1
Photoremovable Protecting Groups:
How fast are they really?
Hassen Boudebous, Dominik Heger, Bruno Hellrung, Yavor Kamdzhilov,
Lubica Klíčová, Marek Mac, Pavel Müller, Markus Schwörer, Peter
Šebej,
Tomáš Šolomek, Jürgen Wintner
Richard Givens, Thomas Kiefhaber, Petr Klán
Photoremovable Protecting Groups:
How fast are they really?
Hassen Boudebous, Dominik Heger, Bruno Hellrung, Yavor Kamdzhilov,
Lubica Klíčová, Marek Mac, Pavel Müller, Markus Schwörer, Peter
Šebej,
Tomáš Šolomek, Jürgen Wintner
Richard Givens, Thomas Kiefhaber, Petr Klán
2
Photoremovable Protecting Groups, Phototriggers, Caged Compounds
Goeldner, Givens, Dynamic Studies in Biology, Wiley-VCH, 2005
3
Activation of an ionotropic receptor with GABA
Kramer, Nat. Methods 5, 2008, 331.
Remote control of ion channels
4
D. Gust, Acc. Chem. Res. 2001, 34, 40
Gust
P. Anfinrud, PNAS. 2010, 107, 7281
Time-resolved X-ray: Myoglobin-CO
X-ray scattering differences recorded over 8 decades of time: 100 ps to 10 ms
5
R. S. Givens, H. Bayley, J. Am. Chem. Soc. 2002, 124, 8220
Caged Protein Kinase A
Monitoring intracellular protein kinase activity
D. H. Lawrence,
JACS, 2003, 13358
6
Kiskin, Eur. Biophys. J. 2002, 30, 588
Caged fluorescers: Transport
phenomena, mixing, motion
flow
deep uv
laser pulse
Near uv lamp
Visible fluorescence
• Focused NIR-pulses of fs-duration
• Fluorescence excitation for imaging or
release of caged reagents in addressable
0.1 fl-volumes (3-d submicron resolution)
• Little radiation damage or scattering in
tissue
• Reagents diffuse out of volume within ~
ms: need fast release!
W.W. Webb, Science Biophys. J. 1999, 76, 489
M. Goeldner, Bioorg. Med. Chem. 2010, 18, 7753
I3-dep.
2- and 3-Photon Excitation
7
Light brings a new dimension to chemistry
Calculations. Rules-of-thumb. Guidelines.
O
O
OAc
CH2
hν
hν
O
OAc
O
8
“Mirror image of GS”
•
•
•
•
Rule: Opposite to ground state reactivity
• PMO (Dewar, Dougherty)
The photoproduct is
often less stable than
the starting material
Rule: Opposite to ground state reactivity
• Charge distributions (Zimmermann)
OH
OAc Δ or hν
OH
OH
+ HOAc
H2O
ground-state: ortho, para excited-state: meta
hν
9
Rule: Opposite to ground state reactivity
• Woodward-Hoffmann rules for pericyclic reactions
(retaining some symmetry elements along the
reaction path)
ground-state allowed
excited-state forbidden
excited-state allowed
ground-state forbidden
No
exceptions!
No
exceptions?
An even simpler use of symmetry:
• Electron count (Salem, Dauben, Turro)
hν
n,π*
A
S
A
O
H
H
H
O
H
H
H
•
• OH
10
Note: The path retaining symmetry is probably
not the best (not the most downhill)!
It is chosen merely because symmetry arguments
are so simple to use.
At geometries with no symmetry (C1), all
wavefunctions belong to the totally symmetric
representation. Do potential energy surfaces
of the same symmetry cross?
!
Adiabatic versus diabatic reaction paths (Förster)
class I
hot ground state reactions
(~never in solution,
common in gas phase)
class II
A B
B*
adiabatic reactions
(exceptional)
OH O–
+ H+hν
*1
H2O
class III
diabatic reactions
conical intersections
(common)
11
Markus Schwörer
Yuri Il‘itchev
Marek Mac
Caged ATP: The „gold standard“
Kaplan/ Trentham/ Corrie/ Mäntele/ Gerwert
JACS 110 (1988) 7170; 117 (1995) 10311; 119, 4149 (1997)
• Time resolved FTIR detection using 13C, 15N, 18O isotopomers
• Release of ATP single exponential, „synchronous“ with
aci-nitro anion decay
• kapp(ATP) ≈ 52 s–1 at pH 7 and 10 ˚C
CH3
NO2
O
O
O–
O
O
O–
O
O
O–
O
O
N
OH OH
N
N
N
NH2
CH3
NO
Ohν
+ ATP
12
Reaction mechanism: common wisdom
C
X
NO2
N
O
O
X
R
S1
C
X
N+
O–
O–
T1
C
NO
+ X –
hν
H
R
R
H
R
O
+ H+
aci-anion decay:
• via nitronic acid
D. R. Trentham, JACS 110, 1988, 7170
• cycliz‘n not reversible
• not necessarily ratedetermining
for release
o-Nitrotoluene: pH–rate profile
Helv. 84, 2001, 1441
13
o-Nitrobenzyl methyl ether: pH–rate profile
JACS 2004,
126, 4581
N+
MeO
O–
OH
N
O
MeO
O
H
NO
OH
– MeOH
MeO
NONO2
MeO
hν
O
aci-decay
hemi decay
aci-decay rate-
determining
cyclo decay
Aci ->isoxazole
∆A x 103
t / 100 ms
ϖ / cm–1
~
MeO
N
+
O–
OH
CD3CN CD3CN
N
O
OH
MeO
H
hν
1248 cm–1
1090 cm–1
k = 6.6 × 103
s–1
14
∆A x 103
t / s
ϖ / cm–1~
Hemiacetal->nitroso
N
O
OH
MeO
H
NO
MeO
HO
H
CD3CN
NO
O H
k = 4.5 × 10–2
s–1
1085 cm–1
1500 cm–1
1504 cm–1
1698 cm–1
not shown
–MeOH
1090 cm–1
k - 0.2 s–1
JACS 2004, 126, 4581
15
Mechanism of release
C
X
NO2
C
X
N+
O–
OH
C
NO
–HX –
hν
B–
R
RH
R
O
C
X
N+
O–
O–
R
+ H+
pKa - 3
general base cat.
specific acid cat.
N
O
O
X R
B– H
C
X
NO
R
OH
general base cat.
H2O
JACS 2004, 126, 4581
OH
N
+
O–
OH
ROH
NO2
R
O
N
OH
OH
RR
N
O
O–H
HO
O
NO
R
hν
path bR = H, Me
– H2O
path a
– H2O
o-Nitrobenzyl alcohol
Photochem. Photobiol. Sci. 2005, 4, 33
16
HO
N
+
O–
OH
CD3CN CD3CN
hν
1247 cm–1
1695 cm–1
(C=O)
k = 6.6 × 104
s–1
O
N
OH
OH
PPS, 2005, 4, 33
O
O
HO
N
COO–
COO–
O O
N
–
OOC
–
OOC
NO2
nitr-5
R. Y. Tsien, JACS 1988, 110, 321.
Caged Ca2+
17
NO2
X
NO2
X
OMe
MeO
Ph
O
X
Ph
O
X
OMe
OMe
O
X
O
O
X
O
X
OMeO O
X
HO O
N
OMe
HO
O
X
Rich Givens, Boris Sket
C. S. Rajesh
Hassen Boudebous
JACS 2000,
122, 611
Benzoin phosphates
250 300 350 400 450 500 550 600 650
0.00
0.04
0.08
0.12
λ / nm
² A
Ph
OO
Ph
D. L. Phillips,
Chem. Eur. J.
2007,13, 2290
18
JPC A 2007, 111, 2811.
Dimethoxybenzoin cages
Corrie, Wan, JOC, 1997, 62, 8278.
Phillips, Chem. Eur. J. 2010, 16, 5102.
Sheehan, JACS, 1971, 93, 7222.Sheehan, JACS, 1971, 93, 7222. Corrie, Wan, JOC, 1997, 62, 8278.
JPC A 2007, 111, 2811.
Picosecond pump–probe spectroscopy (CH3CN)
MeO
OMe
O
Ph
H
MeO
OMe
O
Ph
X
Two-wave appearance of released HX: k = 1 x 109 s–1, 1 x 106 s–1.
MeO
OMe
O
Ph
19
NO2
X
NO2
X
OMe
MeO
Ph
O
X
Ph
O
X
OMe
OMe
O
X
O
O
X
O
X
OMeO O
X
HO O
N
OMe
HO
O
X
Bruno Hellrung,
Jürgen Wintner,
Dominik Heger,
Rich Givens
p-Hydroxyphenacyl (pHP) diethylphosphate
20
JACS, 2000, 122, 9346,
JACS, 2008, 130, 3307.
p-Hydroxyphenacyl (pHP) diethylphosphate
HO
O
X
hν
H2O, air
–HX
HO
CO2H
O
O
H2O
X = OPO(OEt)2 O
– CO
HO
OH
H2O
Kresge et al., JACS, 2002,124, 6349
21
MP2/6-31G* (PCM acetonitrile)
log(k/s–1) = 13 – 4.0/2.3RT = 10
O O
O
O
O
•
•
+ CO
‡
O
O
•
•
T
S
‡
4
101.7
57.4
21.3
T1(1a)
44.3
6 3 5
O
O
O
+ CO
MP2 6-31G*
Ea (g) = 13.3 kcal/mol
Ea (“CH3CN”, PCM) = 4.0 kcal/mol
log(k/s–1) = 13 – 4.0/2.3RT = 10
22
D. L. Phillips, JACS, 2006, 128, 2558
ns-TRIR measurement: appearance of final product
p-Hydroxyphenacyl (pHP) diethylphosphate
23
Why are quantum yields << 1?
p-Hydroxyphenacyl (pHP) diethylphosphate
p-Hydroxyacetophenone
24
p-Hydroxyacetophenone
JACS 2000, 122, 9346
p-Hydroxyacetophenone triplet
D. L. Phillips, JOC 2005, 70, 8661 Tomáš Šolomek
25
p-Hydroxyacetophenone triplet state in water
Dominik Heger, Lubica Klíčová, Peter Šebej, Tomáš Šolomek
Ionization via an ion pair intermediate
Ionization competes with release in pHP cages
26
W. F. van Gunsteren, Angew. Chemie 2001, 40, 351
Van Gunsteren
Protein folding
Energy Landscapes in Protein Folding
Flat Energy Surface Smooth Funnel Rough Landscape
K. Dill, NSB, 4, 10-19, 1997
27
Triplet–triplet energy transfer
350 400 450 500 550 600 650
0.0
0.2
0.4
0.6
0.8
λ/ nm
20 ns
100 ns
1 µs
10 µs
25 µs
S
O
3 * 3 *
² A
S
O
3 * 3 *
Triplet–triplet energy transfer
0
200
400
600
400450
500550
600650
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0
200
400
600
400450
500550
600650
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
Time [ns]Wavelength [nm]
TransientTripletAbsorbance
S
O
O N (Gly–Ser)3–Gly–Ala–(Gly–Ser)–CO2
–
28
Kiefhaber, PNAS 1999, 96, 9597
The speed limit of protein folding
k0=(8.7±0.8)·107 s-1
k ~ n-2.1±0.4
Petr Klán,
Yavor Kamdzhilov
NO2
X
NO2
X
OMe
MeO
Ph
O
X
Ph
O
X
OMe
OMe
O
X
O
O
X
O
X
OMeO O
X
HO O
N
OMe
HO
O
X
29
Photoenolization of 2-Methylacetophenone
Helv. Chim. Acta 1977, 60, 259
O
S1 (n,π*)
T1 (n,π*)
CH2 OH
3E
OH
OH
Z E
slow, requires proton transfer
JACS 2001, 123, 7931
MeOH
CH2
+ OH
O
MeO
– Cl–
MeOH
–H+
CH2 OH
Cl
CH2
+ CH2
O–
CH2
O
Cl
H
– HCl
O
benzene
Z E
+H+
–H+
Formation (inset) and decay kinetics
of the xylylenol transient (λ0bs = 390 nm)
formed by LFP of 1 in benzene solution.
Phenacyl chloride
30
Petr Klán and coworkers, Brno
Dimethyl phenacyl cages
O
X
X = OPO(OEt)2, OSO2R PPS, 2002, 1, 920
MeO
O
Cl hν
O
O
moist solvent dry solvent
MeO
JOC, 2006, 71, 8050
PPS, 2007, 6, 50
PPS, 2005, 4, 43
JPC A, 2001, 105, 10329
O N
O
amines, amino acids
alcohols, phenols
carboxylic acids
O O
R
O
O R
O
5-Ethylnaphthoquinone protecting group
L O
O
OO
O
O
L L
MnO2
+
Δ
L = Br, OAc, OPO(OEt)2
O
O
L–
O
O
hν
+ H+
+
O
O
H
L L
Photochem. Photobiol. Sci. 2007, 6, 865
31
0 2 4 6 8 10 12 14 16 18
0.00
0.05
0.10
0.15
0.20
O
O
OH
O
Br
+ HBr
H
570 nm
385 nm
ΔA
t / μs
350 400 450 500 550 600
0.0
0.1
0.2
0.3
O H
O
O PO(O Et)2
H
O
O
ΔA
λ / nm
quantum yields of release in aqueous solution:
HBr (X=Br): 0.35 ± 0.06 (X=diethyl phosphate): 0.70 ± 0.08
5-Ethylnaphthoquinone protecting group
X
O
O
X
OH
O
O
O
hν
+ XH
X– = Br–, AcO–, (OEt)2OPO–
Bogdan Tokarczyk
NO2
X
NO2
X
OMe
MeO
Ph
O
X
Ph
O
X
OMe
OMe
O
X
O
O
X
O
X
OMeO O
X
HO O
N
OMe
HO
O
X
32
In search of fast alcohol release
N
OMe
N
hν
+ MeO–
+
MeOH
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75
0.00
0.01
0.02
0.03
0.04
0.05
566 nm
fit
490 nm
fit
ΔOD
tim e / ns
440 ± 40 ps
-1 0 1 2 3 4 5 6
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
D a ta
F it
intensity(au)
tim e / n s
480 ± 50 ps
Prof. W. Abraham, Berlin
Jürgen Wintner, Pavel Müller
NO2
X
NO2
X
OMe
MeO
Ph
O
X
Ph
O
X
OMe
OMe
O
X
O
O
X
O
X
OMeO O
X
HO O
N
OMe
HO
O
X
33
Caged fluorescers
Zhao, JACS 2004,
126, 4653
Belov, Angew.
2010, 49, 3520
del Marmol, Anal.
Chem. 2010,
82, 6259
Coumarinmethyl cages
R. Schmidt, V. Hagen,
JOC 2010, 75, 2790;
JPC A, 2007, 111, 5768
34
pKa,1 = 3.44, pKa,2 = 6.31
Titration
1 2 3 4 5 6 7 8 9
0.0
0.2
0.4
0.6
0.8
1.0
pHc/ctot
O
+
OHHO
O OHO
O O–O
400 500
0.0
0.1
0.2
0.3
0.4
0.5
0.6
λ/nmA O OHO
O O–O
O
+
OHHO
O
+
OHHO
O O–O
O OHO
O
+
OHHO
O OHO O OHHO
O O–O O O–HO
O O––O
K
K–
K+
= E+
E
E–
E2–
pKK
a,1 = 3.5
pKE = 2
pK'
E = 3
pKE
a,1 = 4.5
pKK
a,2 = 6.3 pKE
a,2 = 7.3
pKK
a,3 = 13.6 pKE
a,3 = 10.6
rate determining
425 nm
450, 470 nm
482 nm
35
2 4 6 8 10 12 14
-6
-4
-2
0
pH
log(k/s-1)
O OHHO O OHO
Preliminary work
??
Pavel Müller, Jürgen WintnerP. Klán and coworkers
36
Desiderata
Conclusions
• Can YOU use phototriggers?
• Leaving group ability
• Release rate
• Solubility
• Stability in the dark
• Time-resolved X-ray of Enzymes
• Neuronal signal transmission
• Transport phenomena
37
Questions?