Highly efficient photolabile protecting groups with intramolecular energy transfer

Article abstract of DOI:10.1002/anie.200504091

(Chemical Equation Presented) Brought to light: The light sensitivity of photolabile protecting groups of the nitrophenylpropoxycarbonyl (NPPOC) type was enhanced by up to a factor of 25. This was achieved by covalently linking the protecting group to a sensitizer with a much better absorptivity and the ability to transfer its electronic excitation to the protecting group (see scheme).

Full text of DOI:10.1002/anie.200504091

Angewandte
Chemie
principle, the complete human genome can be probed with
[
3]
such a chip. The required high spot densities can be achieved
by photolithographic in situ DNA-chip synthesis. In this
method, the oligonucleotides are synthesized step by step
from protected nucleoside phosphitamides using photolithog-
[
4]
[5]
raphy with masks or micromirror arrays as suitable means
for achieving parallel spatial addressability. This technique
[
6]
requires photolabile protecting groups of high light sensi-
tivity that release their substrate (in this case a nucleotide) in
nearly quantitative yield. For the protecting groups currently
in use for DNA-chip synthesis, the light sensitivity, suitably
characterized by the product ef of the absorption coefficient
and the photochemical quantum yield, reaches only moderate
values. The widely used [(a-methyl-2-nitropiperonyl)oxy]car-
[
7]
bonyl (MeNPOC) group has a reasonable absorption
coefficient at the wavelengths usually applied (in practice
preferentially the mercury line at l = 366 nm, e
MeOH,366 nm
ꢁ1
ꢁ1
ꢀ
2500 m cm ), but the photochemical deprotection yield
is quite small (3% in MeOH). In contrast, the 2-(2-
[
8]
[
9]
nitrophenyl)propoxycarbonyl (NPPOC) protecting group,
[
10]
the reaction of which is represented in Scheme 1, shows a
Intramolecular Sensitization
DOI: 10.1002/anie.200504091
Highly Efficient Photolabile Protecting Groups
with Intramolecular Energy Transfer**
Dominik Wöll, Julia Smirnova, Wolfgang Pfleiderer,
and Ulrich E. Steiner*
Scheme 1. Intramolecular sensitized photocleavage of a protecting
group of the NPPOC type.
[
1]
DNA chips are widely used for genomic analysis. Currently,
high-density DNA chips can be synthesized with up to several
hundred thousand different spots on an area of 1 cm . In
much higher quantum yield (41% in MeOH), but has a
2
[2]
ꢁ1
ꢁ1
significantly lower absorptivity (eMeOH,366 nm ꢀ 230 m cm ).
Thus, for both MeNPOC and NPPOC fairly long irradiation
times are required during which undesired photoreactions
may take place.
[*] D. Wöll, Dr. J. Smirnova, Prof. Dr. Dr. W. Pfleiderer,
Prof. Dr. U. E. Steiner
Sensitization is a suitable method to improve the light
sensitivity of weakly absorbing photolabile protecting
Fachbereich Chemie, Universität Konstanz
Universitätstrasse 10, 78464 Konstanz (Germany)
Fax: (+49)7531-88-3014
[
11,12]
groups.
By using the triplet sensitizer thioxanthone to
sensitize the photocleavage of NPPOC it has been possible to
significantly improve the rate of photodeprotection in homo-
E-mail: ulrich.steiner@uni-konstanz.de
[**] We thank the Deutsche Forschungsgemeinschaft for financial
[
11]
geneous solution as well as on a chip. In this case, most of
the light energy is absorbed by the sensitizer and can be
support (project Ste 283/7-1) and NimbleGen GmbH, Waldkraiburg
(
Germany), for DNA chip experiments.
Angew. Chem. Int. Ed. 2006, 45, 2975 –2978
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2975

Communications
transferred to the protecting group if an encounter with a
triplet-excited sensitizer molecule takes place during the
lifetime of the latter. Diffusion is the limiting factor for the
effectiveness of energy transfer under such intermolecular
sensitization conditions. Various examples of intramolecular
[
13]
triplet–triplet energy transfer have been described.
In this communication, we report the development of
novel photolabile protecting groups wherein a diffusion-
independent enhancement of light sensitivity is achieved by
covalently linking the protecting group to a sensitizer
[
14]
chromophore. Recently the enhancement of photolysis of
a photolabile protecting group by a covalently linked antenna
[
15]
molecule was also reported by the group of Corrie.
The syntheses of the compounds shown in Scheme 2 have
been described. Except for compound 7, the linkage of the
Figure 1. Absorption spectra of compounds 1–7 in methanol.
[
16]
contributes only a fairly weak absorption; the thymidine
moiety does not absorb at all in the spectral range shown.
Compound 6 is the only one exhibiting a direct electronic
coupling between the two chromophores through the p-
electron system. As a result of the thioxanthone moiety the
bichromophoric protecting groups 2–5 and 7 exhibit absorp-
tion coefficients at 366 nm 15 times higher, and compound 6
even about 35 times higher, than that of the NPPOC
protecting group 1. Thus, for unchanged photochemical
quantum yield, an approximately 15 or 35 times higher light
sensitivity would result. To test this expectation, the thymi-
dines capped with the new protecting groups were subject to
continuous irradiation for defined periods of time (condi-
tions: 0.05 and 0.15 mm in methanol, photon irradiance about
ꢁ
8
ꢁ2 ꢁ1
2
” 10 Einsteincm
s
as determined by azobenzene acti-
[
17]
nometry ). The products were separated by HPLC and
analyzed by UV/Vis spectrometry. The decay kinetics of the
starting compound is described by the rate law given in
Equation (1). Here I represents the photon irradiance, F the
0
dc
dt
F d ð1ꢁ10ꢁAðtÞÞ
¼
ꢁI0
e cðtÞ ꢀ
ð1Þ
V
AðtÞ
illuminated cross section, d the optical path length, V the
volume of the solution, A(t) the absorbance of the solution
after irradiation time t, e the molar absorption coefficient of
the protected compound, c its concentration, and f the
quantum yield of the photodeprotection reaction. The
quantum yield f is determined by fitting the result c(t) of
the numerical integration of Equation (1) to the observed
time dependence (Figure 2a).
The data in Table 1 indicate that the quantum yields of the
bichromophoric protecting groups are somewhat lower than
that of the NPPOC protecting group. However, this reduction
of the quantum yield is overcompensated by the increased
absorption coefficient such that the resulting overall enhance-
ment of light sensitivity reaches a factor of up to 21 at 366 nm
and 25 at 405 nm. For compounds 4–7 the yields of depro-
tected substrate range from 66 to 92% (Table 1). A further
improvement of these values by systematic optimization of
the reaction conditions should be possible. The poor thymi-
dine yield of compounds 2 and 3 is a result of specific
photochemical side reactions. Whereas the major reaction of
Scheme 2. Overview of the discussed compounds. R=5’-O-thymidinyl.
o-nitrophenyl and thioxanthone chromophores at different
positions and with different linkers was achieved by C–C
coupling reactions (Sonogashira, Heck, Suzuki). For synthe-
sizing the protected thymidines (R = 5’-O-thymidinyl) con-
nected to the protecting group by means of a carbonate
bridge, the free alcohols (R = H) were first treated with
phosgene or phosgene substitutes and then with thymidine.
Figure 1 shows the absorption spectra of the protected
thymidines. Except for compound 6, the absorptions are
essentially determined by the thioxanthone moiety. Signifi-
cant spectral changes in the substituted thioxanthones appear
in the second absorption band for the 2-vinyl- and the 2-
ethynyl-substituted derivatives. The NPPOC chromophore
2
976 www.angewandte.org
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Angewandte
Chemie
well as the formation of the aci-nitro form of the NPPOC
group, a known intermediate of the deprotection step of
[
10]
NPPOC, could be detected.
In N -saturated solution, the thioxanthone triplet lifetimes
2
for all bichromophoric compounds are much shorter than for
unmodified thioxanthone (see Figure 2 and Table 1). The
combination of this finding and the similarity of the quantum
yields of sensitized and directly excited photoreaction of
NPPOC proves that triplet quenching arises mainly from
energy transfer. Not only the ground-state absorption spec-
trum of compound 6 (see Figure 1) but also its excited-state
absorption spectrum differs significantly from the corre-
sponding ones obtained for the other linked thioxanthones. In
6, the two chromophores are strongly coupled and therefore
cannot be regarded as independent. In this system, the
lifetime of the observed transient is presumably determined
by the intramolecular H-transfer step initiating the photo-
reaction. The photochemical quantum yield of 6 is compara-
ble to that of the other linked thioxanthone–NPPOC
conjugates connected by longer links with less direct coupling.
In aerated solution, molecular oxygen acts as triplet
quencher in a close-to-diffusion-controlled reaction and
reduces the triplet lifetime of all thioxanthone compounds
to a value in the 100-ns range. Since this process competes
with energy transfer it reduces the quantum yield of the
photocleavage. If intramolecular energy transfer is fast or, as
in compound 6, if the lifetime of the triplet is short because of
its fast reaction, the light sensitivity of the photoreaction
depends much less on the oxygen content in solution than
with a free triplet sensitizer. Therefore, the photokinetics of
compounds 4–6 are not very sensitive to the presence of
oxygen. This trend could also be demonstrated for the new
protecting groups under conditions of continuous illumina-
tion in aerated solution. The reduced sensitivity towards
oxygen is an important advantage for their application in the
industrial production of DNA chips because an oxygen-free
process would be technically more demanding.
Figure 2. a) Decomposition kinetics of the protected thymidines 1–7
when continuously irradiated at l=366 nm. The photon irradiance I
0
ꢁ8
ꢁ2 ꢁ1
was on the order of 2”10 Einsteincm
s . Data points were
calculated by integration of the HPLC peaks observed after the
corresponding irradiation times. Curves were fitted to the data points
according to Equation (1). b) Decay kinetics of the thioxanthone triplet
observed by transient absorption at 600 nm after ns laser flash
photolysis (excitation at 355 nm). No triplet signal could be observed
for compound 3.
compound 3 is a trans–cis isomerization, the main product
from compound 2 is a rearrangement product of identical
molar mass but with an as-yet unidentified structure.
Compounds 4 and 6 have already been tested for DNA-
chip synthesis. Under optimized conditions, the cycle times
could be reduced by a factor of 10 compared to the reaction
time with NPPOC-T (compound 1, R = 5’-O-thymidinyl). The
yield per synthesis cycle was about 90%.
To prove that the increased light sensitivity is a result of an
intramolecular triplet energy transfer, nanosecond laser flash
spectroscopic experiments were performed. Transient absorp-
tion spectra as well as decay curves of the thioxanthone triplet
were recorded. Following the change of the transient absorp-
tion spectra in time, the triplet–triplet absorption decay as
Although the involvement of triplet–triplet energy trans-
fer has been clearly demonstrated for the new compounds, it
must be noted that sensitization of the photocleavage reaction
cannot be explained exclusively on the basis of this mecha-
nism. Such a conclusion follows from the observation of
considerable fluorescence quenching, particularly in the cases
with the shortest linkers, which is paralleled by a correspond-
ingly reduced amount of triplet formation, but which does not
[
a]
Table 1: Spectroscopic and photochemical data for compounds 1–7.
ꢁ1
ꢁ1
ꢁ1
ꢁ1
e [m cm
366 nm
]
f
ef [m cm
366 nm
]
Thymidine yield
t [ms]
Air-sat.
Cmpd.
405 nm
366 nm
405 nm
405 nm
366 nm
405 nm
N -sat.
2
1
2
3
4
5
6
7
250
3500
3100
3800
3900
8200
4000
<40
2700
3400
2300
2300
2400
1800
0.41
0.42
0.08
0.21
0.21
0.26
0.09
–
100
1500
250
800
820
<40
1000
170
320
320
650
160
0.90
0.21
0.27
0.86
0.75
0.66
0.92
–
[b]
1.05
[c]
0.11
0.04
0.13
3.6
[b]
0.18
[c]
0.08
0.03
0.12
0.22
0.38
0.05
0.14
0.14
0.27
0.09
0.33
0.38
0.75
0.79
0.89
0.82
2100
360
[
[
a] Molar decadic absorption coefficient e, photochemical quantum yield f, light sensitivity ef, deprotected thymidine yield, and triplet lifetime t.
b] For unsubstituted thioxanthone the triplet lifetime is 22.2 ms in N -saturated MeOH and 0.18 ms in air-saturated MeOH. [c] No transient with a
2
lifetime in the nanosecond time range could be detected.
Angew. Chem. Int. Ed. 2006, 45, 2975 –2978
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
2977

Communications
go along with a decrease of the overall photochemical
quantum yield. Detailed investigations to clarify the under-
lying mechanism are in progress.
[5] S. Singh-Gasson, R. D. Green, Y. Yue, C. Nelson, F. Blattner,
M. R. Sussman, F. Cerrina, Nat. Biotechnol. 1999, 17, 974 – 978.
[
6] “Photoremovable Protecting Groups in DNA Synthesis and
Microarray Fabrication”: M. C. Pirrung, V. S. Rana in Dynamic
Studies in Biology: Phototriggers, Photoswitches, and Caged
Compounds (Eds.: M. Goeldner, R. S. Givens), Wiley, New
York, 2005, p. 341.
In summary, a series of novel, highly light-sensitive
photolabile protecting groups for light-controlled DNA syn-
thesis has been developed. In these compounds the NPPOC
protecting group is covalently linked to thioxanthone as an
intramolecular antenna. The photochemical kinetics of these
compounds under stationary irradiation conditions has been
quantitatively investigated, and photochemical quantum
yields as well as chemical yields of the photodeprotected
substrate were determined for thymidine as a model sub-
strate. The kinetics of triplet–triplet energy transfer between
the antenna molecule and the photolabile protecting group
has been investigated by laser flash spectroscopy. Besides
triplet–triplet energy transfer, a sensitization mechanism
involving the excited sensitizer singlet must be also involved,
particularly in the systems with short linkers. As a result of the
high light sensitivity of these new protecting groups, it should
be possible to reduce the production time for the photolitho-
graphic synthesis of high-density DNA chips.
[7] a) G. H. McGall, A. D. Barone, M. Diggelmann, S. P. A. Fodor,
E. Gentalen, N. Ngo, J. Am. Chem. Soc. 1997, 119, 5081 – 5090;
b) A. C. Pease, D. Solas, E. J. Sullivan, M. T. Cronin, C. P.
Holmes, S. P. A. Fodor, Proc. Natl. Acad. Sci. USA 1994, 91,
5022 – 5026.
[
8] The quantum yield for MeNPOC was determined under
identical condition as for NPPOC (MeOH, irradiation at
366 nm).
[9] a) A. Hasan, K.-P. Stengele, H. Giegrich, P. Cornwell, K. R.
Isham, R. A. Sachleben, W. Pfleiderer, R. S. Foote, Tetrahedron
1997, 53, 4247 – 4264; b) H. Giegrich, S. Eisele-Bühler, C.
Hermann, E. Kvasyuk, R. Charubala, W. Pfleiderer, Nucleosides
Nucleotides 1998, 17, 1987 – 1996; c) S. Bühler, I. Lagoya, H.
Giegrich, K.-P. Stengele, W. Pfleiderer, Helv. Chim. Acta 2004,
87, 620 – 659.
[10] S. Walbert, W. Pfleiderer, U. E. Steiner, Helv. Chim. Acta 2001,
84, 1601 – 1611.
11] D. Wöll, S. Walbert, K.-P. Stengele, T. J. Albert, T. Richmond, J.
Norton, M. Singer, R. D. Green, W. Pfleiderer, U. E. Steiner,
Helv. Chim. Acta 2004, 87, 28 – 45.
[
Received: November 17, 2005
Published online: March 23, 2006
[
12] a) C. Sundararajan, D. E. Falvey, J. Am. Chem. Soc. 2005, 127,
8
000 – 8001; b) C. Sundararajan, D. E. Falvey, Org. Lett. 2005, 7,
2631 – 2634; c) A. Banerjee, K. Lee, D. E. Falvey, Tetrahedron
999, 55, 12699 – 12710.
Keywords: DNA chips · intramolecular energy transfer ·
photochemistry · protecting groups · sensitization
.
1
[
[
[
13] S. Speiser, Chem. Rev. 1996, 96, 1953 – 1976 and references
therein.
[
1] An excellent overview is given in the special issue Nat Genet.
999, 21 (1), 1 – 60..
14] U. E. Steiner, Universitꢀt Konstanz, D. Wöll, DE 10315772 (A1),
WO 2004089529, 2004 [Chem. Abstr. 2004, 141, 366034].
15] G. Papageorgiou, M. Lukeman, P. Wan, J. E. T. Corrie, Photo-
chem. Photobiol. Sci. 2004, 3, 366 – 373; G. Papageorgiou, D.
Ogden, J. E. T. Corrie, J. Org. Chem. 2004, 69, 7228 – 7233.
16] J. Smirnova, D. Wöll, W. Pfleiderer, U. E. Steiner, Helv. Chim.
Acta 2005, 88, 891 – 904.
1
[
2] K.-P. Stengele, U. E. Steiner, D. Wöll, W. Pfleiderer, S. Bühler, J.
Bühler, R. Green, Abstr. Pap. Solid Phase Synthesis & Combi-
natorial Libraries, London, United Kingdom, 2003; http://www.
biocom.uk/sps2003.htm.
[
[
[
[
3] NimbleGen Systems Inc., NimbleGen Releases First Human
Whole-Genome Array, Pressemitteilung, Madison, WI, 28. Juli
17] G. Gauglitz, J. Photochem. 1976, 5, 41 – 47; G. Gauglitz, S. Hubig,
J. Photochem. 1981, 15, 255 – 257.
2003;
http://www.nimblegen.com/news/press_rel/news_2003
_07_28.html.
4] S. P. A. Fodor, J. L. Read, M. C. Pirrung, L. Stryer, A. T. Lu, D.
Solas, Science 1991, 251, 767 – 773.
2
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