPhLi THF -78 to 23°C, 6h 100LiAlH4 THF 23°C, 24h 100NaBH4 THF 23°C, 24h 100H2, Pd/C THF 23°C, 18h 100t-BuOK MeCN 23°C, 24h 100 80°C, 2h 100DDQ MeCN 23°C, 24h 100 80°C, 2h 94CAN MeCN 23°C, 24h 0 80°C, 2h 0AcOH MeCN 23°C, 24h 100 80°C, 2h 100TFA MeCN 23°C, 24h 100 80°C, 2h 91HCl (37%) MeCN 23°C, 24h 100 80°C, 2h 65pTsOH MeCN 23°C, 24h 47 80°C, 2h 0
New PLPGs - Ketones and AldehydesAmino Acetal
Wang, P.; Wang, J.; Hu, H.; Spencer, C.; Liang, X.; Pan, L. J. Org. Chem. 2008, 73, 6152-6157.
R1
R2
O OH Ph
H Ph
O
R1 R2 Yield Time Φ λmaxN(Me)2 H 99 16 0.13 327 OMe OMe 92 30 0.17 297
hν (>280nm)
Med. P Hg lamppyrex filter
Wang, P.; Wang, J.; Hu, H.; Spencer, C.; Liang, X.; Pan, L. J. Org. Chem. 2008, 73, 6152-6157.
Orthogonality Using PLPGs
N
O O
PhPh
OMe
O O
PhPh
H Ph
MeO
H Ph
hνMeCN/
H2O (1:1)
OMe
O O
PhPh
H Ph
MeO H Ph
O
350nm, 20min: 98% >89%
sunlight, 3h : 98% >96%
Kitani, S.; Sugawara, K.; Tsutsumi, K.; Morimoto, T.; Kakiuchi, K. Chem. Comm. 2008, 18, 2103-2105.
Orthogonality Using PLPGs
S
O
O O
OO O
OS
O
O O hν (λ >280nm)
CD3OD, 0.5h99%
S
O
O O
OOH
S
O
O O
CO2
Orthogonality Using PLPGs
Kitani, S.; Sugawara, K.; Tsutsumi, K.; Morimoto, T.; Kakiuchi, K. Chem. Comm. 2008, 18, 2103-2105.
mCPBA85%
S
O
O O
OOH
O O
hν (λ >280nm)
CD3OD, 0.5h99%
HO OHS
O
O O
CO2
>99% 85%
S
O
O O
OOH
Orthogonality Using PLPGs
Blanc, A.; Bochet, C. G., J. Org. Chem., 2002, 67, 5567-5577.
OMe
MeO O
O O
O
O
O2N OMe
OMen
hν, thenCH2N2
420 nm254 nm
MeO
O
O
O
O2N OMe
OMen
OMe
MeO O
O O
OMe
On
n 254 nm 420 nm (%) (%)
0 86 671 94 832 94 853 96 854 78 875 92 70
Summary of Uses of PLPGs in Synthesis• o-Nitrobenzyl is most widely used and has been
applied to total syntheses
• Thiochromone systems could be an alternative although optimization required
• Acetal protection system for aldehydes and ketones is groundbreaking
• Proof of orthogonality makes PLPG more practical
R
OHNO2
R'
O
S
O
OO O
O
OR
OMe
MeOO OR1 R2
PhPh
Caged Biomolecules
• PLPG inactivates the biomolecule until irradiated
• Biomolecules that have been caged include:
• neurotransmitters, ATP, Pi, Glutamate
• An effective method to elucidate cellular functions of biomolecules
• Release occurs on a biologically relevant time scale as compared to diffusion (temporal resolution)
• Release can be controlled to a specific area (spacial resolution)
Furuta, T.; Wang, S. S.; Dantzker, J. L.; Dore, T. M.; Bybee, W. J.; Callaway, E. M.; Denk, W.; Tsien, R. Y.; Proc. Natl. Acad. Sci. 1999, 96, 1193-1200.
Caged Biomolecules - C. Elegans
Li, H.; Avery, L.; Denk, W.; Hess, G. P., Proc. Natl. Acad. Sci. USA, 1997, 94, 5912-5916.
Caged Biomolecules - C. Elegans
Li, H.; Avery, L.; Denk, W.; Hess, G. P., Proc. Natl. Acad. Sci. USA, 1997, 94, 5912-5916.
NO2
COO-
O
O
NH3+
COO-
NO
COO-
O HO
OCOO-
NH3+hν
Light Triggered Molecule-Scale Dosing
McCoy, C. P.; Rooney, C.; Edwards, C. R.; Jones, D. S.; Gorman, S. P.; J. Am. Chem. Soc. 2007, 129, 9572-9573.
O
O
OCH3
OCH3O
O
O
OCH3
OCH3O
O
O
OCH3
OCH3O
O O
OAcetylsalicylic acid Ibuprofen
Ketoprofen
Light Triggered Molecule-Scale Dosing
OOCH3
OCH3
O
O
OCH3
OCH3O
Ibuprofen
hν (365 nm)Hg lamp
quartz cuvette
HO
O
McCoy, C. P.; Rooney, C.; Edwards, C. R.; Jones, D. S.; Gorman, S. P.; J. Am. Chem. Soc. 2007, 129, 9572-9573.
0
30
60
90
0 10 20 30 40
Yie
ld o
f Ib
upro
fen
(%)
Time (min)
Off
Off
Off
Off
OffOff
On
On
On
On
OnOn
Taxol® Prodrug
Skwarczynski, M.; Sohma, Y.; Noguchi, M.; Kimura, M.; Hayashi, Y.; Hamada, Y.; Kiso, Y.; Kimura, T.; J. Med. Chem. 2005, 48, 2655-2666.
OO
O
O
O
HO
O
OH
OH
O
OO
O O
H2N
OHO
O
O
HO
O
OH
OH
O
OO
O O
NH
O
Isotaxel0.45 mg/mL
Taxol®2.5 x 10-4 mg/mL
Skwarczynski, M.; Noguchi, M.; Hirota, S.; Sohma, Y.; Kimura, T.; Hayashi, Y.; Kiso, Y.; Bioorg. Med. Chem. Lett. 2006, 16, 4492-4496.
ON O
O
NHO
OO
O
O
O
HO
O
OH
OH
O
OO
O O
HCl
OO
O
O
O
HO
O
OH
OH
O
OO
O O
H2N
Isotaxel0.45 mg/mL
430 nm
diode laser
Phototaxel< 2.5 x 10-4 mg/mL
Taxol® Prodrug
0
20
40
60
80
100
0 5 10 15 20 25 30
Time (min)
(%)
Skwarczynski, M.; Noguchi, M.; Hirota, S.; Sohma, Y.; Kimura, T.; Hayashi, Y.; Kiso, Y.; Bioorg. Med. Chem. Lett. 2006, 16, 4492-4496.
ON O
O
NHO
OO
O
O
O
HO
O
OH
OHO
OO
O O
HCl
2
OHO
O
O
HO
O
OH
OHO
OO
O O
NH
O
1
ON O
OH5
Taxol® Prodrug
= 1= 2= 5
Skwarczynski, M.; Noguchi, M.; Hirota, S.; Sohma, Y.; Kimura, T.; Hayashi, Y.; Kiso, Y.; Bioorg. Med. Chem. 2008, 16, 5389-5397.
Taxol® Prodrug
ON O
O
NHO
OO
O
O
O
HO
O
OH
OH
O
OO
O O
NHO
N
NH
ON
3 HCl
OO
O
O
O
HO
O
OH
OH
O
OO
O O
H2N
Isotaxel0.45 mg/mL
365 nm
UV lamp
Phototaxel 2> 100mg/mL
0
20
40
60
80
100
0 10 20 30 40 50 60
Time (min)
(%)
Skwarczynski, M.; Noguchi, M.; Hirota, S.; Sohma, Y.; Kimura, T.; Hayashi, Y.; Kiso, Y.; Bioorg. Med. Chem. 2008, 16, 5389-5397.
ON O
O
NHO
OO
O
O
O
HO
O
OH
OH
O
OO
O O
NHO
N
NH
ON
3 HCl
Phototaxel
OHO
O
O
HO
O
OH
OHO
OO
O O
NH
O
Taxol®
Taxol® Prodrug
= Taxol®
= Phototaxel
UV Damage
• Preceding PLPGs required 280 - 365nm radiation for cleavage
Matsumura, Y.; Ananthaswamy, H. N.; Toxicol. Appl. Pharmacol. 2004, 195, 298-308.
X-Rays Visiblelight
100 150 200 250 300 350 400Wavelength (nm)
Ultraviolet radiation
UVC UVB UVA
UV Damage
Matsumura, Y.; Ananthaswamy, H. N.; Toxicol. Appl. Pharmacol. 2004, 195, 298-308.
Acute UV
Sunburn, Suntan, Epidermal Hyperplasia
DNA damage
Immune Suppression
p53 induction
Cell cycle controlApoptosis
DNA Repair
Maintenance of genomic stabilityDNA replication and repairAngiogenesis inhibition
Chronic UV
Skin aging (Photoaging)
DNA damage accumulation
Immune suppression
Genetic mutations
Skin carcinogenesis
Two Photon Excitation
• Maria Göppert-Mayer first described non-linear optics - 1931
• Theodore Maiman developed the ruby laser -1960
• W. Kaiser and C. B. G. Garrett observed two photon excited fluorescence in CaF2:Eu2+ crystals- 1961
• Janis Valdemanis and R. L. Fork described multiphoton microscopy - 1980s
• D. E. Spence, P. N. Kean and W. Sibbert developed Ti-sapphire femtosecond laser - 1990
Masters, B.R.; So, P. T. C., Microscopy Research and Technique, 2004, 63, 3-11.
Two Photon Excitation
Oheim, M.; Michael, D. J.; Geisbauer, M.; Madsen, D.; Chow, R. H.; Adv. Drug Deliv. Rev., 2006, 58, 788–808
S0
S1
S2
T1
Exci
tatio
n
Fluo
resc
ence
Phos
phor
esce
nce
IC
ISC
UVOne Photon Excitation
S0
S1
S2
T1
Exci
tatio
n
Fluo
resc
ence
Phos
phor
esce
nce
IC
ISC
IRTwo Photon Excitation
VirtualState
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.; Adv. Drug Deliv. Rev., 2006, 58, 770-772.
Furuta, T.; Wang, S. S.; Dantzker, J. L.; Dore, T. M.; Bybee, W. J.; Callaway, E. M.; Denk, W.; Tsien, R. Y.; Proc. Natl. Acad. Sci. 1999, 96, 1193-1200.
Released Biomolecule PLPGCaged
Biomolecule
Two Photon ExcitationOne Photon Two Photon
UV IR
Two Photon Excitation
Oheim, M.; Michael, D. J.; Geisbauer, M.; Madsen, D.; Chow, R. H.; Adv. Drug Deliv. Rev., 2006, 58, 788–808.
Two Photon Excitation
Oheim, M.; Michael, D. J.; Geisbauer, M.; Madsen, D.; Chow, R. H.; Adv. Drug Deliv. Rev., 2006, 58, 788–808.
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.; Adv. Drug Deliv. Rev., 2006, 58, 770-772.
Two Photon Excitation
Denk, W.; Helmchen, F.; Nature Methods, 2005, 12, 932-940.
One Photon Two Photon UV IR
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.; Adv. Drug Deliv. Rev., 2006, 58, 770-772.
Two Photon Excitation
• Confocal microscopy depth in brain tissue: 50 - 100μm
• Used for imaging of slices of the brain tissue
• Two Photon Excitation depth in brain tissue: up to 1mm
• Could lead to excitation deeper in a tumor
Denk, W.; Helmchen, F.; Nature Methods, 2005, 12, 932-940.
Coumarin Derivatives
Givens, R. S.; Matuszewski, B., J. Am. Chem. Soc., 1984, 106, 6860-6861.Furuta, T.; Toriagai, H.; Sugimoto, M.; Iwamura, M., J. Org. Chem., 1995, 60, 3953-3956.
OH3CO O
CH2OP(OC2H5)2
O
hν (360nm)
Nu OH3CO O
CH2Nu
Nu = CH3OH, piperidine, cysteine, tyrosine, α-chymotrypsin
OH3CO O
PO
O
O
O OO
N
NN
N
NH2
PO
O
O
O OHO
N
NN
N
NH2
cAMP
Dioxane/Water (1:1)hν (340nm)
Xe lamp1h, Ar, 90%
Furuta, T.; Wang, S. S.; Dantzker, J. L.; Dore, T. M.; Bybee, W. J.; Callaway, E. M.; Denk, W.; Tsien, R. Y.; Proc. Natl. Acad. Sci. 1999, 96, 1193-1200.
Coumarin Derivatives
MeO
MeO
O
OCO2
NH3NO2
1 α-DMNB-gluO
OAc
HO O2
O
OAc
O O3
Cl
Cmpd Φ(365nm) ∂u(740nm (GM)) ∂u(800nm (GM))1 0.006
2 0.025 1.07 ± 0.05 0.13 ± 0.001
3 0.01 1.07 ± 0.07 0.34 ± 0.02
Cross Sectional Area of Uncaging (δu)
• Measures the efficiency of the chromophore to absorb two photons
• 1δu = 10-50 cm4s/photon = 1GM
• Dependent on:
• Cross-sectional area of the chromophore
Albota, M., et. al.; Science, 1998, 281, 1653-1656.
Furuta, T.; Wang, S. S.; Dantzker, J. L.; Dore, T. M.; Bybee, W. J.; Callaway, E. M.; Denk, W.; Tsien, R. Y.; Proc. Natl. Acad. Sci. 1999, 96, 1193-1200.
Coumarin Derivatives
MeO
MeO
O
OCO2
NH3NO2
1 α-DMNB-gluO
OAc
HO O2
O
OAc
O O3
Cl
Cmpd Φ(365nm) ∂u(740nm (GM)) ∂u(800nm (GM))1 0.006
2 0.025 1.07 ± 0.05 0.13 ± 0.001
3 0.01 1.07 ± 0.07 0.34 ± 0.02
Furuta, T.; Wang, S. S.; Dantzker, J. L.; Dore, T. M.; Bybee, W. J.; Callaway, E. M.; Denk, W.; Tsien, R. Y.; Proc. Natl. Acad. Sci. 1999, 96, 1193-1200.
Coumarin Derivatives
Cmpd Φ(365nm) ∂u(740nm (GM)) ∂u(800nm (GM))4 0.037 1.99 ± 0.09 0.42 ± 0.01
5 0.065 0.96 ± 0.05 3.1 ± 0.2
6 0.019 0.89 ± 0.24 0.42 ± 0.11
7 0.019 0.95 ± 0.21 0.37 ± 0.06
O
OAc
O O4
Br
O
OAc
O O5
Br
Br
Br
OO
Br
OHN
O
CO2
O
CO2
7 Bhc-gluOO
Br
O
OCO2
O
NH3
6
Coumarin Derivatives
Furuta, T.; Wang, S. S.; Dantzker, J. L.; Dore, T. M.; Bybee, W. J.; Callaway, E. M.; Denk, W.; Tsien, R. Y.; Proc. Natl. Acad. Sci. 1999, 96, 1193-1200.
= 1=7
0
20
40
60
80
100
0 10 20 30 40 50 60
Time (min)
(%)
MeO
MeO
O
OCO2
NH3NO2
1 α-DMNB-gluOO
Br
OHN
O
CO2
O
CO2
7 Bhc-glu
Coumarin Derivatives - Mechanism of Cleavage
Schmidt, R.; Geisser, D.; Hagen, V.; Bendig, J., J. Phys. Chem. A., 2007, 111, 5768-5774.
OH3CO O
OR
OH3CO O
C OR
OH3CO O
CH2OH
HOR
333nmHH
OH3CO O
CH2H2OOR
SET
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
• Has the potential to revolutionize drug delivery systems
• Combined application with fiber optics to target tumors
Acknowledgements
• Dr. Babak Borhan
• Dr. Ned Jackson
• Dr. Petr Klan
• Toyin, Aman, Arvind, Camille, Dan, Calvin, Xiaoyong, Mercy, Roozbeh, Atefeh, Sarah, Sing, Wenjing, Xiaofei, Chryssoula
• All of you for your attention